How large is an alpha-helix? Studies of the radii of gyration of helical peptides by small-angle X-ray scattering and molecular dynamics

Using synchrotron radiation and the small-angle X-ray scattering technique we have measured the radii of gyration of a series of alanine-based alpha-helix-forming peptides of the composition Ace-(AAKAA)(n)-GY-NH(2), n=2-7, in aqueous solvent at 10(+/-1) degrees C. In contrast to other techniques typically used to study alpha-helices in isolation (such as nuclear magnetic resonance and circular dichroism), small-angle X-ray scattering reports on the global structure of a molecule and, as such, provides complementary information to these other, more sequence-local measuring techniques. The radii of gyration that we measure are, except for the 12-mer, lower than the radii of gyration of ideal alpha-helices or helices with frayed ends of the equivalent sequence-length. For example, the measured radius of gyration of the 37-mer is 14.2(+/-0.6)A, which is to be compared with the radius of gyration of an ideal 37-mer alpha-helix of 17.6A. Attempts are made to analyze the origin of this discrepancy in terms of the analytical Zimm-Bragg-Nagai (ZBN) theory, as well as distributed computing explicit solvent molecular dynamics simulations using two variants of the AMBER force-field. The ZBN theory, which treats helices as cylinders connected by random walk segments, predicts markedly larger radii of gyration than those measured. This is true even when the persistence length of the random walk parts is taken to be extremely short (about one residue). Similarly, the molecular dynamics simulations, at the level of sampling available to us, give inaccurate values of the radii of gyration of the molecules (by overestimating them by around 25% for longer peptides) and/or their helical content. We conclude that even at the short sequences examined here (< or =37 amino acid residues), these alpha-helical peptides behave as fluctuating semi-broken rods rather than straight cylinders with frayed ends.     

Towards a supervised classification of neocortical interneuron morphologies

Background

The challenge of classifying cortical interneurons is yet to be solved. Data-driven classification into established morphological types may provide insight and practical value.

Results

We trained models using 217 high-quality morphologies of rat somatosensory neocortex interneurons reconstructed by a single laboratory and pre-classified into eight types. We quantified 103 axonal and dendritic morphometrics, including novel ones that capture features such as arbor orientation, extent in layer one, and dendritic polarity. We trained a one-versus-rest classifier for each type, combining well-known supervised classification algorithms with feature selection and over- and under-sampling. We accurately classified the nest basket, Martinotti, and basket cell types with the Martinotti model outperforming 39 out of 42 leading neuroscientists. We had moderate accuracy for the double bouquet, small and large basket types, and limited accuracy for the chandelier and bitufted types. We characterized the types with interpretable models or with up to ten morphometrics.

Conclusion

Except for large basket, 50 high-quality reconstructions sufficed to learn an accurate model of a type. Improving these models may require quantifying complex arborization patterns and finding correlates of bouton-related features. Our study brings attention to practical aspects important for neuron classification and is readily reproducible, with all code and data available online.

     

SOME OBSERVATIONS ON THE FINE STRUCTURE OF THE LUTEIN CELLS OF X-IRRADIATED RAT OVARY

The ovaries of 10- to 13-week-old rats were exteriorized and irradiated with sterilizing doses of X-rays. Following treatment, the animals entered a phase of constant vaginal cornification. Animals were killed 8 to 12 wk after the onset of this phase, and their ovaries were prepared for electron microscopy. Tissue was fixed in glutaraldehyde, postfixed in osmium tetroxide (Millonig's, phosphate-buffered), and embedded in Epon. Lutein cells from these ovaries were compared with those from sham-irradiated controls. The cytoplasm of lutein cells from experimental animals was characterized by an increase in the amount of agranular endoplasmic reticulum and by an increase in the number of mitochondria. These mitochondria are more variable in external form and often possess increased numbers of villiform cristae. Other features noted were a decrease in the amount of cytoplasmic lipid granules and an increase in cell size and surface irregularity. The significance of the morphological findings is discussed in relation to ovarian hormone production in animals sterilized by X-irradiation.     

Centaurea zlatiborensis (Asteraceae,Cardueae−Centaureinae), a new endemic species from Zlatibor mountain range,Serbia

A new species of Centaurea sect. Acrocentron (Cass.) DC. (Asteraceae) found growing in dry, steppe‐like habitats on the Zlatibor mountain in western Serbia is described as Centaurea zlatiborensis. This novelity is compared with all other species of C. sect. Acrocentron known from Serbia and a diagnistic key to these is provided. Centaurea zlatiborensis is morphologically close to C. calocephala Willd. but the stems are simple or sparsely branched with monocephalous branches and itis generally smaller in all plant parts. The most important difference is the appendage morphology where C. zlatiborenis has triangular, dark brown appendages with short fimbriae and appendages not completely covering the bracts.     

Six-Minute Walk Test in Renal Failure Patients: Representative Results,Performance Analysis and Perceived Dyspnea Predictors

Objectives

Six-minute walk test in dialysis population hasn’t been consistently evaluated for the isolated impact of renal failure and other predictive factors. We measured six-minute walk distance in patients representative for low level of comorbidity and searched for potentially modifiable predictive factors of performance and dyspnea.

Methods

This was a cross-sectional study with hemodialysis patients (N = 90) and control subjects (N = 140). Main outcome measures: six-minute walk test distance and dyspnea severity using the 10-item Borg scale.

