Thyrotropin Receptor Antagonism by a Novel Small Molecule: Preclinical In Vitro Observations.

Background: Treatment of Graves' hyperthyroidism (GH) and Graves' orbitopathy (GO) is far from adequate, and hence, new substances that specifically target the autoantigens in GH/GO are warranted. This study determined the preclinical in vitro efficacy of SYD5115, a novel low-molecular-weight compound that inhibits the thyrotropin receptor (TSH-R). Methods: The TSH-R inhibiting capability of SYD5115 was tested through stimulation of wild-type and chimeric TSH-R expressed in Chinese hamster ovary (CHO) cells using two functional (stimulatory and blocking) cell-based TSH-R-Ab bioassays. TSH-R expressing human orbital fibroblasts, collected from GH+GO patients (GOF), were stimulated with the monoclonal antibody M22 or with stimulatory TSH-R-Ab (TSAb)-positive sera with cyclic adenosine monophosphate (cAMP) or hyaluronic acid (HA) release as readouts. The effect of SYD5115 on the viability of GOF was tested in 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and scratch cell growth assays. Results: SYD5115 significantly and dose dependently inhibited the TSH-R activation through M22 or TSAb-positive sera in all performed bioassays. Inhibition showed similar levels in the TSAb reporter bioassay and in the cAMP assay with GOF. The % inhibition and compound concentration showed a sigmoidal relationship, with all seven TSAb-positive sera markedly inhibited by SYD5115. An SYD5115 dose-dependent inhibition of M22 (10 ng/mL, 6 hours)-stimulated HA and/or cAMP-release from GOF was observed. Strong SYD5115-induced inhibitions of M22-stimulated cAMP production in GOF were registered with SYD5115 concentrations of 1 (p = 0.0029), 10 (p < 0.0001), 100 (p < 0.0001), 1,000 (p < 0.0001), and 10,000 (p < 0.0001) nM, respectively. SYD5115-induced inhibition of M22-stimulated HA production was noted with SYD5115 concentrations of 100 (p = 0.0392), 1000 (p = 0.0431), and 10,000 (p = 0.0245) nM, respectively. The inhibitory activity of SYD5115 was confirmed in a human osteosarcoma U2OS cell line stably expressing human TSH-R with cAMP as readout. SYD5115 induced 100% inhibition of the M22-induced cAMP levels with a potency of 193 nM. Compared with control, SYD5115 did neither impact the growth nor the migration of cultivated GOF. In addition, SYD5115 did not alter the viability of GOF. Conclusions: SYD5115 blocked M22- and TSAb-induced TSH-R activity with a nanomolar potency in TSH-R-overexpressed CHO cells as well as primary GOF, which demonstrates the ability of this small molecule to block TSH-R overactivity.

Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling.

Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein-protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-l-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-l-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the single-step Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.

Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

Beyond protein tagging: Rewiring the genetic code of fluorescent proteins - A review.

Fluorescent proteins (FP) are an integral part of modern biology due to its diverse biochemical and photophysical properties. The boundaries of FP have been extended through conventional mutagenesis and directed evolution approaches. Engineering of FP based on the standard genetic code consisting of 20 amino acids with limited functional groups restrict its diversification. Degeneracy of genetic code has helped in covering this substantial gap through genetic code engineering, wherein introduction of unnatural amino acid (UAA) analogues resulted in a collection of FP with varying properties. This review features the work carried till date in the area of FP incorporated with UAAs and explores strategies employed for incorporation, impact of UAAs in chromophore and surrounding residues and changes in inherent properties of FP. The long-standing association of FP as a tool for high throughput screening of orthogonal aaRS/tRNA pairs used in site specific incorporation of UAAs is expounded. Insertion of UAAs in FP has enabled their use in contemporary fields such as biophotovoltaics, bioremediation, biosensors, biomaterials and imaging of acidic vesicles. Thus, expansion of genetic code of FP is envisaged to rejig the existing spectra of colors and future research initiative in this direction is expected to glow brighter and brighter.

Clinical Determinants of Myocardial Injury, Detectable and Serial Troponin Levels among Patients with Hypertensive Crisis.

