OrFin: A web tool for detection of putative orthologs

Identification of ortholog is one of the important tasks to understand a novel genome. It helps to assign functional annotations, from one organism to another organism. To identify the putative ortholog, Reciprocal Best BLAST hit (RBBH) method is known to be an efficient approach. OrFin makes use of the same approach to identify pair of orthologous proteins for a given set of sequences of two species. It is a user-friendly web tool which works with user defined parameters to search RBBHs. Results are produced in both html and text format.

Availability

This web tool is freely available at http://bifl.uohyd.ac.in/orfin     

Myelin Lipid Abnormalities in the Aspartoacylase-Deficient Tremor Rat

The high concentration of N-acetylaspartate (NAA) in neurons of the central nervous system and its growing clinical use as an indicator of neuronal viability has intensified interest in the biological function of this amino acid derivative. The biomedical relevance of such inquiries is highlighted by the myelin-associated pathology of Canavan disease, an inherited childhood disorder resulting from mutation of aspartoacylase (ASPA), the NAA-hydrolyzing enzyme. This enzyme is known to be localized in oligodendrocytes with bimodal distribution in cytosol and the myelin sheath, and to produce acetyl groups utilized in myelin lipid synthesis. Loss of this acetyl source in Canavan disease and rodent models such as the tremor rat are thought to account for the observed myelin deficit. This study was undertaken to further define and quantify the specific lipid abnormalities that occur as a result of ASPA deficit in the tremor rat. Employing mass spectrometry together with high performance thin-layer chromatography, we found that myelin from 28-day-old animals showed major reduction in cerebrosides (CB) and sulfatides (Sulf) with unsubstituted fatty acids, and equal if not greater changes in myelin from 7-month-old tremors. Cerebrosides with 2-hydroxyfatty acids showed little if any change at either age; Sulf with 2-hydroxyfatty acids showed no significant change at 28 days, but surprisingly a major increase at 7 months. Two species of phosphatidylcholine, 32:0 and 34:1, also showed significant increase, but only at 28 days. One form of phosphatidylethanolamine, PE36:1, was reduced a modest amount at both ages, whereas the plasmalogen form did not change. The dysmyelination that results from inactivation of ASPA is thus characterized by selective decreases as well as some increases in specific lipids. Special issue article in honor of Dr. George DeVries. Fatty acid designations (e.g. 18:1) indicate carbon number and number of double bonds.     

Modeling the noncovalent interactions at the metabolite binding site in purine riboswitches

We present gas phase quantum chemical studies on the metabolite binding interactions in two important purine riboswitches, the adenine and guanine riboswitches, at the B3LYP/6-31G(d,p) level of theory. In order to gain insights into the strucutral basis of their discriminative abilities of regulating gene expression, the structural properties and binding energies for the gas phase optimized geometries of the metabolite bound binding pocket are analyzed and compared with their respective crystal geometries. Kitaura-Morokuma analysis has been carried out to calculate and decompose the interaction energy into various components. NBO and AIM analysis has been carried out to understand the strength and nature of binding of the individual aptamer bases with their respective purine metabolites. The Y74 base, U in case of adenine riboswitch and C in case of guanine riboswitch constitutes the only differentiating element between the two binding pockets. As expected, with W:W cis G:C74 interaction contributing more than 50% of the total binding energy, the interaction energy for metabolite binding as calculated for guanine (-46.43 Kcal/mol) is nearly double compared to the corresponding value for that of adenine (-24.73 Kcal/mol) in the crystal context. Variations in the optimized geometries for different models and comparison of relative contribution to metabolite binding involving four conserved bases reveal the possible role of U47:U51 W:H trans pair in the conformational transition of the riboswitch from the metabolite free to metabolite bound state. Our results are also indicative of significant contributions from stacking and magnesium ion interactions toward cooperativity effects in metabolite recognition.     

Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys⁷³ only after the formation of a Cys³²-Cys³⁵ disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1^C32S/C35S (a mutant Trx1 with both Cys³² and Cys³⁵ replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys¹⁷³ and Cys⁸³ and protects it from H₂O₂-induced overoxidation. Functionally, we found that Cys⁷³-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.Nitric oxide (NO) is an important second messenger for signal transduction in cells. The production of cGMP by guanylyl cyclase, enabled by the binding of NO onto heme, is considered the primary mechanism responsible for the plethora of functions exerted by NO (1). However, S-nitrosylation, the covalent addition of the NO moiety onto cysteine thiols, is increasingly recognized as an important post-translational modification for regulating protein functions (for reviews, see Refs. 2 and 3). S-Nitrosylation is dynamic, reversible, site-specific, and modulated by selected cellular stimuli (4–7). With improved detection sensitivity, an increasing number of S-nitrosylated proteins have been identified by proteomics technologies (5, 8–13). Among the known modified proteins, nitrosylation occurs only on selected cysteines (4, 6, 14–17). Non-enzymatic mechanisms proposed to determine S-nitrosylation specificity include the availability of specific NO donors and protein microenvironments that stabilize the pK_a of acidic target cysteines (18). Furthermore, several enzymes, including hemoglobin (19, 20), superoxide dismutase 1 (21, 22), S-nitrosoglutathione reductase (23–25), and protein-disulfide isomerase (26), have been shown to possess either transnitrosylase or denitrosylase activities. However, an enzymatic system that governs site-specific transnitrosylation and denitrosylation, analogous to the kinase/phosphatase paradigm for regulating protein phosphorylation, has remained largely uncharacterized.Trx1¹ is an important antioxidant protein with protein reductase activity (27, 28). It has been characterized as an antiapoptotic protein because of its ability to suppress proapoptotic proteins, including apoptosis signal-regulating kinase 1 via disulfide reduction and Casp3 via transnitrosylation of Cys¹⁶³ (14, 29). Conversely, Trx1 can denitrosylate Casp3 at Cys¹⁶³, resulting in Casp3 activation (7). Trx1 appears to govern site-specific reversible nitrosylation of selected protein targets (14, 15), but what are the underlying mechanisms that regulate Trx1 transnitrosylation and denitrosylation activities? Are there additional Trx1-mediated transnitrosylation or denitrosylation targets that have not yet been identified? In this study, we used ESI-Q-TOF mass spectrometry (MS) to analyze the nitrosylation of Trx1 and a Casp3 peptide (Casp3p) under different redox conditions. Because of the labile nature of the S–NO bond, direct identification of S-nitrosylated proteins and their specific nitrosylation sites by MS remains challenging (8). A biotin switch method that is based on the derivatization of protein S–NO with a biotinylating agent is typically used for such analyses (8). However, like any indirect method, both false positive and negative identifications have been reported (30). Recently, we developed a method for direct analysis of protein S-nitrosylation by ESI-Q-TOF MS without prior chemical derivatization (31). Here we applied the same technique to determine the regulation of Trx1 by stepwise oxidative and nitrosative modifications of distinct cysteines and its subsequent ability to transnitrosylate target proteins. Nitrosative modification at Cys⁷³ of Trx1 cannot occur without prior attenuation of the Trx1 disulfide reductase and denitrosylase activities via either disulfide bond formation between Cys³² and Cys³⁵ or their mutation to serines. This is a key observation that has never been previously reported. Consequently, we designed a proteomics approach and discovered over 40 putative Trx1 transnitrosylation target proteins. We further characterized the Trx1 transnitrosylation proteome and identified three consensus motifs surrounding the putative Trx1 transnitrosylation sites, suggesting a protein-protein interaction mechanism for determining transnitrosylation specificity.     

Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute beta-adrenergic stimulation

Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development.     

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila melanogaster

We have examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila melanogaster. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. Here we show that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages.     

