Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Characterization of nonrandom chromosomal gains and losses in multiple myeloma by comparative genomic hybridization

Clonal chromosomal changes in multiple myeloma (MM) and related disorders are not well defined, mainly due to the low in vivo and in vitro mitotic index of plasma cells. This difficulty can be overcome by using comparative genomic hybridization (CGH), a DNA-based technique that gives information about chromosomal copy number changes in tumors. We have performed CGH on 25 cases of MM, 4 cases of monoclonal gammopathy of uncertain significance, and 1 case of Waldenstrom's macroglobulinemia. G-banding analysis of the same group of patients demonstrated clonal chromosomal changes in only 13 (43%), whereas by CGH, the number of cases with clonal chromosomal gains and losses increased to 21 (70%). The most common recurrent changes detected by CGH were gain of chromosome 19 or 19p and complete or partial deletions of chromosome 13. +19, an anomaly that has so far not been detected as primary or recurrent change by G-banding analysis of these tumors, was noted in 2 cases as a unique change. Other recurrent changes included gains of 9q, 11q, 12q, 15q, 17q, and 22q and losses of 6q and 16q. We have been able to narrow the commonly deleted regions on 6q and 13q to bands 6q21 and 13q14-21. Gain of 11q and deletion of 13q, which have previously been associated with poor outcome, can thus be detected by CGH, allowing the use of this technique for prognostic evaluation of patients, without relying on the success of conventional cytogenetic analysis

Clinical and public health implications of acute and early HIV detection and treatment: a scoping review.

INTRODUCTION: The unchanged global HIV incidence may be related to ignoring acute HIV infection (AHI). This scoping review examines diagnostic, clinical, and public health implications of identifying and treating persons with AHI. METHODS: We searched PubMed, in addition to hand-review of key journals identifying research pertaining to AHI detection and treatment. We focused on the relative contribution of AHI to transmission and the diagnostic, clinical, and public health implications. We prioritized research from low- and middle-income countries (LMICs) published in the last fifteen years. RESULTS AND DISCUSSION: Extensive AHI research and limited routine AHI detection and treatment have begun in LMIC. Diagnostic challenges include ease-of-use, suitability for application and distribution in LMIC, and throughput for high-volume testing. Risk score algorithms have been used in LMIC to screen for AHI among individuals with behavioural and clinical characteristics more often associated with AHI. However, algorithms have not been implemented outside research settings. From a clinical perspective, there are substantial immunological and virological benefits to identifying and treating persons with AHI - evading the irreversible damage to host immune systems and seeding of viral reservoirs that occurs during untreated acute infection. The therapeutic benefits require rapid initiation of antiretrovirals, a logistical challenge in the absence of point-of-care testing. From a public health perspective, AHI diagnosis and treatment is critical to: decrease transmission via viral load reduction and behavioural interventions; improve pre-exposure prophylaxis outcomes by avoiding treatment initiation for HIV-seronegative persons with AHI; and, enhance partner services via notification for persons recently exposed or likely transmitting. CONCLUSIONS: There are undeniable clinical and public health benefits to AHI detection and treatment, but also substantial diagnostic and logistical barriers to implementation and scale-up. Effective early ART initiation may be critical for HIV eradication efforts, but widespread use in LMIC requires simple and accurate diagnostic tools. Implementation research is critical to facilitate sustainable integration of AHI detection and treatment into existing health systems and will be essential for prospective evaluation of testing algorithms, point-of-care diagnostics, and efficacious and effective first-line regimens

Assessment of myocardial injury after reperfused infarction by T1ρ cardiovascular magnetic resonance

BACKGROUND: The evolution of T1ρ and of other endogenous contrast methods (T2, T1) in the first month after reperfused myocardial infarction (MI) is uncertain. We conducted a study of reperfused MI in pigs to serially monitor T1ρ, T2 and T1 relaxation, scar size and transmurality at 1 and 4 weeks post-MI. METHODS: Ten Yorkshire swine underwent 90 min of occlusion of the circumflex artery and reperfusion. T1ρ, T2 and native T1 maps and late gadolinium enhanced (LGE) cardiovascular magnetic resonance (CMR) data were collected at 1 week (n = 10) and 4 weeks (n = 5). Semi-automatic FWHM (full width half maximum) thresholding was used to assess scar size and transmurality and compared to histology. Relaxation times and contrast-to-noise ratio were compared in healthy and remote myocardium at 1 and 4 weeks. Linear regression and Bland-Altman was performed to compare infarct size and transmurality. RESULTS: Relaxation time differences between infarcted and remote myocardial tissue were ∆T1 (infarct-remote) = 421.3 ± 108.8 (1 week) and 480.0 ± 33.2 ms (4 week), ∆T1ρ = 68.1 ± 11.6 and 74.3 ± 14.2, and ∆T2 = 51.0 ± 10.1 and 59.2 ± 11.4 ms. Contrast-to-noise ratio was CNR(T1) = 7.0 ± 3.5 (1 week) and 6.9 ± 2.4 (4 week), CNR(T1ρ) = 12.0 ± 6.2 and 12.3 ± 3.2, and CNR(T2) = 8.0 ± 3.6 and 10.3 ± 5.8. Infarct size was not significantly different for T1ρ, T1 and T2 compared to LGE (p = 0.14) and significantly decreased from 1 to 4 weeks (p < 0.01). Individual infarct size changes were ∆T1ρ = −3.8%, ∆T1 = −3.5% and ∆LGE = −2.8% from 1 – 4 weeks, but there was no observed change in infarct size for T2 or histologically. CONCLUSIONS: T1ρ was highly correlated with alterations left ventricle (LV) pathology at 1 and 4 weeks post-MI and therefore it may be a useful method endogenous contrast imaging of infarction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12968-017-0332-z) contains supplementary material, which is available to authorized users