ALBERT

Description: Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms.

Description: Renal cell carcinoma (RCC) is a heterogeneous malignancy which often develops and progresses asymptomatically. Benign oncocytomas are morphologically similar to malignant chromophobe RCC and distinguishing between these two forms on cross-sectional imaging remains a challenge. Therefore, RCC-specific biomarkers are urgently required for accurate and non-invasive, pre-surgical diagnosis of benign lesions. We have previously shown that dysregulation in glycolytic and tricarboxylic acid cycle intermediates can distinguish benign lesions from RCC in a stage-specific manner. In this study, preoperative fasting urine samples from patients with renal masses were assessed by ¹H nuclear magnetic resonance (NMR). Significant alterations in levels of tricarboxylic acid cycle intermediates, carnitines and its derivatives were detected in RCC relative to benign masses and in oncocytomas vs. chromophobe RCC. Orthogonal Partial Least Square Discriminant Analysis plots confirmed stage discrimination between benign vs. pT1 (R2 = 0.42, Q2 = 0.27) and benign vs. pT3 (R2 = 0.48, Q2 = 0.32) and showed separation for oncocytomas vs. chromophobe RCC (R2 = 0.81, Q2 = 0.57) and oncocytomas vs. clear cell RCC (R2 = 0.32, Q2 = 0.20). This study validates our previously described metabolic profile distinguishing benign tumors from RCC and presents a novel metabolic signature for oncocytomas which may be exploited for diagnosis before cross-sectional imaging.

Description: Caffeine-containing, nutritionally fortified energy shots are consumed at high rates by adolescents, yet little is known about their metabolic impact. The purpose of this study was to examine the consequences of small format, caffeinated energy shots on glucose metabolism and gastrointestinal hormone secretion in adolescents. Twenty participants aged 13–19 years participated in a double-blind, randomized cross-over study consisting of two trials separated by 1–4 weeks. Participants consumed a volume-matched caffeinated energy shot (CAF, 5 mg/kg) or a decaffeinated energy shot (DECAF) followed by a 2 h oral glucose tolerance test. Blood samples were collected and area under the curve (AUC) calculated for glucose, insulin and gut and metabolic hormones. Consumption of CAF resulted in a 25% increase in glucose and a 26% increase in insulin area under the curve (AUC, p = 0.037; p 〈 0.0001) compared to DECAF. No impact on gut hormones was observed. To further characterize responses, individuals were classified as either slow or fast caffeine metabolizers based on an allele score. Glucose intolerance was greater in genetically fast vs. slow caffeine metabolizers and differences between groups were supported by distinct serum metabolomics separation. Consumption of caffeine-containing energy shots results in acute impaired glucoregulation in healthy adolescents as characterized by hyperinsulinemia following an oral glucose challenge.

Description: While children with appendicitis often have excellent clinical outcomes, some develop life-threatening complications including sepsis and organ dysfunction requiring pediatric intensive care unit (PICU) support. Our study applied a metabolomics and inflammatory protein mediator (IPM) profiling approach to determine the bio-profiles of children who developed severe appendicitis compared with those that did not. We performed a prospective case-control study of children aged 0–17 years with a diagnosis of appendicitis. Cases had severe disease resulting in PICU admission. Primary controls had moderate appendicitis (perforation without PICU); secondary controls had mild appendicitis (non-perforated). Serum samples were analyzed using Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy and Gas Chromatography-Mass Spectrometry (GC-MS); IPM analysis was performed using plasma bead-based multiplex profiling. Comparisons were made using multivariate data statistical analysis. Fifty-three children were included (15 severe, 38 non-severe). Separation between severe and moderate appendicitis demonstrated excellent sensitivity and specificity (100%, 88%; 14 compounds), separation between severe and mild appendicitis also showed excellent sensitivity and specificity (91%, 90%; 16 compounds). Biomarker patterns derived from metabolomics and IPM profiling are capable of distinguishing children with severe appendicitis from those with less severe disease. These findings provide an important first step towards developing non-invasive diagnostic tools for clinicians in early identification of children who are at a high risk of developing severe appendicitis.