The role of FREM2 and FRAS1 in the development of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.

Targeted Lipid Nanoemulsions Encapsulating Epigenetic Drugs Exhibit Selective Cytotoxicity on CDH1-/FOXM1+ Triple Negative Breast Cancer Cells.

The plasticity of cancer epigenetics makes them plausible candidates for therapeutic intervention. We took advantage of elevated expression of lysophosphatidic acid receptor 1 (LPAR1) in triple negative breast cancer (TNBC) tissues to target decitabine (DAC) and panobinostat (PAN) to breast cancer cells. DAC and PAN were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to increased expression of tumor suppressor genes and decreased expression of oncogenes. Although DAC and PAN have therapeutic benefits, they are limited by chemical instability and systemic toxicity. Herein, we present LPAR1-targeted, lipid nanoemulsions (LNEs) encapsulating both DAC and PAN. Our results demonstrated that the cell uptake and in vivo biodistribution of LNEs was dependent on LPAR1 expression in TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth of mesenchymal breast cancer cells by restoring CDH1/E-cadherin and suppressing forkhead box M1 (FOXM1) expression. Epithelial breast cancer cells that inherently express low FOXM1 and high CDH1 were unaffected by DAC/PAN-LNEs. Overall, we successfully designed LPAR1-targeted LNEs that selectively act on CDH1(low)/FOXM1(high) TNBC cell lines.

Phosphoinositide dependent protein kinase 1 is required for exercise-induced cardiac hypertrophy but not the associated mitochondrial adaptations.

Phosphoinositide-dependent protein kinase-1 (PDPK1) is an important mediator of phosphatidylinositol 3-kinase (PI3K) signaling. We previously reported that PI3K but not Akt signaling mediates the increase in mitochondrial oxidative capacity following physiological cardiac hypertrophy. To determine if PDPK1 regulates these metabolic adaptations we examined mice with cardiomyocyte-specific heterozygous knockout of PDPK1 (cPDPK1(+/-)) after 5 wk. exercise swim training. Akt phosphorylation at Thr308 increased by 43% in wildtype (WT) mice but not in cPDPK1(+/-) mice following exercise training. Ventricular contractile function was not different between WT and cPDPK1(+/-) mice at baseline. In addition, exercise did not influence ventricular function in WT or cPDPK1(+/-) mice. Heart weight normalized to tibia length ratios increased by 13.8% in WT mice (6.2±0.2 vs. 7.1±0.2, P=0.001), but not in cPDPK1(+/-) (6.2±0.3 vs. 6.5±0.2, P=0.20) mice after swim training. Diastolic LV dimension increased in WT mice (3.7±0.1 vs. 4.0±0.1 mm, P=0.01) but not in cPDPK1(+/-) (3.8±0.1 vs. 3.7±0.1 mm, P=0.56) following swim training. Maximal mitochondrial oxygen consumption (VADP, nmol/min/mg) using palmitoyl carnitine as a substrate was significantly increased in mice of all genotypes following swim training (WT: 13.6±0.6 vs.16.1±0.9, P=0.04; cPDPK1(+/-): 12.4±0.6 vs.15.9±1.2, P=0.04). These findings suggest that PDPK1 is required for exercise-induced cardiac hypertrophy but does not contribute to exercise-induced increases in mitochondrial function.

Two-dimensional reactive transport model as a new approach for identifying the origins and contribution of arsenic in a soil and water system.

Distribution of arsenic (As) in the environment can occur through various paths, depending on the interrelationship between soil and surface water, and groundwater. This study aimed to identify the origins and pathways of As among various potential origins in soil and water systems using two-dimensional reactive transport models (2D RTMs) and to evaluate the contribution of each origin to the geochemical distribution of As in the study area, which included a reservoir, two streams, and two abandoned mines. The 2D RTMs were prepared by combining hydraulic and geochemical reactive models. The MODFLOW-2005 package was used to simulate the water flow in the study area. The geochemical reaction and distribution of As were simulated using Geochemist's Workbench. The concentration of As in the reservoir was ∼50 µg/L, while that in the soils collected around the mine X and Y was 15.0-1853.2 mg/kg (median: 126.1 mg/kg) and 3.6-1629.2 mg/kg (median: 60.9 mg/kg), respectively. Hydraulic and geochemical input data were derived from field surveys and laboratory experiments. Based on the geochemical and hydrogeological input data, 2D RTMs determined that 32.0 kg of As accumulated into the reservoir over 18 years through surface water, which is from mines X (2.7 kg) and Y (29.3 kg). Hence, mine Y would be the primary origin of As to the reservoir. Based on 2D RTM, 2.4 kg of As entered the reservoir via groundwater (DO <0.1 mg/L). Also, precipitation of sulfide minerals (e.g., orpiment; As2S3) influences As dispersion and contribution under subsurface conditions. These modeling results suggest that 2D RTMs could be a new approach for identifying and assessing the contribution of potential As origins in soil and water system.

