Dual PI3Kδ/γ Inhibitor Duvelisib Prevents Development of Neuropathic Pain in Model of Paclitaxel-Induced Peripheral Neuropathy.

The development of painful paclitaxel-induced peripheral neuropathy (PIPN) represents a major dose-limiting side effect of paclitaxel chemotherapy. Here we report a promising effect of duvelisib (Copiktra), a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, in preventing paclitaxel-induced pain-like behavior and pronociceptive signaling in DRGs and spinal cord dorsal horn (SCDH) in rat and mouse model of PIPN. Duvelisib blocked the development of mechanical hyperalgesia in both males and females. Moreover, duvelisib prevented paclitaxel-induced sensitization of TRPV1 receptors, and increased PI3K/Akt signaling in small-diameter DRG neurons and an increase of CD68+ cells within DRGs. Specific optogenetic stimulation of inhibitory neurons combined with patch-clamp recording revealed that duvelisib inhibited paclitaxel-induced weakening of inhibitory, mainly glycinergic control on SCDH excitatory neurons. Enhanced excitatory and reduced inhibitory neurotransmission in the SCDH following PIPN was also alleviated by duvelisib application. In summary, duvelisib showed a promising ability to prevent neuropathic pain in PIPN. The potential use of our findings in human medicine may be augmented by the fact that duvelisib is an FDA-approved drug with known side effects.SIGNIFICANCE STATEMENT We show that duvelisib, a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, prevents the development of paclitaxel-induced pain-like behavior in males and females and prevents pronociceptive signaling in DRGs and spinal cord dorsal horn in rat and mouse model of paclitaxel-induced peripheral neuropathy.

A Novel Antigen, Otubain Cysteine Peptidase of Leishmania donovani, for the Serodiagnosis of Visceral Leishmaniasis and for Monitoring Treatment Response.

Tests for visceral leishmaniasis (VL) are not uniformly effective for all endemic regions. In a serological assay, a novel antigen, otubain cysteine peptidase, compared with rK39, showed comparable sensitivity with Indian VL serum samples and prominently increased sensitivity with Brazilian samples, as well as improved monitoring of the treatment response.

Immunoproteomic Identification and Characterization of Leishmania Membrane Proteins as Non-Invasive Diagnostic Candidates for Clinical Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a potentially fatal disease is an outcome of infection caused by the parasite Leishmania donovani. The clinical diagnostic tests for this disease are still related to invasive tissue aspiration or serological immunochromatography. Advancements in immunoproteomics such as two-dimensional gel electrophoresis, mass spectrometry, B cell epitope prediction, and peptide synthesis have enabled researchers to discover newer biomarkers for disease diagnosis. In this study, we have screened several urine-reactive leishmanial membrane proteins as potential biomarker candidates. In the immunoblot assay, three proteins 51, 55 and 63 kDa showed 100% reactivity to the urine of 47 VL patients and nonreactive to 18 healthy and other diseases. Mass spectrometry revealed the identity of 51, 55 and 63 kDa proteins as elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63, respectively. B cell reactive epitopes of these proteins were mapped through bioinformatic tools and one epitope from each protein that had the highest score were synthesized. All the three native electroeluted proteins and their corresponding synthetic peptides were tested through ELISA for reactivity with VL and control urine samples. While all three demonstrated good reactivity, the diagnostic performance of EF1-α was the best. Our findings illustrate the use of urine-based proteomic approach for biomarker discovery in non-invasive clinical diagnosis of VL.

Pulse Oximetry and Arterial Oxygen Saturation during Cardiopulmonary Exercise Testing.

