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Two new bivalve species of the genus Chama, C. subcalcarata and C. ferrata, from the Middle Eocene of Krivoi Rog are described.

The species Chama clavaticostata Klushnikov, 1958, which was originally described based on one juvenile exemplar from the Middle Eocene in the environs of the town of Krivoi Rog (Ukraine), is redescribed.

Three new species of bivalves of the family Cardiidae are described from the Paleogene of Ukraine: Freneixicardia picturata (Upper Eocene, town of Dnepr), Loxocardium marmoreum (Middle Eocene, the town of Kanev), and Schedocardia imperfecta (Middle Eocene, town of Ingulets). The species Loxocardium denticostatum (Berezovsky, 1998) is redescribed, because its first description was based on specimens (besides the types), which are assigned in this work to the new species L. marmoreum.

The article presents data on our study of the epizootiological situation in apiaries with a mixed form of bee diseases in greenhouses of farms in Sumy region. The data showed that of the previously identified infectious diseases of bees, which are one of the most common in the world – it's actually 4–5 major diseases, of which we studied American rot and ascospherosis, which are manifested in a mixed form with varroasis. Effective control of infectious diseases and early preventive work by a beekeeper in apiaries is a guarantee of a prepared strong bee family both for honey collection and preparation of a bee nest for wintering. Earlier, we conducted a study to monitor the epizootic situation of mixed infectious diseases of bees in the north – eastern region of Ukraine, where a large part of the epizootic was due to ascospherosis American and European rot in field work of bees. But in greenhouse conditions, where high humidity and crowding of bees increases the likelihood of damage by both American rot and ascospherosis. Given these factors, there was a suspicion of a synergistic effect of contamination of bee colonies with the above diseases indoors, damp and isolated. Which led us to the necessary detailed study of these diseases in the greenhouse of bee colonies in the Sumy region in the winter – spring periods of 2020. When examining some samples of printed brood, it was found that, with a strong degree of invasion of varroasis, favorable conditions were created for the accumulation of purulent mass at a sufficiently high level when Bac resistance. Larvae White increased to adverse factors and allowed to affect both weakened and healthy larvae, and with low resistance to developing bees associated with extreme greenhouse conditions and the presence of varroa mites, can sometimes affect bee pupae. After analyzing the results of research, we found that varroa mites contribute to the high development of American rot and ascospherosis in all its forms and witness the role of varroa mites as a factor influencing the intensity of the infectious process in closed and open brood of bee nests.

Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both refinement of diagnostics for emerging variants of concern (VOCs) and design of variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high-mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escape mutations via non-competitive inhibition originating from alternative distal locations. The exhaustive allosteric signaling and probing maps presented herein provide a comprehensive picture of allostery in the spike protein, making it possible to locate potential mutations that could work as new VOC “drivers” and to determine binding patches that may be targeted by newly developed allosteric drugs., Graphical abstract, Tan et al. characterize the dynamics of the SARS-CoV-2 Spike protein and derive allosteric signaling maps for the spike protein, which is of interest for drug development. The analysis of allosteric effects of spike protein mutations allows the prediction of potential drivers in emerging variants of concern.

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractable Drosophila border cell collective migration model. Candidates for functional studies were generated using results from a targeted Drosophila genetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress of Drosophila border cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.

new species of the genus Chelotia, C. pervicina sp. nov., from Middle Eocene deposits of the southern outskirts of the town of Krivoi Rog (Ukraine) is described. This species is compared with the most similar species C. concava (Deshayes) based on studying Chelotia specimens from the collection of the National Museum of Natural History (Paris, France).

Activated platelet derived growth factor receptor alpha (PDGFRα) is essential for the maintenance of oligodendrocyte progenitors. Differential signaling results from phosphorylation of specific tyrosine residues in the kinase insert (KI) domain present in most RTKs in variable length. Our goal is to identify the molecular contexts in which PDGFRA activation is an essential driver for maintenance of the malignant phenotype in glioblastoma. RNA sequencing was conducted on 13 glioblastoma mouse orthotopic PDXs (n=3/line). PDGFRA mRNA overexpression was observed for 3 glioblastomas: one carrying PDGFRA extrachromosomal (ecDNA) amplification (HF3253) and two non-amplified (Tukey p=0.41, 0.19). Corresponding PDGFRα KI activation (pY754) was observed only for HF3253. To assess activation as an adaptation to microenvironmental pressures, pY754 was quantified by reverse phase protein array on 4 growth conditions (normal, -growth factors, two serum concentrations) for 8 neurospheres. PDGFRɑ activation was highest in growth factor-depleted conditions in the absence of exogenous ligand (Tukey p=0.02). Taking advantage of genomic heterogeneity in HF3253, we isolated cellular lineages with divergent frequencies of PDGFRA ecDNA. PDGFRA copy number (TaqMan) decreased longitudinally. PDX tumors from higher passage had slower tumor growth due to strong selection for initial low frequency PDGFRA ecDNA-amplified clones. IHC showed that the tumors had comparably high total and activated PDGFRα to low passage tumors. Further exploiting intra-tumoral heterogeneity, we have isolated single cell clones. Tumor growth was reduced in 2 ecDNA(-) tumors compared to parental ecDNA(+) (log-rank test p= 3e-3, 1e-3). In contrast to parental, ecDNA(-) tumors demonstrated diffuse tumor morphology and weak PDGFRα activation. The staining index (mean intensity x area) for the ecDNA(-) clones was significantly lower than the ecDNA(+) (1-wayANOVA p=2e-16). We established dependency on PDGFRα signaling in a patient-derived glioblastoma model. Our data demonstrates that detection of ecDNA-amplified PDGFRA has the potential to be a predictive biomarker of future PDGFRɑ-targeted therapies.

