From Didactics to Disasters: Unveiling CBRNe and Counter-Terrorism Medicine Training in US Medical Schools.

INTRODUCTION: The threat of chemical, biological, radiologic, nuclear, and explosive (CBRNe) terrorist attacks has increased over time. The need for rapid and effective responses to such attacks is paramount. Effective medical counter-measures to CBRNe events are critical and training for such may effectively occur early in physician training. While some medical specialties are more involved than others, counter-terrorism medicine (CTM) spans all medical specialties. METHODS: All United States allopathic medical schools were examined via online curriculums and queries for academic content related to CBRNe and terrorist medical counter-measures. RESULTS: Analysis of 153 United States allopathic medical schools demonstrated that 15 (9.8%) medical schools offered educational content related to CBRNe and terrorist counter-measures. This is in contrast to legislation following the September 11, 2001 attacks that called for high priority for such education. CONCLUSION: Effective CBRNe medical counter-measures are currently in place; however, there is room for improvement in education that may begin during medical school. While certain medical specialties such as emergency medicine, primary care, and dermatology may have specific niches in such events, physicians of all medical specialties have something to offer, and even a basic education in medical school can help best prepare the nation for future attacks.

Transcription Factors: The Fulcrum Between Cell Development and Carcinogenesis.

Higher eukaryotic development is a complex and tightly regulated process, whereby transcription factors (TFs) play a key role in controlling the gene regulatory networks. Dysregulation of these regulatory networks has also been associated with carcinogenesis. Transcription factors are key enablers of cancer stemness, which support the maintenance and function of cancer stem cells that are believed to act as seeds for cancer initiation, progression and metastasis, and treatment resistance. One key area of research is to understand how these factors interact and collaborate to define cellular fate during embryogenesis as well as during tumor development. This review focuses on understanding the role of TFs in cell development and cancer. The molecular mechanisms of cell fate decision are of key importance in efforts towards developing better protocols for directed differentiation of cells in research and medicine. We also discuss the dysregulation of TFs and their role in cancer progression and metastasis, exploring TF networks as direct or indirect targets for therapeutic intervention, as well as specific TFs' potential as biomarkers for predicting and monitoring treatment responses.

Assessment of trace inorganic contaminates in water and sediment to address its impact on common fish varieties along Kuwait Bay.

The impact of the trace elements on selected marine fishes/crustacean in Kuwait (Sheam, Lobster, Speatty, and Nagroor) were investigated (As, Cd, Ni, Pb, and V) using the element concentrations in marine water and sediments. The toxic elements concentrations were measured in water samples (As, Cd, Cr, Cu, Hg, Ni, Pb, V, and Zn) for estimation of toxic levels, heavy metal evaluation index (84-360), and the degree of contamination (77-353). Similarly, sediment samples were analyzed for As, Cd, Cr, Cu, Ni, Pb, V and estimated for contamination factor, Igeo index, and ecological risk factor with respect to each element analyzed in the sample. The modified degree of contamination (0.25-3.67), risk index (6.5-282.27), metal pollution index (5.95-18.21), and pollution load index (0.27-1.2) were calculated for the samples. This study demonstrated that the water was medium to high contaminated with Cd, Hg, Pb, and V. The sediment analyses showed that most of the metals were within the toxic limits except for Cd, Cu, and Pb in few samples. Most samples were in between the effect range low-effect range medium and threshold effect level-probable effect level range of most metals, except for Cr, Cu, and Ni. Average trace elements concentration in fishes varieties investigated in this study indicated high As in all varieties irrespective of the season and high Ni in all fish during summer. The bioaccumulation factor showed that the trace elements in sediments contributed more to the fish than water. Concentrations of trace elements were greater in fish sampled in winter than that sampled in summer due to variations in the planktonic population in the sea. The estimated daily intake and the chronic daily intake for the Kuwaiti male and female were calculated. The hazards studied revealed that the consumption of Lobster and Speatty may lead to cancer and non-cancer hazards, in both male and female, Speatty having higher probability. The major sources of toxic elements contamination of Kuwait Bay water and sediment appear to be oil-based contamination, urban sewage, brine from desalination, and the trace elements released due to the natural oxidation-reduction processes.

Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein-Protein Interactions.

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein-protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.

Stapled Peptides Inhibitors: A New Window for Target Drug Discovery.

