MicroRNAs from urinary extracellular vesicles are non-invasive early biomarkers of diabetic nephropathy in type 2 diabetes patients with the 'Asian Indian phenotype'.

AIMS: MicroRNAs (miRNAs) from extracellular vesicles (EVs) have been proposed as promising biomarkers for a number of diseases. In this study, their potential as urine-based biomarkers of diabetic nephropathy (DN) was assessed. METHODS: MiRNAs were profiled in urinary EVs from 160 fasting subjects with normal glucose tolerance (NGT) and in T2DM patients with either microalbumininuria (MIC) or macroalbuminuria (MAC). RESULTS: A total of 73 miRNAs detected in urinary EVs (NGT) were predicted to target important functions for kidney homoeostasis, thereby validating their use as indicators of kidney dysfunction. Indeed, a urinary EV miRNA signature was found to comprise increased levels of let-7i-3p, miR-24-3p and miR-27b-3p, and decreased levels of miR-15b-5p, to identify patients with MIC. ROC curve analysis confirmed this ability to identify MIC in normo-albuminuria T2DM (T2DM-NA) patients and to differentiate between MAC and T2DM patients. These miRNAs were also predicted to target protein networks involved in the Wnt/ß-catenin signalling cascade, activin receptor signalling and cell differentiation/proliferation, and correlated with eGRF, HbA1c, serum creatinine, urea, albumin and blood pressure. Concentrations of miR-30a-5p were specifically modified in urinary EVs from patients with MAC, but not MIC, suggesting that miR-30a-5p could be related to severe kidney damage. CONCLUSION: Urinary EV miRNAs correlate with the degree of MIC. As they are also thought to regulate pathways that are targets of pharmacological agents to prevent DN (reticulum stress, activin receptors), they may also serve as non-invasive 'liquid biopsies' to stratify patients at risk of developing MAC and to monitor treatment efficacy.

Raw material variability of an active pharmaceutical ingredient and its relevance for processability in secondary continuous pharmaceutical manufacturing.

Active Pharmaceutical Ingredients (API) raw material variability is not always thoroughly considered during pharmaceutical process development, mainly due to low quantities of drug substance available. However, synthesis, crystallization routes and production sites evolve during product development and product life cycle leading to changes in physical material attributes which can potentially affect their processability. Recent literature highlights the need for a global approach to understand the link between material synthesis, material variability, process and product quality. The study described in this article aims at explaining the raw material variability of an API using extensive material characterization on a restricted number of representative batches using multivariate data analysis. It is part of a larger investigation trying to link the API drug substance manufacturing process, the resulting physical API raw material attributes and the drug product continuous manufacturing process. Eight API batches produced using different synthetic routes, crystallization, drying, delumping processes and processing equipment were characterized, extensively. Seventeen properties from seven characterization techniques were retained for further analysis using Principal Component Analysis (PCA). Three principal components (PCs) were sufficient to explain 92.9% of the API raw material variability. The first PC was related to crystal length, agglomerate size and fraction, flowability and electrostatic charging. The second PC was driven by the span of the particle size distribution and the agglomerates strength. The third PC was related to surface energy. Additionally, the PCA allowed to summarize the API batch-to-batch variability in only three PCs which can be used in future drug product development studies to quantitatively evaluate the impact of the API raw material variability upon the drug product process. The approach described in this article could be applied to any other compound which is prone to batch-to-batch variability.

Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process "anatomical structure morphogenesis" contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired-like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5-Aza-2'-deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof-of-concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

Gene expression profiling in peripheral blood cells of patients with rheumatoid arthritis in response to anti-TNF-alpha treatments.

