Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling.

The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling

Synthesis of Improved Catalytic Materials for High-Temperature Water-gas Shift Reaction

In this investigation, we report the preparation and characterization of Co-, Cu- and Mn-substituted iron oxide catalytic materials supported on activated carbon. Co-precipitation method and low temperature treatment were used for their synthesis. The influence of chemical composition, stoichiometry, particle size and dispersity on their catalytic activity was studied. Samples were characterized in all stages of their co-precipitation, heating and spend samples after catalytic tests. The obtained results from room and low temperature Mössbauer spectroscopy were combined with analysis of powder X-ray diffraction patterns (XRD). They revealed the preparation of nano-sized iron oxide materials supported on activated carbon. Relaxation phenomena were registered also for the supported phases. The catalytic performance in the water-gas shift reaction was studied. The activity order was as follows: Cu0.5Fe2.5O4 > Co0.5Fe2.5O4 > Mn0.5Fe2.5O4. Catalytic tests demonstrated very promising results and potential application of studied samples due to their cost-effective composition

Varijabilnost biohemijskih parametara krvnog seruma karakačanske ovce

Karakachan sheep represents an endangered, indigenous sheep breed from Balkan Peninsula. There is relatively little information about the characteristics of this sheep breed in the veterinary scientific literature. The aim of this research was an examination of certain metabolic profile parameters of the Karakachan sheep blood serum, and variability of their concentrations in comparison to age and some other indigenous sheep breeds from Balkans. Examination was conducted on 14 clinically healthy sheep divided in two age groups. Blood samples were collected by puncture of v. jugularis and blood serum was separated after spontaneous coagulation. The concentrations of total protein, albumin, calcium, inorganic phosphorus, aspartate amino transferase (AST) and γ- glutamyl transferase (GGT) were determined. In relation to age of Karakachan sheep, statistically significant difference between the calculated mean values of examined parameters was not observed. A statistically significant difference was found between the mean concentrations of the studied parameters in Karakachan sheep and other breeds in total protein (Tsigai, Dubrovnik and Dalmatian sheep), albumin (Dalmatian), calcium and inorganic phosphorus (Tsigai, Dubrovnik) and AST's (Dalmatian, Karakachan sheep from Bulgaria).Karakačanska ovca je ugrožena autohtona rasa ovaca sa Balkanskog poluostrva, o čijim rasnim karakteristikama postoji relativno malo podataka. Cilj ovog rada je bio da se u uzorcima krvnog seruma ispitaju određeni biohemijski parametri karakačanske ovce i ustanove odstupanja njihovih vrednosti u odnosu na starost karakačanske ovce, kao i u odnosu na vrednosti istih parametara kod drugih autohtonih rasa ovaca Balkana. Ispitivanjem je obuhvaćeno 14 klinički zdravih ovaca podeljenih u dve starosne grupe. Uzorci krvi uzimani su punkcijom v. jugularis iz kojih je, nakon spontane koagulacije i centrifugovanja, izdvojen krvni serum. Određivane su koncentracije ukupnih proteina, albumina, kalcijuma, neorganskog fosfora, aspartat amino transferaze (AST) i γ-glutamil transferaze (GGT). U odnosu na starost karakačanske ovce, nije ustanovljena statistički značajna razlika između izračunatih srednjih vrednosti ispitivanih parametara. Statistički značajna razlika je ustanovljena između srednjih vrednosti koncentracija ispitivanih parametara karakačanske ovce i drugih rasa za: ukupne proteine (cigaja, dubrovačka i dalmatinska ovca), albumin (dalmatinska), kalcijum i neorganski P (cigaja i dubrovačka) i aktivnosti AST-a (dalmatinska, karakačanska ovca iz Bugarske)

Electrical Charge State Manipulation of Single Silicon Vacancies in a Silicon Carbide Quantum Optoelectronic Device

