Association between pre-arrest left ventricular ejection fraction and survival in nontraumatic out-of-hospital cardiac arrest.

BACKGROUND AND IMPORTANCE: Out-of-hospital cardiac arrest (OHCA) poses major public health issues. Pre-arrest heart function is a prognostic factor, but the specific contribution of pre-arrest echocardiographic evaluation in predicting OHCA outcome remains limited. OBJECTIVE: The primary objective was to investigate the association between left ventricular ejection fraction (LVEF) measured in echocardiography prior to OHCA and survival to hospital discharge. DESIGN, SETTINGS, AND PARTICIPANTS: This multicenter retrospective cohort study analyzed data from the National Taiwan University Hospital and its affiliated hospitals. We included adult nontraumatic OHCA patients who were treated by the emergency medical services (EMS) and underwent echocardiography within 6 months prior to the OHCA event from January 2016 to December 2022. Data included demographics, preexisting diseases, resuscitation events, and echocardiographic reports. OUTCOMES MEASURE AND ANALYSIS: The primary outcome was the survival to hospital discharge after post-arrest care. Statistical analysis involved multivariable logistic regression to modify potential confounders, reported as adjusted odds ratio (aOR) and 95% confidence interval (CI), and evaluate the association between echocardiographic findings and survival to hospital discharge. MAIN RESULTS: This study analyzed 950 patients, with 33.6% surviving to discharge. A higher pre-arrest LVEF was independently associated with increased survival. Compared to patients with LVEF < 40%, those with LVEF between 40% and 60% had significantly higher odds of survival (aOR = 3.68, 95% CI = 2.14-6.35, P < 0.001), and those with LVEF > 60% had even greater odds of survival (aOR = 5.46, 95% CI = 3.09-9.66, P < 0.001). There was also an association between lower tricuspid regurgitation pressure gradient and survival (aOR = 0.98, 95% CI = 0.97-1.00, P = 0.015). Younger age, male gender, dyslipidemia, stroke, cancer, witnessed arrest, initial shockable rhythm, and shorter low-flow time are other significant predictors of survival. CONCLUSION: In adult, nontraumatic, EMS-treated OHCA patients, a higher LVEF 6 months prior to OHCA was associated with improved survival at hospital discharge.

Diflunisal versus tafamidis on neuropathy and cardiomyopathy in hereditary transthyretin amyloidosis.

OBJECTIVES: Hereditary transthyretin (TTR) amyloidosis (ATTRv) is frequently complicated by polyneuropathy (ATTRv-PN) and cardiomyopathy (ATTRv-CM). The long-term efficacy of diflunisal on both polyneuropathy and cardiomyopathy in ATTRv patients, especially those with non-V30M genotypes, has not been fully investigated and compared with that of tafamidis. METHODS: We compared the structural and biochemical characteristics of A97S-TTR complexed with tafamidis with those of diflunisal, and prospectively followed up and compared the progression of polyneuropathy and cardiomyopathy between ATTRv-PN patients taking diflunisal and those taking tafamidis. RESULTS: Both diflunisal and tafamidis effectively bind to the two thyroxine-binding sites at the A97S-TTR dimer-dimer interface and equally and almost sufficiently reduce amyloid fibril formation. Thirty-five ATTRv-PN patients receiving diflunisal and 22 patients receiving tafamidis were enrolled. Compared with no treatment, diflunisal treatment significantly delayed the transition of FAP Stage 1 to 2 and Stage 2 to 3 and decreased the deterioration in parameters of the ulnar nerve conduction study (NCS). The progression of FAP stage or NCS parameters did not differ between patients treated with diflunisal and those treated with tafamidis. Both diflunisal and tafamidis treatments significantly decreased radiotracer uptake on 99mTc-PYP SPECT and stabilized cardiac wall thickness and blood pro-B-type natriuretic peptide levels. No significant adverse events occurred during diflunisal or tafamidis treatment. INTERPRETATIONS: The binding patterns of both tafamidis and diflunisal to A97S-TTR closely resembled those observed in the wild type. Diflunisal can effectively delay the progression of polyneuropathy and cardiomyopathy with similar efficacy to tafamidis and may become a cost-effective alternative treatment for late-onset ATTRv-PN.

Disulfiram inhibits coronaviral main protease by conjugating to its substrate entry site.

