Tubulin CFEOM mutations both inhibit or activate kinesin motor activity.

Kinesin-mediated transport along microtubules is critical for axon development and health. Mutations in the kinesin Kif21a, or the microtubule subunit ß-tubulin, inhibit axon growth and/or maintenance resulting in the eye-movement disorder congenital fibrosis of the extraocular muscles (CFEOM). While most examined CFEOM-causing ß-tubulin mutations inhibit kinesin-microtubule interactions, Kif21a mutations activate the motor protein. These contrasting observations have led to opposed models of inhibited or hyperactive Kif21a in CFEOM. We show that, contrary to other CFEOM-causing ß-tubulin mutations, R380C enhances kinesin activity. Expression of ß-tubulin-R380C increases kinesin-mediated peroxisome transport in S2 cells. The binding frequency, percent motile engagements, run length and plus-end dwell time of Kif21a are also elevated on ß-tubulin-R380C compared with wildtype microtubules in vitro. This conserved effect persists across tubulins from multiple species and kinesins from different families. The enhanced activity is independent of tail-mediated kinesin autoinhibition and thus utilizes a mechanism distinct from CFEOM-causing Kif21a mutations. Using molecular dynamics, we visualize how ß-tubulin-R380C allosterically alters critical structural elements within the kinesin motor domain, suggesting a basis for the enhanced motility. These findings resolve the disparate models and confirm that inhibited or increased kinesin activity can both contribute to CFEOM. They also demonstrate the microtubule's role in regulating kinesins and highlight the importance of balanced transport for cellular and organismal health.

Pharmacokinetics and safety of inhaled oxytocin compared with intramuscular oxytocin in women in the third stage of labour: A randomized open-label study.

AIMS: To compare pharmacokinetics (PK) and safety of heat-stable inhaled (IH) oxytocin with intramuscular (IM) oxytocin in women in third stage of labour (TSL), the primary endpoint being PK profiles of oxytocin IH and secondary endpoint of safety. METHODS: A phase 1, randomized, cross-over study was undertaken in 2 UK and 1 Australian centres. Subjects were recruited into 2 groups: Group 1, women in TSL; Group 2, nonpregnant women of childbearing potential (Cohort A, combined oral contraception; Cohort B, nonhormonal contraception). Participants were randomized 1:1 to: Group 1, oxytocin 10 IU (17 µg) IM or oxytocin 240 IU (400 µg) IH immediately after delivery; Group 2, oxytocin 5 IU (8.5 µg) intravenously and oxytocin 240 IU (400 µg) IH at 2 separate dosing sessions. RESULTS: Participants were recruited between 23 November 2016 to 4 March 2019. In Group 1, 17 participants were randomized; received either IH (n = 9) or IM (n = 8) oxytocin. After IH and IM administration, most plasma oxytocin concentrations were below quantification limits (2 pg/mL). In Group 2 (n = 14), oxytocin IH concentrations remained quantifiable ≤3 h postdose. Adverse events were reported in both groups, with no deaths reported: Group 1, IH n = 3 (33%) and IM n = 2 (25%); Group 2, n = 14 (100%). CONCLUSION: Safety profiles of oxytocin IH and IM were similar. However, PK profiles could not be established for oxytocin IH or IM in women in TSL, despite using a highly sensitive and specific assay.

Exploring the role of ex vivo metabolism on blood and plasma measurements of oxytocin among women in the third stage of labour: A post hoc study.

