A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging.

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.

New Derivatization Reagent for Detection of free Thiol-groups in Metabolites and Proteins in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Several diseases are associated with disturbed redox signaling and altered metabolism of sulfur-containing metabolites and proteins. Importantly, oxidative degradation of fresh-frozen tissues begins within the normal time scale of MALDI MSI sample preparation. As a result, analytical methods that preserve the redox state of the tissue are urgently needed for refined studies of the underlying mechanisms. Nevertheless, no derivatization strategy for free sulfhydryl groups in tissue is known for MALDI MSI. Here, we report the first derivatization reagent, (E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)acrylamide (CHC-Mal), for selective detection of free thiols using MALDI MSI. We performed in situ derivatization of free thiol groups from thiol-containing metabolites such as glutathione and cysteine and reduced proteins such as insulin and imaged their spatial distribution in porcine and mouse xenograft tissue. Derivatization of thiol-containing metabolites with CHC-Mal for MALDI MSI was also possible when using aged tissue in the presence of excess reducing agents. Importantly, CHC-Mal-derivatized low mass-metabolites could be detected without the use of a conventional MALDI matrix.

Old subjects with sepsis in the emergency department: trend analysis of case fatality rate.

BACKGROUND: The burden of sepsis represents a global health care problem. We aimed to assess the case fatality rate (CFR) and its predictors in subjects with sepsis admitted to a general Italian hospital from 2009 to 2016, stratified by risk score. METHODS: We performed a retrospective analysis of all sepsis-related hospitalizations after Emergency Department (ED) visit in a public Italian hospital in an 8-year period. A risk score to predict CFR was computed by logistic regression analysis of selected variables in a training set (2009-2012), and then confirmed in the whole study population. A trend analysis of CFR during the study period was performed dividing patient as high-risk (upper tertile of risk score) or low-risk. RESULTS: Two thousand four hundred ninety-two subjects were included. Over time the incidental admission rate (no. of sepsis-related admissions per 100 total admissions) increased from 4.1% (2009-2010) to 5.4% (2015-2016); P < 0.001, accompanied by a reduced CFR (from 38.0 to 18.4%; P < 0.001). A group of 10 variables (admission to intensive care unit, cardio-vascular dysfunction, HIV infection, diabetes, age ≥ 80 years, respiratory diseases, number of organ dysfunction, digestive diseases, dementia and cancer) were selected by the logistic model to predict CFR with good accuracy: AUC 0.873 [0.009]. Along the years CFR decreased from 31.8% (2009-2010) to 25.0% (2015-2016); P = 0.007. The relative proportion of subjects ≥80 years (overall, 52.9% of cases) and classified as high-risk did not change along the years. CFR decreased only in low-risk subjects (from 13.3 to 5.2%; P < 0.001), and particularly in those aged ≥80 (from 18.2 to 6.6%; P = 0.003), but not in high-risk individuals (from 69.9 to 64.2%; P = 0.713). CONCLUSION: Between 2009 and 2016 the incidence of sepsis-related hospitalization increased in a general Italian hospital, with a downward trend in CFR, only limited to low-risk patients and particularly to subjects ≥80 years.

Filling the gap between the OR and virtual simulation: a European study on a basic neurosurgical procedure.

BACKGROUND: Currently available simulators are supposed to allow young neurosurgeons to hone their technical skills in a safe environment, without causing any unnecessary harm to their patients caused by their inexperience. For this training method to be largely accepted in neurosurgery, it is necessary to prove simulation efficacy by means of large-scale clinical validation studies. METHODS: We correlated and analysed the performance at a simulator and the actual operative skills of different neurosurgeons (construct validity). We conducted a study involving 92 residents and attending neurosurgeons from different European Centres; each participant had to perform a virtual task, namely the placement of an external ventricular drain (EVD) at a neurosurgical simulator (ImmersiveTouch). The number of attempts needed to reach the ventricles and the accuracy in positioning the catheter were assessed. RESULTS: Data suggests a positive correlation between subjects who placed more EVDs in the previous year and those who get better scores at the simulator (p = .008) (fewer attempts and better surgical accuracy). The number of attempts to reach the ventricle was also analysed; senior residents needed fewer attempts (mean = 2.26; SD = 1.11) than junior residents (mean = 3.12; SD = 1.05) (p = .007) and staff neurosurgeons (mean = 2.89, SD = 1.23). Scoring results were compared by using the Fisher's test, for the analysis of the variances, and the Student's T test. Surprisingly, having a wider surgical experience overall does not correlate with the best performance at the simulator. CONCLUSION: The performance of an EVD placement on a simulator correlates with the density of the neurosurgical experience for that specific task performed in the OR, suggesting that simulators are able to differentiate neurosurgeons according to their surgical ability. Namely this suggests that the simulation performance reflects the surgeons' consistency in placing EVDs in the last year.