The efficacy of anti-TNF-α therapies highlights the role of TNF-α in the pathogenesis of rheumatoid arthritis (RA). However, the mechanism of action of these agents is poorly understood at the molecular level. The aim of this study was to characterize the effects of anti-TNF-α treatment on the global gene expression profile in peripheral blood mononuclear cells (PBMCs) of responder RA patients. Changes in gene expression were determined using oligonucleotide microarrays (25,341 genes) in PBMCs obtained before and after 12 wk of treatment with either etanercept or adalimumab from responder RA patients. Two hundred fifty-one genes displayed significant changes (false discovery rate < 0.1%) in expression level (178 upregulations with mean fold change = 1.5 and 73 downregulations with mean fold change = -1.50) after 12 wk of treatment. Importantly, the expression of several genes, including those coding for the calcium binding proteins S100A12 and A8, CD14 antigen, Selectin P, or ribosomal protein L39, reported to be upregulated in RA patients, were found to be decreased after anti-TNF-α treatment. Globally, inflammation, immune response, apoptosis, protein synthesis, and mitochondrial oxido-reduction were the most affected pathways in response to anti-TNF-α treatment. The obtained gene expression signature in PBMCs provides new information to better understand the mechanisms of action of anti-TNF-α treatment in RA patients.

Microarray analysis of genes with impaired insulin regulation in the skeletal muscle of type 2 diabetic patients indicates the involvement of basic helix-loop-helix domain-containing, class B, 2 protein (BHLHB2).

AIMS/HYPOTHESIS: One of the major processes by which insulin exerts its multiple biological actions is through gene expression regulation. Thus, the identification of transcription factors affected by insulin in target tissues represents an important challenge. The aim of the present study was to gain a greater insight into this issue through the identification of transcription factor genes with insulin-regulated expression in human skeletal muscle. METHODS: Using microarray analysis, we defined the sets of genes modulated during a 3 h hyperinsulinaemic-euglycaemic clamp (2 mU min(-1) kg(-1)) in the skeletal muscle of insulin-sensitive control volunteers and in moderately obese insulin-resistant type 2 diabetic patients. RESULTS: Of the 1,529 and 1,499 genes regulated during the clamp in control and diabetic volunteers, respectively, we identified 30 transcription factors with impaired insulin-regulation in type 2 diabetic patients. Analysis of the promoters of the genes encoding these factors revealed a possible contribution of the transcriptional repressor basic helix-loop-helix domain-containing, class B, 2 protein (BHLHB2), insulin regulation of which is strongly altered in the muscle of diabetic patients. Gene ontology analysis of BHLHB2 target genes, identified after BHLHB2 overexpression in human primary myotubes, demonstrated that about 10% of the genes regulated in vivo during hyperinsulinaemia are potentially under the control of this repressor. The data also suggested that BHLHB2 is situated at the crossroads of a complex transcriptional network that is able to modulate major metabolic and biological pathways in skeletal muscle, including the regulation of a cluster of genes involved in muscle development and contraction. CONCLUSIONS/INTERPRETATION: We have identified BHLHB2 as a potential novel mediator of insulin transcriptional action in human skeletal muscle.

Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes.

Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+]i in most cells, but always suppressed subsequent rises of [Ca2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system.

Evidence for the absence of an intestinal adaptive mechanism to compensate for C. parvum-induced amino acid malabsorption in suckling rats.

In order to assess the impact of Cryptosporidium parvum on host intestinal physiology, we investigated absorption of the two principal amino acids in dam's milk (leucine, glutamate), using Ussing chambers and RT-PCR analyses. Experiments were performed in both heavily (ileum) and mildly (duodenum) infected segments of the small intestine at the peak of infection [day 8 post-infection (PI)] and after spontaneous clearance of the parasite (day 17 PI). At day 8 PI, amino acid fluxes across the mucosa were decreased throughout the small intestine (P<0.01) and EAAT3 mRNA expression was reduced ( from -49% to -28%). At day 17 PI, leucine and glutamate fluxes were normalized but the decrease in EAAT3 mRNA levels persisted (from -31% to -46%). Our results demonstrate that cryptosporidiosis induces major amino acid malabsorption involving the entire small intestine which is not counterbalanced by any up-regulation, even after spontaneous clearance of the parasite.

The effect of fatigue on abnormal vibration induced illusion of movement in idiopathic focal dystonia.

BACKGROUND: Perception of vibration induced illusionary movement (VIIM) is subnormal in dystonic patients, suggesting abnormal sensory-motor processing in patients with idiopathic focal dystonia. OBJECTIVE: To examine the effects of fatigue on VIIM in patients with idiopathic torticollis. METHODS: An illusionary sensation of arm extension was evoked by an 80 Hz transcutaneous vibratory stimulus applied to the biceps brachii tendon while the arm was restrained. Blindfolded patients attempted to copy the perceived movement of the vibrated arm with the opposite (tracking) arm and the change in elbow angle of the tracking arm was quantified over 45 seconds. The tasks were repeated following volitional fatigue of the vibrated arm. RESULTS: The subnormal perception of VIIM perceived by patients with torticollis, occurring bilaterally and remote from the location of dystonic symptoms, was corrected by fatigue of the vibrated arm compared with prefatigue values (mean (SEM): 19.04 degrees (1.76) degrees v 24.25 degrees (2.41 degrees ); p = 0.01, paired t test). CONCLUSIONS: While a combination of central or peripheral factors may be involved in the correction of abnormal perception of the vibration induced illusion of movement in dystonia, subnormal elasticity of muscle spindles could be implicated in the impaired perception of vibration induced illusionary movement and may predispose an individual towards developing idiopathic focal dystonia.

Intestinal peptide transporter PepT1 is over-expressed during acute cryptosporidiosis in suckling rats as a result of both malnutrition and experimental parasite infection.

Cryptosporidium parvuminfection induces amino acid malnutrition leading to growth retardation in children. Owing to the nutritional efficiency of peptides compared to free amino acids and the resistance of the di-tripeptide transporter PepT1 to mucosal injury, we analyzed the intestinal expression of PepT1 during experimental acute cryptosporidiosis in suckling rats from day 4 to day 50. PepT1 mRNA levels were increased at the peak of infection (day 10) all along the small intestine and normalized after spontaneous clearance of the parasite (day 21). Immunolocalization of PepT1 showed that its expression was maintained in the brush border membrane of enterocytes in infected rats from day 4 to day 50 all along the small intestine. Our results suggest a transcriptional up-regulation during acute cryptosporidiosis in response to both C. parvum-induced malnutrition and parasite implantation. As no treatment is available, a semi-elemental diet should be considered part of the treatment of cryptosporidiosis.

Metabolic evidence for adaptation to a high protein diet in rats.

This study was designed to assess the effects of long-term adaptation to a high protein diet on energy intake, body weight gain, body composition and splanchnic metabolic indicators in rats. For this purpose, adult male Wistar rats were fed either a 50 g/100 g dry matter (DM) protein diet (P50 group) or a 14 g/100 g DM protein diet (P14 group) for 21 d. These two groups were compared with a P14 pair-fed (P14-pf) group that consumed the same daily energy as the P50 group. The energy intake of the P50 group was 16 +/- 1% less than that of the P14 group (P < 0.05), and the P50 group had significantly lower body weight. The P50 group had significantly less adipose tissue compared with both P14 and P14-pf rats. The activities of the brush border membrane enzymes, neutral aminopeptidase and gamma-glutamyl transferase, were significantly higher in the P50 group than in the P14 rats. Similarly, the activities of alanine aminotransferase, arginase and serine dehydratase were significantly higher in the liver of P50 rats compared with P14 rats. Both amino acid transporter system A and X(A,G-) activities, measured in freshly isolated hepatocytes, were significantly higher in the P50 group (8- and 1.5-fold, P < 0.05, respectively) compared with the P14 group. The 1.5-fold increase in the steady-state activity of X(A,G-) was accompanied by a doubling of EAAT2 mRNA, involved in the system X(A,G-). This study provides confirmation that specific biochemical and molecular adaptive processes of the splanchnic area are involved in the response to variations in the protein content of the diet.

Dysfunctional voiding in women.

PURPOSE: We characterized presenting symptoms and urodynamic findings in women with dysfunctional voiding. MATERIALS AND METHODS: We reviewed the charts of 26 women diagnosed with dysfunctional voiding. Those with a known or suspected history of neurological disease before evaluation were excluded from study. All patients completed an American Urological Association symptom index, and scores were classified as total, storage (irritative) and emptying (obstructive). The diagnosis of dysfunctional voiding was made on multichannel video urodynamics. There was increased external sphincter activity during voiding. Presenting symptoms and urodynamic findings in all cases were summarized. In addition, symptoms and urodynamic findings in patients later diagnosed with occult neurological disease were compared with those in patients without neurological disease. RESULTS: Mean patient age was 39.2 years (range 19 to 79). Mean total American Urological Association-7 score was 24.4 of 35. Frequency and urgency were the most common presenting symptoms in 82% of cases. Mean storage score was 11.3 of 15 and mean voiding score was 13.2 of 20. Urge and stress incontinence was noted in 6 (23%) and 4 patients (15%), respectively, while 11 (42%) had a history of recurrent urinary tract infection. Cystometrography revealed detrusor instability in 11 cases (42%), sensory urgency in 11 (42%) and impaired compliance in 2 (8%), including 1 with instability. There was great variability in voiding parameters. Mean maximum urinary flow plus or minus standard deviation was 10.4 +/- 6.2 cc per second, mean detrusor pressure at maximum urinary flow was 50.3 +/- 23.5 cm. water and mean post-void residual urine volume was 103.4 +/- 120.0 cc. Video urodynamics prompted neurological evaluation, which revealed occult neurological disease in 5 patients who were then reclassified with external-detrusor sphincter dyssynergia. CONCLUSIONS: Female patients presenting with lower urinary tract symptoms may have dysfunctional voiding patterns. Storage symptoms appear to be even more common than voiding symptoms in this study group. These patients tend to have decreased flow, increased voiding pressure and high post-void residual urine volume. However, there is wide variation in these parameters among individuals. Therefore, careful review of the voiding phase, including pelvic floor electromyography and the fluoroscopic appearance of the bladder outlet, is critical. Occult neurological disease should be suspected in patients with dysfunctional voiding.

Abnormal perception of the tonic vibration reflex in idiopathic focal dystonia.

Although the pathophysiological basis of idiopathic focal dystonia (IFD) remains unclear, we recently reported abnormal perception of the tonic vibration reflex (TVR) in the biceps brachii in IFD. In this study we examined whether the abnormality affects muscles other than the biceps brachii. A 100-Hz vibration stimulating predominantly the muscle spindle afferent was transcutaneously applied to one muscle tendon of the triceps brachii, the wrist extensor and flexor muscles in 29 subjects with IFD (18 with torticollis, 9 with writer's cramp, 2 with blepharospasm) and 15 control subjects. The blindfolded subjects were instructed to copy any perceived movement with the opposite tracking arm. The elbow or wrist angle changes were quantified electronically. The TVR and subjects' perception of the TVR were evaluated by angle movements of the vibrated joint and of the tracking joint, respectively. Perception of the TVR was significantly reduced in dystonic subjects at both elbow and wrist joints, while magnitude of the TVR did not differ between the two groups. Abnormal central perception of the TVR is a feature of IFD, suggesting a widespread involvement of abnormal muscle spindle afferent processing in IFD.

Abnormal perception of vibration-induced illusion of movement in dystonia.