A Comprehensive Study on EMI Shielding Performance of Carbon Nanomaterials-Embedded CFRP or GFRP Composites.

The rapid advancement of electrical and telecommunication facilities has resulted in increasing requirements for the development of electromagnetic interference (EMI) shielding composites. Accordingly, an experimental study was conducted to evaluate the EMI shielding performance of carbon nanomaterial (CNM)-embedded carbon-fiber-reinforced polymer (CFRP) or glass-fiber-reinforced polymer (GFRP) composites. Nine combinations of CNMs and carbon or glass fibers were used to fabricate the composites. The synergistic effects of CNMs on the EMI shielding performance were systematically investigated. The results indicated that plate-type CNMs (i.e., graphene and graphite nanoplatelets) have more prominent effects than fiber-type CNMs (carbon nanofibers). The composites fabricated with CFRP afforded higher EMI shielding than the GFRP-based composites. Among the eighteen samples, 3% CNT-GNP in CFRP composites, which included plate-typed CNM, exhibited the best EMI shielding performances, showing 38.6 dB at 0.7 GHz. This study helps understand the shielding performance of CNM-embedded CFRP and GFRP composites in electrical and telecommunication facilities.

Formulation of pH-Responsive Methacrylate-Based Polyelectrolyte-Stabilized Nanoparticles for Applications in Drug Delivery.

pH-responsive polyelectrolytes, including methacrylate-based anionic copolymers (MACs), are widely used as enteric coatings and matrices in oral drug delivery. Despite their widespread use in these macroscopic applications, the molecular understanding of their use as stabilizers for nanoparticles (NPs) is lacking. Here, we investigate how MACs can be used to create NPs for therapeutic drug delivery and the role of MAC molecular properties on the assembly of NPs via flash nanoprecipitation. The NP size is tuned from 59 to 454 nm by changing the degree of neutralization, ionic strength, total mass concentration, and the core-to-MAC ratio. The NP size is determined by the volume of hydrophilic domains on the surface relative to the volume of hydrophobic domains in the core. We calculate the dimensions of the hydrophobic NP core relative to the thickness of the polyelectrolyte layer over a range of ionizations. Importantly, the results are shown to apply to both high-molecular-weight polymers as core materials and small-molecule drugs. The pH responsiveness of MAC-stabilized NPs is also demonstrated. Future development of polyelectrolyte copolymer-stabilized nanomedicines will benefit from the guiding principles established in this study.

A novel function of Siglec-9 A391C polymorphism on T cell receptor signaling.

BACKGROUND: Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are the best-characterized immunoglobulin-type lectins. There is a growing amount of data linking Siglec and autoimmune diseases. The recently identified Siglec-9 inhibits T cell receptor (TCR)-mediated signaling which has been demonstrated by site-directed mutagenesis. In human Siglec-9, at least 8 nonsynonymous SNPs have been detected without functional studies. This study examined the SNP(s) related to TCR-mediated signaling. METHODS: Since the functions of Siglecs are modulated by their interaction with sialic-acid-containing carbohydrate groups, a molecular modeling analysis of carbohydrate binding interactions and an RBC binding analysis were performed using the 8 SNPs. The TCR-mediated signaling was analyzed with the downstream signaling molecules ZAP-70 and IL-2. RESULTS: This study revealed that an A391C polymorphism is the only mutant related to the binding. Jurkat T cells transfected with the A391C mutant reduced the inhibition of ZAP-70 phosphorylation and IL-2 production compared to cells transfected with the wild type. CONCLUSIONS: Siglec-9 A391C was the only polymorphism related to TCR-mediated signaling in human Siglec-9, resulting in less inhibition compared to the wild type.

Machine learning guided association of adverse drug reactions with in vitro target-based pharmacology.

BACKGROUND: Adverse drug reactions (ADRs) are one of the leading causes of morbidity and mortality in health care. Understanding which drug targets are linked to ADRs can lead to the development of safer medicines. METHODS: Here, we analyse in vitro secondary pharmacology of common (off) targets for 2134 marketed drugs. To associate these drugs with human ADRs, we utilized FDA Adverse Event Reports and developed random forest models that predict ADR occurrences from in vitro pharmacological profiles. FINDINGS: By evaluating Gini importance scores of model features, we identify 221 target-ADR associations, which co-occur in PubMed abstracts to a greater extent than expected by chance. Amongst these are established relations, such as the association of in vitro hERG binding with cardiac arrhythmias, which further validate our machine learning approach. Evidence on bile acid metabolism supports our identification of associations between the Bile Salt Export Pump and renal, thyroid, lipid metabolism, respiratory tract and central nervous system disorders. Unexpectedly, our model suggests PDE3 is associated with 40 ADRs. INTERPRETATION: These associations provide a comprehensive resource to support drug development and human biology studies. FUNDING: This study was not supported by any formal funding bodies.

Microfluidic pycnometer for in situ analysis of fluids in microchannels.

This paper presents a new particle migration principle and demonstrates an analysis of solution density in a microfluidic channel. A particle situated in a microfluidic interface with different fluid density moves toward the lower-density fluid, driven by the asymmetric hydrostatic pressure acting on the submerged particle. Since the hydrostatic pressure is related to the diameter of a particle submerged in a solution and the density of a solution, we expected that the particle injected into the middle of three inlets would show larger lateral deflection as the particle size and density difference between two-injected solutions increase. In addition to hydrostatic pressure differences on the particle, we were concerned not only about fluid momentum difference caused by solution density and flow rate but also about rotational motion due to asymmetric buoyancy driven by the density gradient. The fluid momentum difference affects an initial position of the injected particles and its influence becomes enlarged according to the flow rate increases. The rotational motion of the particle had been evaluated using a computational tool. The experimental results and numerical expectation proved our theoretical estimation, and here this behavior explains a new principle termed pyklinophoresis (Greek; pyk-, density; -klino-, gradient; -phoresis, migration), which enables in situ analysis of microfluidic liquid samples. An analytical model for pyklinophoresis was provided as a proof-of-concept, and the analytical results of sucrose and volatile solutions were also demonstrated.

Clinical Benefit of Maintenance Therapy for Advanced Biliary Tract Cancer Patients Showing No Progression after First-Line Gemcitabine Plus Cisplatin.

PURPOSE: Gemcitabine plus cisplatin (GemCis) is the standard first-line chemotherapy for patients with advanced biliary tract cancer (BTC). In ABC-02 study, the BTC patients received up to 6-8 cycles of 3-weekly GemCis; however, those without progression often receive more than 6-8 cycles. The clinical benefit of maintenance treatment in patients without progression is uncertain. MATERIALS AND METHODS: Advanced BTC patients treated with GemCis between April 2010 and February 2015 at Asan Medical Center, Seoul, Korea, were retrospectively analysed. The patients without progression after 6-8 cycles were stratified according to further treatment i.e., with or without further cycles of GemCis (maintenance vs. observation groups). The primary endpoint was overall survival (OS) and progression-free survival (PFS). RESULTS: Among the 740 BTC patients in the initial screen, 231 cases (31.2%) were eligible for analysis (111 in the observation group, 120 in the maintenance group). The median OS from the GemCis initiation was 20.5 months (95% confidence interval [CI], 15.4 to 25.6) and 22.4 months (95% CI, 17.0 to 27.8) in the observation and maintenance groups, respectively (p=0.162). The median PFS was 10.4 months (95% CI, 7.0 to 13.8) and 13.2 months (95% CI, 11.3 to 15.2), respectively (p=0.320). CONCLUSIONS: GemCis maintenance is not associated with an improved survival outcome.

Cre recombinase expression controlled by the hematopoietic regulatory domain of Gata-1 is erythroid-specific.

Available data suggest that gene regulation by the Gata-1 Hematopoietic Regulatory Domain (Gata-1-HRD) is limited to cells derived from the erythroid lineage. This characteristic makes Gata-1-HRD a candidate for control of cre expression in conditional knock-in and knock-out models in which erythroid-specific gene expression is essential. To characterize the specificity of Gata-1 HRD regulation of cre, transgenic mice expressing improved cre recombinase (iCre) under the control of Gata-1-HRD were generated. The founders were crossbred with mice that have an inactive loxP-containing beta-galactosidase gene that can be rescued by the cre recombinase. The beta-galactosidase activity was detected in the marrow of this crossbred mouse, but no activity was observed in other organs. To identify the cre expressing cells in marrow, double-immunostaining of marrow sections with anti-beta-galactosidase, and antibodies against various hematopoietic lineage markers or erythropoietin receptor (epor) was performed. The epor positive cells in marrow expressed beta-galactosidase, but megakaryocytic precursors and nonerythroid epor-positive cells in brain and spleen did not. We conclude that when cre is under control of Gata-1-HRD, its expression/function is limited to erythroid progenitors. The knock-in and knock-out models utilizing Gata-1-HRD-iCre, can be explored for the studies of erythroid-specific gene expression.

Thymic PTH Increases After Thyroparathyroidectomy in C57BL/KaLwRij Mice.

We previously reported a substantial correlation between serum parathyroid hormone (PTH) levels and the myeloma response to proteasome inhibition that suggests a crucial role for the PTH receptor 1 system in the control of myeloma tumor growth. While investigating the role of PTH in the antimyeloma effect, we observed the recovery of serum PTH levels after thyroparathyroidectomy (TPTX). Although the presence of thymus-derived PTH has been reported previously, the existence or role of thymic PTH in the serum remains controversial. Here, TPTX was performed in 8- to 12-week-old C57BL/KaLwRij mice to delineate the potential source(s) for the recovery of serum PTH. Immediately after TPTX, the expected loss of measurable serum PTH was observed. Serum PTH levels recovered 3 to 4 weeks after TPTX. Thirteen endocrine organs from mice with recovered serum PTH were examined. The thymus from control mice expressed measurable and detectable Pth transcripts; however, the Pth transcript level was substantially elevated in tissue from TPTX mice. Western blot analysis of the thymus demonstrated a reproducible and distinct PTH band in thymus tissue that was significantly increased after TPTX. To directly confirm the identity of the distinct PTH band, immunoprecipitated proteins were isolated and subjected to tandem mass spectrometry. After fragmentation and direct peptide sequencing, PTH peptides PTH(1-13) and PTH(54-70), diagnostic for PTH, were identified. These data demonstrate that the murine thymus produces PTH and that after TPTX the thymus becomes the major source of serum PTH, compensating for the loss of the parathyroid glands and returning circulating PTH levels to normal.

Electrochemical detection of cardiac troponin I using a microchip with the surface-functionalized poly(dimethylsiloxane) channel.

A sensitive and rapid electrochemical microchip fabricated by assembling a surface-functionalized poly(dimethylsiloxane) (PDMS) microchannel with an interdigitated array (IDA) gold electrode was developed for the detection of human cardiac troponin I (cTnI) in the early diagnosis of acute myocardial infarction. Anti-cTnI was immobilized onto the internal surface of the PDMS channel on which protein G layer had been generated by silanization. To reduce electrode fouling, a PDMS channel was assembled with an IDA chip after surface treatment. The detection experiments were performed with successive injection of cTnI, alkaline phosphatase (AP)-labeled anti-cTnI, and p-aminophenylphosphate. Then, cyclic voltammograms were obtained by the oxidation peak current proportionally to the concentration of enzymatic product, p-aminophenol. The optimal packing density of anti-cTnI on the surface of the PDMS channel was determined at the anti-cTnI concentration of 30 microg/ml for the highest electrochemical signal. These demonstrate that the proper orientation and best packing density of antibody as well as no electrode fouling contributed to the low detection limit (148 pg/ml) of cTnI within 8 min.

Incorporating gold nanoclusters and target-directed liposomes as a synergistic amplified colorimetric sensor for HER2-positive breast cancer cell detection.

Breast cancer is the second leading cause of cancer-related mortality in women. Successful development of sensitive nanoprobes for breast cancer cell detection is of great importance for breast cancer diagnosis and symptomatic treatment. Herein, inspired by the intrinsic peroxidase property of gold nanoclusters, high loading, and targeting ability of ErbB2/Her2 antibody functionalized liposomes, we report that gold nanoclusters-loaded, target-directed, functionalized liposomes can serve as a robust sensing platform for amplified colorimetric detection of HER2-positive breast cancer cells. This approach allows HER2-positive breast cancer cell identification at high sensitivity with high selectivity. In addition, the colorimetric "readout" offers extra advantages in terms of low-cost, portability, and easy-to-use applications. The practicality of this platform was further proved by successful detection of HER2-positive breast cancer cells in human serum samples and in breast cancer tissue, which indicated our proposed method has potential for application in cancer theranostics.

Swelling of Collagen-Hyaluronic Acid Co-Gels: An In Vitro Residual Stress Model.

Tissue-equivalents (TEs), simple model tissues with tunable properties, have been used to explore many features of biological soft tissues. Absent in most formulations however, is the residual stress that arises due to interactions among components with different unloaded levels of stress, which has an important functional role in many biological tissues. To create a pre-stressed model system, co-gels were fabricated from a combination of hyaluronic acid (HA) and reconstituted Type-I collagen (Col). When placed in solutions of varying osmolarity, HA-Col co-gels swell as the HA imbibes water, which in turn stretches (and stresses) the collagen network. In this way, co-gels with residual stress (i.e., collagen fibers in tension and HA in compression) were fabricated. When the three gel types tested here were immersed in hypotonic solutions, pure HA gels swelled the most, followed by HA-Col co-gels; no swelling was observed in pure collagen gels. The greatest swelling rates and swelling ratios occurred in the lowest salt concentration solutions. Tension on the collagen component of HA-Col co-gels was calculated from a stress balance and increased nonlinearly as swelling increased. The swelling experiment results were in good agreement with the stress predicted by a fibril network + non-fibrillar interstitial matrix computational model.

Modulation of the N-type calcium channel gene expression by the alpha subunit of Go.

Go, a heterotrimeric G-protein, is enriched in brain and neuronal growth cones. Although several reports suggest that Go may be involved in modulation of neuronal differentiation, the precise role of Go is not clear. To investigate the function of Go in neuronal differentiation, we determined the effect of Goalpha, the alpha subunit of Go, on the expression of Ca(v)2.2, the pore-forming unit of N-type calcium channels, at the transcription level. Treatment with cyclic AMP (cAMP), which triggers neurite outgrowth in neuroblastoma F11 cells, increased the mRNA level and the promoter activity of the Ca(v)2.2 gene. Overexpression of Goalpha inhibited neurite extension in F11 cells and simultaneously repressed the stimulatory effect of cAMP on the Ca(v)2.2 gene expression to the basal level. Targeted mutation of the Goalpha gene also increased the level of Ca(v)2.2 in the brain. These results suggest that Go may regulate neuronal differentiation through modulation of gene expression of target genes such as N-type calcium channels.

Increased size of solid organs in patients with Chuvash polycythemia and in mice with altered expression of HIF-1alpha and HIF-2alpha.

Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen-sensing to be recognized, is characterized by a homozygous germ-line loss-of-function mutation of the VHL gene (VHL(R200W)) resulting in elevated hypoxia inducible factor (HIF)-1alpha and HIF-2alpha levels, increased red cell mass and propensity to thrombosis. Organ volume is determined by the size and number of cells, and the underlying molecular control mechanisms are not fully elucidated. Work from several groups has demonstrated that the proliferation of cells is regulated in opposite directions by HIF-1alpha and HIF-2alpha. HIF-1alpha inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21(Cip1), thereby inducing its expression. In contrast, HIF-2alpha promotes MYC activity and cell proliferation. Here we report that the volumes of liver, spleen, and kidneys relative to body mass were larger in 30 individuals with Chuvash polycythemia than in 30 matched Chuvash controls. In Hif1a(+/-) mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1alpha, hepatic HIF-2alpha mRNA was increased (2-fold) and the mass of the liver was increased, compared with wild-type littermates, without significant difference in cell volume. Hepatic p21(Cip1) mRNA levels were 9.5-fold lower in Hif1a(+/-) mice compared with wild-type littermates. These data suggest that, in addition to increased red cell mass, the sizes of liver, spleen, and kidneys are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2alpha and decreased p21(Cip1) levels leading to increased hepatocyte proliferation.

The expression pattern of nuclear receptors during cerebellar development.

The cerebellum is essential for fine control of movement and posture, and it has been a useful model for studying many aspects of neural development because of its relatively simple anatomy and developmental program. However, the roles of nuclear receptors (NRs) underlying formation of the cerebellum and maintenance of cerebellar functions are still poorly characterized. As a contribution to the Nuclear Receptor Signaling Atlas (NURSA), we employed immunohistochemistry to investigate the expression pattern of 18 NRs in the cerebellum. Ten receptors were demonstrated to be expressed in the postnatal day 21 (P21) cerebellum. Among them, five receptors (COUP-TFI, COUP-TFII, RORalpha, ERbeta, and ERRgamma) were expressed at all stages (embryonic stage, P0, P7, and P21) examined. Interestingly, COUP-TFI and COUP-TFII show differential anterior-posterior expression patterns during cerebellar development. Taken together, our results suggest that members of the nuclear receptor superfamily might play importantly physiological roles in the cerebellum.