Coronaviruses (CoV) are highly pathogenic single-strand RNA viruses. CoV infections cause fatal respiratory symptoms and lung injuries in humans and significant economic losses in livestock. Since the SARS-2 outbreak in 2019, the highly conserved main protease (Mpro), also termed 3-chymotrypsin-like protease (3CLpro), has been considered an attractive drug target for treating CoV infections. Mpro mediates the proteolytic cleavage of eleven sites in viral polypeptides necessary for virus replication. Here, we report that disulfiram, an FDA-approved drug for alcoholic treatment, exhibits a broad-spectrum inhibitory effect on CoV Mpros. Analytical ultracentrifugation and circular dichroism analyses indicated that disulfiram treatment blocks the dimeric formation of SARS and PEDV Mpros and decreases the thermostability of SARS, SARS-2, and PEDV Mpros, whereas it facilitates the dimerization and stability of MERS Mpro. Furthermore, mass spectrometry and structural alignment revealed that disulfiram targets the Cys44 residue of Mpros, which is located at the substrate entrance and close to the catalytic His41. In addition, molecular docking analysis suggests that disulfiram conjugation interferes with substrate entry to the catalytic center. In agreement, mutation of Cys44 modulates the disulfiram sensitivity of CoV Mpros. Our study suggests a broad-spectrum inhibitory function of disulfiram against CoV Mpros.

Dual dimeric interactions in the nucleic acid-binding protein Sac10b lead to multiple bridging of double-stranded DNA.

Nucleoid-associated proteins play a crucial role in the compaction and regulation of genetic material across organisms. The Sac10b family, also known as Alba, comprises widely distributed and highly conserved nucleoid-associated proteins found in archaea. Sac10b is identified as the first 10 kDa DNA-binding protein in the thermoacidophile Sulfolobus acidocaldarius. Here, we present the crystal structures of two homologous proteins, Sac10b1 and Sac10b2, as well as the Sac10b1 mutant F59A, determined at a resolution of 1.4-2.0 Å. Electron microscopic images reveal the DNA-bridging capabilities of both Sac10b1 and Sac10b2, albeit to varying extents. Analyses of crystal packing and electron microscopic results suggest that Sac10b1 facilitates cooperative DNA binding, forming extensive bridged filaments via the conserved R58 and F59 residues at the dimer-dimer interface. Substitutions at R58 or F59 of Sac10b1 attenuate end-to-end association, resulting in non-cooperative DNA binding, and formation of small, bridged DNA segments in a way similar to Sac10b2. Analytical ultracentrifuge and circular dichroism confirm the presence of thermostable, acid-tolerant dimers in both Sac10b1 and Sac10b2. These findings attest to the functional role of Sac10b in organizing and stabilizing chromosomal DNA through distinct bridging interactions, particularly under extreme growth conditions.

Structural basis for recruitment of peptidoglycan endopeptidase MepS by lipoprotein NlpI.

Peptidoglycan (PG) sacculi surround the cytoplasmic membrane, maintaining cell integrity by withstanding internal turgor pressure. During cell growth, PG endopeptidases cleave the crosslinks of the fully closed sacculi, allowing for the incorporation of new glycan strands and expansion of the peptidoglycan mesh. Outer-membrane-anchored NlpI associates with hydrolases and synthases near PG synthesis complexes, facilitating spatially close PG hydrolysis. Here, we present the structure of adaptor NlpI in complex with the endopeptidase MepS, revealing atomic details of how NlpI recruits multiple MepS molecules and subsequently influences PG expansion. NlpI binding elicits a disorder-to-order transition in the intrinsically disordered N-terminal of MepS, concomitantly promoting the dimerization of monomeric MepS. This results in the alignment of two asymmetric MepS dimers respectively located on the two opposite sides of the dimerization interface of NlpI, thus enhancing MepS activity in PG hydrolysis. Notably, the protein level of MepS is primarily modulated by the tail-specific protease Prc, which is known to interact with NlpI. The structure of the Prc-NlpI-MepS complex demonstrates that NlpI brings together MepS and Prc, leading to the efficient MepS degradation by Prc. Collectively, our results provide structural insights into the NlpI-enabled avidity effect of cellular endopeptidases and NlpI-directed MepS degradation by Prc.

An Intelligent Water Monitoring IoT System for Ecological Environment and Smart Cities.