Self-Regulation of Anterior Insula with Real-Time fMRI and Its Behavioral Effects in Obsessive-Compulsive Disorder: A Feasibility Study

Introduction

Obsessive-compulsive disorder (OCD) is a common and chronic condition that can have disabling effects throughout the patient''s lifespan. Frequent symptoms among OCD patients include fear of contamination and washing compulsions. Several studies have shown a link between contamination fears, disgust over-reactivity, and insula activation in OCD. In concordance with the role of insula in disgust processing, new neural models based on neuroimaging studies suggest that abnormally high activations of insula could be implicated in OCD psychopathology, at least in the subgroup of patients with contamination fears and washing compulsions.

Methods

In the current study, we used a Brain Computer Interface (BCI) based on real-time functional magnetic resonance imaging (rtfMRI) to aid OCD patients to achieve down-regulation of the Blood Oxygenation Level Dependent (BOLD) signal in anterior insula. Our first aim was to investigate whether patients with contamination obsessions and washing compulsions can learn to volitionally decrease (down-regulate) activity in the insula in the presence of disgust/anxiety provoking stimuli. Our second aim was to evaluate the effect of down-regulation on clinical, behavioural and physiological changes pertaining to OCD symptoms. Hence, several pre- and post-training measures were performed, i.e., confronting the patient with a disgust/anxiety inducing real-world object (Ecological Disgust Test), and subjective rating and physiological responses (heart rate, skin conductance level) of disgust towards provoking pictures.

Results

Results of this pilot study, performed in 3 patients (2 females), show that OCD patients can gain self-control of the BOLD activity of insula, albeit to different degrees. In two patients positive changes in behaviour in the EDT were observed following the rtfMRI trainings. Behavioural changes were also confirmed by reductions in the negative valence and in the subjective perception of disgust towards symptom provoking images.

Conclusion

Although preliminary, results of this study confirmed that insula down-regulation is possible in patients suffering from OCD, and that volitional decreases of insula activation could be used for symptom alleviation in this disorder.     

Chronic Electrical Stimulation with a Suprachoroidal Retinal Prosthesis: A Preclinical Safety and Efficacy Study

Purpose

To assess the safety and efficacy of chronic electrical stimulation of the retina with a suprachoroidal visual prosthesis.

Methods

Seven normally-sighted feline subjects were implanted for 96–143 days with a suprachoroidal electrode array and six were chronically stimulated for 70–105 days at levels that activated the visual cortex. Charge balanced, biphasic, current pulses were delivered to platinum electrodes in a monopolar stimulation mode. Retinal integrity/function and the mechanical stability of the implant were assessed monthly using electroretinography (ERG), optical coherence tomography (OCT) and fundus photography. Electrode impedances were measured weekly and electrically-evoked visual cortex potentials (eEVCPs) were measured monthly to verify that chronic stimuli were suprathreshold. At the end of the chronic stimulation period, thresholds were confirmed with multi-unit recordings from the visual cortex. Randomized, blinded histological assessments were performed by two pathologists to compare the stimulated and non-stimulated retina and adjacent tissue.

Results

All subjects tolerated the surgical and stimulation procedure with no evidence of discomfort or unexpected adverse outcomes. After an initial post-operative settling period, electrode arrays were mechanically stable. Mean electrode impedances were stable between 11–15 kΩ during the implantation period. Visually-evoked ERGs & OCT were normal, and mean eEVCP thresholds did not substantially differ over time. In 81 of 84 electrode-adjacent tissue samples examined, there were no discernible histopathological differences between stimulated and unstimulated tissue. In the remaining three tissue samples there were minor focal fibroblastic and acute inflammatory responses.

Conclusions

Chronic suprathreshold electrical stimulation of the retina using a suprachoroidal electrode array evoked a minimal tissue response and no adverse clinical or histological findings. Moreover, thresholds and electrode impedance remained stable for stimulation durations of up to 15 weeks. This study has demonstrated the safety and efficacy of suprachoroidal stimulation with charge balanced stimulus currents.