Urinary C-type natriuretic peptide excretion: a potential novel biomarker for renal fibrosis during aging

Renal aging is characterized by structural changes in the kidney including fibrosis, which contributes to the increased risk of kidney and cardiac failure in the elderly. Studies involving healthy kidney donors demonstrated subclinical age-related nephropathy on renal biopsy that was not detected by standard diagnostic tests. Thus there is a high-priority need for novel noninvasive biomarkers to detect the presence of preclinical age-associated renal structural and functional changes. C-type natriuretic peptide (CNP) possesses renoprotective properties and is present in the kidney; however, its modulation during aging remains undefined. We assessed circulating and urinary CNP in a Fischer rat model of experimental aging and also determined renal structural and functional adaptations to the aging process. Histological and electron microscopic analysis demonstrated significant renal fibrosis, glomerular basement membrane thickening, and mesangial matrix expansion with aging. While plasma CNP levels progressively declined with aging, urinary CNP excretion increased, along with the ratio of urinary to plasma CNP, which preceded significant elevations in proteinuria and blood pressure. Also, CNP immunoreactivity was increased in the distal and proximal tubules in both the aging rat and aging human kidneys. Our findings provide evidence that urinary CNP and its ratio to plasma CNP may represent a novel biomarker for early age-mediated renal structural alterations, particularly fibrosis. Thus urinary CNP could potentially aid in identifying subjects with preclinical structural changes before the onset of symptoms and disease, allowing for the initiation of strategies designed to prevent the progression of chronic kidney disease particularly in the aging population.     

Exercise training normalizes impaired NOS-dependent responses of cerebral arterioles in type 1 diabetic rats

Our goal was to examine whether exercise training (ExT) could normalize impaired nitric oxide synthase (NOS)-dependent dilation of cerebral (pial) arterioles during type 1 diabetes (T1D). We measured the in vivo diameter of pial arterioles in sedentary and exercised nondiabetic and diabetic rats in response to an endothelial NOS (eNOS)-dependent (ADP), an neuronal NOS (nNOS)-dependent [N-methyl-D-aspartate (NMDA)], and a NOS-independent (nitroglycerin) agonist. In addition, we measured superoxide anion levels in brain tissue under basal conditions in sedentary and exercised nondiabetic and diabetic rats. Furthermore, we used Western blot analysis to determine eNOS and nNOS protein levels in cerebral vessels/brain tissue in sedentary and exercised nondiabetic and diabetic rats. We found that ADP and NMDA produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic rats. In contrast, ADP and NMDA produced only minimal vasodilation in sedentary diabetic rats. ExT restored impaired ADP- and NMDA-induced vasodilation observed in diabetic rats to that observed in nondiabetics. Nitroglycerin produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic and diabetic rats. Superoxide levels in cortex tissue were similar in sedentary and exercised nondiabetic rats, were increased in sedentary diabetic rats, and were normalized by ExT in diabetic rats. Finally, we found that eNOS protein was increased in diabetic rats and further increased by ExT and that nNOS protein was not influenced by T1D but was increased by ExT. We conclude that ExT can alleviate impaired eNOS- and nNOS-dependent responses of pial arterioles during T1D.     

Low dose Estrogen Prevents Neuronal Degeneration and Microglial Reactivity in an Acute Model of Spinal Cord Injury: Effect of Dosing,Route of Administration,and Therapy Delay

Spinal cord injury (SCI), depending on the severity of injury, leads to neurological dysfunction and paralysis. Methylprednisolone, the only currently available therapy renders limited protection in SCI. Therefore, other therapeutic agents must be tested to maximize neuroprotection and functional recovery. Previous data from our laboratory indicate that estrogen (17β-estradiol) at a high dose may attenuate multiple damaging pathways involved in SCI and improve locomotor outcome. Since use of high dose estrogen may have detrimental side effects and therefore may never be used in the clinic, the current study investigated the efficacy of this steroid hormone at very low doses in SCI. In particular, we tested the impact of dosing (1–10 μg/kg), mode of delivery (intravenous vs. osmotic pump), and delay in estrogen application (15 min–4 h post-SCI) on microgliosis and neuronal death in acute SCI in rats. Treatment with 17β-estradiol (1–10 μg/kg) significantly reduced microglial activation and also attenuated apoptosis of neurons compared to untreated SCI animals. The attenuation of cell death and inflammation by estrogen was observed regardless of mode and time of delivery following injury. These findings suggest estrogen as a potential agent for the treatment of individuals with SCI.     

Automated Tracing of Horizontal Neuron Processes During Retinal Development

In the developing mammalian retina, horizontal neurons undergo a dramatic reorganization of their processes shortly after they migrate to their appropriate laminar position. This is an important process because it is now understood that the apical processes are important for establishing the regular mosaic of horizontal cells in the retina and proper reorganization during lamination is required for synaptogenesis with photoreceptors and bipolar neurons. However, this process is difficult to study because the analysis of horizontal neuron anatomy is labor intensive and time-consuming. In this paper, we present a computational method for automatically tracing the three-dimensional (3-D) dendritic structure of horizontal retinal neurons in two-photon laser scanning microscope (TPLSM) imagery. Our method is based on 3-D skeletonization and is thus able to preserve the complex structure of the dendritic arbor of these cells. We demonstrate the effectiveness of our approach by comparing our tracing results against two sets of semi-automated traces over a set of 10 horizontal neurons ranging in age from P1 to P5. We observe an average agreement level of 81% between our automated trace and the manual traces. This automated method will serve as an important starting point for further refinement and optimization.     

Evidence for aluminum-binding erythropoietin by size-exclusion chromatography coupled to electrothermal absorption atomic spectrometry

Erythropoietin (EPO) is a glycoprotein that stimulates erythropoiesis and is clinically used for treating anemia during chronic renal failure and for anemia in preterm infants. EPO formulations usually have elevated rates of contamination due to aluminum (Al), which is toxic to both types of patients. Size-exclusion chromatography (SEC) coupled with graphite furnace atomic absorption spectrometry (GF AAS) was employed to separate proteins and to quantify the amount of aluminum present in the elution volume corresponding to EPO and, therefore, to evaluate possible binding. Because EPO formulations contain human serum albumin (HSA), a chromatographic method was optimized for the separation of these proteins. Subsequent to the chromatographic separation, 1-mL fractions of the column effluent were collected, and the Al content in these aliquots was measured by GF AAS. EPO and HSA samples were incubated with Al for 4 h at 4 °C and 37 °C as well as for 16 h at 4 °C and 37 °C. Afterwards, they were injected into the chromatographic system. These samples were also submitted to ultrafiltration (10 and 50 kDa membranes), and Al was measured in the ultrafiltrates. The results showed that Al was present in the eluent volume corresponding to the EPO peak but not in the HSA peak in the chromatograms. Temperature strengthened the interaction because the Al present in the EPO fraction was 3 times higher at 37 °C compared to 4 °C. Thirty-eight percent of the Al present in a 2.4 μg/mL EPO standard solution, and approximately 50% of the Al in formulation samples containing approximately 11 μg/mL EPO and either citrate or phosphate, were non-ultrafiltrable, which suggests that EPO is an effective Al acceptor in vitro.     

A critical review on some closely related species of <Emphasis Type="Italic">Tetranychus</Emphasis> sensu stricto (Acari: Tetranychidae) in the public DNA sequences databases

Taxonomic misidentification of the specimens used to obtain DNA sequences is a growing problem reported for different groups of organisms, which threatens the utility of the deposited sequences in public DNA databases. This paper provides new evidence of misidentifications in molecular DNA public databases in phytophagous mites of the Tetranychidae family belonging to the group Tetranychus (Tetranychus). Several species in this group are of economic and quarantine importance in agriculture and among them Tetranychus urticae, a highly polyphagous mite causing outbreaks in many crops worldwide, is certainly the most studied. We analyzed and evaluated the identity of 105 GenBank accessions of ITS2 rDNA and 138 COI mtDNA sequences which were deposited as T. urticae and those of 14 other taxa morphologically closely related to Tetranychus sensu stricto. In addition, ITS2 and COI sequences of 18 T. urticae samples collected for this study and identified by morphological criteria, were generated and included in the analyzed dataset. Among the deposited sequences in the GenBank database, numerous cases of apparently mistaken identities were identified in the group Tetranychus s. str., especially between T. urticae, T. cinnabarinus, T. kanzawai and T. truncatus. Unreliable sequences (misidentified or dubious) were estimated at nearly 30%. In particular the analysis supports the invalidity of the controversial species status of T. cinnabarinus. More generally, it highlights the need of using combined morphological and molecular approaches to guarantee solid species diagnostics for reliable sequence accessions in public databases.     

Structure, function and membrane interactions of plant annexins: An update

Knowledge accumulated over the past 15 years on plant annexins clearly indicates that this disparate group of proteins builds on the common annexin function of membrane association, but possesses divergent molecular mechanisms. Functionally, the current literature agrees on a key role of plant annexins in stress response processes such as wound healing and drought tolerance. This is contrasted by only few established details of the molecular level mechanisms that are driving these activities.In this review, we appraise the current knowledge of plant annexin molecular, functional and structural properties with a special emphasis on topics of less coverage in recent past overviews. In particular, plant annexin post-translational modification, roles in polar growth and membrane stabilisation processes are discussed.     

Multiscale computational modeling reveals a critical role for TNF-α receptor 1 dynamics in tuberculosis granuloma formation

Multiple immune factors control host responses to Mycobacterium tuberculosis infection, including the formation of granulomas, which are aggregates of immune cells whose function may reflect success or failure of the host to contain infection. One such factor is TNF-α. TNF-α has been experimentally characterized to have the following activities in M. tuberculosis infection: macrophage activation, apoptosis, and chemokine and cytokine production. Availability of TNF-α within a granuloma has been proposed to play a critical role in immunity to M. tuberculosis. However, in vivo measurement of a TNF-α concentration gradient and activities within a granuloma are not experimentally feasible. Further, processes that control TNF-α concentration and activities in a granuloma remain unknown. We developed a multiscale computational model that includes molecular, cellular, and tissue scale events that occur during granuloma formation and maintenance in lung. We use our model to identify processes that regulate TNF-α concentration and cellular behaviors and thus influence the outcome of infection within a granuloma. Our model predicts that TNF-αR1 internalization kinetics play a critical role in infection control within a granuloma, controlling whether there is clearance of bacteria, excessive inflammation, containment of bacteria within a stable granuloma, or uncontrolled growth of bacteria. Our results suggest that there is an interplay between TNF-α and bacterial levels in a granuloma that is controlled by the combined effects of both molecular and cellular scale processes. Finally, our model elucidates processes involved in immunity to M. tuberculosis that may be new targets for therapy.