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CaiB is a DR cluster actinobacteriophage that was isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540 as the host. The genome is 61,620 bp, has a GC content of 68.6%, and contains 85 predicted protein coding genes. CaiB has several putative operons and has repeated intergenic regions that may be involved in gene regulation.

Metastasis is the main cause of cancer-related mortality; therefore, the ability to predict its propensity can remarkably affect survival rate. Metastasis development is predicted nowadays by lymph-node status, tumor size, histopathology, and genetic testing. However, all these methods may have inaccuracies, and some require weeks to complete. Identifying novel prognostic markers will open an essential source for risk prediction, possibly guiding to elevated patient treatment by personalized strategies. Cancer cell invasion is a critical step in metastasis. The cytoskeletal mechanisms used by metastatic cells for the invasion process are very similar to the utilization of actin cytoskeleton in the endocytosis process. In the current study, the adhesion and encapsulation efficiency of low-cost carboxylate-modified fluorescent nanoparticles by breast cancer cells with high (HM) and low metastatic potential (LM) have been evaluated; benign cells were used as control. Using high-content fluorescence imaging and analysis, we have revealed (within a short time of 1h), that efficiency of nanoparticles adherence and encapsulation is sufficiently higher in HM cells compared to LM cells, while benign cells are not encapsulating or adhering the particles during experiment time at all. We have utilized custom-made automatic image analysis algorithms to find quantitative co-localization (Pearson’s coefficients) of the nanoparticles with the imaged cells. The method proposed here is straightforward; it does not require especial equipment or expensive materials nor complicated cell manipulations, it may be potentially applicable for various cells, including patient-derived cells. Effortless and quantitative determination of the metastatic likelihood has the potential to be performed using patient-specific biopsy/surgery sample, which will directly influence the choice of protocols for cancer patient’s treatment and, as a result, increase their life expectancy.

We provide an innovative, bioengineering, mechanobiology-based approach to rapidly (2-h) establish the in vivo metastatic likelihood of patient tumor-samples, where results are in direct agreement with clinical histopathology and patient outcomes. Cancer-related mortality is mostly due to local recurrence or to metastatic disease, thus early prediction of tumor-cell-fate may critically affect treatment protocols and survival rates. Metastasis and recurrence risks are currently predicted by lymph-node status, tumor size, histopathology and genetic testing, however, these are not infallible and results may require days/weeks. We have previously observed that subpopulations of invasive cancer-cells will rapidly (1-2 h) push into the surface of physiological-stiffness, synthetic polyacrylamide gels, reaching to cell-scale depths, while normal or noninvasive cells do not considerably indent gels. Here, we evaluate the mechanical invasiveness of established breast and pancreatic cell lines and of tumor-cells from fresh, suspected pancreatic cancer tumors. The mechanical invasiveness matches the in vitro metastatic potential in cell lines as determined with Boyden chamber assays. Moreover, the mechanical invasiveness directly agrees with the clinical histopathology in primary-site, pancreatic-tumors. Thus, the rapid, patient-specific, early prediction of metastatic likelihood, on the time-scale of initial resection/biopsy, can directly affect disease management and treatment protocols.

Non-melanoma skin cancers, including basal and squamous cell carcinomas (BCC and SCC), are the most common malignancies worldwide. BCC/SCC cancers are generally highly localized and can be surgically excised; however, invasive tumors may be fatal. Current diagnosis of skin cancer and prognosis of potential invasiveness are based mainly on clinical-pathological factors of the biopsied lesions. SCC invasiveness is also predicted by histomorphological factors, such as the degree of differentiation or the mitotic index, while BCCs are typically considered non-invasive. The above subjective measures do not provide direct, objective prognosis of cellular invasiveness in each specific sample. Hence, we have developed a mechanobiology-based approach to rapidly determine sample invasiveness. Here, cells from 15 fresh tissue samples of suspected non-melanoma skin cancer were seeded on physiological-stiffness (2.4 kPa) synthetic gels, and within 1-h invasive cell subsets were observed to push/indent the gel surface; clinicopathological results were separately obtained using standard protocols. The percentage of indenting cells from invasive (26.2 ± 2.4%) and non-invasive (4.8 ± 0.5%) SCC samples differed significantly (p 0.0001), with well-separated invasiveness cutoffs of, respectively, 12% and 5%. The mechanical invasiveness directly agrees with the SCC cell-differentiation state, where over 3.3-fold more (p 0.0001) cells from moderately differentiated samples indent the gels as compared to well-differentiated cell samples. In BCCs, 20% of cells typically indented, and a highly migratory, desmoplastic sample was identified with 46%. By providing rapid, quantitative, early prognosis of invasiveness and potential metastatic risk, our rapid technology may facilitate informed (bed-side) decision making and choice of disease-management protocols on the time-scale of the initial diagnosis and surgical excision.

