Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

DNA origami is an emerging technology that can be used as a nanoscale platform in numerous applications ranging from drug delivery systems to biosensors. The DNA nanostructures are assembled from large single-stranded DNA (ssDNA) scaffolds, ranging from hundreds to thousands of nucleotides and from short staple strands. Scaffolds are usually obtained by asymmetric PCR (aPCR) or Escherichia coli infection/transformation with phages or phagemids. Scaffold quantification is typically based on agarose gel electrophoresis densitometry for molecules obtained by aPCR, or by UV absorbance, in the case of scaffolds obtained by infection or transformation. Although these methods are well-established and easy-to-apply, the results obtained are often inaccurate due to the lack of selectivity and sensitivity in the presence of impurities. Herein, we present an HPLC method based on ion-pair reversed-phase (IP-RP) chromatography to quantify DNA scaffolds. Using IP-RP chromatography, ssDNA products (449 and 1000 nt) prepared by aPCR were separated from impurities and from the double stranded (ds) DNA byproduct. Additionally, both ss and dsDNA were quantified with high accuracy. The method was used to guide the optimization of the production of ssDNA by aPCR, which targeted the maximization of the ratio of ssDNA to dsDNA obtained. Moreover, ssDNA produced from phage infection of E. coli cells was also quantified by IP-RP using commercial ssDNA from the M13mp18 phage as a standard.

The impact of sputum quality on Xpert positivity in active case-finding for TB.

BACKGROUND: Studies evaluating sputum quality and Xpert® MTB/RIF positivity in the context of active case finding are scarce. We aimed to determine whether sputum quality is associated with Xpert positivity and whether the association differed according to demographic and clinical characteristics.METHODS: A cross-sectional analysis using data from a mass screening programme in Brazilian prisons was conducted from 2017 to 2021. We administered a standardised questionnaire, obtained a chest X-ray and collected a spot sputum sample for Xpert testing. Sputum quality was classified as 'salivary', 'mucoid/mucopurulent' or 'blood-stained'. We used log binomial regressions to estimate the relationship between sputum quality and Xpert positivity, assessing interactions with participant characteristics.RESULTS: Among 4,368 participants for whom sputum quality was assessed, 957 (21.9%) produced salivary specimens, 3,379 (77.4%) had mucoid/mucopurulent sputum and 32 (0.7%) had blood-stained sputum. Xpert positivity was higher among those with mucoid/mucopurulent sputum than among those with salivary samples (12.0% vs. 3.7%). Mucopurulent sputum independently predicted Xpert positivity among individuals with and without symptoms, current smoking and abnormal chest radiographs on CAD4TB.CONCLUSIONS: In our study, sputum appearance independently predicted Xpert positivity, and could be used together with chest X-ray and symptom screening to inform use of Xpert in individual or pooled testing.

Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation.

A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (>80%).

Purification of Plasmid DNA by Multimodal Chromatography.

Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.

A Standardized Aspiration-First Approach for Thrombectomy to Increase Speed and Improve Recanalization Rates.

BACKGROUND AND PURPOSE: Direct aspiration is a recognized technique for revascularization in large-vessel ischemic strokes. There is ongoing debate regarding its efficacy compared with stent retrievers. Every delay in achieving revascularization and a decrease in reperfusion rates reduces the likelihood of patients achieving functional independence. We propose a standardized setup technique for aspiration-first for all anterior circulation thrombectomy procedures for increasing speed and recanalization rates. MATERIALS AND METHODS: We analyzed 127 consecutive patients treated by a standardized approach to thrombectomy with an intention to perform aspiration-first compared with 127 consecutive patients treated with a stent retriever-first approach. Key time metrics evaluated included groin to first angiogram, first angiogram to reperfusion, groin to first reperfusion, and length of the procedure. The degree of successful recanalization (TICI 2b-3) and the number of passes were compared between the 2 groups. RESULTS: In 127 patients who underwent the standardized technique, the median time from groin puncture to first reperfusion was 18 minutes compared with 26 minutes (P < .001). The duration of the procedure was shorter compared with the stent retriever group (26 minutes in the aspiration first group versus 47 minutes, P < .001) and required fewer passes (mean, 2.4 versus 3.1; P < .05). A higher proportion of patients had a TICI score of 2b-3 in the aspiration-first group compared with stent retriever group (96.1% versus 85.8%, P < .005). CONCLUSIONS: Our study highlights the increasing speed and recanalization rates achieved with fewer passes in a standardized approach to thrombectomy with an intention to attempt aspiration-first. Any attempt to reduce revascularization time and increase successful recanalization should be used.

Nutritional Risk and Quality of Life in Community-Dwelling Elderly: A Cross-Sectional Study.

OBJECTIVES: To evaluate the association between nutritional risk and quality of life in community-dwelling elderly. METHOD: A cross-sectional study of individuals aged 60 years or older living in the Brazilian state of Minas Gerais was conducted. Nutritional status was evaluated using the Mini Nutritional Assessment, and quality of life was determined using the WHOQOL-bref and WHOQOL-old. The absolute and relative frequency distributions for each domain were calculated, and chi-square tests were used to examine the association between nutritional risk and quality of life (dichotomous variables). Poisson regression was used to perform univariate and multivariate analyses. RESULTS: 789 (27.9%) of the 2823 elderly patients in the sample were at nutritional risk. The risk of malnutrition was associated with the physical, environment, psychological, sensory abilities, social participation and intimacy domains of quality of life [(PR= 1.42, 95%CI 1.23-1.63); (PR= 1.28, 95%CI 1.12-1.47); (PR= 1.18, 95%CI 1.03-1.36); (PR= 1.36, 95%CI 1.20-1.54); (PR= 1.29, 95%CI 1.13-1.46); (PR= 1.14, 95%CI 1.01-1.29); respectively]. CONCLUSION: The present findings contribute to the understanding of nutritional risk, and support the association between quality of life and nutritional status. These factors should be considered in the diagnosis, monitoring and treatment of nutritional disorders, and may contribute to the prevention and reduction of nutritional risk in this especially vulnerable population.

Separation of plasmid DNA topoisomers by multimodal chromatography.

The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.