AIMS: To examine the role of ex vivo oxytocin metabolism in post-dose peptide measurements. METHODS: The stability of oxytocin (Study 1) and oxytocinase activity (Study 2) in late-stage pregnancy blood was quantified using liquid-chromatography tandem mass-spectrometry (LC-MS/MS) and a fluorogenic assay, respectively. Analyses were conducted using blood from pregnant women (>36 weeks gestation) evaluated in lithium heparin (LH), ethylenediaminetetraacetic acid (EDTA) and BD P100 blood collection tubes with or without protease inhibitors. In addition, plasma oxytocin concentrations following administration of oxytocin 240 IU inhaled, 5 IU intravenous or 10 IU intramuscular in women in third stage of labour (TSL) were analysed using enzyme-linked immunosorbent assay (ELISA) and LC-MS/MS to understand how quantified peptide concentrations differ between these analytical methods (Study 3). RESULTS: Study 1: Oxytocin was stable in blood collected into EDTA tubes with or without protease inhibitors but not in LH tubes. Study 2: Blood collected into all EDTA-containing collection tubes led to near-complete inhibition of oxytocinase (≤100 min). In plasma, a 35% reduction in oxytocinase activity was observed in LH tubes with EDTA added. In plasma from late-stage pregnancy compared to nonpregnant participants, the oxytocinase activity was approximately 11-fold higher. Study 3: Plasma oxytocin concentrations from nonpregnant or women in TSL following exogenous oxytocin administration were ≤33 times higher when analysed using ELISA vs. LC-MS/MS methods. CONCLUSIONS: Collection of blood from late-stage pregnant women into tubes containing EDTA inhibits oxytocinase effectively stabilizing oxytocin, suggesting low concentrations of oxytocin after dose administration reflect rapid in vivo metabolism.

Pharmacodynamic evaluation and safety assessment of treatment with antibodies to serum amyloid P component in patients with cardiac amyloidosis: an open-label Phase 2 study and an adjunctive immuno-PET imaging study.

BACKGROUND: In a Phase I study treatment with the serum amyloid P component (SAP) depleter miridesap followed by monoclonal antibody to SAP (dezamizumab) showed removal of amyloid from liver, spleen and kidney in patients with systemic amyloidosis. We report results from a Phase 2 study and concurrent immuno-positron emission tomography (PET) study assessing efficacy, pharmacodynamics, pharmacokinetics, safety and cardiac uptake (of dezamizumab) following the same intervention in patients with cardiac amyloidosis. METHODS: Both were uncontrolled open-label studies. After SAP depletion with miridesap, patients received ≤ 6 monthly doses of dezamizumab in the Phase 2 trial (n = 7), ≤ 2 doses of non-radiolabelled dezamizumab plus [89Zr]Zr-dezamizumab (total mass dose of 80 mg at session 1 and 500 mg at session 2) in the immuno-PET study (n = 2). Primary endpoints of the Phase 2 study were changed from baseline to follow-up (at 8 weeks) in left ventricular mass (LVM) by cardiac magnetic resonance imaging and safety. Primary endpoint of the immuno-PET study was [89Zr]Zr-dezamizumab cardiac uptake assessed via PET. RESULTS: Dezamizumab produced no appreciable or consistent reduction in LVM nor improvement in cardiac function in the Phase 2 study. In the immuno-PET study, measurable cardiac uptake of [89Zr]Zr-dezamizumab, although seen in both patients, was moderate to low. Uptake was notably lower in the patient with higher LVM. Treatment-associated rash with cutaneous small-vessel vasculitis was observed in both studies. Abdominal large-vessel vasculitis after initial dezamizumab dosing (300 mg) occurred in the first patient with immunoglobulin light chain amyloidosis enrolled in the Phase 2 study. Symptom resolution was nearly complete within 24 h of intravenous methylprednisolone and dezamizumab discontinuation; abdominal computed tomography imaging showed vasculitis resolution by 8 weeks. CONCLUSIONS: Unlike previous observations of visceral amyloid reduction, there was no appreciable evidence of amyloid removal in patients with cardiac amyloidosis in this Phase 2 trial, potentially related to limited cardiac uptake of dezamizumab as demonstrated in the immuno-PET study. The benefit-risk assessment for dezamizumab in cardiac amyloidosis was considered unfavourable after the incidence of large-vessel vasculitis and development for this indication was terminated. Trial registration NCT03044353 (2 February 2017) and NCT03417830 (25 January 2018).

Cancer cell migration in collagen-hyaluronan composite extracellular matrices.

