Fibrin Hydrogels Reinforced by Reactive Microgels for Stimulus-Triggered Drug Administration.

Tissue engineering is a steadily growing field of research due to its wide-ranging applicability in the field of regenerative medicine. Application-dependent mechanical properties of a scaffold material as well as its biocompatibility and tailored functionality represent particular challenges. Here the properties of fibrin-based hydrogels reinforced by functional cytocompatible poly(N-vinylcaprolactam)-based (PVCL) microgels are studied and evaluated. The employment of temperature-responsive microgels decorated by epoxy groups for covalent binding to the fibrin is studied as a function of cross-linking degree within the microgels, microgel concentration, as well as temperature. Rheology reveals a strong correlation between the mechanical properties of the reinforced fibrin-based hydrogels and the microgel rigidity and concentration. The incorporated microgels serve as cross-links, which enable temperature-responsive behavior of the hydrogels, and slow down the hydrogel degradation. Microgels can be additionally used as carriers for active drugs, as demonstrated for dexamethasone. The microgels' temperature-responsiveness allows for triggered release of payload, which is monitored using a bioassay. The cytocompatibility of the microgel-reinforced fibrin-based hydrogels is demonstrated by LIVE/DEAD staining experiments using human mesenchymal stem cells. The microgel-reinforced hydrogels are a promising material for tissue engineering, owing to their superior mechanical performance and stability, possibility of drug release, and retained biocompatibility.

Predicting Undesired Treatment Outcomes With Machine Learning in Mental Health Care: Multisite Study.

Background: Predicting which treatment will work for which patient in mental health care remains a challenge. Objective: The aim of this multisite study was 2-fold: (1) to predict patients' response to treatment in Dutch basic mental health care using commonly available data from routine care and (2) to compare the performance of these machine learning models across three different mental health care organizations in the Netherlands by using clinically interpretable models. Methods: Using anonymized data sets from three different mental health care organizations in the Netherlands (n=6452), we applied a least absolute shrinkage and selection operator regression 3 times to predict the treatment outcome. The algorithms were internally validated with cross-validation within each site and externally validated on the data from the other sites. Results: The performance of the algorithms, measured by the area under the curve of the internal validations as well as the corresponding external validations, ranged from 0.77 to 0.80. Conclusions: Machine learning models provide a robust and generalizable approach in automated risk signaling technology to identify cases at risk of poor treatment outcomes. The results of this study hold substantial implications for clinical practice by demonstrating that the performance of a model derived from one site is similar when applied to another site (ie, good external validation).

Trop-2 induces ADAM10-mediated cleavage of E-cadherin and drives EMT-less metastasis in colon cancer.

We recently reported that activation of Trop-2 through its cleavage at R87-T88 by ADAM10 underlies Trop-2-driven progression of colon cancer. However, the mechanism of action and pathological impact of Trop-2 in metastatic diffusion remain unexplored. Through searches for molecular determinants of cancer metastasis, we identified TROP2 as unique in its up-regulation across independent colon cancer metastasis models. Overexpression of wild-type Trop-2 in KM12SM human colon cancer cells increased liver metastasis rates in vivo in immunosuppressed mice. Metastatic growth was further enhanced by a tail-less, activated ΔcytoTrop-2 mutant, indicating the Trop-2 tail as a pivotal inhibitory signaling element. In primary tumors and metastases, transcriptome analysis showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 is through alternative mechanisms. Trop-2 can tightly interact with ADAM10. Here, Trop-2 bound E-cadherin and stimulated ADAM10-mediated proteolytic cleavage of E-cadherin intracellular domain. This induced detachment of E-cadherin from ß-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of ß-catenin signaling, which were further enhanced by the ΔcytoTrop-2 mutant. This Trop-2/E-cadherin/ß-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival. In patients with colon cancer, activation of this Trop-2-centered program led to significantly reduced relapse-free and overall survival, indicating a major impact on progression to metastatic disease. Recently, the anti-Trop-2 mAb Sacituzumab govitecan-hziy was shown to be active against metastatic breast cancer. Our findings define the key relevance of Trop-2 as a target in metastatic colon cancer.

Genotype scores predict drug efficacy in subtypes of female sexual interest/arousal disorder: A double-blind, randomized, placebo-controlled cross-over trial.