Colour centres with long-lived spins are established platforms for quantum sensing and quantum information applications. Colour centres exist in different charge states, each of them with distinct optical and spin properties. Application to quantum technology requires the capability to access and stabilize charge states for each specific task. Here, we investigate charge state manipulation of individual silicon vacancies in silicon carbide, a system which has recently shown a unique combination of long spin coherence time and ultrastable spin-selective optical transitions. In particular, we demonstrate charge state switching through the bias applied to the colour centre in an integrated silicon carbide opto-electronic device. We show that the electronic environment defined by the doping profile and the distribution of other defects in the device plays a key role for charge state control. Our experimental results and numerical modeling evidence that control of these complex interactions can, under certain conditions, enhance the photon emission rate. These findings open the way for deterministic control over the charge state of spin-active colour centres for quantum technology and provide novel techniques for monitoring doping profiles and voltage sensing in microscopic devices

Identification of Genomic Loci Associated with Rhodococcus equi Susceptibility in Foals

Pneumonia caused by Rhodococcus equi is a common cause of disease and death in foals. Although agent and environmental factors contribute to the incidence of this disease, the genetic factors influencing the clinical outcomes of R. equi pneumonia are ill-defined. Here, we performed independent single nucleotide polymorphism (SNP)- and copy number variant (CNV)-based genome-wide association studies to identify genomic loci associated with R. equi pneumonia in foals. Foals at a large Quarter Horse breeding farm were categorized into 3 groups: 1) foals with R. equi pneumonia (clinical group [N = 43]); 2) foals with ultrasonographic evidence of pulmonary lesions that never developed clinical signs of pneumonia (subclinical group [N = 156]); and, 3) foals without clinical signs or ultrasonographic evidence of pneumonia (unaffected group [N = 49]). From each group, 24 foals were randomly selected and used for independent SNP- and CNV-based genome-wide association studies (GWAS). The SNP-based GWAS identified a region on chromosome 26 that had moderate evidence of association with R. equi pneumonia when comparing clinical and subclinical foals. A joint analysis including all study foals revealed a 3- to 4-fold increase in odds of disease for a homozygous SNP within the associated region when comparing the clinical group with either of the other 2 groups of foals or their combination. The region contains the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene, which is involved in neutrophil function. No associations were identified in the CNV-based GWAS. Collectively, these data identify a region on chromosome 26 associated with R. equi pneumonia in foals, providing evidence that genetic factors may indeed contribute to this important disease of foals.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Ocean-bottom seismographs based on broadband MET sensors: architecture and deployment case study in the Arctic

The Arctic seas are now of particular interest due to their prospects in terms of hydrocarbon extraction, development of marine transport routes, etc. Thus, various geohazards, including those related to seismicity, require detailed studies, especially by instrumental methods. This paper is devoted to the ocean-bottom seismographs (OBS) based on broadband molecular–electronic transfer (MET) sensors and a deployment case study in the Laptev Sea. The purpose of the study is to introduce the architecture of several modifications of OBS and to demonstrate their applicability in solving different tasks in the framework of seismic hazard assessment for the Arctic seas. To do this, we used the first results of several pilot deployments of the OBS developed by Shirshov Institute of Oceanology of the Russian Academy of Sciences (IO RAS) and IP Ilyinskiy A.D. in the Laptev Sea that took place in 2018–2020. We highlighted various seismological applications of OBS based on broadband MET sensors CME-4311 (60 s) and CME-4111 (120 s), including the analysis of ambient seismic noise, registering the signals of large remote earthquakes and weak local microearthquakes, and the instrumental approach of the site response assessment. The main characteristics of the broadband MET sensors and OBS architectures turned out to be suitable for obtaining high-quality OBS records under the Arctic conditions to solve seismological problems. In addition, the obtained case study results showed the prospects in a broader context, such as the possible influence of the seismotectonic factor on the bottom-up thawing of subsea permafrost and massive methane release, probably from decaying hydrates and deep geological sources. The described OBS will be actively used in further Arctic expeditions