General Taq PCR Master Mix -- CHEM 384/584 v2

SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.

Skeletal Growth Rates in Porites lutea Corals from Pulau Tinggi, Malaysia

Skeletal records of massive Porites lutea corals sampled from reefs around Malaysia have previously shown average decadal declines in growth rates associated with sea warming. However, there was a variability in growth declines between sites that warrant the need for investigations into more site-specific variations. This study analyzed decade-long (December 2004–November 2014) annual growth records (annual linear extension rate, skeletal bulk density, calcification rate) reconstructed from five massive P. lutea colonies from Pulau Tinggi, Malaysia. Significant non-linear changes in inter-annual trends of linear extension and calcification rates were found, with notable decreases that corresponded to the 2010 El Niño thermal stress episode and a pan-tropical mass coral bleaching event. Coral linear extension and calcification were observed to return to pre-2010 rates by 2012, suggesting the post-stress recovery of P. lutea corals at the study site within 2 years. Although no long-term declines in linear extension and calcification rates were detected, a linear decrease in annual skeletal bulk density by ≈9.5% over the 10-year study period was found. This suggests that although coral calcification rates are retained, the skeletal integrity of P. lutea corals may be compromised with potential implications for the strength of the overall reef carbonate framework. The correlation of coral calcification rates with sea surface temperature also demonstrated site-specific thermal threshold at 29 °C, which is comparable to the regional thermal threshold previously found for the Thai-Malay Peninsula.

Kinetic Explanations for the Sequence Biases Observed in the Nonenzymatic Copying of RNA Templates

ABSTRACTThe identification of nonenzymatic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymatic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates containing all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at saturating intermediate concentrations still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.

Towards a universal shear correction factor in filament stretching rheometry

Filament stretching rheometry is a prominent experimental method to determine rheological properties in extensional flow whereby the separating plates determine the extension rate. In literature, several correction factors that can compensate for the errors introduced by the shear contribution near the plates have been introduced and validated in the linear viscoelastic regime. In this work, a systematic analysis is conducted to determine if a material-independent correction factor can be found for non-linear viscoelastic polymers. To this end, a finite element model is presented to describe the flow and resulting stresses in the filament stretching rheometer. The model incorporates non-linear viscoelasticity and a radius-based controller for the plate speed is added to mimic the typical extensional flow in filament stretching rheometry. The model is validated by comparing force simulations with analytical solutions. The effects of the end-plates on the extensional flow and resulting force measurements are investigated, and a modification of the shear correction factor is proposed for the non-linear viscoelastic flow regime. This shows good agreement with simulations performed at multiple initial aspect ratios and strain rates and is shown to be valid for a range of polymers with non-linear rheological behaviour.

Nutrients attenuate the negative effect of ocean acidification on reef coral calcification in the Arabian Sea upwelling zone (Masirah Island, Oman)

Tropical shallow-water reefs are the most diverse ecosystem in the ocean. Its persistence rests upon adequate calcification rates of the reef building biota, such as reef corals. Optimum calcification rates of reef corals occur in oligotrophic environments with high seawater saturation states of aragonite (Ωsw), which leads to increased vulnerability to anthropogenic ocean acidification and eutrophication. The calcification response of reef corals to this changing environment is largely unknown, however. Here, we present annually and sub-annually resolved records of calcification rates (n = 3) of the coral Porites from the nutrient rich and low Ωsw Arabian Sea upwelling zone (Masirah Island, Oman). Calcification rates were determined from the product of skeletal extension and bulk density derived from X-ray densitometry. Compared to a reference data set of coral skeletons from typical reef environments (Great Barrier Reef, Hawaii), mean annual skeletal bulk density of Porites from Masirah Island is reduced by 28 %. This density deficit prevails over the entire year and probably reflects a year-round low saturation state of aragonite at the site of calcification (Ωcf), independent of seasonal variations in Ωsw (e.g. upwelling). Mean annual extension rate is 20 % higher than for the reference data set. In particular, extension rate is strongly enhanced during the seasons with the lowest water temperatures, presumably due to a high PO43−/NO3−-ratio promoting rapid upward growth of the skeleton. Enhanced annual extension attenuates the negative effect of low density on calcification rate from −25 % to −11 %, while sub-annual calcification rates during the cool seasons even exceed those of the reference corals. We anticipate optimal nutrient environments (e.g. high PO43−/NO3−-ratios) to have significant potential to compensate the negative effect of ocean acidification on reef coral calcification, thereby allowing to maintain adequate rates of carbonate accumulation, which are essential for preserving this unique ecosystem.

