The Sensitivity of HIV-1 gp120 Polymorphs to Inhibition by Temsavir Correlates to Temsavir Binding On-rate.

Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. The fold-change in half-maximal inhibitory concentration (IC50) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC50 and temsavir binding affinity (r=0.7332; P=0.0246) as well as temsavir binding on-rate (r=-0.8940; P=0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations.

Next-generation ABACUS biosensors reveal cellular ABA dynamics driving root growth at low aerial humidity.

The plant hormone abscisic acid (ABA) accumulates under abiotic stress to recast water relations and development. To overcome a lack of high-resolution sensitive reporters, we developed ABACUS2s-next-generation Förster resonance energy transfer (FRET) biosensors for ABA with high affinity, signal-to-noise ratio and orthogonality-that reveal endogenous ABA patterns in Arabidopsis thaliana. We mapped stress-induced ABA dynamics in high resolution to reveal the cellular basis for local and systemic ABA functions. At reduced foliar humidity, root cells accumulated ABA in the elongation zone, the site of phloem-transported ABA unloading. Phloem ABA and root ABA signalling were both essential to maintain root growth at low humidity. ABA coordinates a root response to foliar stresses, enabling plants to maintain foraging of deeper soil for water uptake.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

The Effect of Adding Procalcitonin to the Systemic Inflammatory Response Syndrome (Sirs) and Quick Sepsis-Related Organ Failure Assessment (qSOFA) Scoring System in Predicting Sepsis Mortality.

OBJECTIVE:  The primary objective of this study was to determine if the addition of procalcitonin to the existing systemic inflammatory response syndrome (SIRS) and quick Sepsis-related Organ Failure Assessment (qSOFA) scoring systems could improve the predictability of in-hospital sepsis-related mortality. Secondarily, we sought to determine if the addition of procalcitonin could predict the likelihood of ICU admission and discharge home. DESIGN: This is a retrospective, single-center, observational study that looked at data from January 1, 2017 to January 1, 2019. Patients were stratified into four groups: SIRS-positive + procalcitonin >2 ng/mL (pSIRS+), SIRS-positive + procalcitonin ≤2 ng/mL (pSIRS-), qSOFA-positive + procalcitonin >2 ng/mL (pqSOFA+), and qSOFA-positive + procalcitonin ≤2 ng/mL (pqSOFA-). SETTING: The study was conducted at a community hospital in Las Vegas, Nevada. PATIENTS: Patients were included in the study if they were >18 years of age and had hospital admission diagnosis of sepsis with at least one value of procalcitonin level. INTERVENTIONS: After patients which met the inclusion criteria, patients were divided into subgroups of SIRS, SIRS + procalcitonin > 2 ng/mL, qSOFA, qSOFA + procalcitonin >2 ng/mL. Primary outcomes were in-hospital mortality and secondary outcomes were ICU admission, length of stay, and discharge to home. RESULTS:  933 patients were included in the study with an overall mortality rate of 21.22%, an overall ICU admission rate of 56.15%, and an overall discharge home rate of 29.58%. In those identified with a sepsis-related diagnosis code, pSIRS+ predicted an in-hospital mortality rate of 31.89% compared to pSIRS- 16.15% (P < 0.0001). In regards to qSOFA, the addition of procalcitonin added no statistically significant difference in predicting in-hospital mortality. pSIRS+ patients were found to have an ICU admission rate of 76.16% and a discharge home rate of 19.20% compared to pSIRS- who had 47.40% and 34.90%, respectively (P < 0.0001). Like in our primary outcome, our data for qSOFA was not statistically significant. CONCLUSIONS:  Procalcitonin added utility to the SIRS scoring system in predicting sepsis-related in-hospital mortality, ICU admission, and discharge home. Procalcitonin did not add statistically significant benefit to the qSOFA scoring system in predicting sepsis-related in-hospital mortality, ICU admission, and discharge home.

A Rare Case of Intraparenchymal Cerebrospinal Fluid Cyst Associated With Ventriculoperitoneal Shunt in an Adult Patient.

Here we are reporting a rare phenomenon associated with ventriculoperitoneal (VP) shunt in the adult patient, namely, the development and finding of intraparenchymal pericatheter cerebrospinal fluid cyst. Our patient had a VP shunt placed for idiopathic intracranial hypertension 16 years ago before presentation to the hospital. The patient was admitted to the hospital for headache for past three weeks with the initial CT scan showing encephalomalacia and vasogenic edema. MRI showed the presence of a 4-cm intraparenchymal cyst in the right frontal lobe with surrounding vasogenic edema. The patient underwent two surgeries with the initial surgery for the drainage of cyst and second surgery for the placement of the cystoperitoneal shunt. Catheter-associated cysts are easily misdiagnosed due to their similarity in appearance to abscesses and other malignancies on imaging, and there are no guidelines yet on their evaluation and management. This is a unique case as the pericatheter cyst developed 16 years after the initial VP shunt placed. Given the rarity of this presentation, we hope that our case report can contribute to the development of guidelines and treatment options in adults with long-standing VP shunts.

Fluorescent biosensors illuminating plant hormone research.

Phytohormones act as key regulators of plant growth that coordinate developmental and physiological processes across cells, tissues and organs. As such, their levels and distribution are highly dynamic owing to changes in their biosynthesis, transport, modification and degradation that occur over space and time. Fluorescent biosensors represent ideal tools to track these dynamics with high spatiotemporal resolution in a minimally invasive manner. Substantial progress has been made in generating a diverse set of hormone sensors with recent FRET biosensors for visualising hormone concentrations complementing information provided by transcriptional, translational and degron-based reporters. In this review, we provide an update on fluorescent biosensor designs, examine the key properties that constitute an ideal hormone biosensor, discuss the use of these sensors in conjunction with in vivo hormone perturbations and highlight the latest discoveries made using these tools.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Beta-blockers do not provide survival benefit in a population with angiographic coronary artery disease without myocardial infarction or reduced ejection fraction: A meta-analysis.

BACKGROUND: While beta-blockers have been widely used for patients with stable coronary artery disease (CAD), some concerns have been raised that beta blockers do not have survival benefit in this population. We conducted a meta-analysis to determine the effects of beta blockers on all-cause and cardiac mortality in adults with CAD without previous myocardial infarction (MI) or reduced ejection fraction. METHODS: A systematic search of PubMed, Web of Science, Medline/Ovid and Google Scholar through March 2016 identified 4 studies that reported angiographic CAD without previous myocardial infarction or reduced ejection fraction. Fixed-effects pooled odds ratios and 95% confidence intervals of all-cause and cardiac mortality were estimated. We used the Grading of Recommendations Assessment, Development, and Evaluation system to assess overall quality of evidence. RESULTS: A total of 17,397 patients were analyzed. In both all-cause and cardiac mortality analysis, no serious limitation was identified. Beta-blockers were not associated with reductions in all-cause mortality (odds ratios=0.910, 95% confidence intervals 0.797-1.039, p=.163) or cardiac mortality (odds ratio=0.926, 95% confidence interval 0.773-1.110, p=.407). CONCLUSION: Beta-blockers do not provide any survival benefit in patients with angiographic CAD without history of MI or reduced ejection fraction.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

Crystal structure of a ß-prism II lectin from Remusatia vivipara.

The crystal structure of a ß-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 Å. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin.

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented.

Recurrent intra-cardiac thrombosis-A unique presentation of prothrombin G20210 mutation.

We report a 49-year-old female patient with recurrent large left ventricular thrombus on echocardiogram in an apparently normal heart and insignificant cardiac past medical history. She underwent an excision of the left ventricular mass, final biopsy on which proved it to be a thrombus. Postoperative anti-coagulation was initiated with enoxaparin and warfarin and the patient was followed up at a cardiology clinic 6 weeks later. A repeat trans-thoracic echocardiogram revealed a new mass arising from the left atrium. Considering the increased risk of repeat ventriculostomy, she was treated conservatively with her current management. During this time the patient's pro-thrombotic work-up revealed positive prothrombin G20210 mutation. A follow up trans-thoracic echocardiogram done 2 months later surprisingly revealed complete resolution of the intracardiac mass. Our patient had prothrombin G20210 mutation, an entity primarily known for deep venous thrombosis, which rarely causes intra-arterial thrombus, intra-cardiac being unreported. There are no established protocols for management of these cases. The rate of embolic episodes in mobile pedunculated thrombi is reported as high as 60%. Patients with prior embolism must be offered immediate surgery, especially if the thrombus is large with an irregular surface, pedunculated, and multiple in number. Aggressive anti-coagulation with close monitoring is essential.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity.

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80 degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 microg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.