Synthesis of Novel Chain‐End‐Functionalized Polyethylenes via Thiol‐ene Click Chemistry

Chain‐end‐functionalized polyethylenes (Cef‐PEs: PE? S? SH (Cef₁), PE? S? furfuryl (Cef₂), PE? S? NH₂ (Cef₄), PE? S? NH₂?HCl (Cef₅), PE? S? COONa (Cef₆), PE? S? CHA(Cef₇), PE? S? SO₃Na (Cef₈), PE? S? OH (Cef₉) and PE? S? COOH (Cef₁₀)) are synthesized by thiol‐ene addition of vinyl‐terminated polyethylene (v‐PE) with 1,2‐ethanedithiol, furfurylmercaptan, cysteamine, cysteamine hydrochloride, sodium thioglycolate, L‐cysteine hydrochloride, sodium 2‐mercaptoethanesulfonate, mercaptoethanol, and thioglycolic acid, respectively. PE? S? t? NCO (Cef₃) is obtained by subsequent reaction of Cef₉ with anhydrous 4,4′‐MDI. Cef‐PE_1–8 are reported for the first time. The conversions of all the reactions are up to 95%–100%. All the Cef‐PEs are characterized by NMR, GPC, DSC, FT‐IR, and TGA. In the experiments, it is found that alkaline mercaptans and solvents are adverse to the thiol‐ene click reactions.

     

Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.Neuronal migration and maturation is a key step in brain development. Defects in this process have been implicated in many disorders, including autism (1) and schizophrenia (2). Thoroughly understanding how neural progenitor cell (NPC) migration is affected in neurodevelopmental disorders requires a means of dissecting the process using cells with genetic alterations matching those in patients. Existing in vitro assays of migration generally involve measurement of cell movement across a scratch or gap or through a membrane toward a chemoattractant in 2D culture systems. Although widely used, such assays may not accurately reveal in vivo differences, as neuronal migration is tightly regulated by physical and chemical cues in the extracellular matrix (ECM) that NPCs encounter as they migrate.In vitro 3D culture systems offer a solution to these limitations (3–7). Compared with 2D culture, a 3D arrangement allows neuronal cells to interact with many more cells (4); this similarity to the in vivo setting has been shown to lengthen viability, enhance survival, and allow formation of longer neurites and more dense networks in primary neurons in uniform matrices or aggregate culture (8, 9). Indeed, 3D culture systems have been used to study nerve regeneration, neuronal and glial development (10–12), and amyloid-β and tau pathology (13). Thus, measuring neuronal migration through a soft 3D matrix would continue this trend toward using 3D systems to study neuronal development and pathology.We sought to develop a 3D assay to examine potential migration and neuronal maturation defects in Rett syndrome (RTT), a genetic neurodevelopmental disorder that affects 1 in 10,000 children in the United States and is caused by mutations in the X-linked methyl-CpG-binding protein-2 (MECP2) gene (14). Studies using induced pluripotent stem cells (iPSCs) from RTT patients in traditional 2D adherent culture have revealed reduced neurite outgrowth and synapse number, as well as altered calcium transients and spontaneous postsynaptic currents (1). However, 2D migration assays seemed unlikely to reveal inherent defects in this developmental process, which could be affected because MeCP2 regulates multiple developmental related genes (15). Migration of RTT iPSC-derived NPCs has not previously been studied.Using a previously unidentified 3D tissue culture system that allows creation of layered architectures, we studied differences in migration of MeCP2-mutant iPSC-derived versus control iPSC-derived NPCs. This approach revealed a defect in migration of MeCP2-mutant iPSC-derived NPCs induced by either astrocytes or neurons. Further, this 3D system accelerated maturation of neurons from human iPSC-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. With mature neurons derived from RTT patients and controls, we further confirmed defective neurite outgrowth and synaptogenesis in MeCP2-mutant neurons. Thus, this 3D system enables study of morphological features accessible in 2D system as well as previously unexamined phenotypes.     