We demonstrate the relative roles of the cell cytoskeleton, and specific importance of actin in facilitating mechanical aspects of metastatic invasion. A crucial step in metastasis, the typically lethal spread of cancer to distant body-sites, is cell invasion through dense tissues composed of extracellular matrix and various non-cancerous cells. Cell invasion requires cell-cytoskeleton remodeling to facilitate dynamic morphological changes and force application. We have previously shown invasive cell subsets in heterogeneous samples can rapidly (2 h) and forcefully indent non-degradable, impenetrable, synthetic gels to cell-scale depths. The amounts of indenting cells and their attained depths provide the mechanical invasiveness of the sample, which as we have shown agrees with the in vitro metastatic potential and the in vivo metastatic risk in humans. To identify invasive force-application mechanisms, we evaluated changes in mechanical invasiveness following chemical perturbations targeting the structure and function of cytoskeleton elements and associated proteins. We evaluate effects on short-term (2-hr) indentations of single, well-spaced or closely situated cells as compared to long-time-scale Boyden chamber migration. We show that actomyosin inhibition may be used to reduce (mechanical) invasiveness of single or collectively invading cells, while actin-disruption may induce escape-response of treated single-cells, which may promote metastasis.

Tumor microenvironment-mechanics greatly affect tumor-cell characteristics such as invasion and proliferation. We and others have previously shown that after chemotherapy, tumor cells shed more extracellular vesicles (EVs), leading to tumor growth and even spread, via angiogenesis and the mobilization of specific bone-marrow-derived cells contributing to metastasis. However, physical, mechanobiological and mechanostructural changes at premetastatic sites that may support tumor cell seeding, have yet to be determined. Here, we collected tumor-derived extracellular vesicles (tEV) from breast carcinoma cells exposed to paclitaxel chemotherapy, and tested their effects on tissue mechanics (eg, elasticity and stiffness) of likely metastatic organs in cancer-free mice, using shear rheometry. Cancer-free mice were injected with saline or with tEVs from untreated cells and lung tissue demonstrated widely variable, viscoelastic mechanics, being more elastic than viscous. Contrastingly, tEVs from chemotherapy-exposed cells induced more uniform, viscoelastic lung mechanics, with lower stiffness and viscosity; interestingly, livers were significantly stiffer than both controls. We observe statistically significant differences in softening of lung samples from all three groups under increasing strain-amplitudes and in their stiffening under increasing strain-frequencies; the groups reach similar values at high strain amplitudes and frequencies, indicating local changes in tissue microstructure. Evaluation of genes associated with the extracellular matrix and fibronectin protein-expression revealed potential compositional changes underlying the altered mechanics. Thus, we propose that tEVs, even without cancer cells, contribute to metastasis by changing microstructures at distant organs. This is done partially by altering the composition and mechanostructure of tissues to support tumor cell invasion and seeding.

A critical step in metastases formation is cancer-cell invasion through tissue. During invasion, cells change morphology and apply forces to their surroundings. We have previously shown that single, metastatic breast-cancer cells will mechanically indent a synthetic, impenetrable polyacrylamide gel with physiological-stiffness in attempted invasion; benign breast cells do not indent the gels. In solid tumors, e.g., breast cancers, metastases occur predominantly by collective cell-invasion. Thus, here we evaluate the effects of cell proximity on mechanical invasiveness, specifically through changes in gel indention. Gel indentation is induced by 56, 33 and 2% (in >1000 cells), respectively, of adjacent high metastatic potential (MP), low MP and benign breast cells, being double the amounts observed in single, well-separated cells. Single cells exhibited a distribution of indentation depths below 10 µm, while adjacent cells also showed a second peak of deeper indentations. The second peak included 65% of indenting high MP cells as compared to 15% in the low MP cells, illustrating the difference in their invasiveness. Thus, proximity of the metastatic cells enhances their mechanical ability to invade, demonstrating why collective cancer-cell migration is likely more efficient. This could potentially provide a rapid, quantitative approach to identify metastatic cells, and to determine their metastatic potential.

The main cause of cancer-related mortality is metastasis; thus, its prediction can critically affect the survival rate. Metastases are currently predicted by lymph-node status, tumor size, histopathology and genetic testing, however, all these are not infallible and getting the results may require weeks. The identification new potential prognostic factors will be an important source of risk information for the practicing oncologist, potentially leading to enhanced patient care through the proactive optimization of treatment strategies. We propose to use fluorescent nanoparticles for metastasis detection. Nowadays nanoparticles are widely used for targeted drug delivery, while particles with specific coatings are encapsulated by cancer cells via endocytosis. We have used low-cost carboxylate-modified fluorescent 200 nm particles to achieve the adhesion and encapsulation efficiency of breast cancer cells with high (MDA-MB-231) and low (MCF7) metastatic potential. Using high-content fluorescence imaging microscope (ImageXpress Micro XL), we have discovered that during short time (up to 1h) highly metastatic cells are able to adhere and encapsulate sufficiently more (p Citation Format: Yulia Merkher, Elizaveta Kontareva, Anna Melekhova, Sergey Leonov. Nanoparticles imaging for cancer metastasis diagnosis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-042.

Background This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Methods Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. Results It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%–24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. Conclusions The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. General significance The study for the first time shows that prolyl hydroxylation is highly regulated in elastin.

The main cause of cancer-related deaths is metastasis-spreading of cancer cells to different sites in the body. A critical step in metastasis formation is invasion of cells through the surrounding tissue. During invasion, cancer cells change their shape and apply forces. We have previously identified that about 30% of single, metastatic, breast cancer cells will indent impenetrable synthetic, non-degradable, polyacrylamide gels, when gel stiffness is in the range 1–10 kPa. By measuring the depth of indentation of an initially flat gel and monitoring time-dependent microscopic changes in cell morphology, we were able to distinguish between benign and metastatic cells, also identifying their metastatic potential (MP); benign cells do not indent the gels. Recent works have indicated that metastases from solid tumors occur predominantly by collective cell invasion. Hence, in the current study we evaluate the mechanical interactions of cell clusters with the impenetrable gel. We observe that indenting subpopulations of metastatic cells are doubled in clusters, and cells are also indent more deeply; this increases likelihood to successfully form metastasis in the body. Concurrently, double the fraction of high MP cells indent gels as compared to low MP cells, while benign cells do not indent even in clusters. We also show that the gel platform can be used to determine the time-dependent impact of chemotherapeutics on the cells’ ability to apply forces and indent gels. Our approach can provide a rapid, mechanical prediction of the likelihood for invasiveness of cancer cells and can further be applied in a patient-specific approach, thus providing a personalized prognosis that may improve treatment of cancer patients and increase their life expectancy.

Purpose: One possible source of chronic low back pain is a degenerated intervertebral disc. In this review, various diagnostic methods for the assessment of the presence of degenerative changes are described. These include clinical MRI, a number of novel MRI techniques and nuclear magnetic resonance spectroscopy. Methods: Non-systematic literature review. Results: Clinical MRI is the most commonly employed technique to determine the general "health status" of the intervertebral disc. Novel MRI techniques, such as quantitative MRI, T1¿ MRI, sodium MRI and nuclear magnetic resonance spectroscopy, are more sensitive in quantifying the biochemical changes of disc degeneration, as measured by alteration in collagen structure, as well as water and proteoglycan loss. As potential future diagnostic alternatives, miniature sensors are currently being developed to measure parameters associated with the disc degeneration cascade, such as intradiscal pressure and PG concentration. However, none of the methods listed above show sufficient specificity to identify a degenerated disc as the actual source of the pain. Provocative discography is the only test aimed at a direct diagnosis of discogenic pain, but it has a high false positive rate and there is some evidence of long-term adverse effects. Imaging techniques have also been tested for this purpose, but their validity has not been confirmed and they do appear to be problematic. Conclusions: A reliable diagnostic tool that could help a clinician to determine if a disc is the source of the pain in patients with chronic LBP is still not available. New MRI techniques are under investigation that could result in a significant improvement over current methods, particularly as they can allow monitoring, not only of morphological but also of biochemical changes. © 2013 Springer-Verlag.

