Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin "switches." The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptidebased molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechanotransduction events within the cellular microenvironment.
A discovery study was carried out where serum samples from 22 systemic lupus erythematosus (SLE) patients and matched healthy controls were hybridized to antibody-coated glass slide arrays that interrogated the level of 274 human proteins. On the basis of these screens, 48 proteins were selected for ELISA-based validation in an independent cohort of 28 SLE patients. Whereas AXL, ferritin, and sTNFRII were significantly elevated in patients with active lupus nephritis (LN) relative to SLE patients who were quiescent, other molecules such as OPN, sTNFRI, sTNFRII, IGFBP2, SIGLEC5, FAS, and MMP10 exhibited the capacity to distinguish SLE from healthy controls with ROC AUC exceeding 90%, all with p < 0.001 significance. These serum markers were next tested in a cohort of 45 LN patients, where serum was obtained at the time of renal biopsy. In these patients, sTNFRII exhibited the strongest correlation with eGFR (r = −0.50, p = 0.0014) and serum creatinine (r = 0.57, p = 0.0001), although AXL, FAS, and IGFBP2 also correlated with these clinical measures of renal function. When concurrent renal biopsies from these patients were examined, serum FAS, IGFBP2, and TNFRII showed significant positive correlations with renal pathology activity index, while sTNFRII displayed the highest correlation with concurrently scored renal pathology chronicity index (r = 0.57, p = 0.001). Finally, in a longitudinal cohort of seven SLE patients examined at ∼3 month intervals, AXL, ICAM-1, IGFBP2, SIGLEC5, sTNFRII, and VCAM-1 demonstrated the ability to track with concurrent disease flare, with significant subject to subject variation. In summary, serum proteins have the capacity to identify patients with active nephritis, flares, and renal pathology activity or chronicity changes, although larger longitudinal cohort studies are warranted.
Inflammation and altered glutamate metabolism are two pathways implicated in the pathophysiology of depression. Interestingly, these pathways may be linked given that administration of inflammatory cytokines such as interferon-α to otherwise non-depressed controls increased glutamate in the basal ganglia and dorsal anterior cingulate cortex (dACC) as measured by magnetic resonance spectroscopy (MRS). Whether increased inflammation is associated with increased glutamate among patients with major depression is unknown. Accordingly, we conducted a cross-sectional study of 50 medication-free, depressed outpatients using single-voxel MRS, to measure absolute glutamate concentrations in basal ganglia and dACC. Multivoxel chemical shift imaging (CSI) was used to explore creatine-normalized measures of other metabolites in basal ganglia. Plasma and cerebrospinal fluid (CSF) inflammatory markers were assessed along with anhedonia and psychomotor speed. Increased log plasma C-reactive protein (CRP) was significantly associated with increased log left basal ganglia glutamate controlling for age, sex, race, body mass index, smoking status and depression severity. In turn, log left basal ganglia glutamate was associated with anhedonia and psychomotor slowing measured by the finger-tapping test, simple reaction time task and the Digit Symbol Substitution Task. Plasma CRP was not associated with dACC glutamate. Plasma and CSF CRP were also associated with CSI measures of basal ganglia glutamate and the glial marker myoinositol. These data indicate that increased inflammation in major depression may lead to increased glutamate in the basal ganglia in association with glial dysfunction and suggest that therapeutic strategies targeting glutamate may be preferentially effective in depressed patients with increased inflammation as measured by CRP.
Atherosclerosis is the leading cause of morbidity and mortality in the U.S., and is a multifactorial disease that preferentially occurs in regions of the arterial tree exposed to disturbed blood flow. The detailed mechanisms by which d-flow induces atherosclerosis involve changes in the expression of genes, epigenetic patterns, and metabolites of multiple vascular cells, especially endothelial cells. This review presents an overview of endothelial mechanobiology and its relation to the pathogenesis of atherosclerosis with special reference to the anatomy of the artery and the underlying fluid mechanics, followed by a discussion of a variety of experimental models to study the role of fluid mechanics and atherosclerosis. Various in vitro and in vivo models to study the role of flow in endothelial biology and pathobiology are discussed in this review. Furthermore, strategies used for the global profiling of the genome, transcriptome, miR-nome, DNA methylome, and metabolome, as they are important to define the biological and pathophysiological mechanisms of atherosclerosis. These "omics" approaches, especially those which derive data based on a single animal model, provide unprecedented opportunities to not only better understand the pathophysiology of atherosclerosis development in a holistic and integrative manner, but also to identify novel molecular and diagnostic targets.
