Patellofemoral interactions in walking,stair ascent,and stair descent using a virtual patella model

Restoration of normal patella kinematics is an important clinical outcome of total knee arthroplasty. Failure of the patella within total knee systems has been documented and, upon occurrence, often necessitates revision surgery. It is thus important to understand patella mechanics following implantation, subject to load states that are typically realized during walking and other gaits. Here, a computational model of the patella is developed and used to examine the effects of walking, stair ascent, and stair descent on the development of stress and contact pressure in the patella throughout the gait cycle. Motion of the patella was governed by a combination of kinematic and force control, based on knee flexion and patellofemoral joint reaction force data from the literature. Unlike most previous analyses of full gait, quasi-static equilibrium was enforced throughout the cycle. Results indicate that, though peak forces vary greatly between the three gaits, maximum contact pressure and von Mises stress are roughly equivalent. However, contact area is larger in stair ascent and descent than walking, as patellofemoral loading, implant geometry, and polyethylene yield increase conformity between the femoral component and patella. Additionally, maximum contact pressure does not coincide with maximum load except for the case of walking. Though specific to the implant design considered here, this result has important ramifications for patella testing and emphasizes the need to characterize patella mechanics throughout gait.     

Influence of clotting time on the protein composition of serum samples based on LC-MS data

Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classifications especially with respect to long-term follow-up that is otherwise very difficult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may influence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the influence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label-free analysis of trypsin-digested serum showed that the overall pattern of LC-MS data is not affected by clotting times varying from 2 to 8h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived fibrinopeptides, change significantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of fibrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC-MS data using serum sample collections of which the clotting time is not known. Next to the fibrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than 1h. For prospective studies, we recommend to let blood clot for at least 2h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting.     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.     

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

Background

A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.

Results

We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip^® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).

Conclusion

Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.     

Production of lactic acid from hemicellulose extracts by <Emphasis Type="Italic">Bacillus coagulans</Emphasis> MXL-9

Bacillus coagulans MXL-9 was found capable of growing on pre-pulping hemicellulose extracts, utilizing all of the principle monosugars found in woody biomass. This organism is a moderate thermophile isolated from compost for its pentose-utilizing capabilities. It was found to have high tolerance for inhibitors such as acetic acid and sodium, which are present in pre-pulping hemicellulose extracts. Fermentation of 20 g/l xylose in the presence of 30 g/l acetic acid required a longer lag phase but overall lactic acid yield was not diminished. Similarly, fermentation of xylose in the presence of 20 g/l sodium increased the lag time but did not affect overall product yield, though 30 g/l sodium proved completely inhibitory. Fermentation of hot water-extracted Siberian larch containing 45 g/l total monosaccharides, mainly galactose and arabinose, produced 33 g/l lactic acid in 60 h and completely consumed all sugars. Small amounts of co-products were formed, including acetic acid, formic acid, and ethanol. Hemicellulose extract formed during autohydrolysis of mixed hardwoods contained mainly xylose and was converted into lactic acid with a 94% yield. Green liquor-extracted hardwood hemicellulose containing 10 g/l acetic acid and 6 g/l sodium was also completely converted into lactic acid at a 72% yield. The Bacillus coagulans MXL-9 strain was found to be well suited to production of lactic acid from lignocellulosic biomass due to its compatibility with conditions favorable to industrial enzymes and its ability to withstand inhibitors while rapidly consuming all pentose and hexose sugars of interest at high product yields.     

3-Amino-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: a new class of CDK2 inhibitors

We have recently reported about a new class of Aurora-A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. Here we describe the synthesis and early expansion of CDK2/cyclin A-E inhibitors belonging to the same chemical class. Synthesis of the compounds was accomplished using a solution-phase protocol amenable to rapid parallel expansion. Compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit CDK2-mediated tumor cell proliferation have been obtained.     

Interaction between CK2α and CK2β, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.     

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2?? and its paralogous isoform CK2????

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open β4/β5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2β. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.