Multifunctional Properties of Multistage Spark Plasma Sintered HA-BaTiO3-Based Piezobiocomposites for Bone Replacement Applications

This work reports the processing-microstructure-property correlation of novel HA-BaTiO3-based piezobiocomposites, which demonstrated the bone-mimicking functional properties. A series of composites of hydroxyapatite (HA) with varying amounts of piezoelectric BaTiO3 (BT) were optimally processed using uniquely designed multistage spark plasma sintering (SPS) route. Transmission electron microscopy imaging during in situ heating provides complementary information on the real-time observation of sintering behavior. Ultrafine grains (0.50m) of HA and BT phases were predominantly retained in the SPSed samples. The experimental results revealed that dielectric constant, AC conductivity, piezoelectric strain coefficient, compressive strength, and modulus values of HA-40wt% BT closely resembles with that of the natural bone. The addition of 40wt% BT enhances the long-crack fracture toughness, compressive strength, and modulus by 132%, 200%, and 165%, respectively, with respect to HA. The above-mentioned exceptional combination of functional properties potentially establishes HA-40wt% BT piezocomposite as a new-generation composite for orthopedic implant applications.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-beta and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation. Published by Elsevier Ltd.

Perovskite ceramic nanoparticles in polymer composites for augmenting bone tissue regeneration

There is increasing interest in the use of nanoparticles as fillers in polymer matrices to develop biomaterials which mimic the mechanical, chemical and electrical properties of bone tissue for orthopaedic applications. The objective of this study was to prepare poly(epsilon-caprolactone) (PCL) nanocomposites incorporating three different perovskite ceramic nanoparticles, namely, calcium titanate (CT), strontium titanate (ST) and barium titanate (BT). The tensile strength and modulus of the composites increased with the addition of nanoparticles. Scanning electron microscopy indicated that dispersion of the nanoparticles scaled with the density of the ceramics, which in turn played an important role in determining the enhancement in mechanical properties of the composite. Dielectric spectroscopy revealed improved permittivity and reduced losses in the composites when compared to neat PCL. Nanofibrous scaffolds were fabricated via electrospinning. Induction coupled plasma-optical emission spectroscopy indicated the release of small quantities of Ca+2, Sr+2, Ba+2 ions from the scaffolds. Piezo-force microscopy revealed that BT nanoparticles imparted piezoelectric properties to the scaffolds. In vitro studies revealed that all composites support osteoblast proliferation. Expression of osteogenic genes was enhanced on the nanocomposites in the following order: PCL/CT>PCL/ST>PCL/BT>PCL. This study demonstrates that the use of perovskite nanoparticles could be a promising technique to engineer better polymeric scaffolds for bone tissue engineering.

Retinoic Acid and Iron Metabolism: A Step Towards Design of a Novel Antitubercular Drug

The scenario of tuberculosis has gone deadly due to its high prevalence and emergence of widespread drug resistance. It is now high time to develop novel antimycobacterial strategies and to understand novel mechanisms of existing antimycobacterial compounds so that we are equipped with newer tuberculosis controlling molecules in the days to come. Iron has proven to be essential for pathogenesis of tuberculosis and retinoic acid is known to influence the iron metabolism pathway. Retenoic acid is also known to exhibit antitubercular effect in in vivo system. Therefore there is every possibility that retinoic acid by affecting the iron metabolism pathway exhibits its antimycobacterial effect. These aspects are reviewed in the present manuscript for understanding the antimycobacterial role of retinoic acid in the context of iron metabolism and other immunological aspects.

Cells Producing Their Own Nemesis: Understanding Methylglyoxal Metabolism

Methylglyoxal, which is technically known as 2-oxopropanal or pyruvaldehyde, shows typical reactions of carbonyl compounds as it has both an aldehyde and a ketone functional group. It is an extremely cytotoxic physiological metabolite, which is generated by both enzymatic and nonenzymatic reactions. The deleterious nature of the compound is due to its ability to glycate and crosslink macromolecules like protein and DNA, respectively. However, despite having toxic effects on cellular processes, methylglyoxal retains its efficacy as an anticancer drug. Indeed, methylglyoxal is one of the well-known anticancer therapeutic agents used in the treatment. Several studies on methylglyoxal biology revolve around the manifestations of its inhibitory effects and toxicity in microbial growth and diabetic complications, respectively. Here, we have revisited the chronology of methylglyoxal research with emphasis on metabolism of methylglyoxal and implications of methylglyoxal production or detoxification on bacterial pathogenesis and disease progression. (C) 2014 IUBMB Life, 66(10): 667-678, 2014

