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Ex vivo expansion is critical in facilitating the application of hematopoietic/progenitor stem cells (HSPCs) for regenerative therapies. Wnt signaling is implicated in the expansion and self-renewal maintenance of HSPCs. However, a reasonable method to regulate Wnt signaling in ex vivo cultures to achieve robust expansion of HSPCs has not yet been investigated. Here, cord blood-derived CD34+ cells were cultured with the activator of Wnt signaling 6-bromoindirubin-3′-oxime (BIO) under the following conditions: vehicle control (group A); BIO was added to the culture on days 0, 4, and 7 (group B); and BIO was added to the culture on days 0 and 7 (group C). Initial BIO treatment promoted the expansion of CD34+ cells on day 4. However, BIO supplementation on days 0 and 4 in group B attenuated HSPC expansion on day 7, while enhancing the multilineage commit potential and secondary expansion ability of expanded CD34+ cells. Based on this finding, an optimized BIO feeding strategy (group C) was proposed to support substantial expansion of HSPCs. After 10 days of culture, the expansion fold of CD34+ cells was 28.70 ± 0.46-folds, which was significantly higher than group A (16.20 ± 0.72-folds, p

Hematopoietic stem/progenitor cells (HSPCs) ex vivo expansion is critical in facilitating their widespread clinical application. NF-κB pathway is implicated in the energy homeostasis and metabolic adaptation. To explore the effect of NF-κB pathway on the ex vivo HSPC expansion and metabolism, the 50 nM–1 μM inhibitor of NF-κB pathway TPCA-1 was used to expand cord blood derived CD34+ cells in serum-free culture. The expansion folds, function, mitochondrial profile and metabolism of HSPCs were determined. After 10 days of culture with 100 nM TPCA-1, the expansion of total cells， CD34+CD38− cells， and CD34+CD38−CD45RA−CD90+CD49f+ cells were significantly increased compared to the cytokine priming alone. Notably, TPCA-1 treatment generated ~ 2-fold greater percentage of CD34+EPCR+ and CD34+CD38−CD45RA−CD90+CD49f+ cells compared to cytokine only conditions. Moreover, TPCA-1 expanded CD34+ cells displayed enhanced serial colonies forming potential and secondary expansion capability. NF-κB inhibition increased the expression of self-renewal related genes, while downregulated the expression of mitochondrial biogenesis regulator (Pgc1α) and mitochondrial chaperones and proteases (ClpP, Hsp10, Hsp60). Mitochondrial mass and membrane potential were markedly decreased with TPCA-1 treatment, leading to the reduced mitochondrial reactive oxygen species (ROS) level in HSPCs. NF-κB inhibition displayed augmented glycolysis rate with compromising mitochondrial metabolism. This study demonstrated that NF-κB pathway inhibition improved glycolysis and limited ROS production that promoted the ex vivo expansion and maintenance of functional HSPCs.

Geomagnetic field is widely used for remote sensing, environment observation, geoscience and geology research. A portable geomagnetic field vector measurement system is designed, but misalignment greatly influences measurement accuracy. It is calibrated by establishing interim coordinate without measuring the projection of geomagnetic field and gravity. In addition, earth's magnetic dip, the accurate attitude of equipment and careful operation in collection process are not need. It is easy to implement the experiment, and misalignment angles between sensors are calculated. Magnetometer, inertial navigation system, host platform frame and perpendicular platform are used in experiment. After calibration, North, Vertical and East vector measurement error are reduced to 3.9%, 4.7% and 6.6% respectively. Inclination and declination error are reduced from 4.374° and 4.974° to 0.173° and 0.309° respectively. Thus, it is convenient and effective for misalignment calibration.

Magnetic interference is one key factor reducing the vector measurement accuracy of geomagnetic field measurement instrument, where scalar suppression algorithm is ineffective. A convenient component suppression method is proposed to avoid complex equipments and strict operations in previous component methods. The key factor is to overcome the problem that additive unknown parameters are added when attitude changes. System attitude change information is proposed for component suppression, where the attitude change is provided by inertial navigation system (INS). After suppression, magnetic interference of north, vertical and east direction are suppressed to 2.1%, 4.28% and 2.53% respectively. It provides a new and convenient way to improve the accuracy of geomagnetic field vector measurement.

