Acceleration of Antimicrobial Susceptibility Testing of Positive Blood Cultures by Inoculation of Vitek 2 Cards with Briefly Incubated Solid Medium Cultures

Briefly incubated agar cultures from positive blood cultures were used for antimicrobial susceptibility testing (AST) by Vitek 2. The cultivation time until inoculation was 3.8 h for Gram-positive cocci and 2.4 h for Gram-negative rods. The error rates were low, providing early and reliable AST without additional time or cost expenditure.     

New poly-imidazolium–triazole particles by CuAAC cross-linking of calix[4]arene bis-azide/alkyne amphiphiles – a prospective support for Pd in the Mizoroki–Heck reaction

A new imidazolium amphiphilic calix[4]arene with terminal acetylene fragments in the polar region was synthesized according to a two step scheme including regioselective chloromethylation of distal di-O-butyl calix[4]arene and subsequent interaction with 1-(hex-5-yn-1-yl)-1H-imidazole. The aggregation properties (CAC, the size and zeta potential of aggregates) of alkynyl calix[4]arene as well as of previously synthesized azidopropyl calix[4]arene and their 1 : 1 mixture were disclosed. Macrocycles with azide and alkyne fragments in the polar region were covalently cross-linked under CuAAC conditions in water. Successful cross-linking of molecules has been proven by IR spectroscopy and MALDI-TOF spectrometry. The obtained polymeric particles were studied both in solution and the solid state and the presence of submicron (∼200 nm) and micron (∼1–5 μm) particles with the prevalence of the latter was found. The average molecular weight of the polymer according to the static light scattering data was found to be 639 ± 44 kDa. The obtained polymeric imidazolium–triazole particles were tested as a support for Pd(OAc)₂ in the Mizoroki–Heck reaction carried out in both organic and water media. In both solvents (especially in water) the addition of imidazolium–triazole particles to Pd(OAc)₂ increased the conversion of 4-iodanisole. It was found that the ratio between the products (1,1 and 1,2-substituted ethylenes) changes drastically on going from DMF to water from 1 : 5 to 1 : 40 when using supported Pd(OAc)₂.

A new supramolecular approach to the formation of polytriazole–imidazolium particles, promising supports for catalysis, based on self-assembly of amphiphilic bis-azides and bis alkynes and their linkage using CuAAC is presented.     

Linked versus unlinked estimates of mortality and length of life by education and marital status: evidence from the first record linkage study in Lithuania

Earlier studies have found large and increasing with time differences in mortality by education and marital status in post-Soviet countries. Their results are based on independent tabulations of population and deaths counts (unlinked data). The present study provides the first census-linked estimates of group-specific mortality and the first comparison between census-linked and unlinked mortality estimates for a post-Soviet country. The study is based on a data set linking 140,000 deaths occurring in 2001-2004 in Lithuania with the population census of 2001. The same socio-demographic information about the deceased is available from both the census and death records. Cross-tabulations and Poisson regressions are used to compare linked and unlinked data. Linked and unlinked estimates of life expectancies and mortality rate ratios are calculated with standard life table techniques and Poisson regressions. For the two socio-demographic variables under study, the values from the death records partly differ from those from the census records. The deviations are especially significant for education, with 72-73%, 66-67%, and 82-84% matching for higher education, secondary education, and lower education, respectively. For marital status, deviations are less frequent. For education and marital status, unlinked estimates tend to overstate mortality in disadvantaged groups and they understate mortality in advantaged groups. The differences in inter-group life expectancy and the mortality rate ratios thus are significantly overestimated in the unlinked data. Socio-demographic differences in mortality previously observed in Lithuania and possibly other post-Soviet countries are overestimated. The growth in inequalities over the 1990s is real but might be overstated. The results of this study confirm the existence of large and widening health inequalities but call for better data.     