Global precipitation is becoming increasingly intense due to the extreme climate. Therefore, creating new technology to manage water resources is crucial. To create a sustainable urban and ecological environment, a water level and water quality control system implementing artificial intelligence is presented in this research. The proposed smart monitoring system consists of four sensors (two different liquid level sensors, a turbidity and pH sensor, and a water oxygen sensor), a control module (an MCU, a motor, a pump, and a drain), and a power and communication system (a solar panel, a battery, and a wireless communication module). The system focuses on low-cost Internet of Things (IoT) devices along with low power consumption and high precision. This proposal collects rainfall from the preceding 10 years in the application region as well as the region's meteorological bureau's weekly weather report and uses artificial intelligence to compute the appropriate water level. More importantly, the adoption of dynamic adjustment systems can reserve and modify water resources in the application region more efficiently. Compared to existing technologies, the measurement approach utilized in this study not only achieves cost savings exceeding 60% but also enhances water level measurement accuracy by over 15% through the successful implementation of water level calibration decisions utilizing multiple distinct sensors. Of greater significance, the dynamic adjustment systems proposed in this research offer the potential for conserving water resources by more than 15% in an effective manner. As a result, the adoption of this technology may efficiently reserve and distribute water resources for smart cities as well as reduce substantial losses caused by anomalous water resources, such as floods, droughts, and ecological concerns.

Colonic Diverticulitis Location Affects the Diagnostic Accuracy of Point-of-Care Ultrasound: A Multicenter, 10-Year Study.

OBJECTIVE: Point-of-care ultrasound (PoCUS) has high sensitivity and specificity in diagnosing uncomplicated colonic diverticulitis in Western patients. Evidence regarding the accuracy of PoCUS in Asian patients in which diverticulitis frequently occurs in the right-side colon is lacking. This multicenter, 10-y study was aimed at evaluating the diagnostic accuracy of PoCUS in various locations of uncomplicated diverticulitis among Asians. METHODS: A convenience sample of patients with suspected colonic diverticulitis who had undergone computed tomography (CT) were eligible. Patients undergoing PoCUS before CT were included. The primary outcome was the diagnostic accuracy of PoCUS in the various locations, compared with the final diagnosis made by the expert physicians. The sensitivity, specificity, positive predictive value and negative predictive value were computed. The logistic regression model was used to investigate the possible factors related to the accuracy of PoCUS. RESULTS: A total of 326 patients were included. The overall accuracy of PoCUS was 92% (95% confidence interval [CI]: 89.1%-95.0%) and was lower in the cecum (84.3%, 95% CI: 77.8%-90.8%), compared with other locations (p < 0.0001). Nine of 10 false positives had the final diagnosis of appendicitis: 5 had an outpouching structure whose origin in the cecum could not be traced and 4 had elongated "diverticula." Moreover, body mass index was negatively associated with the accuracy of PoCUS in cecal diverticulitis (odds ratio: 0.79, 95% CI: 0.64-0.97) after adjusting for other covariates. CONCLUSION: Point-of-care ultrasound exhibits high diagnostic accuracy in diagnosing uncomplicated diverticulitis in the Asian population. However, the accuracy varies according to location, and was relatively low in the cecum.

A molecular basis for tetramer destabilization and aggregation of transthyretin Ala97Ser.

Transthyretin (TTR)-related amyloidosis (ATTR) is a syndrome of diseases characterized by the extracellular deposition of fibrillar materials containing TTR variants. Ala97Ser (A97S) is the major mutation reported in Taiwanese ATTR patients. Here, we combine atomic resolution structural information together with the biochemical data to demonstrate that substitution of polar Ser for a small hydrophobic side chain of Ala at residue 97 of TTR largely influences the local packing density of the FG-loop, thus leading to the conformational instability of native tetramer, the increased monomeric species, and thus the enhanced amyloidogenicity of apo-A97S. Based on calorimetric studies, the tetramer destabilization of A97S can be substantially altered by interacting with native stabilizers via similarly energetic patterns compared to that of wild-type (WT) TTR; however, stabilizer binding partially rearranges the networks of hydrogen bonding in TTR variants while FG-loops of tetrameric A97S still remain relatively flexible. Moreover, TTR in complexed with holo-retinol binding protein 4 is slightly influenced by the structural and dynamic changes of FG-loop caused by A97S substitution with an approximately five-fold difference in binding affinity. Collectively, our findings suggest that the amyloidogenic A97S mutation destabilizes TTR by increasing the flexibility of the FG-loop in the monomer, thus modulating the rate of amyloid fibrillization.

