Methodological artefacts cause counter-intuitive evolutionary conclusions in a simulation study.

In their simulation study, Garcia-Costoya et al. (2023) conclude that evolutionary constraints might aid populations facing climate change. However, we are concerned that this conclusion is largely a consequence of the simulated temperature variation being too small, and, most importantly, that uneven limitations to standing variation disadvantage unconstrained populations.

A time course analysis through diapause reveals dynamic temporal patterns of microRNAs associated with endocrine regulation in the butterfly Pieris napi.

Organisms inhabiting highly seasonal environments must cope with a wide range of environmentally induced challenges. Many seasonal challenges require extensive physiological modification to survive. In winter, to survive extreme cold and limited resources, insects commonly enter diapause, which is an endogenously derived dormant state associated with minimized cellular processes and low energetic expenditure. Due to the high degree of complexity involved in diapause, substantial cellular regulation is required, of which our understanding primarily derives from the transcriptome via messenger RNA expression dynamics. Here we aim to advance our understanding of diapause by investigating microRNA (miRNA) expression in diapausing and direct developing pupae of the butterfly Pieris napi. We identified coordinated patterns of miRNA expression throughout diapause in both head and abdomen tissues of pupae, and via miRNA target identification, found several expression patterns to be enriched for relevant diapause-related physiological processes. We also identified two candidate miRNAs, miR-14-5p and miR-2a-3p, that are likely involved in diapause progression through their activity in the ecdysone pathway, a critical regulator of diapause termination. miR-14-5p targets phantom, a gene in the ecdysone synthesis pathway, and is upregulated early in diapause. miR-2a-3p has been found to be expressed in response to ecdysone, and is upregulated during diapause termination. Together, the expression patterns of these two miRNAs match our current understanding of the timing of hormonal regulation of diapause in P. napi and provide interesting candidates to further explore the mechanistic role of microRNAs in diapause regulation.

Influence of Solvent Selection on the Crystallizability and Polymorphic Selectivity Associated with the Formation of the "Disappeared" Form I Polymorph of Ritonavir.

The comparative crystallizability and polymorphic selectivity of ritonavir, a novel protease inhibitor for the treatment of acquired immune-deficiency syndrome, as a function of solvent selection are examined through an integrated and self-consistent experimental and computational molecular modeling study. Recrystallization at high supersaturation by rapid cooling at 283.15 K is found to produce the metastable "disappeared" polymorphic form I from acetone, ethyl acetate, acetonitrile, and toluene solutions in contrast to ethanol which produces the stable form II. Concomitant crystallization of the other known solid forms is not found under these conditions. Isothermal crystallization studies using turbidometric detection based upon classical nucleation theory reveal that, for an equal induction time, the required driving force needed to initiate solution nucleation decreases with solubility in the order of ethanol, acetone, acetonitrile, ethyl acetate, and toluene consistent with the expected desolvation behavior predicted from the calculated solute solvation free energies. Molecular dynamics simulations of the molecular and intermolecular chemistry reveal the presence of conformational interplay between intramolecular and intermolecular interactions within the solution phase. These encompass the solvent-dependent formation of intramolecular O-H...O hydrogen bonding between the hydroxyl and carbamate groups coupled with differing conformations of the hydroxyl's shielding phenyl groups. These conformational preferences and their relative interaction propensities, as a function of solvent selection, may play a rate-limiting role in the crystallization behavior by not only inhibiting to different degrees the nucleation process but also restricting the assembly of the optimal intermolecular hydrogen bonding network needed for the formation of the stable form II polymorph.

Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

Bumblebee thermoregulation at increasing temperatures is affected by behavioral state.

Over the past decades, increasing environmental temperatures have been identified as one of the causes of major insect population declines and biodiversity loss. However, it is unclear how these rising temperatures affect endoheterothermic insects, like bumblebees, that have evolved thermoregulatory capacities to exploit cold and temperate habitats. To investigate this, we measured head, thoracic, and abdominal temperature of bumblebee (Bombus terrestris) workers across a range of temperatures (24 °C-32 °C) during three distinct behaviors. In resting bumblebees, the head, abdomen, and thorax conformed to the environmental temperature. In pre-flight bumblebees, the head and abdominal temperatures were elevated with respect to the environmental temperature, while the thoracic temperature was maintained, indicating a pre-flight muscle warming stage. In post-flight bumblebees, abdominal temperature increased at the same rate as environmental temperature, but the head and the thoracic temperature did not. By calculating the excess temperature ratio, we show that thermoregulation in bumblebees during flight is partially achieved by the active transfer of heat produced in the thorax to the abdomen, where it can more easily be dissipated. These results provide the first indication that the thermoregulatory abilities of bumblebees are plastic and behavior dependent. We also show that the flight speed and number of workers foraging increase with increasing temperature, suggesting that bees do not avoid flying at these temperatures despite its impact on behavioral performance.