Results

Median distance decreased from 600m below the 6^th decade to 420m in the 8^th decade of age. Dialysis dependence predicted 101.5m shorter distance in the adjusted model that explained 70% of variability in results. Adjusted for significant covariates of age, height and spontaneous gait speed, fat mass (but not lean body mass) and serum total iron binding capacity were significantly associated with distance (95% CI for B coefficients -4.6 to –1.4 m/kg and 0.1 to 5 m/μmol/l, respectively). Serum total iron binding capacity as an explanatory variable was superior to C-reactive protein and albumin. Dialysis dependence, odds ratio (OR) 2.97 (1.11–7.94), spontaneous gait speed, OR 0.08 (0.02–0.41), rate-pressure product, OR 1.15 (1.08–1.23) and hemoglobin, OR 0.95 (0.92–0.98) predicted dyspnea in the adjusted model.

Conclusions

Renal failure without the confounding effect of comorbidity is a significant negative predictor of performance at six-minute walk test and perceived level of dyspnea. Body fat mass and serum total iron binding capacity are the main potentially modifiable predictors of performance, total iron binding capacity being superior to C-reactive protein and albumin. Although hemoglobin is not associated with test performance, it negatively predicts perceived shortness of breath.     

The electronic medical-student exchange: a low-cost alternative to overseas electives.

The authors report on an international collaboration that uses the Internet for medical education. In addition to introducing the students to electronic communication, the project aims to further international collaboration and understanding and to emphasize the importance of a population perspective for health and disease. Students and professors in Canada, England and Hungary participated.     

Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1δ, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1δ and RAE-1γ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1δ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1δ compared to RAE-1γ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1δ. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.Cytomegaloviruses (CMVs) are ubiquitous pathogens causing morbidity in immune suppressed and immunodeficient hosts (34). Since CMVs are strictly species-specific viruses, the infection of mice with murine CMV (MCMV) represents a widely used model for studying CMV infection and disease (22, 40).Natural killer (NK) cells play a crucial role in the control of many viruses and are among the first cells to sense proinflammatory cytokines, as well as the perturbations in the expression of major histocompatibility complex (MHC) class I molecules and other surface molecules induced by viral infection (13). Both human CMV (HCMV) and MCMV have evolved strategies to compromise innate immunity-mediated by NK cells (20, 49).Although proinflammatory cytokines released during the early stage of MCMV infection induce NK cell activation, this is usually not sufficient for virus control (11). Namely, most mouse strains fail to mount an effector phase of NK cell response against infected cells (42), in spite of the fact that MCMV infection causes the downmodulation of MHC I molecules (17), which should activate NK cells via a “missing-self” mechanism (28). The lack of NK cell activation by MCMV is even more puzzling considering that NK cells possess activating receptors that recognize cellular ligands induced by infection. Among these is the activating receptor NKG2D, a C-type lectinlike receptor encoded by a single gene in humans and rodents (39). Engagement of NKG2D transduces a strong activating signal to promote NK cell stimulation. NKG2D also serves as a costimulatory receptor on CD8⁺ T cells (2). Several NKG2D ligands have been described in mice: MULT-1, H60a, H60b, H60c, and RAE-1α, -1β, -1γ, -1δ, and -1ɛ isoforms (4-6, 10, 14, 32, 35, 44). What prevents the activation of NK cells via the NKG2D receptor during MCMV infection? We and others have characterized four MCMV proteins involved in the downmodulation of NKG2D ligands (15, 23, 24, 26, 29, 30). Furthermore, the deletion of any of the four MCMV inhibitors of NKG2D ligands rendered virus mutants susceptible to NK cell control in vivo. The MCMV immunoevasin of NKG2D described first was the glycoprotein gp40, encoded by the gene m152 (23). Note that m152 also compromises the CD8⁺ T-cell response by downregulation of MHC class I molecules (25, 54). Later, it was noticed that m152 also affects the expression of RAE-1 proteins (29). It is important to point out that mouse strains express different RAE-1 isoforms. Some strains, such as BALB/c, express RAE-1α, -1β, and -1γ, while others, such as C57BL/6, express RAE-1δ and -1ɛ (29). All five RAE-1 isoforms are glycosylphosphatidylinositol (GPI)-linked proteins and contain MHC class I-like α1 and α2 domains (6, 10, 14, 35).Based on our initial observation that there is NKG2D-dependent control of wild type (WT) MCMV in certain mouse strains, we postulated NKG2D ligands that resist virus mediated downmodulation. We show here that the RAE-1 proteins differ in their susceptibility to downregulation by MCMV. In contrast to RAE-1γ, representing the sensitive isoform, surface-resident RAE-1δ remains present on MCMV-infected cells. The differential downmodulation of RAE-1 isoforms during MCMV infection is caused by differences in the stability of the mature RAE-1 molecules associated with a sequence motif absent in RAE-1δ.     

Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal demosponge Lubomirskia baicalensis

Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical-basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of the branches and comparably low at the top, the pro-survival gene follows an opposite gradient. Parallel with the tuned expression of these genes, the activities of the apoptosis-executing enzymes caspase-8 (IETDase activity) and caspase-3 (DEVDase activity) are lowest at the top of the branch and highest at their base. This characteristic expression/activity pattern of the genes/enzymes, which had been determined in a few specimens, collected from an unpolluted, natural site, appears reversed in specimens collected from an anthropogenically polluted site. These findings indicate the involvement of apoptotic proteins in the axis formation (branches) in L. baicalensis.