Selected Topics in Network Optimization: Aligning Binary Decision Diagrams for a Facility Location Problem and a Search Method for Dynamic Shortest Path Interdiction

This work deals with three different combinatorial optimization problems: minimizing the total size of a pair of binary decision diagrams (BDDs) under a certain structural property, a variant of the facility location problem, and a dynamic version of the Shortest-Path Interdiction (DSPI) problem. However, these problems all have the following core idea in common: They all stem from representing an optimization problem as a decision diagram. We begin from cases in which such a diagram representation of reasonable size might exist, but finding a small diagram is difficult to achieve. The first problem develops a heuristic for enforcing a structural property for a collection of BDDs, which allows them to be merged into a single one efficiently. In the second problem, we consider a specific combinatorial problem that allows for a natural representation by a pair of BDDs. We use the previous result and ideas developed earlier in the literature to reformulate this problem as a linear program over a single BDD. This approach enables us to obtain sensitivity information, while often enjoying runtimes comparable to a mixed integer program solved with a commercial solver, after we pay the computational overhead of building the diagram (e.g., when re-solving the problem using different costs, but the same graph topology). In the last part, we examine DSPI, for which building the full decision diagram is generally impractical. We formalize the concept of a game tree for the DSPI and design a heuristic based on the idea of building only selected parts of this exponentially-sized decision diagram (which is not binary any more). We use a Monte Carlo Tree Search framework to establish policies that are near optimal. To mitigate the size of the game tree, we leverage previously derived bounds for the DSPI and employ an alpha–beta pruning technique for minimax optimization. We highlight the practicality of these ideas in a series of numerical experiments

The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA

DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag

Structural and Chemical Profiling of the Human Cytosolic Sulfotransferases

The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone

Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA

The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8–10 nucleotides (nt)] and stable (∼30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 Å structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and flanked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix–turn–helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8–10 to 30 nt mode is mediated by DNA binding with the trimerization core

Morphological Differentiation, Mitochondrial and Nuclear DNA Variability Between Geographically Distant Populations of Daphnia galeata and Daphnia cucullata (Anomopoda, Daphniidae)

Несмотря на то, что представители р. Daphnia (Anomopoda, Daphniidae) являются одними из наиболее распространенных водных беспозвоночных и используются в качестве модельных организмов в таксономических, экологических и эволюционных исследованиях, их систематика остается весьма запутанной. Настоящее исследование посвящено изучению морфологической дифференциации и генетической изменчивости географически удаленных популяций сестринских видов Daphnia galeata Sars, 1864 и Daphnia cucullata Sars, 1862 (Anomopoda, Daphniidae) из пресноводной части Балтийского моря - Куршского залива (Россия, Калининградская область) и Новосибирского водохранилища (Россия, Новосибирская область). Морфологическая дивергенция между видами и их популяциями оценивалась по диагностическим признакам и на основании анализа изменчивости формы тела по набору морфометрических признаков. Самыми изменчивыми были признаки, характеризующие форму головы, шлема и хвостовой иглы. Реконструкция филогенетических отношений между видами выполнена на основе изменчивости 16S и 12S генов митохондриальной ДНК и фрагмента ITS2 ядерной ДНК. Дивергенция между видами D. galeata и D. cucullata на основе генов митохондриальной ДНК была значительной и свидетельствует об их монофилетическом происхождении, тогда как внутривидовые генетические дистанции оцениваются как незначительные.Although members of genus Daphnia (Anomopoda, Daphniidae) are the most common water invertebrates and are considered as model organisms for many taxonomic, ecological and evolutionary studies their systematics remains unresolved. Here, morphological differentiation and genetic polymorphism between the geographically distant populations of the sister species Daphnia galeata Sars, 1864 and Daphnia cucullata Sars, 1862 in the Curonian Lagoon, a large shallow freshwater lagoon of the Baltic Sea (Russia, Kaliningrad Oblast) and Novosibirsk Reservoir (Russia, Novosibirsk Oblast) are presented. The divergence between species and their populations was analyzed based on traditional morphological traits and a large set of morphometric traits describing the body shape. The traits describing the shape of head and helmet, and spine were the most variable morphological characters. Phylogenetic relationships between species and populations were constructed based on variation in mitochondrial 16S and 12S rRNA genes and nuclear ITS2 rDNA sequences. The mitochondrial DNA divergence between D. galeata and D. cucullata species was significant and reflected their monophyletic origin, whereas intraspecific genetic distances are estimated as insignificant

Unexpected endemism in the Daphnia longispina complex (Crustacea: Cladocera) in Southern Siberia.