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3^CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3^CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3^CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3^CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Cellulose, a linear homopolymer of d-glucopyranose linked by β-1,4-glycosidic bonds, is the major structural component of the cell walls of plants, oomycetes, and algae and constitute the most abundant biopolymer on Earth (1). Cellulose is synthesized by cellulose synthases (CESAs) that belongs to the glycosyltransferase GT-2 superfamily (1, 2). In land plants, cellulose is produced at the plasma membrane by six-lobed rosette-shaped CESA complexes (CSCs) where each CESA is thought to synthesize one cellulose chain (3). The precise number of CESAs per CSC is unresolved but estimated to range between 18 and 36 (4–6).Plants contain multiple cesa genes, with 10 found in the Arabidopsis genome (7). Of these, CESA1, CESA3, CESA6, and the CESA6-like CESAs (i.e., CESA2, CESA5, and CESA9) are involved in primary cell wall formation, whereas CESA4, CESA7, and CESA8 participate in secondary cell wall formation (8–12). These two types of CSCs form heterotrimeric complexes with a ratio of 1:1:1 (13, 14). The Arabidopsis CESAs share an overall sequence identity of ∼60% and have seven transmembrane helices (15). In plants, the catalytic domain (CatD) of the CESAs is located between the second and third transmembrane helices and contains a canonical D, D, D, QxxRW motif (1). While there are similarities between the plant CatD and its counterpart in bacterial cellulose synthases, the CatD is flanked by two plant-specific domains, the so-called plant-conserved region (P-CR) and class-specific region (C-SR) (16). These domains are proposed to have important functions in cellulose synthesis and CESA oligomerization (17).The oligomerization of plant CESAs is thought to be important for the final CSC assembly, and multiple oligomeric states of CESAs, including homodimers, have been reported (18, 19). For example, immunoprecipitation assays using CESA7 fused to a dual His/STRP-tag demonstrated that CESA4, CESA7, and CESA8 could form independent homodimers, and it was hypothesized that the CESA homodimerization may contribute to early stages of CSC assembly. These homodimers might then be converted into CSC heterotrimeric configurations (19). This feature poses a marked difference from the bacterial cellulose synthase complex. However, how CESA homodimers are formed and how they function in cellulose synthesis are unknown.To comprehend the mechanisms behind plant cellulose synthesis, it is essential to acquire structural information about plant CESAs. Indeed, the BcsA–BcsB complex structure from Rhodobacter greatly aided our understanding of the cellulose synthesis in bacteria (20). Nevertheless, there are many differences between bacterial and plant CESAs and the corresponding protein complexes. Extensive efforts have been undertaken to acquire plant CESA structural information, including homology modeling and small-angle X-ray scattering analyses (5, 6, 16, 21, 22). While these efforts have been important to form new hypotheses, they did not reveal significant insights into substrate coordination, cellulose chain extrusion, and complex assembly. Recently, a homotrimeric CESA8 structure from Populus tremula × tremuloides was resolved by cryogenic electron microscopy (cryo-EM), which offered significant new molecular understanding of cellulose microfibril biosynthesis and CESA coordination within the CSC (15). Here we report the crystal structures of Arabidopsis CESA3 CatD (AtCESA3^CatD) in apo and uridine diphosphate (UDP)-glucose (UDP-Glc) bound forms and outline how the CatD might contribute to CESA homodimerization and substrate coordination.     

Mimicry between neurokinin-1 and fibronectin may explain the transport and stability of increased substance P immunoreactivity in patients with bone marrow fibrosis

Bone marrow (BM) fibrosis may occur in myeloproliferative diseases, lymphoma, myelodysplastic syndrome, myeloma, and infectious diseases. In this study, the role of substance P (SP), a peptide with pleiotropic functions, was examined. Some of its functions-angiogenesis, fibroblast proliferation, and stimulation of BM progenitors-are amenable to inducing BM fibrosis. Indeed, a significant increase was found in SP-immunoreactivity (SP-IR) in the sera of patients with BM fibrosis (n = 44) compared with the sera of patients with hematologic disorders and no histologic evidence of fibrosis (n = 46) (140 +/-12 vs 18 +/-3; P <.01). Immunoprecipitation of sera SP indicated that this peptide exists in the form of a complex with other molecule(s). It was, therefore, hypothesized that SP might be complexed with NK-1, its natural receptor, or with a molecule homologous to NK-1. To address this, 3 cDNA libraries were screened that were constructed from pooled BM stroma or mononuclear cells with an NK-1 cDNA probe. A partial clone (clone 1) was retrieved that was 97% homologous to the ED-A region of fibronectin (FN). Furthermore, sequence analyses indicated that clone 1 shared significant homology with exon 5 of NK-1. Immunoprecipitation and Western blot analysis indicated co-migration of SP and FN in 27 of 31 patients with BM fibrosis. Computer-assisted molecular modeling suggested that similar secondary structural features between FN and NK-1 and the relative electrostatic charge might explain a complex formed between FN (negative) and SP (positive). This study suggests that SP may be implicated in the pathophysiology of myelofibrosis, though its role would have to be substantiated in future research. (Blood. 2001;97:3025-3031)     

Comprehensive health status assessment of centenarians: results from the 1999 large health survey of veteran enrollees

BACKGROUND: Information on the health status of centenarians provides a means for understanding the health care needs of this growing population. Therefore, we examined the health status of a national cohort of centenarian veteran enrollees. METHODS: Ninety-three centenarian veteran enrollees returned a complete health history questionnaire, which included questions about sociodemographic information, age-associated conditions, health behaviors, health-related quality of life as measured by the Veterans SF-36, and change in health status. RESULTS: Centenarian veteran enrollees are a group with major impairment across multiple dimensions of health-related quality of life despite having a relatively low prevalence of diseases. They had considerable physical limitations as reflected by their physical health summary scores (26.2 +/- 8.3). However, their mental health was comparatively good (mental health summary score 44.1 +/- 12.5). Compared to younger elderly veterans (ages 85-99), centenarians had a lower prevalence of hypertension, angina or myocardial infarction, diabetes, and chronic low back pain (p <.05). Centenarians had significantly worse physical functioning, role physical, vitality, and social functioning scores than did younger elderly veterans. The two groups did not differ in their general health, bodily pain, role emotional, and mental health scores. Centenarians did not perceive much decline in their physical or mental health during the preceding year. CONCLUSIONS: Centenarian veteran enrollees are a group with a low number of age-associated diseases and good mental health despite substantial physical limitations. These results support future studies of services directed toward improvement of function as opposed to those focused solely on the treatment of diseases.     

Distinct microRNA expression profiles in acute myeloid leukemia with common translocations

MicroRNAs (miRNAs) are postulated to be important regulators in cancers. Here, we report a genome-wide miRNA expression analysis in 52 acute myeloid leukemia (AML) samples with common translocations, including t(8;21)/AML1(RUNX1)-ETO(RUNX1T1), inv(16)/CBFB-MYH11, t(15;17)/PML-RARA, and MLL rearrangements. Distinct miRNA expression patterns were observed for t(15;17), MLL rearrangements, and core-binding factor (CBF) AMLs including both t(8;21) and inv(16) samples. Expression signatures of a minimum of two (i.e., miR-126/126*), three (i.e., miR-224, miR-368, and miR-382), and seven (miR-17-5p and miR-20a, plus the aforementioned five) miRNAs could accurately discriminate CBF, t(15;17), and MLL-rearrangement AMLs, respectively, from each other. We further showed that the elevated expression of miR-126/126* in CBF AMLs was associated with promoter demethylation but not with amplification or mutation of the genomic locus. Our gain- and loss-of-function experiments showed that miR-126/126* inhibited apoptosis and increased the viability of AML cells and enhanced the colony-forming ability of mouse normal bone marrow progenitor cells alone and particularly, in cooperation with AML1-ETO, likely through targeting Polo-like kinase 2 (PLK2), a tumor suppressor. Our results demonstrate that specific alterations in miRNA expression distinguish AMLs with common translocations and imply that the deregulation of specific miRNAs may play a role in the development of leukemia with these associated genetic rearrangements.     

Role of N-methyl-D-aspartate receptor in hyperoxia-induced lung injury

Glutamate (Glu) N-methyl-D-aspartate (NMDA) receptor is present in the lungs, and NMDA receptor antagonist MK-801 attenuates oxidant lung injury. We hypothesized that Glu excitotoxicity may participate in the pathogenesis of hyperoxia-induced lung injury. To determine possible pulmonary protective effects, we administered 0.05 ml/kg MK-801 or saline intraperitoneally daily to neonatal rats exposed to more than 95% oxygen in air. After 7 days, MK-801 decreased the hyperoxia-associated elevation of wet-to-dry lung weight, total leukocyte and neutrophil counts, total protein and lactate dehydroase in BAL fluid, total myeloperoxidase activity, and lung pathological injury. MK-801 inhibited hyperoxia-associated increments in reactive oxygen species production and NF-kappaB production. Hence, NMDA receptor antagonist MK-801 ameliorates hyperoxia-induced lung injury in neonatal rats, and is associated with decreased reactive oxygen species and NF-kappaB. We conclude that Glu may play an important role in hyperoxia-induced lung injury by activation of NMDA receptor.     

