Elevated labile iron in castration-resistant prostate cancer is targetable with ferrous iron-activatable antiandrogen therapy.

Clinical responses to second generation androgen signaling inhibitors (e.g., enzalutamide) in metastatic castration-resistant prostate cancer (mCRPC) are variable and transient, and are associated with dose limiting toxicities, including rare but severe CNS effects. We hypothesized that changes to iron metabolism coincident with more advanced disease might be leveraged for tumor-selective delivery of antiandrogen therapy. Using the recently described chemical probes SiRhoNox and 18F-TRX in mCRPC models, we found elevated Fe2+ to be a common feature of mCRPC in vitro and in vivo. We next synthesized ferrous-iron activatable drug conjugates of second and third-generation antiandrogens and found these conjugates possessed comparable or enhanced antiproliferative activity across mCRPC cell line models. Mouse pharmacokinetic studies showed that these prototype antiandrogen conjugates are stable in vivo and limited exposure to conjugate or free antiandrogen in the brain. Our results reveal elevated Fe2+ to be a feature of mCRPC that might be leveraged to improve the tolerability and efficacy of antiandrogen therapy.

Correlation of Serum Nt-Pro Bnp Levels and Myocardial Performance Index in Asymptomatic Type 2 Diabetics for Assessment of Myocardial Ischaemia.

Diabetes mellitus is a global health problem. Cardiovascular events accounts for approximately 50 percent of mortality amongst the diabetics. Diabetics often present with silent myocardial ischaemia, since myocardial ischaemia is a strong trigger of N-terminal pro-B-type natriuretic peptide (NT-proBNP) release, we hypothesized that NT-proBNP might be useful in the early diagnosis. This study also evaluates whether any correlation exits between NT-proBNP levels and myocardial performance index (MPI). MATERIAL: This case control study was done on 30 patients with type 2 diabetes and similar matched group of 30 controls. Investigations like fasting and postprandial sugar level, kidney function test, HBA1c, lipid profile and NT-ProBNP levels were done. TMT was done to diagnose silent MI. Echocardiography was performed to assess MPI. The outcome measures were cut off levels of NT-ProBNP for predicting silent myocardial ischaemia based on TMT. P<0.05 was considered as significant. OBSERVATION: NT-proBNP (pg/mL) was significantly increased in Diabetics in comparison to controls (595.33±464.48 vs 110.97 ± 34.81, p <.0001). MPI was significantly higher in diabetics (0.54±0.08 vs 0.33±0.02, p<.0001), and NT-proBNP showed significant positive correlation myocardial performance index (r= 0.726, p<0.0001). Silent MI was seen in 18(60%) patients which was based on TMT testing. Compared to those without silent MI, patients with silent MI had higher NTproBNP levels (795.5 vs 106, p value=0.0001) and higher MPI (0.59 ± 0.06 vs 0.46 ± 0.05, p value<.0001). A cut off of NT-pro BNP(pg/ mL) of >152 predicted silent MI in 93.33% cases with 100% sensitivity, 83.33% specificity, 90% PPV and 100% NPV. CONCLUSION: NT-proBNP is a novel marker with positive correlation with MPI. It may be used as an independent predictor for silent MI in diabetics, thereby helping in reduction and treatment of CVD among them. Thus, it is suggested to screen all asymptomatic diabetics for underlying myocardial ischaemia by MPI, preferably, or by NTproBNP levels.

Role of Hemoglobin Content of Reticulocyte to Evaluate Anemia in Patients with Malignancy.

Anemia is common in patients with cancer and it's pathophysiology is complex and multifactorial . Conventional methods (Serum Iron, serum ferritin, TIBC, TSAT) to diagnosing iron deficiency anemia in cancer patients is affected by cancer type, duration, treatment, infection and inflammation related to cancer. RET-He measure the recent functional availability of iron and the correlation well with iron deficient / restricted erythropoiesis, and it is not affected by infection and inflammation related to cancer so it can be useful marker to rapidly rule out iron deficiency in cancer patients. Material: This is observation longitudinal study and study subjects including all type of diagnosed cancer patients with anemia (Hb <13 gm % in males and <12gm% in females) with or without treatment. Study duration was 18 month and 200 sample size was taken. Complete blood count (Hb, TLC, platelets, MCV, MCH, MCHC, reticulocyte hemoglobin) were analysed on SYSMEX XN 1000i. Serum Ferritin was estimated using AVANTOR CL-1000i and Serum iron, TIBC, TSAT was run on EBRA MANHEIN CHEM 5X machine. Bone marrow examination was done for diagnosis / staging . Iron stores were evaluated by Perl's Prussian blue stain and graded as per criteria laid down by Gale et al. Observation: At a cut off of 28.4 pg, RET-He achieved sensitivity of 96.77 % and specificity of 81.66% with NPV of 99.3% and PPV of 49.2% for iron deficient state in cancer patients. This cut off value rules out iron deficient erythropoiesis, reduces unnecessary iron studies and encourage early treatment of iron deficiency. There is also moderate agreement exist between iron stores of bone marrow and RET-He with Kappa 0.411 and p value <.0001. Conclusion: RET-He is better indicator of IDA in cancer patients as compared to other conventional methods of diagnosing IDA.This study also revealed a direct correlation between RET-He and bone marrow iron stores. In future it is advisable to use RET-He as a predictor of IDA, which is sensitive and specific at particular cut off points in routine evaluation in IDA in cancer patients.

Correlation of Prognostic Markers {CEA, Ca19.9, Neutrophil To Lymphocyte Ratio (NLR) and Platelet to Lymphocyte Ratio (PLR)} With Progression Free Survival (PFS) in Advanced/Metastatic Gallbladder Carcinoma.

Gallbladder carcinoma (GBC) is likely to be diagnosed at progressive stages and shows a very poor prognosis. Combination of gemcitabine and cisplatin (GEMCIS) has been widely used as first line palliative chemotherapy. Prognostic significance of inflammatory markers Neutrophil to lymphocyte ratio (NLR) and Platelet to lymphocyte ratio (PLR) in advanced gallbladder carcinoma (GBC) is not well established. MATERIAL: 30 patients who were diagnosed as advanced/ metastatic gallbladder carcinoma with age more than 18 years were included. All patients who were fit to receive chemotherapy was started on gemcitabine 1000mg/m2 and cisplatin 25mg/m2 (GEMCIS) administered intravenously on days 1 and 8 every 3 weeks. The treatment was repeated for a total of 6 cycles or until the occurrence of unacceptable toxicity, loss to follow up, confirmation of disease progression or death. All the patients underwent Contrast enhanced CT scan along with markers NLR, PLR, carcinoembryonic antigen (CEA), carbohydrate antigen 19.9 (CA19.9) at the baseline and at the end of 3 and 6 cycles of chemotherapy and the tumour response was assessed based on Response Evaluation Criteria in Solid Tumours RECIST 1.1 criteria. Progression free survival was calculated from the date of documentation of best response. The response was correlated with markers CEA, CA19.9, NLR and PLR. OBSERVATION: At a cut-off of NLR (>3 ng/ml) and PLR (>190) predicts progression with a sensitivity of 91% and 100% respectively and both with a specificity of 100%. Out of 30 participants 11 (36.7%) had disease progression (p<0.001). Disease progression noted in 8 participants (26.7%) after 3 cycles (P<0.001) and in 3 participants (13.6%) after 6 cycles of chemotherapy (p <0.001). Disease control rate was 63.33%: 01(3.3%) patient with complete response, 07(23.3%) patients with partial response, 11(36.7%) patients with stable disease. Mean progression free survival in participants associated with progressive disease was 11.45±5.54 weeks (p <0.001). Neutrophil to lymphocyte ratio (NLR) >3 (95%CI 7.6-13.6; log rank test P<0.01) and Platelet to lymphocyte ratio (PLR) >190 (95%CI 7.67-8.83; log rank test p<0.001) were significantly associated with worse progression free survival. CONCLUSION: Increased levels of NLR (>3) and PLR (>190) have prognostic value to predict progression free survival (PFS) in advanced gallbladder carcinoma patients on palliative chemotherapy. NLR and PLR can be used as prognostic markers in advanced gallbladder carcinoma.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

Physicochemical and Structural Parameters Contributing to the Antibacterial Activity and Efflux Susceptibility of Small-Molecule Inhibitors of Escherichia coli.