Magnetic field distortion of INS is the major factor influencing the accuracy of geomagnetic field information measurement system. Simulation and experiment results show that traditional scalar compensation methods are disabled for component compensation. A component compensation method is proposed, in which parallelepiped frame and perpendicular platform are used with designed operation process. Comparing with traditional methods, the component compensation method is effective for distortion parameter estimation, and it shows better component compensation performance. Experimental test result demonstrates that distortion field components of INS are suppressed approximately two orders after compensation, and the North, Vertical and East component measurement error of the geomagnetic field are reduced to 2.3%, 3.3% and 4.5% of the former values respectively. Declination error is reduced from 7.074° to 0.331° (4.6% of the former value). This compensation method contributes to the accuracy improvement of geomagnetic field vector measurement system.

On April 20, 2013, an earthquake of M7.0 occurred in Lushan, Sichuan province, China. This paper investigates the coseismic ionospheric anomalies using GPS (Global Positioning System) data from 23 reference stations in Sichuan province that are a part of the Crustal Movement Observation Network of China (CMONOC). The recorded results show that a clear ionospheric anomaly occurred within 15 min after the earthquake near the epicenter, and the occurrence time of the anomalies recorded by various stations is related to the distance from the epicenter. The maximum anomaly is 0.25 TECu, with a 2 min duration and the distance of the recording station to the epicenter is 83 km. Acoustic waves generated by the crustal vertical movement during the earthquake propagate up to the height of the ionosphere lead to the ionospheric anomaly, and the propagation speed of the acoustic wave is calculated as 0.72 ± 0.04 km/s based on the propagation time and propagation distance, consistent with the average speed of sound waves within a 0–450 km atmospheric height.

Olmsted syndrome (OS) is a rare congenital disorder characterized by palmoplantar and periorificial keratoderma, alopecia in most cases, and severe itching. The genetic basis for OS remained unidentified. Using whole-exome sequencing of case-parents trios, we have identified a de novo missense mutation in TRPV3 that produces p.Gly573Ser in an individual with OS. Nucleotide sequencing of five additional affected individuals also revealed missense mutations in TRPV3 (which produced p.Gly573Ser in three cases and p.Gly573Cys and p.Trp692Gly in one case each). Encoding a transient receptor potential vanilloid-3 cation channel, TRPV3 is primarily expressed in the skin, hair follicles, brain, and spinal cord. In transfected HEK293 cells expressing TRPV3 mutants, much larger inward currents were recorded, probably because of the constitutive opening of the mutants. These gain-of-function mutations might lead to elevated apoptosis of keratinocytes and consequent skin hyperkeratosis in the affected individuals. Our findings suggest that TRPV3 plays essential roles in skin keratinization, hair growth, and possibly itching sensation in humans and selectively targeting TRPV3 could provide therapeutic potential for keratinization or itching-related skin disorders.

We propose a simple but reliable technology for the generation of oxygen gradient in microfluidic devices, which is useful for cell aerotaxis study. The technology is based on single layer patterning of polydimethylsiloxane (PDMS) and integration of a thin layer polyvinylidene chloride (PVDC). The PDMS layer was patterned to define a cell chamber embedded by two micro-channels, whereas the PVDC layer acts as barrier to prevent the diffusion of gas outside into the layer, thus forcing gas to diffuse along the PDMS layer. As a result, a linear oxygen gradient in the cell chamber can be established by controlling the gas contents inside the two micro-channels. By using 3-way valves, such a linear gradient could be changed from one state to another. To demonstrate the application potential of this approach, we used this device in the study of the aerotactic behaviour of magnetospirillum sp. strain AMB-1 and observed a stable and reversible cell migration by changing the gas distribution in the cell chamber. This system should be useful for better understanding of cell aerotactic mechanism.

We propose a programmed syringe pumps system for diffusion-based concentration control in microcavities. Fluorescein is used to demonstrate the influence of different durations of unvarying injecting speed and different sizes of microcavities on the concentration control and distribution in the microcavities. With longer duration of unvarying injecting speed and smaller size of microcavities, the concentration control of the microcavities will be more consistent with the input signal. Using this system, we can precisely control sudden as well as gradual change of the microenvironment of cells within a period from several minutes to days by re-setting parameters of the program. As a demonstration, we apply this system to culture budding yeast cells in the microfluidic device and follow the growth status in precisely controlled nutritional environment. This system will be useful for high-throughput cell stimulation and differentiation studies.