The nematode Caenorhabditis elegans uses striated muscle in its body wall for locomotion. The myofilament lattice is organized such that all the thin filament attachment structures (dense bodies, analogous to Z-disks) and thick filament organizing centers (M-lines) are attached to the muscle cell membrane. Thus, the force of muscle contraction is transmitted through these structures and allows locomotion of the worm. Dense bodies and M-lines are compositionally similar to focal adhesions and costameres, and are based on integrin and associated proteins. Null mutants for many of the newly discovered dense body and M-line proteins do not have obvious locomotion defects when observed casually, or when assayed by counting the number of times a worm moves back and forth in liquid. We hypothesized that many of these proteins, located as they are in muscle focal adhesions, function in force transmission, but we had not used an appropriate or sufficiently sensitive assay to reveal this function. Recently, we have developed a new quantitative assay of C. elegans locomotion that measures the maximum bending amplitude of an adult worm as it moves backwards. The assay had been used to reveal locomotion defects for null mutants of genes encoding ATN-1 (α-actinin) and PKN-1 (protein kinase N). Here, we describe the details of this method, and apply it to 21 loss of function mutants in 17 additional genes, most of which encode components of muscle attachment structures. As compared to wild type, mutants in 11 genes were found to have less ability to bend, and mutants in one gene were found to have greater ability to bend. Loss of function mutants for eight proteins had been reported to have normal locomotion (ZYX-1 (zyxin), ALP-1 (Enigma), DIM-1, SCPL-1), or locomotion that was not previously investigated (FRG-1 (FRG1), KIN-32 (focal adhesion kinase), LIM-8), or had only slightly decreased locomotion (PFN-3 (profilin)).
miR-21, as an oncogene that overexpresses in most human tumors, is involved in radioresistance; however, the mechanism remains unclear. Here, we demonstrate that miR-21-mediated radioresistance occurs through promoting repair of DNA double strand breaks, which includes facilitating both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). The miR-21-promoted NHEJ occurs through targeting GSK3B (a novel target of miR-21), which affects the CRY2/PP5 pathway and in turn increases DNA-PKcs activity. The miR-21-promoted HRR occurs through targeting both GSK3B and CDC25A (a known target of miR-21), which neutralizes the effects of targeting GSK3B-induced CDC25A increase because GSK3B promotes degradation of both CDC25A and cyclin D1, but CDC25A and cyclin D1 have an opposite effect on HRR. A negative correlation of expression levels between miR-21 and GSK3β exists in a subset of human tumors. Our results not only elucidate miR-21-mediated radioresistance, but also provide potential new targets for improving radiotherapy.
Magnetic resonance imaging (MRI) is routinely used to obtain anatomical images that have greatly advanced biomedical research and clinical health care today, but the full potential of MRI in providing functional, physiological, and molecular information is only beginning to emerge. In this work, we sought to provide a gene expression marker for MRI based on bacterial magnetosomes, tiny magnets produced by naturally occurring magnetotactic bacteria. Specifically, magA, a gene in magnetotactic bacteria known to be involved with iron transport, is expressed in a commonly used human cell line, 293FT, resulting in the production of magnetic, iron-oxide nanoparticles by these cells and leading to increased transverse relaxivity. MRI shows that these particles can be formed in vivo utilizing endogenous iron and can be used to visualize cells positive for magA. These results demonstrate that magA alone is sufficient to produce magnetic nanoparticles and that it is an appropriate candidate for an MRI reporter gene.