More on how and why: a response to commentaries

We are grateful to the commentators for taking the time to respond to our article. Too many interesting and important points have been raised for us to tackle them all in this response, and so in the below we have sought to draw out the major themes. These include problems with both the term ‘ultimate causation’ and the proximate-ultimate causation dichotomy more generally, clarification of the meaning of reciprocal causation, discussion of issues related to the nature of development and phenotypic plasticity and their roles in evolution, and consideration of the need for an extended evolutionary synthesis.     

Enzyme-catalyzed protein crosslinking

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.     

Exponential Size Distribution of von Willebrand Factor

Von Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications.     

THE EVOLUTION OF LOCOMOTOR RHYTHMICITY IN TETRAPODS

Differences in rhythmicity (relative variance in cycle period) among mammal, fish, and lizard feeding systems have been hypothesized to be associated with differences in their sensorimotor control systems. We tested this hypothesis by examining whether the locomotion of tachymetabolic tetrapods (birds and mammals) is more rhythmic than that of bradymetabolic tetrapods (lizards, alligators, turtles, salamanders). Species averages of intraindividual coefficients of variation in cycle period were compared while controlling for gait and substrate. Variance in locomotor cycle periods is significantly lower in tachymetabolic than in bradymetabolic animals for datasets that include treadmill locomotion, non‐treadmill locomotion, or both. When phylogenetic relationships are taken into account the pooled analyses remain significant, whereas the non‐treadmill and the treadmill analyses become nonsignificant. The co‐occurrence of relatively high rhythmicity in both feeding and locomotor systems of tachymetabolic tetrapods suggests that the anatomical substrate of rhythmicity is in the motor control system, not in the musculoskeletal components.     

Old‐male paternity advantage is a function of accumulating sperm and last‐male precedence in a butterfly

Old‐male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double‐mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2–3 days old) and afterwards an older virgin male (12–13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last‐male precedence and the number of sperm transferred upon mating, both increasing paternity success.     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.     

Additive effects of beta-alanine and sodium bicarbonate on upper-body intermittent performance

We examined the isolated and combined effects of beta-alanine (BA) and sodium bicarbonate (SB) on high-intensity intermittent upper-body performance in judo and jiu-jitsu competitors. 37 athletes were assigned to one of four groups: (1) placebo (PL)+PL; (2) BA+PL; (3) PL+SB or (4) BA+SB. BA or dextrose (placebo) (6.4 g day^?1) was ingested for 4 weeks and 500 mg kg^?1 BM of SB or calcium carbonate (placebo) was ingested for 7 days during the 4th week. Before and after 4 weeks of supplementation, the athletes completed four 30-s upper-body Wingate tests, separated by 3 min. Blood lactate was determined at rest, immediately after and 5 min after the 4th exercise bout, with perceived exertion reported immediately after the 4th bout. BA and SB alone increased the total work done in +7 and 8 %, respectively. The co-ingestion resulted in an additive effect (+14 %, p < 0.05 vs. BA and SB alone). BA alone significantly improved mean power in the 2nd and 3rd bouts and tended to improve the 4th bout. SB alone significantly improved mean power in the 4th bout and tended to improve in the 2nd and 3rd bouts. BA+SB enhanced mean power in all four bouts. PL+PL did not elicit any alteration on mean and peak power. Post-exercise blood lactate increased with all treatments except with PL+PL. Only BA+SB resulted in lower ratings of perceived exertion (p = 0.05). Chronic BA and SB supplementation alone equally enhanced high-intensity intermittent upper-body performance in well-trained athletes. Combined BA and SB promoted a clear additive ergogenic effect.