The backbone of prokaryotic adaptive immunity

CRISPR/Cas is the prokaryotic adaptive immune response to viral invasion. Its mechanism is reminiscent of the eukaryotic RNA interference. The host actively incorporates short sequences from invading genetic elements (viruses or plasmids) into a region of its genome that is characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) and a number of CRISPR-associated (cas) genes. The molecular memory of previous infections can be transcribed and processed into small RNAs (crRNAs) that guide a multiprotein–nucleic acid interference complex to recognize and cleave incoming foreign genetic material. Three pathways (I, II, III) are defined by their protein machinery and target specificity (DNA vs. RNA). In types I and III, the main protagonist of the interference complex is the Cas7 protein. Up to six copies of Cas7 constitute the complex’s main building block that assembles around the crRNA and provides a platform for protein interactions and target binding. During my PhD work, I solved the crystal structures of two Cas7 orthologs from different archaeal species, at 1.8 Å for Thermofilum pendens (Tp) Csc2 and at 2.37 Å for Meth- anopyrus kandleri (Mk) Csm3. The crystal structures of Mk Csm3 and Tp Csc2 were solved by experimental phasing and revealed a core RRM-like domain with a β1-α1-β2-β3-α2-β4 arrangement of secondary structure elements. The core is flanked by three peripheral domains that are defined by insertions within the core. Structural superposition of the RRM-like core domains of Mk Csm3 and Tp Csc2 with the representatives of other Cas families (5/6/7) revealed the highest homology beyond the RRM with a Cas7 family homolog. Thus I showed that Cas7 family proteins share equivalent insertions, forming homologous peripheral domains. Using the information obtained from structural data, I investigated the RNA binding properties Mk Csm3, Tp Csc2 and a Cas7 protein from subtype I-A, Thermoproteus tenax (Tt) Csa2. All orthologs bound RNA in a sequence-independent manner, according to their physiological function of spacer binding. Furthermore, a combined approach consisting of mutation analysis, UV-based protein–RNA crosslinking, mass spectrometry and fluorescence anisotropy mapped the RNA interacting regions to two structurally highly conserved positively charged surfaces. Taken together, this thesis describes a comprehensive structural study of the Cas7 family, defining the family’s structural features. These structural data from single proteins and the mapped RNA binding interfaces agree with protein–RNA interactions observed in the Escherichia coli interference complex

The backbone of prokaryotic adaptive immunity

CRISPR/Cas is the prokaryotic adaptive immune response to viral invasion. Its mechanism is reminiscent of the eukaryotic RNA interference. The host actively incorporates short sequences from invading genetic elements (viruses or plasmids) into a region of its genome that is characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) and a number of CRISPR-associated (cas) genes. The molecular memory of previous infections can be transcribed and processed into small RNAs (crRNAs) that guide a multiprotein–nucleic acid interference complex to recognize and cleave incoming foreign genetic material. Three pathways (I, II, III) are defined by their protein machinery and target specificity (DNA vs. RNA). In types I and III, the main protagonist of the interference complex is the Cas7 protein. Up to six copies of Cas7 constitute the complex’s main building block that assembles around the crRNA and provides a platform for protein interactions and target binding. During my PhD work, I solved the crystal structures of two Cas7 orthologs from different archaeal species, at 1.8 Å for Thermofilum pendens (Tp) Csc2 and at 2.37 Å for Meth- anopyrus kandleri (Mk) Csm3. The crystal structures of Mk Csm3 and Tp Csc2 were solved by experimental phasing and revealed a core RRM-like domain with a β1-α1-β2-β3-α2-β4 arrangement of secondary structure elements. The core is flanked by three peripheral domains that are defined by insertions within the core. Structural superposition of the RRM-like core domains of Mk Csm3 and Tp Csc2 with the representatives of other Cas families (5/6/7) revealed the highest homology beyond the RRM with a Cas7 family homolog. Thus I showed that Cas7 family proteins share equivalent insertions, forming homologous peripheral domains. Using the information obtained from structural data, I investigated the RNA binding properties Mk Csm3, Tp Csc2 and a Cas7 protein from subtype I-A, Thermoproteus tenax (Tt) Csa2. All orthologs bound RNA in a sequence-independent manner, according to their physiological function of spacer binding. Furthermore, a combined approach consisting of mutation analysis, UV-based protein–RNA crosslinking, mass spectrometry and fluorescence anisotropy mapped the RNA interacting regions to two structurally highly conserved positively charged surfaces. Taken together, this thesis describes a comprehensive structural study of the Cas7 family, defining the family’s structural features. These structural data from single proteins and the mapped RNA binding interfaces agree with protein–RNA interactions observed in the Escherichia coli interference complex

DEA Window Analysis for Measuring Port Efficiencies in Serbia

The aim of the paper is to apply Data Envelopment Analysis (DEA) method in measuring and analyzing the efficiencies of ports on the river Danube. DEA window analysis is used to determine the efficiency of ports and to observe the possibility of changes in the port efficiency over time. A study is conducted to evaluate the efficiencies of ports on the territory of Serbia in order to identify the sources of inefficiencies and formulate proposals for improving the services of those ports and their operations through a four-year window analysis with port efficiency trends and average efficiencies. The progress is made in the measurement of port efficiency in relation to port productive activities - total area of warehouses, quay length, number of cranes and port throughput, for the Serbian river ports. Keywords: river ports, total area of warehouses, quay length, number of cranes, port throughput, port efficiency, DEA window analysis</p

Application of dea method to intermodal container transport

In order to make better assessment of intermodal container transportation, the authors have analyzed DEA (Data Envelopment Analysis) method in deciding the most favorable container line from Serbian ports to the near East ports. To allow for applications to a wide variety of activities, term organization unit (Decision Making Unit – DMU) refers to container line. DEA Method has been used to give the estimation of efficiency of operations in organization units. Organization units are container lines in which containers are loaded with mineral water in bottles. The main points in transportation net are factories of mineral water (Knjaz Milos Arandjelovac and Minaqua Novi Sad), Serbian ports ( the Port of Belgrade, the Port of Danube Pancevo and the Port of Prahovo) and nine Mediterranean ports in the near East area. Amount of mineral water that is dispatching from factories to Serbian ports is equal to amount that can be loaded to one container. Having containerized in Serbian ports mineral water is reloaded to the river–sea ships with unified dimensions, so the ships are continuing to travel to the near East ports. The near East ports are the ending points. Input variables in DEA problem are transportation costs and strategy resistance factor in function of container line. Time traveling of one container is in a function of container line and transportation capabilities of container lines are output variables. Transportation costs are counted on the base of integer programming method and Dijkstra algorithm

Changes of intracellular sodium and potassium ion concentrations in frog spinal motoneurons induced by repetitive synaptic stimulation

A post-tetanic membrane hyperpolarization following repetitive neuronal activity is a commonly observed phenomenon in the isolated frog spinal cord as well as in neurons of other nervous tissues. We have now used double-barrelled Na+- and K+-ion-sensitive microelectrodes to measure the intracellular Na+- and K+-concentrations and also the extracellular K+-concentration of lumbar spinal motoneurons during and after repetitive stimulation of a dorsal root. The results show that the posttetanic membrane hyperpolarization occurred at a time when the intracellular [Na+] reached its maximal value, intracellular [K+] had its lowest level and extracellular [K+] was still elevated. The hyperpolarization was blocked by ouabain and reduced by Li+. These data support the previous suggestion that an electrogenic Na+/K+ pump mode may be the mechanism underlying the post-tetanic membrane hyperpolarization

In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits

Programmable RNA Shredding by the Type III-A CRISPR-Cas System of Streptococcus thermophilus

Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA. Highlights • Streptococcus thermophilus type III-A Csm (StCsm) complex targets RNA •Multiple cuts are introduced in the target RNA at 6 nt intervals •Csm3 protein subunits are responsible for endoribonuclease activity of the complex •StCsm complex offers a programmable tool for RNA degradatio

Business Analytics in (a) Blink

The Blink project’s ambitious goal is to answer all Business Intelligence (BI) queries in mere seconds, regardless of the database size, with an extremely low total cost of ownership. Blink is a new DBMS aimed primarily at read-mostly BI query processing that exploits scale-out of commodity multi-core processors and cheap DRAM to retain a (copy of a) data mart completely in main memory. Additionally, it exploits proprietary compression technology and cache-conscious algorithms that reduce memory bandwidth consumption and allow most SQL query processing to be performed on the compressed data. Blink always scans (portions of) the data mart in parallel on all nodes, without using any indexes or materialized views, and without any query optimizer to choose among them. The Blink technology has thus far been incorp