Enhanced anti-HIV-1 activity of G-quadruplexes comprising locked nucleic acids and intercalating nucleic acids

Two G-quadruplex forming sequences, 5′-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5′-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge–charge repulsion. Contrary to that, we found that attachment of the 5′-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation—forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed

The clinical utility of the continuous performance test and objective measures of activity for diagnosing and monitoring ADHD in children: a systematic review

Attention deficit hyperactivity disorder (ADHD) is typically diagnosed using clinical observation and subjective informant reports. Once children commence ADHD medication, robust monitoring is required to detect partial or non-responses. The extent to which neuropsychological continuous performance tests (CPTs) and objective measures of activity can clinically aid the assessment and titration process in ADHD is not fully understood. This review describes the current evidence base for the use of CPTs and objectively measured activity to support the diagnostic procedure and medication management for children with ADHD. Four databases (PsycINFO, Medline, Allied and Complementary Medicine (AMED) and PsycARTICLES) were systematically searched to understand the current evidence base for: (1) the use of CPTs to aid clinical assessment of ADHD; (2) the use of CPTs to aid medication management; (3) the clinical utility of objective measures of activity in ADHD. Sixty relevant articles were identified. The search revealed six commercially available CPTs that had been reported on for their clinical use. There were mixed findings with regard to the use of CPTs to assess and manage medication, with contrasting evidence on their ability to support clinical decision making. There was a strong evidence base for the use of objective measures of activity to aid ADHD/non-ADHD group differentiation, which appears sensitive to medication effects and would also benefit from further research on their clinical utility. The findings suggest that combining CPTs and an objective measure of activity may be particularly useful as a clinical tool and worthy of further pursuit

Long-term survival differences in gastric cancer after D2 lymphadenectomy and an improved assessment of resectability

Objective. The purpose of this work is to compare the long-term survival between a control group with mostly D1 lymphadenectomy and a study group with mostly D2 lymphadenectomy. Materials and Methods. We analyzed consecutive cases operated by a single surgeon performing mostly D2 lymphadenectomy and a control group of consecutive gastric cancer patients operated by a group of surgeons, performing mostly D1 lymphadenectomy, in a period when D2 lymphadenectomy was not the standard and was introduced as a new technique in our center. Results. We found 30,56% D2 lymphadenectomy in the control groups and 85,1% in the study group. At a five years interval from surgery the control group had no survivors, while the study group had 8 (20%) survivors. Conclusions. The spleen and pancreas preserving D2 lymphadenectomy done by experienced surgeons, according to the current guidelines in order to select operable patients improves the overall survival of such gastric cancer patients

Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis

A Flexibility Market Platform for Electricity System Operators Using Blockchain Technology

The paper’s main objective is to demonstrate the trading and flexibility of services amongst TSOs, DSOs, and Prosumers in a transparent, secure, and cost-effective manner using Blockchain-based TSO-DSO flexibility marketplace (EFLEX). The aim is to look for ways to help DSOs/TSOs be more flexible and more directly engaged in managing energy flows on the network. EFLEX will streamline the needs of both TSO and DSO on the same platform. Based on the paper’s proposed services, the pilot service demonstration will be carried out in Bulgaria and Romania, and the main focus will be on congestion management, TSO-DSO Coordination, and Marketplace. The proposed objective is achieved by using Blockchain-based smart contracts and distributed ledger technology. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Spontaneous network activity visualized by ultra-sensitive Ca2+ indicators, yellow Cameleon-Nano

We report a new set of ultra-sensitive Ca2+ indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca2+-sensing domain of a genetically encoded Ca2+ indicator, YC2.60 or YC3.60. Their high Ca2+ affinities (Kd = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca2+ transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multi-cellular networks