Clusters of alpha satellite on human chromosome 21 are dispersed far onto the short arm and lack ancient layers

Human alpha satellite (AS) sequence domains that currently function as centromeres are typically flanked by layers of evolutionarily older AS that presumably represent the remnants of earlier primate centromeres. Studies on several human chromosomes reveal that these older AS arrays are arranged in an age gradient, with the oldest arrays farthest from the functional centromere and arrays progressively closer to the centromere being progressively younger. The organization of AS on human chromosome 21 (HC21) has not been well-characterized. We have used newly available HC21 sequence data and an HC21p YAC map to determine the size, organization, and location of the AS arrays, and compared them to AS arrays found on other chromosomes. We find that the majority of the HC21 AS sequences are present on the p-arm of the chromosome and are organized into at least five distinct isolated clusters which are distributed over a larger distance from the functional centromere than that typically seen for AS on other chromosomes. Using both phylogenetic and L1 element age estimations, we found that all of the HC21 AS clusters outside the functional centromere are of a similar relatively recent evolutionary origin. HC21 contains none of the ancient AS layers associated with early primate evolution which is present on other chromosomes, possibly due to the fact that the p-arm of HC21 and the other acrocentric chromosomes underwent substantial reorganization about 20 million years ago.     

Characterization of Clinical Cases of Advanced Papillary Renal Cell Carcinoma via Comprehensive Genomic Profiling

Background

Papillary renal cell carcinoma (PRCC) is a rare subset of RCC. The Cancer Genome Atlas (TCGA) data largely reflect localized disease, and there are limited data for advanced PRCC.

Early spectral changes of cellular malignant transformation using Fourier transform infrared microspectroscopy

Fourier transform infrared microspectroscopy (FTIR-MSP) is potentially a powerful analytical method for identifying the spectral properties of biological activity in cells. The goal of the present research is the implementation of FTIR-MSP to study early spectral changes accompanying malignant transformation of cells. As a model system, cells in culture are infected by the murine sarcoma virus (MuSV), which induces malignant transformation. The spectral measurements are taken at various postinfection time intervals. To follow up systematically the progress of the spectral changes at early stages of cell transformation, it is essential first to determine and validate consistent and significant spectral parameters (biomarkers), which can evidently discriminate between normal and cancerous cells. Early stages of cell transformation are classified by an array of spectral biomarkers utilizing cluster analysis and discriminant classification function techniques. The classifications indicate that the first spectral changes are detectable much earlier than the first morphological signs of cell transformation. Our results point out that the first spectral signs of malignant transformation are observed on the first and third day of postinfection (PI) (for NIH/3T3 and MEF cell cultures, respectively), while the first visible morphological alterations are observed only on the third and seventh day, respectively. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent-free method for early detection of malignancy.     

Correction to: Clinical-Dynamic Features of Affective Disorders Comorbid with Alcohol Dependence

International Journal of Mental Health and Addiction - The following Acknowledgement is missing from this article as published.     

Genotoxic effect of 4-aroyl-1-(2-chloroethyl)- 1-nitrosohydrazinecarboxamides on Saccharomyces cerevisiae cells

The study of some 4-aroyl-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamides with a Saccharomyces cerevisiae mutagenicity test of increased sensitivity defined two of them, 4-(4-bromobenzoyl)-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamide and 4-(4-fluorophenyl)-1-(2-chloroethyl)-1-nitrosohydrazine carboxamide as typical cytostatic agents. At concentrations of 2–5 μg/ml the substances kill up to 60%–70% of cells without having any detectable recombinogenic and mutagenic effects. At the same concentrations, lomustine, well known as a cytostatic reference, demonstrated recombinogenic and mutagenic activity on yeast cells. The advantage of the newly synthesized substances is that, in a certain concentration range, their biological activity is mainly cytotoxic without induction of recombinogenic and mutagenic events in surviving cells. Received: 25 February 1997 / Accepted: 14 January 1998     

Prespacers formed during primed adaptation associate with the Cas1–Cas2 adaptation complex and the Cas3 interference nuclease–helicase

For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease–helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1–Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1–Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.

CRISPR-Cas systems of adaptive immunity provide prokaryotes with resistance against bacteriophages and plasmids (1–4). They consist of CRISPR DNA arrays and cas genes. Functionally, CRISPR defense can be subdivided into the interference and adaptation steps. The interference step involves specific recognition of regions in foreign nucleic acids, named protospacers, based on their complementarity to CRISPR arrays spacers followed by their destruction (5). The CRISPR adaptation step leads to integration of new spacers into the array (6, 7), forming inheritable memory that allows the entire lineage of cells derived from a founder that acquired a particular spacer to do away with genetic invaders carrying matching protospacers (8).Both interference and adaptation can be subdivided into multiple steps. For interference to occur, the CRISPR array is transcribed from a promoter located in the upstream leader region. The resulting pre-CRISPR RNA (pre-crRNA) is processed into short CRISPR RNAs (crRNAs), each containing a spacer flanked by repeat fragments (9). Individual crRNAs are bound by Cas proteins forming the effector complex, which is capable of recognizing sequences complementary to the spacer part of crRNA (10). Upon protospacer recognition, the target is destroyed either by a protein component of the effector complex or by additional recruitable Cas nucleases (3, 11–14). In a well-studied Type I-E CRISPR-Cas system of Escherichia coli, the effector comprises a multisubunit Cascade protein complex bound to a crRNA (11, 12, 15). The complementary interaction of Cascade-bound crRNA with a target protospacer leads to localized protospacer DNA melting and formation of an R-loop complex, where the crRNA spacer is annealed to the protospacer “target” strand, while the opposing “nontarget” strand is displaced and is present in a single-stranded form (16, 17). To avoid potentially suicidal recognition of CRISPR array spacers from which crRNAs originate, target recognition and R-loop complex formation require, in addition to complementarity with the crRNA spacer, the presence of a three-nucleotide long PAM (protospacer adjacent motif) preceding the protospacer (15, 18, 19). For E. coli type I-E system, the consensus PAM sequence is 5′-AAG-3′ on the nontarget strand. Some other trinucleotides also allow target recognition, though with decreased efficiency (15, 20). Below, we will refer to consensus PAM as “PAM^AAG.” The Cas3 nuclease-helicase is recruited to the R-loop complex and is responsible for target destruction (21–24). Cas3 first introduces a single-stranded break in the nontarget protospacer strand 11 to 15 nucleotides downstream of the PAM on the nontarget strand (25). Next, Cas3 unwinds and cleaves DNA in the 3′-5′ direction from the PAM (26–29). In vitro, Cas3-dependent degradation of DNA at the other side of the protospacer was also detected (16). Bidirectional Cas3-dependent degradation of DNA was also detected in vivo (30). The details of Cas3 “molecular gymnastics” required for such bidirectional destruction of DNA around the R-loop complex are not known.The main proteins of CRISPR adaptation are Cas1 and Cas2. In vitro, these proteins interact with each other, and the resulting complex is capable of inserting spacer-sized fragments in substrate DNA molecules containing at least one CRISPR repeat and a repeat-proximal leader region (31, 32). In the course of spacer integration, the Cas1–Cas2 complex first catalyzes a direct nucleophilic attack by the 3′-OH end of the incoming spacer at a phosphodiester bond between the leader and the first repeat in the top CRISPR strand (32, 33). This reaction proceeds via concurrent cleavage of the leader-repeat junction and covalent joining of one spacer strand to the 5′ end of the repeat. Subsequently, the 3′-OH on the second spacer strand attacks the phosphodiester bond at the repeat-spacer junction in the bottom CRISPR strand leading to full integration (32, 33). As a result, an intermediate with the newly incorporated spacer flanked by single-stranded repeat sequences is formed (32, 34). The gaps are filled in by a DNA polymerase, possibly DNA polymerase I (35).When overexpressed, E. coli Cas1 and Cas2 can integrate new spacers into the array in the absence of other Cas proteins (7, 36). During such “naive” adaptation, ∼50% of newly acquired spacers are selected from sequences flanked by the 5′-AAG-3′ trinucleotide, that is, consensus interference-proficient PAM^AAG. It thus follows that at least 50% of spacers acquired by Cas1 and Cas2 alone will be defensive during the interference step. The adaptation process must be tightly controlled, activated in the presence of the infecting mobile genetic elements, and directed toward foreign DNA, for otherwise, spacers acquired from host DNA will lead to suicidal self-interference. The details of the activation of CRISPR