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Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

BACKGROUND: Diabetes mellitus is associated with an increased risk of development of non-alcoholic fatty liver disease (NAFLD). However, the risk posed by diabetes mellitus in progression of liver disease is uncertain. This study compared the severity of hepatic fibrosis in patients with NAFLD with and without diabetes mellitus. METHODS: Consecutive adult patients with NAFLD undergoing transient elastography [FibroScan Touch 502 (Echosens, Paris, France)] at a tertiary care center in north India were analyzed for severity of hepatic fibrosis. The aspartate aminotransferase (AST) to platelet ratio index (APRI), fibrosis index based on 4 factors (FIB-4), and NAFLD Fibrosis Score (NFS) were calculated. The degree of hepatic fibrosis as determined by FibroScan and non-invasive serum fibrosis models in patients with and without diabetes mellitus were compared. RESULTS: A total of two hundred patients [118 (59%) males, mean age 50.30 ± 11.13 years] were enrolled. Significant hepatic fibrosis was present in 86 (43%) patients [mean age 50.66 ± 10.96 years, 56 (65.11%) males]. The mean FibroScan, APRI, FIB-4, and NFS scores were 9.86 ± 2.97, 0.75 ± 0.47, 2.41 ± 1.41 and −0.24 ± 1.43 in patients with diabetes compared to 5.31 ± 1.09, 0.49 ± 0.27, 1.55 ± 0.85, and −2.12 ± 1.88 in patients without diabetes, respectively (P=

Detecting 15N-TROSY in the direct dimension at high magnetic field yields narrow linewidth, alleviating spectral crowding in IDRs and facilitating resonance assignments.

We introduce μVAST, a high-throughput acoustic microstreaming platform using second-order microstreaming to induce fluid transport and measure the viscosity of 16 samples, automating process flows in drug development, materials manufacturing and production.

Coronary artery disease (CAD) is one of the predominant types of cardiovascular disease (CVD). The aim of present study was to study various factors that are causing difference in prevalence of coronary risk factors among siblings.This cross-sectional study was conducted in Dayanand Medical College and Hospital, among the healthy individuals (not known CAD) attending regular health care outpatient department (OPD) and their siblings over a period of 1½ years. All individuals coming for regular health checkup (not known CAD) of age more than 30 years or above and their siblings (with or without known CAD).This was a cross-sectional study, conducted among 100 pairs of healthy siblings (not known cases of CAD) who came for health checkup at health center of Dayanand Medical College and Hospital, a tertiary care hospital in North India. Prevalence of obesity was more in siblings living in urban area than their counter siblings living in rural area, but it was statistically insignificant. Six had impaired fasting blood sugar (FBS) and two were diabetic. Among their siblings living in urban area, 21 were nondiabetic, 10 had impaired FBS, and seven were diabetic. This correlation was statistically significant with p-value of 0.02. Among the CAD negative, out of 23 subjects, two subjects (9.0%) had heavy stress level, while remaining four subjects (17.0%) and 17 subjects (74.0%) had light and moderate stress levels, respectively. Among the CAD negative, out of 23 subjects, 10 subjects (43.0%) had high stress level, while remaining zero subject (0%) and 13 subjects (57.0%) had light and moderate stress levels, respectively. Significant results were obtained while comparing the CAD findings of subjects divided on the basis of stress level.In our study, among siblings (CAD positive and CAD negative), significant results were obtained for residence, socioeconomic class, physical activity, stress levels, smoking, waist-to-hip ratio (WHR), and diabetes, that is, all these factors have correlation in increasing CAD among siblings.

Several Conus-derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABAB receptor agonist that inhibits Cav2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABABRs expressed in dorsal root ganglion (DRG) sensory neurons.

