Digital Isothermal Quantification of Nucleic Acids via Simultaneous Chemical Initiation of Recombinase Polymerase Amplification Reactions on SlipChip

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for “yes or no” digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37−42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications

Time course of lymphocyte profile after femoral bone fracture

Isolated fractures of femur account for more than 10% of all road traffic injuries. Traumatic injury of femoral bone triggers a cascade of interrelated neuroendocrine reactions at systemic level, primarily at the hypothalamic-pituitary-adrenal axis, systemic response of immune system, initiated by release of tissue degradation products, cytokines and other mediators of damage into systemic blood circulation. Specific cellular reactions in response to traumatic injury to bone tissue include both innate and adaptive immune responses. In this regard, there is still scarce information on changes in blood lymphocyte subpopulations observed after closed isolated fracture of the femoral diaphysis at the middle third, before and after surgery. The aim of the present study was to evaluate the subpopulations of peripheral blood lymphocytes following closed isolated fracture of the femoral diaphysis with bone displacement in thecourse dynamics of surgical treatment, thus being required for studies in pathogenesis, development of diagnostic criteria and creating innovative treatment approaches. The study included 20 apparently healthy men and 36 men with closed isolated fracture of the femoral diaphysis of the middle third (32A and 32B, by AO/ASIF clinical classification, coded according to ICD-10 S72.3). The exclusion criteria were as follows: exacerbation of chronic comorbidities, diseases of lymphatic system and haematopoietic organs, oncological diseases, and evidence of osteoporosis. The spectrum of blood lymphocyte subsets was assessed on days 5, 7 (immediately after surgery) and on day 18 after closed isolated fracture of femoral diaphysis. We have found that, on the day 5 after IPBC along with leukocytosis in peripheral blood, the number of T-regulatory cells, cells with markers of early (CD25+) and late activation (HLA-DR+) proved to be increased, whereas representation of NK cells was decreased. On the day 7 after IPBC and immediately after surgery, leukocytosis persisted in blood, along with increased number of T-regulatory cells, CD3+ cells with early and late activation markers. On the day 18 after closed isolated fracture of the femoral diaphysis, the total numbers of leukocytes, T-lymphocytes, T-helpers, T-regulatory cells, T cells with an early activation marker are restored in peripheral blood, whereas the number of T-lymphocytes expressing HLA-DR+ molecules showed a significant increase

New materials based on polylactide modified with silver and carbon ions

An integrated study of poly-L-lactide (PL) synthesis and the physicochemical properties of film surfaces, both modified by silver and carbon ion implantation and also unmodified PL surfaces, has been carried out. Surface modification was done using aMevva-5.Ru metal ion source with ion implantation doses of 1·1014, 1·1015 and 1·1016 ion/cm2. Material characterization was done using NMR, IRS, XPS and AFM. The molecular weight (MW), micro-hardness, surface resistivity, and limiting wetting angle of both un-implanted and implanted samples were measured. The results reveal that degradation of PL macromolecules occurs during ion implantation, followed by CO or CO2 removal and MW decrease. With increasing implantation dose, the glycerol wettability of the PL surface increases but the water affinity decreases (hydrophobic behavior). After silver and carbon ion implantation into the PL samples, the surface resistivity is reduced by several orders of magnitude and a tendency to micro-hardness reductionis induced

Lymphocyte apoptosis and immune response in patients with drug-resistant fibro-cavernous tuberculosis with different prevalence of destructive changes in the lungs

