Protease Inhibition Mechanism of Camelid-like Synthetic Human Antibodies.

Macromolecular protease inhibitors and camelid single-domain antibodies achieve their enzymic inhibition functions often through protruded structures that directly interact with catalytic centers of targeted proteases. Inspired by this phenomenon, we constructed synthetic human antibody libraries encoding long CDR-H3s, from which highly selective monoclonal antibodies (mAbs) that inhibit multiple proteases were discovered. To elucidate their molecular mechanisms, we performed in-depth biochemical characterizations on a panel of matrix metalloproteinase (MMP)-14 inhibitory mAbs. Assays included affinity and potency measurements, enzymatic kinetics, a competitive enzyme-linked immunosorbent assay, proteolytic stability, and epitope mapping followed by quantitative analysis of binding energy changes. The results collectively indicated that these mAbs of convex paratopes were competitive inhibitors recognizing the vicinity of the active cleft, with their significant epitopes scattered across the north and south rims of the cleft. Remarkably, identified epitopes were the surface loops that were highly diverse among MMPs and predominately located at the prime side of the proteolytic site, shedding light on the mechanisms of target selectivity and proteolytic resistance. Substrate sequence profiling and paratope mutagenesis further suggested that mAb 3A2 bound to the active-site cleft in a canonical (substrate-like) manner, by direct interactions between 100hNLVATP100m of its CDR-H3 and subsites S1-S5' of MMP-14. Overall, synthetic mAbs carrying convex paratopes can achieve efficient inhibition and thus hold great therapeutic promise for effectively and safely targeting biomedically important proteases.

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries.

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 109 individual variants) that carried the extended, 23- to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes.

Identification of highly selective MMP-14 inhibitory Fabs by deep sequencing.

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10-4,000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. Biotechnol. Bioeng. 2017;114: 1140-1150. © 2017 Wiley Periodicals, Inc.

Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli.

BACKGROUND: As regulators of multifunctional metalloproteinases including MMP, ADAM and ADAMTS families, tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in extracellular matrix remodeling, which is involved in a wide variety of physiological processes. Since abnormal metalloproteinase activities are related to numerous diseases such as arthritis, cancer, atherosclerosis, and neurological disorders, TIMPs and their engineered mutants hold therapeutic potential and thus have been extensively studied. Traditional productions of functional TIMPs and their N-terminal inhibitory domains (N-TIMPs) rely on costly and time-consuming insect and mammalian cell systems, or tedious and inefficient refolding from denatured inclusion bodies. The later process is also associated with heterogeneous products and batch-to-batch variation. RESULTS: In this study, we developed a simple approach to directly produce high yields of active TIMPs in the periplasmic space of Escherichia coli without refolding. Facilitated by disulfide isomerase (DsbC) co-expression in protease-deficient strain BL21 (DE3), N-TIMP-1/-2 and TIMP-2 which contain multiple disulfide bonds were produced without unwanted truncations. 0.2-1.4 mg purified monomeric TIMPs were typically yielded per liter of culture media. Periplasmically produced TIMPs exhibited expected inhibition potencies towards MMP-1/2/7/14, and were functional in competitive ELISA to elucidate the binding epitopes of MMP specific antibodies. In addition, prepared N-TIMPs were fully active in a cellular context, i.e. regulating cancer cell morphology and migration in 2D and 3D bioassays. CONCLUSION: Periplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.

Direct production of functional matrix metalloproteinase--14 without refolding or activation and its application for in vitro inhibition assays.

