Implementing a Memory Clinic Model to Facilitate Recruitment into Early Phase Clinical Trials for Mild Cognitive Impairment and Alzheimer's Disease.

BACKGROUND: The recruitment challenges for MCI and AD subjects into clinical trials are well known, and this is particularly true for early phase studies. Currently, only 10-20% of all patients who are referred for research from the community are trial eligible (Grill and Karlawish, 2011). Due to the limited and specific study objectives in early phase study designs, these rates drop to approximately one patient every two months. Barriers to research recruitment are multi-factorial, involving patient centered factors, issues related to caregiver/study partner participation, and aspects related to the involvement of their treating physicians. To address this challenge, we implemented a Memory Clinic within PAREXEL's Early Phase Clinical Pharmacology Unit. Our objective was to significantly facilitate recruitment into AD clinical trials by providing resources and education to patients, their treating physicians, and caregivers in the community. METHOD: The Clinic's primary goals were to increase research visibility and partnerships with local organizations and referring physicians. Members of the research team co-sponsored community outreach events with local organizations, thereby increasing awareness about the services of this memory clinic. Secondly, physician outreach was expanded to include those who were not previously amenable to clinical trial referrals. Finally, Memory Clinic patients were given clinical evaluations, free of charge and the results were discussed with the patients and their caregivers. If the patients were interested in hearing more about possible research opportunities, they were referred to the early phase unit for a screening visit. RESULTS: We found that new referrals for research participation significantly increased as a result of this new paradigm. In 2016, 12 patients diagnosed with MCI or AD per protocol, were referred to a research study and 3 were randomized. In 2017, 98 patients were referred and 16 were enrolled In addition, our referral network increased with 30 physicians over a 20 mile radius. Collaborations with national non-profit organizations also increased, thereby increasing public awareness about the importance of research participation in the development of new treatments for Alzheimer's Disease. CONCLUSIONS: In summary, community engagement and providing referring physicians with a clinical service improved recruitment significantly for our phase 1 unit. Resource education, staff training, and dedicated medical professionals can significantly improve awareness about clinical research participation and provide additional participants over and above traditional recruitment methods and trial registry enrollment in a large urban area.

Knockout of histamine receptor H3 alters adaptation to sudden darkness and monoamine levels in the zebrafish.

AIM: Histamine receptor H3 (HRH3) has substantial neuropharmacological potential. Currently, knockout models of this receptor have been investigated only in mice. We characterized the expression of this receptor in the zebrafish and generated a zebrafish HRH3 knockout line. Using this model, we studied the role of HRH3 in important behaviours. We also analysed the effect of HRH3 knockout on monoaminergic systems, which has not been thoroughly studied in any animal model. METHODS: Generation of a mutant zebrafish line using the CRISPR-Cas9 system. Analysis of locomotor and social behaviour. Expression of HRH3 was characterized using in situ hybridization. Analysis of monoamine networks using HPLC, immunohistochemistry and quantitative PCR. RESULTS: We found that HRH3 knockout zebrafish larvae showed a shorter period of increased locomotion after a sudden onset of darkness, while the knockout larvae had a wild-type-like acute response to sudden darkness. Adult knockout fish showed decreased swimming velocity, although locomotor activity of knockout larvae was unaltered. Additionally, levels of dopamine and serotonin were significantly decreased in the knockout fish, while monoamine-related gene expression and immunohistochemistry patterns were unchanged. CONCLUSIONS: Our results show that HRH3 knockout larvae adapt faster to sudden darkness, suggesting a role for this receptor in regulating responses to changes in the environment. The decreased levels of dopamine and serotonin provide the first direct evidence that knockout of HRH3 alters these systems.

TRP ÑHANNELS IN THE ENDOSOMAL PATHWAY.

The TRP (transient receptor potential) superfamily consists of a wide range of cation channels with different physiological functions and different cellular distribution. The TRP channels are implicated in pain perception, hearing and temperature sensation, photoreception, mechanotransduction, Ca2+-(re)absorption, cell proliferation and differentiation. Initially it was believed that TRP channels exist exclusively in the plasma membrane of the cells. Now this view is changing, since many members of this superfamily were found in the intracellular organelles where they successfully fulfill their conductive function. This review presents the current data concerning the members of the superfamily of TRP, which are integrated into the intracellular compartments. The role of TRP channels in the complicated and multicomponent machinery of endosomal pathways is analyzed.

[Possibilities of treatment optimization in children and adolescents with epilepsy and disturbances of emotion and volition (disphoria)]. / Vozmozhnosti optimizatsii terapii detei i podrostkov, stradayushchikh epilepsiei s emotsional'no-volevymi narusheniyami (disforii).