Potent and selective CDK4/6 inhibitors (CDK4/6i) have potential for treating glioblastoma. Although genomic abnormalities affecting CDK4/6-RB1 signaling axis, such as CDK4 or CDK6 amplification and CDKN2A deletion, which are frequent in GBM, have been proposed to predict response of RB1-wildtype GBM to CDK4/6i, there is not enough data validating these biomarkers as sufficient for patient selection. Here we employ a panel of GBM patient derived cancer stem cells (CSC) representing the most frequent somatic genomic alterations in GBM to test their response to CDK4/6i. Twelve CSC lines were treated in quintuplicate for 4–7 days with 0–10 mM Abemaciclib, Ribociclib or vehicle control. Cell viability was measured using CellTiterGlo and dose response curves were analyzed using GRmetrics in R to determine Area Above the Curve (AAC) and IC50, factoring variable growth rates among cell lines. Experiments were repeated 2–4 times. Abemaciclib was more potent in reducing cell viability, based on the RB1-null CSC line measured resistance (mean AAC = 0.3 +/- 0.06), ribociclib was more specific (mean AAC = 0.05 +/-0.01). 4/7 CSCs with CDKN2A homozygous deletion were sensitive (mean AAC > 0.7) while 3/7 were resistant (mean AAC < 0.33) to 4 and 7-day treatments of abemaciclib. One CSC from a newly diagnosed GBM bearing CDK4, MYC and EGFR amplifications was sensitive to both inhibitors (mean AAC = 0.5 abemaciclib and ribociclib), while CSC from the matched recurrent tumor presenting the same driver genomic alterations was significantly more resistant (mean AAC = 0.2 for abemaciclib and ribociclib) (p < 0.05, t-test). Additionally, we exposed a sensitive cell line to conditioned media from a resistant cohort, resulting in significantly reduced proliferation and increased resistance to CDK4/6i (p< 0.05, Dunn). These findings underscore the importance of a utilizing a robust molecular profiling approach in evaluating which patients will benefit from CDK4/6i therapy.

The AlloSigMA 2 server provides an interactive platform for exploring the allosteric signaling caused by ligand binding and/or mutations, for analyzing the allosteric effects of mutations and for detecting potential cancer drivers and pathogenic nsSNPs. It can also be used for searching latent allosteric sites and for computationally designing allosteric effectors for these sites with required agonist/antagonist activity. The server is based on the implementation of the Structure-Based Statistical Mechanical Model of Allostery (SBSMMA), which allows one to evaluate the allosteric free energy as a result of the perturbation at per-residue resolution. The Allosteric Signaling Map (ASM) providing a comprehensive residue-by-residue allosteric control over the protein activity can be obtained for any structure of interest. The Allosteric Probing Map (APM), in turn, allows one to perform the fragment-based-like computational design experiment aimed at finding leads for potential allosteric effectors. The server can be instrumental in elucidating of allosteric mechanisms and actions of allosteric mutations, and in the efforts on design of new elements of allosteric control. The server is freely available at: http://allosigma.bii.a-star.edu.sg

In the second part of the monograph, bivalves of the family Crassatellidae from the Upper Eocene Mandrikovka Beds of Dnepr (former city of Dnepropetrovsk, Ukraine) and its environs are described. The family Crassatellidae is represented by 25 species, 21 of which were established by the author. These species belong to the genera Crassatella (Crassatella) (seven species), Crassatella (Furvusa subgen. nov.) (one species), Crassatella (Bathytormus) (five species), Crassatina (Crassatina) (four species), Crassovella gen. nov. (four species), and Chattonia (three species). All of these taxa are described and provided with figures of type species. Species are figured in 15 plates. These taxa are mostly established on the shell material from the Upper Eocene beds exposed by the Rybalsky quarry in the city of Dnepr. Valves belonging to the new genus Crassovella and new subgenus Crassatella (Furvusa) come from the same beds. Three out of 25 species are described from a core of the same age, which was obtained during drilling in the vicinity of Dnepr (near the towns of Sinel’nikovo and Kamensky and the village of Vasil’kovka).

Biological specifics of Trichuris оvis Abildgaard, 1795 and T. globulosa Linstow, 1901 parasitizing domestic sheep were analyzed enhancing the species identification of these nematode species. The embryonic development of nematodes was timed, survival of embryonic stages was determined taking into account their morphological and metric specifics in laboratory culture. Trichuris eggs were isolated from gonads of adult female nematodes collected from caeca of dissected sheep. Then the eggs were cultured at optimal temperature to the formation of eggs with mobile larvae. Six morphologically distinct stages of embryogenesis were established in T. оvis and T. globulosa nematodes. The protoplast stage lasted from the 1st to the 12th day in T. оvis and to the 18th day in T. globulosa. Blastomeric formation occurred from the 3rd to the 18th day in T. оvis and from the 3rd to the 21st day in T. globulosa. Bean-shaped embryos formed from the 6th to the 21st day in T. оvis, and from the 9th to the 30th day in T. globulosa. Tadpole-like embryos developed from the 12th to the 24th day in T. оvis and from the 18th to the 33rd day in T. globulosa. Larvae formed in eggs of T. оvis from the 18th to the 27th day, and in eggs of T. globulosa from the 21st to the 36th day. Mobile larvae formed from the 21st to the 30th day in T. оvis, and from the 30th to the 39th day in T. globulosa. At 27 °С, mature eggs with mobile larvae developed in 30 days in T. оvis and in 39 in T. globulosa. The egg survival in laboratory culture was 84.3 ± 4.2 % and 76.3 ± 1.5%, respectively. Developmental changes of metric parameters in Trichuris nematode eggs (length and width of eggs, plug length, eggshell thickness) were species-specific.

AlloMAPS database provides data on the causality and energetics of allosteric communication obtained with the structure-based statistical mechanical model of allostery (SBSMMA). The database contains data on allosteric signaling in three sets of proteins and protein chains: (i) 46 proteins with comprehensively annotated functional and allosteric sites; (ii) 1908 protein chains from PDBselect set of chains with low (

Three new species of Crassatellidae: Crassatella ballista sp. nov., C. calva sp. nov., and Eucrassatella depulsoris sp. nov. from the Middle Eocene deposits of the western slope of the a quarry of the Ingulets Ore Mining and Processing Enterprise (Ingulets, Ukraine), which are correlated with the Lutetian Stage, are described. The study of valves of Crassatella parisiensis Orbigny, 1852 from the Middle Eocene deposits of the Parisian Basin has shown that it differs from the species C. illustris Berezovsky, 2004, which was previously described as a subspecies of C. parisiensis. The study of variation of Crassatellidae provided substantiation of the species rank of C. artacosta Berezovsky, 2004, which was previously described as a subspecies of C. densicostata Berezovsky, 1998.