The intervertebral disc (IVD) is a complex cartilaginous structure which functions to resist biomechanical loads during spinal movement. It consists of the highly viscous cartilaginous nucleus pulposus, which is surrounded laterally by a thick outer ring of fibrous cartilage—the annulus fibrosus—and sandwiched inferiorly and superiorly by the cartilage end-plates. The main extracellular matrix molecules of the disc are collagens, proteoglycans, glycoproteins and elastin. The disc also contains appreciable amounts of water, matrix-degrading protease enzymes and their inhibitors, soluble signalling molecules and various metabolic breakdown products. This review provides a comprehensive description of the biochemical composition of the extracellular matrix of the IVD and, specifically, the proteases involved in its molecular turnover. Quantitation of the turnover rates using racemization of aspartic acid as a molecular clock is also discussed. Molecular turnover rates of the major constituent matrix macromolecules of the IVD are found to be particularly slow, especially in the case of collagen. Over a normal human life span, this slow turnover may compromise the structural integrity of the IVD extracellular matrix essential for normal physiological functioning.

The load-bearing biomechanical role of the intervertebral disc is governed by the composition and organization of its major macromolecular components, collagen and aggrecan. The major function of aggrecan is to maintain tissue hydration, and hence disc height, under the high loads imposed by muscle activity and body weight. Key to this role is the high negative fixed charge of its glycosaminoglycan side chains, which impart a high osmotic pressure to the tissue, thus regulating and maintaining tissue hydration and hence disc height under load. In degenerate discs, aggrecan degrades and is lost from the disc, particularly centrally from the nucleus pulposus. This loss of fixed charge results in reduced hydration and loss of disc height; such changes are closely associated with low back pain. The present authors developed biomimetic glycosaminoglycan analogues based on sulphonate-containing polymers. These biomimetics are deliverable via injection into the disc where they polymerize in situ, forming a non-degradable, nuclear "implant" aimed at restoring disc height to degenerate discs, thereby relieving back pain. In vitro, these glycosaminoglycan analogues possess appropriate fixed charge density, hydration and osmotic responsiveness, thereby displaying the capacity to restore disc height and function. Preliminary biomechanical tests using a degenerate explant model showed that the implant adapts to the space into which it is injected and restores stiffness. These hydrogels mimic the role taken by glycosaminoglycans in vivo and, unlike other hydrogels, provide an intrinsic swelling pressure, which can maintain disc hydration and height under the high and variable compressive loads encountered in vivo. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Using a surface force balance, normal and shear interactions have been measured between two atomically smooth surfaces coated with hyaluronan (HA), and with HA/aggrecan (Agg) complexes stabilized by cartilage link protein (LP). Such HA/Agg/LP complexes are the most abundant mobile macromolecular species permeating articular cartilage in synovial joints and have been conjectured to be present as boundary lubricants at its surface. The aim of the present study is to gain insight into the extremely efficient lubrication when two cartilage surfaces slide past each other in healthy joints, and in particular to elucidate the possible role in this of the HA/Agg/LP complexes. Within the range of our parameters, our results reveal that the HA/Agg/LP macromolecular surface complexes are much better boundary lubricants than HA alone, likely because of the higher level of hydration, due to the higher charge density, of the HA/Agg/LP layers with respect to the HA alone. However, the friction coefficients (μ) associated with the mutual interactions and sliding of opposing HA/Agg/LP layers (μ ≈ 0.01 up to pressure P of ca. 12 atm, increasing sharply at higher P) suggest that such complexes by themselves cannot account for the remarkable boundary lubrication observed in mammalian joints (up to P > 50 atm).

