Preparation of Polyacrylonitrile-Based Immobilized Copper-Ion Affinity Membranes for Protein Adsorption.

A polyacrylonitrile (PAN)-based immobilized metal-ion affinity membrane (IMAM) was prepared with a high capacity for protein adsorption. PAN was selected as the substrate due to its excellent thermal and chemical stability. The cyano groups on the PAN membrane were substituted with carboxyl groups, followed by reactions with ethylenediamine (EDA) and ethylene glycol diglycidyl ether (EGDGE) to produce the terminal epoxy groups. The chelating agent iminodiacetic acid (IDA) was then bound to the modified PAN membrane and further chelated with copper ions. The immobilized copper ion amount of membrane was analyzed to obtain the optimal reaction conditions, which were 60 °C/3 h for EDA coupling and 60 °C/4 h for EGDGE grafting. Furthermore, under the use of minor IDA and copper ion concentrations, the immobilized copper ion capacity of the IMAM was 4.8 µmol/cm2 (253.4 µmol/mL, or 1.47 µmol/mg). At a neutral pH, the cationic lysozyme exhibited a large adsorption capacity with the IMAM (1.96 µmol/mL), which was most likely multilayer binding, whereas the adsorption capacity for bovine serum albumin (BSA) and histidine-tagged green fluorescent protein (GFP-His6) was 0.053 µmol/mL and 0.135 µmol/mL, respectively, with a monolayer adsorption arrangement. The protein desorption efficiency was greater than 95%, implying that the prepared IMAM could be reused for protein adsorption.

Clinical and biological relevance of CREB3L1 in Philadelphia chromosome-negative myeloproliferative neoplasms.

Cyclic AMP-response element-binding protein 3-like 1 (CREB3L1) is a gene involved in the unfolded protein response (UPR). Recently, we demonstrated that CREB3L1 is specifically overexpressed in the platelets of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In this study, we aimed to show the clinical and biological relevance of CREB3L1 in these hematological diseases. Overexpression of CREB3L1 was specific to platelets in MPNs and associated with a higher risk of thrombosis and fibrotic transformation in essential thrombocythemia (ET) and polycythemia vera (PV) cases, respectively. Furthermore, we found that UPR genes were downregulated in platelets of patients with ET and PV, which were more pronounced in patients harboring the JAK2 V617F mutation. However, CREB3L1 overexpression does not alter UPR gene expression or cell proliferation in UT-7/TPO/CALRm cells exogenously expressing mutated calreticulin and HEL cells harboring endogenous JAK2 V617F. Furthermore, CREB3L1 overexpression did not modulate sensitivity to endoplasmic reticulum stress in these cell lines. Taken together, our data show 1) a potential role of CREB3L1 expression in platelets as a new marker of high-risk MPNs and 2) an association between CREB3L1 overexpression and UPR gene downregulation in these patients' platelets, with CREB3L1 not altering UPR in our in vitro models and possibly further in vivo mechanisms being involved.

Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.

A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.

The challenge of micropollutants in surface water of the Yangtze River.

The Yangtze River, the third largest river and supporting nearly one-third of Chinese population, has been severely polluted in recent decades. Among the numerous pollutants, organic micropollutants, as one kind of important emerging contaminants, are currently key contaminants of concern. However, few studies have focused on their mixture environmental impacts, especially for the complex environmental mixtures. In the current study, four categories of organic micropollutants, including 16 polycyclic aromatic hydrocarbons (PAHs), 32 polychlorinated biphenyls (PCBs), 27 organochlorine pesticides (OCPs) and 20 pharmaceutical and personal care products (PPCPs) are analyzed in 10 study sites on the Yangtze River. Subsequently, comprehensive risk assessment for micropollutant mixtures was conducted by risk quotient based on the sum of PEC/PNEC values (RQMEC/PNEC) and risk quotient based on the toxic units (RQSTU). The mixture risk evaluation based on the detected environmental concentrations indicates that micropollutant mixtures in surface water of the Yangtze River exhibited relative high risks for aquatic organisms. The observed results revealed that mixture risk assessments have to consider the complexity of environmental samples; PCBs dominated main mixture risks in the upper stream; PAHs contributed major comprehensive risks in the middle stream; and OCPs were the key micropollutants in the downstream. The outcomes of the present study here can serve for pollution control in the Yangtze River, which provide the scientific underpinnings and regulatory reference for risk management and river protection.

Phase transformation behaviors and mechanical properties of NiTi endodontic files after gold heat treatment and blue heat treatment.

WaveOne Gold, ProTaper Gold, Reciproc Blue, ProTaper Next, WaveOne and ProTaper files were selected to compare the phase transformation behaviors and mechanical properties of nickel-titanium (NiTi) rotary files after gold heat and blue heat treatments. The reverse transformation finishing point temperatures of WaveOne Gold, ProTaper Gold, ProTaper Next and WaveOne were higher than those of the other two instruments investigated. At a deflection of 0.5 mm, the loads were significantly varied except for ProTaper Next and ProTaper Gold. At a deflection of 3.0 mm, the loads of Reciproc Blue and WaveOne Gold were significantly varied compared to WaveOne and ProTaper. Cycles to failure were reduced in the order of WaveOne Gold, Reciproc Blue, ProTaper Gold, ProTaper Next, WaveOne and ProTaper. NiTi instruments after gold heat and blue heat treatments exhibited significantly higher bending properties and cyclic fatigue resistances, representing an improved performance over traditional and M-wire instruments.

Mutant calreticulin interacts with MPL in the secretion pathway for activation on the cell surface.

Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.

Homomultimerization of mutant calreticulin is a prerequisite for MPL binding and activation.

Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.

Loss of microRNA-7a2 induces hypogonadotropic hypogonadism and infertility.

MicroRNAs (miRNAs) are negative modulators of gene expression that fine-tune numerous biological processes. miRNA loss-of-function rarely results in highly penetrant phenotypes, but rather, influences cellular responses to physiologic and pathophysiologic stresses. Here, we have reported that a single member of the evolutionarily conserved miR-7 family, miR-7a2, is essential for normal pituitary development and hypothalamic-pituitary-gonadal (HPG) function in adulthood. Genetic deletion of mir-7a2 causes infertility, with low levels of gonadotropic and sex steroid hormones, small testes or ovaries, impaired spermatogenesis, and lack of ovulation in male and female mice, respectively. We found that miR-7a2 is highly expressed in the pituitary, where it suppresses golgi glycoprotein 1 (GLG1) expression and downstream bone morphogenetic protein 4 (BMP4) signaling and also reduces expression of the prostaglandin F2a receptor negative regulator (PTGFRN), an inhibitor of prostaglandin signaling and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Our results reveal that miR-7a2 critically regulates sexual maturation and reproductive function by interconnecting miR-7 genomic circuits that regulate FSH and LH synthesis and secretion through their effects on pituitary prostaglandin and BMP4 signaling.

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

Systemic injection of low-dose lipopolysaccharide fails to break down the blood-brain barrier or activate the TLR4-MyD88 pathway in neonatal rat brain.

We aimed to investigate whether peripheral low-dose lipopolysaccharide (LPS) induces the breakdown of the blood-brain barrier (BBB) and/or the activation of toll-like receptor 4 (TLR4) in the neonatal rat brain. Neonatal rats received intraperitoneal injections of low-dose LPS (0.3 mg/kg∙bw), and the BBB compromise was detected by Evans Blue extravasation and electron microscopy. Meanwhile, TLR4, adaptin myeloid differentiation factor 88 (MyD88), nuclear transcription factor kappa-B (NF-κB) p50 and tumor necrosis factor alpha (TNFα) in the neonatal rat brain were determined by quantitative real-time polymerase chain reaction (PCR) and Western Blot. Immunohistochemistry was used to determine the distribution and activation of microglia in the brain after LPS administration. It was demonstrated that Evans Blue extravasation was not observed in the brain parenchyma, and that tight junctions of cerebral endothelial cells remained intact after systemic injections of LPS in neonatal rats. Although intracerebroventricular injections of LPS activated microglia and up-regulated the expression of TLR4, MyD88, NF-κB p50 and TNFα in the neonatal rat brain, systemic LPS did not induce these responses. These findings indicate that while the neonatal rat brain responds to the direct intra-cerebral administration of LPS through robust TLR4 activation, systemic low-dose LPS does not induce the innate immune reaction or compromise the BBB in neonatal rats.

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures.

Several autophagy proteins contain an LC3-interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B-positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP-ALFY-LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR-binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.

The possible interplanetary transfer of microbes: assessing the viability of Deinococcus spp. under the ISS Environmental conditions for performing exposure experiments of microbes in the Tanpopo mission.

To investigate the possible interplanetary transfer of life, numerous exposure experiments have been carried out on various microbes in space since the 1960s. In the Tanpopo mission, we have proposed to carry out experiments on capture and space exposure of microbes at the Exposure Facility of the Japanese Experimental Module of the International Space Station (ISS). Microbial candidates for the exposure experiments in space include Deinococcus spp.: Deinococcus radiodurans, D. aerius and D. aetherius. In this paper, we have examined the survivability of Deinococcus spp. under the environmental conditions in ISS in orbit (i.e., long exposure to heavy-ion beams, temperature cycles, vacuum and UV irradiation). A One-year dose of heavy-ion beam irradiation did not affect the viability of Deinococcus spp. within the detection limit. Vacuum (10(-1) Pa) also had little effect on the cell viability. Experiments to test the effects of changes in temperature from 80 °C to -80 °C in 90 min (± 80 °C/90 min cycle) or from 60 °C to -60 °C in 90 min (± 60 °C/90 min cycle) on cell viability revealed that the survival rate decreased severely by the ± 80 °C/90 min temperature cycle. Exposure of various thicknesses of deinococcal cell aggregates to UV radiation (172 nm and 254 nm, respectively) revealed that a few hundred micrometer thick aggregate of deinococcal cells would be able to withstand the solar UV radiation on ISS for 1 year. We concluded that aggregated deinococcal cells will survive the yearlong exposure experiments. We propose that microbial cells can aggregate as an ark for the interplanetary transfer of microbes, and we named it 'massapanspermia'.

Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy.

The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62's binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.

Deinococcus aetherius sp. nov., isolated from the stratosphere.

A pink-red pigmented, non-motile, coccoid bacterial strain, ST0316(T), was isolated from dust samples collected from the stratosphere in Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that it belonged to the genus Deinococcus. DNA G+C content (69.8 mol%), desiccation tolerance, and resistance to gamma-rays [D(10) (dose required to reduce the bacterial population by 10-fold) >8 kGy] and UV radiation (D(10) 1000 J m(-2)) supported the affiliation of strain ST0316(T) to the genus Deinococcus. The major peptidoglycan amino acids were d-glutamic acid, glycine, d-alanine, l-alanine and ornithine. Predominant fatty acids were C(16 : 1)omega7c, C(16 : 0), C(17 : 0) and iso-C(17 : 0). Strain ST0316(T) diverged from recognized species of the genus Deinococcus, showing less than 93.0 % similarity values to its closest relatives Deinococcus apachensis, D. aerius, D. geothermalis and D. murrayi. Strain ST0316(T) also differed from the type strains of closely related species in its polar lipid profile, nitrate reduction and carbon-source assimilation tests. Therefore, we propose a new species of the genus Deinococcus, Deinococcus aetherius sp. nov. (type strain, ST0316(T) =JCM 11751(T) =DSM 21230(T)).

Deinococcus aerius sp. nov., isolated from the high atmosphere.

An orange-pigmented, non-motile, coccoid bacterial strain, designated TR0125T, was isolated from dust samples collected in the high atmosphere above Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was within the radiation of Deinococcus species. The major peptidoglycan amino acids were D-glutamic acid, glycine, D-alanine, L-alanine and ornithine. The predominant fatty acids were iso-C17:0, iso-C17:1omega9c and iso-C15:0. Strong resistance to desiccation, UV-C and gamma radiation and high DNA G+C content also supported the affiliation of strain TR0125T to the genus Deinococcus. Strain TR0125T showed the highest 16S rRNA gene sequence similarity value (95.7%) to the type strain of Deinococcus apachensis, and phylogenetic analysis showed that it was further separated from D. apachensis than from Deinococcus geothermalis, indicating that strain TR0125T was not a member of these two Deinococcus species. In addition, phenotypic differences were found between strain TR0125T and the type strains of these two Deinococcus species. Therefore, a novel species of the genus Deinococcus, Deinococcus aerius sp. nov. (type strain, TR0125T=JCM 11750T=DSM 21212T), is proposed to accommodate this isolate.