Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured “superfolder” mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

This study assesses the effect of temperature and fluctuations in salinity on the nitrogen excretion and osmoregulation of Penaeus vannamei juveniles to determine the lowest stress combination so that these can be used to optimize production of the Mexican strain in culture. The ammonium excretion rate of juveniles acclimated to 20, 24, 28 and 32°C was measured. Fluctuating salinity levels were applied to these animals in a sequence of 40%o, 33, 25, 18, 11, 18, 25, 33 and back to 40%o. The results indicate that when the salinity was reduced from 40 to 11%> the ammonium excretion of the shrimp was reduced. The osmotic concentration of the animals was hyposmotic as the salinity decreased from 40 to 25%o, hyperosmotic during the 18-11-18 in %> interval and hyposmotic as the salinity increased from 25 to 40%> again. The range of isomotic points over this range of salinity was 712-777mmol Kg -1 . The ammonium excretion of P. vannamei exposed to these experimental conditions can be attributed to the process of osmoregulation because excretion was increased when the shrimp were hyper-regulating and reduced when they were hypo-regulating. Based on our results, the animals experience the lowest stress in a temperature between 27 to 30°C and a salinity close to the isosmotic point between 25 -27%o. We propose that this should prove to be the optimal temperature and salinity regime for culturing the Mexican strain of P. vannamei.

The e¡ects of dietary astaxanthin supplemented at 0, 40, 80 or 150 mg astaxanthin kg À1 on growth, survival, moult frequency, osmoregulatory capacity (OC) and selected metabolic and haematological variables in Litopenaeus vannamei acclimated to low-salinity water (3 g L À 1 ) were evaluated. Supplemented astaxanthin at 80 mg kg À1 improved growth, survival and moult frequency in shrimp. The lowest OC was also exhibited in shrimp fed with dietary astaxanthin at 80 mg kg À1 . Shrimp haemolymph concentrations of glucose, lactate, haemocyanin and total haemocyte count were all signi¢cantly enhanced by feeding the diet supplemented with 80 mg astaxanthin kg À1 compared with shrimp fed with the other diets. On the basis of these results, dietary astaxanthin supplementation of 80 mg kg À1 is recommended for juvenile L. vannamei cultured in low-salinity water. Physiological, metabolic and haematological responses in L. vannamei M Flores et al. r 2007 The Authors Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 740^747 Median values and con¢dence interval (95%) in parenthesis. Di¡erent letters indicate a signi¢cant di¡erence on responses (Po0.05). DGC, daily growth coe⁄cient; OC, osmoregulatory capacity; THC, total haemocyte count. Aquaculture Research, 2007, 38, 740^747 Physiological, metabolic and haematological responses in L. vannamei M Flores et al.

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The behavioural and metabolic responses of the largemouth bass Micropterus salmoides (Lacépède) to temperature were determined to define optimal thermal conditions. The final preferendum of largemouth bass juveniles determined with acute and gravitation methods was independent of the method (29.0–28.1 °C). The displacement velocity in the horizontal thermal gradient of bass juveniles was 22.4 cm h−1 in the light phase and 22.6 cm h−1 in the dark phase. Oxygen consumption rates in the largemouth bass increased significantly (P<0.05) from 48.8 to 69.4 mg O2 Kg−1 h−1 with an increase in the acclimation temperature from 20 to 33 °C. The temperature quotient (Q10) in the juveniles was 1.37–2.00 in the range of acclimation temperatures of 26–29 and 29–32 °C. The optimum temperature range for growth calculated using Jobling's equation was 28.1–28.6 °C and for Q10 values 26–29 °C. The results are discussed in relation to the use of this information in aquaculture.

Considering that swim-flume or chasing methods fail in the estimation of maximum metabolic rate and in the estimation of Aerobic Scope (AS) of sedentary or sluggish aquatic ectotherms, we propose a novel conceptual approach in which high metabolic rates can be obtained through stimulation of organism metabolic activity using high and low non-lethal temperatures that induce high (HMR) and low metabolic rates (LMR), This method was defined as TIMR: Temperature Induced Metabolic Rate, designed to obtain an aerobic power budget based on temperature-induced metabolic scope which may mirror thermal metabolic scope (TMS = HMR—LMR). Prior to use, the researcher should know the critical thermal maximum (CT max) and minimum (CT min) of animals, and calculate temperature TIMR max (at temperatures −5–10% below CT max) and TIMR min (at temperatures +5–10% above CT min), or choose a high and low non-lethal temperature that provoke a higher and lower metabolic rate than observed in routine conditions. Two sets of experiments were carried out. The first compared swim-flume open respirometry and the TIMR protocol using Centropomus undecimalis (snook), an endurance swimmer, acclimated at different temperatures. Results showed that independent of the method used and of the magnitude of the metabolic response, a similar relationship between maximum metabolic budget and acclimation temperature was observed, demonstrating that the TIMR method allows the identification of TMS. The second evaluated the effect of acclimation temperature in snook, semi-sedentary yellow tail (Ocyurus chrysurus), and sedentary clownfish (Amphiprion ocellaris), using TIMR and the chasing method. Both methods produced similar maximum metabolic rates in snook and yellowtail fish, but strong differences became visible in clownfish. In clownfish, the TIMR method led to a significantly higher TMS than the chasing method indicating that chasing may not fully exploit the aerobic power budget in sedentary species. Thus, the TIMR method provides an alternative way to estimate the difference between high and low metabolic activity under different acclimation conditions that, although not equivalent to AS may allow the standardized estimation of TMS that is relevant for sedentary species where measurement of AS via maximal swimming is inappropriate.