The Utility of Urinary Titin to Diagnose and Predict the Prognosis of Acute Myocardial Infarction.

Early detection and management are crucial for better prognosis in acute myocardial infarction (AMI). Serum titin, a component of the sarcomere in cardiac and skeletal muscle, was associated with AMI. Thus, we hypothesized that urinary N-fragment titin may be a biomarker for its diagnosis and prognosis. Between January 2021 and November 2021, we prospectively enrolled 83 patients with suspected AMI. Their urinary N-fragment titin, serum high-sensitivity troponin I (hsTnI), creatine kinase (CK), and creatine kinase-MB (CK-MB) were measured on admission. Then, urinary titin was assessed as diagnostic and prognostic biomarker in AMI. Among 83 enrolled patients, 51 patients were diagnosed as AMI. In AMI patients who were admitted as early as 3 h or longer after symptom onset, their urinary titin levels were significantly higher than non-AMI patients who are also admitted 3 h or longer after symptom onset (12.76 [IQR 5.87-16.68] pmol/mgCr (creatinine) and 5.13 [IQR 3.93-11.25] pmol/mgCr, p = 0.045, respectively). Moreover, the urinary titin levels in patients who died during hospitalization were incredibly higher than in those who were discharged (15.90 [IQR 13.46-22.61] pmol/mgCr and 4.90 [IQR 3.55-11.95] pmol/mgCr, p = 0.023). Urinary N-fragment titin can be used as non-invasive early diagnostic biomarker in AMI. Furthermore, it associates with hospital discharge disposition, providing prognostic utility.

Mild traumatic brain injury: not always a mild injury.

PURPOSE: In general, risk of mortality after trauma correlates with injury severity. Despite arriving in relatively stable clinical condition, however, some patients are at risk of death following mild traumatic brain injury (TBI). The study objective was delineation of patients who die in-hospital following mild isolated TBI in order to inform Emergency Department (ED) disposition and care discussions with patients and families. METHODS: In this retrospective cohort study, patients from the National Trauma Data Bank (NTDB) (2007-2018) were included if they were injured by blunt trauma and sustained a mild TBI (defined as Head Abbreviated Injury Scale [AIS] score of 1 or 2 and arrival Glasgow Coma Scale [GCS] score of 13-15). Exclusions were severe associated injuries (extracranial AIS > 2); transfers; and missing data. Patients were defined by in-hospital mortality: Survivors vs. Mortalities. Demographics, clinical/injury data, and the outcomes were collected and compared with univariate analysis. Multivariate analysis established independent factors associated with in-hospital mortality following mild TBI. RESULTS: In total, 932,107 patients (10% of NTDB population) met study criteria: 928,542 (99.6%) Survivors and 3,565 (0.4%) Mortalities. In general, comorbidities (including home anticoagulation, cardiac disease, and diabetes mellitus) were significantly more common among patients who died (p < 0.001), although drug and alcohol intoxication on arrival were more common among Survivors (16% vs. 7%, p < 0.001; 13% vs. 10%, p < 0.001). In terms of insurance status, Private/Commercial insurance was more common among Survivors (39% vs. 20%, p < 0.001) while Governmental Insurance was more common among Mortalities (55% vs. 36%, p < 0.001). On multivariate analysis, age ≥ 65 was most strongly associated with death (OR 26.43, p < 0.001), followed by ED intubation (OR 10.08, p < 0.001), admission hypotension (OR 4.55, p < 0.001), and comorbidities, particularly end-stage renal disease (ESRD) (OR 3.03, p < 0.001) and immunosuppression (OR 2.18, p < 0.001). CONCLUSIONS: Survivors differed substantially from Mortalities after mild TBI in terms of comorbidities, intoxicants, and insurance status. Independent variables most strongly associated with in-hospital death following mild head injury included age ≥ 65, intubation in the ED, admission hypotension, and comorbidities (particularly ESRD and immunosuppression). Increased clinical vigilance, including a mandatory period of clinical observation, for patients with these risk factors should be considered to optimize outcomes and potentially mitigate death after mild TBI.

Resuscitative Endovascular Balloon Occlusion of the Aorta is Associated with Increased Risk of Extremity Compartment Syndrome.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) has been used as a temporizing procedure to control intra-abdominal or pelvic bleeding. Theoretically, occlusion of the aorta and the resulting ischemia-reperfusion of the lower extremities may increase the risk of extremity compartment syndrome (CS). To date, no study has addressed systematically the incidence and risk factors of CS following REBOA intervention. The purpose of this study was to address this knowledge gap. METHODS: Adult trauma patients from the American College of Surgeons Trauma Quality Improvement Program (ACS-TQIP) database (2016-2019) were included. Patients who received REBOA within 4 h of admission were compared to patients without REBOA after propensity score matching for demographics, vital signs on admission, comorbidities, injury severity of different body regions, pelvic and lower extremity fractures, vascular trauma to the lower extremities, fixation for fractures, angioembolization (AE) for pelvis, preperitoneal pelvic packing (PPP), laparotomy, and venous thromboembolism (VTE) prophylaxis. The primary outcomes were rates of lower extremity CS and fasciotomy and acute kidney injury (AKI). Secondary outcomes included mortality. RESULTS: There were 534 patients who received REBOA matched with 1043 patients without REBOA. Overall, patients in the REBOA group had significantly higher rates of CS than no REBOA patients [5.4% vs 1.1%, p < 0.001, OR: 5.39]. The risk of CS remained significantly higher in the subgroups of patients with or without pelvic or lower extremity fractures, as well as in the subgroup of patients with associated extremity vascular injury [11.2% vs 1.5%, p < 0.001, OR: 8.12].The fasciotomy and AKI rates were significantly higher in the REBOA group (5.8% vs 1.2%, p < 0.001 and 12.9% vs 7.4%, p< 0.001 respectively). CONCLUSION: REBOA use is associated with a higher risk of lower extremity CS, fasciotomy and AKI, especially in patients with associated lower extremity vascular injuries. These complications should be taken into account when considering REBOA use, and close observation for this complication should always be part of the routine monitoring.

Clinical Utility of Overlap Time for Incomplete Relaxation to Predict Cardiac Events in Heart Failure.

BACKGROUND: The overlap time of transmitral flow can be a novel marker of subclinical left ventricular dysfunction for predicting adverse events in heart failure (HF). We aimed to (1) investigate the role of overlap time of the E-A wave in association with clinical parameters and (2) evaluate whether the overlap time could add prognostic information with respect to other conventional clinical prognosticators in HF. METHODS: We prospectively evaluated 153 patients hospitalized with HF (mean age 68 ± 15 years; 63% male). The primary endpoint was readmission following HF or cardiac death. RESULTS: During a median period of 25 months, 43 patients were readmitted or died. Overlap time appeared to be associated with worse outcomes. After adjustment for readmission scores and ratios of diastolic filling period and cardiac cycle length in a Cox proportional-hazards model, overlap time was associated with event-free survival, independent of elevated left atrial pressure based on guidelines. When overlap time was added to the model based on clinical variables and elevated left atrial pressure, the C-statistic significantly improved from 0.70 (95% CI: 0.63-0.77) to 0.77 (95% CI: 0.69-0.83, compared) (P = 0.035). CONCLUSION: This preliminary study suggested that prolonged overlap time may have potential for predicting readmission and cardiac mortality risk assessment in patients with HF.

Cardiac reserve by 6-minute walk stress echocardiography in systemic sclerosis.

OBJECTIVES: There is a high prevalence of left ventricular diastolic dysfunction (LVDD) in systemic sclerosis (SSc) which is associated with high mortality. Thus, early detection of LVDD could be important in management of SSc. We hypothesised that exercise echocardiography in SSc patients with normal resting haemodynamics may expose early phase LVDD, which could affect its prognosis, defined as cardiovascular death and unplanned hospitalisation for heart failure. METHODS: Between January 2014 and December 2018, we prospectively enrolled 140 patients with SSc who underwent 6-minute walk (6MW) stress echocardiographic studies with normal range of estimated mean pulmonary arterial pressure (mPAP) (<25 mm Hg) and mean pulmonary artery wedge pressure (mPAWP) (<15 mm Hg) at rest. We used ΔmPAP/Δcardiac output (CO) to assess pulmonary vascular reserve and ΔmPAWP/ΔCO to assess LV cardiac reserve between resting and post-6MW. RESULTS: During a median period of 3.6 years (IQR 2.0-5.1 years), 25 patients (18%) reached the composite outcome. Both ΔmPAP/ΔCO and ΔmPAWP/ΔCO in patients with events were significantly greater than in those without events (8.9±3.8 mm Hg/L/min vs 3.0±1.7 mm Hg/L/min; p=0.002, and 2.2±0.9 mm Hg/L/min vs 0.9±0.5 mm Hg/L/min; p<0.001, respectively). Patients with both impaired LV cardiac reserve (ΔmPAWP/ΔCO>1.4 mm Hg/L/min) and impaired pulmonary vascular reserve (ΔmPAP/ΔCO>3.0 mm Hg/L/min) had worse outcomes compared with those without these abnormalities (p<0.001). CONCLUSION: The 6MW stress echocardiography revealed impaired LV cardiac reserve in SSc patients with normal resting haemodynamics. Furthermore, LV cardiac reserve independently associates with clinical worsening in SSc, providing incremental prognostic utility, in addition to pulmonary vascular parameters.