Disturbances of programmed cell death are at the heart of many immunopathological processes in tuberculosis. The relationship between activity of apoptosis and severity of immune response is of particular interest in the patients with fibrous-cavernous drug-resistant pulmonary tuberculosis at different extent of the process. The paper concerns features of apoptosis, proliferative activity of lymphocytes, cytokine’s production and subpopulation composition of peripheral blood lymphocytes in the patients with uni- and bilateral fibrous-cavernous drug-resistant pulmonary tuberculosis. It was shown that apoptotic rates in the examined patients is closely related to extent of pathological process. Extent of early and late apoptosis and, accordingly, the number of living cells reflected the progression degree of destructive process in the lungs affected by fibrous-cavernous tuberculosis. The possibility of predicting the extent of destructive changes in affected lungs based on expression of apoptosis markers is presumed. Index of activity for early apoptosis of T lymphocytes, exceeding normal values by 25% and higher were clinically significant. A clear relationship between the immune response and apoptosis level was revealed. Ambiguous changes of immunological parameters were shown with increasing apoptosis associated with the severity of destructive changes. Increased apoptotic cell death in all patients with fibrous-cavernous tuberculosis, regardless of extent of the process, was associated with inhibition of antigen-specific proliferative response, decrease in CD25+ lymphocytes, increased numbers of B cells, along with decreased production of IFNγ, IL-8, and increased IL-2 response to PPD. In cases of unilateral destruction, increased apoptotic rates were accompanied by a decrease in the CD95+ cell numbers, and a decrease in TNFα production. On the contrary, in patients with bilateral destruction it was characterized by a high content of CD95+ lymphocytes, increased production of TNFα and IL-10. An index of extremely unfavorable course of the process is a combination of high apoptosis levels and low antigen-specific response with low expression of CD25+ cells, increased number of CD19+ and CD95+ lymphocytes, decreased production of IFNγ, IL-8 and increased production of IL-2, TNFα, IL-10. The relationships found in the work indicate that the combined assessment of apoptosis indexes, together with immunological parameters, has a higher informative value when assessing the state of immunocompetent cells, the origin of the process and trends for its development. Detecting the features of programmed lymphocyte death, in conjunction with immune parameters, allows to evaluate the role of apoptosis in each single case and to predict the course of the process, with subsequent justification of immunotherapy administration

The influence of a combined strain-heat treatment on the features of electromagnetic testing of fatigue degradation of quenched constructional steel

The possibilities of the magnetic and eddy-current methods for testing fatigue degradation during low-cycle loading of quenched steel 50 (0.51% C) that was subjected to a combined strain-heat treatment according to an optimal regime that included friction treatment with subsequent tempering at T = 350 C, were investigated. It is shown that for steel that was subjected to a combined nanostructuring treatment, the accumulation of a plastic strain under "hard" cyclic loading can be tested using the coercimetric method and values of the residual magnetic induction on the major and minor magnetic-hysteresis loops, values of the maximum and initial magnetic permeabilities, and readings of an eddy-current instrument at a low excitation frequency of the eddy-current transducer. The appearance of surface fatigue cracks can be tested via eddy-current measurements at high frequencies, when the contribution of the crack formation in the hardened layer to the eddy-current characteristics is considerable. © 2013 Pleiades Publishing, Ltd

Eddy-current testing of fatigue degradation upon contact fatigue loading of gas powder laser clad NiCrBSi-Cr 3 C 2 composite coating

The possibilities of the eddy-current method for testing the fatigue degradation under contact loading of gas powder laser clad NiCrBSi-Cr 3 C 2 composite coating with 15 wt.% of Cr 3 C 2 additive have been investigated. It is shown that the eddy-current testing of the fatigue degradation under contact loading of the NiCrBSi-15%Cr 3 C 2 composite coating can be performed at high excitation frequencies 72-120 kHz of the eddy-current transducer. At that, the dependences of the eddy-current instrument readings on the number of loading cycles have both downward and upward branches, with the boundary between the branches being 3×10 5 cycles in the given loading conditions. This is caused, on the one hand, by cracking, and, on the other hand, by cohesive spalling and compaction of the composite coating, which affect oppositely the material resistivity and, correspondingly, the eddy-current instrument readings. The downward branch can be used to monitor the processes of crack formation and growth, the upward branch - to monitor the degree of cohesive spalling, while taking into account in the testing methodology an ambiguous character of the dependences of the eddy-current instrument readings on the number of loading cycles. © 2017 Author(s)