Human matrix metalloproteinase (MMP)-14, a membrane-bound zinc endopeptidase, is one of the most important cancer targets because it plays central roles in tumor growth and invasion. Large amounts of active MMP-14 are required for cancer research and the development of chemical or biological MMP-14 inhibitors. Current methods of MMP-14 production through refolding and activation are labor-intensive, time-consuming, and often associated with low recovery rates, lot-to-lot variation and heterogeneous products. Here, we report direct production of the catalytic domain of MMP-14 in the periplasmic space of Escherichia coli. 0.5 mg/L of functional MMP-14 was produced without tedious refolding or problematic activation process. MMP-14 prepared by simple periplasmic treatment can be readily utilized to evaluate the potencies of chemical and antibody-based inhibitors. Furthermore, co-expression of both MMP-14 and antibody Fab fragments in the periplasm facilitated inhibitory antibody screening by avoiding purification of MMP-14 or Fabs. We expect this MMP-14 expression strategy can expedite the development of therapeutic drugs targeting MMPs with biological significance.

Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.

Circadian Rhythms in Tongue Features.

(1) Background: The aim of this study was to investigate the circadian rhythms of tongue features according to the effects of physiological phases over a 24 h period. (2) Methods: Fifteen healthy participants aged 20 to 69 years were recruited. The participants did not have current chronic diseases or past diseases and had to meet the inclusion and exclusion criteria. The participants stayed at the Gil Hospital for a duration of 2 nights and 3 days. On the first day, at 18:00, they consumed their allocated portions of food and water and then completed a questionnaire. At approximately 21:00, their tongue images were acquired using a computerized tongue image acquisition system, following which they slept for 8 h, commencing at 23:00. Measurements were taken from 07:00 through 21:00 on the second day, and the final acquisition was taken at 07:00 on the following morning, resulting in a total of eight images. The circadian rhythm was authenticated and quantified utilizing the single cosinor analysis, a technique for periodic regression analysis for fitting a 24 h cosine curve. (3) Results: Cosinor analysis revealed that all tongue features were significantly related to circadian rhythm. (4) Conclusions: The results of this study may be important for considering the time of day at which the tongue is observed and tongue status is evaluated.

Development of a periplasmic FRET screening method for protease inhibitory antibodies.

Proteases play critical roles in numerous physiological processes and thus represent one of the largest families of potential pharmaceutical targets. Previous failure of broad-spectrum small molecule inhibitors toward tumorigenic metalloproteinases in clinical trials emphasizes that selectivity is the key for a successful protease-inhibition therapy. With exquisite specificity, antibody-based inhibitors are emerging as promising therapeutics. However, the majority of current antibody selection technologies are based on binding and not on inhibition. Here, we report the development of a function-based inhibitory antibody screening method, which combines a simple periplasmic preparation and an ultra sensitive FRET assay, both processes are amenable to high-throughput applications. Using this method, inhibitory antibodies can be rapidly distinguished from non-inhibitory clones with satisfactory Z-factors. Coupled with ELISA, this method also provides a fast semi-quantitative estimation of IC50 values without antibody purification. We expect this technology to greatly facilitate the generation of highly selective biologic inhibitors, targeting many proteases that are important to medical research and therapeutic development.

Measurement of spatial pulse wave velocity by using a clip-type pulsimeter equipped with a Hall sensor and photoplethysmography.

A prototype of a clip-type pulsimeter equipped with a magnetic field-sensing semiconductor Hall sensor was developed. It has a permanent magnet attached in the "Chwan" position to the center of a radial artery. The clip-type pulsimeter is composed of a hardware system measuring voltage signals. To measure spatial pulse wave velocity (SPWV), the signal from the radial artery pulsimeter and that from the photoplethysmography (PPG) were simultaneously compared. The pulse wave data from a clinical test of 39 clinical participants (male:female = 25:14) with a mean age of 24.36 (±2.35) years was analyzed. The mean SPWV, which was simultaneously measured from the radial artery pulsimeter and PPG, was 0.8 m/s. We suggest the SPWV results were higher for men than women, because of the better vascularity of terminal tissue in men. The findings of this research may be useful for developing a biomedical signal storage device for a U-health-care system.

Predicting of tunneling resistivity between adjacent nanosheets in graphene-polymer systems.