Fluorogenic probes for live-cell imaging of the cytoskeleton.

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.

Divergent solid-phase synthesis of natural product-inspired bipartite cyclodepsipeptides: total synthesis of seragamide†A.

Macrocyclic natural products (NPs) and analogues thereof often show high affinity, selectivity, and metabolic stability, and methods for the synthesis of NP-like macrocycle collections are of major current interest. We report an efficient solid-phase/cyclorelease method for the synthesis of a collection of macrocyclic depsipeptides with bipartite peptide/polyketide structure inspired by the very potent F-actin stabilizing depsipeptides of the jasplakinolide/geodiamolide class. The method includes the assembly of an acyclic precursor chain on a polymeric carrier, terminated by olefins that constitute complementary fragments of the polyketide section and cyclization by means of a relay-ring-closing metathesis (RRCM). The method was validated in the first total synthesis of the actin-stabilizing cyclodepsipeptide seragamide A and the synthesis of a collection of structurally diverse bipartite depsipeptides.

Biology-oriented synthesis of a natural-product inspired oxepane collection yields a small-molecule activator of the Wnt-pathway.

In Biology Oriented Synthesis the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is met by the structurally complex scaffolds of natural products (NPs) selected in evolution. The synthesis of NP-inspired compound collections approaching the complexity of NPs calls for the development of efficient synthetic methods. We have developed a one pot 4-7 step synthesis of mono-, bi-, and tricyclic oxepanes that resemble the core scaffolds of numerous NPs with diverse bioactivities. This sequence entails a ring-closing ene-yne metathesis reaction as key step and makes productive use of polymer-immobilized scavenger reagents. Biological profiling of a corresponding focused compound collection in a reporter gene assay monitoring for Wnt-signaling modulation revealed active Wntepanes. This unique class of small-molecule activators of the Wnt pathway modulates the van-Gogh-like receptor proteins (Vangl), which were previously identified in noncanonical Wnt signaling, and acts in synergy with the canonical activator protein (Wnt-3a).

Influence of Hematocrit Level and Integrin αIIbßIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.

Purpose: Shear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated-specifically the effect of elevated hematocrit has not yet been demonstrated. Methods: Here, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s-1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored. Results: Increasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbßIII function and the presence of red blood cells. Inhibition of αIIbßIII results in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1 compared to 40% and 60%. Inhibition of αIIbßIII triggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbßIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbßIII blocking antibody, is transient and did not lead to sustained thrombus formation. Conclusions: This study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbßIII-mediated adhesion at supraphysiological shear rates. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00796-0.

Rectus sheath block added to parasternal block may improve postoperative pain control and respiratory performance after cardiac surgery: a superiority single-blinded randomized controlled clinical trial.

BACKGROUND: The population undergoing cardiac surgery confronts challenges from uncontrolled post-sternotomy pain, with possible adverse effects on outcome. While the parasternal block can improve analgesia, its coverage may be insufficient to cover epigastric area. In this non-blinded randomized controlled study, we evaluated the analgesic and respiratory effect of adding a rectus sheath block to a parasternal block. METHODS: 58 patients undergoing cardiac surgery via median sternotomy were randomly assigned to receive parasternal block with rectus sheath block (experimental) or parasternal block with epigastric exit sites of chest drains receiving surgical infiltration of local anesthetic (control). The primary outcome of this study was pain at rest at extubation. We also assessed pain scores at rest and during respiratory exercises, opiate consumption and respiratory performance during the first 24 hours after extubation. RESULTS: The median (IQR) maximum pain scores (on a 0-10 Numeric Rate Scale (NRS)) at extubation were 4 (4, 4) in the rectus sheath group and 5 (4, 5) in the control group (difference 1, p value=0.03). Rectus sheath block reduced opioid utilization by 2 mg over 24 hours (IC 95% 0.0 to 2.0; p<0.01), reduced NRS scores at other time points, and improved respiratory performance at 6, 12, and 24 hours after extubation. CONCLUSION: The addition of a rectus sheath block with a parasternal block improves analgesia for cardiac surgery requiring chest drains emerging in the epigastric area. TRIAL REGISTRATION NUMBER: NCT05764616.

