Implementation of a Multimodal Knowledge-Exchange Platform to Provide Trauma Critical Care Education During the Ongoing Conflict in Ukraine.

Importance: The conflict in Ukraine has forced civilian hospitals with limited trauma and battlefield medicine experience to care for casualties of war, placing significant strain on the health care system. Using the Checklist for Early Recognition and Treatment of Acute Illness and Injury (CERTAIN) program, a multimodal trauma critical care knowledge-exchange platform was created for clinicians practicing in these institutions. Objectives: To describe the development and implementation of the CERTAIN for Ukraine program and to evaluate the reach of this intervention, together with participant engagement and satisfaction. Design, Setting, and Participants: This quality improvement study included clinicians caring for critically ill patients during the ongoing Ukrainian conflict who were part of a community developed using a messaging app. The program was implemented by a group of international trauma and critical care experts in collaboration with critical care leaders from the Shupyk National Healthcare University in Kyiv, Ukraine. This study evaluates data collected from the CERTAIN for Ukraine program from its launch on April 9, 2022, to August 31, 2022. Interventions: The initiative comprised a longitudinal series of interactive tele-education sessions, a webpage containing the CERTAIN approach and current trauma critical care guidelines translated into Ukrainian and Russian, and a private messaging chat for asynchronous discussion. Main Outcomes and Measures: Participant engagement and satisfaction were tracked using multimedia analytics and a post-session survey. Results: Since program launch, 838 participants have joined the messaging group, and 6 tele-education sessions have been delivered, with 1835 total views. The CERTAIN website has had 3527 visits, mainly from Ukraine (1378 [39%]) and the United States (1060 [30%]). Of the 74 completed postsession surveys, 65 respondents (88%) rated the course content excellent or very good, and 73 (99%) recommended it to others. Conclusions and Relevance: The findings of this quality improvement study indicate that, using widely available and low-cost platforms, knowledge was shared rapidly and efficiently to a large community of clinicians practicing in a wartime environment with broad-based engagement and a high level of learner satisfaction.

Thienoguanosine, a unique non-perturbing reporter for investigating rotational dynamics of DNA duplexes and their complexes with proteins.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine (thG), a fluorescent analogue of guanosine, overcomes all these limitations. We recorded the TRFA decays of thG-labelled dsDNA of different lengths. thG behaved as a rigid, non-perturbing reporter, since no fast correlation time was recorded for any tested dsDNA. Due to its perfect stacking, only two correlation times, instead of the typical three, describe thG-labelled dsDNA rotational dynamics. Thanks to these features, we provided a complete description of the tumbling of the different dsDNA and their complexes with the Set and Ring Associated (SRA) domain of UHRF1, a key epigenetic regulator, obtaining values in excellent agreement with theoretical predictions. Moreover, thG was also found sensitive to SRA-induced base flipping of neighboring nucleobases. In the DNA label toolbox, thG thus stands out as a unique reporter for investigating the rotational dynamics of dsDNA and protein/dsDNA complexes.

Environmentally sensitive fluorescent nucleoside analogues as probes for nucleic acid - protein interactions: molecular design and biosensing applications.

Fluorescent nucleoside analogues (FNAs) are indispensable in studying the interactions of nucleic acids with nucleic acid-binding proteins. By replacing one of the poorly emissive natural nucleosides, FNAs enable real-time optical monitoring of the binding interactions in solutions, under physiologically relevant conditions, with high sensitivity. Besides that, FNAs are widely used to probe conformational dynamics of biomolecular complexes using time-resolved fluorescence methods. Because of that, FNAs are tools of high utility for fundamental biological research, with potential applications in molecular diagnostics and drug discovery. Here I review the structural and physical factors that can be used for the conversion of the molecular binding events into a detectable fluorescence output. Typical environmentally sensitive FNAs, their properties and applications, and future challenges in the field are discussed.

Scientists without borders: lessons from Ukraine.

