CATS: linearizability and partition tolerance in scalable and self-organizing key-value stores

Distributed key-value stores provide scalable, fault-tolerant, and self-organizing storage services, but fall short of guaranteeing linearizable consistency in partially synchronous, lossy, partitionable, and dynamic networks, when data is distributed and replicated automatically by the principle of consistent hashing. This paper introduces consistent quorums as a solution for achieving atomic consistency. We present the design and implementation of CATS, a distributed key-value store which uses consistent quorums to guarantee linearizability and partition tolerance in such adverse and dynamic network conditions. CATS is scalable, elastic, and self-organizing; key properties for modern cloud storage middleware. Our system shows that consistency can be achieved with practical performance and modest throughput overhead (5%) for read-intensive workloads

Handling Network Partitions and Mergers in Structured Overlay Networks

Structured overlay networks form a major class of peer-to-peer systems, which are touted for their abilities to scale, tolerate failures, and self-manage. Any long-lived Internet-scale distributed system is destined to face network partitions. Although the problem of network partitions and mergers is highly related to fault-tolerance and self-management in large-scale systems, it has hardly been studied in the context of structured peer-to-peer systems. These systems have mainly been studied under churn (frequent joins/failures), which as a side effect solves the problem of network partitions, as it is similar to massive node failures. Yet, the crucial aspect of network mergers has been ignored. In fact, it has been claimed that ring-based structured overlay networks, which constitute the majority of the structured overlays, are intrinsically ill-suited for merging rings. In this paper, we present an algorithm for merging multiple similar ring-based overlays when the underlying network merges. We examine the solution in dynamic conditions, showing how our solution is resilient to churn during the merger, something widely believed to be difficult or impossible. We evaluate the algorithm for various scenarios and show that even when falsely detecting a merger, the algorithm quickly terminates and does not clutter the network with many messages. The algorithm is flexible as the tradeoff between message complexity and time complexity can be adjusted by a parameter

Thioester Synthesis By a Designed Nickel Enzyme Models Prebiotic Energy Conversion

The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetylCoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the NiI species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation

A Biochemical Nickel(I) State Supports Nucleophilic Alkyl Addition: A Roadmap for Methyl Reactivity in Acetyl Coenzyme A Synthase

Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic NiIII-CH3 species initially generated can be rapidly reduced to a high-spin NiII-CH3 species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. NiIII-CH3 Az is only the second example of two-electron addition of methyl iodide to a NiI center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving–Williams series (MnII ZnII). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving–Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine–valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate

Development and validation of a novel panel of 16 STR markers for simultaneous diagnosis of β-thalassemia, aneuploidy screening, maternal cell contamination detection and fetal sample authenticity in PND and PGD/PGS cases

Prenatal diagnosis (PND) may be complicated with sample mix-up; maternal cell contamination, non-paternity and allele drop out at different stages of diagnosis. Aneuploidy screening if combined with PND for a given single gene disorder, can help to detect any common aneuploidy as well as aiding sample authenticity and other probable complications which may arise during such procedures. This study was carried out to evaluate the effectiveness of a novel panel of STR markers combined as a multiplex PCR kit (HapScreen� kit) for the detection of β-thalassemia, aneuploidy screening, ruling in/out maternal cell contamination (MCC), and sample authenticity. The kit uses 7 STR markers linked to β-globin gene (HBB) as well as using 9 markers for quantitative analysis of chromosomes 21, 18, 13, X and Y. Selection of the markers was to do linkage analysis with β-globin gene, segregation analysis and to perform a preliminary aneuploidy screening of fetal samples respectively. These markers (linked to the β-globin gene) were tested on more than 2185 samples and showed high heterozygosity values (68.4�91.4). From 2185 fetal cases we found 3 cases of non-paternity, 5 cases of MCC, one case of sample mix-up and one case of trisomy 21 which otherwise may have end up to misdiagnosis. This kit was also successfully used on 231 blastomeres for 29 cases of pre-implantation genetic diagnosis (PGD) and screening (PGS). The markers used for simultaneous analysis of haplotype segregation and aneuploidy screening proved to be very valuable to confirm results obtained from direct mutation detection methods (i.e. ARMS, MLPA and sequencing) and aneuploidy screening. © 2019, The Author(s)

Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics

Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual B. subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade sporesâ peptidoglycan cortex. Conclusions from these measurements include: 1) CaDPA release from individual wild-type germinating spores was biphasic; in a first heterogeneous slow phase, Tlag, CaDPA levels decreased â ¼15% and in the second phase ending at Trelease, remaining CaDPA was released rapidly; 2) in L-alanine germination of wild-type spores and spores lacking SleB: a) the ESLI rose â ¼2-fold shortly before Tlag at T1; b) following Tlag, the ESLI again rose â ¼2-fold at T2 when CaDPA levels had decreased â ¼50%; and c) the ESLI reached its maximum value at â ¼Trelease and then decreased; 3) in CaDPA germination of wild-type spores: a) Tlag increased and the first increase in ESLI occurred well before Tlag, consistent with different pathways for CaDPA and L-alanine germination; b) at Trelease the ESLI again reached its maximum value; 4) in L-alanine germination of spores lacking both CLEs and unable to degrade their cortex, the time Î Trelease (Treleaseâ Tlag) for excretion of â ¥75% of CaDPA was â ¼15-fold higher than that for wild-type or sleB spores; and 5) spores lacking only CwlJ exhibited a similar, but not identical ESLI pattern during L-alanine germination to that seen with cwlJ sleB spores, and the high value for Î Trelease. Originally published Analytical Chemistry, Vol. 81, No. 10, May 200

Construction of copy number variation landscape and characterization of associated genes in a Bangladeshi cohort of neurodevelopmental disorders

Introduction: Copy number variations (CNVs) play a critical role in the pathogenesis of neurodevelopmental disorders (NDD) among children. In this study, we aim to identify clinically relevant CNVs, genes and their phenotypic characteristics in an ethnically underrepresented homogenous population of Bangladesh. Methods: We have conducted chromosomal microarray analysis (CMA) for 212 NDD patients with male to female ratio of 2.2:1.0 to identify rare CNVs. To identify candidate genes within the rare CNVs, gene constraint metrics [i.e., “Critical-Exon Genes (CEGs)”] were applied to the population data. Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) was followed in a subset of 95 NDD patients to assess the severity of autism and all statistical tests were performed using the R package. Results: Of all the samples assayed, 12.26% (26/212) and 57.08% (121/212) patients carried pathogenic and variant of uncertain significance (VOUS) CNVs, respectively. While 2.83% (6/212) patients’ pathogenic CNVs were found to be located in the subtelomeric regions. Further burden test identified females are significant carriers of pathogenic CNVs compared to males (OR = 4.2; p = 0.0007). We have observed an increased number of Loss of heterozygosity (LOH) within cases with 23.85% (26/109) consanguineous parents. Our analyses on imprinting genes show, 36 LOH variants disrupting 69 unique imprinted genes and classified these variants as VOUS. ADOS-2 subset shows severe social communication deficit (p = 0.014) and overall ASD symptoms severity (p = 0.026) among the patients carrying duplication CNV compared to the CNV negative group. Candidate gene analysis identified 153 unique CEGs in pathogenic CNVs and 31 in VOUS. Of the unique genes, 18 genes were found to be in smaller (<1 MB) focal CNVs in our NDD cohort and we identified PSMC3 gene as a strong candidate gene for Autism Spectrum Disorder (ASD). Moreover, we hypothesized that KMT2B gene duplication might be associated with intellectual disability. Conclusion: Our results show the utility of CMA for precise genetic diagnosis and its integration into the diagnosis, therapy and management of NDD patients