Direct comparison of solution and solid phase synthesis of sequence-defined macromolecules

The synthesis of perfectly defined, monodisperse macromolecules is one of the challenges faced by polymer chemists today. Such precision synthesis requires a fundamentally different approach to conventional polymer synthesis, but in turn can unlock the door to many new applications. Therefore, we introduce here the combination of ultra-fast "click" reactions using 1,2,4-triazoline-3,5-diones (TAD) with the highly efficient and versatile Passerini three-component reaction. This new approach not only resulted in the synthesis of monodisperse, sequence-defined macromolecules of high purity and molecular weight (>7000 Da), but also offered new insights into the iterative synthesis of sequence-defined macromolecules in general, as we present a detailed comparative study of the same chemistry protocols carried out on solid phase as well as in solution

Motion in the north Iceland volcanic rift zone accommodated by bookshelf faulting

Along mid-ocean ridges the extending crust is segmented1 on length scales of 10–1,000 km. Where rift segments are offset from one another, motion between segments is accommodated by transform faults that are oriented orthogonally to the main rift axis. Where segments overlap, non-transform offsets with a variety of geometries2 accommodate shear motions. Here we use micro-seismic data to analyse the geometries of faults at two overlapping rift segments exposed on land in north Iceland. Between the rift segments, we identify a series of faults that are aligned sub-parallel to the orientation of the main rift. These faults slip through left-lateral strike-slip motion. Yet, movement between the overlapping rift segments is through right-lateral motion. Together, these motions induce a clockwise rotation of the faults and intervening crustal blocks in a motion that is consistent with a bookshelf-faulting mechanism, named after its resemblance to a tilting row of books on a shelf3. The faults probably reactivated existing crustal weaknesses, such as dyke intrusions, that were originally oriented parallel to the main rift and have since rotated about 15° clockwise. Reactivation of pre-existing, rift-parallel weaknesses contrasts with typical mid-ocean ridge transform faults and is an important illustration of a non-transform offset accommodating shear motion between overlapping rift segments

The middle ear of the pink fairy armadillo Chlamyphorus truncatus (Xenarthra, Cingulata, Chlamyphoridae): comparison with armadillo relatives using computed tomography.

The pink fairy armadillo Chlamyphorus truncatus is the smallest extant armadillo and one of the least-known fossorial mammals. The aim of this study was to establish if its middle ear is specially adapted to the subterranean environment, through comparison with more epigeic relatives of the groups Euphractinae (Chaetophractus villosus, Chaetophractus vellerosus, Zaedyus pichiy) and Dasypodinae (Dasypus hybridus). We examined the middle ears using micro-computed tomography and subsequent three-dimensional reconstructions. D. hybridus has a relatively small middle ear cavity, an incomplete bulla and 'ancestral' ossicular morphology. The other species, including Chlamyphorus, have fully ossified bullae and middle ear ossicles, with a morphology between 'transitional' and 'freely mobile', but in all armadillos the malleus retains a long anterior process. Unusual features of armadillo ears include the lack of a pedicellate lenticular apophysis and the presence, in some species, of an element of Paaw within the stapedius muscle. In common with many subterranean mammals, Chlamyphorus has a relatively flattened malleo-incudal articulation and appears to lack a functional tensor tympani muscle. Its middle ear cavity is not unusually enlarged, and its middle ear ossicles seem less robust than those of the other armadillos studied. In comparison with the euphractines, there is no reason to believe that the middle ear of this species is specially adapted to the subterranean environment; some aspects may even be indicative of degeneration. The screaming hairy armadillo, Chaetophractus vellerosus, has the most voluminous middle ear in both relative and absolute terms. Its hypertrophied middle ear cavity likely represents an adaptation to low-frequency hearing in arid rather than subterranean conditions.Argentinian research grants: Secretaría General de Ciencia y Tecnología, UNS (Project PGI 24/B243); Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) through a PhD fellowship to APB; Subsecretaría de Relaciones Internacionales, UNS, through a grant to APB

Development and implementation of an herbal and natural product elective in undergraduate medical education