Conflicts and natural disasters affect entire populations of the countries involved and, in addition to the thousands of lives destroyed, have a substantial negative impact on the scientific advances these countries provide. The unprovoked invasion of Ukraine by Russia, the devastating earthquake in Turkey and Syria, and the ongoing conflicts in the Middle East are just a few examples. Millions of people have been killed or displaced, their futures uncertain. These events have resulted in extensive infrastructure collapse, with loss of electricity, transportation, and access to services. Schools, universities, and research centers have been destroyed along with decades' worth of data, samples, and findings. Scholars in disaster areas face short- and long-term problems in terms of what they can accomplish now for obtaining grants and for employment in the long run. In our interconnected world, conflicts and disasters are no longer a local problem but have wide-ranging impacts on the entire world, both now and in the future. Here, we focus on the current and ongoing impact of war on the scientific community within Ukraine and from this draw lessons that can be applied to all affected countries where scientists at risk are facing hardship. We present and classify examples of effective and feasible mechanisms used to support researchers in countries facing hardship and discuss how these can be implemented with help from the international scientific community and what more is desperately needed. Reaching out, providing accessible training opportunities, and developing collaborations should increase inclusion and connectivity, support scientific advancements within affected communities, and expedite postwar and disaster recovery.

Fundamental photophysics of isomorphic and expanded fluorescent nucleoside analogues.

Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.

ACUTE ISCHEMIC STROKE IN WOMEN: EFFICACY OF THE FREE RADICAL SCAVENGER EDARAVONE.

OBJECTIVE: The aim: To investigate the effectiveness of usage of the free radical scavenger Edaravone in the therapy of women with AIS. PATIENTS AND METHODS: Materials and methods: A prospective study was conducted of 48 women with AIS, divided into two groups. Patients in the first group (n = 36) were treated with edaravone 30 mg twice a day intravenously. Neuroprotectors were not used in the control group (n = 12). Clinical-instrumental and neurological examination (Glasgow scale (SCG), FOUR, NIHSS, and neuronspecific enolase (NSE) levels) were performed on all patients. RESULTS: Results: The mean FOUR score in the 1th group increased from 11.04±0.85 to 15.47±0.63 points against 11.39±0.56 to 13.46±1.49 in the control group (pp<0.05). The level of NSE in control group patients increased 10-fold (from 9.2 to 96.4 ng/ml, p<0.01). Subsequently, there was a rapid decrease in NSE level in 1th group, and in the control group until 10 days of treatment, the level of NSE did not reach the reference values (p p<0.05). CONCLUSION: Conclusions: The introduction of edaravone in women with AIS results in positive results already in the acute period of the disease. The use of edaravon was significantly effective on the FOUR scale and the dynamics of NSE levels.

Extracorporeal Membrane Oxygenation during Percutaneous Coronary Intervention in Patients with Coronary Heart Disease.

Extracorporeal membrane oxygenation (ECMO) has become an effective method in the treatment of adults and children with severe cardiac and pulmonary dysfunction that is resistant to conventional therapy. The aim of this article was to summarize an experience of ECMO usage for cardiac dysfunction, which develops in patients with coronary heart disease (CHD) during percutaneous transluminal coronary angioplasty. The study comprised a retrospective, single-center analysis of 23 patients with CHD (19 men and four women, average age 65.7 ± 12.3 years), who undertook the ECMO technique during percutaneous transluminal coronary angioplasty. A total of 13 (56.52%) patients died directly in the hospital, or 30 days after a discharge. Independent predictors of fatal outcomes were diabetes mellitus (odds ratio [OR] = 17.58; 95% confidence interval [CI] = 6.47-47.48; p = .00125), chronic renal failure (CRF) (OR = 20.81; 95% CI = 5.95-72.21; p = .00014), and damage to the right coronary artery (RCA) (OR = 25.51; 95% CI = 8.27-79.12; p = .00013). For deceased patients, the "no reflow" phenomenon was indicated in a larger percentage of cases (23.1% in the group of deceased, vs. 10% in the group of survivors). A routine connection to ECMO before the occurrence of cardiac events was significantly more often used in the group of survived patients (90% of cases) than in the deceased (p = .0000001). Diabetes mellitus, CRF, and damage to the RCA were independent predictors of mortality during percutaneous transluminal coronary angioplasty in patients with CHD. The routine use of ECMO in high-risk patients with percutaneous transluminal coronary angioplasty was a positive prognostic factor of patient survival.

Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels.

Fluorescence labeling and probing are fundamental techniques for nucleic acid analysis and quantification. However, new fluorescent probes and approaches are urgently needed in order to accurately determine structural and conformational dynamics of DNA and RNA at the level of single nucleobases/base pairs, and to probe the interactions between nucleic acids with proteins. This review describes the means by which to achieve these goals using nucleobase replacement or modification with advanced fluorescent dyes that respond by the changing of their fluorescence parameters to their local environment (altered polarity, hydration, flipping dynamics, and formation/breaking of hydrogen bonds).

Suppression of mICAT in Mouse Small Intestinal Myocytes by General Anaesthetic Ketamine and its Recovery by TRPC4 Agonist (-)-englerin A.

A better understanding of the negative impact of general anesthetics on gastrointestinal motility requires thorough knowledge of their molecular targets. In this respect the muscarinic cationic current (mICAT carried mainly via TRPC4 channels) that initiates cholinergic excitation-contraction coupling in the gut is of special interest. Here we aimed to characterize the effects of one of the most commonly used "dissociative anesthetics", ketamine, on mICAT. Patch-clamp and tensiometry techniques were used to investigate the mechanisms of the inhibitory effects of ketamine on mICAT in single mouse ileal myocytes, as well as on intestinal motility. Ketamine (100 µM) strongly inhibited both carbachol- and GTPγS-induced mICAT. The inhibition was slow (time constant of about 1 min) and practically irreversible. It was associated with altered voltage dependence and kinetics of mICAT. In functional tests, ketamine suppressed both spontaneous and carbachol-induced contractions of small intestine. Importantly, inhibited by ketamine mICAT could be restored by direct TRPC4 agonist (-)-englerin A. We identified mICAT as a novel target for ketamine. Signal transduction leading to TRPC4 channel opening is disrupted by ketamine mainly downstream of muscarinic receptor activation, but does not involve TRPC4 per se. Direct TRPC4 agonists may be used for the correction of gastrointestinal disorders provoked by general anesthesia.

A Genetically Encoded Diazirine Analogue for RNA-Protein Photo-crosslinking.

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA-protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA-protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA-protein interactions.

The Small Non-coding Vault RNA1-1 Acts as a Riboregulator of Autophagy.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Brightly Fluorescent 2'-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry.

Synthesis of cytosine, uracil, and 7-deazaadenine 2'-deoxyribonucleosides and triphosphates (dNTPs) bearing hexamethylated phenyl-bodipy fluorophore attached at position 5 of pyrimidines or at position 7 of 7-deazapurine was developed. All the title labeled nucleosides and dNTPs displayed bright green fluorescence with very high quantum yields. The modified dNmBdpTPs were good substrates to diverse DNA polymerases and were used for in vitro enzymatic synthesis of labeled DNA by primer extension or PCR. In combination with cationic cyclodextrin-peptide-based dNTP transporter, the dNmBdpTPs were successfully used for staining of genomic DNA in living cells for applications in confocal microscopy and in flow cytometry. The best performing cytosine nucleotide dCmBdpTP was used to monitor mitosis in live cells.

Environmentally Sensitive Fluorescent Nucleoside Analogues for Surveying Dynamic Interconversions of Nucleic Acid Structures.