磁共振形态学半定量评分对新生儿细菌性脑膜炎出院结局的评估价值

目的 分析MRI形态学半定量评分对新生儿细菌性脑膜炎出院结局的评估价值。方法 收集复旦大学附属儿科医院2011年7月至2013年12月NICU收治的出院诊断为新生儿细菌性脑膜炎的病例,采用基于大脑损伤MRI形态学分析的半定量评分,对头颅MRI图像进行回顾性分析。MRI形态学评价包括脑室扩大、脑室旁白质容积丢失、脑白质囊性病灶、内囊后肢髓鞘化异常、皮质信号异常、颅内脑外间隙异常、基底节信号异常、脑白质非囊性信号异常、脑室内出血、脑室积脓、脑膜异常强化、室管膜异常强化和脑脓肿。将上述13项评分归纳为脑白质异常（WMA）、脑灰质异常（GMA）和非脑实质异常（NPA）。同时采集患儿出生孕周、发病时间、MRI检查时间、发病至MRI检查间隔时间和出院结局。按照出生孕周分为早产儿组和足月儿组,再按照出院结局分为预后良好和预后不良亚组,在各组内比较亚组之间时间因素、MRI单项评分和综合评分的差异。结果 63例新生儿细菌性脑膜炎病例进入分析（早产儿组18例,足月儿组45例）。MRI单项评分构成预后良好和预后不良亚组间差异有统计学意义的指标：早产儿组中有脑室扩大(P=0.012)和脑室旁白质容积丢失(P=0.004);足月儿组有脑室扩大(P=0.002)、脑室旁容积丢失(P=0.040)、颅内脑外间隙异常(P=0.005)和脑室内出血(P=0.038)。MRI综合评分中,早产儿组WMA评分(P=0.001)和NPA评分（P=0.039）、足月儿组NPA评分(P=0.018)在预后不良和预后良好亚组之间分布差异有统计学意义。足月儿组和早产儿组内不同预后亚组的各时间因素差异未发现统计学意义或临床意义。结论 新生儿细菌性脑膜炎MRI脑室扩大和脑室旁白质容积丢失预示早产儿出院不良结局;脑室扩大、脑室旁白质容积丢失、颅内脑外间隙异常和脑室内出血预示足月儿出院不良结局。WMA评分高预示早产儿出院不良结局,NPA评分高预示早产儿和足月儿出院不良结局。     

Hemangiopoietin, a novel human growth factor for the primitive cells of both hematopoietic and endothelial cell lineages

The cells of hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblast. However, the existence of a growth factor acting relatively specifically on hemangioblasts remains unclear. Here we report the identification of hemangiopoietin (HAPO), a novel growth factor acting on both hematopoietic and endothelial cell lineages. In vitro in the human system, recombinant human HAPO (rhHAPO) significantly stimulated the proliferation and hematopoietic and/or endothelial differentiation of human bone marrow mononuclear cells and of purified CD34+, CD133+, kinase domain receptor-positive (KDR+), or CD34+/KDR+ cell populations. In the murine system, rhHAPO stimulated the proliferation of long-term culture-initiating cells (LTC-ICs) as well as CD34+ and stem cell antigen-1 (Sca-1+) cell subsets. In vivo, subcutaneous injection of rhHAPO into normal mice resulted in a significant increase in bone marrow hematopoietic cells. Furthermore, irradiated mice injected with rhHAPO had an enhanced survival rate and accelerated hematopoiesis. Our data suggest that HAPO is a novel growth factor acting on the primitive cells of both hematopoietic and endothelial cell lineages and that HAPO may have a clinical potential in the treatment of various cytopenias and radiation injury and in the expansion of hematopoietic and endothelial stem/progenitor cells.