Honeycomb-like porous chitosan films prepared via phase transition of poly(N-isopropylacrylamide) during water evaporation under ambient conditions.

Honeycomb-like porous chitosan (CS) films are attractive tools for developing functional materials for filters, catalyses, adsorbents, biomaterials, etc. A simple method for fabricating honeycomb-like porous CS films without special reagents, facilities, and techniques would make them accessible. Here we introduce an easily available method for fabricating honeycomb-like CS films without a strong acid/base, toxic reagents, or special facilities/techniques. An aqueous solution containing CS and poly(N-isopropylacrylamide) (PNIPAm) was allowed to stand at 25 °C to evaporate water. After 3 days, CS-PNIPAm composite films with homogenously phase-separated PNIPAm particles were obtained. The PNIPAm particles were removed by immersion in methanol, and the resulting films dried under reduced pressure to become honeycomb-like porous CS films. The pore size could be varied in the range of 0.5-3.0 µm by altering the CS concentration and the molecular weight of CS where the pore size was reduced under conditions with stronger interaction between CS molecules. We reveal that the key to success with this system is the decrease of lower critical solution temperature (LCST) of PNIPAm with water evaporation. In addition, we confirmed the removed PNIPAm was recyclable in this system. Furthermore, we found this method was also applicable to alginate. The proposed facile method for fabricating honeycomb-like porous polymeric films could provide various functional porous materials.

Correction: Synergistic anti-AML effects of the LSD1 inhibitor T-3775440 and the NEDD8-activating enzyme inhibitor pevonedistat via transdifferentiation and DNA rereplication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expression of Stem Cell Factor in Feline Mast Cell Tumour.

Stem cell factor (SCF) is a ligand of the molecule Kit, which is expressed in mast cells and is important for mast cell proliferation, migration and survival. Mast cell tumours (MCTs) are associated with mutations of c-kit, a proto-oncogene encoding the Kit protein. In this study, we examined SCF expression in 23 samples of feline MCTs. SCF expression was detected in 10 cutaneous MCTs and a case of splenic mastocytosis. In the cutaneous MCTs, SCF-positive tumour cells were located at the margins. Kit was expressed in eight of the 10 cutaneous cases of SCF-expressing MCTs. In these cases, Kit-positive cells were located near to SCF-positive cells, and SCF/Kit double-positive tumour cells were found. Ki67-positive tumour cells were not found near to SCF-positive cells. These results suggest that SCF autocrine/paracrine mechanisms are involved in the expansion of cutaneous MCTs, but not in tumour proliferation.

Detection of copy number variations in epilepsy using exome data.

Epilepsies are common neurological disorders and genetic factors contribute to their pathogenesis. Copy number variations (CNVs) are increasingly recognized as an important etiology of many human diseases including epilepsy. Whole-exome sequencing (WES) is becoming a standard tool for detecting pathogenic mutations and has recently been applied to detecting CNVs. Here, we analyzed 294 families with epilepsy using WES, and focused on 168 families with no causative single nucleotide variants in known epilepsy-associated genes to further validate CNVs using 2 different CNV detection tools using WES data. We confirmed 18 pathogenic CNVs, and 2 deletions and 2 duplications at chr15q11.2 of clinically unknown significance. Of note, we were able to identify small CNVs less than 10 kb in size, which might be difficult to detect by conventional microarray. We revealed 2 cases with pathogenic CNVs that one of the 2 CNV detection tools failed to find, suggesting that using different CNV tools is recommended to increase diagnostic yield. Considering a relatively high discovery rate of CNVs (18 out of 168 families, 10.7%) and successful detection of CNV with <10 kb in size, CNV detection by WES may be able to surrogate, or at least complement, conventional microarray analysis.

Marked efficacy of combined three-drug therapy (Sodium Valproate, Topiramate and Stiripentol) in a patient with Dravet syndrome.

WHAT IS KNOWN AND OBJECTIVE: Dravet syndrome (DS) is an intractable epilepsy syndrome. The three-drug combination therapy of sodium valproate (VPA), clobazam (CLB) and stiripentol (STP) is recommended worldwide. CASE SUMMARY: We present a case of DS, in which treatment with CLB could not be continued because of the appearance of adverse reactions to it. Replacement with topiramate (TPM) proved to be markedly effective. WHAT IS NEW AND CONCLUSION: It is suggested that combination therapy with VPA, TPM and STP is for DS epilepsy.

Identification of novel BCL11A variants in patients with epileptic encephalopathy: Expanding the phenotypic spectrum.

BCL11A encodes a zinc finger protein that is highly expressed in hematopoietic tissues and the brain, and that is known to function as a transcriptional repressor of fetal hemoglobin (HbF). Recently, de novo variants in BCL11A have been reported in individuals with intellectual disability syndrome without epilepsy. In this study, we performed whole-exome sequencing of 302 patients with epileptic encephalopathies (EEs), and identified 2 novel BCL11A variants, c.577delC (p.His193Metfs*3) and c.2351A>C (p.Lys784Thr). Both the patients shared major physical features characteristic of BCL11A-related intellectual disability syndrome, suggesting that characteristic physical features and the persistence of HbF should lead clinicians to suspect EEs caused by BCL11A pathogenic variants. Patient 1, with a frameshift variant, presented with Lennox-Gastaut syndrome, which expands the phenotypic spectrum of BCL11A haploinsufficiency. Patient 2, with a p.Lys784Thr variant, presented with West syndrome followed by drug-resistant focal seizures and more severe developmental disability. These 2 newly described patients contribute to delineating the associated, yet uncertain phenotypic characteristics of BCL11A disease-causing variants.

Synergistic anti-AML effects of the LSD1 inhibitor T-3775440 and the NEDD8-activating enzyme inhibitor pevonedistat via transdifferentiation and DNA rereplication.

Lysine-specific demethylase 1A (LSD1, KDM1A) specifically demethylates di- and monomethylated histones H3K4 and K9, resulting in context-dependent transcriptional repression or activation. We previously identified an irreversible LSD1 inhibitor T-3775440, which exerts antileukemic activities in a subset of acute myeloid leukemia (AML) cell lines by inducing cell transdifferentiation. The NEDD8-activating enzyme inhibitor pevonedistat (MLN4924, TAK-924) is an investigational drug with antiproliferative activities in AML, and is also reported to induce cell differentiation. We therefore tested the combination of these two agents in AML models. The combination treatment resulted in synergistic growth inhibition of AML cells, accompanied by enhanced transdifferentiation of an erythroid leukemia lineage into granulomonocytic-like lineage cells. In addition, pevonedistat-induced rereplication stress during the S phase was greatly augmented by concomitant treatment with T-3775440, as reflected by the increased induction of apoptosis. We further demonstrated that the combination treatment was markedly effective in subcutaneous tumor xenograft models as well as in a disseminated model of AML, leading to tumor eradication or prolonged survival in T-3775440/pevonedistat cotreated mice. Our findings indicate the therapeutic potential of the combination of LSD1 inhibitors and pevonedistat for the treatment of AML.

Sorafenib plus hepatic arterial infusion chemotherapy with cisplatin versus sorafenib for advanced hepatocellular carcinoma: randomized phase II trial.

BACKGROUND: Sorafenib (Sor) is acknowledged as a standard therapy for advanced hepatocellular carcinoma (HCC). This trial was conducted to evaluate the effect of addition of hepatic arterial infusion chemotherapy with cisplatin (SorCDDP) to Sor for the treatment of advanced HCC. PATIENTS AND METHODS: We conducted a multicenter open-labeled randomized phase II trial in chemo-naïve patients with advanced HCC with Child-Pugh scores of 5-7. Eligible patients were randomly assigned 2:1 to receive SorCDDP (sorafenib: 400 mg bid; cisplatin: 65 mg/m2, day 1, every 4-6 weeks) or Sor (400 mg bid). The primary end point was overall survival. RESULTS: A total of 108 patients were randomized (Sor, n = 42; SorCDDP, n = 66). The median survival in the Sor and SorCDDP arms were 8.7 and 10.6 months, respectively [stratified hazard ratio (95% confidence interval), 0.60 (0.38-0.96), P = 0.031]. The median time to progression and the response rate were, respectively, 2.8 months and 7.3% in the Sor arm and 3.1 months and 21.7% in the SorCDDP arm. The adverse events were more frequent in the SorCDDP arm than in the Sor arm, but well-tolerated. CONCLUSION: SorCDDP yielded favorable overall survival when compared with Sor in patients with advanced HCC. CLINICAL TRIAL REGISTRATION: UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number: UMIN000005703.

Eicosapentaenoic acid ameliorates hyperglycemia in high-fat diet-sensitive diabetes mice in conjunction with restoration of hypoadiponectinemia.

