Computational Approaches and Observer Variation in the 3D Musculoskeletal Modeling of the Heads of Anolis.

High-resolution imaging, 3D modeling, and quantitative analyses are equipping evolutionary biologists with new approaches to understanding the variation and evolution of the musculoskeletal system. However, challenges with interpreting DiceCT data and higher order use of modeled muscles have not yet been fully explored, and the error in and accuracy of some digital methods remain unclear. West Indian Anolis lizards are a model clade for exploring patterns in functional adaptation, ecomorphology, and sexual size dimorphism in vertebrates. These lizards possess numerous jaw muscles with potentially different anatomies that sculpt the adductor chamber of the skull. Here we test approaches to quantifying the musculoskeletal shape of the heads of two species of Anolis: A. pulchellus and A. sagrei. We employ comparative approaches such as DiceCT segmentation of jaw muscles, 3D surface attachment mapping, and 3D landmarking with the aim of exploring muscle volumes, 3D muscle fiber architecture, and sexual dimorphism of the skull. We then compare sources of measurement error in these 3D analyses while also presenting new 3D musculoskeletal data from the Anolis feeding apparatus. These findings demonstrate the accessibility and repeatability of these emerging techniques as well as provide details regarding the musculoskeletal anatomy of the heads of A. pulchellus and A. sagrei which show potential for further research of comparative biomechanics and evolution in the clade.

Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.

Evidence against the presence of A2 adenosine receptors on guinea pig ventricular myocytes.

The effects of different adenosine agonists on cAMP levels in isolated adult guinea pig ventricular myocytes were investigated. 2-Chloro-N6-cyclopentyladenosine (CCPA), R-N6-phenylisopropyl-adenosine (R-PIA), S-N6-phenylisopropyladenosine (S-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA) reduced isoprenaline-stimulated cAMP Both the nonselective adenosine antagonist 8-[4-[( [[(2-aminoethyl)amino]carbonyl]methyl)oxy]-phenyl]-1, 3-dipropylxanthine ('xanthine amine congener'; XAC) and the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the inhibitory effect of NECA. Basal cAMP levels were not altered by NECA or the highly A2-selective 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680), either alone or in the presence of DPCPX or XAC. These results provide evidence against the existence of A2 receptors on the ventricular myocyte but support the presence of A1 receptors.

Pharmacological characterization of the adenylate cyclase-coupled adenosine receptor in isolated guinea pig atrial myocytes.

Although adenosine is known to activate K+ conduction in atrial tissue, there is still debate as to the involvement of cAMP-dependent mechanisms. In isolated adult guinea pig atrial myocytes, we demonstrate that the highly A1-selective adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine reduced basal cAMP levels by 30-40% in the absence and presence of the nonxanthine phosphodiesterase inhibitor Ro 20-1724. Isoprenaline caused a concentration-dependent increase in cAMP levels, which was more pronounced in the presence of the phosphodiesterase inhibitor. Several adenosine derivatives suppressed the isoprenaline-induced cAMP increase by approximately 80%. The rank order of potency was 2-chloro-N6-cyclopentyladenosine (IC50, 93 nM) greater than (R)-N6-phenylisopropyladenosine (IC50, 309 nM) greater than 5'-N-ethylcarboxamidoadenosine (IC50, 813 nM) much greater than (S)-N6-phenylisopropyladenosine (IC50, 26,300 nM). A similar but complete suppression of the isoprenaline-induced cAMP increase was produced by the muscarinic receptor agonist carbachol (IC50, 398 nM), which like adenosine is known to activate atrial K+ channels. The A1-adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine antagonized the effect of 2-chloro-N6-cyclopentyladenosine concentration-dependently, with a KB value of 9.6 nM. In atrial myocytes isolated from guinea pigs pretreated with pertussis toxin, the inhibitory effects of adenosine analogs on basal and isoprenaline-stimulated cAMP accumulation were markedly attenuated. It is concluded that the adenosine receptor in guinea pig atrial myocytes, which is known to be linked to K+ channels, is also coupled to adenylate cyclase via a pertussis toxin-sensitive guanine nucleotide-binding protein and shows the characteristics of the A1-adenosine receptor subtype.

Pertussis toxin prevents adenosine receptor- and m-cholinoceptor-mediated sinus rate slowing and AV conduction block in the guinea-pig heart.

The influence of pertussis toxin on the effects of adenosine, the adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA) and the m-cholinoceptor agonist carbachol on heart rate and atrioventricular (AV) conduction was investigated in spontaneously beating isolated perfused guinea-pig hearts. In addition, the effects of the agents on the electrocardiogram recorded from anesthetized guinea pigs were studied. Adenosine (0.1-100 mumol/l) and PIA (0.001-100 mumol/l) had concentration-dependent negative chronotropic and negative dromotropic effects. These effects were prevented by pretreatment of the animals with pertussis toxin (150 micrograms/kg; i.v.). Carbachol (0.001-100 mumol/l) had similar cardiac depressant effects. These effects were also abolished by pertussis toxin. In contrast, the negative chronotropic and negative dromotropic effects of the calcium antagonist verapamil which was investigated for comparison were not influenced by pretreatment with pertussis toxin. Since the cardiac depressant effects mediated via adenosine receptors or via m-cholinoceptors are most probably due to an activation of a K+ conductance, it is concluded that both receptors in the sinus node and in the AV node may be coupled via a common pertussis toxin-sensitive guanine nucleotide-binding protein to the K+ channel. It remains to be elucidated whether an additional inhibitory coupling to Ca2+ channels also plays a role.