Introduction There is a high prevalence of hypertensive crisis with myocardial injury, as evidenced by elevation in cardiac troponin levels. The risk factors predisposing patients to developing a myocardial injury, detectable troponin, and increase in serial troponin in this population are not known. Methods A retrospective study was designed to include all patients, presenting to the emergency room, diagnosed with hypertensive crisis, using International Classification of Diseases, 10th revision, Clinical Modification (ICD-10-CM) codes between 2016-2018 (n=467). Logistic regression was used to determine the important predictors of myocardial injury evidenced by troponin elevation >99th percentile of upper reference level (URL), detectable troponin (> 0.015 ng/ml), and increase in serial troponin levels. Results The 99th percentile of the initial troponin level among all patients was 0.433 ng/ml. A total of 15% had a myocardial injury, and the significant risk factors associated with it were body mass index (BMI) < 30 kg/m2 (odds ratio [OR] 0.50, confidence interval [CI] 0.28-0.89), congestive heart failure (CHF; OR 4.28, CI 2.21-8.25) and prior use of aspirin (OR 1.98, CI 1.08-3.63). About 35% had detectable troponin, and BMI < 30 kg/m2 (OR 0.62, CI 0.40-0.97), CHF (OR 3.49, CI 2.06-5.9), elevated creatinine (OR 1.17, CI 1.02-1.34) and age <61 years (OR 0.59, CI 0.38-0.94) were associated with it. The factors associated with an increase in serial troponin were BMI < 30 Kg/m2 (OR 0.56, CI 0.36-0.87), CHF (OR 1.78, CI 1.06-3.0), coronary artery disease (CAD; OR 2.08, CI 1.28-3.36) and non-Caucasian race (OR 0.52, CI 0.29-0.93). Conclusion About one-third of patients with the hypertensive crisis have detectable troponin. Still, among these, less than half have troponin levels >99th percentile URL, and the majority of these patients have minimal changes in serial troponin. Low BMI was associated with higher initial and serial troponin levels, and this obesity paradox was stronger among females and older patients.

Correction: The asymmetric syntheses of cryptocaryols A and B.

Correction for 'The asymmetric syntheses of cryptocaryols A and B' by Alhanouf Zakaria Aljahdali et al., Chem. Commun., 2018, 54, 3428-3435.

The asymmetric syntheses of cryptocaryols A and B.

The recent total syntheses of cryptocaryols A and B are reviewed. These efforts include the correction of the initially assigned absolute and relative stereochemistry of this class of natural products. In addition to enabling the initial structure activity relationships for this class of natural products, these syntheses demonstrated the practical utility of several novel synthetic approaches.

Feasibility of early mechanical circulatory support in acute myocardial infarction complicated by cardiogenic shock: The Detroit cardiogenic shock initiative.

OBJECTIVE: The 'Detroit Cardiogenic Shock Initiative' is a single-arm, multicenter study to assess the feasibility of early mechanical circulatory support (MCS) in patients who present with acute myocardial infarction complicated by cardiogenic shock (AMICS) who undergo percutaneous coronary intervention. METHODS: Between July 2016 and February 2017, 4 metro Detroit sites participated in the study. The centers agreed to treat patients with AMICS using a mutually agreed-upon protocol emphasizing invasive hemodynamic monitoring and rapid initiation of MCS. Inclusion and exclusion criteria mimicked those from the 'SHOCK' trial with an additional exclusion criteria being use of intra-aortic balloon pump counter pulsation prior to MCS. RESULTS: A total of 41 consecutive patients were included. Patients had an average age of 65 ± 14 years, 71% were male and 59% of patients were admitted to the hospital in cardiogenic shock. Prior to receiving MCS, 93% of patients were on vasopressors or inotropes, 15% of patients had a witnessed out of hospital cardiac arrest, 27% of patients had an in-hospital cardiac arrest, and 17% were under active cardiopulmonary resuscitation while MCS was being implanted. In accordance to the protocol recommendation, 66% of patients had a MCS device inserted prior to PCI. Right heart catheterization and hemodynamic monitoring was performed in 83% of patients. Door to support times averaged 83 ± 58 minutes and 71% of patients were able to reduce the levels of inotropes and vasopressors within the first 24-hours of their index procedure. Pre-procedure cardiac power output (CPO) was 0.57 W and post-procedure CPO was 0.95 W, a 67% increase (p < 0.001). Survival to explant for the entire cohort was 85% a significant improvement from institutional historical controls (85% vs 51% p < 0.001) and survival to discharge was 76%. CONCLUSION: Centers who adopted a regional shock protocol emphasizing the delivery of early MCS with invasive hemodynamic monitoring can achieve rapid door to support times and can improve survival in patients who present with AMICS. Larger national studies will be needed to further validate this pilot feasibility study.

Development, implementation, and initial evaluation of a foundational open interoperability standard for oncology treatment planning and summarization.

OBJECTIVE: Develop and evaluate a foundational oncology-specific standard for the communication and coordination of care throughout the cancer journey, with early-stage breast cancer as the use case. MATERIALS AND METHODS: Owing to broad uptake of the Health Level Seven (HL7) Consolidated Clinical Document Architecture (C-CDA) by health information exchanges and large provider organizations, we developed an implementation guide in congruence with C-CDA. The resultant product was balloted through the HL7 process and subsequently implemented by two groups: the Health Story Project (Health Story) and the Athena Breast Health Network (Athena). RESULTS: The HL7 Implementation Guide for CDA, Release 2: Clinical Oncology Treatment Plan and Summary, DSTU Release 1 (eCOTPS) was successfully balloted and published as a Draft Standard for Trial Use (DSTU) in October 2013. Health Story successfully implemented the eCOTPS the 2014 meeting of the Healthcare Information and Management Systems Society (HIMSS) in a clinical vignette. During the evaluation and implementation of eCOPS, Athena identified two practical concerns: (1) the need for additional CDA templates specific to their use case; (2) the many-to-many mapping of Athena-defined data elements to eCOTPS. DISCUSSION: Early implementation of eCOTPS has demonstrated successful vendor-agnostic transmission of oncology-specific data. The modularity enabled by the C-CDA framework ensures the relatively straightforward expansion of the eCOTPS to include other cancer subtypes. Lessons learned during the process will strengthen future versions of the standard. CONCLUSION: eCOTPS is the first oncology-specific CDA standard to achieve HL7 DSTU status. Oncology standards will improve care throughout the cancer journey by allowing the efficient transmission of reliable, meaningful, and current clinical data between the many involved stakeholders.

Development and validation of a prognostic index for risk stratification of patients with early repolarization.

BACKGROUND: Early repolarization (ER) is associated with increased mortality in the general population. We sought to develop and validate a prognostic index (PI) of mortality in patients with ER. METHODS: We identified 852 consecutive patients (mean age 49 ± 12 years) with ER (J-point elevation ≥0.1 mV in inferior or lateral leads), from the VA electronic electrocardiogram (ECG) database. A random sample of age-matched patients with normal ECG was used as control (n = 257). The initial cohort was randomly split into a derivation and a validation cohort (2/3 and 1/3 of patients, respectively). A PI was derived from the weighed sum of the regression coefficients of each independent risk factor in the final model using Cox regression analysis. RESULTS: During a median follow-up of 6.4 years, 170 patients died. ER was associated with increased mortality compared to control (HR 1.49, 95% CI 1.05-2.12; P = 0.03). Older age, lower body mass index, non-African American race, current use of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers or sulfonyureas, prolonged corrected QT (QTc), and higher ER amplitude independently predicted all-cause mortality. Annualized mortality rates were 1.3%, 2.2%, and 3.7% in the low, intermediate, and high-risk groups, respectively, in the derivation cohort (log rank P < 0.0001) and 0.8%, 1.9%, and 4.1% in the low, intermediate, and high-risk groups, respectively, in the validation cohort (log rank P < 0.0001). Model discrimination was very good (c-statistic = 0.85 and 0.80 for derivation and validation cohort, respectively). CONCLUSIONS: A PI derived from simple clinical and ECG characteristics predicts mortality in patients with ER and may be used clinically for risk stratification.

Arteriovenous Malformation in a Child with Extensive Involvement of the Mandible and Maxilla.

Arteriovenous malformation of both the maxilla and mandible in a pediatric patient is a very rare phenomenon that carries with it a high mortality rate. Arteriovenous malformations, sometimes known as simple vascular tumors, can be present from birth or acquired over time due to trauma, surgery, or any kind of vascular manipulation of an area of the body.