INTRODUCTION/PURPOSE: Peripheral capillary oxygen saturation (SpO2) is used as surrogate for arterial blood oxygen saturation. We studied the degree of discrepancy between SpO2 and arterial oxygen (SaO2) and identified parameters that may explain this difference. METHODS: We included patients who underwent cardiopulmonary exercise testing at Cleveland Clinic. Pulse oximeters with forehead probes measured SpO2 and arterial blood gas samples provided the SaO2 both at rest and peak exercise. RESULTS: We included 751 patients, 54 ± 16 yr old with 53% of female gender. Bland-Altman analysis revealed a bias of 3.8% with limits of agreement of 0.3% to 7.9% between SpO2 and SaO2 at rest. A total of 174 (23%) patients had SpO2 ≥ 5% of SaO2, and these individuals were older, current smokers with lower forced expiratory volume in the first second and higher partial pressure of carbon dioxide and carboxyhemoglobin. At peak exercise (n = 631), 75 (12%) SpO2 values were lower than the SaO2 determinations reflecting difficulties in the SpO2 measurement in some patients. The bias between SpO2 and SaO2 was 2.6% with limits of agreement between -2.9% and 8.1%. Values of SpO2 ≥ 5% of SaO2 (n = 78, 12%) were associated with the significant resting variables plus lower heart rate, oxygen consumption, and oxygen pulse. In multivariate analyses, carboxyhemoglobin remained significantly associated with the difference between SpO2 and SaO2 both at rest and peak exercise. CONCLUSIONS: In the present study, pulse oximetry commonly overestimated the SaO2. Increased carboxyhemoglobin levels are independently associated with the difference between SpO2 and SaO2, a finding particularly relevant in smokers.

Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis.

BACKGROUND: Aurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important. METHODS: Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy. RESULTS: Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12µM and 82.9pmoles·min(-1)mg(-1) respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK-/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis. CONCLUSION: We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability. GENERAL SIGNIFICANCE: Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.

Cyclooxygenase-2 expression in non-metastatic triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) is characterised by lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER)2/neu gene amplification. TNBC patients typically present at a younger age, with a larger average tumor size, higher grade and higher rates of lymph node positivity compared to patients with ER/PR-positive tumors. Cyclooxygenase (COX)-2 regulates the production of prostaglandins and is overexpressed in a variety of solid tumors. In breast cancer, the overexpression of COX-2 is associated with indicators of poor prognosis, such as lymph node metastasis, poor differentiation and large tumor size. Since both TNBC status and COX-2 overexpression are known poor prognostic markers in primary breast cancer, we hypothesized that the COX-2 protein is overexpressed in the primary tumors of TNBC patients. The purpose of this study was to determine whether there exists an association between TNBC status and COX-2 protein overexpression in primary breast cancer. We prospectively evaluated COX-2 expression levels in primary tumor samples obtained from 125 patients with stage I-III breast cancer treated between February, 2005 and October, 2007. Information on clinicopathological factors was obtained from a prospective database. Baseline tumor characteristics and patient demographics were compared between TNBC and non-TNBC patients using the Chi-square and Fisher's exact tests. In total, 60.8% of the patients were classified as having ER-positive tumors, 51.2% were PR-positive, 14.4% had HER-2/neu amplification and 28.0% were classified as TNBC. COX-2 overexpression was found in 33.0% of the patients. TNBC was associated with COX-2 overexpression (P=0.009), PR expression (P=0.048) and high tumor grade (P=0.001). After adjusting for age, menopausal status, body mass index (BMI), lymph node status and neoadjuvant chemotherapy (NACT), TNBC was an independent predictor of COX-2 overexpression (P=0.01). In conclusion, the association between TNBC and COX-2 overexpression in operable breast cancer supports further investigation into COX-2-targeted therapy for patients with TNBC.

Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer.

Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5-10 mL) were collected prospectively from patients with clinical stages I-IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK-/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45-/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2- primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2- primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.

Regulation of miR-155 biogenesis in cystic fibrosis lung epithelial cells: antagonistic role of two mRNA-destabilizing proteins, KSRP and TTP.

Cystic fibrosis (CF) is characterized by a massive pro-inflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyper-expression of IL-8 protein through elevated expression of miR-155. We therefore investigated what factors promote the enhanced aberrant expression of miR-155 in CF. Here we report for the first time, the role of mRNA-destabilizing inflammatory RNA-binding proteins, KSRP and TTP, in the regulation of miR-155 biogenesis in CF lung epithelial cells. We find that KSRP and TTP have an antagonistic role in miR-155 biogenesis. While KSRP promotes enhanced processing of miR-155 precursors to mature miR-155, over-expression of TTP in the CF lung epithelial cells suppresses expression of miR-155. We find that TTP induces the expression of miR-1, which appears to be a regulator of miR-155 biogenesis in CF lung epithelial cells. These data provide novel insights into the mechanisms that induce hyper-inflammatory phenotype of CF, and are potential candidates for anti-inflammatory therapeutics for CF.