BACKGROUND/OBJECTIVE: Eicosapentaenoic acid (EPA) exerts pleiotropic effects on metabolic disorders such as atherosclerosis and dyslipidemia, but its effectiveness in the treatment of type 2 diabetes mellitus remains controversial. METHODS: We examined the antidiabetic effect of EPA in insulin receptor mutant (Insr(P1195L/+)) mice that exhibit high-fat diet (HFD)-dependent hyperglycemia. RESULTS: EPA supplementation was found to alleviate hyperglycemia of Insr(P1195L/+) mice fed HFD (Insr(P1195L/+)/HFD mice), which was accompanied by amelioration of increased gluconeogenesis and impaired insulin signaling, as assessed by glucose-6-phosphatase (G6pc) expression on refeeding and insulin-induced phosphorylation of Akt in the liver, respectively. We found that serum levels of adiponectin, the antidiabetic adipokine, were decreased by HFD along with the body weight gain in Insr(P1195L/+) mice but not in wild-type mice, suggesting that Insr(P1195L/+) mice are prone to hypoadiponectinemia in response to obesity. Interestingly, the blood glucose levels of Insr(P1195L/+) mice were in reverse proportion to their serum adiponectin levels and EPA supplementation ameliorated their hyperglycemia in conjunction with the restoration of hypoadiponectinemia. CONCLUSIONS: EPA exerts an antidiabetic effect in Insr(P1195L/+)/HFD mice, an HFD-sensitive, insulin-resistant animal model, possibly through its action against hypoadiponectinemia.

Facile preparation of surface N-halamine chitin nanofiber to endow antibacterial and antifungal activities.

N-halamine chitin nanofiber (NF) film was prepared by the reaction of chitin NF film with sodium hypochlorite solution to endow the film with antibacterial and antifungal activities. The amount of active chlorine content loaded on the chitin NF film depended on the sodium hypochlorite concentration and reaction time. FT-IR, UV-vis, XRD, and TG analyses showed that the N-H bond was substituted to the N-Cl bond and that the reaction took place at the chitin NF surface. After chlorination, the characteristic nanochitin morphology was maintained. Although the active chlorine content of the film gradually decreased by disassociation of the N-Cl bond, chlorine was rechargeable into chitin NF by another sodium hypochlorite solution treatment. The chlorinated chitin NF film showed strong efficacies against Gram-negative and -positive bacteria of Escherichia coli and Staphylococcus aureus, respectively. Moreover, the films showed 100% and 80% inhibition of spore germination when faced against Alternaria alternata and Penicillium digitatum fungi, respectively.

Impairment of host resistance to helminthes with age in murine small intestine.

Age-associated alterations of Th2 immune responses against nematode parasites are largely unknown. We investigated primary and memory responses against two types of gastrointestinal nematode parasites, Heligmosomoides polygyrus (Hp) and Nippostrongylus brasiliensis (Nb), in aged mice. The small intestinal gene expression of Th2 cytokines was almost unchanged after primary (Nb and Hp) and secondary infection (Hp) in aged mice in contrast to strongly increased small intestinal gene expression of Th2 cytokines in young (3-month-old) mice. Mucus production decreased (Nb), and worm expulsion was impaired (Nb and Hp) compared with the young mice. Immunofluorescent staining revealed that after Hp infection, the number of alternatively activated macrophages, which are induced by Th2 cytokines, was lower in the aged mice. On the other hand, the number of CD4(+) T cells recruited to the worm cysts was normal compared with the young mice. These results suggest that migration of CD4(+) T cells to the host-parasite interface is not affected by ageing. Alterations in Th2 immune responses in aged mice might be due to inappropriate or insufficient activation of CD4(+) T cells in the submucosa.

Evaluation of nanodispersion of iron oxides using various polymers.

In order to create Fe2O3 and Fe2O3·H2O nanoparticles, various polymers were used as dispersing agents, and the resulting effects on the dispersibility and nanoparticulation of the iron oxides were evaluated. It was revealed that not only the solution viscosity but also the molecular length of the polymers and the surface tension of the particles affected the dispersibility of Fe2O3 and Fe2O3·H2O particles. Using the dispersing agents 7.5% hydroxypropylcellulose-SSL, 6.0% Pharmacoat 603, 5.0% and 6.5% Pharmacoat 904 and 7.0% Metolose SM-4, Fe2O3 nanoparticles were successfully fabricated by wet milling using Ultra Apex Mill. Fe2O3·H2O nanoparticles could also be produced using 5.0% hydroxypropylcellulose-SSL and 4.0 and 7.0% Pharmacoat 904. The index for dispersibility developed in this study appears to be an effective indicator of success in fabricating nanoparticles of iron oxides by wet milling using Ultra Apex Mill.

Altered somatosensory barrel cortex refinement in the developing brain of Mecp2-null mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 (MeCP2) gene. In previous studies, monoaminergic dysfunctions have been detected in patients with RTT and in a murine model of RTT, the Mecp2-null mouse. Therefore, the pathogenesis of RTT is thought to involve impairments in the monoaminergic systems. However, there have been limited data showing that the impairment of monoamines leads to early symptoms during development. We used histochemistry to study the somatosensory barrel cortex in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The barrel cortex is widely used to investigate neuronal development and its regulation by various neurotransmitters including 5-HT. 5-HT levels were measured by high performance liquid chromatography with electrochemical detection (HPLC/EC), and serotonin transporter (SERT) and 5-HT1B receptor mRNAs were measured in the somatosensory cortex, thalamus and striatum on postnatal days (P) 10, P20 and P40. Mecp2-null mice (Mecp2-/y) had significantly smaller barrel fields than age-matched wild-type controls (Mecp2+/y) on P10 and P40, but the topographic map was accurately formed. Levels of 5-HT, and SERT and 5-HT1B receptor mRNA expression in the somatosensory cortex did not differ significantly between the Mecp2-null and wild-type mice on P10. However, thalamic 5-HT was reduced in Mecp2-null mice. Our data indicate that a lack of MeCP2 may disturb the refinement of the barrel cortex in the early postnatal period. Our findings suggest that a decrease in thalamic 5-HT might be involved in this phenomenon.