AIM: To optimize the treatment of dysphoriain children and adolescents in regard to sex and disease severity. MATERIAL AND METHODS: Seventy children and adolescents (boys - 45, girls - 25), aged from 6 to 18 years, with different forms of epilepsy and emotion and dysphoric disturbances were studied using CPRS andGCIscales Depending on dysphoria severity, patients were stratified into three groups: mild (n=19 (27.1%), moderate (n=27 (38.6%)) and severe (n=24 (34.3%)). RESULTS AND CONCLUSION: Dysphoric disorders were significantly more prevalent in boys, hostility and aggression were characteristic of boys as well. These facts impactedtreatment options. Neuroleptics were more frequently used in boys (35.5%) compared to girls(16%).Mild dysphoria didn't require additional treatment besides AED in 78,4%. In 75% cases of moderate dysphoria,systemic treatment with neuroleptics for 6 months was necessary. One-time recommendations for neuroleptic treatment were made in all three groups with the prevalence in a groupof children with severe and moderate dysphoria.

The tyrosine hydroxylase 2 (TH2) system in zebrafish brain and stress activation of hypothalamic cells.

Two tyrosine hydroxylases (TH1 and TH2) are found in teleost fish, but no antibodies are available for TH2 protein to analyze the detailed structure of the system. We generated antibodies targeting TH2 and used them to characterize the TH2-producing cells in larval and adult zebrafish brain. The rabbit antisera reliably detected two bands corresponding to TH1 and TH2 close to 55 kDa in brain homogenates. The antisera detected neurons in brain nuclei which express th1 and th2 mRNA; knockdown of th2 expression by morpholino oligonucleotide injection abolished both the th2 mRNA signal and immunoreactivity with the rabbit antisera in TH2 cells. Double staining of samples with the rabbit antiserum made against TH2 and a monoclonal antibody which detects only TH1 allowed identification of cell groups expressing either one of the proteins. Cell groups in preoptic area, anterior, intermediate, and posterior part of the paraventricular organ contained neurons stained with the new TH2 antisera but not with the characterized monoclonal TH1 antibody. Neurons immunoreactive for TH2 and 5-HT were distinct. In situ hybridization for the mRNA of the immediate early gene c-fos combined with TH1/TH2 immunohistochemistry was used to characterize the cells of the zebrafish brain reacting to handling stress and a noxious chemical stimulus. Strong upregulation of c-fos expression was detected in hypothalamic nuclei containing TH2 cells, but few of the c-fos-expressing cells were positive for TH2, suggesting that these stressors do not directly activate a large proportion of TH2 cells.

[Polymorphism of the cytochrome oxidase b gene (cyt b) in Russian populations of common carp and wild common carp (Cyprinus carpio L.)].

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (pi) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, pi = 0.67%, h = 0.7; and BB, pi = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (pi = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (pi = 0.035%, h = 0.4; and pi = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (pi = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.

[Comparative analysis of variability of three mitochondrial genes of cytochrome oxidase complex (cox1, cox2, and cox3) in wild and domestic carp (Cyprinus carpio L.)].

For the first time, we studied the polymorphism of three mitochondrial genes of the cytochrome oxidase complex (cox1, cox2, and cox3) in natural populations of wild carp living in the Volga, Amur, and Don River Basins, as well as in European Hungarian carp and two pedigree lines of Ropsha carp of domestic breeding. The highest level of nucleotide and haplotype diversity in the studied samples was detected for the cox1 gene (pi = 0.61, h = 100%). Two lines of the Ropsha carp (pi = 0.61, h = 100%) and the Far East population of Amur wild carp from Shershikh strait (Am: pi = 0.20, h = 70%) were the most polymorphic for three genes. The second sample of Amur wild carp from the Amur River (Ac), as well as the samples of Volga and Don wild carp and Hungarian carp had lower values of variability. The presence of two main genealogical lines of the wild carp and carp was demonstrated based on the total sequence of three genes, as well as the corresponding amino acid sequences in the studied area. One of these lines (line I) is typical of the sample of Amur wild carp (Am) and three members of the Ropsha carp. Line II is developed by sequences of Volga, Don, and Amur wild carp (Ac), as well as European Hungarian carp and seven other members of the Ropsha carp. Three to four sublines, which differ in nucleotide and amino acid substitutions, were found within the lines. Possible reasons for the origin of genomic variability in wild carp, as well as in European and Russian breeds of carp, are discussed.

