Challenges of continuous global optimization in molecular structure prediction

The molecular geometry, the three dimensional arrangement of atoms in space, is a major factor determining the properties and reactivity of molecules, biomolecules and macromolecules. Computation of stable molecular conformations can be done by locating minima on the potential energy surface (PES). This is a very challenging global optimization problem because of extremely large numbers of shallow local minima and complicated landscape of PES. This paper illustrates the mathematical and computational challenges on one important instance of the problem, computation of molecular geometry of oligopeptides, and proposes the use of the Extended Cutting Angle Method (ECAM) to solve this problem.

ECAM is a deterministic global optimization technique, which computes tight lower bounds on the values of the objective function and fathoms those part of the domain where the global minimum cannot reside. As with any domain partitioning scheme, its challenge is an extremely large partition of the domain required for accurate lower bounds. We address this challenge by providing an efficient combinatorial algorithm for calculating the lower bounds, and by combining ECAM with a local optimization method, while preserving the deterministic character of ECAM.

Changes in the composition of the extracellular matrix in patellar tendinopathy

Objective: To compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy.

Methods: Sulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase.

Results: There was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue.

Conclusion: The changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.

A novel nanoplatform for oral delivery of anti-cancer biomacromolecules

Oral administration of bio–macromolecules is an uphill task and the challenges from varying pH and enzymatic activity are difficult to overcome. In this regard, nanotechnology promises the new hope and offers advantages such as controlled release, target specific delivery, combinatorial therapy and many more. In this study, we demonstrate the formulation of a novel alginate enclosed, chitosan coated ceramic, anti cancer nano carrier (ACSC NC). These NC were loaded with multi functional anti cancer bovine lactoferrin (Lf), a natural milk based protein, for improvement of intestinal absorption, in order to develop a novel platform to carry anti cancer protein and/or peptides for oral therapy. Here we demonstrate the size, morphology, internalisation and release profiles of the nanoparticles (NC) under varying pH as perceived in human digestive system. We further determine the uptake of these particles by colon cancer cell lines by measuring the endocytosis and transcytosis of the NC. These NC can be used for future targeted protein/peptide or nucleic acid based drug delivery to treat difficult diseases including cancer.

Uncoupling the mechanisms that facilitate cell survival in hormone-deprived bovine mammary explants

Mammary explants can be hormonally stimulated to mimic the biochemical changes that occur during lactogenesis. Previous studies using mammary explants concluded that the addition of exogenous macromolecules were required for mammary epithelial cells to remain viable in culture. The present study examines the survival of mammary explants from the dairy cow using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants cultured from late pregnant cows mimicked lactogenesis and showed significantly elevated milk protein gene expression after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. During this time, the mammary explants remained hormone responsive, the alveolar architecture was maintained and the expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. We report that a population of bovine mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. In addition, we found mammary explant viability was dependent on de novo protein and RNA synthesis. Global functional microarray analysis showed that differential expression of genes involved in energy production, immune responses, oxidative stress and apoptosis signalling might contribute to cell survival. As the decline in milk production in dairy cattle after peak lactation results in considerable economic loss, the identification of novel survival genes may be used as genetic markers for breeding programmes to improve lactational persistency in dairy cows.

Structure and biodegradation mechanism of milled Bombyx mori silk particles

The aim of this study was to understand the structure and biodegradation relationships of silk particles intended for targeted biomedical applications. Such a study is also useful in understanding structural remodelling of silk debris that may be generated from silk-based implants. Ultrafine silk particles were prepared using a combination of efficient wet-milling and spray-drying processes with no addition of chemicals other than those used in degumming. Milling reduced the intermolecular stacking forces within the β-sheet crystallites without changing the intramolecular binding energy. Because of the rough morphology and the ultrafine size of the particles, degradation of silk particles by protease XIV was increased by about 3-fold compared to silk fibers. Upon biodegradation, the thermal degradation temperature of silk increased, which was attributed to the formation of tight aggregates by the hydrolyzed residual macromolecules. A model of the biodegradation mechanism of silk particles was developed based on the experimental data. The model explains the process of disintegration of β-sheets, supported by quantitative secondary structural analysis and microscopic images. © 2012 American Chemical Society.

A cartilage-inspired lubrication system

Articular cartilage is an example of a highly efficacious water-based, natural lubrication system that is optimized to provide low friction and wear protection at both low and high loads and sliding velocities. One of the secrets of cartilage's superior tribology comes from a unique, multimodal lubrication strategy consisting of both a fluid pressurization mediated lubrication mechanism and a boundary lubrication mechanism supported by surface bound macromolecules. Using a reconstituted network of highly interconnected cellulose fibers and simple modification through the immobilization of polyelectrolytes, we have recreated many of the mechanical and chemical properties of cartilage and the cartilage lubrication system to produce a purely synthetic material system that exhibits some of the same lubrication mechanisms, time dependent friction response, and high wear resistance as natural cartilage tissue. Friction and wear studies demonstrate how the properties of the cellulose fiber network can be used to control and optimize the lubrication and wear resistance of the material surfaces and highlight what key features of cartilage should be duplicated in order to produce a cartilage-mimetic lubrication system.

Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein

X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering.

Attenuated total reflectance fourier transform infrared spectroscopy: an analytical technique to understand therapeutic responses at the molecular level

Rapid monitoring of the response to treatment in cancer patients is essential to predict the outcome of the therapeutic regimen early in the course of the treatment. The conventional methods are laborious, time-consuming, subjective and lack the ability to study different biomolecules and their interactions, simultaneously. Since; mechanisms of cancer and its response to therapy is dependent on molecular interactions and not on single biomolecules, an assay capable of studying molecular interactions as a whole, is preferred. Fourier Transform Infrared (FTIR) spectroscopy has become a popular technique in the field of cancer therapy with an ability to elucidate molecular interactions. The aim of this study, was to explore the utility of the FTIR technique along with multivariate analysis to understand whether the method has the resolution to identify the differences in the mechanism of therapeutic response. Towards achieving the aim, we utilized the mouse xenograft model of retinoblastoma and nanoparticle mediated targeted therapy. The results indicate that the mechanism underlying the response differed between the treated and untreated group which can be elucidated by unique spectral signatures generated by each group. The study establishes the efficiency of non-invasive, label-free and rapid FTIR method in assessing the interactions of nanoparticles with cellular macromolecules towards monitoring the response to cancer therapeutics.

Analysis of mammalian cell proliferation and macromolecule synthesis using deuterated water and gas chromatography-mass spectrometry.

Deuterated water (²H₂O), a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules), thus permitting the calculation of their synthesis rates. Here, we have combined ²H₂O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids) from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation), protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines). Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both 'self-made' and exogenously-derived fatty acid.