The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30

The low efficiency of knock-in, especially in primary human cells, limits the use of genome editing technology for therapeutic purposes, which makes it important to develop approaches for increasing knock-in levels. In this work, using a knock-in model of the peptide fusion inhibitor of HIV MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line, we analyzed the effectiveness of several approaches to increasing knock-in levels. First, donor DNA modification aimed at improving the efficiency of plasmid transport into the nucleus was evaluated, namely the introduction into the donor plasmid of the SV40 DNA transport sequence (DTS) or the binding sites for the transcription factor NF-κB, whose effects on knock-in levels have not been described. In the MT-C34 knock-in model into the CXCR4 locus, this modification was ineffective. The second approach, modifying the Cas9 nuclease by introducing two additional nuclear localization signals (NLS), increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining pathway using DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. The combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the nonhomologous end joining pathway significantly increased the level of knock-in of the HIV-1 peptide fusion inhibitor into the clinically relevant locus CXCR4, which can be used to develop effective gene therapy approaches for the treatment of HIV infection.

To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo.

Gene editing using the CRISPR/Cas9 system provides new opportunities to treat human diseases. Approaches aimed at increasing the efficiency of genome editing are therefore important to develop. To increase the level of editing of the CXCR4 locus, which is a target for gene therapy of HIV infection, the Cas9 protein was modified by introducing additional NLS signals and ribonucleoprotein complexes of Cas9 and guide RNA were stabilized with poly-L-glutamic acid. The approach allowed a 1.8-fold increase in the level of CXCR4 knockout in the CEM/R5 T cell line and a 2-fold increase in the level of knock-in of the HIV-1 fusion peptide inhibitor MT-C34 in primary CD4+ T lymphocytes.

The Josephson junction is a building block of quantum circuits. Its behavior, well understood when treated as an isolated entity, is strongly affected by coupling to an electromagnetic environment. In 1983 Schmid predicted that a Josephson junction shunted by a resistance exceeding the resistance quantum $\mathbf{\textit{R}}_\mathrm{Q} = h/4e^2 \approx 6.45$ k$\mathbf{\Omega}$ for Cooper pairs would become insulating since the phase fluctuations would destroy the coherent Josephson coupling. Although this prediction has been confirmed in charge transport experiments, recent microwave measurements have questioned this interpretation. Here, we insert a small junction in a Johnson-Nyquist type setup, where it is driven by weak current noise arising from thermal fluctuations. Our heat probe minimally perturbs the junction's equilibrium, shedding light on features not visible in charge transport. We find that while charge transport through the junction is dissipative as expected, thermal transport is determined by the inductive-like Josephson response, unambiguously demonstrating that a supercurrent survives even deep into the expected insulating regime. The discrepancy between these two measurements highlights the difference between the low frequency and the high frequency response of a junction and calls for further theoretical and experimental inputs on the dynamics of Josephson junctions in a highly dissipative environment.

Nonlinear phenomena in superconducting resonator circuits are of great significance in the field of quantum technology. We observe thermal self-oscillations in a monolayer graphene flake coupled to Molybdenum-Rhenium superconducting resonator. The graphene flake forms a SINIS junction coupled to the resonator with strong temperature dependent resistance. In certain conditions of pump power and frequency, this nonlinearity leads to thermal self-oscillations appearing as sidebands in cavity transmission measurements with strong temperature dependence and gate tunability. The experimental observations fit well with theoretical model based on thermal instability. The modelling of the oscillation sidebands provides a method to evaluate electron phonon coupling in disordered graphene sample at low energies.

Generation of electric voltage in a conductor by applying a temperature gradient is a fundamental phenomenon called the Seebeck effect. This effect and its inverse is widely exploited in diverse applications ranging from thermoelectric power generators to temperature sensing. Recently, a possibility of thermoelectricity arising from the interplay of the non-local Cooper pair splitting and the elastic co-tunneling in the hybrid normal metal-superconductor-normal metal structures was predicted. Here, we report the observation of the non-local Seebeck effect in a graphene-based Cooper pair splitting device comprising two quantum dots connected to an aluminum superconductor and present a theoretical description of this phenomenon. The observed non-local Seebeck effect offers an efficient tool for producing entangled electrons.