Attempts to develop a drug treatment for female sexual interest/arousal disorder have so far been guided by the principle of 'one size fits all', and have failed to acknowledge the complexity of female sexuality. Guided by personalized medicine, we designed two on-demand drugs targeting two distinct hypothesized causal mechanisms for this sexual disorder. The objective of this study was to design and test a novel procedure, based on genotyping, that predicts which of the two on-demand drugs will yield a positive treatment response. In a double-blind, randomized, placebo-controlled cross-over experiment, 139 women with female sexual interest/arousal disorder received three different on-demand drug-combination treatments during three 2-week periods: testosterone 0.5 mg + sildenafil 50 mg, testosterone 0.5 mg + buspirone 10 mg, and matching placebo. The primary endpoint was change in satisfactory sexual events. Subjects' genetic profile was assessed using a microarray chip that measures 300,000 single-nucleotide polymorphisms. A preselection of single-nucleotide polymorphisms associated with genes that are shown to be involved in sexual behaviour were combined into a Phenotype Prediction Score. The Phenotype Prediction Score demarcation formula was developed and subsequently validated on separate data sets. Prediction of drug-responders with the Phenotype Prediction Score demarcation formula gave large effect sizes (d = 0.66 through 1.06) in the true drug-responders, and medium effect sizes (d = 0.51 and d = 0.47) in all patients (including identified double, and non-responders). Accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the Phenotype Prediction Score demarcation formula were all between 0.78 and 0.79, and thus sufficient. The resulting Phenotype Prediction Score was validated and shown to effectively and reliably predict which women would benefit from which on-demand drug, and could therefore also be useful in clinical practice, as a companion diagnostic establishing the way to a true personalized medicine approach.

Efficacy and Safety of On-Demand Use of 2 Treatments Designed for Different Etiologies of Female Sexual Interest/Arousal Disorder: 3 Randomized Clinical Trials.

BACKGROUND: In women, low sexual desire and/or sexual arousal can lead to sexual dissatisfaction and emotional distress, collectively defined as female sexual interest/arousal disorder (FSIAD). Few pharmaceutical treatment options are currently available. AIM: To investigate the efficacy and safety of 2 novel on-demand pharmacologic treatments that have been designed to treat 2 FSIAD subgroups (women with low sensitivity for sexual cues and women with dysfunctional over-activation of sexual inhibition) using a personalized medicine approach using an allocation formula based on genetic, hormonal, and psychological variables developed to predict drug efficacy in the subgroups. METHODS: 497 women (21-70 years old) with FSIAD were randomized to 1 of 12 8-week treatment regimens in 3 double-blinded, randomized, placebo-controlled, dose-finding studies conducted at 16 research sites in the United States. Efficacy and safety of the following on-demand treatments was tested: placebo, testosterone (T; 0.5 mg), sildenafil (S; 50 mg), buspirone (B; 10 mg) and combination therapies (T 0.25 mg + S 25 mg, T 0.25 mg + S 50 mg, T 0.5 mg + S 25 mg, T 0.5 mg + S 50 mg, and T 0.25 mg + B 5 mg, T 0.25 mg + B 10 mg, T 0.5 mg + B 5 mg, T 0.5 mg + B 10 mg). OUTCOMES: The primary efficacy measure was the change in satisfying sexual events (SSEs) from the 4-week baseline to the 4-week average of the 8-week active treatment period after medication intake. For the primary end points, the combination treatments were compared with placebo and the respective monotherapies on this measure. RESULTS: In women with low sensitivity for sexual cues, 0.5 mg T + 50 mg S increased the number of SSEs from baseline compared with placebo (difference in change [Δ] = 1.70, 95% CI = 0.57-2.84, P = .004) and monotherapies (S: Δ = 1.95, 95% CI = 0.44-3.45, P = .012; T: Δ = 1.69, 95% CI = 0.58-2.80, P = .003). In women with overactive inhibition, 0.5 mg T + 10 mg B increased the number of SSEs from baseline compared with placebo (Δ = 0.99, 95% CI = 0.17-1.82, P = .019) and monotherapies (B: Δ = 1.52, 95% CI = 0.57-2.46, P = .002; T: Δ = 0.98, 95% CI = 0.17-1.78, P = .018). Secondary end points followed this pattern of results. The most common drug-related side effects were flushing (T + S treatment, 3%; T + B treatment, 2%), headache (placebo treatment, 2%; T + S treatment, 9%), dizziness (T + B treatment, 3%), and nausea (T + S treatment, 3%; T + B treatment, 2%). CLINICAL IMPLICATIONS: T + S and T + B are promising treatments for women with FSIAD. STRENGTHS AND LIMITATIONS: The data were collected in 3 well-designed randomized clinical trials that tested multiple doses in a substantial number of women. The influence of T + S and T + B on distress and the potentially sustained improvements after medication cessation were not investigated. CONCLUSIONS: T + S and T + B are well tolerated and safe and significantly increase the number of SSEs in different FSIAD subgroups. Tuiten A, van Rooij K, Bloemers J, et al. Efficacy and Safety of On-Demand Use of 2 Treatments Designed for Different Etiologies of Female Sexual Interest/Arousal Disorder: 3 Randomized Clinical Trials. J Sex Med 2018;15:201-216.