Context: COVID-19 patients undergo myriad of psychological problems such as mood swings, depression, fear of isolation, fear of dying, feeling helpless, insomnia, anxious forebodings, and nervousness. These are commonly seen among isolated and quarantined patients who experience notable levels of anxiety, uncertainty, anger, confusion, stress and insecurity. Aims: This study aims to determine the prevalence of anxiety and depression among these infected patients admitted to tertiary care center. Settings and Design: This cross-sectional study was conducted on 100 COVID-19 patients admitted to DMC&H, Ludhiana. Subjects and Methods: The data were collected on sociodemographic parameters and assessment was done using Hamilton Depression Rating Scale (HDRS) and Hamilton Anxiety Rating Scale (HARS) at the time of discharge from the hospital. Statistical Analysis Used: SPSS 21 version for Microsoft Windows. Results: Majority of the patients were males and in the age group of 31–50 years. 48% patients had comorbid depression. Moderate-to-severe levels of depression were found more in males (25%) as compared to females (15%). Comorbid anxiety was seen commonly in females (60%) than male patients (28.75%), though the moderate-to-severe level of anxiety was more in males (71.25%) as compared to females (40%). On symptom checklist of HDRS and HARS, patients had high scores on insomnia (75%), psychic anxiety (45%–50%), somatic symptoms (gastrointestinal [50%], muscular [56%], and respiratory [81%]) and loss of weight (40%). Conclusions: COVID-19 patients score higher on comorbid anxiety and depression. Moderate-to-severe level of anxiety and depression is commonly seen among male patients than female patients.

Energy is a basic necessity of survival on the planet earth. One of the most promising and excellent way of harnessing the solar energy is the implications of solar collectors. Sun is readily available for such kind of applications throughout the year. A large scale solar concentrator plant is required to convert sun energy into heat energy. This task is accomplished by various kind of solar collectors loaded with various kinds of nanofluids. In this work, the effect of introduction of different kinds of nano-particles mixed with base fluids in various kinds of solar collectors has been discussed. The search for the increment of heat gain of solar collectors lead the researchers to the use of various kinds of nanofluids such as carbon nanofluid, aluminum oxides nanofluid, silver nanofluid, copper oxide nanofluid and many more together with working liquid. It has been observed that the output increases several times by the inclusion of nanofluids as a working fluid in solar collectors. Therefore, various studies are underway to prove the importance of nanofluids as a base fluid in different types of collectors to seek the optimal action of nanofluids in different collectors. Lots of nanofluids are categorized according to the performance they give when they are used as an alternate fluid along with base fluids in solar stills. The action of some available nanofluids as a working fluid in different collectors has been reviewed in this article followed by the conclusion based on study.

The source of solar energy is unlimited and it has the potential of reducing the contemporary shortage of fresh water prevalent throughout the globe if the solar energy is used wisely. This source of energy has emerged as one of the best alternatives to mitigate the crisis of energy as well as fresh water which are the two important factors for the survival of human beings on the earth. The research is going on throughout the world and new type of solar energy technology based water purifiers are being reported. The various studies have been reported in the literature with various designs of the basin liner with an objective of increasing the fresh water output. In this work, a review on the performance investigation of solar still by incorporating the different shapes of basin liner has been carried out. Authors have critically reviewed the solar stills with fins, corrugates wick, hemispherical basin and stepped still. It has been concluded that the evaporation area can be increased by changing the design of basin liner which provides higher fresh water output.

Background: India recently encountered fierce second wave of coronavirus disease (COVID-19), and scarcity of novel medications added to the management challenges. Various studies have highlighted the effectiveness of tocilizumab and high-dose steroids in severe COVIDs, but none has compared their efficacy. Materials and Methods: This retrospective multi-centric analysis compares intravenous tocilizumab (8 mg/kg/day, maximum dose-800 mg), and intravenous Methylprednisolone Pulse (MPS-1 g/day for 3 days) in severe COVID-19. Both the groups had additionally received the standard of care COVID treatment as per protocol. Outcomes were assessed at 30 days. Results: A total of 336 patients, with 249 receiving MPS and 87 receiving tocilizumab were compared. Majority of these were males (72.9%) with a mean age of 57.4 ± 13.6 years. Diabetes was the most common comorbidity. Patients in both groups had comparable age distribution, comorbidities, presenting mean-arterial pressures, d-Dimer levels, serum ferritin, serum leukocyte-dehydrogenase, and procalcitonin. However, the tocilizumab group had more number of males, higher incidence of coronary artery disease, more tachypnea and leukocytosis, more number of patients with severe acute respiratory disease syndrome (PaO2/FiO2 ratio

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site‐specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell‐free expression systems without modification of the expression protocol. The method relies on simple stereo‐selective 13C‐labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of “local” deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell‐free synthesis of bacteriorhodopsin and in the insect‐cell expression of the RRM2 domain of human RBM39., Methyl‐based magnetization usage can strongly benefit investigations of large protein systems via NMR spectroscopy. Here, a simple and cost‐effective method to obtain locally deuterated leucine suitable for methyl NMR and to quantify its benefits is presented. The approach substantially expands the applicability of methyl labeling in emerging expression systems including cell‐free and eukaryotic systems.

Severe acute respiratory syndrome coronavirus 2, caused by the novel coronavirus disease 2019 (COVID-19), led to a devastating pandemic that hit majority of the countries globally in a wave-like pattern. The characteristics of the disease varied in different geographical areas and different populations. This study highlights the epidemiological and clinical characteristics of COVID-19 during two major waves in North India.Clinical characteristics and outcomes of all COVID-19-reverse transcription-polymerase chain reaction-positive patients, admitted from March 2020 to June 2021, to a tertiary care center in North India, were studied retrospectively.During this period, total of 5652 patients were diagnosed having COVID. Patients who were incidentally diagnosed as COVID-positive (Majority of the patients with COVID-19 infection presenting to our hospital were young during the second wave. Fever was noted as presenting manifestation. Mortality was low during the second wave as compared to the first wave, likely to be due to proper protocol-based treatment resulting in better outcomes.

Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-μm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

Introduction: Anemia is a common complication of pulmonary tuberculosis (TB). Nutritional deficiency and malabsorption syndrome can deepen the severity of anemia. The aim of the present study was to study anemia and nutritional status in patients with TB at a tertiary care center. Materials and Methods: All patients diagnosed with TB (pulmonary and extrapulmonary), registered with Revised National TB Control Programme (RNTCP), taking DOTS regimen, attending the outpatient department as well as those admitted in the medicine and chest units of a tertiary care hospital, were enrolled in the present study. Results: Anemia was more common between the age groups of 51 and 60 years, with distribution of males (87 [58%]), patients with diabetes (49 [32%]), and hypertensive patients (29 [19.3%]). In our study, 48 (85.7%) out of 56 patients diagnosed with extrapulmonary TB were found to have anemia; similarly, 77 (88.5%) out of 87 patients diagnosed to have pulmonary TB were anemic. Anemia of chronic disease (128 [97.17%]) was higher when compared to that of those with iron-deficiency anemia (3 [2.29%]). According to body mass index (BMI), 135 (90%) patients were underweight; according to mid-arm circumference (MAC), 131 (87.3%) patients had severe malnutrition; and according to waist–hip ratio [WHR], 96 (64%) patients were underweight. Conclusion: Anemia was common in males and alcoholics, and there was a high prevalence of anemia of chronic disease. In addition, anemia was associated with high erythrocyte sedimentation rate and C-reactive protein. High proportions of TB patients (pulmonary and extrapulmonary) were classified as underweight and malnourished on the basis of different parameters (BMI, MAC, and WHR); in addition, the degree of malnutrition was higher in patients with anemia than in those without.

Coupling two copies of an iminodiacetic acid-cysteine hybrid ligand to a pair of cysteine residues positioned in an i, i+4 arrangement within a protein α-helix leads to generation of an EDTA-like metal ion-binding motif. Rigid binding of a Co(II) ion by this motif produces pseudo-contact shifts suitable for paramagnetic NMR structural studies.

Background: The physiological changes associated with menopause are responsible for increase in cardiovascular disease after menopause. BMI, physical inactivity, metabolic syndrome, hypertension and diabetes mellitus increase in post-menopausal women which are all powerful predictors of cardiovascular events. The risk factor profile between pre-menopausal and post-menopausal women presenting with acute coronary syndrome was thus studied.Methods: This study was a prospective cross-sectional hospital based study and was conducted in the Department of Medicine and Department of Cardiology, Dayanand Medical College and Hospital, Ludhiana wherein 50 pre-menopausal women and 50 post- menopausal women who presented with acute coronary syndrome were enrolled. The risk factor profile and angiography findings amongst the 2 groups were compared.Results: There was no difference in the incidence of Diabetes Mellitus and Hypertension among the pre & post-menopausal groups. The prevalence of family history of cardiovascular disease was higher in the pre-menopausal group. The post-menopausal women showed a significant decrease in physical activity due to sedentary lifestyle. Both the groups had high BMI and increased waist circumference. Lipid parameters did not show any significant difference between the two groups. However, pre-menopausal women had higher LDL-C and triglyceride levels and lower HDL-C levels. On analysis of the angiographic findings in diabetics and non-diabetics, of both the groups diabetes mellitus was much higher in subjects presenting with triple vessel disease suggesting extensive atherosclerosis.Conclusions: study showed physical inactivity as an important cardiovascular risk factor in post-menopausal women. Obesity is an important risk factor for coronary artery disease in both pre- and post-menopausal women.

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1 H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15 N-detected experiments that take advantage of the slower transverse relaxation property of 15 N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1 H and 13 C resonances. The six 3D experiments described here start with aliphatic 1 H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131–294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine–proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

The voltage-gated potassium channel Kv1.3 is an important target for the treatment of autoimmune diseases and asthma. Blockade of Kv1.3 by the sea anemone peptide K+-channel toxin from Stichodactyla helianthus (ShK) inhibits the proliferation of effector memory T lymphocytes and ameliorates autoimmune diseases in animal models. However, the lack of selectivity of ShK for Kv1.3 over the Kv1.1 subtype has driven a search for Kv1.3-selective analogues. In the present study, we describe N-terminally extended analogues of ShK that contain a negatively-charged Glu, designed to mimic the phosphonate adduct in earlier Kv1.3-selective analogues, and consist entirely of common protein amino acids. Molecular dynamics simulations indicated that a Trp residue at position [-3] of the tetrapeptide extension could form stable interactions with Pro377 of Kv1.3 and best discriminates between Kv1.3 and Kv1.1. This led to the development of ShK with an N-terminal Glu-Trp-Ser-Ser extension ([EWSS]ShK), which inhibits Kv1.3 with an IC50 of 34 pm and is 158-fold selective for Kv1.3 over Kv1.1. In addition, [EWSS]ShK is more than 2900-fold more selective for Kv1.3 over Kv1.2 and KCa3.1 channels. As a highly Kv1.3-selective analogue of ShK based entirely on protein amino acids, which can be produced by recombinant expression, this peptide is a valuable addition to the complement of therapeutic candidates for the treatment of autoimmune diseases.

Objectives To investigate the long-term prognostic significance of baseline plasma PLTP levels in a group of well-characterized male patients with diabetes mellitus and known or suspected coronary artery disease referred for coronary angiography. Background PLTP is a plasma protein that mediates the net transfer and exchange of phospholipids between lipoproteins. It has been implicated in the pathogenesis of atherosclerosis and elevated plasma levels have been reported in patients with diabetes mellitus. Methods Baseline plasma PLTP levels were measured in 154 male patients with diabetes mellitus who were referred for coronary angiography and followed prospectively for 5 years for the development of all-cause mortality. Results After adjustment for a variety of baseline clinical, angiographic and laboratory parameters, plasma PLTP levels (analyzed as a continuous variable) were an independent predictor of all-cause mortality at 5 years (HR, 1.55; 95% CI, 1.22–2.00; P = 0.0009). Furthermore, in 3 additional multivariate models that also included a wide variety of contemporary biomarkers with established prognostic efficacy (i.e., ST2, GDF-15, Cystatin C, Fibrinogen, and NT-proBNP), PLTP remained an independent predictor of all-cause mortality at 5 years. Conclusions Elevated baseline plasma levels of PLTP are associated with an increased risk of long-term all-cause mortality in patients with diabetes and known or suspected coronary disease. Furthermore, this association is independent of a variety of clinical, angiographic, and laboratory variables, including a whole host of contemporary biomarkers with established prognostic efficacy.

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a &gt, 100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC50s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of &gt, 10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

Background Neurological status of comatose patients in ICU cannot be assessed clinically and certain factors affecting the management and prognosis can be missed on routine neurological examination like non-convulsive seizures, secondary brain insults and increased intracranial pressure. Structural derangements are usually evaluated by imaging techniques and functional status by EEG. Objectives A 24-hour continuous EEG monitoring was done to detect non-convulsive status epilepticus (NCSE) and non-convulsive seizures (NCS) in non-traumatic comatose patients. Methods This prospective study included 50 patients with non-traumatic coma who were divided into Group A (30 patients with seizures) and Group B (20 patients without seizures). A 24-hour continuous EEG monitoring was done to detect NCSE and NCS. The data was collected and the results statistically analyzed. Results There was a statistically significant difference in the number of patients with altered sensorium ( p p p p p Conclusions Continuous EEG is of great help in detecting NCS/NCSE which are not detected clinically. Certain risk factors are associated with the presence of NCS/NCSE.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies. Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216–33. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Jenkins et al., p. 196. This article is highlighted in the In This Issue feature, p. 127

Torsades de pointes with prolonged QTc interval is a form of ventricular tachycardia. Many predisposing factors have been identified and hypocalcemia is among the rare ones. Our case illustrates that though rare, hypocalcemia might manifest as torsades de pointes with prolongation of QTc interval. Early diagnosis and management of dyselectrolytemia can prevent these patients from catastrophic torsades de pointes.

ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value ∼50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.

ShK is a 35-residue peptide that binds with high affinity to human voltage-gated potassium channels through a conserved K-Y dyad. Here we have employed NMR measurements of backbone-amide (15)N spin-relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps to ns timescale, increased linewidths observed for 11 backbone-amide (15)N resonances identify chemical or conformational exchange contributions to the spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub-millisecond timescale. Affected residues are mostly clustered around the central helix-kink-helix structure and the critical K22-Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.

Inhibitors of the α-subunit of the voltage-gated sodium channel subtype 1.3 (NaV 1.3) are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described μ-conotoxin BuIIIB (μ-BuIIIB) from Conus bullatus was shown to block NaV 1.3 with submicromolar potency (Kd = 0.2 μm), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of μ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure-activity studies. In the present study, we report the synthesis and characterization of μ-BuIIIB analogs incorporating a disulfide-deficient, diselenide-containing scaffold designed to simplify synthesis and facilitate structure-activity studies directed at identifying amino acid residues involved in NaV 1.3 blockade. Our results indicate that, similar to other μ-conotoxins, the C-terminal residues (Trp16, Arg18 and His20) are most crucial for NaV 1 blockade. At the N-terminus, replacement of Glu3 by Ala resulted in an analog with an increased potency for NaV 1.3 (Kd = 0.07 μm), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ-carboxyglutamate replacement, decreased potency (Kd = 0.33 μm), whereas replacement with positively-charged 2,4-diamonobutyric acid increased potency (Kd = 0.036 μm). These results provide a foundation for the design and synthesis of μ-BuIIIB-based analogs with increased potency against NaV 1.3.

© FASEB. The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7-effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases. - Chhabra, S., Chang, S. C., Nguyen, H. M., Huq, R., Tanner, M. R., Londono, L. M., Estrada, R., Dhawan, V., Chauhan, S., Upadhyay, S. K., Gindin, M., Hotez, P. J., Valenzuela, J. G., Mohanty, B., Swarbrick, J. D., Wulff, H., Iadonato, S. P., Gutman, G. A., Beeton, C., Pennington, M. W., Norton, R. S., Chandy, K. G. Kv1.3 channelblocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.

A facile stereoselective synthesis of cis and trans unsaturated dicarba peptides has been established using preformed diaminosuberic acid derivatives as bridging units. In addition, characteristic spectral differences in the (13)C-NMR spectra of the cis- and trans-isomers show that the chemical shift of carbons in the Δ4,5-diaminosuberic acid residue can be used to assign stereochemistry in unsaturated dicarba peptides formed from ring closing metathesis of linear peptide sequences.

International audience; Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.

Phospholipid transfer protein (PLTP) is an important modulator of phospholipid transfer and exchange among proteins. It also plays a role in inflammation and oxidative stress. Accordingly, PLTP has been implicated in the development of atherosclerosis. Left ventricular (LV) systolic dysfunction is common in patients with atherosclerosis, and both inflammation and oxidative stress have also been implicated in its development and progression. The goal of the present study was to examine the relation between plasma PLTP activity and LV systolic function. Baseline plasma PLTP activity was measured in 389 male patients referred for coronary angiography for a variety of indications. Detailed clinical, angiographic and laboratory characteristics were available for the patients. Compared to those patients with normal LV function (defined as an ejection fraction of ≥55% on ventriculography), patients with any degree of LV dysfunction had elevated PLTP activity (median PLTP 17.8 pmol/μl/h versus 15.9 pmol/μl/h, p = 0.0038). Using multivariate analysis, and adjusting for a variety of confounding variables known to affect both LV function and PLTP activity, PLTP activity was an independent predictor of the presence of any left ventricular systolic dysfunction in the entire population (OR 1.47, 95% CI 1.12–1.93, p = 0.0052). Furthermore, PLTP activity was an independent predictor of the presence of LV dysfunction in both patients with and without myocardial infarction on presentation (OR 2.39, 95% CI 1.18–4.86, p = 0.0161 and OR 1.41, 95% CI 1.05–1.89, p = 0.0206, respectively). In conclusion, PLTP activity may represent a novel marker of LV systolic dysfunction in patients with known or suspected coronary artery disease.

Tebuquine is a 4-aminoquinoline that shows significantly more potency as an antimalarial than amodiaquine and chloroquine both in vitro and in vivo. To explore the conformation in the rate-limiting step and to elucidate pharmacophoric properties of tebuquine-related analogues, molecular shape analysis (MSA) along with molecular field analysis (MFA) methods were applied on a series of 5-[(7-chloro-4-quinolinyl)amino]-3-[(alkylamino)methyl][1,1'-biphenyl]-2-ol analogues and their Nomega-oxides possessing antimalarial activity. The study was performed using 45 compounds in which 37 molecules were taken as a training set for the derivation of the 3D quantitative structure-activity relationship models and eight molecules were kept as a test set to evaluate the predictive ability of the derived models. Both methods were analyzed in terms of their predictive abilities and produced comparable results with good conventional and cross-validated r2 values (0.908 and 0.886, respectively, for the MFA model and 0.846 and 0.812, respectively, for the MSA model). In external data set prediction, the MSA model scored much better than MFA. Steric, electrostatic, and hydrogen-bond donor/acceptor fields of molecules were found to be relevant descriptors for structure-activity relationships. The inclusion of polar solvent-accessible charged surface area and spatial descriptors in the MSA model generation resulted in a model with significant predictive ability for the test set molecules. This indicates the importance of the orientation of conformationally favored molecules inside the receptor site and solvation of the charged surfaces of the molecule by a polar solvent for the activity of the molecule. The results provided the appropriate tools for predicting the affinity of related compounds using a ligand-based approach, and for guiding the design and synthesis of novel and more potent antimalarial agents.