The study was performed for miscanthus introduced and grown in West Siberia. Transformation of miscanthus into bacterial nanocellulose was accomplished for the first time. The microbiological synthesis of bacterial nanocellulose using the Medusomyces gisevii Sa-12 symbiotic culture gave chemically pure bacterial nanocellulose with exceptionally high crystallinity index and content of Iα allomorph.

The Seebeck effect producing voltage difference from temperature gradient has a wide spectrum of applications. Recent theoretical studies show that the Cooper pair splitting and the elastic co-tunneling can give rise to the nonlocal Seebeck effect in hybrid normal metal-superconductor-normal metal systems. Here we propose a coherent transport description of this nonlocal effect and validate its experimental observation in a graphene-based Cooper pair splitter.

Miscanthus introduced to and grown in West Siberia was used herein. The possibility of transformation of miscanthus into bacterial nanocellulose has been shown for the first time. It has been established that micro-biological synthesis of bacterial nanocellulose using a symbiotic culture of Medusomyces gisevii Sa-12 makes it possible to obtain chemically pure bacterial nanocellulose with exceptionally high values of the crystallinity index and the content of allomorph I  .

We study one-dimensional chains of superconducting islands with a particular emphasis on the regime in which every second island is switched into its normal state, thus forming a superconductor-insulator-normal metal (S-I-N) repetition pattern. As is known since Giaever tunneling experiments, tunneling charge transport between a superconductor and a normal metal becomes exponentially suppressed, and zero-bias resistance diverges, as the temperature is reduced and the energy gap of the superconductor grows larger than the thermal energy. Here we demonstrate that this physical phenomenon strongly impacts transport properties of inhomogeneous superconductors made of weakly coupled islands with fluctuating values of the critical temperature. We observe a nonmonotonous dependence of the chain resistance on both temperature and magnetic field, with a pronounced resistance peak at temperatures at which some but not all islands are superconducting. We explain this phenomenon by the inhomogeneity of the chains, in which neighboring superconducting islands have slightly different critical temperatures. We argue that the Giaever's resistance divergence can also occur in the zero-temperature limit. Such quantum transition can occur if the magnetic field is tuned such that it suppresses superconductivity in the islands with the weaker critical field, while the islands with stronger energy gap remain superconducting. In such a field, the system acts as a chain of S-I-N junctions.

The performance of low temperature detectors utilizing thermal effects is determined by their energy relaxation properties. Usually, heat transport experiments in mesoscopic structures are carried out in the steady-state, where temperature gradients do not change in time. Here, we present an experimental study of dynamic thermal relaxation in a mesoscopic system – thin metallic film. We find that the thermal relaxation of hot electrons in copper and silver films is characterized by several time constants, and that the annealing of the films changes them. In most cases, two time constants are observed, and we can model the system by introducing an additional thermal reservoir coupled to the film electrons. We determine the specific heat of this reservoir and its coupling to the electrons. The experiments point at the importance of grain structure on the thermal relaxation of electrons in metallic films.

Бактериальная наноцеллюлоза (БНЦ) обладает уникальными физико-химическими свойствами и является многообещающим материалом для широкого круга применения. Целью данной работы является исследование зависимости физико-механических характеристик образцов БНЦ, синтезированных продуцентом Medusomyces gisevii Sa-12, от способа получения питательных сред – ферментативных гидролизатов из плодовых оболочек овса. Образцы БНЦ были высушены на воздухе. Термогравиметрический анализ образцов БНЦ проведён на термогравиметрическом анализаторе ДТЖ-60; физико-механический анализ – на термомеханическом анализаторе ТМА-60. Методом термогравиметрического анализа показано, что в исследуемых образцах температура начала пиролиза варьирует от 268 °С до 334 °С, при этом при пиролизе теряется от 52 % до 78 %, чем выше данные показатели, тем чище образцы БНЦ и выше их термостабильность. Методом испытания образцов БНЦ на растяжение установлено, что механические свойства полимера существенно зависят от способа получения субстрата для ферментативного гидролиза и последующего биосинтеза БНЦ на полученной питательной среде. По модулю Юнга образцы БНЦ в зависимости от способа получения питательной среды из плодовых оболочек овса отличаются в 13,4 раза: от 933,3 МПа до 69,8 МПа; при этом, чем выше модуль Юнга, тем меньше величина относительного удлинения при пределе текучести, а его значения изменяются в 8,8 раза: от 0,6 до 5,3 %.

