Oligomeric structure of the alpha1b-adrenoceptor: comparisons with rhodopsin

The structural basis of the quaternary organization of rhodopsin has recently been explored and modeled. Because information obtained from studying rhodopsin has frequently been directly applicable to other G protein-coupled receptors we wished to ascertain if dimeric and/or oligomeric forms of the alpha(1b)-adrenoceptor could be observed and if so whether rhodopsin might provide insights into the quaternary structure of this receptor. Co-immunoprecipitation and both conventional and time-resolved fluorescence resonance energy transfer studies demonstrated quaternary structure of the alpha(1b)-adrenoceptor and, in concert with the reconstitution of fragments of this receptor, provided information on the molecular basis of these interactions. Development of three color fluorescence resonance energy transfer (FRET) allowed the imaging of alpha(1b)-adrenoceptor oligomers in single living cells. Mutation of hydrophobic residues in transmembrane domains I and IV of the receptor resulted in marked reduction in three color FRET suggesting an alteration in oligomeric organization and potential similarities with rhodopsin. The mutated alpha(1b)-adrenoceptor was unable to reach the cell surface, did not become terminally N-glycosylated and was unable to signal.     

Resistance to ceftazidime is associated with a S220Y substitution in the omega loop of the AmpC beta-lactamase of a Serratia marcescens clinical isolate

OBJECTIVES: The aim of this study was to characterize the ampC beta-lactamase gene of a clinical isolate of Serratia marcescens resistant to ceftazidime. METHODS: S. marcescens SMSA was isolated from an intra-abdominal wound of a patient previously treated with ceftazidime. A susceptible strain, SLS73, was used as a control. Susceptibility testing, PCR, DNA sequencing, molecular cloning, site-directed mutagenesis and determination of kinetic parameters were carried out to investigate the mechanism of resistance to ceftazidime. RESULTS: MICs of ceftazidime were 64 and 0.2 mg/L for SMSA and SLS73, respectively. Sequencing of the ampC gene of SMSA was carried out. When compared with the closest AmpC enzyme, the S. marcescens S3 beta-lactamase, the novel protein showed E57Q, Q129K and S220Y substitutions. The S220Y substitution is located in the omega loop. Introduced by mutagenesis in the ampC gene of SLS73, this substitution conferred the same level of resistance to ceftazidime. The catalytic efficiency (k(cat)/K(m)) of the mutated enzyme toward ceftazidime was increased by about 100-fold. CONCLUSIONS: We present another example of in vivo selection of broad-spectrum resistance by amino acid substitution in the omega loop of chromosomal AmpC beta-lactamase in S. marcescens.     

Protective effect of superoxide dismutase in radiation-induced intestinal inflammation

PURPOSE: To analyze the therapeutic value of Cu/Zn-superoxide dismutase (SOD1) supplementation in an experimental model of radiation-induced intestinal inflammation and explore its mechanistic effects. METHODS AND MATERIALS: Mice were subjected to abdominal irradiation with 10 Gy or sham irradiation and studied 24 or 72 hours after radiation. Groups of mice were treated with 0.1, 4, or 6 mg/kg/day of SOD1 or vehicle. Leukocyte-endothelial cell interactions in intestinal venules were assessed by intravital microscopy. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was determined with radiolabeled antibodies. Effects of SOD1 on histologic damage and levels of lipid hydroperoxides were also measured. RESULTS: A significant increase in the flux of rolling leukocytes and number of firmly adherent leukocytes in intestinal venules was observed at 24 and 72 hours after irradiation. Treatment with SOD1 had no effect on leukocyte rolling but significantly and dose-dependently decreased firm leukocyte adhesion to intestinal venules. Treatment with SOD1 at doses that reduced leukocyte recruitment abrogated the increase in hydroperoxides in intestinal tissue and ICAM-1 upregulation in intestinal endothelial cells. The inflammatory score, but not a combined histology damage score, was also significantly reduced by SOD1. CONCLUSIONS: Treatment with SOD1 decreases oxidative stress and adhesion molecule upregulation in response to abdominal irradiation. This is associated with an attenuation of the radiation-induced intestinal inflammatory response.     

Delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor

The treatment of Alzheimer's disease and many other brain-related disorders is limited because of the presence of the blood-brain barrier, which highly regulate the crossing of drugs. Metal nanoparticles have unique features that could contribute to the development of new therapies for these diseases. Nanoparticles have the capacity to carry several molecules of a drug; furthermore, their unique physico-chemical properties allow, for example, photothermal therapy to produce molecular surgery to destroy tumor cells and toxic structures. Recently, we demonstrated that gold nanoparticles conjugated to the peptide CLPFFD are useful to destroy the toxic aggregates of β-amyloid, similar to the ones found in the brains of patients with Alzheimer's disease. However, nanoparticles, like many other compounds, have null or very low capacity to cross the blood-brain barrier. In order to devise a strategy to improve drug delivery to the brain, here we introduced the peptide sequence THRPPMWSPVWP into the gold nanoparticle-CLPFFD conjugate. This peptide sequence interacts with the transferrin receptor present in the microvascular endothelial cells of the blood-brain barrier, thus causing an increase in the permeability of the conjugate in brain, as shown by experiments in?vitro and in?vivo. Our results are highly relevant for the therapeutic applications of gold nanoparticles for molecular surgery in the treatment of neurodegenerative diseases such as Alzheimer's disease.     

A new method for the determination of arylesterase activity in human serum using simulated body fluid

Arylesterase activity (EC 3.1.1.2), one of the three functions of the paraoxonase enzyme (PON1), is thought to protect low-density lipoproteins (LDL) and high-density lipoprotein (HDL) from oxidation and to facilitate reverse cholesterol transport. The Eckerson et al. method, which employs Tris/HCl buffer, has been extensively used and could be considered as the reference method, although it shows relatively low precision and reproducibility. Using simulated body fluid (SBF), which simulates blood electrolyte composition, a significant improvement in arylesterase activity determination was achieved. Modifications of SBF bicarbonate and chloride concentrations did not result in further improvements. To validate this method arylesterase activity was measured using SBF and Tris/HCl in 23 subjects with increased cardiovascular disease risk. Precision was significantly higher using SBF than with Tris/HCl. Data from both methods do not significantly differ in samples with arylesterase activity > or = 30.6 U/L using SBF, but do differ significantly when very low activity samples (< 30.6 U/L) were included. Differences suggest that the Tris/HCl buffer gives misleading activity results, especially in very low activity samples. For the first time, results suggest that SBF can successfully be used instead of Tris/HCl in arylesterase activity determination.     

Targeting the CYP2B 1/cyclophosphamide suicide system to fibroblast growth factor receptors results in a potent antitumoral response in pancreatic cancer models

The CYP2B1/cyclophosphamide (CPA) suicide gene therapy approach has been shown to be highly promising in clinical trials for the treatment of pancreatic cancer. However, delivering the therapeutic gene to a sufficient number of tumor cells able to trigger a complete response remains a challenge. Target-specific delivery of adenovirus to fibroblast growth factor receptors (FGFRs) has been obtained in a variety of tumor models and has been shown to highly increase transduction efficiency. In the present paper we have tested the therapeutic outcome of retargeting the adenoviral vector, Ad-CYP2B1, to FGFRs, using an FGF2-Fab' conjugate, in pancreatic cancer models. First, we show a heterogeneous subcellular distribution of overexpressed FGFR-1 in pancreatic cancer cells. Higher transduction efficiency was observed in five of the six cell lines studied after FGF2-AdGFPLuc infection. Interestingly, an association between FGFR-1 membrane cell expression and viral entry was found. Moreover, tumors injected with FGF2-AdGFPLuc showed enhanced and persistent transgene expression. Importantly, we demonstrate the relevant enhanced cytotoxic effect of the FGF2-Ad-CYP2B]/CPA system in four of the six cell lines studied. Moreover, retargeting Ad-CYP2B1/CPA to FGFRs resulted in a potent antitumoral effect and in an increased survival rate, in two human pancreatic xenograft models. Thus, our results indicate that redirecting adenoviruses to FGFRs highly increases the potency of the suicide system CYP2B1/CPA. Consequently, it may constitute a promising approach to the treatment of patients with pancreatic tumors, in which a high proportion of FGF receptors precisely localize to the plasma membrane.     

Bosutinib in Combination With the Aromatase Inhibitor Exemestane: A Phase II Trial in Postmenopausal Women With Previously Treated Locally Advanced or Metastatic Hormone Receptor‐Positive/HER2‐Negative Breast Cancer

Background.

Bosutinib is an oral, selective Src/Abl tyrosine kinase inhibitor with activity in breast cancer (BC). We evaluated bosutinib plus exemestane as second-line therapy in previously treated hormone receptor-positive (HR+) locally advanced or metastatic BC.

Methods.

This was a phase II study with patients enrolled in a single-arm safety lead-in phase. Patients receiving bosutinib at 400 mg or 300 mg/day (based on toxicity) plus exemestane at 25 mg/day were monitored for adverse events (AEs) and dose-limiting toxicities for 28 days, and initial efficacy was assessed. After the lead-in and dose-determination phase, randomized evaluation of combination therapy versus exemestane was planned.

Results.

Thirty-nine of 42 patients (93%) experienced treatment-related AEs including diarrhea in 28 (67%) and hepatotoxicity in 11 (26%); overall serious treatment-related AEs were recorded in 4 (10%). No liver toxicity met Hy’s law criteria. Dose-limiting toxicities occurred in 5 of 13 patients receiving 400 mg (38%) and 3 of 26 patients receiving 300 mg (12%) of bosutinib; all resolved on treatment discontinuation. One patient (300 mg/day) achieved confirmed partial response; three (400 mg/day, n = 2; 300 mg/day, n = 1) maintained stable disease for >24 weeks; a best response of progressive disease occurred in 15 of 42 patients (36%). Median progression-free survival was 12.3 weeks (80% confidence interval: 11.0–15.6).

Conclusion.

The risk-benefit profile of bosutinib at 300 mg/day plus exemestane resulted in early study termination before the randomized portion. Alternative bosutinib regimens merit investigation in BC.     

Microbiome signatures of progression toward celiac disease onset in at-risk children in a longitudinal prospective cohort study

Other than exposure to gluten and genetic compatibility, the gut microbiome has been suggested to be involved in celiac disease (CD) pathogenesis by mediating interactions between gluten/environmental factors and the host immune system. However, to establish disease progression markers, it is essential to assess alterations in the gut microbiota before disease onset. Here, a prospective metagenomic analysis of the gut microbiota of infants at risk of CD was done to track shifts in the microbiota before CD development. We performed cross-sectional and longitudinal analyses of gut microbiota, functional pathways, and metabolites, starting from 18 mo before CD onset, in 10 infants who developed CD and 10 matched nonaffected infants. Cross-sectional analysis at CD onset identified altered abundance of six microbial strains and several metabolites between cases and controls but no change in microbial species or pathway abundance. Conversely, results of longitudinal analysis revealed several microbial species/strains/pathways/metabolites occurring in increased abundance and detected before CD onset. These had previously been linked to autoimmune and inflammatory conditions (e.g., Dialister invisus, Parabacteroides sp., Lachnospiraceae, tryptophan metabolism, and metabolites serine and threonine). Others occurred in decreased abundance before CD onset and are known to have anti-inflammatory effects (e.g., Streptococcus thermophilus, Faecalibacterium prausnitzii, and Clostridium clostridioforme). Additionally, we uncovered previously unreported microbes/pathways/metabolites (e.g., Porphyromonas sp., high mannose–type N-glycan biosynthesis, and serine) that point to CD-specific biomarkers. Our study establishes a road map for prospective longitudinal study designs to better understand the role of gut microbiota in disease pathogenesis and therapeutic targets to reestablish tolerance and/or prevent autoimmunity.

Celiac disease (CD) is a chronic systemic autoimmune disorder that occurs in genetically predisposed individuals and is characterized by loss of tolerance to dietary gluten protein. CD affects ∼1% of the global population, with regional variations depending on human leukocyte antigen (HLA) presence and dietary gluten consumption (1). The incidence of CD most likely will continue to increase, along with other autoimmune conditions, despite the fact that its associated genes, HLA, and the trigger, gluten, have not changed (1). Nevertheless, more than 30% of the population carries the predisposing gene and is exposed to gluten, yet only 2 to 3% develop CD in their lifetime (2). This suggests that other factors such as the intestinal microbiota may also contribute to CD pathogenesis.The inflammatory process underlying CD involves both innate and adaptive immune systems (3). While the adaptive immune response in CD has been described, less is known about the innate immune response following gluten exposure, which drives early steps in CD pathogenesis and eventually leads to loss of gluten tolerance (4). Previous work has linked the trigger of CD, gluten, the intestinal microbiota, and the innate immune response (5–8).Given the cross-talk between the gut microbiota and immune system, alterations in the gut microbiota have been linked to several autoimmune conditions (9) such as inflammatory bowel disease (10), type 1 diabetes (T1D) (11), multiple sclerosis (12), and CD (13–18). We, and others, have looked for changes in the gut microbiota of infants at risk for CD (15, 17–19). For example, using 16S ribsosomal ribonucelic acid (rRNA) amplicon sequencing, we previously reported higher abundance of Lactobacillus up to 12 mo of age in one infant who later developed CD compared with 15 at-risk infants who did not (15). Other studies of gut microbiota and CD assessed changes in gut microbiota composition of individuals during the first year after birth who later developed CD compared with controls (17, 18). For example, Olivares et al. (17) used a prospective cohort of 200 infants at risk for CD to compare, with 16S rRNA sequencing, the intestinal microbiota of 10 cases who developed CD during the 5-y study period and 10 matched controls at 4 and 6 mo of age. They reported increases in abundance of Firmicutes, Enterococcaceae, and Peptostreptococcaceae in controls from 4 to 6 mo (17). Rintala et al. (18) also examined intestinal microbiota of infants at risk for CD, using 16S rRNA sequencing, at 9 and 12 mo of age in nine subjects who developed CD by ages 4 and 18 and matched controls, but did not identify any significant differences in microbiota composition. Huang et al. (20) examined intestinal microbiota, using 16S rRNA sequencing, at ages 1, 2.5, and 5 y in 16 subjects with CD (11 of whom developed CD after age 5) and 16 controls and found significant differences in taxonomic composition of the microbiota in cases compared with controls at all of the time points. Finally, a recent study using 16S rRNA sequencing to examine differences in the gut microbiota of children with untreated CD compared with children with treated CD and healthy control subjects did not identify changes in alpha diversity at CD diagnosis (21) but did identify differences in taxonomic composition, such as a lower abundance in Alistipes in subjects with CD compared with healthy controls (21). A separate study utilizing 16S rRNA sequencing also identified significant differences in taxonomic composition between patients with newly diagnosed CD and healthy control subjects, with subjects with recently diagnosed CD having a lower abundance of Bacteroides–Prevotella, Akkermansia, and Staphylococcaceae (22).While these studies provide an important foundation concerning alterations in gut microbiota of subjects at risk for CD early in life, they analyzed only up to three time points in the first year after birth (17, 18, 20) or were restricted to only one subject with CD (15). In addition, the studies used 16S rRNA sequencing to analyze intestinal microbiota, which cannot provide information about functional characteristics of the microbiota nor provide taxonomic data at the strain level, both of which are necessary to design effective treatment for CD. Furthermore, metabolomic analysis (if any) in these studies was generally limited to serum (as opposed to fecal) metabolites, which do not provide direct information about metabolic activity of the gut microbiota. More importantly, here we argue that, to gain mechanistic insight into the pathogenesis of CD and other autoimmune diseases, we need to transition from case–control microbiome studies to prospective longitudinal studies, which prospectively examine subjects at multiple time points before disease development (23). Studies aimed at identifying changes in the microbiome (11, 24) and intestinal permeability (25) have been performed and identified taxonomic changes prior to T1D (11) and necrotizing enterocolitis (24) as well as increased intestinal permeability up to 3 y prior to the development of Crohn’s disease (25). However, longitudinal birth cohort studies focused on multiomic data collection and analysis are limited and have not been developed for CD.The first step toward achieving this goal was to establish a prospective cohort study for CD, the Celiac Disease Genomic, Environmental, Microbiome, and Metabolomic (CDGEMM) study (26), where we have been following approximately 500 infants in the United States, Italy, and Spain who have a first-degree relative with CD and therefore, are at a high risk of developing CD. We have previously utilized other study subjects from this cohort and multiomics analysis to investigate the impact of genetic and environmental risk factors on the development of the gut microbiota in infants at risk for CD (19). In the current study, we present proof of concept intersubject and intrasubject analyses using fecal metagenomic and metabolomic data collected at multiple time points before onset of CD in 10 cases and 10 matched controls in order to identify alterations in the intestinal microbiota and metabolome, which may serve as markers of progression toward CD onset.     

Polycondensation of Metal Salts of 6‐(2‐Chloroacetate)hexanoic Acid: A New Method to Synthesize Alternating Copolyesters Constituted by Glycolic Acid Units

A new copolyester was prepared from glycolic acid and 6‐hydroxyhexanoic acid units. The monomer was prepared by a thermal polycondensation method and its reactivity evaluated by means of DSC and IR spectroscopy. The driving force of the reaction was the formation of a metal chloride salt. Thus, the reactivity depended on the ionization potential of the alkaline cation; while the cesium salt started to polymerize at room temperature, the sodium salt did so only above 120 °C. The low reaction temperature and the absence of catalysts allowed transesterification and other secondary reactions to be avoided. DSC, optical microscopy and XRD techniques demonstrated that the new polymer was highly crystalline. The final molecular weight was sufficient to obtain fiber‐forming properties.