Protein-protein interaction (PPI) is a hot topic in clinical research as protein networking has a major impact in human disease. Such PPIs are potential drugs targets, leading to the need to inhibit/block specific PPIs. While small molecule inhibitors have had some success and reached clinical trials, they have generally failed to address the flat and large nature of PPI surfaces. As a result, larger biologics were developed for PPI surfaces and they have successfully targeted PPIs located outside the cell. However, biologics have low bioavailability and cannot reach intracellular targets. A novel class -hydrocarbon-stapled α-helical peptides that are synthetic mini-proteins locked into their bioactive structure through site-specific introduction of a chemical linker- has shown promise. Stapled peptides show an ability to inhibit intracellular PPIs that previously have been intractable with traditional small molecule or biologics, suggesting that they offer a novel therapeutic modality. In this review, we highlight what stapling adds to natural-mimicking peptides, describe the revolution of synthetic chemistry techniques and how current drug discovery approaches have been adapted to stabilize active peptide conformations, including ring-closing metathesis (RCM), lactamisation, cycloadditions and reversible reactions. We provide an overview on the available stapled peptide high-resolution structures in the protein data bank, with four selected structures discussed in details due to remarkable interactions of their staple with the target surface. We believe that stapled peptides are promising drug candidates and open the doors for peptide therapeutics to reach currently "undruggable" space.

A fluorinated indole-based MDM2 antagonist selectively inhibits the growth of p53wt osteosarcoma cells.

The p53 protein is engaged in the repair of DNA mutations and elimination of heavily damaged cells, providing anticancer protection. Dysregulation of p53 activity is a crucial step in carcinogenesis. This dysregulation is often caused by the overexpression of negative regulators of p53, among which MDM2 is the most prominent one. Antagonizing MDM2 with small molecules restores the activity of p53 in p53 wild-type (p53wt ) cells and thus provides positive outcomes in the treatment of p53wt cancers. Previously, we have reported the discovery of a panel of fluoro-substituted indole-based antagonists of MDM2. Here, we demonstrate the biological activity and stereoselectivity of the most active compound from this series. Both enantiomers of the esterified form of the compound, as well as its corresponding carboxylic acids, were found active in fluorescence polarization (FP) assay, nuclear magnetic resonance (NMR) and microscale thermophoresis (MST) assay, with Ki and KD values around 1 µm. From these four compounds, the esterified enantiomer (R)-5a was active in cells, which was evidenced by the increase of p53 levels, the induced expression of p53-target genes (CDKN1A and MDM2), the selective induction of cell cycle arrest, and selective growth inhibition of p53wt U-2 OS and SJSA-1 compared to p53del SAOS-2 cells. The analysis of the crystal structure of human MDM2 in complex with the compound (R)-6a (carboxylic acid of the active (R)-5a compound) revealed the classical three-finger binding mode. Altogether, our data demonstrate the activity of the compound and provide the structural basis for further structure optimization.

Impact of Storage Conditions on the Stability of Predominant Phenolic Constituents and Antioxidant Activity of Dried Piper betle Extracts.

The phenolic constituents in Piper betle are well known for their antioxidant potential; however, current literature has very little information on their stability under the influence of storage factors. Present study evaluated the stability of total phenolic content (TPC) and antioxidant activity together with individual phenolic constituents (hydroxychavicol, eugenol, isoeugenol and allylpyrocatechol 3,4-diacetate) present in dried Piper betle's extract under different storage temperature of 5 and 25 °C with and without light for a period of six months. Both light and temperature significantly influenced TPC and its corresponding antioxidant activity over time. More than 95% TPC and antioxidant activity was retained at 5 °C in dark condition after 180 days of storage. Hydroxychavicol demonstrated the best stability with no degradation while eugenol and isoeugenol displayed moderate stability in low temperature (5 °C) and dark conditions. 4-allyl-1,2-diacetoxybenzene was the only compound that underwent complete degradation. A new compound, 2,4-di-tert-butylphenol, was detected after five weeks of storage only in the extracts exposed to light. Both zero-order and first-order kinetic models were adopted to describe the degradation kinetics of the extract's antioxidant activity. Zero-order displayed better fit with higher correlation coefficients (R² = 0.9046) and the half-life was determined as 62 days for the optimised storage conditions (5 °C in dark conditions).

The Pex4p-Pex22p complex from Hansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization.

Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Šresolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.

Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex.

Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation. Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

Assessment of urinary iodine excretion among normal Kuwaiti adults.

This study was performed to investigate the status of iodine intake among the Kuwaiti population and its effect on thyroid function. The study group was comprised of 139 females and 86 males with a mean age of 33 and 35 years, respectively. Urinary iodine excretion (UIE) and serum free T4 (FT4), thyrotropin hormone (TSH), antiperoxidase antibodies (anti- TPOAb), and antithyroglobulin antibodies (anti-TGAb) were determined. Median UIE was 148 µg/L (within the recommended level by the World Health Organization [WHO]). However, UIE levels of <100 and <50 µg/L were detected in both male and female groups, respectively. Serum levels of TSH and FT4 were normal for all except one of the participants who suffered from hyperthyroidism, possibly as a result of elevated iodine intake, which was reflected in an increased UIE of 590 µg/L. Elevated anti-TPOAb >75 IU/mL and anti-TGAb >150 IU/mL were detected in 15% and 34% of subjects; only 10% of them had elevated levels of both anti-TPOAb and anti-TGAb. Thus, based on the WHO recommendations, the iodine intake for the Kuwaiti population is adequate. However, it is recommended that a national study be conducted by the appropriate authority in order to eliminate any artifacts which may have appeared in this study.