<p>Abstract</p> <p>Background</p> <p>Medical students have consistently expressed interest in learning about alternative healing modalities, especially herbal and natural products. To fill this void in medical education at our institution, a novel elective was developed and implemented for fourth year medical students. This herbal/natural product course uses guest lecturers, classroom presentations, and active learning mechanisms that include experiential rotations, case-based learning, and team-based learning to increase student knowledge of herbal/natural product safety and efficacy.</p> <p>Methods</p> <p>Knowledge outcomes were evaluated via administration of a pre- and post-course test (paired student <it>t</it>-test). End-of-course evaluations (Likert-type questions and narrative responses) were used to assess student opinion of knowledge and skills imparted by the elective and overall course content (mean, standard deviation).</p> <p>Results</p> <p>Over three academic years, 23 students have enrolled in this elective. More than 60% of participants have been female and nearly half of the students (43%) have pursued residencies in primary care. Completion of the course significantly increased student knowledge of common herbal/natural product mechanisms, uses, adverse effects, and drug-interactions as determined by a pre- and post-course knowledge assessment (45% ± 10% versus 78% ± 6%; p < 0.0001). The course was highly rated by enrollees (overall course quality, 4.6 of 5.0 ± 0.48) who appreciated the variety of activities to which they were exposed and the open classroom discussions that resulted. While students tended to view some alternative medical systems with skepticism, they still believed it was valuable to learn what these modalities encompass.</p> <p>Conclusions</p> <p>Development and implementation of a herbal/natural product elective that engages undergraduate medical students through active learning mechanisms and critical analysis of the literature has proven effective in increasing knowledge outcomes and is deemed to be a valuable curricular addition by student participants. In the future, it will be of interest to explore mechanisms for expanding the course to reach a larger number of students within the time, financial, and logistical constraints that currently exist.</p

Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys

Background Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion. Methods Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology. Results Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment. Conclusion In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation

Modulation of Cellular Hsp72 Levels in Undifferentiated and Neuron-Like SH-SY5Y Cells Determines Resistance to Staurosporine-Induced Apoptosis

Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis

The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures

Repeat-proteins are made up of near repetitions of 20– to 40–amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi–one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete “domain” (the stability and cooperativity of the repeating array) can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (ΔGwater) and the cooperativity of denaturation (m-value), which do not ordinarily apply for globular proteins. We show how the parameters for the “coarse-grained” description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR) repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are “poised” at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.This work was supported by grants BIO2009-09562, CSD2009-00088 from the Spanish Ministry of Science and Innovation, and FEDER Funds (JMS-R)

X-ray emission from the Sombrero galaxy: discrete sources

We present a study of discrete X-ray sources in and around the bulge-dominated, massive Sa galaxy, Sombrero (M104), based on new and archival Chandra observations with a total exposure of ~200 ks. With a detection limit of L_X = 1E37 erg/s and a field of view covering a galactocentric radius of ~30 kpc (11.5 arcminute), 383 sources are detected. Cross-correlation with Spitler et al.'s catalogue of Sombrero globular clusters (GCs) identified from HST/ACS observations reveals 41 X-rays sources in GCs, presumably low-mass X-ray binaries (LMXBs). We quantify the differential luminosity functions (LFs) for both the detected GC and field LMXBs, whose power-low indices (~1.1 for the GC-LF and ~1.6 for field-LF) are consistent with previous studies for elliptical galaxies. With precise sky positions of the GCs without a detected X-ray source, we further quantify, through a fluctuation analysis, the GC LF at fainter luminosities down to 1E35 erg/s. The derived index rules out a faint-end slope flatter than 1.1 at a 2 sigma significance, contrary to recent findings in several elliptical galaxies and the bulge of M31. On the other hand, the 2-6 keV unresolved emission places a tight constraint on the field LF, implying a flattened index of ~1.0 below 1E37 erg/s. We also detect 101 sources in the halo of Sombrero. The presence of these sources cannot be interpreted as galactic LMXBs whose spatial distribution empirically follows the starlight. Their number is also higher than the expected number of cosmic AGNs (52+/-11 [1 sigma]) whose surface density is constrained by deep X-ray surveys. We suggest that either the cosmic X-ray background is unusually high in the direction of Sombrero, or a distinct population of X-ray sources is present in the halo of Sombrero.Comment: 11 figures, 5 tables, ApJ in pres