In this work, the tunneling resistivity between neighboring nanosheets in grapheme-polymer nanocomposites is expressed by a simple equation as a function of the characteristics of graphene and tunnels. This expression is obtained by connecting two advanced models for the conductivity of graphene-filled materials reflecting tunneling role and interphase area. The predictions of the applied models are linked to the tested data of several samples. The impressions of all factors on the tunneling resistivity are evaluated and interpreted using the suggested equation. The calculations of tunneling resistivity for the studied examples by the model and suggested equation demonstrate the same levels, which confirm the presented methodology. The results indicate that the tunneling resistivity decreases by super-conductive graphene, small tunneling width, numerous contacts among nanosheets and short tunneling length.

Decellularized allogeneic cartilage paste with human costal cartilage and crosslinked hyaluronic acid-carboxymethyl cellulose carrier augments microfracture for improved articular cartilage repair.

Articular cartilage lacks natural healing abilities and necessitates surgical treatments for injuries. While microfracture (MF) is a primary surgical approach, it often results in the formation of unstable fibrocartilage. Delivering hyaline cartilage directly to defects poses challenges due to the limited availability of autologous cartilage and difficulties associated with allogeneic cartilage delivery. We developed a decellularized allogeneic cartilage paste (DACP) using human costal cartilage mixed with a crosslinked hyaluronic acid (HA)-carboxymethyl cellulose (CMC) carrier. The decellularized allogeneic cartilage preserved the extracellular matrix and the nanostructure of native hyaline cartilage. The crosslinked HA-CMC carrier provided shape retention and moldability. In vitro studies confirmed that DACP did not cause cytotoxicity and promoted migration, proliferation, and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. After 6 months of implantation in rabbit knee osteochondral defects, DACP combined with MF outperformed MF alone, demonstrating improved gait performance, defect filling, morphology, extracellular matrix deposition, and biomechanical properties similar to native cartilage. Thus, DACP offers a safe and effective method for articular cartilage repair, representing a promising augmentation to MF. STATEMENT OF SIGNIFICANCE: Directly delivering hyaline cartilage to repair articular cartilage defects is an ideal treatment. However, current allogeneic cartilage products face delivery challenges. In this study, we developed a decellularized allogeneic cartilage paste (DACP) by mixing human costal cartilage with crosslinked hyaluronic acid (HA)-carboxymethyl cellulose (CMC). DACP preserves extracellular matrix components and nanostructures similar to native cartilage, with HA-CMC ensuring shape retention and moldability. Our study demonstrates improved cartilage repair by combining DACP with microfracture, compared to microfracture alone, in rabbit knee defects over 6 months. This is the first report showing better articular cartilage repair using decellularized allogeneic cartilage with microfracture, without the need for exogenous cells or bioactive substances.

Polydopamine-Functionalized Bacterial Cellulose as Hydrogel Scaffolds for Skin Tissue Engineering.