Three cases of paediatric pancreatic injury involving the main pancreatic duct.

We report on 3 patients aged 9-12 years with pancreatic injury involving the main pancreatic duct. None of them presented with shock. They were initially transported to secondary emergency care facilities, leading to delays in diagnosis and treatment. Two patients underwent organ (spleen and pancreatic tail)-preserving surgery and one underwent non-operative management (NOM). They recovered and were discharged without major complications. Although the indications for NOM for paediatric pancreatic injury might increase in the future, we believe that it is preferable for patients to be transferred to the tertiary care hospital from the very beginning to recieve appropriate diagnosis and treatment.

The ferrous-oxy complex of human aromatase.

In this communication, we document the self-assembly of heterologously expressed truncated human aromatase (CYP19) into nanometer scale phospholipids bilayers (Nanodiscs). The resulting P450 CYP19 preparation is stable and can tightly associate with the substrate androstenedione to form a nearly complete high-spin ferric protein. Ferrous CYP19 in Nanodiscs was mixed anaerobically in a rapid-scan stopped-flow with atmospheric dioxygen and the formation of the ferrous-oxy complex observed. First order decay of the oxy-complex to release superoxide and regenerate the ferric enzyme was monitored kinetically. Surprisingly, the ferrous-oxy complex of aromatase is more stable than that of hepatic CYP3A4, opening the path to precisely determine the biochemical and biophysical properties of the reaction cycle intermediates in this important human drug target.

Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy.

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli.

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.

CYP51 from Trypanosoma cruzi: a phyla-specific residue in the B' helix defines substrate preferences of sterol 14alpha-demethylase.

A potential drug target for treatment of Chagas disease, sterol 14alpha-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from approximately 25% between phyla to approximately 80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B' helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14alpha-demethylation of obtusifoliol and its 24-demethyl analog 4alpha-,4alpha-dimethylcholesta-8,24-dien-3beta-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced approximately 3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites.

Comparison of the 1.85 A structure of CYP154A1 from Streptomyces coelicolor A3(2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways.

The genus Streptomyces produces two-thirds of microbially derived antibiotics. Polyketides form the largest and most diverse group of these natural products. Antibiotic diversity of polyketides is generated during their biosynthesis by several means, including postpolyketide modification performed by oxidoreductases, a broad group of enzymes including cytochrome P450 monooxygenases (CYPs). CYPs catalyze site-specific oxidation of macrolide antibiotic precursors significantly affecting antibiotic activity. Efficient manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of monooxygenases with activities toward a diverse array of chemical substrates. To begin to link structure to function of CYPs involved in secondary metabolic pathways of industrially important species, we determined the X-ray structure of Streptomyces coelicolor A3(2) CYP154A1 at 1.85 A and analyzed it in the context of the closely related CYP154C1 and more distant CYPs from polyketide synthase (EryF) and nonribosomal peptide synthetase (OxyB) biosynthetic pathways. In contrast to CYP154C1, CYP154A1 reveals an active site inaccessible from the molecular surface, and an absence of catalytic activities observed for CYP154C1. Systematic variations in the amino acid patterns and length of the surface HI loop correlate with degree of rotation of the F and G helices relative to the active site in CYP154A1-related CYPs, presumably regulating the degree of active site accessibility and its dimensions. Heme in CYP154A1 is in a 180 degrees flipped orientation compared with most other structurally determined CYPs.

The 1.92-A structure of Streptomyces coelicolor A3(2) CYP154C1. A new monooxygenase that functionalizes macrolide ring systems.

Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O(2)-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code ). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional beta-strand transforming the five-stranded beta-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactones in vitro.