De Novo Mutations of CCNK Cause a Syndromic Neurodevelopmental Disorder with Distinctive Facial Dysmorphism.

Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK, is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency.

Improved gastrointestinal stability of solid lipid nanoparticles using comb-shaped amphiphilic inulin derivatives.

In order to reduce enzymatic degradation and thereby enhance the stability of solid lipid nanoparticles (SLNs) in the gastrointestinal tract, comb-shaped amphiphilic macromolecular material (CAM) of dodecyl inulin (Inu12) and octadecyl inulin (Inu18) were designed as the emulsifier and stabilizer to modify SLNs (Inu12/Inu18-SLNs). Inu12-SLNs and Inu18-SLNs had similar particle size as the control SLNs (P188-SLNs and Tween-SLNs) prepared with the straight chain surfactants, poloxamer 188 and tween-80 as the emulsifier, which ranged from 220 nm to 270 nm. The zeta potentials of all the SLNs formulations were slightly negative. Cyclosporine A (CsA)-loaded Inu12-SLNs and Inu18-SLNs showed a much lower drug release than CsA-loaded Tween-SLNs at pH 6.8 PBS containing 0.1% sodium dodecylsulfate and all the three SLNs exhibited biphasic release profiles. The results of cytotoxicity test showed that the toxic effects of Inu12-SLNs and Inu18-SLNs on cell viability had no significant difference in comparison to P188-SLNs and Tween-SLNs. Both CAM-modified SLNs (Inu12/Inu18-SLNs) showed a significant reduced lipolysis in vitro. As compared to P188-SLNs and Tween-SLNs, the total lipolysis of Inu18-SLNs during 4 h was decreased by 31.51 % and 45.67 % and that of Inu12-SLNs was decreased by 24.13 % and 38.29 %, respectively. Besides, the cumulative drug precipitations for CsA-loaded Inu12-SLNs and Inu18-SLNs during 4 h lipolysis were dramatically declined, which were 64% and 42% of that for Tween-SLNs, respectively. Therefore, it can be concluded that both alkylated inulin-derived CAM-modified SLNs, especially the Inu18-SLNs had the improved gastrointestinal stability to resist the lipid degradation by lipase enzyme.

Aqueous remote loading of model cationic peptides in uncapped poly(lactide-co-glycolide) microspheres for long-term controlled release.

Remote loading microencapsulation of peptides into polymer microspheres without organic solvent represents a promising alternative to develop long-acting release depots relative to conventional encapsulation methods. Here, we formulated drug-free microspheres from two kinds of uncapped poly(lactide-co-glycolides) (PLGAs), i.e., ring-opening polymerized Expansorb® DLG 50-2A (50/50, 11.2 kDa) and Expansorb® DLG 75-2A (75/25, 9.0 kDa), and evaluated their potential capacity to remote-load and control the release of two model peptides, leuprolide and octreotide. Degradation and erosion kinetics, release mechanism, and storage stability was also assessed. As control formulations, peptide was loaded in the same PLGA 75/25 polymer by the conventional double emulsion-solvent evaporation method (W/O/W) and remote loaded in polycondensation poly(lactic-co-glycolic acid) 75/25 (Wako 7515, 14.3 kDa). Loading content of 6.7%-8.9% w/w (~ 67%-89% encapsulation efficiency (EE)) was attained for octreotide, and that of 9.5% w/w loading (~ 95% EE) was observed for leuprolide, by the remote loading paradigm. Octreotide and leuprolide were both slowly and continuously released in vitro from the remote-loaded Expansorb® DLG 75-2A MPs for over 56 days, which was highly similar to that observed from traditionally-loaded formulations by W/O/W (8.8% loading, 52.8% EE). The faster release kinetics was observed for the faster degrading PLGA 50/50 remote-loaded Expansorb® DLG 50-2A MPs relative to microspheres from the PLGA 75/25 Expansorb® DLG 75-2A. Despite slight differences in degradation kinetics, the release mechanism of octreotide from the Expansorb® microspheres, whether remote loaded or by W/O/W, was identical as determined by release vs. mass loss curves. Octreotide acylation was also minimal (< ~ 10%) for this polymer. Finally, drug-free Expansorb® DLG 75-2A MPs displayed excellent storage stability over 3 months. Overall, this work offers support for the use of ring-opening Expansorb® PLGA-based microspheres to remote load peptides to create simple and effective long-acting release depots.

In vitro degradation and erosion behavior of commercial PLGAs used for controlled drug delivery.