Bacterial cellulose (BC) is a natural polysaccharide polymer hydrogel produced sustainably by the strain Gluconacetobacter hansenii under static conditions. Due to their biocompatibility, easy functionalization, and necessary physicochemical and mechanical properties, BC nanocomposites are attracting interest in therapeutic applications. In this study, we functionalized BC hydrogel with polydopamine (PDA) without toxic crosslinkers and used it in skin tissue engineering. The BC nanofibers in the hydrogel had a thickness of 77.8 ± 20.3 nm, and they could be used to produce hydrophilic, adhesive, and cytocompatible composite biomaterials for skin tissue engineering applications using PDA. Characterization techniques, namely Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), and Raman spectroscopy, were performed to investigate the formation of polydopamine on the BC nanofibers. The XRD peaks for BC occur at 2θ = 14.65°, 16.69°, and 22.39°, which correspond to the planes of (100), (010), and (110) of cellulose type Iα. Raman spectroscopy confirmed the formation of PDA, as indicated by the presence of bands corresponding to the vibration of aromatic rings and aliphatic C-C and C-O stretching at 1336 and 1567 cm-1, respectively. FTIR confirmed the presence of peaks corresponding to PDA and BC in the BC/PDA hydrogel scaffolds at 3673, 3348, 2900, and 1052 cm-1, indicating the successful interaction of PDA with BC nanofibers, which was further corroborated by the SEM images. The tensile strength, swelling ratio, degradation, and surface wettability characteristics of the composite BC biomaterials were also investigated. The BC/PDA hydrogels with PDA-functionalized BC nanofibers demonstrated excellent tensile strength and water-wetting ability while maintaining the stability of the BC fibers. The enhanced cytocompatibility of the BC/PDA hydrogels was studied using the PrestoBlue assay. Culturing murine NIH/3T3 fibroblasts on BC/PDA hydrogels showed higher metabolic activity and enhanced proliferation. Additionally, it improved cell viability when using BC/PDA hydrogels. Thus, these BC/PDA composite biomaterials can be used as biocompatible natural alternatives to synthetic substitutes for skin tissue engineering and wound-dressing applications.

A Clinical Study on the Relationship Among Insomnia, Tongue Diagnosis, and Oral Microbiome.

Currently, there is a lack of adequate methods to assess insomnia objectively. This study addresses the usefulness of tongue features and oral microbial profile as a potential diagnostic biomarker of insomnia. One hundred insomniac patients and 20 healthy control subjects were selected. Their demographic and clinical characteristics, as well as the tongue diagnostic indices and oral microbial profile, were examined. Compared to the control group, insomniac patients showed a higher abnormal low-frequency/high-frequency (LF/HF) ratio. In tongue diagnosis, the indices related to lightness of tongue body and tongue coating were higher in the insomniac group vs. the control group. Furthermore, linear discriminant analysis (LDA) of oral microbial population revealed that the relative abundances of Clostridia, Veillonella, Bacillus and Lachnospiraceae were significantly higher in the insomniac patients than the control group. Additionally, the tongue features of the insomniac group exhibited that the non-coating group had a poor sleep condition compared to the thick-coating group, although the difference was insignificant. On the other hand, the oral microbial communities of the insomniac patients revealed greater alpha and beta diversities in the non-coating group vs. the thick-coating group. The alpha and beta diversities were higher in orotype1 than orotype2. Collectively, this study highlighted that the lightness of tongue body and tongue coating as well as oral microbial profiles of SR1, Actinobacteria, Clostridia and Lachnospiraceae_unclassified could be considered potential biomarkers of insomnia.

Measurement of blood pressure using an arterial pulsimeter equipped with a Hall device.

To measure precise blood pressure (BP) and pulse rate without using a cuff, we have developed an arterial pulsimeter consisting of a small, portable apparatus incorporating a Hall device. Regression analysis of the pulse wave measured during testing of the arterial pulsimeter was conducted using two equations of the BP algorithm. The estimated values of BP obtained by the cuffless arterial pulsimeter over 5 s were compared with values obtained using electronic or liquid mercury BP meters. The standard deviation between the estimated values and the measured values for systolic and diastolic BP were 8.3 and 4.9, respectively, which are close to the range of values of the BP International Standard. Detailed analysis of the pulse wave measured by the cuffless radial artery pulsimeter by detecting changes in the magnetic field can be used to develop a new diagnostic algorithm for BP, which can be applied to new medical apparatus such as the radial artery pulsimeter.

The Herbal Formula CWBSD Improves Sleep Quality Dependent on Oral Microbial Type and Tongue Diagnostic Features in Insomnia.

