Magnetic hydrogel particles improve nanopore sequencing of SARS-CoV-2 and other respiratory viruses.

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with Nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40 × improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher automation platform.

Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types.

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100-1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.

Estimating insulin demand for protein-containing foods using the food insulin index.

BACKGROUND/OBJECTIVE: The Food Insulin Index (FII) is a novel algorithm for ranking foods on the basis of insulin responses in healthy subjects relative to an isoenergetic reference food. Our aim was to compare postprandial glycemic responses in adults with type 1 diabetes who used both carbohydrate counting and the FII algorithm to estimate the insulin dosage for a variety of protein-containing foods. SUBJECTS/METHODS: A total of 11 adults on insulin pump therapy consumed six individual foods (steak, battered fish, poached eggs, low-fat yoghurt, baked beans and peanuts) on two occasions in random order, with the insulin dose determined once by the FII algorithm and once with carbohydrate counting. Postprandial glycemia was measured in capillary blood glucose samples at 15-30 min intervals over 3 h. Researchers and participants were blinded to treatment. RESULTS: Compared with carbohydrate counting, the FII algorithm significantly reduced the mean blood glucose level (5.7±0.2 vs 6.5±0.2 mmol/l, P=0.003) and the mean change in blood glucose level (-0.7±0.2 vs 0.1±0.2 mmol/l, P=0.001). Peak blood glucose was reached earlier using the FII algorithm than using carbohydrate counting (34±5 vs 56±7 min, P=0.007). The risk of hypoglycemia was similar in both treatments (48% vs 33% for FII vs carbohydrate counting, respectively, P=0.155). CONCLUSIONS: In adults with type 1 diabetes, compared with carbohydrate counting, the novel FII algorithm improved postprandial hyperglycemia after consumption of protein-containing foods.

Adsorption of Al3+ to phosphatidylcholine vesicles.

Aluminum toxicity to soil and aquatic organisms is widespread, but the mechanisms of toxicity are unknown. To understand these mechanisms, it is important to know how aluminum reacts with cell surfaces. In this report, we studied adsorption of Al3+ to liposomes composed of phosphatidylcholine, the most abundant phospholipid in plasma membranes of eukaryotic cells. Our equilibrium dialysis and electrophoresis experiments both showed that Al3+ has a 560-fold higher affinity for the phosphatidylcholine surface than Ca2+. Unlike previous reports for adsorption of divalent metals, adsorption of Al3+ to phosphatidylcholine was predicted only approximately by the Stern model. Adsorption of AlF2+ and AlF2+ to the surface was not detectable at the activities we used. From our data, we calculate that Al3+ at 5 x 10(-6) activity could neutralize the surface charge on plant cell plasma membranes and cause a surface potential shift from -30 to +11 mV. This is consistent with non-specific Al3+ inhibition of cation uptake by root cells. Al3+ adsorption to phosphatidylcholine may also play a role in aluminum uptake into cytoplasm by endocytosis.

Lipid bilayer permeation by neutral aluminum citrate and by three alpha-hydroxy carboxylic acids.

Several groups have proposed that aluminum (Al) may permeate biological membranes as a neutral complex with citrate. We tested this hypothesis by measuring aluminum citrate flux across unilamellar phospholipid vesicles (liposomes). Results from two independent procedures show that lipid bilayer permeation by the neutral aluminum-citrate complex is slow (P approximately equal to 1 x 10(-11) cm.s-1). We then compared aluminum-citrate permeation with permeation by a series of alpha-hydroxy carboxylic acids and by trimethylcitrate. This comparison showed that the aluminum-citrate flux is limited by diffusion across the water/lipid interface. This is due to hydrogen bonding between water and the citrate carboxyl groups, and by hydration of the bound metal in the aqueous phase. By analogy with citric acid, steric hindrance of diffusion within the bilayer does not affect the permeation rate of aluminum citrate. Elevated tissue levels of Al in subjects fed a diet supplemented with citric acid and Al(OH)3 cannot be explained by lipid bilayer permeation of the neutral complex.

A validation of the defining characteristics of the nursing diagnosis "potential for violence".

This descriptive study provides validation for the list of characteristics and etiologic factors that currently define the nursing diagnosis "potential for violence." The study also identifies a new characteristic, those characteristics which are most critical, and suggests grouping characteristics which occur in clusters.

Phosphate Nutrition of Rhizobium spp.

This study was conducted to determine the behavior of 40 strains from six species of Rhizobium in liquid defined media containing orthophosphate at levels likely to be encountered naturally, ranging from the high concentrations expected in nodules and artificial media to the low concentrations of soil solutions. Storage capacity in strains with high levels (2 mM) of P and ability to utilize this stored P for growth after transfer to low levels (0.06 muM) of P varied with each strain. Storage varied from about 1 to 2% P (dry weight) for all strains, with the number of generations supported dependent on the quantity of P stored and on the utilization efficiency. The ability to store P at high levels is probably less important than the uptake and utilization efficiency of P supplied at low levels. Strains varied greatly in tolerance to low levels of P maintained in solution by an iron oxide buffering system. Differences in growth rate at low levels of P were large enough to be agronomically important.

Unerupted incisors.

Fifty consecutive cases presenting with unerupted maxillary incisors at orthodontic clinics were recorded and progress of the unerupted tooth followed up over a 4-year period following the exposure of the unerupted tooth (including removal of supernumerary tooth, etc., if present). The occlusal level, rotation, tilting, and general appearance were assessed at the conclusion of the investigation and the results are presented in this report. The earlier the removal of the causative factor preventing eruption of the incisor the better is the prognosis, and it is suggested that all teeth that have not erupted 6 months after the normal eruption date should be subject to radiological examination to ascertain any possible cause for the delayed eruption.

Nodulation and Nitrogen Fixation in Cliffrose (Cowania mexicana var. stansburiana (Torr.) Jeps.).

Nodulation and acetylene reduction were demonstrated for cliffrose (Cowania mexicana var. stansburiana [Torr.] Jeps.) in a greenhouse trial. Nitrogen fixation was also verified with (15)N. Seedlings were grown in a soil known to nodulate both bitterbrush species (Purshia tridentata (Pursh) D.C. and P. glandulosa [Curran]) to which a suspension of crushed bitterbrush nodules had been added. Bitterbrush seedlings grew faster and reduced more acetylene than cliffrose seedlings planted in the same pots, but the specific activity of the nodules and the nodule mass to root mass ratios were approximately the same.