Selective chemical imaging of static actin in live cells.

We have characterized rationally designed and optimized analogues of the actin-stabilizing natural products jasplakinolide and chondramide C. Efficient actin staining was achieved in fixed permeabilized and non-permeabilized cells using different combinations of dye and linker length, thus highlighting the degree of molecular flexibility of the natural product scaffold. Investigations into synthetically accessible, non-toxic analogues have led to the characterization of a powerful cell-permeable probe to selectively image static, long-lived actin filaments against dynamic F-actin and monomeric G-actin populations in live cells, with negligible disruption of rapid actin dynamics.

Triple Monitoring May Avoid Intraneural Injection during Interscalene Brachial Plexus Block for Arthroscopic Shoulder Surgery: A Prospective Preliminary Study.

Nerve injury is a feared complication of peripheral nerve blockade. The aim of this study was to test the effectiveness of a triple monitoring (TM), i.e., a combination of ultrasound (US), nerve stimulation (NS) and opening injection pressure (OIP) during interscalene brachial plexus block (IBPB) for surgery of the shoulder. Sixty patients undergoing IBPB for shoulder arthroscopy received TM. BSmart®, an inline injection device connected to a 10 mL syringe, was used to detect OIP during IBPB. Nerve stimulation was set to 0.5 mA to rule out any motor response, and if OIP was below 15 PSI, 10 mL of local anaesthetic was injected under US guidance between the C5 and C6 roots. The main outcome was the ability of TM to detect a needle-nerve contact. Other outcomes including the duration of IBPB; pain during injection; postoperative neurologic dysfunction. Triple monitoring revealed needle-nerve contact in 33 patients (55%). In 18 patients, NS evoked motor responses despite first control with US; in a further 15 patients, BSmart® detected an OIP higher than 15 PSI, despite the absence of motor response to NS. Mean duration of IBPB was 67.2 ± 5.3 seconds; neither pain during injection nor postoperative neurologic dysfunctions were detected. Clinical follow up excluded the presence of postoperative neuropathies. Triple monitoring showed to be a useful and feasible tool while performing IBPB for arthroscopic shoulder surgery. Future studies will be needed to confirm our findings.

Targeting Alzheimer's disease: Novel indanone hybrids bearing a pharmacophoric fragment of AP2238.

We report on a series of hybrid compounds structurally derived from donepezil and AP2238. This study was aimed at improving the activities of the reference compounds, donepezil and AP2238, and at broadening the range of activities of new derivatives as, due to the multifactorial nature of AD, molecules that modulate the activity of a single protein target are unable to significantly modify the progression of the disease. In particular, the indanone core from donepezil was linked to the phenyl-N-methylbenzylamino moiety from AP2238, through a double bond that was kept to evaluate the role of a lower flexibility in the biological activities. Moreover, SAR studies were performed to evaluate the role of different substituents in position 5 or 6 of the indanone ring in the interaction with the PAS, introducing also alkyl chains of different lengths carrying different amines at one end. Derivatives 21 and 22 proved to be the most active within the series and their potencies against AChE were in the same order of magnitude of the reference compounds. Compounds 15, 21-22, with a 5-carbon alkyl chain bearing an amino moiety at one end, better contacting the PAS, remarkably improved the inhibition of AChE-induced Abeta aggregation with respect to the reference compounds. They also showed activity against self-aggregation of Abeta(42) peptide, the most amyloidogenic form of amyloid produced in AD brains, while the reference compounds resulted completely ineffective.

Stress Hyperglycemia and Mortality in Subjects With Diabetes and Sepsis.

