Enamino-oxindole HIV protease inhibitors

We have designed and synthesized a series of HIV protease inhibitors (PIs) with enamino-oxindole substituents optimized to interact with the S2' subsite of the HIV protease binding pocket. Several of these inhibitors have sub-nanomolar K(i) and antiviral IC(50) in the low nM range against WT HIV and against a panel of multi-drug resistant (MDR) strains.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

Bridging the information gap: computational tools for intermediate resolution structure interpretation

Due to large sizes and complex nature, few large macromolecular complexes have been solved to atomic resolution. This has lead to an under-representation of these structures, which are composed of novel and/or homologous folds, in the library of known structures and folds. While it is often difficult to achieve a high-resolution model for these structures, X-ray crystallography and electron cryomicroscopy are capable of determining structures of large assemblies at low to intermediate resolutions. To aid in the interpretation and analysis of such structures, we have developed two programs: helixhunter and foldhunter. Helixhunter is capable of reliably identifying helix position, orientation and length using a five-dimensional cross-correlation search of a three-dimensional density map followed by feature extraction. Helixhunter's results can in turn be used to probe a library of secondary structure elements derived from the structures in the Protein Data Bank (PDB). From this analysis, it is then possible to identify potential homologous folds or suggest novel folds based on the arrangement of alpha helix elements, resulting in a structure-based recognition of folds containing alpha helices. Foldhunter uses a six-dimensional cross-correlation search allowing a probe structure to be fitted within a region or component of a target structure. The structural fitting therefore provides a quantitative means to further examine the architecture and organization of large, complex assemblies. These two methods have been successfully tested with simulated structures modeled from the PDB at resolutions between 6 and 12 A. With the integration of helixhunter and foldhunter into sequence and structural informatics techniques, we have the potential to deduce or confirm known or novel folds in domains or components within large complexes.     

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.     

EMAN: semiautomated software for high-resolution single-particle reconstructions

We present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN's extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data.     

The extended Price equation quantifies species selection on mammalian body size across the Palaeocene/Eocene Thermal Maximum

Species selection, covariation of species’ traits with their net diversification rates, is an important component of macroevolution. Most studies have relied on indirect evidence for its operation and have not quantified its strength relative to other macroevolutionary forces. We use an extension of the Price equation to quantify the mechanisms of body size macroevolution in mammals from the latest Palaeocene and earliest Eocene of the Bighorn and Clarks Fork Basins of Wyoming. Dwarfing of mammalian taxa across the Palaeocene/Eocene Thermal Maximum (PETM), an intense, brief warming event that occurred at approximately 56 Ma, has been suggested to reflect anagenetic change and the immigration of small bodied-mammals, but might also be attributable to species selection. Using previously reconstructed ancestor–descendant relationships, we partitioned change in mean mammalian body size into three distinct mechanisms: species selection operating on resident mammals, anagenetic change within resident mammalian lineages and change due to immigrants. The remarkable decrease in mean body size across the warming event occurred through anagenetic change and immigration. Species selection also was strong across the PETM but, intriguingly, favoured larger-bodied species, implying some unknown mechanism(s) by which warming events affect macroevolution.