BackgroundUveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.MethodsA series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.ResultsUsing dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.ConclusionMolecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.

A dual physical crosslinking (DPC) strategy is used to construct moldable hydrogels with ultrahigh strength and toughness. First, polyelectrolyte complex (PEC) hydrogels are prepared through the in situ polymerization of acrylic acid monomers in the concentrated chitosan (Ch) solutions. Subsequently, Ag + ions are introduced into the PEC hydrogels to form coordination bonds between NH 2 and COOH groups. High-density electrostatic interaction and coordination bonds endow the DPC hydrogels with high strength and toughness. The mechanical properties of the DPC hydrogels strongly depend on the weight ratio of Ch to poly(acrylic acid) and the loading concentration of Ag + ions. When the loading concentration of Ag + ions is in the range of 1.0-1.5 mol L −1 , DPC 0.10-0.25 hydrogels display the maximum tensile strength (24.0 MPa) and toughness (84.7 MJ m −3 ) with a strain of more than 600%. Specially, the DPC hydrogels display an excellent moldable behavior due to the reversible properties of the electrostatic interaction and coordination bonds. The DPC strategy can also be applied in various other systems and opens a new avenue to fabricate hydrogels with outstanding mechanical properties and antibacterial activities.

Quaternized chitosan (QCh) was homogeneously synthesized by reacting chitosan with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in alkali/urea aqueous solution for the first time. The structure and solution properties of QCh were characterized by using element analysis, FT-IR, 13 C NMR, SEC-LLS, rheology, viscometer, and ξ-potential measurements. Subsequently, polyelectrolyte complex (PEC) hydrogels were constructed by in situ polymerization of acrylic acid (AA) monomers in the concentrated QCh solution. The structure and mechanical behavior of the prepared hydrogels were systematic studied. Because of the high charge density and solubility of QCh, strong electrostatic interactions were formed in the hydrogels and endowed them tough with self-recovery properties. The mechanical behavior of the hydrogels was accurately tuned from stiff and viscoelastic to soft and elastic by changing the poly(acrylic acid) (PAA) content. The regulation mechanism relied on the remarkable difference in the chain segmental mobility between QCh and PAA. Moreover, the QCh/PAA PEC hydrogels displayed excellent solvent-induced shape-memory behavior due to the reversible properties of the ionic bonds. In summary, we offered a novel modification method for chitosan and opened up a new avenue to construct chitosan-based hydrogels with outstanding mechanical properties.

The human gut-resident commensal microbiota is a unique ecosystem associated with various bodily functions, especially immunity. Gut microbiota dysbiosis plays a crucial role in autoimmune disease pathogenesis as well as in bowel-related diseases. However, the role of the gut microbiota, which causes or influences systemic immunity in autoimmune diseases, remains elusive. Aryl hydrocarbon receptor, a ligand-activated transcription factor, is a master moderator of host-microbiota interactions because it shapes the immune system and impacts host metabolism. In addition, treatment optimization while minimizing potential adverse effects in autoimmune diseases remains essential, and modulation of the gut microbiota constitutes a potential clinical therapy. Here, we present evidence linking gut microbiota dysbiosis with autoimmune mechanisms involved in disease development to identify future effective approaches based on the gut microbiota for preventing autoimmune diseases.

Herein, a new type of injectable carboxymethyl chitosan (CMCh) hydrogel wound dressing with self-healing properties is constructed. First, CMCh samples are homogeneously synthesized in alkali/urea aqueous solutions. Subsequently, trivalent metal ions of Fe3+ and Al3+ are introduced to form coordination bonds with CMCh, leading to an ultrafast gelation process. A series of hydrogels can be obtained by altering the concentration of CMCh and the relative content of metal ions. Owing to the dynamic and reversible characteristics of the coordination bonds, the hydrogel exhibits self-healing, self-adaption, and thermoresponsive ability. Moreover, due to the interaction between the amino groups on CMCh and SO4 2–, the hydrogel undergoes phase separation and can be painlessly detached from the skin with little residue. Taking advantage of all these characteristics, the hydrogel is used as a wound dressing and can significantly accelerate skin tissue regeneration and wound closure. This hydrogel has great potential in the application of tissue engineering.

IMPORTANCE Uveal melanoma (UM) is an intraocular primary malignant neoplasm that often gives rise to metastatic disease for which there are no effective therapies. A substantial proportion of UMs express the cancer-testis antigen PRAME (preferentially expressed antigen in melanoma), which can potentially be targeted by adoptive T-cell therapy.OBJECTIVE To determine whether there may be a rationale for PRAME-directed T-cell therapy for metastatic UM.DESIGN, SETTING, AND PARTICIPANTS An experimental study using a retrospective cohort of 64 patients with UM (median follow-up, 62 months) was conducted from January 8, 2015, to November 20, 2016, at the Leiden University Medical Center. Clinical, histopathologic, and genetic parameters were compared between 64 PRAME-positive and PRAME-negative UMs. HLA class I restricted, PRAME-specific T cells were stimulated with UM cell lines to measure their antigen-specific reactivity against these cell lines, which were analyzed for PRAME expression by real-time quantitative polymerase chain reaction. Uveal melanoma metastases from 16 unrelated patients were assessed for PRAME expression by messenger RNA fluorescence in situ hybridization and for HLA class I expression by immunofluorescence staining.MAIN OUTCOMES AND MEASURES Interferon γ production for antigen-specific reactivity and detection of PRAME and HLA class I expression in primary and metastatic UM. RESULTSOf the 64 patients in the study (31 women and 33 men; mean [SD] age at the time of enucleation, 60.6 [15.6] years), PRAME expression was negative in 35 primary UMs and positive in 29 primary UMs. Positive PRAME expression was associated with a high largest basal diameter (15.0 vs 12.0 mm; P = .005), ciliary body involvement (59% vs 26%; P = .008), and amplification of chromosome 8q (66% vs 23%; P = .002). PRAME-specific T cells reacted against 4 of 7 UM cell lines, demonstrating that T-cell reactivity correlated with PRAME expression. Metastatic UM samples were positive for PRAME messenger RNA in 11 of 16 patients and for HLA class I in 10 of 16 patients, with 8 of 16 patients demonstrating coexpression of both PRAME and HLA class I.CONCLUSIONS AND RELEVANCE PRAME is expressed in many primary and metastatic UMs, and about half of the metastatic UMs coexpress PRAME and HLA class I. The finding that PRAME-specific T cells in this study reacted against PRAME-positive UM cell lines suggests a potential role for PRAME-directed immunotherapy for selected patients with metastatic UM.

PurposeConjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS. We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro.Methods131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206).ResultsThe BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines.ConclusionERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma.

Polysaccharide-based injectable hydrogels have several advantages in the context of biomedical use. However, the main obstruction associated with the utilization of these hydrogels in clinical application is their poor mechanical properties. Herein, we describe in situ gelling of nanocomposite hydrogels based on quaternized cellulose (QC) and rigid rod-like cationic cellulose nanocrystals (CCNCs), which can overcome this challenge. In all cases, gelation immediately occurred with an increase of temperature, and the CCNCs were evenly distributed throughout the hydrogels. The nanocomposite hydrogels exhibited increasing orders-of-magnitude in the mechanical strength, high extension in degradation and the sustained release time, because of the strong interaction between CCNCs and QC chains mediated by the cross-linking agent (β-glycerophosphate, β-GP). The results of the in vitro toxicity and in vivo biocompatibility tests revealed that the hydrogels did not show obvious cytotoxicity and inflammatory reaction to cells and tissue. Moreover, DOX-encapsulated hydrogels were injected beside the tumors of mice bearing liver cancer xenografts to assess the potential utility as localized and sustained drug delivery depot systems for anticancer therapy. The results suggested that the QC/CCNC/β-GP nanocomposite hydrogels had great potential for application in subcutaneous and sustained delivery of anticancer drug to increase therapeutic efficacy and improve patient compliance.