Representatives of the genus Crassatina from the Upper Eocene Mandrikovka Beds in the vicinity of Dnepr (former Dnepropetrovsk) are discussed and figured. From the Mandrikovka assemblage, two new species, C. conquisita and C. insolita, are described. One more new species, C. subcostata, is described from the Middle Eocene deposits of southern suburbs of Krivoi Rog. Two species, C. raricostata (Klushnikov) and C. expolita (Klushnikov), which were originally established as varieties, are redescribed and their species rank is substantiated.

Evaluation of livestock genomes to identify gene complexes which lead to expression of the quantitative trait desired for breeding is impossible without knowledge of its fine molecular structure and characteristics of "work" of this complex system – the interaction of genes at the level of their protein products, regulation of gene activity, and so on. To address such complex issues focused efforts of leading scientists in the world who have worked within the mapping and sequencing the genomes of the most economically important species of farm animals – "transcript" of the genome of some dog breeds was completed in 2003, the structure of the genome of cattle, pigs, rabbits, horses and turkeys was identified only in 2009, and the noticeabout completion of international program "Honeybee Genome" appeared only in 2010. The rapid genesis of methods of molecular genetic analysis of farm animals’ genomes is not in vain called "DNA Revolution" and the number of methodological approaches and developments in this field is huge, so we consider it appropriate to be limited to developments of a leading scientific center of Ukraine to address these important issues – Institute of Animal Breeding and Genetics nd. a. M.V.Zubets of NAAS of Ukraine. The main focus of the institute is developments of scientists which help to solve an important problem of the state –preservation of the existing gene pool of domestic breeds. In this sense, the developments of genetic testing of cattle on the "major" genes of quantitative traits, involved in forming the qualitative indicators of milk and meat productivity (k-Cn, βLG, GH, TG5, CAPN1 530, MSTN), have shown uniqueness of domestic breeds and high frequency of genotypes desired for breeding aimed at improving quality of milk and meat products. For example, k-Cn (kappa-casein) gene can be determined in a herd of animals with genotypes determining the presence of milk protein with desirable properties for cheese-making and selection of animals on some genotypes of βLG, GH, TG5 not only helps to intensify breeding towards milk yield increase, but will increase its fat content. Some mutation, identified in CAPN1 530 (calpain) gene, will provide for its carrier –a representative of beef cattle breeds by expressed marbling of muscle and individuals with abnormalities in MSTN (myostatin) gene will have "double" hypertrophied muscle structure. By the way, convenience to address practical issues of production of molecular genetic markers is that any genetic material (blood, pinched hair from an ear at marking of animals, buccal scrape of mucous membranes, urine, manure, semen, hair with hair follicles and even a few cells of embryos, etc.) can be used for researches at any age of animals. The value of this information is an early assessment of a genotype of an animal, that is, its genetic potential immediately after birth. Using crossbreeding of different local populations of farm animals with imported ones has helped to improve the genetic potential of productivity of many populations of animals in very short term and the widespread artificial insemination has created conditions for economically useful programmable transmission of genes from parents to offspring. But now, as a result of intensive breeding and breed formation the accumulated reserve of variability has been reduced that can’t affect the possibilityofstrategic breeding. Intensive processes of "holsteinisation" and use of the limited number of sires of leading lines of this breed at moderate inbreeding and limitation of effective population size have led to the accumulation of "genetic load" in herds of local breeds–mutant variants of gene with lethal and semi-lethal effects. Determination of hidden genetic abnormalities in cattle (BLAD – Bovine Leukocyte Adhesion Deficiency, CVM – Complex Vertebral Malformation, DUMPS – Deficiency of Uridine Monophosphate Synthase) using DNA diagnostics is carried out to prevent the accumulation of genetic load and reproductive losses. The vast number of spontaneous abortions and reasonsof early death of calves with birth defects is caused by gene mutations appearing phenotypically only if carriers of mutant alleles are both parents of a descendant. Identification of animals with heterozygous genotype as carriers of hidden abnormalities, is possible only if using proposed DNA technology. Another type of testing breeding animals which is obligatory under law "On Livestock Breeding" is a cytogenetic analysis. In the selection and breeding work it is important not only to use methods for assessing the genetic potential of animals on the basis of productivity traits and availability of genetic diseases caused by mutations in specific genes. We must also take into account the sensitivity of animals’ genetic apparatus to different mutagenic environmental factors; it makes possible to select animals to improve genetic stability of their offspring and creating genetically stable populations at some farms that will significantly reduce the likelihood of chromosomal or genetic reconstructions in animals. Reliable ways to assess genetic and population situation, genetic differences at interspecies, within species and individual level include a method of ISSR-PCR. Effective criteria for determining the impact of mutagenic environmental factors and selection factors on the structure of the investigated samples are implemented via this method and it is used as a tool for search of genetic loci associated with expression of the desired quantitative trait. This development is intended for use primarily in pig breeding for obtaining heterosis of offspring with high potential fattening and meat qualities. As a result of long-term molecular genetic monitoring with use of own-created informative DNA markers in technology ISSR, RAPD, the confirmation of selection achievements in beekeeping was received– creating Khmelnytsky intra-breed type of Ukrainian Steppe bees with construction of unique genetic passport. The basic genetic criteria of "pure breed" of three bee species – Ukrainian, Carpathian and Grey Caucasian Mountain were determined, thus the degree of "pure breed"of bees can be determined accurately on the basis of morpho-metric and molecular genetic indicators. Using ISSR-S4 primer is able to identify the genome fragment of Ukrainian bees with size 950 bp, which is significantly associated with the expression of honey productivity; the research is carried out in this field. Conducting DNA certification of bees to determine the purebred Ukrainian Steppe bees and Carpathian bees is a necessary part of the breeding work in beekeeping for the prevention and elimination of consequences of undesirable interbreed crossbreeding, linear consolidation, increase of traits of honey productivity. In 2014,"Guidelines for morphological and genetic evaluation of Ukrainian bees" were formed and approved by the Ministry of Agrarian Policy of Ukraine as a result of long-term comprehensive studies on the genetics of honey bees. Despite the difficult economic situation, a significant reduction in funding scientific support, reduction of scientific staff, low technological level of existing equipment, the work towards the development of effective technologies for the needs of the agricultural sector is conducted. The unique researches of peculiarities of genetic structure of major histocompatibility complex of cattle and pigs are carried out to identify animals with the best heredity for reproductive capacity and resistance to infectious diseases. Further study of protective and hygienic behaviour of bees, which is currently underway, will enable to improve breeding work in beekeeping with getting environmentally friendly products which can be exported and receive significant foreign exchange earnings to the state budget. In general, livestock of Ukraine is the most important part of the socio-economic development and food security, as it is a manufacturer of biologically important products in the human diet. It is decidedly, that the progressive development of agriculture which defines export potential, quality of life and prosperity of citizens of Ukraine is impossible without the development of modern technologies and appropriate scientific support.

Three new bivalve species of Crassatella (C. necopina sp. nov., C. personata sp. nov., C. singulata sp. nov.) from detrital sand of the Rybal’sky quarry of Dnepropetrovsk are described. Based on the study of additional material, the subspecies C. (C.) parisiensis Orbigny duplex Berezovsky, 2004 is ranked species (Upper Eocene; Ukraine Dnepropetrovsk, Rybal’sky quarry, Mandrikovka Beds).

PDGFRA is the second most frequently amplified gene encoding receptor tyrosine kinase in adult glioblastoma (GBM), oftentimes as extrachromosomal elements (ecDNA). Our overall objective is to elucidate mechanisms underlying PDGFRα dependency in GBM tumor maintenance. We have isolated distinct subpopulations from a GBM model (HF3253), harboring two alterations in PDGFRA: constitutively active genomic rearrangement and extrachromosomal amplification, that differ in the frequency of PDGFRA ecDNA. HF3253 tumor growth rate correlates with the initial proportion of ecDNA+ population implanted. Furthermore, slower tumor growth is due to selection for initially low-frequency PDGFRA ecDNA amplified clones based on histology and TaqMan Copy Number assay. Further exploiting intra-tumoral heterogeneity, we have isolated single cell clones from bulk cells. Compared to bulk cells, single cell clones do not express PDGFRα, PDGFRA mRNA and exhibit diploid PDGFRA copy number. Tumor growth was reduced in 4 ecDNA(-) clones compared to parental ecDNA(+) (log-rank test p= 0.00772, 0.00379, 0.00076, 0.00379). In contrast to parental HF3253, ecDNA(-) tumors demonstrated diffuse tumor morphology and weak PDGFRα activation. HF3253 ecDNA(-) PDX tumors lack detectable PDGFRα. Correspondingly, HF3253 ecDNA(-) cell populations do not exhibit de novo PDGFRA copy number gains post-implant. We conducted paired, whole RNA-sequencing on 20 HF3253 populations (ecDNA+/-: 6 clones from 3 biological replicates PDXs and 4 clones from 4 in vitro technical replicates). Employing a false discovery rate of 0.05, we identified 785 differentially expressed genes. Platelet-derived growth factor binding (GO:0048407) and central carbon metabolism were down-regulated in ecDNA(-) while genes significantly associated with astrocytic processes were upregulated. We demonstrated the dependency on PDGFRα signaling in a patient-derived GBM model carrying ecDNA PDGFRA amplification. Our data validates PDGFRɑ as a therapeutic target in a subset of GBM patients and demonstrates that detection of ecDNA-amplified PDGFRA has the potential to be a predictive biomarker of future PDGFRɑ targeted therapies.

Two new bivalve genera and three new species of the family Limidae from the Upper Eocene of Ukraine are described.

Prognosis of patients diagnosed with IDHwt glioblastoma is influenced by known clinical and demographic factors, and likely by physiological characteristics. Our goal is to determine tumor-intrinsic gene expression signatures associated with aggressive tumor growth in patient-derived xenografts (PDXs). Cancer stem cell (CSC) lines established from 10 IDHwt glioblastoma tumors were implanted orthotopically in cohorts of 10 to 15 nude mice (3x10E5 viable cells/mouse), for development of PDX under uniform conditions. Mice were monitored and sacrificed when symptomatic. Five PDX lines, presenting median survival of 29 to 59 days were classified as short (S) survivors, and 5 lines with median survival between 111 and 134 days as long (L) survivors. RNA was isolated from terminal PDX tumors (n=3/line) and sequenced using Illumina HiSeq 2000. Differential gene expression analysis between tumors in S and L survival groups was conducted using the lmFit and eBayes, and genes were ranked by Benjamini-Hochberg adjusted P-values, set to adj.p< 0.05, resulting in 1663 genes upregulated and 1539 genes downregulated in the aggressive S group. Gene ontology analysis was performed using Metacore (Clarivate Analytics) and Metascape (http://metascape.org). Chromatin modification was significantly enriched in the aggressive tumor group (Metacore, p= 1x 10–12). Remarkably, 40% (654/1663) of the genes upregulated in the aggressive PDX tumors were co-expressed with an epigenetic master regulator in the TCGA glioblastoma RNAseq dataset (cBio Portal, q< 10E-15), and this subset was also highly enriched in chromatin modification, stemness transcriptional regulation and DNA repair (q=10E-45 to q=10E-13). These results indicate that novel host-independent prognostic gene expression signatures can be derived from the PDX models and underline the potential of epigenetic regulators as therapeutic target for aggressive glioblastomas. Our results further indicate that these models are suitable for testing a new generation of epigenetic drugs currently in pre-clinical and clinical development.

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractableDrosophilaborder cell collective migration model. Candidates for functional studies were generated using results from a targetedDrosophilagenetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress ofDrosophilaborder cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.

The molecular mechanisms of pathological non-synonymous single-nucleotide polymorphisms are still the object of intensive research. To this end, we explore here whether non-synonymous single-nucleotide polymorphisms can work via allosteric mechanisms. Using structure-based statistical mechanical model of allostery and analyzing energetics of the effects of mutations in a set of 27 proteins with at least 50 pathological SNPs in each molecule, we found that, indeed, some SNPs can work allosterically. We illustrate the molecular basis of disease phenotypes caused by allosteric SNPs with the case studies of human galactose 1-phosphate uridyltransferase (GALT) and glucose-6-phosphate dehydrogenase (G6PD). We also found that mutations of a number of other residues in the protein may cause modulation comparable to those observed for known pathological SNPs. In order to explain this, we propose a notion of allosteric polymorphism, which implies the presence of a number of critical positions in the protein sequence, whose mutations can allosterically disrupt the protein function and result in a disease phenotype. We conclude that the emerging importance of allosteric polymorphism calls for the development of computational framework for analyzing the allosteric effects of mutations and their role in the modulation of protein activity.

Background Oncogene activation through somatic gene amplification happens frequently in GBM, with over 70% of these tumors presenting amplification of at least one putative driver gene, oftentimes in small extrachromosomal circular DNA segments composed of chromatin (ecDNA). A molecularly diverse and representative panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX), which retain the original genomic abnormalities and ecDNA amplifications, was employed to assess adaptive response to the absence of ecDNA amplification. Methods We have isolated ecDNA negative cell populations from two patient-derived models. HF3035 harbors a MET amplification and HF3253 harbors a PDGFRA constitutively active genomic rearrangement and extrachromosomal amplification. We conducted paired, whole RNA-sequencing on 20 HF3253 populations (ecDNA+/-: 6 clones from 3 biological replicate PDXs and 4 clones from 4 in vitro technical replicates) and 12 HF3035 population (ecDNA+/-: 6 clones from 3 biological replicate PDXs). Results Nonparametric differentially expressed gene (DEG) analysis using NOISeqBio (R/Bioconductor), identified 564 differentially expressed genes (482 upregulated in ecDNA(-)) employing a stringent false discovery rate of 0.05. Genes significantly associated with PDGF stimulation, central carbon metabolism, and H3K27me3 were downregulated in ecDNA(-), while genes significantly associated with astrocytic processes, neuronal differentiation, and EGFR signaling were upregulated in ecDNA(-) (EnrichR). We employed an additive linear model with PDX serving as a blocking factor to compare ecDNA+ and ecDNA- populations in both models (R/edgeR). 2071 genes were upregulated in ecDNA+ PDX specimens and 2365 genes were downregulated. Specifically, E2F targets were highly enriched in ecDNA+ populations, in addition to mRNA pre-processing. ecDNA loss primarily targeted glycogen metabolism, NTRK signaling, and inositol phosphate catabolism. Conclusions We have identified PDX-specific and non-specific features to an adaptive response to the loss of ecDNA amplification. Notably, a signature adaptation is an upregulation of seemingly redundant receptor tyrosine kinases.

Two new species of the genus Crassatella, C. imitatoris sp. nov. and C. pseudolamellosa sp. nov., from Upper Eocene detrital sands of the Rybal’sky quarry of Dnepropetrovsk are described. The species Crassatella oblongula Klushnikov, 1958 is redescribed as a variety.

The aim of the study was to investigate and compare the effect of exogenous melatonin administration on the morphofunctional state of the exocrine and endocrine parts of the pancreases of spontaneously-hypertensive rats (SHR line) in spring and autumn. The research was conducted on 48 male SHR line rats, age 4 months. The rats of the experimental group received melatonin (Unipharm Inc.,USA) every day (at 10 am) orally at a dose of 5 mg/kg of body weight. The duration of the experiment was 28 days. Morphofunctional activity of the exocrine and endocrine parts of the pancreas was assessed using morphological and morphometric methods. The histological preparations were made by the standard methods. The slides were photographed using a digital camera on a Nikon microscope. The morphometric analysis was performed on digital images using the computer program Image J. The morphological signs of the exocrine pancreas activity were greater in the control rats in spring compared to autumn. Conversely, the activity of the endocrine part of the pancreas in control animals was higher in autumn, than in spring. Exocrine pancreas activity increases after administration of melatonin regardless of time of the year. This was evidenced by the increase in size and height of the epithelial acini, and the number of exocrinocytes placed in them. The width of the interlobular and interacinus connective tissue layers in the pancreas decreased in spring (mostly) and in autumn. The activity of the endocrine part of the pancreas increased after administration of melatonin in spring. Proof of this was an increase in the number and size of islets of Langerhans and the number of endocrinocytes they contained, compared with control values. The number and size of islets of Langerhans, the number and density of the endocrinocytes they contained decreased in the rats which received melatonin in autumn. This indicates a reduction of the endocrine pancreas activity. These results may have implications for therapeutic dosing of melatonin in patients with hypertension in different seasons of the year.

The presence of multiple allosteric sites in proteins motivates development of allosteric drugs-modulators of protein activity with potentially higher specificity and less toxicity than traditional orthosteric compounds. A quest for allosteric control of any protein starts from the identification and characterization of allosteric sites. Protein dynamics is the basis for allosteric communication. Binding of effector molecules to allosteric sites modulates structural dynamics, thus affecting activity of remote functional sites. We review here theoretical concepts and experimental approaches for exploring allosteric sites, their role in allosteric regulation, and ways to assess their druggability. Key steps of the design procedure aimed at obtaining allosteric drugs with required agonistic/antagonistic effect are proposed, and their computational and experimental elements are discussed.

SummaryGap junction-mediated cell-cell communication enables tumor cells to synchronize the execution of complex processes. Despite the connexin family of gap junction proteins being considered tumor suppressors, we previously found that glioblastoma cancer stem cells (CSCs) express higher levels of Cx46 compared to non-stem tumor cells, and this was necessary and sufficient for CSC maintenance. To develop a Cx46 targeting strategy, we utilized point mutants to disrupt specific functions of Cx46 and found that gap junction coupling was the critical function of Cx46 for CSCs. Based on this finding, we screened a clinically relevant library of small molecules and identified clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuated proliferation, self-renewal, and tumor growth and synergized with temozolomide to induce apoptosis. These data suggest that combining clofazimine with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.