Hydroxyapatite-titanium bulk composites for bone tissue engineering applications

The research work on bulk hydroxyapatite (HA)-based composites are driven by the need to develop biomaterials with better mechanical properties without compromising its bioactivity and biocompatibility properties. Despite several years of research, the mechanical properties of the HA-based composites still need to be enhanced to match the properties of natural cortical bone. In this regard, the scope of this review on the HA-based bulk biomaterials is limited to the processing and the mechanical as well as biocompatibility properties for bone tissue engineering applications of a model system that is hydroxyapatite-titanium (HA-Ti) bulk composites. It will be discussed in this review how HA-Ti based bulk composites can be processed to have better fracture toughness and strength without compromising biocompatibility. The advantages of the functionally gradient materials to integrate the mechanical and biocompatibility properties is a promising approach in hard tissue engineering and has been emphasized here in reference to the limited literature reports. On the biomaterials fabrication aspect, the recent results are discussed to demonstrate that advanced manufacturing techniques, like spark plasma sintering can be adopted as a processing route to restrict the sintering reactions, while enhancing the mechanical properties. Various toughening mechanisms related to careful tailoring of microstructure are discussed. The in vitro cytocompatibilty, cell fate processes as well as in vivo biocompatibility results are also reviewed and the use of flow cytometry to quantify in vitro cell fate processes is being emphasized. (C) 2014 Wiley Periodicals, Inc.

Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

A new approach to depict bone surfaces in finger imaging using photoacoustic tomography

Imaging the vasculature close around the finger joints is of interest in the field of rheumatology. Locally increased vasculature in the synovial membrane of these joints can be a marker for rheumatoid arthritis. In previous work we showed that part of the photoacoustically induced ultrasound from the epidermis reflects on the bone surface within the finger. These reflected signals could be wrongly interpreted as new photoacoustic sources. In this work we show that a conventional ultrasound reconstruction algorithm, that considers the skin as a collection of ultrasound transmitters and the PA tomography probe as the detector array, can be used to delineate bone surfaces of a finger. This can in the future assist in the localization of the joint gaps. This can provide us with a landmark to localize the region of the inflamed synovial membrane. We test the approach on finger mimicking phantoms.

Linking carbon metabolism to carotenoid production in mycobacteria using Raman spectroscopy

Bacteria can utilize multiple sources of carbon for growth, and for pathogenic bacteria like Mycobacterium tuberculosis, this ability is crucial for survival within the host. In addition, phenotypic changes are seen in mycobacteria grown under different carbon sources. In this study, we use Raman spectroscopy to analyze the biochemical components present in M. smegmatis cells when grown in three differently metabolized carbon sources. Our results show that carotenoid biosynthesis is enhanced when M. smegmatis is grown in glucose compared to glycerol and acetate. We demonstrate that this difference is most likely due to transcriptional upregulation of the carotenoid biosynthesis operon (crt) mediated by higher levels of the stress-responsive sigma factor SigF. Moreover, we find that increased SigF and carotenoid levels correlate with greater resistance of glucose-grown cells to oxidative stress. Thus, we demonstrate the use of Raman spectroscopy in unraveling unknown aspects of mycobacterial physiology and describe a novel effect of carbon source variation on mycobacteria.

A Method for Delineation of Bone Surfaces in Photoacoustic Computed Tomography of the Finger

Photoacoustic (PA) imaging of interphalangeal peripheral joints is of interest in the context of using the synovial membrane as a surrogate marker of rheumatoid arthritis. Previous work has shown that ultrasound (US) produced by absorption of light at the epidermis reflects on the bone surfaces within the finger. When the reflected signals are backprojected in the region of interest, artifacts are produced, confounding interpretation of the images. In this work, we present an approach where the PA signals known to originate from the epidermis are treated as virtual US transmitters, and a separate reconstruction is performed as in US reflection imaging. This allows us to identify the bone surfaces. Furthermore, the identification of the joint space is important as this provides a landmark to localize a region-of-interest in seeking the inflamed synovial membrane. The ability to delineate bone surfaces allows us to identify not only the artifacts but also the interphalangeal joint space without recourse to new US hardware or a new measurement. We test the approach on phantoms and on a healthy human finger.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M-tuberculosis

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

Conceptual design of three-dimensional scaffolds of powder-based materials for bone tissue engineering applications

Purpose - The purpose of this paper is to investigate the possibility to construct tissue-engineered bone repair scaffolds with pore size distributions using rapid prototyping techniques. Design/methodology/approach - The fabrication of porous scaffolds with complex porous architectures represents a major challenge in tissue engineering and the design aspects to mimic complex pore shape as well as spatial distribution of pore sizes of natural hard tissue remain unexplored. In this context, this work aims to evaluate the three-dimensional printing process to study its potential for scaffold fabrication as well as some innovative design of homogeneously porous or gradient porous scaffolds is described and such design has wider implication in the field of bone tissue engineering. Findings - The present work discusses biomedically relevant various design strategies with spatial/radial gradient in pore sizes as well as with different pore sizes and with different pore geometries. Originality/value - One of the important implications of the proposed novel design scheme would be the development of porous bioactive/biodegradable composites with gradient pore size, porosity, composition and with spatially distributed biochemical stimuli so that stem cells loaded into scaffolds would develop into complex tissues such as those at the bone-cartilage interface.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.

Dual role of arginine metabolism in establishing pathogenesis

Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential.