Systematic analysis of Hyperpolarization-activated and Cyclic Nucleotide-gated (HCN) channels in photoreceptors and bipolar cells of the rat retina.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels were studied in the rat retina. The expression pattern of the four HCN channel isoforms HCN1 - 4 was studied using immunocytochemistry with isoform-specific antibodies and confocal microscopy. Using the patch-clamp technique, functional properties of HCN currents were recorded in 240 voltage-clamped bipolar cells and 20 photoreceptor cells in a retinal slice preparation. While recording, cells were filled with Lucifer Yellow. On the basis of morphological and electrophysiological criteria, recorded and filled bipolar cells could be classified as 11 types of cone bipolar cells and one type of rod bipolar cell. Two of the cone bipolar cell types had not been identified in previous studies. Based on immunohistochemical and electrophysiological results, HCN1 was found in rod and cone photoreceptors. Cones also expressed HCN3 at the cone pedicle base. Type 3 bipolar cells expressed HCN4, type 5 and type 6a bipolar cells HCN1 and HCN4. These isoforms were found throughout the bipolar cells. Strikingly, HCN2 was highly concentrated at the axon terminal systems of type 3, 5, 6, 7, 8, and 9 cone bipolar cells and the rod bipolar cell. No HCN immunoreactivity was found in type 1, 2, and 4 cone bipolar cells. In the electrophysiological recordings, the activation kinetics and the mid-point potential of activation were studied in detail. In general, the properties of HCN currents recorded from the different bipolar cell types in the retinal slice agreed reasonably well with those of the respective isoforms expressed heterologously in HEK293 cells. All HCN currents could be blocked by application of external cesium. Cones and cone bipolar cells type 5 and 6a expressed more than one HCN isoform, however, electrophysiologically, no evidence for the formation of heterooligomeric channels was found. In some instances, functional properties of HCN channels in retinal cells differed from those of the heterologously expressed channel isoforms, indicating that the channels might be modulated in the retina. Preliminary experiments suggest that intracellular calcium and CAMP might be involved in the modulation of the channels. The widespread expression of HCN channels in the retina suggests that they are involved in retinal information processing at several stages. The most striking feature of HCN channel distribution was the synaptic localization of HCN2 and HCN3. This might indicate that the channels are involved in regulation of synaptic transmission

Computational Modeling of S-Thiolation Reaction: Toward Discovering the Enzymatic Mechanisms of Endogenous HNO Formation

S-nitrosothiols (RSNOs) have long been proposed as potential sources of the elusive endogenous nitroxyl (HNO) via S-thiolation reaction with thiols. It is however not clear how S-thiolation can compete with the trans-S-nitrosation pathway commonly observed in vitro. Based on the insights into the highly unusual, antagonistic chemical nature of RSNO molecules, we hypothesize that, while difficult in vitro, S-thiolation could easily lend itself to enzymatic catalysis. To explore this possibility, we adopted a bottom-up computational approach that aims to identify possible catalytic mechanisms able to steer the RSNO + thiol reaction toward HNO production. Inspired by recent discovery of small bioactive HSNO molecule and its involvement in HNO production, we first study HSNO molecule and its subsequent isomerization reactions that can possibly lead to other potentially bioactive small molecules or reactive intermediates that can undergo S-thiolation reaction. Then, we mapped out the profile of the uncatalysed S-thiolation reaction that suggests that the most difficult step of S-thiolation is the R’SH to RSNO proton transfer, and that the reaction proceeds through unusual highly polar zwitterionic species R’SS+(R)N(H)O– that further decomposes yielding HNO and disulfide RSSR’. Therefore, facilitating the proton transfer and stabilizing this zwitterionic intermediate could lead to dramatic acceleration of this reaction. Further, we identified the required arrangements of amino acid residues using theozyme (‘theoretical enzyme’) modeling. These calculations showed that several of these putative active site models can drop the energetic barrier for S-thiolation/HNO formation to less than 10 kcal/mol. An extensive search in the RCSB protein data bank yielded over 600 structures that match one of these successful theozyme models. Remarkably, among these proteins we found DJ-1 protein known to be involved in RSNO-related processes. Furthermore, full-scale modeling of S-thiolation between an incoming RSNO and DJ-1 Cys106 using a hybrid quantum mechanics/molecular mechanics approach have shown that the reaction can be indeed efficiently catalyzed by the His126–Glu18 dyad, a prediction whose relevance to the DJ-1/RSNO biochemistry presents an intriguing question to the experimentalists