Orphan nuclear receptor HNF4G promotes bladder cancer growth and invasion through the regulation of the hyaluronan synthase 2 gene.

Nuclear receptors (NRs) are a class of transcription factors that are closely involved in the progression of certain types of cancer. We aimed to study the relation between bladder cancer and NRs, with special focus on orphan NRs whose ligands and functions have not been identified. First, we examined the expression levels of 22 genes encoding orphan NRs in clinical bladder cancer and found that hepatocyte nuclear factor 4γ (HNF4G; NR2A2) and NR2F6 were the genes that were upregulated most frequently in cancer tissues compared with their paired normal tissues. Knockdown and overexpression of each of these orphan NRs suppressed and stimulated the growth of bladder cancer cells in vitro, respectively. HNF4G also promoted tumor growth in bladder cancer xenograft models in vivo. Furthermore, HNF4G was both necessary and sufficient for the invasion of bladder cancer cells in vitro. Moreover, using microarray analyses, we identified hyaluronan synthase 2 (HAS2) as one of the genes induced by HNF4G in bladder cancer cells. Transcription was activated by HNF4G in reporter assays using the promoter/enhancer region of the HAS2 gene. The endogenous expression of the HAS2 gene was suppressed by knockdown of HNF4G. In turn, knockdown of HAS2 inhibited the growth and invasion of bladder cancer cells. Taken together, our data suggest that some orphan NRs are involved in bladder cancer progression and that, among them, HNF4G promotes the growth and invasion of bladder cancer, at least in part, via the regulation of the HAS2 gene.

Delaying embryo development by storing at 4°C for synchronization to recipients in microinjection technique in rabbits.

Short-term storage of embryos at low temperature induces developmental arrest of the embryo and would appear to be a valuable aid in embryo-transfer techniques to avoid wasting embryos. Embryo storage at 4°C was examined to allow synchronization with embryo-transfer recipients using the microinjection technique. Superovulation was induced in female Japanese White donor rabbits four days before mating with males. At the same time, control recipients were injected with human chorionic gonadotropin (hCG) to allow synchronization (R1); the hCG injections were delayed by 24 h in the experimental group (R2). DNA constructs for expressing human C-reactive protein or apolipoprotein AII were microinjected into the male pronuclei of the ova. The microinjected embryos were immediately transferred to recipients (R1) or stored at 4°C in phosphate-buffered saline containing 10% fetal bovine serum. After 17-20 h, the stored embryos were incubated at 37°C for one hour, and the morphologically normal embryos were transferred to recipients (R2). In the R1 rabbits, 855 embryos were transferred to 29 recipients, and 72.4% of the recipients became pregnant. Seven of the 84 offspring were transgenic. In the R2 rabbits, 478 embryos were transferred to 16 recipients, and 62.5% of the recipients became pregnant. Two of the 39 offspring were transgenic. There were no differences in pregnancy rate, litter size and transgenic integration rate between R1 and R2. These results suggest that the short-term 4°C storage of microinjected embryos can be a valuable method for synchronization with recipients, and reducing wastage of embryos and the sacrifice of rabbits.

Thermoresponsive chitosan/N-isopropylacrylamide copolymer through atom transfer radical polymerization.

Regioseletive copolymerization of N-isopropylacrylamide (NIPAM) onto chitosan was achieved by atom transfer radical polymerization (ATRP) by using a regioselective 3,6-di-O-bromoisobutyryl-2-N-phthaloyl chitosan as a macroinitiator. The degree of polymerization (DP) of polyNIPAM onto the chitosan derivative changed by varying the ratio between NIPAM monomer, macroinitiator, ligand, and transition metal. ATRP successfully proceeded and a DP of polyNIPAM up to 110.5 was obtained. The thermal decomposition temperature of the 3,6-di-O-bromoisobutyryl-2-N-phthaloyl chitosan was significantly improved by increasing the DP of the NIPAM component. The polyNIPAM-g-chitosan derivative showed a thermoresponsive property. Although it formed a stable suspension in water at room temperature, it caused a hydrophilic-to-hydrophobic transition at around 32°C, resulting in precipitate formation.