A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

<p>Abstract</p> <p>Background</p> <p>The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V <it>versus </it>L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.</p> <p>Methods</p> <p>Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.</p> <p>Results</p> <p>Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.</p> <p>Conclusions</p> <p>The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.</p

Downstream hydrochemistry and irrigation water quality of the Syr Darya, Aral Sea Basin, South Kazakhstan

New hydrochemical measurements from the Syr Darya provide insights into factors affecting the composition and quality of a major freshwater source replenishing the Aral Sea. This river is heavily used for power and irrigation and crosses territories of four Central Asia republics. It is intensely managed, draining several major tributaries, many reservoirs, and numerous irrigation distribution systems and canals. Analysis of seasonal changes in dissolved ion concentrations using geochemical diagrams, elemental ratios, statistical correlation, and equilibrium modeling allowed the characterization of mineral formation processes that control the dissolved chemical composition. Measured water hydrochemistry and composition type differs substantially from previous reports from the upper reaches of the Syr Darya in Kyrgyzstan. Element ratios, statistical correlation, and the presence of NO3- and NO2- suggest that the downstream trend of increasing total dissolved solids (TDS) from Zhetysay city to the Aral Sea in June is controlled by evaporation–crystallization processes, which contribute to the river dissolved load through soil runoff and return irrigation waters following leaching of secondary salts. Downstream sample composition during the growing season shows significant changes in magnesium-to-calcium ratios in the river water. Elevated magnesium levels in Syr Darya waters may pose a problem to sustainable uses for irrigation

Treatment of atopic dermatitis with upadacitinib: adcare single center experience

IntroductionThe role of upadacitinib in the management of moderate to severe atopic dermatitis seems promising, but more data on its efficacy and safety are needed. This study endeavors to assess the practical impact and safety of upadacitinib in patients with moderate to severe atopic dermatitis. The study aims to evaluate the efficacy and safety of upadacitinib in the treatment of moderate to severe atopic dermatitis, focusing on analyzing patient responses to the treatment.MethodsIn this study, adult patients diagnosed with moderate to severe atopic dermatitis received upadacitinib at daily doses of 15 mg or 30 mg, as prescribed by their attending physicians. The therapeutic efficacy of upadacitinib was meticulously assessed using established clinical metrics. Simultaneously, a comprehensive safety assessment was conducted through monthly monitoring, including the evaluation of potential effects of upadacitinib intake on hepatic function, lipid profile, and hematopoiesis using the pertinent laboratory tests.ResultsSixteen participants were enrolled in the study. At 1month follow-up, there was a significant reduction in the mean Eczema Area and Severity Index (EASI) score to 18.8 points, which further increased to 24 points at the 4-month mark. Additionally, 9 participants (56%) demonstrated an EASI-50 response after 1 month of treatment, with this response increasing to 9 participants (90%) after 4 months. Furthermore, enhanced therapeutic responses were observed at 4 months, with 6 patients (38%) achieving an EASI-75 response at 1month and 8 patients (80%) achieving this milestone at the 4-month follow-up. This study highlights the potential of upadacitinib as an effective treatment option for moderate to severe atopic dermatitis. While it demonstrates improved symptom management, close monitoring for potential adverse events, particularly infections and the known risks of Janus kinase inhibitors, is essential. Further research is essential to determine the long-term safety and efficacy of upadacitinib

Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method

The C2H2 zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities

S.F. Udartsev. Constitution and evolution of a society (issues of theory and legal philosophy)

There are a few works in scientific writings that contain theoretical and philosophical-legal comprehension of the constitution and the constitutional development. The article includes the analysis and the value of fundamental theoretical deductions contained in the monograph “Constitution and evolution of a society (issues of theory and legal philosophy)” written by S.F. Udartsev (Saint-Petersburg: Universitetskii izdatel'skii konsortsium, 2015. 388 p.). It is dedicated specially to the aforementioned aspects of the constitutional evolution. The work under review observes the search for basis of law in the human history and etymology of a term «constitution». Due to the fruitful using the method of moving from the abstract to the concrete the monograph presents a proved, theoretically multidimensional picture of evolution of the constitution during the stages of development and stagnation of the human civilization. It shows the place of the basic law in mechanisms of economical and political development. The constitution is interpreted as a basis as well as apex of the national legal system that connects it with the international law. The impact of the constitution on the social-political and social-economical life is presented by its regulative activity as well as ideological influence through contemporary forms of political, legal, moral and religious consciousness. The work shows its back influence on the forming and acting constitutions. It marks general tendencies and perspectives of political and legal development and the role of the constitution in this process. Globalization contributes to including of whole countries and peoples into space of the developed civilization and this is accompanied by appearance of supranational, planetary regulative tools. One of the latter shall be planetary law that already has some ideological and legal background in the modern world society

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184

HYDATID CYST IN THE CERVICAL SPINAL CORD COMPLICATED BY POTENTIALLY LIFE-THREATENING DIFFICULT AIRWAY: A CASE REPORT

Hydatid cyst in the cervical region is an extremely rare condition that can create challenges for anesthesiologists. Timely recognition of difficult airway and preparing the management plan is crucial to avoid life‐threatening complications such as hypoxic brain damage. We describe a case of difficult airway management in a patient with massive cervical hydatid cyst. We used a low‐dose ketamine‐propofol sedation and lidocaine spray for local oropharyngeal anesthesia. Muscular relaxants were not used, and spontaneous breathing was maintained during intubation. Recognition, assessment, and perioperative planning are essential for difficult airway management in patients with cervical hydatid cyst

Downstream hydrochemistry and irrigation water quality of the Syr Darya, Aral Sea Basin, South Kazakhstan

New hydrochemical measurements from the Syr Darya provide insights into factors affecting the composition and quality of a major freshwater source replenishing the Aral Sea. This river is heavily used for power and irrigation and crosses territories of four Central Asia republics. It is intensely managed, draining several major tributaries, many reservoirs, and numerous irrigation distribution systems and canals. Analysis of seasonal changes in dissolved ion concentrations using geochemical diagrams, elemental ratios, statistical correlation, and equilibrium modeling allowed the characterization of mineral formation processes that control the dissolved chemical composition. Measured water hydrochemistry and composition type differs substantially from previous reports from the upper reaches of the Syr Darya in Kyrgyzstan. Element ratios, statistical correlation, and the presence of NO3- and NO2- suggest that the downstream trend of increasing total dissolved solids (TDS) from Zhetysay city to the Aral Sea in June is controlled by evaporation–crystallization processes, which contribute to the river dissolved load through soil runoff and return irrigation waters following leaching of secondary salts. Downstream sample composition during the growing season shows significant changes in magnesium-to-calcium ratios in the river water. Elevated magnesium levels in Syr Darya waters may pose a problem to sustainable uses for irrigation. HIGHLIGHTS Water composition transitions from Ca–Mg–HCO3 to Ca–Mg–SO4–Cl to Na–SO4–Cl downstream the Syr Darya.; Seasonal and downstream differences in composition suggest that irrigation water inputs are likely controlled by different mineral equilibria.; Water quality indices showed that, generally, Syr Darya water is suitable for irrigation.