Risk factors associated with day-30 mortality in patients over 60 years old admitted in ICU for severe COVID-19: the Senior-COVID-Rea Multicentre Survey protocol

IntroductionWith the spread of COVID-19 epidemic, health plans must be adapted continuously. There is an urgent need to define the best care courses of patients with COVID-19, especially in intensive care units (ICUs), according to their individualised benefit/risk ratio. Since older age is associated with poorer short-term and long-term outcomes, prediction models are needed, that may assist clinicians in their ICU admission decision. Senior-COVID-Rea was designed to evaluate, in patients over 60 years old admitted in ICU for severe COVID-19 disease, the impact of age and geriatric and paraclinical parameters on their mortality 30 days after ICU admission.Methods and analysisThis is a multicentre survey protocol to be conducted in seven hospitals of the Auvergne-Rhône-Alpes region, France. All patients over 60 years old admitted in ICU for severe COVID-19 infection (or their legally acceptable representative) will be proposed to enter the study and to fill in a questionnaire regarding their functional and nutritional parameters 1 month before COVID-19 infection. Paraclinical parameters at ICU admission will be collected: lymphocytes and neutrophils counts, high-fluorescent lymphoid cells and immature granulocytes percentages (Sysmex data), D-dimers, C-reactive protein, lactate dehydrogenase (LDH), creatinine, CT scan for lung extension rate as well as clinical resuscitation scores, and the delay between the first signs of infection and ICU admission. The primary outcome will be the overall survival at day 30 post-ICU admission. The analysis of factors predicting mortality at day 30 will be carried out using univariate and multivariate logistic regressions. Multivariate logistic regression will consider up to 15 factors.The ambition of this trial, which takes into account the different approaches of geriatric vulnerability, is to define the respective abilities of different operational criteria of frailty to predict patients’ outcomes.Ethics and disseminationThe study protocol was ethically approved. The results of the primary and secondary objectives will be published in peer-reviewed journals.Trial registration numberNCT04422340.

Determination of Critical Hydrogen Concentration and Its Effect on Mechanical Performance of 2200 MPa and 600 HBW Martensitic Ultra-High-Strength Steel

The influence of hydrogen on the mechanical performance of a hot-rolled martensitic steel was studied by means of constant extension rate test (CERT) and constant load test (CLT) followed with thermal desorption spectroscopy measurements. The steel shows a reduction in tensile strength up to 25% of ultimate tensile strength (UTS) at critical hydrogen concentrations determined to be about 1.1 wt.ppm and 50% of UTS at hydrogen concentrations of 2 wt.ppm. No further strength degradation was observed up to hydrogen concentrations of 4.8 wt.ppm. It was observed that the interplay between local hydrogen concentrations and local stress states, accompanied with the presence of total average hydrogen reducing the general plasticity of the specimen are responsible for the observed strength degradation of the steel at the critical concentrations of hydrogen. Under CLT, the steel does not show sensitivity to hydrogen at applied loads below 50% of UTS under continuous electrochemical hydrogen charging up to 85 h. Hydrogen enhanced creep rates during constant load increased linearly with increasing hydrogen concentration in the steel.

Scaling of subcellular actin structures with cell length through decelerated growth

How cells tune the size of their subcellular parts to scale with cell size is a fundamental question in cell biology. Until now, most studies on the size control of organelles and other subcellular structures have focused on scaling relationships with cell volume, which can be explained by limiting pool mechanisms. Here, we uncover a distinct scaling relationship with cell length rather than volume, revealed by mathematical modeling and quantitative imaging of yeast actin cables. The extension rate of cables decelerates as they approach the rear of the cell, until cable length matches cell length. Further, the deceleration rate scales with cell length. These observations are quantitatively explained by a 'balance-point' model, which stands in contrast to the limiting pool mechanisms and that senses the linear dimensions of the cell.