Survival factors in patients of high fall - A 10-year level-I multi-trauma center study.

OBJECTIVES: This study aims to investigate the characteristics of patients after free falls at the Level-I trauma centers. The factors associated with survival were differentiated. METHODS: This retrospective study was conducted at the National Taiwan University Hospital, the Hsin-Chu branch, and the Yun-Lin branch, all accredited as Level-I trauma centers between January 2010 and September 2020. Adult patients with falls from height of more than one story (i.e. 3.6 m) were included. Clinical data were obtained from electronic medical records. Odds ratios (OR) were computed with 95% confidence intervals (CIs) for significant parameters for survival. RESULTS: A total of 371 patients were included. Only 2 survived to discharge with poor neurologic outcomes in 101 patients with OHCA. The overall mortality rate was 98% and 11% in patients with and without OHCA. A higher falling height with a one-meter increase (OR, 1.14, 95% CI, 1.10-1.19) was significantly related to OHCA, especially the height over 6 m (OR, 3.07, 95% CI, 1.19-7.94). A higher trauma injury severity score (TRISS) was significantly related to survival among patients without OHCA (OR, 1.07, 95% CI, 1.04-1.11), especially TRISS≧0.945 (OR, 5.21, 95% CI, 1.28-21.24). Patients without severe head/neck injury of Abbreviated Injury Scale (AIS)≧3 (OR, 0.17, 95% CI, 0.07-0.42) were positively associated with survivors among patients without OHCA. CONCLUSION: Patients with traumatic OHCA following falls had a high mortality rate of 98% and dismal outcomes, compared with non-traumatic OHCA. Falling heights, especially over 6 m was associated with OHCA. Patients without OHCA had a mortality rate of 11%. Patients with a higher TRISS, especially more than 0.945, or without severe head injury had more chances to survive in the non-OHCA group. The study provided the evidence to guide termination of high futility resuscitation for traumatic OHCA secondary to falls to conserve the clinical resources.

N-terminal Backbone Pairing Shifts in CCL5-12AAA14 Dimer Interface: Structural Significance of the FAY Sequence.

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 (12FAY14) sequence is involved in CCL5 aggregation. The CCL5-12AAA14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5-12AAA14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5-12AAA14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12FAY14 sequence.

Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions.

Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4-70 °C, and show that structures of both the Mg2+ form (KARI:2Mg2+) and its ternary complex (KARI:2Mg2+:NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure-function relationships and enhance the mechanistic analysis of enzymatic functions.

Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase.

While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg2+. The results showed that the distances of the two catalytic Mg2+ ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.

NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 µM for NADPH and 6.0 µM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

The Arginine Pairs and C-Termini of the Sso7c4 from Sulfolobus solfataricus Participate in Binding and Bending DNA.

The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22' and R11'/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP, EM and fluorescence resonance energy transfer (FRET) data indicated that the two basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its similarity to Escherichia coli Ecrep 6.8 and Ecrep 7.3 as well as Agrobacterium tumefaciens ACCR in amino acid sequence. Based on these data, we proposed a model of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator protein-DNA complex. The DNA end-to-end distance measured with FRET upon wild-type Sso7c4 binding is almost equal to the distance measured in the model, which supports the fidelity of the proposed model. The FRET data also confirm the EM observation showing that the binding of wild-type Sso7c4 reduces the DNA length while the C-terminal truncation does not. A functional role for Sso7c4 in the organization of chromosomal DNA and/or the regulation of gene expression through bridging and bending interactions is suggested.

Moenomycin Biosynthesis: Structure and Mechanism of Action of the Prenyltransferase MoeN5.

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C25) moenocinyl-sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are "bent" and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics.

Structures of Trypanosome Vacuolar Soluble Pyrophosphatases: Antiparasitic Drug Targets.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases, including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and, as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruzi and T. brucei, together with that of the T. cruzi protein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. On the basis of Cytoscape and other bioinformatics analyses, it is apparent that similar folds will be found in most if not all trypanosomatid VSP1s, including those found in insects (Angomonas deanei, Strigomonas culicis), plant pathogens (Phytomonas spp.), and Leishmania spp. Overall, the results are of general interest since they open the way to structure-based drug design for many of the neglected tropical diseases.