Cheonwangbosim-dan (CWBSD) is a traditional Korean herb formula that has been widely prescribed for insomnia patients with a heart-yin deficiency (HYD) pattern. Several studies have reported that heart function and insomnia are interrelated, and few have explored associations between insomnia, oral microbiota, and tongue diagnosis. This study aimed to evaluate the effects of CWBSD on primary insomnia, tongue diagnosis, and oral microbiota. At baseline, 56 patients with primary insomnia were assigned to two groups, a HYD group and a non-HYD (NHYD) group and they took CWBSD for 6 weeks. During the study, Pittsburgh Sleep Quality Indices (PSQIs) and Insomnia Severity Indices (ISIs) decreased significantly in both groups. However, the PSQI reduction observed in the HYD group was greater than in the NHYD group and sleep times increased only in the HYD group. As sleep quality improved, the amount of tongue coating increased at the posterior tongue, where heart function appears. At baseline, the HYD and NHYD group had a specific oral microbiota (Veillonella at genus level), but no significant change was observed after taking CWBSD. Additionally, subjects were divided into two oral microbiota types ("orotypes"). The genera Prevotella, Veillonella, or Neisseria were abundant in each orotype. The reduction in PSQI in orotype 1 during the 6-week treatment period was greater than in orotype 2. In conclusion, this study shows that CWBSD could be used to treat primary insomnia in patients with a HYD pattern as determined using tongue diagnosis and oral microbiota distributional patterns.

Reliability, Accuracy, and Use Frequency of Evaluation Methods for Amount of Tongue Coating.

OBJECTIVE: To classify the evaluation methods for amount of tongue coating (TC) and investigate their reliability, accuracy, and frequency of use. METHODS: Articles published from 1985 to 2015 were searched for evaluation methods for the amount of TC in PubMed and the Cochrane Library. Only clinical researches were included except protocol articles. The methods were classified according to their characteristics. RESULTS: Finally, 113 articles were selected. The evaluation method for the amount of TC from the articles was classified into 4 types: intuitive, specificative, computerized, and weighing TC. The reliability in the intuitive and specificative methods (κ =0.33-0.92) showed varying levels among the studies. In general, the amount of TC calculated by the specificative method (Spearman's r=0.68-0.80) was more strongly related to the directly measured value than to the value estimated by the computerized method (Pearson's r=0.442). The number of articles published on this topic has increased consistently, and the specificative method was the most frequently used. Despite the higher reliability of the computerized method, it has not been widely used. CONCLUSIONS: The high prevalence of the specificative method would continue in clinical practice because of its convenience and accuracy. However, to establish higher reliability, the limitation of the subjectivity of the assessors should be overcome through calibration training. In the computerized method, novel algorithms are needed to obtain a higher accuracy so that it can help the practitioners confidently estimate the amount of TC.

Comparing Verum and Sham Acupuncture in Fibromyalgia Syndrome: A Systematic Review and Meta-Analysis.

OBJECTIVES: Acupuncture is often used for relieving symptoms of fibromyalgia syndrome (FMS). Our aim is to ascertain whether verum acupuncture is more effective than sham acupuncture in FMS. METHODS: We collected RCTs to investigate the effects of verum acupuncture and sham acupuncture on pain, sleep quality, fatigue, and general status in FMS patients. The databases used for data retrieval were PubMed, Central Cochrane, EMBASE, PsycINFO, CNKI, VIP, OASIS, KoreaMed, and RISS. Selection/exclusion from the retrieved records was performed according to prespecified criteria, and the final selected records were assessed according to the Cochrane risk of bias tool. The results of the included trials were synthesized on the basis of outcomes, and subgroup analysis depended on the type of add-on sham acupuncture that was performed. RESULTS: Ten RCTs (690 participants) were eligible, and eight RCTs were eventually included in the meta-analysis. The synthesis showed a sizable effect of verum acupuncture compared with sham acupuncture on pain relief (standardized mean difference (SMD) -0.49, Z = 3.26, P=0.001; I 2 = 59%), improving sleep quality (SMD -0.46, Z = 3.24, P=0.001; I 2 = 0%), and reforming general status (SMD -0.69, Z = 6.27, P < 0.00001; I 2 = 4%). However, efficacy on fatigue was insignificant (SMD -0.10, Z = 0.51, P=0.61; I 2 = 46%). When compared with a combination of simulation and improper location of needling, the effect of verum acupuncture for pain relief was the most obvious. CONCLUSIONS: Verum acupuncture is more effective than sham acupuncture for pain relief, improving sleep quality, and reforming general status in FMS posttreatment. However, evidence that it reduces fatigue was not found.