BACKGROUND Somatic oncogene amplification happens frequently in glioblastoma (GBM). The second most frequently amplified gene encoding receptor tyrosine kinases in GBMs is platelet derived growth factor alpha (PDGFRA) (15%). In contrast, MYC and MYCN amplification occurs in 1.6% and 2.9%, respectively. Our goal was to characterize the role of PDGFRɑ and Myc in GBM. MATERIAL AND METHODS Neurosphere cultures were implanted in cohorts of 10–15 nude mice. 5 PDX lines, presenting median survival of 29–59 days were classified as short survivors, and 5 lines with median survival between 104–134 days classified as long survivors. Total RNA was extracted from PDX terminal tumors (3 biological replicates) and sequenced in a paired-end read format. Mouse reads were filtered out using Xenome. MYC and PDGFRA expression patterns were analyzed in tissue microarrays representing duplicated samples from 40 glioma neurosphere-derived PDX lines by IHC (1 anaplastic oligodendroglioma, 8 recurrent GBM with 2 newly diagnosed/recurrent pairs). Normalized staining intensity (MI) and area (A) were quantified using Fiji/ImageJ. RESULTS PDGFRA, MYC, MYCN gene amplifications were represented in a molecularly diverse panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX). Transforming to a normal distribution (log10), 4/13 of cell lines had a PDGFRA mRNA expression (RPKM) higher than 1.5. Similarly, one PDX line had a staining index of greater than 10, 11 (27.5%) had an index between 5–10. The range of intra-tumoral variance, represented by standard deviation, was 0.09–24.25 highlighting the heterogeneity of PDGFRɑ expression. PDGFRɑ phosphorylation (Y754) did not differ between 8 cell lines cultured in NMGF, but deviated in alternate medias without growth factors, supplemented with FBS. In comparison, MYC(N) mRNA expression is only elevated in the context of a known amplification. Furthermore, a a MYC activity signature consisting of 18 target genes was only evident in the 5 amplified CSC lines. Taking advantage of genomic heterogeneity, we have isolated subclones lacking PDGFRA amplification from a PDGFRA amplified GBM CSC. The absence of PDGFRA amplification reduced the self-renewal potential to 37% of the PDGFRA amplified cell population (p=0.001) in clone 1 and 57% in clone 2 (p=0.013). Pertaining to determinants of in vivo survival, MYC was altered in 80% of short survivors (2/5 MYC, 2/5 MYCN amplification) and in 0% of long survivors. Myc signature was highly correlated with in vivo survival (Pearsons’ corr. = -0.77) and MYC gene expression was correlated with in vivo TMZ resistance (corr. = 0.7). CONCLUSION These results suggest that PDGFRɑ expression and activity can occur in the absence of gene amplification, while Myc activity is dependent on gene amplification. Both oncogenes drive oncogenic pathways that should be explored as therapeutic targets.

All presently known bivalve species of the orders Nuculida and Arcida with the taxodont hinge from the Upper Eocene of Dnepropetrovsk (Ukraine) are described and figured. The material includes private bivalve collection of the author collected from 1989 to 2013 and specimens collected by N.A. Sokolov, M.N. Klushnikov, S.V. Popov, O.V. Amitrov, I.A. Goncharova, L.S. Belokrys, V.V. Ablets, V.L. Stefansky, V.V. Dovben’, and V.V. Dem’yanov, housed in the museums, scientific institutes, and private collections. The material comes from three localities: Rybal’sky quarry, village of Mandrikovka, and channel of the Dnieper River under the Amurskii railroad bridge. Revision is based on representative materials from the Rybal’sky quarry and specimens collected by N.A. Sokolov and M.N. Klushnikov. Previously published identification of species from the last two localities are revised. Photographs arranged in 33 plates display 56 species of the genera Arca, Barbatia, Asperarca, Acar, Venignia, Cucullaria, Siptionella, Arcopsis, Trigonodesma, Limopsis, Nucunella, Nucinella, Glycymeris, Nucula, Nuculana, and Yoldia. Of them 32 species are new. Two new genera, Venignia and Siptionella, are established. An extensive collection of shells from the Middle and Upper Eocene of Western Europe kindly placed at my disposal by the Netherlands paleontologist R. Hessel was of great importance for revision.

Two new genera and two new species of Mitilidae, Admytilus alius sp. nov. and Assytilus alpha sp. nov., from the Middle Eocene of a quarry near the town of Ingulets, Ukraine, are described.

NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand-protein interactions found in crystallized ligand-protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions.

Aminoacyl-tRNA synthetases (ARSs) play an essential role in the protein synthesis by catalyzing an attachment of their cognate amino acids to tRNAs. Unlike their prokaryotic counterparts, ARSs in higher eukaryotes form a multiaminoacyl-tRNA synthetase complex (MARS), consisting of the subset of ARS polypeptides and three auxiliary proteins. The intriguing feature of MARS complex is the presence of only nine out of twenty ARSs, specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro, regardless of the organism, cell, or tissue types. Although existence of MARSs complex in higher eukaryotes has been already known for more than four decades, its functional significance remains elusive. We found that seven of the nine corresponding amino acids (Arg, Gln, Glu, Ile, Leu, Lys, and Met) together with Ala form a predictor of the protein α-helicity. Remarkably, all amino acids (besides Ala) in the predictor have the highest possible number of side-chain rotamers. Therefore, compositional bias of a typical α-helix can contribute to the helix's stability by increasing the entropy of the folded state. It also appears that position-specific α-helical propensity, specifically periodic alternation of charged and hydrophobic residues in the helices, may well be provided by the structural organization of the complex. Considering characteristics of MARS complex from the perspective of the α-helicity, we hypothesize that specific composition and structure of the complex represents a functional mechanism for coordination of translation with the fast and correct folding of amphiphilic α-helices.

Five new bivalve species (Limopsis obscura, L. indubia, L. lacunosa, L. pretiosa, and L. quadrata) from the Upper Eocene of Ukraine (Dnepropetrovsk, Rybal’sky quarry, Mandrikovka Beds) are described.

A new bivalve genus and species, Venignia arcula gen. et sp. nov. of the family Arcidae from the Upper Eocene of Ukraine is described.