The biological significance of regional cladoceran morphotypes in the montane regions of the central Palearctic remains poorly understood. In the Holarctic Daphnia longispina complex (Cladocera: Daphniidae), several variants, lineages and species have been proposed as endemic for Southern Siberia. Daphnia turbinata Sars, for example, named after its unusual head shape, is known only from Southern Siberia. Here we sequence DNA of Daphnia from three mitochondrial genes (12S rRNA, 16S rRNA, and NADH dehydrogenase subunit 2, ND2) from 57 localities in Russia and Mongolia (the majority being from Southern Siberia) and place them in evolutionary context with existing data. Our aim was to examine regional endemism of the Daphnia longispina complex in Southern Siberian; to improve the phylogenetic understanding with improved taxonomic and regional sampling, and to better understand the influence of Pleistocene glaciation on the biogeography of these lineages. At least three lineages showed genetic evidence for endemism in Southern Siberia. There was strong support for D. turbinata as a sister lineage to to D. longispina/D. dentifera. Another endemic, Siberian D. cf. longispina, is a sister group to the longispina group in general. Within D. longispina s. str. there was an endemic Siberian clade with a western range boundary near the Yenisei River Basin. Gene flow estimates among populations (based on FST values) were very low for clades of D. longispina on a regional (the original 12S dataset), and on a pan-Eurasian (the extended 12S dataset) scale. Negative values of Fu's FS and Tajima's D tests prevailed for the species examined with significant values found for two D. longispina clades, D. dentifera, D. galeata and D. cristata. Our results support the notion that Southern Siberia is an important biogeographic region for cladocerans as it contained unexpected diversity of endemics (such as D. turbinata, D. cf. longispina and lineages of D. umbra and D. longsipina s.str.) and from being the geographic meeting place of expanding postglacial lineages from eastern and western refugia

2′-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro

Antisense oligodeoxynucleotides (ODNs) have biological activity in treating various forms of cancer. The antisense effects of two types of 20mer ODNs, phosphorothioate-modified ODNs (S-ODNs) and S-ODNs with 12 2′-O-methyl groups (Me-S-ODNs), targeted to sites 109 and 277 of bcl-2 mRNA, were compared. Both types were at least as effective as G3139 (Genta, Inc.) in reducing the level of Bcl-2 protein in T24 cells following a 4 h transfection at a dose of 0.1 µM. Circular dichroism spectra showed that both types formed A-form duplexes with the complementary RNA, and the melting temperatures were in the order of Me-S-ODN·RNA > normal DNA·RNA > S-ODN·RNA. In comparison with the S-ODN, the Me-S-ODN had reduced toxic growth inhibitory effects, was less prone to bind the DNA-binding domain A of human replication protein A, and was as resistant to serum nucleases. Neither type of oligomer induced apoptosis, according to a PARP-cleavage assay. Hybrids formed with Me-S-ODN sequences were less sensitive to RNase H degradation than those formed with S-ODN sequences. Despite this latter disadvantage, the addition of 2′-O-methyl groups to a phosphorothioate-modified ODN is advantageous because of increased stability of binding and reduced non-specific effects

Human replication protein A (RPA) binds a primer–template junction in the absence of its major ssDNA-binding domains

The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein–DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5′-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a ‘zero-length’ photoreactive group (4-thio-2′-deoxyuridine-5′-monophosphate) embedded in the 3′ end of the DNA primer, or a group connected to the 3′ end by a lengthy linker (5-{N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2′-deoxyuridine-5′-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C·p32D·p14) was capable of correctly recognizing the primer– template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D·p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process