Association between tongue coating thickness and ultraviolet fluorescence in patients with functional dyspepsia: A prospective observational study.

The aim of this study was to examine the correlation between the tongue coating thickness (TCT) and ultraviolet (UV) fluorescence and propose a new method for the estimation of TCT using a computerized tongue image acquisition system (CTIS).In this prospective and observational single-center study, we acquired tongue images under visible light and near-UV light for 60 patients with functional dyspepsia. Tongue images were acquired twice within a 30-minute interval to assess the reliability of CTIS. Then, the tongue coating was scraped and weighed to derive the wet weight of the tongue coating (WWTC). The percentage of the tongue coating area was calculated from the tongue images acquired under visible light. Mean color values (mCVs) for the UV fluorescence of the dorsal surface of the tongue were also computed.The reliabilities of the derived mCVs and percentage of the tongue coating area were acceptable (intraclass correlation coefficients, 0.907-0.947). The mCVs were more strongly correlated with WWTC than with the area, with mCV of modified lightness showing the strongest association (r = 0.785, P < .01). Finally, we suggested an estimation model for TCT based on the results.The results of this study suggest that both UV fluorescence of the dorsal tongue and the distribution area of tongue coating are useful parameters for the quantitative assessment of tongue coating. We believe that these findings will contribute to the development of a clinically useful CTIS.

Degradation and Remodeling of Epitaxially Grown Collagen Fibrils.

INTRODUCTION­: The extracellular matrix (ECM) in the tumor microenvironment contains high densities of collagen that are highly aligned, resulting in directional migration called contact guidance that facilitates efficient migration out of the tumor. Cancer cells can remodel the ECM through traction force controlled by myosin contractility or proteolytic activity controlled by matrix metalloproteinase (MMP) activity, leading to either enhanced or diminished contact guidance. METHODS­: Recently, we have leveraged the ability of mica to epitaxially grow aligned collagen fibrils in order to assess contact guidance. In this article, we probe the mechanisms of remodeling of aligned collagen fibrils on mica by breast cancer cells. RESULTS­: We show that cells that contact guide with high fidelity (MDA-MB-231 cells) exert more force on the underlying collagen fibrils than do cells that contact guide with low fidelity (MTLn3 cells). These high traction cells (MDA-MB-231 cells) remodel collagen fibrils over hours, pulling so hard that the collagen fibrils detach from the surface, effectively delaminating the entire contact guidance cue. Myosin or MMP inhibition decreases this effect. Interestingly, blocking MMP appears to increase the alignment of cells on these substrates, potentially allowing the alignment through myosin contractility to be uninhibited. Finally, amplification or dampening of contact guidance with respect to a particular collagen fibril organization is seen under different conditions. CONCLUSIONS­: Both myosin II contractility and MMP activity allow MDA-MB-231 cells to remodel and eventually destroy epitaxially grown aligned collagen fibrils.

Generation of Highly Selective MMP Antibody Inhibitors.

Inhibiting individual MMPs of biomedical importance with high selectivity is critical for both fundamental research and therapy development. Here we describe the methods for discovery of inhibitory monoclonal antibodies from synthetic human antibody phage display libraries carrying convex paratopes encoded by long complementarity-determining region (CDR)-H3 segments. We demonstrate the application of this technique for isolation of highly specific and potent antibody inhibitors of human MMP-14.