We observe a crossover fromelectron-phonon (e-ph) coupling limited energy relaxation to that governed by thermal boundary resistance (phonon-phonon coupling, ph-ph) in copper films at subkelvin temperatures. Our measurement yields a quantitative picture of heat currents, in terms of temperature dependences and magnitudes, in both e-ph and pp limited regimes, respectively. We show that by adding a third layer in between the copper film and the substrate, the thermal boundary resistance is increased fourfold, consistent with an assumed series connection of thermal resistances.

The development of quantum limited magnetic flux sensors has recently gained a lot of attention for the possibility of detecting the magnetic moment of nanoscaled systems. Here, the ultimate goal is the observation of a single spin. Such sensors are of fundamental importance for applications, ranging from spintronics and spin-based quantum information processing, to fundamental studies of nano-magnetism in molecules and magnetic nanoclusters. A nano-scale superconducting quantum interference device (nanoSQUID) is indeed a promising candidate to reach this ambitious goal. Nanowires, fabricated of high critical temperature superconductors (HTS), have been shown to be a valid candidate for the realization of nanoSQUIDs. A crucial requirement to achieve the necessary flux sensitivity and spatial resolution, is a SQUID loop on the nanometer scale. Moreover, HTS nanowire-based SQUIDs in combination with large area pickup loops or flux transformers might become instrumental in magnetometer applications, such as magneto encephalography and low field magnetic resonance imaging, where low intrinsic magnetic field noise is required. In this review we will give a survey on the state of the art of YBa2Cu3O7−δ thin film nanowires and their implementation in low noise nanoSQUIDs and magnetometers.

Bacterial cellulose (BC) finds multiple applications due to unique physicochemical properties and biocompatibility. The mechanical characteristics of hydrated BC such as Young modulus, tear strength, and tensile elongation under maximum load are crucial in some instances. The diversity of test methods does not allow correct comparison of the results of BC thermomechanical analysis (TMA) obtained by different researchers. However, current standards for determination of the mechanical characteristics are not intended for highly hydrated samples and do not take into account conditions for their use. The goal of the study is to develop a tensile test method for hydrated gel films of bacterial cellulose and to compare their relative elongation when tested in air and in an aqueous medium. Test samples were produced in a synthetic nutrient broth using Medusomyces gisevii Sa-12 symbiont. Physico-mechanical analysis was performed on a TMA-60 thermomechanical analyzer. The loading rate was selected proceeding from the requirement that the specimen will not dry out when tested in air. The microfibrillar structure of BC samples was studied before and after stretching using scanning electron microscope (JSM-840). The results showed that at different loading rate, tensile strength varies by a factor of 16, Young’s modulus, and elongation at maximum load by a factor of 1.3 and 1.5, respectively. The maximum tensile elongation of hydrated BC in an aqueous medium (51.4%) is 3.1 times larger compared to that determined for the test specimen tested in air. The recommended loading rate is 20 g/min. The BC structure changes during tension: after testing the BC fibers line up along the load vector and thus structured bacterial cellulose acquires the anisotropic properties.

Bacterial nanocellulose (BNC) samples were produced by the symbiotic culture Мedusomyces gisevii Sa-12 in synthetic nutrient medium and enzymatic hydrolysates of chemically treated oat hulls and studied by X-ray diffraction. The structural characteristics of the crystalline component of BNC samples were determined. A comparison of the X-ray diffraction patterns recorded in reflection and transmission geometry shows that the samples have an anisotropic structure. All BNC samples are characterized by a high degree of crystallinity (from 86 to 93%) and are mainly composed of the low-symmetry metastable phase Iα (its fraction is 93.6–100%). The Мedusomyces gisevii Sa-12 culture was found to produce highly crystalline BNC, with the low-symmetry phase Iα prevailing, regardless of the composition of the nutrient medium.

In this paper, we demonstrate that the hybrid normal-superconducting-normal (NSN) structure has potential for a multifunctional thermal device which could serve for heat flux control and cooling of microstructures. By adopting the scattering matrix approach, we theoretically investigate thermal and electrical effects emerging in such structures due to the Cooper pair splitting (CPS) and elastic cotunneling phenomena. We show that a finite superconductor can, in principle, mediate heat flow between normal leads, and we further clarify special cases when this seems contradictory to the second law of thermodynamics. Among other things, we demonstrate that the CPS phenomenon can appear even in the simple case of a ballistic NSN structure.