Altered neuropeptide Y and neurokinin messenger RNA expression and receptor binding in stress-sensitised rats.

A single session of footshocks in rats causes long-lasting sensitisation of behavioural, hormonal and autonomic responses to subsequent novel stressful challenges as well as altered pain sensitivity. These changes mimic aspects of post-traumatic stress disorder in humans. Our aim was to identify neuropeptide substrates in the central nervous system involved in stress sensitisation. Male Wistar rats were exposed to ten footshocks in 15 min (preshocked) or placed in the same cage without shocks (control). Two weeks later, rats were placed in a novel cage, subjected to 5 min of 85 dB noise, and returned to their home cage. Rats were killed either before or 1 h after noise and their brains processed for in situ hybridization for neuropeptide Y (NPY) and beta-preprotachykinin-I (PPT) mRNA. Additional groups of rats were killed under basal conditions and brains processed for NPY and neurokinin receptor binding with radiolabelled ligands. Two weeks after footshock treatment NPY mRNA expression was increased in the basolateral amygdala and showed preshockxnoise interaction in the locus coeruleus (down after noise in controls, lower basal and unchanged after noise in preshocked). PPT expression in the lateral parabrachial nucleus also showed preshockxnoise interaction (up after noise in controls, higher basal and down after noise in preshocked), and was increased after noise in the periaquaeductal grey. NK1 receptor binding in the agranular insular cortex and arcuate nucleus of the hypothalamus and NK2 receptor binding in the amygdala was lower in preshocked rats than in controls. Altered expression of NPY in the basolateral amygdala and locus coeruleus could contribute to or compensate for behavioural and autonomic sensitisation in preshocked rats. Altered PPT expression in the parabrachial nucleus may be involved in the altered pain processing seen in this model. Lower NK1 and NK2 receptor numbers in cortex, hypothalamus and amygdala may reflect secondary adaptations to altered neuropeptide release. These long-term changes in brain neuropeptide systems could offer novel leads for pharmacological modulation of long-term stress-induced sensitisation.

Involvement of group II metabotropic glutamate receptors in stress-induced behavioural sensitization.

RATIONALE: A short session of repeated foot shocks in rats causes long-lasting sensitization of behavioural, hormonal and autonomic responses to novel stressful challenges. The behavioural sensitization can be reduced by anxiolytics and mimics aspects of stress-induced changes in patients with post-traumatic stress disorder. OBJECTIVES: The aim of this study was to test the efficacy of a group II metabotropic glutamate receptor (mGluR) agonist and assess altered brain mGluR receptor expression in shock-sensitized rats. MATERIALS AND METHODS: Male Wistar rats were exposed to a 15-min session with ten 6-s foot shocks (preshocked). One and 2 weeks later, rats were intraperitoneally injected with the group II metabotropic glutamate receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC) or vehicle, and 30 min later exposed to 5 min of 85 dB noise. For in situ hybridization with probes for mGluR1, mGluR2, mGluR3 and mGluR5, preshocked and control rats were killed under basal conditions 2 weeks after foot shocks and their brains cryosectioned. RESULTS: APDC had no clear effect in controls, but dose-dependently reduced high immobility and increased low locomotion and rearing seen in preshocked rats to the levels of controls. mGluR3 expression was increased in the basolateral nucleus of the amygdala, and mGluR2 expression was increased in the agranular insular cortex of preshocked rats compared to controls. CONCLUSIONS: Shock-induced behavioural sensitization in rats is reduced by acute treatment with a group II metabotropic glutamate receptor agonist. This effect may depend on the increased expression of amygdala mGluR3, which could be hypothesized as an endogenous mechanism to counteract stress-induced neuronal sensitization.

Higher sensitivity secondary ion mass spectrometry of biological molecules for high resolution, chemically specific imaging.

To expand the role of high spatial resolution secondary ion mass spectrometry (SIMS) in biological studies, numerous developments have been reported in recent years for enhancing the molecular ion yield of high mass molecules. These include both surface modification, including matrix-enhanced SIMS and metal-assisted SIMS, and polyatomic primary ions. Using rat brain tissue sections and a bismuth primary ion gun able to produce atomic and polyatomic primary ions, we report here how the sensitivity enhancements provided by these developments are additive. Combined surface modification and polyatomic primary ions provided approximately 15.8 times more signal than using atomic primary ions on the raw sample, whereas surface modification and polyatomic primary ions yield approximately 3.8 and approximately 8.4 times more signal. This higher sensitivity is used to generate chemically specific images of higher mass biomolecules using a single molecular ion peak.

Branching of calyceal afferents during postnatal development in the rat auditory brainstem.

Cells in the anteroventral cochlear nucleus (aVCN) send out calyceal axons that form large excitatory somatic terminals, the calyces of Held, onto principal cells of the contralateral medial nucleus of the trapezoid body (MNTB). It is unclear which fraction of these axons might form more than one calyx and whether this fraction changes during development. We combined in vitro anterograde tracing, stereological cell counts, analysis of apoptosis, and immunohistochemistry to study the development of calyceal afferents in rats of different postnatal ages. We found that some principal cells were contacted by multiple large axosomatic inputs, but these invariably originated from the same axon. Conversely, at least 18% of traced afferents branched to form multiple calyces, independently of age. Calyces from the same axon generally innervated nearby principal cells, and most of these branch points were <50 microm away from the synaptic terminals. Our results show that the projection from the aVCN to the MNTB is divergent, both when calyces have just been formed and in the adult. Cell counts did not provide evidence for principal cell loss during development, although analysis of apoptosis showed a large increase in nonneuronal cell death around the onset of hearing. Our data suggest that, once a calyceal synapse forms in the MNTB, it stays.

Gold-enhanced biomolecular surface imaging of cells and tissue by SIMS and MALDI mass spectrometry.

Surface metallization by plasma coating enhances desorption/ionization of membrane components such as lipids and sterols in imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) of tissues and cells. High-resolution images of cholesterol and other membrane components were obtained for neuroblastoma cells and revealed subcellular details (resolving power 1.5 mum). Alternatively, in matrix-enhanced SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allowed intact molecular ion imaging of phosphatidylcholine and sphingomyelin at the cellular level. Gold deposition on top of matrix-coated rat brain tissue sections strongly enhanced image quality and signal intensity in stigmatic matrix-assisted laser desorption/ionization imaging mass spectrometry. High-quality total ion count images were acquired, and the neuropeptide vasopressin was localized in the rat brain tissue section at the hypothalamic area around the third ventricle. Although the mechanism of signal enhancement by gold deposition is under debate, the results we have obtained for cells and tissue sections illustrate the potential of this sample preparation technique for biomolecular surface imaging by mass spectrometry.

Identification of genes associated with enhanced metastasis of a large cell lung carcinoma cell line.

We have compared the transcriptional profile of large cell lung tumor cell lines NIH-H460 and H460-M making use of the Affymetrix GeneChip system. H460-M is derived from NIH-H460 and displays enhanced experimental and spontaneous metastasis in nude mice. Out of the 12,600 genes investigated, 73 (0.6%) were up-regulated and 114 (0.9%) were down-regulated on the basis of the scoring criteria. We have classified the de-regulated genes according to the following categories: immune response, enzymes, modulation of transcription, signal transduction, cytoskeleton/adhesion and extracellular matrix associated proteins, cell-cycle/apoptosis, transporters and 'others'. Among the remarkable features of this system are the up-regulation of the steady-state mRNA levels of neuroendocrine markers such as neurotensin (NTS), neuroendocrine-specific protein (NSP), neural cell adhesion molecule 1 (NCAM1) and gamma-aminobutyric acid B-type receptor (GPR51) in cell line H460-M. Semaphorin 3B (SEMA3B) was dramatically down-regulated in cell line H460-M and emerged as the most interesting gene for target validation.