adaptation upon the entry of foreign DNA into the cell remain elusive. Some data indicate that active replication and/or a small size of phage or plasmid DNA may be responsible for a preferential selection of spacers from these molecules compared to selection of self-targeting spacers from host chromosomes (19). In addition, DNA repair/recombination signals present in host DNA, but lacking in foreign DNA may also increase the bias of the adaptation machinery to the latter (37).The bias of spacer acquisition machinery toward foreign DNA does not have to be significant, for acquisition of a self-targeting spacer by an infected cell will lead to the demise of such a cell in an act of altruism that would help control the spread of the infectious agent through the population. In contrast, acquisition of interference-proficient spacers from foreign DNA may allow the infected cell to survive, clear the infection, and endow its progeny with inheritable resistance—clearly an advantageous trait.To overcome CRISPR resistance, viruses and plasmids accumulate “escaper” mutations in the targeted protospacer or its PAM (36, 38). Given that the acquisition of protective spacers in infected cells is likely to be a rare event and the ease with which escaper mutations accumulate, the complex multistage CRISPR defense could become costly and ineffective (39). To increase the efficiency of CRISPR defense and counter the spread of mobile genetic elements with escaper mutations, CRISPR-Cas systems have evolved a specialized mode of spacer acquisition referred to as “primed adaptation” or “priming” (36, 40–47). Unlike the naive adaptation, in Type I CRISPR-Cas systems, priming requires, in addition to Cas1 and Cas2, a Cascade charged with crRNA recognizing the foreign target and the Cas3 nuclease–helicase. Spacers acquired during priming originate almost exclusively from DNA located in cis with the protospacer initially recognized by the effector complex (referred to hereafter as the “priming protospacer” or “PPS”). Furthermore, 90% or more of spacers acquired during priming by the I-E system of E. coli originate from protospacers with PAM^AAG and are therefore capable of efficient interference. Another hallmark of primed adaptation is the following: spacers acquired from DNA located at different sides of the PPS map to opposite DNA strands. The mapping of spacers acquired during naive adaptation shows no strand bias (48). Thus, the strand bias of spacers acquired during priming is probably related to Cas3 nuclease activity; however, exact details are lacking.The overall yield of spacers acquired during priming is increased when the PPS is imperfectly matched with a Cascade-bound crRNA spacer or when the PAM of the PPS is suboptimal (49). Thus, escaper protospacers serve as PPS, and priming initiated by inefficient recognition of such protospacers allows cells to quickly update their immunological memory by specific and efficient acquisition of additional interference-proficient spacers from mobile genetic elements that accumulated escaper mutations to earlier acquired spacers.The exact molecular mechanism of primed adaptation is not fully understood. Clearly, it should involve tight coordination between suboptimal interference against escaper targets and the spacer acquisition process. The DNA fragments produced by Cas3, a nuclease responsible for target degradation during interference, may feed primed adaptation, directly or indirectly, providing a functional link between the interference and adaptation arms of the CRISPR-Cas response. Based on results of in vitro experiments, it has been proposed that Cas3-generated degradation products may be used as substrates for the generation of prespacers (50)—DNA fragments that can be incorporated by the Cas1–Cas2 complex into arrays. However, no Cas3-generated products were detected in cells undergoing interference only, suggesting that Cas3 may degrade DNA to very short, subspacer length products (30). On the other hand, mutations abolishing the Cas3 nuclease activity lead to very little primed adaptation, indicating that priming requires the Cas3 nuclease activity (51). A possible way out from this impasse would be the existence of a “priming complex” that includes both Cas1–Cas2 and Cas3 and is responsible for the generation of prespacers by the Cas1–Cas2 complex from DNA along which Cas3 moves. Single-molecule analysis supports the existence of such a complex and even suggests that PPS-bound Cascade may be part of the priming complex (52). Here, we show that both Cas1–Cas2 and Cas3 associate with the same set of prespacers in cells undergoing primed adaptation, functionally linking CRISPR interference and adaptation machineries during priming. We also investigate the phenomenon of strand bias of spacer acquisition during priming and show that this bias does not depend on the orientation of PPS.