Hyaluronan (HA) is a key component in the tumor microenvironment (TME) that participates in cancer growth and invasiveness. While the molecular weight (MW) dependent properties of HA can cause tumor-promoting and -repressing effects, the elevated levels of HA in the TME impedes drug delivery. The degradation of HA using hyaluronidases (HYALs), resulting in fragments of HA, is a way to overcome this, but the consequences of changes in HA molecular weight and concentration is currently unknown. Therefore, it is critical to understand the MW-dependent biological effects of HA. Here we examine the influence of HA molecular weight on biophysical properties that regulate cell migration and extracellular matrix (ECM) remodeling. In our study, we used vLMW, LMW and HMW HA at different physiologically relevant concentrations, with a particular interest in correlating the mechanical and structural properties to different cell functions. The elastic modulus, collagen network pore size and collagen fiber diameter increased with increasing HA concentration. Although the collagen network pore size increased, these pores were filled with the bulky HA molecules. Consequently, cell migration decreased with increase in HA concentration due to multiple, long-lived and unproductive protrusions, suggesting the influence of steric factors. Surprisingly, even though elastic modulus increased with HA molecular weight and concentration, gel compaction assays showed an increased degree of ECM compaction among HMW HA gels at high concentrations (2 and 4 mg mL-1 [0.2 and 0.4%]). These results were not seen in collagen gels that lacked HA, but had similar stiffness. HA appears to have the effect of decreasing migration and increasing collagen network contraction, but only at high HA molecular weight. Consequently, changes in HA molecular weight can have relatively large effects on cancer cell behavior. STATEMENT OF SIGNIFICANCE: Hyaluronan (HA) is a critical component of the tumor microenvironment (TME). Overproduction of HA in the TME results in poor prognosis and collapse of blood vessels, inhibiting drug delivery. Hyaluronidases have been used to enhance drug delivery. However, they lead to low molecular weight (MW) HA, altering the mechanical and structural properties of the TME and cancer cell behavior. Understanding how HA degradation affects cancer cell behavior is critical for uncovering detrimental effects of this therapy. Very little is known about how HA MW affects cancer cell behavior in tumor-mimicking collagen-HA composite networks. Here we examine how MW and HA content in collagen-HA networks alter structural and mechanical properties to regulate cell migration and matrix remodeling in 3D TME-mimicking environments.

On chip detection of glial cell-derived neurotrophic factor secreted from dopaminergic cells under magnetic stimulation.

Glial cell-derived neurotrophic factor (GDNF) is a small protein potently promoting the survival of dopaminergic and motor neurons. GDNF can be secreted from different types of cells including the dopaminergic neural cell line, N27. N27 cells, a rat dopaminergic neural cell line, is regarded as a suitable in vitro model for Parkinson's disease (PD) research. For PD treatment, transcranial magnetic stimulation (TMS), a noninvasive therapeutic method, showed beneficial clinical effects, but the mechanism for its benefit is not understood. Because GDNF is a potent neurotrophic factor, it is of great value to evaluate if GDNF secretion from N27 cells can be affected by magnetic stimulation (MS). However, the current methods for detecting GDNF are time-consuming and expensive. In this paper we outline the detection of GDNF secretion from N27 cells by ultrasensitive nanopore thin film sensors (nanosensor) for the first time. As low as 2 pg/mL GDNF can be readily detected by the nanosensor. Furthermore, we show that MS can promote GDNF secretion from N27 cells. Specifically, the GDNF concentration in N27 cell-conditioned media under MS treatment shows statistically significant increase up to 2-fold after 5 days in vitro in comparison with the control. This nanosensor along with the in vitro PD model N27 cells provides a low-cost, easy-to-use, sensitive approach for studying potential cell biological mechanisms of the clinical benefits of MS on PD.

A Phase I Study to Show the Relative Bioavailability and Bioequivalence of Fixed-Dose Combinations of Ambrisentan and Tadalafil in Healthy Subjects.

PURPOSE: Pulmonary arterial hypertension (PAH) is a life-threatening disease that typically causes shortness of breath and exercise intolerance. Combination therapy with ambrisentan and tadalafil has proven to be more effective at preventing clinical failure events in patients with PAH than either drug alone. The aim of this study was to evaluate the bioequivalence of an ambrisentan/tadalafil fixed-dose combination (FDC) compared with co-administration of the 2 monotherapies. METHODS: This 3-part, randomized, single-dose, open-label crossover study was conducted in healthy volunteers. The first part of the study consisted of a 5-way crossover that compared the relative bioavailability of 4 FDC formulations (10-mg ambrisentan + 40-mg tadalafil) with co-administered reference monotherapies. One formulation was selected and its relative bioavailability was assessed when produced in 3 different granulation sizes during the second part of the study. In the third part of the study, the bioequivalence of the candidate FDC with the reference monotherapies was evaluated for the 10-mg/40-mg dose strength, in addition to 2 other dose strengths (5 mg/20 mg and 5 mg/40 mg). For all parts of the study, blood samples were taken at regular intervals after each dose, ambrisentan and tadalafil concentrations determined, and pharmacokinetic (PK) parameters (Cmax, AUC0-∞, and AUC0-t) obtained. Test/reference ratios of the geometric means of PK parameters were used to evaluate bioequivalence. Safety and tolerability were assessed by recording adverse events and monitoring vital signs, ECGs, and clinical laboratory data. FINDINGS: Of the 174 subjects screened for eligibility, 112 were allocated to a randomized treatment sequence across all study parts, and 100 completed their full assigned treatments. All 4 FDC formulations tested during part 1 of the study yielded PK parameters similar those of the reference treatments. In part 2, granulation size was found to not affect the relative bioavailability of the selected formulation. In part 3, the selected FDC was found to be bioequivalent to co-administration of the monotherapies in both the fasted and fed states. The FDC was also found to be bioequivalent to the reference treatments at the 2 additional dose strengths. All but one of the adverse events was mild to moderate in intensity, and no serious adverse events were reported. IMPLICATIONS: An ambrisentan/tadalafil FDC was bioequivalent to concurrently administered monotherapies and therefore represents a viable alternative treatment to co-administration. Use of an FDC is likely to be associated with reduced costs and improved patient compliance. ClinicalTrials.gov identifier: NCT02688387.

Tuning surface functionalization and collagen gel thickness to regulate cancer cell migration.

Cancer cells have a tremendous ability to sense and respond to extracellular matrix (ECM) stiffness, modulating invasion. The magnitude of the sensed stiffness can either promote or inhibit the migration of cancer cells out of the primary tumor into surrounding tissue. Work has been done on examining the role of stiffness in tuning cancer cell migration by controlling elastic modulus in the bulk. However, a powerful and complementary approach for controlling stiffness is to leverage interactions between stiff-soft (e.g. glass-hydrogel) interfaces. Unfortunately, most work in this area probes cells in 2D environments. Of the reports that probe 3D environments, none have assessed the role of mechanical linkage to the interface as a potential handle in controlling local stiffness and cell behavior. In this paper, we examine the migration of cancer cells embedded in a collagen fiber network between two flat plates. We examine the role of both surface attachment of the collagen network to the stiff interface as well as thickness (50-540 µm) of the collagen gel in driving collagen organization, cell morphology and cell migration. We find that surface attachment and thickness do not operate overlapping mechanisms, because they elicit different cell responses. While thickness and surface chemistry appear to control morphology, only thickness regulates collagen organization and cell migration speed. This suggests that surface attachment and thickness of the collagen gel control cell behavior through both collagen structure and local stiffness in confined fiber-forming networks.

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.

Pentablock Copolymer Micelle Nanoadjuvants Enhance Cytosolic Delivery of Antigen and Improve Vaccine Efficacy while Inducing Low Inflammation.

As the focus has shifted from traditional killed or live, attenuated vaccines toward subunit vaccines, improvements in vaccine safety have been confronted with low immunogenicity of protein antigens. This issue has been addressed by synthesizing and designing a wide variety of antigen carriers and adjuvants, such as Toll-like receptor agonists (e.g., MPLA, CpG). Studies have focused on optimizing adjuvants for improved cellular trafficking, cytosolic availability, and improved antigen presentation. In this work, we describe the design of novel amphiphilic pentablock copolymer (PBC) adjuvants that exhibit high biocompatibility and reversible pH- and temperature-sensitive micelle formation. We demonstrate improved humoral immunity in mice in response to single-dose immunization with PBC micelle adjuvants compared with soluble antigen alone. With the motive of exploring the mechanism of action of these PBC micelles, we studied intracellular trafficking of these PBC micelles with a model antigen and demonstrated that the PBC micelles associate with the antigen and enhance its cytosolic delivery to antigen-presenting cells. We posit that these PBC micelles operate via immune-enhancing mechanisms that are different from that of traditional Toll-like receptor activating adjuvants. The metabolic profile of antigen-presenting cells stimulated with traditional adjuvants and the PBC micelles also suggests distinct mechanisms of action. A key finding from this study is the low production of nitric oxide and reactive oxygen species by antigen-presenting cells when stimulated by PBC micelle adjuvants in sharp contrast to TLR adjuvants. Together, these studies provide a basis for rationally developing novel vaccine adjuvants that are safe, that induce low inflammation, and that can efficiently deliver antigen to the cytosol.

Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues.

Contact guidance or bidirectional migration along aligned fibers modulates many physiological and pathological processes such as wound healing and cancer invasion. Aligned 2D collagen fibrils epitaxially grown on mica substrates replicate many features of contact guidance seen in aligned 3D collagen fiber networks. However, these 2D collagen self-assembled substrates are difficult to image through, do not have known or tunable mechanical properties and cells degrade and mechanically detach collagen fibrils from the surface, leading to an inability to assess contact guidance over long times. Here, we describe the transfer of aligned collagen fibrils from mica substrates to three different functionalized target substrates: glass, polydimethylsiloxane (PDMS) and polyacrylamide (PA). Aligned collagen fibrils can be efficiently transferred to all three substrates. This transfer resulted in substrates that were to varying degrees resistant to cell-mediated collagen fibril deformation that resulted in detachment of the collagen fibril field, allowing for contact guidance to be observed over longer time periods. On these transferred substrates, cell speed is lowest on softer contact guidance cues for both MDA-MB-231 and MTLn3 cells. Intermediate stiffness resulted in the fastest migration. MTLn3 cell directionality was low on soft contact guidance cues, whereas MDA-MB-231 cell directionality marginally increased. It appears that the stiffness of the contact guidance cue regulates contact guidance differently between cell types. The development of this collagen fibril transfer method allows for the attachment of aligned collagen fibrils on substrates, particularly flexible substrates, that do not normally promote aligned collagen fibril growth, increasing the utility of this collagen self-assembly system for the fundamental examination of mechanical regulation of contact guidance.

Correction to: Microtissue size and cell-cell communication modulate cell migration in arrayed 3D collagen gels.

The original version of this article unfortunately contained a mistake. One line indicating statistical significance was improperly placed in Fig. 5.

On-Chip Studies of Magnetic Stimulation Effect on Single Neural Cell Viability and Proliferation on Glass and Nanoporous Surfaces.

Transcranial magnetic stimulation (TMS) is a noninvasive neuromodulation technique, an FDA-approved treatment method for various neurological disorders such as depressive disorder, Parkinson's disease, post-traumatic stress disorder, and migraine. However, information concerning the molecular/cellular-level mechanisms of neurons under magnetic simulation (MS), particularly at the single neural cell level, is still lacking, resulting in very little knowledge of the effects of MS on neural cells. In this paper, the effects of MS on the behaviors of neural cell N27 at the single-cell level on coverslip glass substrate and anodic aluminum oxide (AAO) nanoporous substrate are reported for the first time. First, it has been found that the MS has a negligible cytotoxic effect on N27 cells. Second, MS decreases nuclear localization of paxillin, a focal adhesion protein that is known to enter the nucleus and modulate transcription. Third, the effect of MS on N27 cells can be clearly observed over 24 h, the duration of one cell cycle, after MS is applied to the cells. The size of cells under MS was found to be statistically smaller than that of cells without MS after one cell cycle. Furthermore, directly monitoring cell division process in the microholders on a chip revealed that the cells under MS generated statistically more daughter cells in one average cell cycle time than those without MS. All these results indicate that MS can affect the behavior of N27 cells, promoting their proliferation and regeneration.

Microtissue size and cell-cell communication modulate cell migration in arrayed 3D collagen gels.

Cells communicate through the extracellular matrix (ECM) in many physiological and pathological processes. This is particularly important during cell migration, where cell communication can alter both the speed and the direction of migration. However, most cell culture systems operate with large volumes relative to cell numbers, creating low cell densities and diluting factors that mediate cell communication. Furthermore, they lack the ability to isolate single cells or small groups of cells. Droplet forming devices allow for an ability to embed single or small groups of cells into small volume segregated 3D environments, increasing the cell density to physiological levels. In this paper we show a microfluidic droplet device for fabricating 3D collagen-based microtissues to study breast cancer cell motility. MDA-MB-231 cells fail to spread and divide in small, thin chambers. Cell migration is also stunted as compared to thick 3D gels. However, larger chambers formed by a thicker devices promote cell spreading, cell division and faster migration. In the large devices, both cell-ECM and cell-cell interactions affect cell motility. Increasing collagen density decreases cell migration and increasing the number of cells per chamber increases cell migration speed. Furthermore, cells appear to sense both the ECM-chamber wall interface as well as other cells. Cells migrate towards the ECM-chamber interface if within roughly 150 µm, whereas cells further than 150 µm tend to move towards the center of the chamber. Finally, while cells do not show enhanced movement towards the center of mass of a cell cluster, their migration speed is more variable when further away from the cell cluster center of mass. These results show that microfluidic droplet devices can array 3D collagen gels and promote cell spreading, division and migration similar to what is seen in thick 3D collagen gels. Furthermore, they can provide a new avenue to study cell migration and cell-cell communication at physiologically relevant cell densities.