OBJECTIVES: To examine the illusional sensation of movement evoked by vibration of an immobilized arm. METHODS: Patients with idiopathic focal dystonia were compared with those with idiopathic PD and with patients with dystonia secondary to PD. A 100-Hz transcutaneous vibratory stimulus was applied to the biceps brachii tendon to elicit an illusional sensation of arm extension. Blindfolded patients were instructed to copy any perceived movement of the vibrated arm with the opposite (tracking) arm. By recording movement of reflective markers on the tracking arm using infrared video cameras, the change in elbow angle was quantified over 45 seconds. The effect of treatment with botulinum toxin was examined by retesting previously untreated patients after commencing therapy. These results were also compared with patients with hemifacial spasm who had ongoing treatment with botulinum toxin. RESULTS: Vibration of the biceps in dystonic patients produced a smaller sensation of arm extension than in control subjects unaffected by botulinum toxin treatment. There were no differences between the types of idiopathic focal dystonia examined, including patients with dystonia in sites other than the arm. Those with idiopathic PD and hemifacial spasm did not differ from healthy control subjects. Patients with dystonia secondary to PD showed a unilateral abnormality on the side of dystonic symptoms. CONCLUSION: Bilateral abnormal perception of the illusion of vibration-induced movement is a feature of idiopathic focal dystonia but not idiopathic PD, and is independent of treatment with botulinum toxin. Unilateral, abnormal sensorimotor integration is implicated in dystonia secondary to PD. These results may reflect abnormal sensorimotor integration of Ia afferent activity.

Rapid identification of Medicago nodulating strains by using two oligonucleotide probes complementary to 16S rDNA sequences.

Symbiotic bacteria associated with the Medicago genus are separated into two closely related species named Sinorhizobium meliloti and Sinorhizobium medicae. To discriminate rapidly between these two bacterial species, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiate S. meliloti from S. medicae. Their specificities were evaluated by dot-blot hybridization experiments on 25 reference strains representing 13 species of Rhizobium and Sinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific for S. medicae. By contrast, as expected according to the 16S rDNA sequence alignment, the labeled 16Smfs probe cross-hybridized with the DNAs of S. meliloti, Sinorhizobium fredii, and Sinorhizobium saheli but not with the DNA of S. medicae. Since S. saheli and S. fredii do not nodulate Medicago, 16Smed and 16Smfs can be routinely used to characterize the two Sinorhizobium species nodulating Medicago from pure cultures or from Medicago root nodules. Fifty strains isolated from eight annual Medicago species were then characterized by using colony hybridizations. Sinorhizobium meliloti was more frequently obtained (> 80% isolates) than was S. medicae. Both Sinorhizobium species seemed to be trapped by annual Medicago and no plant-host specificity was detected.

Sinorhizobium medicae sp. nov., isolated from annual Medicago spp.

The taxonomic position of isolates of a new genomic species (designated genomic species 2) obtained from several annual Medicago species and originating from different geographical locations was established through the results of phenotypic tests (including the results of auxanographic and biochemical tests and symbiotic properties) and 16S rRNA phylogenetic inferences. A comparison of the complete 16S rRNA sequence of a representative of genomic species 2 (strain A 321T [T = type strain]) with the 16S rRNA sequences of other members of the Rhizobiaceae and closely related taxa showed that genomic species 2 was phylogenetically related to Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, and Sinorhizobium teranga. The levels of sequence similarity and observed numbers of nucleotide substitutions in Sinorhizobium strains indicated that A 321T and S. meliloti exhibited the highest level of sequence similarity (99.7%), with four nucleotide substitutions and one deletion. The results of a numerical analysis based on data from 63 auxanographic and biochemical tests clearly separated genomic species 2 isolates from S. meliloti. Genomic species 2 isolates nodulated and fixed nitrogen with Medicago polymorpha, whereas S. meliloti isolates were ineffective and formed rudimentary nodules on this host plant. On the basis of phenotypic and 16S sequence analysis data, genomic species 2 isolates cannot be assigned to a previously described species. We propose that these isolates belong to a new species, Sinorhizobium medicae.

Evidence that two genomic species of Rhizobium are associated with Medicago truncatula.

Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer). Two genotypic groups were found, regardless of the DNA region targeted. These two groups were given the status of genomic species based on results of DNA/DNA hybridization.