INTERNATIONAL ASPECTS OF HUMAN TRAFFICKING – ESPECIALLY TRAFFICKING WITH MINORS

Liberalization of understanding and relations, the liberation of sexuality from the constrains of primitivism and tradition, leads to rapid growth of prostitution as a socio-pathological phenomenon, which necessarily stimulate the emergence of crime - trafficking with human beings. Particularly worrying is the emergence of a new dimension of human trafficking - trafficking in minors. Children, in addition to women, in international documents, often receive the status of “specially protected persons” which, following the direction in which this crime is developing is more than justified. We can freely say that trafficking in minors is industry in development

Rehabilitation students‘ awareness of unconventional medicine and willingness to integrate it in practice

In the last years there have been numerous studies of unconventional methods as a result of increasing interest to its possibilities for additional treatment of chronic disease.This aim of this article is to investigate the level of awareness of rehabilitation students of unconventional medicine and their readiness to apply it in practice.Material and MethodsThe study was done among third year Rehabilitation students at the Medical Colleges of Varna and Sofia.Results and DiscussionThe study revealed that a large number of the participants are familiar with the methods of unconventional medicine. They used social media as the main resource to get information on the subject. Half of the respondents are willing to integrate unconventional medicine in their practice.ConclusionThe analysis of the result presents the need of incorporating unconventional medicine in the educational program of Rehabilitation students

The Problem of Child Trafficking Enriched with Its Newest Purpose - Establishing Illegal Adoption

The emergence of new ways of committing the crime of human trafficking have increased the prevelance and variety of this crime so that it has become necessary to establish a separate incrimination of the crime, to enable its detailed and comprehensive regulation. Trading with people not only means simple exploitation or the simple (ab)use of their services. It means much more than that. It has developed into a game with human destinies and it involves the whole life of a human. Situations become alarming when a child’s life is put into the center of the crime. More and more often, internationally speaking, children are declared as missing who either are found dead or are not found at all. Their innocence and naivety is used to more easily approach to them and take them away. Children are used around the world for begging, labor exploitation, sexual exploitation, and even as camel – riders in the United Arab Emirates.  In addition their organs and tissues are sometimes harvested and they are used for medical experiments, so their life ends tragically. Trading children so that they are taken from their place of belonging for illegal and unlawful adoption, is a crime which is becoming more and more interesting for the perpetrators because of the enormous profits. It is a painful reality for the biological parents  as well, and a new direction for the crime of human trafficking on a global scale. It is regulated often as child trafficking or trafficking in humans in general, but its impact requires separate regulation and treatment. Objectifying a child's life by making it a commercial entity greatly harms the child

Study Of Patient’s Attitude Towards Use Of Unconventional Medicine In Complex Rehabilitation

Introduction: Unconventional medicine encompasses a field of unofficially accepted medical methods and practices for diagnosis and treatment, which have arisen in ancient times. Unconventional medicine coexists in parallel with the officially accepted traditional medical sciences. Rehabilitation is part of the conventional medical school which focuses on treatment methods that allow the patient to restore normal bodily functions following an illness.Aim: This study aims to research the patients’ attitude towards the application of unconventional methods for beneficial effects over an individual’s complex rehabilitation.Materials and Methods: This study has been conducted via an anonymous questionnaire targeted at rehabilitation patients. The data has been processed and analyzed using statistical methods.Results and Discussion: The survey results point that a third of the study participants have used at some point unconventional medical methods. It is worth noting that a large portion of the subjects who have not used unconventional methods believe that these methods can be included in complex specialized rehabilitation programs. More than half of the subjects have expressed willingness to use unconventional medicine and the same group would recommend such treatment to relatives and friends.Conclusion: The study results show that an increasing percentage of people seek and use unconventional medicine. This in turn will require new legislation to address the issue of legalizing the practicing of unconventional medicine