Osteoarthritis (OA) is a disease of joints, affecting a large number of people worldwide. One of the symptoms of OA is wear of articular cartilage; it is thought that among other factors this may be due to failure of lubrication. Injection of bio-lubricants into a joint may remedy this problem. Wear of cartilage and its prevention is a focus of much interest. The present paper describes wear tests performed using human cartilage on cartilage under various working conditions. Several techniques assessing wear are described, such as changes in surface morphology using optical profilometry and variation in the content of collagen and proteoglycans (PG) in the lubricating solution. Of all these techniques the PG content analysis was found to be the most efficient one.

Fluid balance in the intervertebral disc under applied load is determined primarily by its swelling pressure, that is, the external pressure at which it neither loses nor gains water. This depends on the composition of the tissue, in particular on its proteoglycan concentration. Proteoglycans develop a high osmotic pressure due to their fixed negatively charged groups. Because of their size, proteoglycans are excluded from the collagen's intrafibrillar volume; hence their osmotic activity is determined only by the extrafibrillar water. Here, we show that in order to evaluate correctly the swelling pressure in the annuli fibrosi of human intervertebral disc, it is essential to evaluate its proportion of intrafibrillar water. We used low-angle X-ray scattering and osmotic stress techniques to determine the lateral packing of the collagen molecules in the fibrils of the annuli fibrosi (ages: 25-77). It was found that the lateral packing and, hence, the intrafibrillar water content depends on age, external osmotic pressure, and location in the tissue. Subtracting intrafibrillar water from total hydration yields the amount of extrafibrillar water, from which the true fixed charge density of the tissue could be estimated. From a force balance, it would appear that collagen tension plays only a minor role in the equilibrium of the human intervertebral disc under load, in contrast to articular cartilage, where collagen tension is important for load bearing.

Efficient lubrication is essential for synovial joint mobility in both health and disease. It is well known that extremely low friction is required for proper functioning of synovial joints. In several medical treatments, bio-lubricants are injected into human joints to maintain their proper functioning. In the course of developing and screening such bio-lubricants, it is important to measure their effect under conditions similar to the ones in vivo. To this end, a first attempt was made to test the friction of two slices of human articular cartilage sliding over each other under various working conditions in the presence of different lubricating fluids. The results can be used for future research in the field of joint lubrication.

Introduction: Paclitaxel (Taxol) is chemotherapy drug widely used to treat different types of solid tumors (e.g. ovarian, breast, and pancreatic). Taxol acts by hyper-stabilizing microtubules, inhibiting mitosis, and eventually causing cell apoptosis. The change in cytoskeleton dynamics likely influences the cell's ability to apply force and change shape; both are required for metastatic invasion. We have previously shown that the mechanobiological invasiveness of metastatic cells can be determined through their mechanical interactions with a synthetic polyacrylamide gel; metastatic cells indent gels in attempted invasion, while benign cells do not. Objectives: In the current study, we reveal reduction due to Taxol treatment in the mechanobiological invasiveness of metastatic cancer cells, as measured by their ability to forcefully indent elastic gels. Methods: We evaluate the mechanical interaction of two human, metastatic breast cell lines, MDA-MB-231 and MDA-MB-468, with a physiological-stiffness, synthetic, non-degradable and impenetrable polyacrylamide gel. We evaluate the effects of Taxol at concentrations of 5-25μM. Using an inverted, epifluorescence microscope we obtain images of the cells and the fluorescent particles embedded at the gel surface. When cells indent the gels, the particles at the indentation location (beneath the cells) are pushed to lower focal depths. The indentation depth is then calculated by the difference in focal depths for each individual cell within a group. Results: Treatment with Taxol rapidly (l 2 hours) reduces the number of indenting cells as well as the indentation depth. Specifically, the cells exhibit smaller indentations and lose their ability to synergistically and collectively apply force. In addition, the effect of Taxol increases linearly with drug concentration, and depends on the cancer cells' metastatic potential. Discussion: Our synthetic gel-platform shows drug-induced reduction in the mechanobiological invasiveness capacity of metastatic cells. This approach may be extended to patient-personalized testing of drugs and optimization of drug-treatment protocols.