Discovering new Gram-negative antibiotics has been a challenge for decades. This has been largely attributed to a limited understanding of the molecular descriptors governing Gram-negative permeation and efflux evasion. Herein, we address the contribution of efflux using a novel approach that applies multivariate analysis, machine learning, and structure-based clustering to some 4,500 molecules (actives) from a small-molecule screen in efflux-compromised Escherichia coli We employed principal-component analysis and trained two decision tree-based machine learning models to investigate descriptors contributing to the antibacterial activity and efflux susceptibility of these actives. This approach revealed that the Gram-negative activity of hydrophobic and planar small molecules with low molecular stability is limited to efflux-compromised E. coli Furthermore, molecules with reduced branching and compactness showed increased susceptibility to efflux. Given these distinct properties that govern efflux, we developed the first efflux susceptibility machine learning model, called Susceptibility to Efflux Random Forest (SERF), as a tool to analyze the molecular descriptors of small molecules and predict those that could be susceptible to efflux pumps in silico Here, SERF demonstrated high accuracy in identifying such molecules. Furthermore, we clustered all 4,500 actives based on their core structures and identified distinct clusters highlighting side-chain moieties that cause marked changes in efflux susceptibility. In all, our work reveals a role for physicochemical and structural parameters in governing efflux, presents a machine learning tool for rapid in silico analysis of efflux susceptibility, and provides a proof of principle for the potential of exploiting side-chain modification to design novel antimicrobials evading efflux pumps.

Structure and mechanism of TagA, a novel membrane-associated glycosyltransferase that produces wall teichoic acids in pathogenic bacteria.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to ß-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme's lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

Nutrient Stress Small-Molecule Screening Platform for Escherichia coli.

Clinically approved antibiotics target a narrow spectrum of cellular processes, namely cell wall synthesis, DNA replication, and protein synthesis. Numerous screens have been designed to identify inhibitors that target one of these cellular processes. Indeed, this narrow range of drug mechanisms and a reliance on chemical classes discovered many decades ago are thought to be principally responsible for the current crisis of antibiotic drug resistance. Seeking to expand the target base of antibacterial drug discovery, we developed a nutrient stress screening platform that identifies inhibitors of the growth of in Escherichia coli under nutrient limitation. Under nutrient stress, bacteria require an expanded biosynthetic capacity that includes the synthesis of amino acids, vitamins, and nucleobases. Growing evidence suggests that these processes may be indispensable to certain pathogens and at particular sites of infection. Indeed, more than 100 biosynthetic enzymes become indispensable to E. coli grown under nutrient stress in vitro. The screening platform described here puts a focus on these novel targets for new antibiotics and prioritizes growth inhibitory compounds that can be suppressed by individual nutrients and pools thereof.

Exploiting the Sensitivity of Nutrient Transporter Deletion Strains in Discovery of Natural Product Antimetabolites.

Actinomycete secondary metabolites are a renowned source of antibacterial chemical scaffolds. Herein, we present a target-specific approach that increases the detection of antimetabolites from natural sources by screening actinomycete-derived extracts against nutrient transporter deletion strains. On the basis of the growth rescue patterns of a collection of 22 Escherichia coli (E. coli) auxotrophic deletion strains representative of the major nutrient biosynthetic pathways, we demonstrate that antimetabolite detection from actinomycete-derived extracts prepared using traditional extraction platforms is masked by nutrient supplementation. In particular, we find poor sensitivity for the detection of antimetabolites targeting vitamin biosynthesis. To circumvent this and as a proof of principle, we exploit the differential activity of actinomycete extracts against E. coli ΔyigM, a biotin transporter deletion strain versus wildtype E. coli. We achieve more than a 100-fold increase in antimetabolite sensitivity using this method and demonstrate a successful bioassay-guided purification of the known biotin antimetabolite, amiclenomycin. Our findings provide a unique solution to uncover the full potential of naturally derived antibiotics.

Entropy as an Indicator to Measure Depth of Anaesthesia for Laryngeal Mask Airway (LMA) Insertion during Sevoflurane and Propofol Anaesthesia.

INTRODUCTION: Assessment of the depth of anaesthesia is fundamental to the anaesthetic practice. Entropy measurement is an objective monitoring and is of two types- Response Entropy (RE) and State Entropy (SE) indicating analgesic and hypnotic levels during general anaesthesia. AIM: The aim of our study was to assess the depth of anaesthesia for LMA placement using entropy as a tool. The assessment of entropy as an indicator of depth of anaesthesia in the form of haemodynamic variations and success rate of LMA placement. MATERIALS AND METHODS: A prospective study was carried out after ethical committee approval in 100 patients, aged 20-50 years, with ASA grade I and II of either gender undergoing elective surgery lasting less than two hours under general anaesthesia. Anaesthesia was induced with sevoflurane 2.5% and IV propofol at 6 ml/min until entropy value fell from baseline values to 30-40 and then LMA insertion was performed. SE and RE values were noted every 30 seconds for five minutes. Mean blood pressure and heart rate were recorded every minute after induction for 5 minutes. RESULTS: There was a significant change in RE and SE values within 30 seconds from start of induction. Desired values of RE (40.10±2.52) and SE (39.2±2.47) were achieved at 120 seconds to 150 seconds. Mean dose of propofol used during surgery was 86.5±3.5 mg and mean insertion time was 110±12 seconds Patients in study group had a stable haemodynamics throughout the procedure, (p-value -0.8). CONCLUSION: Entropy is a reliable indicator to assess depth of anaesthesia for LMA placement during sevoflurane and propofol anaesthesia.

Improving Functional Annotation in the DRE-TIM Metallolyase Superfamily through Identification of Active Site Fingerprints.

Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C-C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth ß-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the ß3-5 and ß4-7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the ß3-5 and ß4-7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the ß3-5 and ß4-7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup.

Biochemical characterization of the retaining glycosyltransferase glucosyl-3-phosphoglycerate synthase from Mycobacterium tuberculosis.

Glucosyl-3-phosphoglycerate synthase (GpgS) catalyzes the first step in the biosynthesis of glucosyl glycerate, the putative precursor used in building methylated polysaccharides in mycobacteria. Enzymes from Mycobacterium tuberculosis (MtGpgS) and related species have been structurally characterized and subjected to basic kinetic analyses, but more in-depth kinetic analysis is currently lacking. Dead-end inhibition studies with MtGpgS suggest an ordered kinetic mechanism with 3-phosphoglycerate (3-PGA) binding first, followed by UDP-glucose, in contrast to previous reports. At higher concentrations, 3-PGA exhibits competitive substrate inhibition vs. UDP-glucose, suggesting 3-PGA can bind to either binding site on the enzyme. Parabolic noncompetitive inhibition plots by a 3-PGA analog also support this conclusion. The effect of varying pH on the catalytic parameters indicates single ionizable residue involved catalysis (pKa=6.3) that must be deprotonated for full activity. A solvent kinetic isotope effect of 2.0±0.3 on kcat is consistent with a proton in flight during the rate-determining step. Site-directed mutagenesis studies identify several residues critical for interactions with substrates. Although the residues are conserved among other glycosyltransferase families catalyzing similar reactions, the effect of substitutions varies between families suggesting that conserved areas play different catalytic roles in each family.

Evolutionarily distinct versions of the multidomain enzyme α-isopropylmalate synthase share discrete mechanisms of V-type allosteric regulation.

Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme α-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of (D2O)kcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS ((D2O)kcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes.