Discovery and Structure Optimization of a Series of Isatin Derivatives as Mycobacterium tuberculosis Chorismate Mutase Inhibitors

In this study, the crystal structure of the Mycobacterium tuberculosis (MTB) enzyme chorismate mutase (CM) bound to transition state analogue (PDB: 2FP2) was used as a framework for virtual screening of the BITS‐Pilani in‐house database (2500 compounds) to identify new scaffold. We identified isatin as novel small molecule MTB CM inhibitors; further twenty‐four isatin derivatives were synthesized and evaluated in vitro for their ability to inhibit MTB CM, and activity against M. tuberculosis as steps towards the derivation of structure–activity relationships (SAR) and lead optimization. Compound 3‐(4‐nitrobenzylidene)indolin‐2‐one, 24 emerged as the most promising lead with an IC₅₀ of 1.01 ± 0.22 μm for purified CM and MIC of 23.5 μm for M. tuberculosis, with little or no cytotoxicity.     

Effect of orange juice and beverage with phytosterols on cytokines and PAI-1 activity

Background & aims

Inflammation is pivotal in all phases of atherosclerosis. Dietary options which lower inflammatory biomarkers would be an attractive strategy to reduce risk from cardiovascular diseases and cancer. Indeed, fruit and vegetable intake or fruit juice consumption is associated with health and wellness. However, there is a paucity of data examining the effect of orange juice on biomarkers of inflammation in healthy volunteers. We have previously conducted the first placebo-controlled randomized studies examining the effect of sterol fortified orange juice or sterol fortified reduced calorie orange juice beverage supplementation (2 g sterols/day) compared to Placebo OJ or Placebo OJBev, and showed significant benefits on the lipid profile as well as significant reduction in hsCRP, the prototypic marker of inflammation and a cardiovascular risk marker. The aim of this study was to examine the effect of orange juice (OJ) or OJ beverage (Bev) alone and fortified with plant sterols (1g/240 ml juice or beverage twice a day) on pro-inflammatory cytokines and PAI-1, a marker of impaired fibrinolysis in healthy human volunteers.

Methods

In the first study, 72 healthy human volunteers received Placebo OJ or Sterol OJ and in the second study, 72 volunteers received OJBev or Sterol OJBev for 8 weeks and blood was drawn at baseline and following supplementation for 8 weeks. Biomarkers of Inflammation (IL-1b, IL-6, TNF, IL-8, IL-10) were assessed in serum using the BD Human Inflammatory Cytokine Cytometric Bead Array and PAI-1 activity was assessed in citrated plasma.

Results

OJ or OJBev alone failed to result in any significant effects on circulating cytokine levels or PAI-1 activity. There was a significant reduction in IL-1b with sterol fortified OJ (p < 0.05) compared to baseline. In addition, both sterol fortified OJ as well as sterol fortified OJBev resulted in significant reductions in serum IL-6 levels (p < 0.01).

Conclusion

Thus, sterol fortified OJ and OJ Beverage are able to effectively lower biomarkers of inflammation in healthy human volunteers in addition to providing lipid profile benefits and may thus contribute to decreasing cardiovascular risk.     

Variations in Diterpenes-Cafestol and Kahweol Content in Beans of Robusta Coffee Grown at Different Altitudes

Cafestol and kahweol are the two anti-nutritional diterpenes unique to coffee. They are associated with lipids of the coffee and are reported to be responsible for hypercholesterolemic effect in humans. In the present study, diterpene profiles in beans of ripened Robusta coffee (Coffea canephora) fruits collected from trees grown at different altitudes were analysed by High performance liquid chromatography and mass spectrum. The levels of free diterpenes cafestol and kahweol were found to be more in beans collected from low elevations (0.12 % of cafestol and 0.045 % of kahweol) than that of high elevation grown trees (0.042 % of cafestol and 0.014 % of kahweol). Apart from these, some major cafestol and kahweol esters viz., cafestol linoleate, cafestol oleate, cafestol palmitate, cafestol stearate, kahweol linoleate, kahweol oleate and kahweol palmitate were identified. The present study shows that, the altitude at which coffee trees are grown had significant influence on their diterpene content.     

Therapeutic mechanisms of high-frequency stimulation in Parkinson’s disease and neural restoration via loop-based reinforcement

High-frequency deep brain stimulation (HFS) is clinically recognized to treat parkinsonian movement disorders, but its mechanisms remain elusive. Current hypotheses suggest that the therapeutic merit of HFS stems from increasing the regularity of the firing patterns in the basal ganglia (BG). Although this is consistent with experiments in humans and animal models of Parkinsonism, it is unclear how the pattern regularization would originate from HFS. To address this question, we built a computational model of the cortico-BG-thalamo-cortical loop in normal and parkinsonian conditions. We simulated the effects of subthalamic deep brain stimulation both proximally to the stimulation site and distally through orthodromic and antidromic mechanisms for several stimulation frequencies (20–180 Hz) and, correspondingly, we studied the evolution of the firing patterns in the loop. The model closely reproduced experimental evidence for each structure in the loop and showed that neither the proximal effects nor the distal effects individually account for the observed pattern changes, whereas the combined impact of these effects increases with the stimulation frequency and becomes significant for HFS. Perturbations evoked proximally and distally propagate along the loop, rendezvous in the striatum, and, for HFS, positively overlap (reinforcement), thus causing larger poststimulus activation and more regular patterns in striatum. Reinforcement is maximal for the clinically relevant 130-Hz stimulation and restores a more normal activity in the nuclei downstream. These results suggest that reinforcement may be pivotal to achieve pattern regularization and restore the neural activity in the nuclei downstream and may stem from frequency-selective resonant properties of the loop.High-frequency (i.e., above 100 Hz) deep brain stimulation (HFS) of the basal ganglia (BG) and thalamus is clinically recognized to treat movement disorders in Parkinson’s disease (PD) (1–4), but its therapeutic mechanisms remain unclear (5, 6).Early hypotheses about HFS were derived from the rate-based model of the BG function (7, 8) and postulated the disruption of the output of the BG-thalamic system via either the inactivation of neurons in the stimulated site (target) (9–15), which would provide an effect similar to a surgical lesion, or the abnormal excitation of axons projecting out of the target (16–19), which would disrupt the neuronal activity in the structures downstream, including any pathophysiological activity (20).More recently, an ever-growing number of experiments in PD humans and animal models of Parkinsonism has indicated that HFS affects the firing patterns of the neurons rather than the mean firing rate both in the target and the structures downstream (18, 19, 21–31) and it replaces repetitive low-frequency (i.e., ≤50 Hz) bursting patterns with regularized (i.e., more tonic) patterns at higher frequencies (25, 26). It has been proposed that increased pattern regularity of neurons in the target may be therapeutic (5, 32–37), but it is still unknown how this regularity comes about with HFS.It has been suggested that an increased pattern regularity can deplete the information content of the target output and this lack of information would act as an “information lesion” (33) and prevent the pathological activity from being transmitted within the BG-thalamic system (22, 33, 36). As a result, an information lesion in the target [typically, one among the subthalamic nucleus (STN), internal globus pallidus (GPi), or thalamus] would have effects similar to those of a destructive lesion in the same site, which has been reported to alleviate the movement disorders (38).Instead, studies (32, 34, 35, 37) have suggested that an increased pattern regularity of the BG output partly compensates the PD-evoked impairment of the information-processing capabilities of the thalamo-cortical system, and this restores a more faithful thalamic relay of the sensorimotor information (35, 39).Although intriguing, these hypotheses remain elusive on (i) the neuronal mechanisms that would elicit pattern regularization (e.g., why regularization would be relevant only for HFS) and (ii) the effects that increased regularity would have on the cortico-BG-thalamo-cortical loop.It has been hypothesized that pattern regularization occurs because axons projecting out of the target follow the pattern of the stimulus pulses (40, 41) and, given the segregated organization of the BG-thalamic connections (42), it has been assumed that pattern regularization percolates straightforward from the target to the structures immediately downstream (34, 36). However, this representation of the pattern regularization as a “local” effect can hardly be reconciled with the fact that HFS of any structure of the cortico-BG-thalamo-cortical loop is therapeutic for at least some movement disorders (1–4, 43–47), nor does it explain why stimulation at frequencies above 160–180 Hz is not necessarily therapeutic despite the fact that the regularity of the axonal patterns may increase (48, 49). Moreover, coherence in the 8–30-Hz band among neurons across different structures may decrease under HFS but not for lower frequencies (26, 50–52), which suggests the emergence of diffused changes in neuronal activity that would be hardly accounted for with purely local effects.There is emerging evidence, instead, that HFS affects multiple structures simultaneously. First, it has been shown that deep brain stimulation (DBS) may antidromically activate afferent axons and fibers of passage (53–59), thus reaching structures not immediately downstream. Second, studies (57, 58) observed in 6-hydroxydopamine (6-OHDA)-intoxicated rats that the antidromic effects increase with the stimulation frequency and peak around 110–130 Hz. Third, it has been shown in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated nonhuman primates (NHPs) that STN DBS may evoke similar poststimulus responses in different BG structures, both downstream from and upstream to the STN (5, 27, 28, 30, 60). Finally, it has been reported that the cortico-BG-thalamo-cortical system consists of multiple sets of reentrant, interconnected, and partially overlapping neuronal loops (5, 42, 61, 62), which means that the structures upstream to the target (e.g., the striatum) may play an important role in the therapeutic mechanisms of HFS.Altogether, these results suggest that (A) pattern regularization is a global effect that exploits the closed-loop nature of the cortico-BG-thalamo-cortical system and selectively emerges only for specific HFS values, and that (B) the therapeutic merit of pattern regularization has to deal with the restoration of a more normal functionality of the entire cortico-BG-thalamo-cortical loop rather than with variations in the information content of one specific structure.We explored hypotheses (A) and (B) and assessed the system-wide effects of DBS by constructing a computational model of the cortico-BG-thalamo-cortical loop in both normal and parkinsonian conditions and by simulating the effects of STN DBS both at low (20–80 Hz) and high (100–180 Hz) frequencies. The model includes populations of single-compartment neurons and interneurons from motor cortex, striatum, GPi, and thalamus according to a network topology derived from the NHP anatomy, and it simulates both the orthodromic and antidromic effects of DBS. As a result, this model reproduced both average activity and discharge patterns of single units in NHP and rats under normal and parkinsonian conditions, with and without DBS, for all modeled structures.We show through numerical simulation that hypothesis (A) is significantly contributed by reinforcement mechanisms in the striatum. These mechanisms are selectively elicited by HFS, facilitate the percolation of regularized discharge patterns from the striatum to the GPi, and have a primary role in (B), because the percolated striato-pallidal input combines with the local effects of STN DBS to restore the thalamic relay function (63).     

Immature myeloid cells directly contribute to skin tumor development by recruiting IL-17–producing CD4+ T cells

Evidence links chronic inflammation with cancer, but cellular mechanisms involved in this process remain unclear. We have demonstrated that in humans, inflammatory conditions that predispose to development of skin and colon tumors are associated with accumulation in tissues of CD33⁺S100A9⁺ cells, the phenotype typical for myeloid-derived suppressor cells in cancer or immature myeloid cells (IMCs) in tumor-free hosts. To identify the direct role of these cells in tumor development, we used S100A9 transgenic mice to create the conditions for topical accumulation of these cells in the skin in the absence of infection or tissue damage. These mice demonstrated accumulation of granulocytic IMCs in the skin upon topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in a dramatic increase in the formation of papillomas during epidermal carcinogenesis. The effect of IMCs on tumorigenesis was not associated with immune suppression, but with CCL4 (chemokine [C-C motif] ligand 4)-mediated recruitment of IL-17–producing CD4⁺ T cells. This chemokine was released by activated IMCs. Elimination of CD4⁺ T cells or blockade of CCL4 or IL-17 abrogated the increase in tumor formation caused by myeloid cells. Thus, this study implicates accumulation of IMCs as an initial step in facilitation of tumor formation, followed by the recruitment of CD4⁺ T cells.Inflammation develops in response to foreign agents or injuries and is crucial to the healing process. However, persistent unresolved inflammation can contribute to the development of cancer (Coussens et al., 2013). Despite the wealth of information implicating inflammation in tumor development, the specific role of different cells, especially myeloid cells, in this process remains largely unclear. Myeloid cells are an important component of inflammation. There is now ample evidence of abnormalities in the myeloid compartment in cancer, which manifests in inhibition of differentiation of DCs, polarization of macrophages (MΦs) toward M2 functional state, and dramatic expansion of myeloid-derived suppressor cells (MDSCs; Gabrilovich et al., 2012). MDSCs represent a heterogeneous population of pathologically activated myeloid cells that includes precursors of neutrophils (polymorphonuclear neutrophils [PMNs]), MΦs, DCs, and cells at earlier stages of myeloid cell differentiation (Gabrilovich and Nagaraj, 2009; Peranzoni et al., 2010; Youn et al., 2012). In mice, these cells are defined as Gr-1⁺CD11b⁺ cells with the recognition of polymorphonuclear (PMN-MDSC) and mononuclear (M-MDSC) subsets based on the expression of Ly6C and Ly6G markers (Fridlender et al., 2009; Peranzoni et al., 2010; Brandau et al., 2011; Youn et al., 2012). MDSCs are characterized by a potent immune-suppressive activity and the ability to promote tumor angiogenesis, tumor cell invasion, and metastases (Bierie and Moses, 2010; Gabrilovich et al., 2012; Talmadge and Gabrilovich, 2013). In tumor-free mice, cells with the same phenotype represent immature myeloid cells (IMCs) lacking immune-suppressive activity. Expansion of MDSCs is considered a consequence of tumor progression. However, in recent years, it has become clear that the cells with phenotypes and functions attributed to MDSCs are readily detectable in different conditions associated with chronic inflammation not directly linked to cancer (Cuenca et al., 2011; Nagaraj et al., 2013). We hypothesize that these cells can contribute to tumor development associated with inflammation. Understanding the role of specific components of inflammation in tumor development is difficult because of the fact that inflammation is a multicomponent complex process. To address this question, we focused on S100A9 protein. This is the member of the S100 family of Ca²⁺-binding proteins with diverse biological activity, including chemotaxis of myeloid cells, fatty acid transport, production of reactive oxygen species, etc. (Markowitz and Carson, 2013). Expression of S100A9 together with its dimerization partner S100A8 is found predominantly in cells of the myeloid lineage. Differentiation of myelocytes/granulocytes is associated with an increase of S100A8/A9 expression, whereas differentiation of MΦs and DCs is associated with loss of their expression (Leder et al., 1990; Ehrchen et al., 2009; Srivastava et al., 2012; Markowitz and Carson, 2013). Cells of the lymphoid lineage do not express these proteins. We and others have previously found that accumulation of MDSCs and inhibition of DC differentiation in cancer were closely associated with up-regulation of S100A8/A9 (Cheng et al., 2008; Sinha et al., 2008; Ichikawa et al., 2011). The expansion of MDSCs was significantly reduced in S100A9-deficient mice treated with complete Freund’s adjuvant or tumor-bearing mice (Cheng et al., 2008). In contrast, overexpression of S100A9 in mice resulted in accumulation of cells with MDSC phenotype (Chen et al., 2013). Furthermore, these data were consistent with a recent report that DCs derived from S100A9-deficient mice induced stronger response of allogeneic T cells (Shimizu et al., 2011). In recent years, S100A9 was identified as a marker of MDSCs in peripheral blood of cancer patients and tumor-bearing mice (Feng et al., 2012; Källberg et al., 2012; Zhao et al., 2012). We asked whether regulation of S100A9 level in myeloid cells could be used to dissect the possible role of MDSCs during early stages of tumor development. Here, we report that cells with the MDSC phenotype accumulate in tissues of patients with precancerous inflammation and that these cells play a major role in tumor development in mice. Importantly, this effect was not directly mediated by immune-suppressive mechanisms but by the recruitment of IL-17–producing CD4⁺ T cells.     

Cardiopulmonary and Noninvasive Hemodynamic Responses to Exercise Predict Outcomes in Heart Failure

BackgroundAn impaired cardiac output response to exercise is a hallmark of chronic heart failure (HF). We determined the extent to which noninvasive estimates of cardiac hemodynamics during exercise in combination with cardiopulmonary exercise test (CPX) responses improved the estimation of risk for adverse events in patients with HF.Methods and ResultsCPX and impedance cardiography were performed in 639 consecutive patients (mean age 48 ± 14 years), evaluated for HF. Clinical, hemodynamic, and CPX variables were acquired at baseline and subjects were followed for a mean of 460 ± 332 days. Patients were followed for the composite outcome of cardiac-related death, hospitalization for worsening HF, cardiac transplantation, and left ventricular assist device implantation. Cox proportional hazards analyses including clinical, noninvasive hemodynamic, and CPX variables were performed to determine their association with the composite endpoint. There were 113 events. Among CPX variables, peak oxygen uptake (VO₂) and the minute ventilation (VE)/carbon dioxide production (VCO₂) slope were significant predictors of risk for adverse events (age-adjusted hazard ratio [HR] 1.08, 95% confidence interval [CI] 1.05–1.11 for both; P < .001). Among hemodynamic variables, peak cardiac index was the strongest predictor of risk (HR 1.08, 95% CI 1.0–1.16; P = .01). In a multivariate analysis including CPX and noninvasively determined hemodynamic variables, the most powerful predictive model included the combination of peak VO₂, peak cardiac index, and the VE/VCO₂ slope, with each contributing significantly and independently to predicting risk; an abnormal response for all 3 yielded an HR of 5.1 (P < .001).ConclusionsThese findings suggest that noninvasive indices of cardiac hemodynamics complement established CPX measures in quantifying risk in patients with HF.