Nucleic acids are characterized by a variety of dynamically interconverting structures that play a major role in transcriptional and translational regulation as well as recombination and repair. To monitor these interconversions, Förster resonance energy transfer (FRET)-based techniques can be used, but require two fluorophores that are typically large and can alter the DNA/RNA structure and protein binding. Additionally, events that do not alter the donor/acceptor distance and/or angular relationship are frequently left undetected. A more benign approach relies on fluorescent nucleobases that can substitute their native counterparts with minimal perturbation, such as the recently developed 2-thienyl-3-hydroxychromone (3HCnt) and thienoguanosine (th G). To demonstrate the potency of 3HCnt and th G in deciphering interconversion mechanisms, we used the conversion of the (-)DNA copy of the HIV-1 primer binding site (-)PBS stem-loop into (+)/(-)PBS duplex, as a model system. When incorporated into the (-)PBS loop, the two probes were found to be highly sensitive to the individual steps both in the absence and the presence of a nucleic acid chaperone, providing the first complete mechanistic description of this critical process in HIV-1 replication. The combination of the two distinct probes appears to be instrumental for characterizing structural transitions of nucleic acids under various stimuli.

A Bifunctional Noncanonical Amino Acid: Synthesis, Expression, and Residue-Specific Proteome-wide Incorporation.

Mapping of weak and hence transient interactions between low-abundance interacting molecules is still a major challenge in systems biology and protein biochemistry. Therefore, additional system-wide acting tools are needed to determine protein interactomics. Most important are reagents that can be applied at any kind of protein interface and the possibility to enrich cross-linked fragments with high efficiency. In this study, we report the synthesis of a novel noncanonical amino acid that features a diazirine group for ultraviolet cross-linking as well as an alkyne group for labeling by click chemistry. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal structural perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell cultures, and we show that PrDiAzK allows protein labeling as well as cross-linking. In addition, we tested PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation, implying potential applications in system-wide mapping of protein-protein interactions in the future.

Environmentally sensitive probes for monitoring protein-membrane interactions at nanomolar concentrations.

Solvatochromic probes are suitable tools for quantitative characterization of protein-membrane interactions. Based on diverse fluorophores these probes have different fluorescent properties and therefore demonstrate different responses when applied for sensing the interactions of biomolecules. Surprisingly, to the best of our knowledge, no systematic comparison of the sensitivities of solvatochromic dyes for monitoring protein-membrane interactions was described. Hence, a rational choice of an optimal environmentally sensitive probe for such experiments is usually not a straightforward task. In this work we developed a series of thiol-reactive fluorescent probes based on the fluorophores with high sensitivity to their environment and compared them with two widely used DNS and DMN probes. We investigated the responses of these probes to the interaction of probe-labeled presynaptic protein α-synuclein with lipid membranes. We observed that newly synthesized probes based on fluorene and chromone dyes, which combine the strongest brightness and significant changes of fluorescence intensity, demonstrated the highest sensitivity to interaction of α-synuclein with lipid membranes. They are especially beneficial for sensing in scattering media such as solutions of lipid vesicles. We show that the described probes permit quantitative measurements of α-synuclein binding to lipid membranes at low nanomolar concentrations. We developed a detailed protocol for measuring Kd and binding stoichiometry for interaction of soluble peripheral proteins with membranes based on the response of the environmentally sensitive fluorescent probes. We applied this protocol for quantification of the affinity of α-synuclein to anionic membranes and found that it is substantially higher than it was earlier reported.

A Rotational BODIPY Nucleotide: An Environment-Sensitive Fluorescence-Lifetime Probe for DNA Interactions and Applications in Live-Cell Microscopy.

Fluorescent probes for detecting the physical properties of cellular structures have become valuable tools in life sciences. The fluorescence lifetime of molecular rotors can be used to report on variations in local molecular packing or viscosity. We used a nucleoside linked to a meso-substituted BODIPY fluorescent molecular rotor (dC(bdp)) to sense changes in DNA microenvironment both in vitro and in living cells. DNA incorporating dC(bdp) can respond to interactions with DNA-binding proteins and lipids by changes in the fluorescence lifetimes in the range 0.5-2.2 ns. We can directly visualize changes in the local environment of exogenous DNA during transfection of living cells. Relatively long fluorescence lifetimes and extensive contrast for detecting changes in the microenvironment together with good photostability and versatility for DNA synthesis make this probe suitable for analysis of DNA-associated processes, cellular structures, and also DNA-based nanomaterials.