Copolymers of lactic (or lactide) and glycolic (or glycolide) acids (PLGAs) are among the most commonly used materials in biomedical applications, such as parenteral controlled drug delivery, due to their biocompatibility, predictable degradation rate, and ease of processing. Besides manufacturing variables of drug delivery vehicles, changes in PLGA raw material properties can affect product behavior. Accordingly, an in-depth understanding of polymer-related "critical quality attributes" can improve selection and predictability of PLGA performance. Here, we selected 19 different PLGAs from five manufacturers to form drug-free films, submillimeter implants, and microspheres and evaluated differences in their water uptake, degradation, and erosion during in vitro incubation as a function of L/G ratio, polymerization method, molecular weight, end-capping, and geometry. Uncapped PLGA 50/50 films from different manufacturers with similar molecular weights and higher glycolic unit blockiness and/or block length values showed faster initial degradation rates. Geometrically, larger implants of 75/25, uncapped PLGA showed higher water uptake and faster degradation rates in the first week compared to microspheres of the same polymers, likely due to enhanced effects of acid-catalyzed degradation from PLGA acidic byproducts unable to escape as efficiently from larger geometries. Manufacturer differences such as increased residual monomer appeared to increase water uptake and degradation in uncapped 50/50 PLGA films and poly(lactide) implants. This dataset of different polymer manufacturers could be useful in selecting desired PLGAs for controlled release applications or comparing differences in behavior during product development, and these techniques to further compare differences in less reported properties such as sequence distribution may be useful for future analyses of PLGA performance in drug delivery.

Legumain protease-sheddable PEGylated, tuftsin-modified nanoparticles for selective targeting to tumour-associated macrophages.

Tumour-associated macrophages (TAMs) represent an attractive cell target for anticancer therapy. However, selective and efficient targeting of TAMs remains difficult. Here, we constructed a novel dually functionalised nanoparticle platform (s-Tpep-NPs) by surface co-modification of nanoparticles (NPs) with tuftsin (Tpep) and legumain protease-sheddable polyethylene glycol 5k (PEG5k) to achieve selective targeted delivery to TAMs. The fluorescence resonance energy transfer experiment and in vitro cellular uptake assay confirmed that s-Tpep-NPs can responsively shed PEG5k and transform into active Tpep-NPs upon the cleavage of legumain that is overexpressed on TAM surfaces, which then promotes TAM phagocytosis through Fc receptor-mediated pathways. Owing to the shielding effect by legumain-sheddable PEG5k, s-Tpep-NPs can effectively decrease the Tpep-induced non-specific accumulation in mononuclear phagocyte system (MPS) organs during systemic circulation. Moreover, s-Tpep-NPs can significantly enhance the tumoural accumulation and improve the specificity and efficiency of targeting to TAMs, as compared with both controls of Tpep-NPs and non-sheddable ns-Tpep-NPs. Overall, this study provides a robust nanoplatform with a novel avenue for improved selectivity of targeted delivery to TAMs.

A novel GJB1 mutation associated with X-linked Charcot-Marie-Tooth disease in a large Chinese family pedigree.

BACKGROUND: Charcot-Marie-Tooth (CMT) disease is a group of hereditary neuropathies with high phenotypic and genetic heterogeneity. In this study, we report a large family with X-linked CMT (CMTX) caused by a novel GJB1 mutation. METHODS: A family with the clinical diagnosis of CMTX was investigated. For mutation analysis, the coding region of GJB1 was sequenced using DNA from 15 family members. The identified GJB1 mutation was investigated by DHPLC in 120 normal controls. Mutation reanalysis was performed based on whole-exome sequencing (WES). Cell transfection studies were performed to characterize the function of the novel mutation. RESULTS: A missense mutation (c.605T>A) in GJB1 was detected in five patients and eight female carriers but not in two unaffected members of the family. The mutation was not found in 120 healthy controls and has not been previously reported. WES excluded other pathogenic mutations in the family. The pathogenicity of the mutation was confirmed by disrupting the membrane localization of the encoded proteins. CONCLUSION: Our findings demonstrate that a novel mutation (c.605T>A) in GJB1 is associated with CMTX and adds to the repertoire of GJB1 mutations related to CMTX.

Treating metastatic triple negative breast cancer with CD44/neuropilin dual molecular targets of multifunctional nanoparticles.

Metastasis of cancer makes up the vast majority of cancer-related deaths, and it usually initiates from tumor cells invasiveness and develops through tumor neovasculature. In this work, we have fabricated a CD44/neuropilin dual receptor-targeting nanoparticulate system (tLyP-1-HT NPs) with endogenous or FDA approved components for treating metastatic triple negative breast cancer (TNBC). The enhanced specific targeting of tLyP-1-HT NPs to both metastatic tumor cells and metastasis-supporting tumor neovasculature was contributed by means of CD44/neuropilin dual receptor-mediated interaction. The NPs not only effectively suppress the invasive capability of tumor cells themselves, but also significantly restrain the metastasis incidence via extravasation as well as the eventual colonization in lungs. In all the three types of TNBC-bearing mice models, orthotopic, post-metastasis and metastasis prevention models, the docetaxel-loaded tLyP-1-HT NPs exhibited markedly enhanced anti-tumor and anti-metastasis efficacy. The inhibitory rates of tLyP-1-HT NPs against orthotopic tumor growth and lung metastasis achieved 79.6% and 100%, respectively. The metastasis inhibition rate and life extension rate of the tLyP-1-HT NPs against post-pulmonary metastasis mice reached 85.1% and up to 62.5%, respectively. All the results demonstrated the designed dual receptor-targeting multifunctional NPs hold great potential in treating metastatic TNBC and lung metastasis.

A novel MSX1 intronic mutation associated with autosomal dominant non-syndromic oligodontia in a large Chinese family pedigree.

BACKGROUND: Tooth agenesis is a common developmental dental anomaly. The aim of the study was to identify the causal genetic mutation in a four-generation Chinese family affected with non-syndromic autosomal dominant tooth agenesis. METHODS: Genome-wide scanning was performed using the Illumina Linkage-12 array. Genotyping of short tandem repeat markers was used to finely map the causative locus. Haplotype analysis and Sanger sequencing was performed to precisely locate the position and nature of the gene defect. RESULTS: Clinical examination of the available 23 family members showed variable tooth agenesis in 10 subjects, ranging from oligodontia to mild hypodontia. Genome-wide scanning and haplotype analyses identified the 4p16.1-p16.3 region with a maximum multi-point LOD score of 3.50, which overlapped with the MSX1 gene. A single heterozygous point mutation IVS1-5 G>A in the MSX1 gene was exclusively detected in the 10 family members affected with tooth agenesis. Sequencing of MSX1 cDNA revealed that the intronic mutation did not affect the normal splicing pattern of the pre-mRNA. However, real-time qPCR analysis of lymphocyte RNA showed that the level of MSX1 mRNA was significantly decreased in individuals heterozygous for the mutation. CONCLUSIONS: We identified and characterized a novel intronic mutation in the MSX1 gene in a large Chinese pedigree, adding to the small repertoire of MSX1 mutations associated with autosomal dominant tooth agenesis. We hypothesize that the variable degree of tooth agenesis observed in each affected individual may be due to sub-optimal levels of MSX1 expression during critical stages tooth development.

Roles of ligand and TPGS of micelles in regulating internalization, penetration and accumulation against sensitive or resistant tumor and therapy for multidrug resistant tumors.

There are several obstacles in the process of successful treatment of malignant tumors, including toxicity to normal cells, inefficiency of drug permeation and accumulation into the deep tissue of solid tumor, and multidrug resistance (MDR). In this work, we prepared docetaxel (DTX)-loaded hybrid micelles with DSPE-PEG and TPGS (TPGS/DTX-M), where TPGS serves as an effective P-gp inhibitor for overcoming MDR, and active targeting hybrid micelles (FA@TPGS/DTX-M) with targeting ligand of folate on the hybrid micelles surface offering active targeting to folate receptor-overexpressed tumor cells. A systematic comparative evaluation of these micelles on cellular internalization, sub-cellular distribution, antiproliferation, mitochondrial membrane potential, cell apoptosis and cell cycle, permeation and inhibition on 3-dimensional multicellular tumor spheroids, as well as antitumor efficacy and safety assay in vivo were well performed between sensitive KB tumors and resistant KBv tumors, and among P-gp substrate or not. We found that the roles of folate and TPGS varied due to the sensitivity of tumors and the loaded molecules in the micelles. Folate and folate receptor-mediated endocytosis played a leading role in internalization, permeation and accumulation for sensitive tumors and non-substrates of P-gp. On the contrary, TPGS played the predominant role which dramatically decreased the efflux of drugs both when the tumor is resistant and for P-gp substrate. These findings are very meaningful for guiding the design of carrier delivery system to treat tumors. The antitumor efficacy in xenograft nude mice model and safety assay showed that the TPGS/DTX-M and FA@TPGS/DTX-M significantly exhibited higher antitumor activity against resistant KBv tumors than the marketed formulation and normal micelles owing to the small size (approximately 20 nm), hydrophilic PEGylation, TPGS inhibition of P-gp function, and folate receptor-modified endocytosis, permeation and accumulation in solid tumor, as well as synergistic effects of DTX-induced cell division inhibition, growth restraint and TPGS-triggered mitochondrial apoptosis in tumor cells. In conclusion, folate-modified TPGS hybrid micelles provide a synergistic strategy for effective delivery of DTX into KBv cells and overcoming MDR.

Novel de novo nonsense mutation of the PHEX gene (p.Lys50Ter) in a Chinese patient with hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLHR), the most common form of inherited rickets, is a dominant disorder characterized by hypophosphatemia, abnormal bone mineralization, and short stature. Mutations in the PHEX gene are major causes of XLHR. Herein, we clinically characterized four unrelated families with hypophosphatemia, bone abnormalities, short stature, and dentin malformation. Mutational analysis of the PHEX gene using Sanger sequencing revealed three recurrent mutations (c.2197T>C, c.1646G>C, and c.2198G>A) and a de novo nonsense mutation (c.148A>T). The novel mutation was not found in any of the unaffected family members or in the 100 healthy controls and was predicted to produce a truncated protein (p.K50X), a truncated form of the PHEX protein caused by nonsense mutations has been frequently detected in XLHR individuals. Thus, our work indicated that the c.148A>T (p.K50X) mutation was the likely pathogenic mutation in individual III-2 in family 2, and that PHEX gene mutations were responsible for XLHR in these Chinese families. These findings expand the mutation spectrum of PHEX and may help us to understand the molecular basis of XLHR in order to facilitate genetic counseling.