OBJECTIVES: Poor glycemic control is associated with mortality in critical patients with diabetes. The aim of the study was to assess the predicting value of stress hyperglycemia in patients with diabetes following hospital admission for sepsis. DESIGN: Retrospective observational study. SETTING: Adult, emergency department, and critical care in a district hospital. PATIENTS: In a 10-year retrospective analysis of sepsis-related hospitalizations in the emergency department, we carried out a secondary analysis of 915 patients with diabetes (males, 54.0%) in whom both fasting glucose at entry and glycosylated hemoglobin were available. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients' mean age was 79.0 (sd 11.0), glucose at admission was 174.0 mg/dL (74.3 mg/dL), and glycosylated hemoglobin was 7.7% (1.7%). Stress hyperglycemia was defined by the stress hyperglycemia ratio, that is, fasting glucose concentration at admission divided by the estimated average glucose derived from glycosylated hemoglobin. A total of 305 patients died (33.3%) in hospital. Factors associated with in-hospital case fatality rate were tested by multivariable logistic model. Ten variables predicting outcomes in the general population were confirmed in the presence of diabetes (male sex, older age, number of organ dysfunction diagnoses, in particular cardiovascular dysfunction, infection/parasitic, circulatory, respiratory, digestive diseases diagnosis, and Charlson Comorbidity Index). In addition, also glycemic control (glycosylated hemoglobin: odds ratio, 1.17; 95% CI, 1.15-1.40) and stress hyperglycemia (stress hyperglycemia ratio: 5.25; 3.62-7.63) were significant case fatality rate predictors. High stress hyperglycemia ratio (≥ 1.14) significantly increased the discriminant capacity (area under the receiver operating characteristic curve, 0.864; se, 0.013; p < 0.001). CONCLUSIONS: Stress hyperglycemia, even in the presence of diabetes, is predictive of mortality following admission for sepsis. Stress hyperglycemia ratio may be used to refine prediction of an unfavorable outcome.

Benzofuran-based hybrid compounds for the inhibition of cholinesterase activity, beta amyloid aggregation, and abeta neurotoxicity.

The complex etiology of Alzheimer's disease (AD) prompts scientists to develop multitarget strategies to combat causes and symptoms. We therefore designed, synthesized, and tested new hybrid molecules linking a benzofuran ring to a N-methyl- N-benzylamine through a heptyloxy chain, affording a series of potential multifunctional drugs for AD. The cholinesterase inhibitory activity was extended to the inhibition of Abeta fibril formation for 1, 3, and 5. Compound 3 showed an additional neuroprotective effect.

Extensive SAR and computational studies of 3-{4-[(benzylmethylamino)methyl]phenyl}-6,7-dimethoxy-2H-2-chromenone (AP2238) derivatives.

AP2238 was the first compound published to bind both anionic sites of the human acetylcholinesterase, allowing the simultaneous inhibition of the catalytic and the amyloid-beta pro-aggregating activities of AChE. Here we attempted to derive a comprehensive structure-activity relationship picture for this molecule, affording 28 derivatives for which AChE and BChE inhibitory activities were evaluated. Selected compounds were also tested for their ability to prevent the AChE-induced Abeta-aggregation. Moreover, docking simulations and molecular orbital calculations were performed.

A preliminary investigation into the physical and chemical properties of biomass ashes used as aggregate fillers for bituminous mixtures.

Fly and bottom ashes are the main by-products arising from the combustion of solid biomass. Since the production of energy from this source is increasing, the processing and disposal of the resulting ashes has become an environmental and economic issue. Such ashes are of interest as a construction material because they are composed of very fine particles similar to fillers normally employed in bituminous and cementitious mixtures. This research investigates the potential use of ash from biomass as filler in bituminous mixtures. The morphological, physical and chemical characteristics of 21 different ashes and two traditional fillers (calcium carbonate and "recovered" plant filler) were evaluated and discussed. Leaching tests, performed in order to quantify the release of pollutants, revealed that five ashes do not comply with the Italian environmental re-use limits. Experimental results show a wide range of values for almost all the investigated properties and a low correlation with biomass type in terms of origin and chemical composition. Furthermore, sieving and milling processes were found to improve the properties of the raw material in terms of grading and sample porosity. The effectiveness of these treatments and the low content of organic matter and harmful fines suggest that most of the biomass ashes investigated may be regarded as potential replacements for natural filler in bituminous mixtures.