[Polymorphism of the coxl mtDNA gene from cercarian isolates of the avian schistosome Bilharziella polonica (Trematoda: Schistosomatidae) from Belarussian lakes].

We have studied the phylogeographic structure of avian schistosomes Bilharziella polonica (class Trematoda, family Schistosomatidae), parasites of 18 molluscs Planorbius corneus (family Planorbidae) from three Belarussian lakes. Low nucleotide (pi<0.5%) and high haplotype (h=85.6%) diversity of the gene encoding the cytochrome C oxidase first subunit (coxl) was found on the part of the species range studied. The phylogeographic reconstructions showed that the sample examined consists of at least two lineages ofhaplotypes A and C. Haplotype diversity was somewhat higher in lineage C. The genetic divergence between these genealogical lines reached 0.55%, while the time of possible divergence was 180,000-270,000 years. Possible evolutionary scenarios for differentiation of the B. polonica lines and effectiveness of using coxl for barcoding trematode populations and finding coevolutionary parasite-host relationships are discussed.

[Polymorphism of the cox1 gene in bird schistosome cercaria isolates (Trematoda, Schistosomatidae) from ponds of Moscow and Moscow oblast].

Polymorphism of a 8 10-bp mitochondrial cox1 gene region was studied in 16 cercaria isolates of bird schistosomes (family Schistosomatidae), which were collected in water bodies of Moscow and Moscow oblast and represented three species: Trichobilharzia szidati, T. franki, and T. regenti. A substantial predominance of AT (65.4%) was characteristic of the cox1 sequences in all three species. Rare single nucleotide substitutions determined low (0.2-0.9%) intraspecific nucleotide and amino acid sequence diversity. Haplotype diversity h was high (80-100%) in all three species, suggesting a unique character for almost all cox1 sequences in the sample. Phylogenetic trees based on the nucleotide and amino acid sequence variations were constructed to study the relationships of the three schistosome species. A high support was observed for the main branching node that reflects differentiation of the monophyletic group Trichobilharzia and species of the genera Bilharziella (B. polonica), Dendritobilharzia (D. pulverulenta), and Gigantobilharzia (G. huronensis). Based on the nucleotide substitutions and amino acid polymorphisms, two groups of isolates, which infect Lymnaea stagnalis (T. szidati) and snails of the group Radix (T. franki and T. regenti) respectively, were isolated in the genus Trichobilharzia. The time of divergence between the two schistosome groups infecting snails of the genera Radix and Lymnaea was calculated from the cox1 nucleotide substitution rate, which is known for Asian and Indian blood flukes from the genus Schistosoma and is 2-3% per million years on average. Divergence of the three bird schistosome species under study and divergence of the Asian species of mammalian schistosome were almost concurrent, dating back to 2.5-3.8 Myr ago. Factors responsible for the lack of intraspecific subdivision with respect to the cox1 gene in bird schistosomes and the lack of separation between two species (T. franki and T. regenti) are discussed.

[Phylogenetic relationships among schistosomatidae].

According updates on molecular genetics, the development of human schistosomes in Asia with subsequent migration to the African continent is considered to be the most probable course of events. Generally, there are 2 hypotheses of the genus Schistoma. A hypothesis of Gondvana origin was based on snail host phylogeny and paleonthology and considered first schistosomes to originate on this continent to and appear in Asia from the Indian subcontinent platform 70-150 million years ago and in South America before Gondvana's split 60-120 million years ago. The recent data of molecular genetics show a high similarity between ITS2 sequences of S.bovis and S.intercalatum, with slightly lesser one between S.bovis and S.matthei. The similar pattern with slightly fewer differences can be seen in variability of cytochrome C subunit 1. Webster et al. 2006 noted a generally high similarity among species of the African group of schistosomes and considered it to originate from interspecies hybridization inside this group. Such hydridization occurring in both nature and a laboratory can make uncertain the determination of schistosome species based on a certain single gene marker.

[Polymorphism of microsatellite markers in breeds of common carp (Cyprinus carpio L.) of Russian breeding].

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian common carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (4.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha common carp, Stavropol common carp, Amur wild common carp, and the two samples of Cherepets common carp. The second cluster included Altai common carp (Cis-Ob' and Chumysh populations), two Angelinskii common carp breeds (mirror and scaly), and Hungarian common carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai common carp, two strains of Ropsha common carp, as well as the breeds of Angelinskii and Cherepets common carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.

The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases.

Modulatory neurotransmitters which signal through G protein-coupled receptors control brain functions which deteriorate in degenerative brain diseases. During the past decade many of these systems have been mapped in the zebrafish brain. The main architecture of the systems in zebrafish brain resembles that of the mammals, despite differences in the development of the telencephalon and mesodiencephalon. Modulatory neurotransmitters systems which degenerate in human diseases include dopamine, noradrenaline, serotonin, histamine, acetylcholine and orexin/hypocretin. Although the number of G protein-coupled receptors in zebrafish is clearly larger than in mammals, many receptors have similar expression patterns, binding and signaling properties as in mammals. Distinct differences between mammals and zebrafish include duplication of the tyrosine hydroxylase gene in zebrafish, and presence of one instead of two monoamine oxidase genes. Zebrafish are sensitive to neurotoxins including MPTP, and exposure to this neurotoxin induces a decline in dopamine content and number of detectable tyrosine hydroxylase immunoreactive neurons in distinct nuclei. Sensitivity to important neurotoxins, many available genetic methods, rapid development and large-scale quantitative behavioral methods in addition to advanced quantitative anatomical methods render zebrafish an optimal organism for studies on disease mechanisms.

[Study of the genetic markers of thrombosis in hypertensive patients of the native population in the Republic of Mordovia].

The genetic markers (encoding factor of blood coagulation, the PAI-1 gene, the prothrombin gene, the GP IIb/IIIa gene, and the MTHFR gene) of thrombosis were studied in the Mordovians and Russians with essential hypertension (EH), who were residents of the Republic of Mordovia. One hundred and forty patients with TH and 90 healthy individuals of three nationalities, such as Russian, Moksha-Mordovian, and Erzya-Mordovian, were examined. Along with the routine clinical and instrumental studies, alleles of polymorphic markers were identified by the polymerase chain reaction (PCR). The polymorphic markers of the Arg 506 Gln gene, encoding factor V of blood coagulation, 4G5G of the PAI-1 gene, the G 20210 A prothrombin gene, the GP IIb/IIIa gene, and the A 1298 C MTHFR gene were also studied. The data were statistically analyzed using a package of the programs: Statistica for Windows 6.0 (Stat Soft), SPSS (version 14.0), and MS Excel XP (Microsoft). The authors used a chi-2 (chi-square) test was used to compare the incidence of genotypes and alleles in the patient groups. Hypertensive and normotensive persons were found to have no significant differences in the distribution of genotypes (encoding factor of blood coagulation, the PAI-1 gene, the prothrombin gene, and the GP IIb/IIIa gene) with regard to ethnicity in the Russian, and Moksha-Mordovian, and Erzya-Mordovian groups). Meanwhile, there was a significant preponderance of MTHFR gene mutation with the unfavorable genotype for Moksha-Mordovian patients with EH. The findings suggest that drug therapy for preventing venous thrombosis in patients with EH should be adjusted in relation to ethnicity.

[The polymorphism and structural peculiarities of two noncoding regions of liver fluke Fasciola hepatica (Plathelminthes: Trematoda) mitochondrial genome].

Structural characteristics and polymorphism of long (LNR) and short (SNR) mitochondrial non-coding regions of the liver fluke Fasciola hepatica were studied. The flukes were obtained from several populations of Russia and Belarus. The amplification of LNR yielded a set of 10 fragments with the length of neighbouring ones differing in one tandem repeat (85 bp; published earlier for Australian fluke). LNR amplification fragments of different length were cloned and sequenced. Comparison of the LNR sequences of Australian and Belarussian flukes revealed 3 nucleotide substitutions and one point heteroplasmy of the first nucleotides in the imperfect repeat and four adjacent perfect repeats. Positions of the three mutations coincide in perfect and imperfect repeats and the frequency of mutations is 4-4.7% while the frequency of heteroplasmic sites varies from 0.1 to 1.2%. It was shown that the presence of mutations and the heteroplasmy of one site can change the structure and stability of the putative secondary structures of the perfect and imperfect re- peats. The amplification of SNR of F. hepatica from several populations yielded fragments which differed from the published SNR sequence of Australian F. hepatica in the single transversion. Both non-coding regions have several conservative and potentially regulatory sequences. Probable cause of heteroplasmy and concerted origin of substitutions in different repeats are discussed.

[Clonal and population RAPD variation of cercariae obtained from Bucephalus polymorphus sporocysts (Trematoda: Bucephalidae)].

Three arbitrary primers produced 114 RAPD markers for 37 cercariae from nine Bucephalus polymorphus sporocysts obtained from three Dreissena polymorpha mollusks, which were collected in two water reservoirs of the Volga basin. Analysis of the RAPD patterns established a unique genotype for each cercaria. The topology of an UPGMA dendrogram did not reliably differentiate the cercaria according to the corresponding sporocysts. However, three groups of genotypes were isolated and corresponded to the host mollusks, indicating that each cercaria clone had a different genotype set. A within-sporocyst variation made the greatest contribution (53.0%) to the total RAPD diversity, while the contributions of within-host and between-host variations to the total diversity were equal (23.5%). Cercariae isolated from two mollusks of the Rybinsk Water Reservoir were more similar to each other than to cercariae from the geographically distant Gor'kovskoe Water Reservoir. Possible causes and distribution specifics of the observed genetic diversity of B. polymorphus are discussed.

[The astacin family of metalloproteinases].

The review deals with the properties of astacin family of zinc-dependent metalloproteinases. One of the remarkable features of these enzymes is their ability to cleave peptidyl-arylamides, which is not typical to other metalloproteinases. Special attention is paid to physiological functions of the astacins and to the influence of domain composition and posttranslational modifications on the activity and stability of these enzymes.

Isolation and properties of collagenolytic serine proteinase isoenzyme from King Crab Paralithodes camtschatica.

An electrophoretically homogeneous isoenzyme CSP-2 of collagenolytic serine proteinase has been isolated from the total preparation of king crab digestive enzymes. The molecular mass of the proteinase is 24.8 +/- 0.3 kD, pH optimum for activity is 8.5, the temperature optimum for activity is 38-40 degrees C, and the pH range of stability is 7-10. The enzyme has dual substrate specificity, but preference for positively charged amino acid residues in P(1)-position is more pronounced than in the case of the major isoenzyme. The temperature dependence of kinetic constants for synthetic substrate hydrolysis by CSP-2 has been investigated. Inhibition specificity of the enzyme is characteristic of serine proteinases but more like that of crab trypsin than that of the major CSP isoenzyme. The isolated collagenolytic proteinase also cleaves fibrinogen and fibrin and activates plasminogen. The amino acid sequence of the CSP-2 proteinase, which has been partially determined by tandem mass spectrometry, displays some similarity to the sequence of the major CSP isoenzyme.

[Polymorphism of the 12S rRNA gene and phylogeography of the Central Asian tortoises Agrionemys horsfieldii gray, 1844].

Based on intraspecific polymorphism of 12S rRNA gene, genetic variation of isolated populations of the Central Asian tortoise, Agrionemys horsfieldii, was for the first time investigated on a large part of the species distribution range, encompassing Uzbekistan, southern Kazakhstan, and northern and eastern Iran. In 59 tortoises, four haplotypes were discovered, including two (AH1 and AH2), described earlier. Haplotype AH1 was detected in 52 tortoises, inhabiting southern Kazakhstan and Uzbekistan. Haplotype AH2 was found in four tortoises from the border territory between Uzbekistan, Tajikistan, and Afghanistan. Two novel haplotypes, AH3 and AH4, were detected in the three tortoises from Iran. Based on nucleotide substitutions in the 12S rDNA sequence, the possible divergence time between the tortoises from different parts of the range was estimated. Possible pathways of the formation of modern intraspecific groups of A. horsfieldii are discussed.

[Antimutagenes' protection action in human repair-defected cells].

The action of natural (garlick extract, retinol) and of synthetic (crown-compound) antimutagenes in lymphotytes with gamma-radiation-induced inhibition of DNA-damages repair in cases of Elers-Danlos, syndrom, progeria and gomocystinurea was studied. Antimutagen cells defence from mutagenes was shown at all cases except one: progeria cells treated by retinol. Thus the repair-deficient cells resistance against mutagenes could be increased by antimutagenes.

[A mass-spectrometric approach to primary screening of collagenolytic enzymes].

A comparative analysis of MALDI TOF mass spectra of low-molecular products resulting from the hydrolysis of native collagen I by collagenases of various classes (bacterial metallocollagenase from Clostridium histolyticum, serine collagenase from the Morikrasa commercial preparation, cysteine collagenase from Serratia proteomaculans, and cysteine collagenases from larvae of beetles Dermestesfrischi and D. maculates) was carried out. The spectra contain a number of ion peaks common for all collagenases; nevertheless, the mass spectra of each hydrolysate contains a unique set of peaks ("fingerprint") characteristic of each enzyme. This is especially true for the peaks of major products with relative intensities of more than 50%. At the same time, the enzymes of one class (cysteine collagenases) exhibit in their mass spectra peaks of identical major products. The results show a potential opportunity for MALDI TOF application in the primary screening of collagenases according to the fingerprints of collagen hydrolysis products.