Freezing And Mechanical Failure of A Habitat-forming Kelp in The Rocky Intertidal Zone.

Kelp and other habitat-forming seaweeds in the intertidal zone are exposed to a suite of environmental factors, including temperature and hydrodynamic forces, that can influence their growth, survival, and ecological function. Relatively little is known about the interactive effect of temperature and hydrodynamic forces on kelp, especially the effect of cold stress on biomechanical resistance to hydrodynamic forces. We used the intertidal kelp Egregia menziesii to investigate how freezing in air during a low tide changes the kelp's resistance to breaking from hydrodynamic forces. We conducted a laboratory experiment to test how short-term freezing, mimicking a brief low tide freezing event, affected the kelp's mechanical properties. We also characterized daily minimum winter temperatures in an intertidal E. menziesii population on San Juan Island, WA near the center of the species' geographic range. In the laboratory, acute freezing events decreased the strength and toughness of kelp tissue by 8 to 20% (change in medians). During low tides in the field, we documented sub-zero temperatures, snow, and low canopy cover (compared to summer surveys). These results suggest that freezing can contribute to frond breakage and decreased canopy cover in intertidal kelp. Further work is needed to understand whether freezing and the biomechanical performance in cold temperatures influence the fitness and ecological function of kelp, and whether this will change as winter conditions, such as freezing events and storms, change in frequency and intensity.

Determinants of tofacitinib discontinuation in adult patients with rheumatoid arthritis during long-term extension studies up to 9.5 years.

Objectives: To examine determinants of tofacitinib discontinuation due to voluntary (i.e. patient-driven) or involuntary reasons (i.e. protocol mandated) in long-term extension (LTE) studies of patients with RA to inform clinical practice, clinical study execution and data capture. Methods: This post hoc analysis used pooled data from patients receiving tofacitinib 5 or 10 mg twice daily (BID) in LTE studies. Outcomes included time to voluntary/involuntary discontinuation (and baseline predictors), including by geographic region. Exposure-adjusted event rates (EAERs) were calculated for adverse events (AEs), serious AEs (SAEs) and discontinuations due to AEs/SAEs. Results: Of 4967 patients, 2463 (49.6%) discontinued [1552/4967 (31.2%) voluntarily, 911/4967 (18.3%) involuntarily] and 55 (1.1%) died over the course of 9.5 years. When involuntary discontinuation was present as a competing risk for voluntary discontinuation, patients who stayed on combination therapy and with higher patient-assessed pain were significantly more likely to discontinue for voluntary reasons (P < 0.05). Older patients, those enrolled in Asia, Europe or Latin America (vs USA or Canada) or with RF+/anti-CCP+ status were significantly less likely to discontinue for voluntary reasons (P < 0.05). Small numeric differences in disease activity were observed between geographic regions in patients who discontinued or completed the studies. EAERs were generally higher for tofacitinib 10 vs 5 mg BID, irrespective of discontinuation reason. Conclusion: The factors associated with voluntary/involuntary discontinuation of tofacitinib suggest that treatment persistence in RA studies is partly predictable, which may be reflected in clinical practice. Applying these results may improve our understanding of attrition and inform future study design/execution. Trial registrations: ClinicalTrials.gov (http://clinicaltrials.gov): NCT00413699 and NCT00661661.

Automated Growth Rate Measurement of the Facet Surfaces of Single Crystals of the ß-Form of l-Glutamic Acid Using Machine Learning Image Processing.

Precision measurement of the growth rate of individual single crystal facets (hkl) represents an important component in the design of industrial crystallization processes. Current approaches for crystal growth measurement using optical microscopy are labor intensive and prone to error. An automated process using state-of-the-art computer vision and machine learning to segment and measure the crystal images is presented. The accuracies and efficiencies of the new crystal sizing approach are evaluated against existing manual and semi-automatic methods, demonstrating equivalent accuracy but over a much shorter time, thereby enabling a more complete kinematic analysis of the overall crystallization process. This is applied to measure in situ the crystal growth rates and through this determining the associated kinetic mechanisms for the crystallization of ß-form l-glutamic acid from the solution phase. Growth on the {101} capping faces is consistent with a Birth and Spread mechanism, in agreement with the literature, while the growth rate of the {021} prismatic faces, previously not available in the literature, is consistent with a Burton-Cabrera-Frank screw dislocation mechanism. At a typical supersaturation of σ = 0.78, the growth rate of the {101} capping faces (3.2 × 10-8 m s-1) is found to be 17 times that of the {021} prismatic faces (1.9 × 10-9 m s-1). Both capping and prismatic faces are found to have dead zones in their growth kinetic profiles, with the capping faces (σc = 0.23) being about half that of the prismatic faces (σc = 0.46). The importance of this overall approach as an integral component of the digital design of industrial crystallization processes is highlighted.