Contact guidance diversity in rotationally aligned collagen matrices.

Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures. Contact guidance environments in 3D more closely resemble in vivo conditions and model cell-ECM interactions better than 2D environments. While most cells engage in directed migration on potent 2D contact guidance cues, there is diversity in response to contact guidance cues based on whether the cell migrates with a mesenchymal or amoeboid migration mode. In this paper, rotational alignment of collagen gels was used to study the differences in contact guidance between MDA-MB-231 (mesenchymal) and MTLn3 (amoeboid) cells. MDA-MB-231 cells migrate with high directional fidelity in aligned collagen gels, while MTLn3 cells show no directional migration. The collagen stiffness was increased through glycation, resulting in decreased MDA-MB-231 directionality in aligned collagen gels. Interestingly, partial inhibition of cell contractility dramatically decreased directionality in MDA-MB-231 cells. The directionality of MDA-MB-231 cells was most sensitive to ROCK inhibition, but unlike in 2D contact guidance environments, cell directionality and speed are more tightly coupled. Modulation of the contractile apparatus appears to more potently affect contact guidance than modulation of extracellular mechanical properties of the contact guidance cue. STATEMENT OF SIGNIFICANCE: Collagen fiber alignment in the tumor microenvironment directs migration, a process called contact guidance, enhancing the efficiency of cancer invasion and metastasis. 3D systems that assess contact guidance by locally orienting collagen fiber alignment are lacking. Furthermore, cell type differences and the role of extracellular matrix stiffness in tuning contact guidance fidelity are not well characterized. In this paper rotational alignment of collagen fibers is used as a 3D contact guidance cue to illuminate cell type differences and the role of extracellular matrix stiffness in guiding cell migration along aligned fibers of collagen. This local alignment offers a simple approach by which to couple collagen alignment with gradients in other directional cues in devices such as microfluidic chambers.

Safety, Tolerability and Pharmacokinetics of Single Doses of Oxytocin Administered via an Inhaled Route in Healthy Females: Randomized, Single-blind, Phase 1 Study.

BACKGROUND: The utility of intramuscular (IM) oxytocin for the prevention of postpartum hemorrhage in resource-poor settings is limited by the requirement for temperature-controlled storage and skilled staff to administer the injection. We evaluated the safety, tolerability and pharmacokinetics (PK) of a heat-stable, inhaled (IH) oxytocin formulation. METHODS: This phase 1, randomized, single-center, single-blind, dose-escalation, fixed-sequence study (NCT02542813) was conducted in healthy, premenopausal, non-pregnant, non-lactating women aged 18-45years. Subjects initially received IM oxytocin 10 international units (IU) on day 1, IH placebo on day 2, and IH oxytocin 50µg on day 3. Subjects were then randomized 4:1 using validated GSK internal software to IH placebo or ascending doses of IH oxytocin (200, 400, 600µg). PK was assessed by comparing systemic exposure (maximum observed plasma concentration, area under the concentration-time curve, and plasma concentrations at 10 and 30min post dose) for IH versus IM oxytocin. Adverse events (AEs), spirometry, laboratory tests, vital signs, electrocardiograms, physical examinations, and cardiac telemetry were assessed. FINDINGS: Subjects were recruited between September 14, 2015 and October 12, 2015. Of the 16 subjects randomized following initial dosing, 15 (IH placebo n=3; IH oxytocin n=12) completed the study. IH (all doses) and IM oxytocin PK profiles were comparable in shape. However, systemic exposure with IH oxytocin 400µg most closely matched IM oxytocin 10IU. Systemic exposure was approximately dose proportional for IH oxytocin. No serious AEs were reported. No clinically significant findings were observed for any safety parameters. INTERPRETATION: These data suggest that similar oxytocin systemic exposure can be achieved with IM and IH administration routes, and no safety concerns were identified with either route. The inhalation route may offer the opportunity to increase access to oxytocin for women giving birth in resource-poor settings.

