Articular cartilage surface roughness as an imaging‐based morphological indicator of osteoarthritis: A preliminary investigation of osteoarthritis initiative subjects

Current imaging‐based morphometric indicators of osteoarthritis (OA) using whole‐compartment mean cartilage thickness (MCT) and volume changes can be insensitive to mild degenerative changes of articular cartilage (AC) due to areas of adjacent thickening and thinning. The purpose of this preliminary study was to evaluate cartilage thickness‐based surface roughness as a morphometric indicator of OA. 3D magnetic resonance imaging (MRI) datasets were collected from osteoarthritis initiative (OAI) subjects with Kellgren–Lawrence (KL) OA grades of 0, 2, and 4 (n = 10/group). Femoral and tibial AC volumes were converted to two‐dimensional thickness maps, and MCT, arithmetic surface roughness (Sa), and anatomically normalized Sa (normSa) were calculated. Thickness maps enabled visualization of degenerative changes with increasing KL grade, including adjacent thinning and thickening on the femoral condyles. No significant differences were observed in MCT between KL grades. Sa was significantly higher in KL4 compared to KL0 and KL2 in the whole femur (KL0: 0.55 ± 0.10 mm, KL2: 0.53 ± 0.09 mm, KL4: 0.79 ± 0.18 mm), medial femoral condyle (KL0: 0.42 ± 0.07 mm, KL2: 0.48 ± 0.07 mm, KL4: 0.76 ± 0.22 mm), and medial tibial plateau (KL0: 0.42 ± 0.07 mm, KL2: 0.43 ± 0.09 mm, KL4: 0.68 ± 0.27 mm). normSa was significantly higher in KL4 compared to KL0 and KL2 in the whole femur (KL0: 0.22 ± 0.02, KL2: 0.22 ± 0.02, KL4: 0.30 ± 0.03), medial condyle (KL0: 0.17 ± 0.02, KL2: 0.20 ± 0.03, KL4: 0.29 ± 0.06), whole tibia (KL0: 0.34 ± 0.04, KL2: 0.33 ± 0.05, KL4: 0.48 ± 0.11) and medial plateau (KL0: 0.23 ± 0.03, KL2: 0.24 ± 0.04, KL4: 0.40 ± 0.10), and significantly higher in KL2 compared to KL0 in the medial femoral condyle. Surface roughness metrics were sensitive to degenerative morphologic changes, and may be useful in OA characterization and early diagnosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2755–2764, 2017.A custom algorithm was used to create two‐dimensional articular cartilage thickness maps of patients from the Osteoarthritis Initiative. Thickness maps demonstrate significantly increased surface roughness as a function of increasing Kellgren–Lawrence (KL) osteoarthritis (OA) grade, particularly in the medial femoral condyle, though mean cartilage thickness was not found to differ significantly between KL grades. Surface roughness‐based metrics have potential utility as morphological indicators of OA.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141486/1/jor23588_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141486/2/jor23588.pd

High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets

Intracellular Symbionts and Other Bacteria Associated with Deer Ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts

The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass. The majority of sequences obtained originated from gram-negative proteobacteria. Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group. Several strains of members of the Sphingomonadaceae were also detected in all but one tick. The results provide a view of the diversity of bacteria associated with I. scapularis ticks in the field

High-throughput insertion tracking by deep sequencing for the analysis of bacterial pathogens

Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis

Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale “negative selection” technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis