Temperature-Responsive "Nano-to-Micro" Transformed Polymersomes for Enhanced Ultrasound/Fluorescence Dual Imaging-Guided Tumor Phototherapy.

The perfect integration of microbubbles for efficient ultrasound imaging and nanocarriers for intelligent tumor-targeting delivery remains a challenge in precise tumor theranostics. Herein, we exquisitely fabricated laser-activated and targeted polymersomes (abbreviated as FIP-NPs) for simultaneously encapsulating the photosensitizer indocyanine green (ICG) and the phase change agent perfluorohexane (PFH). The formulated FIP-NPs were nanosize and effectively accumulated into tumors as observed by ICG fluorescence imaging. When the temperature rose above 56 °C, the encapsulated PFH transformed from liquid to gas and the FIP-NPs underwent balloon-like enlargement without structure destruction. Impressively, the enlarged FIP-NPs fused with adjacent polymersomes to form even larger microparticles. This temperature-responsive "nano-to-micro" transformation and fusion process was clearly demonstrated, and FIP-NPs showed greatly improved ultrasound signals. More importantly, FIP-NPs achieved dramatic antitumor efficacy through ICG-mediated phototherapy. Taken together, the novel polymersomes achieved excellent ultrasound/fluorescence dual imaging-guided tumor phototherapy, providing an optimistic candidate for the application of tumor theranostics.

A luciferase reporter mouse model to optimize in vivo gene editing validated by lipid nanoparticle delivery of adenine base editors.

The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4 months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5% ± 17.5% and 8.4% ± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.

Chlorinated Narrow Bandgap Polymer Suppresses Non-Radiative Recombination Energy Loss Enabling Perylene Diimides-Based Organic Solar Cells Exceeding 10% Efficiency.

The scarcity of narrow bandgap donor polymers matched with perylene diimides (PDI)-based nonfullerene acceptors (NFAs) hinders improvement of the power conversion efficiency (PCE) value of organic solar cells (OSCs). Here, it is reported that a narrow bandgap donor polymer PDX, the chlorinated derivative of the famous polymer donor PTB7-Th, blended with PDI-based NFA boosts the PCE value exceeding 10%. The electroluminescent quantum efficiency of PDX-based OSCs is two orders of magnitude higher than that of PTB7-Th-based OSCs;therefore, the nonradiative energy loss is 0.103 eV lower. This is the highest PCE value for OSCs with the lowest energy loss using the blend of PTB7-Th derivatives and PDI-based NFAs as the active layer. Besides, PDX-based devices showed larger phase separation, faster charge mobilities, higher exciton dissociation probability, suppressed charge recombination, elevated charge transfer state, and decreased energetic disorder compared with the PTB7-Th-based OSCs. All these factors contribute to the simultaneously improved short circuit current density, open circuit voltage, and fill factor, thus significantly improving PCE. These results prove that chlorinated conjugated side thienyl groups can efficiently suppress the non-radiative energy loss and highlight the importance of fine-modifying or developing novel narrow bandgap polymers to further elevate the PCE value of PDI-based OSCs.

Estimation of Wind Speed Based on Schlieren Machine Vision System Inspired by Greenhouse Top Vent.

Greenhouse ventilation has always been an important concern for agricultural workers. This paper aims to introduce a low-cost wind speed estimating method based on SURF (Speeded Up Robust Feature) feature matching and the schlieren technique for airflow mixing with large temperature differences and density differences like conditions on the vent of the greenhouse. The fluid motion is directly described by the pixel displacement through the fluid kinematics analysis. Combining the algorithm with the corresponding image morphology analysis and SURF feature matching algorithm, the schlieren image with feature points is used to match the changes in air flow images in adjacent frames to estimate the velocity from pixel change. Through experiments, this method is suitable for the speed estimation of turbulent or disturbed fluid images. When the supply air speed remains constant, the method in this article obtains 760 sets of effective feature matching point groups from 150 frames of video, and approximately 500 sets of effective feature matching point groups are within 0.1 difference of the theoretical dimensionless speed. Under the supply conditions of high-frequency wind speed changes and compared with the digital signal of fan speed and data from wind speed sensors, the trend of wind speed changes is basically in line with the actual changes. The estimation error of wind speed is basically within 10%, except when the wind speed supply suddenly stops or the wind speed is 0 m/s. This method involves the ability to estimate the wind speed of air mixing with different densities, but further research is still needed in terms of statistical methods and experimental equipment.

Structure-Function Analysis of RBP7910: An Editosome Z-Binding Protein in Trypanosomatids.

RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-ß nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix-turn-helix (HTH) superfamily of proteins with an α1α2α3ß1ß2 topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands.

Visible light-driven self-powered aptasensors for ultrasensitive Microcystin-LR detection based on the carrier density effect of N-doped graphene hydrogel/hematite Schottky junctions.

In this work, a novel visible light-driven self-powered photoelectrochemical (PEC) platform was designed based on 3D N-doped graphene hydrogel/hematite nanocomposites (NGH/Fe2O3) via a facile one-pot hydrothermal route. The coupling NGH with Fe2O3 could generate a Schottky junction, which promoted the separation of charges. Moreover, Mott-Schottky measurements validated that the carrier concentration achieved by NGH/Fe2O3 was about 3.4 × 103 times in comparison to that of pure Fe2O3, which was beneficial for efficient charge transfer. Owing to the carrier density effect and Schottky junction, the photocurrent of the as-fabricated NGH/Fe2O3 nanocomposites was 6.9-fold higher than that of pure Fe2O3. On the basis of such excellent Schottky junctions, an ultrasensitive visible light-induced self-powered PEC aptasensor was developed using a Microcystin-LR (MC-LR) aptamer. The as-fabricated PEC aptasensor displayed good analytical performance toward MC-LR detection in terms of wide linear range (1 pM-5 nM), low detection limit (0.23 pM, S/N = 3), excellent selectivity and high stability. This new strategy can provide a way for regulating nanostructures for more sensitive PEC sensors by increasing the carrier density.

Zwitterionic Unimolecular Micelles with pH and Temperature Response: Enhanced In Vivo Circulation Stability and Tumor Therapeutic Efficiency.

Circulation stability in vivo and stimuli-responsiveness under a tumor microenvironment of the polymeric prodrug micellar drug delivery systems are very critical to improve the tumor therapeutic efficiency. In this study, a series of polyamidoamine (PAMAM)-graft-poly(2-(diethylamino) ethyl methacrylate) (PDEAEMA)-block-poly(betaine sulfonate) (PSBMA) (PDS) unimolecular micelles were prepared via atom transfer radical polymerization. PAMAM served as a hydrophobic core to load the drug, the PDMAEMA segment was a middle layer to provide both thermo- and pH-sensitivity, whereas the PSMBA shell layer was used to improve the stability of the unimolecular micelles. The PDS exhibited a spherical structure with the size of 10-20 nm at pH 7.4. PDS micelles had excellent stability to resist the large volume liquid dilution. Moreover, it exhibited excellent stability in a complex biological microenvironment because of a superhigh antiprotein adhesion capacity of the PSBMA shell layer compared with PAMAM micelles. Drug release studies confirmed that the DOX can remain in the PDS micelles at pH 7.4 and 37 °C, whereas it can rapidly be released when the pH decreases to 5.0 and/or the temperature increases to 40 °C. In vitro studies suggested that the PDS drug delivery system can effectivity induce apoptosis and inhibit the proliferation of cancer cells. In vivo studies suggested that the PDS micelles prolonged the circulation time, decreased the side effects, and increased the antitumor efficacy. Therefore, the prepared PDS micelles are a potential anticancer drug delivery carrier for cancer therapy.

Targeted Codelivery of an Antigen and Dual Agonists by Hybrid Nanoparticles for Enhanced Cancer Immunotherapy.

Among approaches of current cancer immunotherapy, a dendritic cell (DC)-targeted vaccine based on nanotechnology could be a promising way to efficiently induce potent immune responses. To enhance DC targeting and vaccine efficiency, we included imiquimod (IMQ), a toll-like receptor 7/8 (TLR 7/8) agonist, and monophosphoryl lipid A (MPLA), a TLR4 agonist, to synthesize lipid-polymer hybrid nanoparticles using PCL-PEG-PCL and DOTAP (IMNPs) as well as DSPE-PEG-mannose (MAN-IMNPS). The spatiotemporal delivery of MPLA (within the outer lipid layer) to extracellular TLR4 and IMQ (in the hydrophobic core of NPs) to intracellular TLR7/8 can activate DCs synergistically to improve vaccine efficacy. Ovalbumin (OVA) as a model antigen was readily absorbed by positively charged DOTAP and showed a quick release in vitro. Our results demonstrated that this novel nanovaccine enhanced cellular uptake, cytokine production, and maturation of DCs. Compared with the quick metabolism of free OVA-agonists, the depot effect of OVA-IMNPs was observed, whereas MAN-OVA-IMNPs promoted trafficking to secondary lymphoid organs. After immunization with a subcutaneous injection, the nanovaccine, especially MAN-OVA-IMNPs, induced more antigen-specific CD8+ T cells, greater lymphocyte activation, stronger cross-presentation, and more generation of memory T cells, antibody, IFN-γ, and granzyme B. Prophylactic vaccination of MAN-OVA-IMNPs significantly delayed tumor development and prolonged the survival in mice. The therapeutic tumor challenge indicated that MAN-OVA-IMNPs prohibited tumor progression more efficiently than other formulations, and the combination with an immune checkpoint blockade further enhanced antitumor effects. Hence, the DC-targeted vaccine codelivery with IMQ and MPLA adjuvants by hybrid cationic nanoparticles in a spatiotemporal manner is a promising multifunctional antigen delivery system in cancer immunotherapy.

Exome Sequencing and the Management of Neurometabolic Disorders.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

ABCA8 Regulates Cholesterol Efflux and High-Density Lipoprotein Cholesterol Levels.

OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

Mitochondrial carbonic anhydrase VA deficiency resulting from CA5A alterations presents with hyperammonemia in early childhood.

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Novel homozygous PCK1 mutation causing cytosolic phosphoenolpyruvate carboxykinase deficiency presenting as childhood hypoglycemia, an abnormal pattern of urine metabolites and liver dysfunction.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy.

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

Preparation and rebinding properties of protein-imprinted polysiloxane using mesoporous calcium silicate grafted non-woven polypropylene as matrix.

Calcium silicate particle containing mesoporous SiO2 (CaSiO3@SiO2) was grafted on the surface of non-woven polypropylene. The PP non-woven grafted calcium silicate containing mesoporous SiO2 (PP-g-CaSiO3@SiO2) was used as the matrix to prepare bovine serum albumin (BSA) molecularly imprinted polysiloxane (MIP) by using silanes as the functional monomers and BSA as the template. PP non-woven grafted BSA-imprinted polysiloxane (PP-g-CaSiO3@SiO2 MIP) was characterized by scanning electron microscope (SEM), Fourier transform infrared spectometry (FTIR) and drilling string compensator (DSC). Influence factors on the rebinding capacity of the MIP were investigated, such as grafting degree, the pH in treating CaSiO3 and the type and proportion of silanes. The rebinding properties of BSA on PP-g-CaSiO3@SiO2 and MIP were investigated under different conditions. The results indicated that the rebinding capacity of MIP for BSA reached 56.32 mg/g, which was 2.65 times of NIP. The non-woven polypropylene grafted BSA-imprinted polysiloxane could recognize the template protein and the selectivity factor (ß) was above 2.4 when using ovalbumin, hemoglobin and γ-globulin as control proteins. The PP-g-CaSiO3@SiO2 MIP has favorable reusability.

Cytosolic phosphoenolpyruvate carboxykinase deficiency presenting with acute liver failure following gastroenteritis.

We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic and metabolic investigations for acute liver failure failed to yield a diagnosis. Whole exome sequencing revealed a homozygous 12-bp deletion in PCK1 (MIM 614168) encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK); enzymatic studies subsequently confirmed its pathogenic nature. We propose that PEPCK deficiency should be considered in the young child with unexplained liver failure, especially where there are marked, accumulations of TCA cycle metabolites on urine organic acid analysis and/or an amino acid profile with hyperammonaemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-)physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes.

Secondary neurotransmitter deficiencies in epilepsy caused by voltage-gated sodium channelopathies: A potential treatment target?

We describe neurotransmitter abnormalities in two patients with drug-resistant epilepsy resulting from deleterious de novo mutations in sodium channel genes. Whole exome sequencing identified a de novo SCN2A splice-site mutation (c.2379+1G>A, p.Glu717Gly.fs*30) resulting in deletion of exon 14, in a 10-year old male with early onset global developmental delay, intermittent ataxia, autism, hypotonia, epileptic encephalopathy and cerebral/cerebellar atrophy. In the cerebrospinal fluid both homovanillic acid and 5-hydroxyindoleacetic acid were significantly decreased; extensive biochemical and genetic investigations ruled out primary neurotransmitter deficiencies and other known inborn errors of metabolism. In an 8-year old female with an early onset intractable epileptic encephalopathy, developmental regression, and progressive cerebellar atrophy, a previously unreported de novo missense mutation was identified in SCN8A (c.5615G>A; p.Arg1872Gln), affecting a highly conserved residue located in the C-terminal of the Nav1.6 protein. Aside from decreased homovanillic acid and 5-hydroxyindoleacetic acid, 5-methyltetrahydrofolate was also found to be low. We hypothesize that these channelopathies cause abnormal synaptic mono-amine metabolite secretion/uptake via impaired vesicular release and imbalance in electrochemical ion gradients, which in turn aggravate the seizures. Treatment with oral 5-hydroxytryptophan, l-Dopa/Carbidopa, and a dopa agonist resulted in mild improvement of seizure control in the male case, most likely via dopamine and serotonin receptor activated signal transduction and modulation of glutamatergic, GABA-ergic and glycinergic neurotransmission. Neurotransmitter analysis in other sodium channelopathy patients will help validate our findings, potentially yielding novel treatment opportunities.

Unravelling 5-oxoprolinuria (pyroglutamic aciduria) due to bi-allelic OPLAH mutations: 20 new mutations in 14 families.

Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the γ-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology.

Doxorubicin-loaded micelles based on multiarm star-shaped PLGA-PEG block copolymers: influence of arm numbers on drug delivery.

Star-shaped block copolymers based on poly(D,L-lactide-co-glycolide) (PLGA) and poly(ethylene glycol) (PEG) (st-PLGA-PEG) were synthesized with structural variation on arm numbers in order to investigate the relationship between the arm numbers of st-PLGA-PEG copolymers and their micelle properties. st-PLGA-PEG copolymers with arm numbers 3, 4 and 6 were synthesized by using different cores such as trimethylolpropane, pentaerythritol and dipentaerythritol, and were characterized by nuclear magnetic resonance and gel permeation chromatography. The critical micelle concentration decreased with increasing arm numbers in st-PLGA-PEG copolymers. The doxorubicin-loaded st-PLGA-PEG micelles were prepared by a modified nanoprecipitation method. Micellar properties such as particle size, drug loading content and in vitro drug release behavior were investigated as a function of the number of arms and compared with each other. The doxorubicin-loaded 4-arm PLGA-PEG micelles were found to have the highest cellular uptake efficiency and cytotoxicity compared with 3-arm PLGA-PEG micelles and 6-arm PLGA-PEG micelles. The results suggest that structural tailoring of arm numbers from st-PLGA-PEG copolymers could provide a new strategy for designing drug carriers of high efficiency. Structural tailoring of arm numbers from star shaped-PLGA-PEG copolymers (3-arm/4-arm/6-arm-PLGA-PEG) could provide a new strategy for designing drug carriers of high efficiency.

Cloning and characterization of a novel dehydrin gene, SiDhn2, from Saussurea involucrata Kar. et Kir.

Saussurea involucrata Kar. et Kir. is a hardy dicotyledonous plant capable of tolerating severe abiotic stress conditions. In a previous study, we created a cDNA library to determine what factors are associated with the cold acclimation response in S. involucrata. From this, a full-length cDNA of a dehydrin-like gene (SiDhn2) was obtained by RT-PCR. The SiDhn2 gene was characterized in this study. The full-length SiDhn2 cDNA comprised 693 bp containing an open reading frame of 345 bp specifying a protein of 115 amino acids. An alignment of the deduced amino acid sequence showed that SiDhn2 shared 55 % identity with two Brassica dehydrins. Agrobacterium tumefaciens was used to transform RD29A:SiDhn2 and 35S:SiDhn2 constructs into tobacco to investigate the germination and resistance to freezing and drought stress of transgenic plants. The RD29A:SiDhn2 transgenic plants showed greater resistance to freezing and drought stress than 35S:SiDhn2 transgenic plants or the wild-type. This study demonstrates that SiDhn2 confers cold hardiness and drought resistance, and may be a candidate resistance gene for genetic improvement of crops to increase stress resistance.

Regulation of ABCA1 protein expression and function in hepatic and pancreatic islet cells by miR-145.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) protein maintains cellular cholesterol homeostasis in several different tissues. In the liver, ABCA1 is crucial for high-density lipoprotein biogenesis, and in the pancreas ABCA1 can regulate insulin secretion. In this study, our aim was to identify novel microRNAs that regulate ABCA1 expression in these tissues. APPROACH AND RESULTS: We combined multiple microRNA prediction programs to identify 8 microRNAs that potentially regulate ABCA1. A luciferase reporter assay demonstrated that 5 of these microRNAs (miR-148, miR-27, miR-144, miR-145, and miR-33a/33b) significantly repressed ABCA1 3'-untranslated region activity with miR-145 resulting in one of the larger decreases. In hepatic HepG2 cells, miR-145 can regulate both ABCA1 protein expression levels and cholesterol efflux function. In murine islets, an increase in miR-145 expression decreased ABCA1 protein expression, increased total islet cholesterol levels, and decreased glucose-stimulated insulin secretion. Inhibiting miR-145 produced the opposite effect of increasing ABCA1 protein levels and improving glucose-stimulated insulin secretion. Finally, increased glucose levels in media significantly decreased miR-145 levels in cultured pancreatic beta cells. These findings suggest that miR-145 is involved in glucose homeostasis and is regulated by glucose concentration. CONCLUSIONS: Our studies demonstrate that miR-145 regulates ABCA1 expression and function, and inhibiting this microRNA represents a novel strategy for increasing ABCA1 expression, promoting high-density lipoprotein biogenesis in the liver, and improving glucose-stimulated insulin secretion in islets.