Circulating tumour cells in non-metastatic breast cancer: a prospective study.

BACKGROUND: The identification of circulating tumour cells correlate with poor prognosis in metastatic breast cancer, but there are few data describing the importance of circulating tumour cells in patients with non-metastatic disease. Our aim was to establish if circulating tumour cells predicted worse outcome in patients with non-metastatic breast cancer. METHODS: We prospectively collected data on circulating tumour cells at the time of definitive surgery from chemonaive patients with stage 1-3 breast cancer from February, 2005, to December, 2010. We deemed eligible all patients with operable breast cancer presenting at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients were ineligible if they had bilateral breast cancer or any other malignancy within 5 years of the diagnosis of the present cancer. We measured circulating tumour cells with the CellSearch System (Veridex, Raritan, NJ). We correlated findings of circulating tumour cells with standard tumour characteristics, including tumour size and grade; oestrogen and progesterone receptor and human epidural growth factor receptor 2 (HER2) status; and axillary lymph node status with χ(2) or Fisher exact tests. We assessed outcomes at a median follow-up of 35 months. Log-rank test and Cox regression analysis was applied to establish the association of circulating tumour cells with progression-free and overall survival. FINDINGS: No patients reported adverse events or complications from blood collections. We identified one or more circulating tumour cells in 73 (24%) of 302 patients. Detection of one or more circulating tumour cells predicted both decreased progression-free survival (log-rank p=0·005; hazard ratio [HR] 4·62, 95% CI 1·79-11·9) and overall survival (log-rank p=0·01; HR 4·04, 1·28-12·8). INTERPRETATION: The presence of one or more circulating tumour cells predicted early recurrence and decreased overall survival in chemonaive patients with non-metastatic breast cancer. These results suggest that assessment of circulating tumour cells might provide important prognostic information in these patients. FUNDING: Society of Surgical Oncology, Morgan Welch Inflammatory Breast Cancer Program, The University of Texas MD Anderson Cancer Center, and the State of Texas Rare and Aggressive Breast Cancer Research Program.

Invasive lobular carcinoma predicts micrometastasis in breast cancer.

BACKGROUND: Invasive lobular carcinomas (ILCs) are almost always estrogen receptor (ER) positive. Most delayed breast cancer recurrences occur in ER-positive patients. Disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) have been implicated in recurrence. The purpose of this study was to determine whether DTCs and CTCs are associated with ILCs in stage I-III breast cancer. MATERIALS AND METHODS: Clinical stage I-III breast cancer patients consented to participate in an institutional review board-approved study involving collection of bone marrow and blood at surgery for primary breast cancer. We assessed DTCs by anti-CK antibody cocktail after cytospin. We detected CTCs using CellSearch, and defined them as nucleated cells lacking CD45 but expressing cytokeratin. One or more cells per 5 mL bone marrow or 7.5 mL blood was considered positive. We performed statistical analyses using chi-square and Fisher's exact tests. RESULTS: We prospectively enrolled 422 patients, 64 with ILC and 358 with invasive ductal carcinoma. Estrogen receptor positivity was higher in ILCs (92.2% versus 66.2%) {P = .001}. We identified DTCs to be 43.4% with ILC compared with 28.9% with IDC {P = 0.03}. The CTC rates were similar. Either DTCs or CTCs were identified in 75.6% with ILC, compared with 51.7% with invasive ductal carcinoma {P = .002}. We observed no correlation between the presence of DTCs and CTCs in ILC patients and tumor size, grade, hormone receptor status, stage, lymph node status, or administration of NACT. CONCLUSIONS: Invasive lobular carcinomas independently predicted micrometastatic disease. Because most late recurrences are ER positive, this raises the question of whether DTCs and CTCs are indeed responsible for late breast cancer recurrence.

Peripartum cardiomyopathy: a review.

Peripartum cardiomyopathy is idiopathic heart failure occurring in the absence of any determinable heart disease during the last month of pregnancy or the first 5 months postpartum. The incidence varies worldwide but is high in developing nations; the cause of the disease might be a combination of environmental and genetic factors. Diagnostic echocardiographic criteria include left ventricular ejection fraction <0.45 or M-mode fractional shortening <30% (or both) and end-diastolic dimension >2.7 cm/m(2). Electrocardiography, magnetic resonance imaging, endomyocardial biopsy, and cardiac catheterization aid in the diagnosis and management of peripartum cardiomyopathy. Cardiac protein assays can also be useful, as suggested by reports of high levels of NT-proBNP, cardiac troponin, tumor necrosis factor-α, interleukin-6, interferon-γ, and C-reactive protein in peripartum cardiomyopathy. The prevalence of mutations associated with familial dilated-cardiomyopathy genes in patients with peripartum cardiomyopathy suggests an overlap in the clinical spectrum of these 2 diseases.Treatment for peripartum cardiomyopathy includes conventional pharmacologic heart-failure therapies-principally diuretics, angiotensin-converting enzyme inhibitors, vasodilators, digoxin, ß-blockers, anticoagulants, and peripartum cardiomyopathy-targeted therapies. Therapeutic decisions are influenced by drug-safety profiles during pregnancy and lactation. Mechanical support and transplantation might be necessary in severe cases. Targeted therapies (such as intravenous immunoglobulin, pentoxifylline, and bromocriptine) have shown promise in small trials but require further evaluation. Fortunately, despite a mortality rate of up to 10% and a high risk of relapse in subsequent pregnancies, many patients with peripartum cardiomyopathy recover within 3 to 6 months of disease onset.

Utilization of vinasse for the production of polyhydroxybutyrate by Haloarcula marismortui.

Vinasse, a recalcitrant waste of the ethanol industry was employed for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloarcula marismortui in shake flasks. The PHA was recovered by osmotic lysis of the cells and subsequent purification by sodium hypochlorite and organic solvents. Through UV-vis spectroscopy, differential scanning calorimetry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy, the PHA was found to have characteristics very similar to that of the standard polyhydroxybutyrate (PHB) from Sigma. Inhibitory effect of polyphenols contained in vinasse was assessed by a quick and reliable cup-plate agar-diffusion method. Raw vinasse (10%) was utilized leading to accumulation of 23% PHA (of cell dry weight) and following an efficacious pre-treatment process through adsorption on activated carbon, 100% pre-treated vinasse could be utilized leading to 30% accumulation of PHB by H. marismortui. Maximum specific growth rate, specific production rate, and volumetric productivity attained using 10% raw vinasse were comparable to that obtained using a previously reported nutrient deficient medium (NDM), while the values with 100% pre-treated vinasse were higher than that determined using NDM medium. This is the first report of polyhydroxybutyrate production by a halophilic microorganism utilizing vinasse.

Disseminated tumor cells predict survival after neoadjuvant therapy in primary breast cancer.

BACKGROUND: Tumor cells that disseminate to the bone marrow (disseminated tumor cells [DTCs]) have been identified in 30% of patients with stage I through II breast cancer (BC) and predict outcome. Neoadjuvant chemotherapy (NACT) is effective in reducing the size of primary tumors or eradicating lymph node metastases before surgery, but little is known regarding the presence or significance of DTCs after NACT. METHODS: The authors evaluated DTCs in 95 patients with clinical stage I through III BC. Bone marrow samples were collected after completion of NACT at the time they underwent surgery for primary BC. DTCs were assessed using an anticytokeratin antibody cocktail. Primary tumor markers, the extent of lymph node (LN) involvement, they type of NACT administered, and response to NACT were compared with presence of DTCs. Chi-square and Fisher exact tests were used for statistical analyses. RESULTS: The median patient age at diagnosis was 51 years, and the median follow-up was 24 months. Forty-six percent of patients had tumors classified as T1/T2, 20% had T3 tumors, 34.5% had T4 tumors, and 81% had lymph node metastasis before NACT. DTCs were identified in 26% of patients after NACT. No associations were observed between DTCs and primary tumor characteristics or LN involvement. A pathologic complete response was observed in 25 patients (26%) but was not predictive of DTCs after NACT (P = .83). DTCs after NACT predicted worse BC-specific survival (P < .02). CONCLUSIONS: The presence of DTCs was an independent predictor of outcome after NACT. The current results indicated that monitoring hematogenous micrometastatic disease after NACT may be useful in selecting patients who might benefit from additional systemic adjuvant therapies.