Gap junction-mediated cell-cell communication enables tumor cells to synchronize the execution of complex processes. Despite the connexin family of gap junction proteins being considered tumor suppressors, we previously found that glioblastoma cancer stem cells (CSCs) express higher levels of Cx46 compared to non-stem tumor cells, and this was necessary and sufficient for CSC maintenance. To develop a Cx46 targeting strategy, we utilized point mutants to disrupt specific functions of Cx46 and found that gap junction coupling was the critical function of Cx46 for CSCs. Based on this finding, we screened a clinically relevant library of small molecules and identified clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuated proliferation, self-renewal, and tumor growth and synergized with temozolomide to induce apoptosis. These data suggest that combining clofazimine with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.

After decades of research and clinical trials there is still no cure for Alzheimer's disease (AD). While impaired clearance of amyloid beta (Aβ) peptides is considered as one of the major causes of AD, it was recently complemented by a potential role of other toxic amyloidogenic species. Insulin-degrading enzyme (IDE) is the proteolytic culprit of various β-forming peptides, both extracellular and intracellular. On the basis of demonstrated allosteric activation of IDE against Aβ, it is possible to propose a new strategy for the targeted IDE-based cleansing of different toxic aggregation-prone peptides. Consequently, specific allosteric activation of IDE coupled with state-of-the-art compound delivery and CRISP-Cas9 technique of transgene insertion can be instrumental in the fight against AD and related neurodegenerative maladies.

Two new species of Asperarca, A. acuta and A. microida, from the Upper Eocene of the Ukraine (Dnepropetrovsk, Mandrikovka Beds) are described. This genus is recorded for the first time in the Paleogene. The most ancient previously known records of Asperarca were dated Miocene.

A new genus of the family Parallelodontidae (Bivalvia: Arcoidea), Siptionella gen. nov., with four species from the Upper Eocene of Ukraine and Germany and the Maestrichtian of Denmark is described. From the Upper Eocene of Ukraine, two species, the type species of the genus S. prompta (Berezovsky, 2002), previously described by the author within the genus Porterius, and the new species S. demissa sp. nov. are described and figured.

Aim. To analyze the genetic structure of the Holstein and Simmental breeding bulls from various breeding stock-rearing farms of Ukraine by polymorphisms of kappa-casein, β-lactoglobulin, growth hormone, leptin, pituitary-specifi c transcription factor, myostatin loci genes. Methods. Individual genotypes of the animals were determined using the polymerase chain reaction (PCR) followed by determination of restriction fragment length polymorphism (RFLP analysis). Results. A complex model genotype for increasing milk yield of the animals of the Ukrainian Black and White Dairy breed – κ-Cn АВ , βLG АВ , GH LV , Pit-1 АА , LEP АА , butterfat content – κ-Cn АВ , βLG АВ , GH VV , Pit-1 АА , LEP BB/AB ; Ukrainian Red and White Dairy breed – Cn АА , βLG АА , GH L , PIt-1 AB and κ-Cn AA , βLG AB , GH LV , Pit-1 AB ; Simmental breed – κ-Cn ВВ , βLG ВВ , GH LL , LEP AB and κ-Cn ВВ , βLG ВВ , GH LV , LEP AA was determined respectively. Genetic certifi cation of the breeding bulls of 25 various breeds on standard microsatellite panels (ВМ1824, ВМ2113, INRA023, SPS115, TGLA122, TGLA126, TGLA227, ETH10, ETH225, ETH3) was held for their biological material long-term storage rationale in the National Bank of Genetic Resources. Conclusions. Comprehensive monitoring of cattle breeding resources predetermines the implementation of genetic examination of the breeding animals’ origin according to ISAG international guidelines in Ukraine. This will determine the specifi c type or breed gene pool as a whole, also characterize the vector of micro-evolutionary processes in the populations of animals and allow to solve other breeding and genetic problems.

New bivalve species of the genus Acar from the Upper Eocene of Dnepropetrovsk (Ukraine) are described. The differences in ontogenetic development of these species from the close genus Barbatia are discussed.

SUMMARY Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy., Graphical Abstract, In Brief Cx46 was previously shown to be essential for glioblastoma cancer stem cell maintenance. Here, Mulkearns-Hubert et al. show that cancer stem cells depend on Cx46-mediated cell-cell communication and identify a Cx46 inhibitor, clofazimine. Clofazimine preferentially inhibits Cx46-mediated communication and targets cancer stem cells to decrease tumor growth.

Leber’s hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.

The ability to isolate, characterize, and expand distinct tumor cell populations from primary tissue or xenografts is vital to identifying molecular mechanisms specific to cancer stem cells. Once cells have been extracted from tissue, there are multiple methods by which they can be sorted and cultured. We will describe the approaches that can be taken from cancer stem cell isolation through expansion, including Magnetic-activated Cell Sorting (MACS), Fluorescence-activated Cell Sorting (FACS), the use of reporter systems, and various cell culture methods.

Study of the hierarchy of domain structure with alternative sets of domains and analysis of discontinuous domains, consisting of remote segments of the polypeptide chain, raised a question about the minimal structural unit of the protein domain. The hypothesis on the decisive role of the polypeptide backbone in determining the elementary units of globular proteins have led to the discovery of closed loops. It is reviewed here how closed loops form the loop-n-lock structure of proteins, providing the foundation for stability and designability of protein folds/domain and underlying their co-translational folding. Simplified protein sequences are considered here with the aim to explore the basic principles that presumably dominated the folding and stability of proteins in the early stages of structural evolution. Elementary functional loops (EFLs), closed loops with one or few catalytic residues, are, in turn, units of the protein function. They are apparent descendants of the prebiotic ring-like peptides, which gave rise to the first functional folds/domains being fused in the beginning of the evolution of protein structure. It is also shown how evolutionary relations between protein functional superfamilies and folds delineated with the help of EFLs can contribute to establishing the rules for design of desired enzymatic functions. Generalized descriptors of the elementary functions are proposed to be used as basic units in the future computational design.

Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design., Author Summary The 50th anniversary of Monod-Changeux-Jacob seminal paper “Allosteric proteins and cellular control systems” became the hallmark of a new wave in the allostery studies and the turning point in our vision of allostery and its implications in protein engineering and drug design. Recent experimental and theoretical works clearly show relevance of allosteric phenomenon to drug design, unraveling advantages of allosteric drugs in comparison to traditional orthosteric compounds. Remarkable simplicity of allosteric effectors and, at the same time, their potentially high specificity is one of the most important traits. The non conserved nature of allosteric ligands is a basis for avoiding drug resistance, and existence of latent regulatory sites make them attractive drug targets. The model presented in this work provides a theoretical framework for the quantification of the causality and energetics of allosteric regulation, which is a prerequisite for design of effector molecules with required characteristics. The synthesis between the thermodynamics of allostery and the intrinsic atomic nature of proteins and their interactions with the allosteric effectors accomplished in this work is a small initial step in the long endeavor towards future allosteric drugs.