Recent advances in genetics of aggressive behavior

One of the most important problems of modern neurobiology and medicine is an understanding of the mechanisms of normal and pathological behavior of a person. Aggressive behavior is an integral part of the human psyche. However, environmental risk factors, mental illness and somatic diseases can lead to increased aggression to be the biological basis of antisocial behavior in a human society. An important role in development of aggressive behavior belongs to the hereditary factors that may be linked to abnormal functioning of neurotransmitter systems in the brain yet the underlying genetic mechanisms remain unclear, which is due to a large number of single nucleotide polymorphisms, insertions and deletions in the structure of genes that encode the components of the neurotransmitter systems. The most studied candidate genes for aggressive behavior are serotonergic (TPH1, TPH2, HTR2A, SLC6A4) and dopaminergic (DRD4, SLC6A3) system genes, as well as the serotonin or catecholamine metabolizing enzyme genes (COMT, MAOA). In addition, there is evidence that the hypothalamic-pituitary system genes (OXT, OXTR, AVPR1A, AVPR1B), the sex hormone receptors genes (ER1, AR), neurotrophin (BDNF) and neuronal apoptosis genes (CASP3, BAX) may also be involved in development of aggressive behavior. The results of Genome-Wide Association Studies (GWAS) have demonstrated that FYN, LRRTM4, NTM, CDH13, DYRK1A and other genes are involved in regulation of aggressive behavior. These and other evidence suggest that genetic predisposition to aggressive behavior may be a very complex process

Collapse arrest and soliton stabilization in nonlocal nonlinear media

We investigate the properties of localized waves in systems governed by nonlocal nonlinear Schrodinger type equations. We prove rigorously by bounding the Hamiltonian that nonlocality of the nonlinearity prevents collapse in, e.g., Bose-Einstein condensates and optical Kerr media in all physical dimensions. The nonlocal nonlinear response must be symmetric, but can be of completely arbitrary shape. We use variational techniques to find the soliton solutions and illustrate the stabilizing effect of nonlocality.Comment: 4 pages with 3 figure

Genetic basis of depressive disorders

Depression is a common mental disorder being one of the main causes of disability and mortality worldwide. Despite an intensive research during the past decades, the etiology of depressive disorders (DDs) remains incompletely understood; however, genetic factors are significantly involved in the liability to depression. The present review is focused on the studies based on a candidate gene approach, genome-wide association studies (GWAS) and whole exome sequencing (WES), which previously demonstrated associations between gene polymorphisms and DDs. According to the first approach, DD development is affected by serotonergic (TPH1, TPH2, HTR1A, HTR2A, and SLC6A4), dopaminergic (DRD4, SLC6A3) and noradrenergic (SLC6A2) system genes, and genes of enzymatic degradation (MAOA, COMT). In addition, there is evidence of the involvement of HPA-axis genes (OXTR, AVPR1A, and AVPR1B), sex hormone receptors genes (ESR1, ESR2, and AR), neurotrophin (BDNF) and methylenetetrahydrofolate reductase (MTHFR) genes, neuronal apoptosis (CASP3, BCL-XL, BAX, NPY, APP, and GRIN1) and inflammatory system (TNF, CRP, IL6, IL1B, PSMB4, PSMD9, and STAT3) genes in DD development. The results of the second approach (GWAS and WES) revealed that the PCLO, SIRT1, GNL3, GLT8D1, ITIH3, MTNR1A, BMP5, FHIT, KSR2, PCDH9, and AUTS2 genes predominantly responsible for neurogenesis and cell adhesion are involved in liability to depression. Therefore, the findings discussed suggest that genetic liability to DD is a complex process, which assumes simultaneous functioning of multiple genes including those reported previously, and requires future research in this field