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.

Myosin phosphorylation on stress fibers predicts contact guidance behavior across diverse breast cancer cells.

During cancer progression the extracellular matrix is remodeled, forming aligned collagen fibers that proceed radially from the tumor, resulting in invasion. We have recently shown that different invasive breast cancer cells respond to epitaxially grown, aligned collagen fibrils differently. This article develops insight into why these cells differ in their contact guidance fidelity. Small changes in contractility or adhesion dramatically alter directional persistence on aligned collagen fibrils, while migration speed remains constant. The directionality of highly contractile and adhesive MDA-MB-231 cells can be diminished by inhibiting Rho kinase or ß1 integrin binding. Inversely, the directionality of less contractile and adhesive MTLn3 cells can be enhanced by activating contractility or integrins. Subtle, but quantifiable alterations in myosin II regulatory light chain phosphorylation on stress fibers explain the tuning of contact guidance fidelity, separate from migration per se indicating that the contractile and adhesive state of the cell in combination with collagen organization in the tumor microenvironment determine the efficiency of migration. Understanding how distinct cells respond to contact guidance cues will not only illuminate mechanisms for cancer invasion, but will also allow for the design of environments to separate specific subpopulations of cells from patient-derived tissues by leveraging differences in responses to directional migration cues.

Pharmacological guidelines for schizophrenia: a systematic review and comparison of recommendations for the first episode.

OBJECTIVES: Clinical practice guidelines (CPGs) support the translation of research evidence into clinical practice. Key health questions in CPGs ensure that recommendations will be applicable to the clinical context in which the guideline is used. The objectives of this study were to identify CPGs for the pharmacological treatment of first-episode schizophrenia; assess the quality of these guidelines using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) instrument; and compare recommendations in relation to the key health questions that are relevant to the pharmacological treatment of first-episode schizophrenia. METHODS: A multidisciplinary group identified key health questions that are relevant to the pharmacological treatment of first-episode schizophrenia. The MEDLINE and EMBASE databases, websites of professional organisations and international guideline repositories, were searched for CPGs that met the inclusion criteria. The AGREE II instrument was applied by three raters and data were extracted from the guidelines in relation to the key health questions. RESULTS: In total, 3299 records were screened. 10 guidelines met the inclusion criteria. 3 guidelines scored well across all domains. Recommendations varied in specificity. Side effect concerns, rather than comparative efficacy benefits, were a key consideration in antipsychotic choice. Antipsychotic medication is recommended for maintenance of remission following a first episode of schizophrenia but there is a paucity of evidence to guide duration of treatment. Clozapine is universally regarded as the medication of choice for treatment resistance. There is less evidence to guide care for those who do not respond to clozapine. CONCLUSIONS: An individual's experience of using antipsychotic medication for the initial treatment of first-episode schizophrenia may have implications for future engagement, adherence and outcome. While guidelines of good quality exist to assist in medicines optimisation, the evidence base required to answer key health questions relevant to the pharmacological treatment of first-episode schizophrenia is limited.

High Throughput Studies of Cell Migration in 3D Microtissues Fabricated by a Droplet Microfluidic Chip.

Arrayed three-dimensional (3D) micro-sized tissues with encapsulated cells (microtissues) have been fabricated by a droplet microfluidic chip. The extracellular matrix (ECM) is a polymerized collagen network. One or multiple breast cancer cells were embedded within the microtissues, which were stored in arrayed microchambers on the same chip without ECM droplet shrinkage over 48 h. The migration trajectory of the cells was recorded by optical microscopy. The migration speed was calculated in the range of 3⁻6 µm/h. Interestingly, cells in devices filled with a continuous collagen network migrated faster than those where only droplets were arrayed in the chambers. This is likely due to differences in the length scales of the ECM network, as cells embedded in thin collagen slabs also migrate slower than those in thick collagen slabs. In addition to migration, this technical platform can be potentially used to study cancer cell-stromal cell interactions and ECM remodeling in 3D tumor-mimicking environments.