Motivation: Earlier studies of protein structure revealed closed loops with a characteristic size 25–30 residues and ring-like shape as a basic universal structural element of globular proteins. Elementary functional loops (EFLs) have specific signatures and provide functional residues important for binding/activation and principal chemical transformation steps of the enzymatic reaction. The goal of this work is to show how these functional loops evolved from pre-domain peptides and to find a set of prototypes from which the EFLs of contemporary proteins originated. Results: This article describes a computational method for deriving prototypes of EFLs based on the sequences of complete genomes. The procedure comprises the iterative derivation of sequence profiles followed by their hierarchical clustering. The scoring function takes into account information content on profile positions, thus preserving the signature. The statistical significance of scores is evaluated from the empirical distribution of scores of the background model. A set of prototypes of EFLs from archaeal proteomes is derived. This set delineates evolutionary connections between major functions and illuminates how folds and functions emerged in pre-domain evolution as a combination of prototypes. Contact: Igor.Berezovsky@uni.no

Background & Objective: Receptor tyrosine kinase (RTK) signaling is altered in over 80% of glioblastoma (GBM) cases, frequently through gene amplification. About 14% of GBMs carry amplification in the gene coding for platelet-derived growth factor receptor A (PDGFRA), according to TCGA. PDGFRA plays a key role in brain development and is associated with GBM proneural (PN) subtype. Despite the frequency of oncogenic RTK signaling in GBMs, RTK inhibitors have not yet achieved sufficient efficacy in clinical trials to earn FDA approval. Imatinib, a multi-kinase inhibitor that targets PDGFRA, has not shown efficacy in earlier clinical trials for GBM, in which amplification status was not an inclusion criteria. It has been reported that treatment of GBMs carrying extrachromosomal (ecDNA) amplification with EGFR inhibitors leads to a decrease in ecDNA copy number as a mechanism of resistance. Here, our objective was to assess the heterogeneity of ecDNA PDGFRA amplification in a newly diagnosed PN GBM tumor (HF3253), and to evaluate whether similar modulation of ecDNA copy number could be attained by Imatinib treatment of patient-derived xenografts (PDX). Experimental Approaches & Results: PDGFRA amplification was detected by low pass whole genome sequencing and confirmed to be extrachromosomal by fluorescent in situ hybridization (FISH) in metaphase spreads using PDGFRA and centromere 4 labeled DNA probes. The amplified PDGFRA also harbored a novel missense mutation corresponding to the extracellular domain. PDGFRA FISH signals/number ranged from 3 to 100 in the HF3253 samples, with high signal (> 20) evident in 67%, 39%, and 43% and low amplification signal (6 < x < 9) evident in only 0%, 6%, and 19% of nuclei from biopsy, neurosphere, and xenograft respectively. HF3253 neurospheres were orthotopically implanted into the brains of immunocompromised nude mice. Imatinib mesylate was administered by oral gavage at a 75 mg/kg/day dosage in 5-day cycles with 2-day drug holiday intervals, starting 2 weeks post implant. Control mice received vehicle gavage under the same schedule. Mice were monitored daily and sacrificed on the basis of weight loss or neurological symptoms. HF3253 PDX treated with Imatinib mesylate did not have a significant survival advantage (median survival: 47.2 days) relative to control mice (median survival: 45.8 days; log rank test p value = 0.7825). The untreated and treated PDXs exhibited high levels of phospho-PDGFRA by IHC. Two independent in vitro dose response assays showed no difference in the IC50 for Imatinib between PDGFRA positive and negative GBM neurospheres: (9.447 µM, 8.384 µM) and (8.972 µM, 6.896 µM). Conclusions: We have developed a patient derived model of glioblastoma that retains ecDNA PDGFRA amplification and high levels of expression and RTK activation. Although PDGFRA is a driver of malignancy, our results show that better inhibitors are needed. Citation Format: Artem D. Berezovsky, Andrea D. Transou, Susan M. Irtenkauf, Laura A. Hasselbach, Julie Koeman, Hoon Kim, Roel G. Verhaak, Tom Mikkelsen, Laila M. Poisson, Ana C. deCarvalho. Heterogeneous extrachromosomal amplification of mutant PDGFRA is associated with an aggressive phenotype in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3491.

As protein complexes must remain in their native conformations at habitat temperatures, thermal adaptation requires adjustment of their parts and interactions between them. Based on independent sets of structural templates and sequences of 127 complete prokaryotic proteomes with optimal growth temperatures from 8 degrees C to 100 degrees C, we performed proteomic homology modeling of complexes and analyzed peculiarities in their traits related to thermal adaptation. We explore compositional determinants of thermostability of protein complexes based on the model of stability including negative and positive components of design. We show that positively charged amino acids play an important role in protein complexes, working in negative design against misfolded conformations and aberrant assemblies and contributing to positive design by stabilizing both the native interface and the overall structure of the complex. Aggregation propensity of interfaces is higher than that of surfaces and the difference between them increases with optimal growth temperature securing native complexes in hot environments.

Domain hierarchy and closed loops (DHcL) (http://sitron.bccs.uib.no/dhcl/) is a web server that delineates energy hierarchy of protein domain structure and detects domains at different levels of this hierarchy. The server also identifies closed loops and van der Waals locks, which constitute a structural basis for the protein domain hierarchy. The DHcL can be a useful tool for an express analysis of protein structures and their alternative domain decompositions. The user submits a PDB identifier(s) or uploads a 3D protein structure in a PDB format. The results of the analysis are the location of domains at different levels of hierarchy, closed loops, van der Waals locks and their interactive visualization. The server maintains a regularly updated database of domains, closed loop and van der Waals locks for all X-ray structures in PDB. DHcL server is available at: http://sitron.bccs.uib.no/dhcl.