Polarity-Dependent Transcranial Direct Current Stimulation Effects on Central Auditory Processing

Given the polarity dependent effects of transcranial direct current stimulation (tDCS) in facilitating or inhibiting neuronal processing, and tDCS effects on pitch perception, we tested the effects of tDCS on temporal aspects of auditory processing. We aimed to change baseline activity of the auditory cortex using tDCS as to modulate temporal aspects of auditory processing in healthy subjects without hearing impairment. Eleven subjects received 2mA bilateral anodal, cathodal and sham tDCS over auditory cortex in a randomized and counterbalanced order. Subjects were evaluated by the Random Gap Detection Test (RGDT), a test measuring temporal processing abilities in the auditory domain, before and during the stimulation. Statistical analysis revealed a significant interaction effect of time vs. tDCS condition for 4000 Hz and for clicks. Post-hoc tests showed significant differences according to stimulation polarity on RGDT performance: anodal improved 22.5% and cathodal decreased 54.5% subjects' performance, as compared to baseline. For clicks, anodal also increased performance in 29.4% when compared to baseline. tDCS presented polarity-dependent effects on the activity of the auditory cortex, which results in a positive or negative impact in a temporal resolution task performance. These results encourage further studies exploring tDCS in central auditory processing disorders

A comparative study on optimisation of protein extraction methods for Saccharomonospora azurea

To establish the optimal cell disruption and protein extraction protocol for achieving the most efficient whole-cell protein extraction of Saccharomonospora azurea, four commonly used methods (X-Press, bead-vortexing, freezing-throwing and TCA/acetone/phenol extraction) were compared. Total protein content, as well as 1D and 2D SDS-PAGE protein patterns were assayed in the extracts to study the efficacy of these methods. Accordingly, of the four methods the X-Press proved the most effective for all initial weight (maximum 21.523 ± 0.23 mg/ml protein) followed by TCA/acetone/phenol method (maximum 13.682 ± 0.15 mg/ml protein), while the effectiveness of the two other methods were substantially inferior (maximum 3.188 ± 0.03 mg/ml protein). The analysis of protein gels proved that the X-Press method revealed a protein pattern characterised by the presence of the highest number of protein bands (on average of 52 and 385, on 1D and 2D gels, respectively). The TCA/acetone/phenol extraction provided similar effectiveness for only 100-300 mg initial bacteria mass, whereas bead-vortexing produced maximum 35 and 227 separated protein bands, on 1D and 2D gels, respectively. It can be stated that of the four methods the X-Press was the most effective one for all initial weight of bacteria, while the TCA/acetone/phenol method provides interpretable results for the 100-300 mg weight-range of bacteria

Accelerated retinal aging in PACAP knock-out mice

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Calpha for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Muller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system

Proteomic insight into the primycin fermentation process of Saccharomonospora azurea

Saccharomonospora azurea SZMC 14600 is a member of the family Pseudonocardiaceae exclusively used for industrial scale production of primycin a large 36-membered non-polyene macrolide lactone antibiotic belonging to the polyketide class of natural products. Even though maximum antibiotic yield has been achieved by empirically optimized two-step fermentation process, little is known about the molecular components and mechanisms underlying the efficient antibiotic production. In order to identify differentially expressed proteins (DEPs) between the pre- and main-fermentation stages of primycin, comparative 2D-PAGE experiments were performed. In total, 98 DEP spots were reproducibly detected, out of which four spots were excised from gels, and identified through MALDI-TOF/TOF mass spectrometry. Peptide mass fingerprint analysis revealed peptide matches to HicB antitoxin for the HicAB toxin-antitoxin system (EHK86651), to a nucleoside diphosphate kinase regulator ((Ndk; EHK81899) and two other proteins with unknown function (EHK88946 and EHK86777)

SELECTIVE MEASUREMENT OF α SMOOTH MUSCLE ACTIN: WHY β-ACTIN CAN NOT BE USED AS A HOUSEKEEPING GENE WHEN TISSUE FIBROSIS OCCURS

Abstract Background Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms. Results Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO. Conclusion We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur

Complementary Approaches to Retinal Health Focusing on Diabetic Retinopathy

Diabetes mellitus affects carbohydrate homeostasis but also influences fat and protein metabolism. Due to ophthalmic complications, it is a leading cause of blindness worldwide. The molecular pathology reveals that nuclear factor kappa B (NFκB) has a central role in the progression of diabetic retinopathy, sharing this signaling pathway with another major retinal disorder, glaucoma. Therefore, new therapeutic approaches can be elaborated to decelerate the ever-emerging “epidemics” of diabetic retinopathy and glaucoma targeting this critical node. In our review, we emphasize the role of an improvement of lifestyle in its prevention as well as the use of phytomedicals associated with evidence-based protocols. A balanced personalized therapy requires an integrative approach to be more successful for prevention and early treatment

PACAP for Retinal Health: Model for Cellular Aging and Rescue

Retinal aging is the result of accumulating molecular and cellular damage with a manifest decline in visual functions. Somatostatin (SST) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in neuroprotection through regulating disparate aspects of neuronal activity (survival, proliferation and renewal). The aim of the present study was to validate a transgenic model for SST-expressing amacrine cells and to investigate the chronic effect of PACAP on the aging of SSTergic and dopaminergic cells of the retina. SST-tdTomato transgenic mice that were 6, 12 and 18 months old were treated intravitreally with 100 pmol of PACAP every 3 months. The density of SST and dopaminergic amacrine cells was assessed in whole-mounted retinas. Cells displaying the transgenic red fluorescence were identified as SST-immunopositive amacrine cells. By comparing the three age groups. PACAP treatment was shown to induce a moderate elevation of cell densities in both the SST and dopaminergic cell populations in the 12- and 18-month-old animals. By contrast, the control untreated and saline-treated retinas showed a minor cell loss. In conclusion, we report a reliable transgenic model for examining SSTergic amacrine cells. The fundamental novelty of this study is that PACAP could increase the cell density in matured retinal tissue, anticipating new therapeutic potential in age-related pathological processes