A dimensionless mathematical model is proposed to predict the local heat transfer coefficients between vibrated fluidized beds and immersed horizontal tubes, and the effects of the thickness of gas film and the contact time of particle packets are well considered. Experiments using the glass beads (the average diameter d ¯ p = 1.83 mm ) were conducted in a two-dimensional vibrated fluidized bed (240 mm × 80 mm). The local heat transfer law between vibrated fluidized bed and horizontal tube surface has been investigated. The results show that the values of theoretical prediction are in good agreement with experimental data, so the model is able to predict the local heat transfer coefficients between vibrated fluidized beds and immersed horizontal tubes reasonably well, and the error is in range of ±15%. The results can provide references for future designing and researching on the vibrated fluidized beds with immersed horizontal tubes.

Sera from paraneoplastic pemphigus (PNP) immunoprecipitate multiple antigens from human epidermal protein extract. In this study, we further characterized the autoantibodies in 12 PNP sera. Immunoblotting using recombinant linker subdomains of envoplakin, periplakin, desmoplakin, and bullous pemphigoid antigen I found that 11 of the 12 sera recognized linker subdomains of envoplakin and periplakin. We then synthesized 12 peptides covering the linker subdomain of envoplakin for ELISA. One of the peptides, peptide no. 8, was recognized by nine out of the 12 sera with a higher affinity. A method of ligand–receptor binding assay was designed and performed using this peptide labeled with fluorescence as the ligand. Peptide no. 8 bound to CD20+ cells in Castleman's tumors from the patients whose sera were positive to this peptide by ELISA. Our data suggest that the linker subdomain of plakin proteins may be one of the major areas recognized by PNP autoantibodies, and epitopes in the linker subdomain of envoplakin recognized by PNP autoantibodies with a high affinity are dispersed in several areas and are variable among PNP patients. We also demonstrate that B-lymphocyte clones specifically reacting to epidermal proteins exist in Castleman's tumors from PNP.

Summary Background Paraneoplastic pemphigus is an autoimmune mucocutaneous disease associated with Castleman's tumours, which when surgically removed often result in great improvement of mucocutaneous lesions. An IgG autoantibody against epidermal proteins is often used as a diagnostic marker for disease. Our aim was to ascertain the role of Castleman's tumours in production of the autoantibody and pathogenesis of paraneoplastic pemphigus. Methods We enrolled seven patients with paraneoplastic pemphigus associated with Castleman's disease and assessed the effect of removal of tumours on mucocutaneous lesions in six individuals and on autoantibody titre with indirect immunofluorescence in four patients. We cultured tumour cells from one patient and assayed the secreted autoantibody. Finally, we characterised the gene sequence and expression of the variable region of the immunoglobulin heavy chain (IgV H ) in tumour B cells from all patients by reverse transcription-PCR, DNA sequencing, and in-situ hybridisation. Findings Cutaneous lesions disappeared within 6–11 weeks after resection of tumours. Mucosal lesions also improved in this period, but lasted for 5–10 months overall. Autoantibody titre decreased and became undetectable within 5–9 weeks in three of four patients assessed. We identified secreted autoantibody, similar to that identified in patients' serum, in cultured tumour cells. The tumour B-cells of the seven patients shared and expressed two rearrangement patterns of complementarity determining region 3 (CDR3) of IgV H . Interpretation Secreted autoantibody from Castleman's tumours, which reacts against epidermal proteins, could be an essential factor in the pathogenesis of paraneoplastic pemphigus. We noted clonal rearrangement, resulting in similar variable regions of IgV H , in tumour B cells isolated from all seven patients. However, whether this pattern is associated with autoimmunity remains to be ascertained.

Epidermolytic hyperkeratosis (EHK) is a rare dominantly inherited skin disorder with erythroderma and hyperkeratosis. Mutations have been found in keratin 1 (K1) or keratin 10 (K10) gene. In the present study, we reported three sporadic and one familial Chinese EHK patients with their mutation findings. All the mutations turned out to be single heterozygous point substitutions. A novel mutation designated as E477K of K1 was identified in one patient, and previous reported mutations in codon 156 of K10, i.e. R156S, R156P, R156H were found in other patients. This is the first report of the keratin mutations in Chinese kindreds. The results showed that the possible correlation between the genotype and phenotype in these patients was complex, not only depended on the position of the mutation but also on the actual amino acid substitution. And palmoplantar keratoderma (PPKD) can be an accompanied symptom caused by either K1 or K10 mutation.

We describe a Chinese family with generalized atrophic benign epidermolysis bullosa (GABEB), a non-lethal variant of junctional epidermolysis bullosa. The proband was an offspring of consanguineous parents, had generalized blisters since birth and developed severe alopecia during early childhood. Ultrastructural examination of the proband's skin revealed fissures in the lamina lucida. Immunofluorescence assays using a monoclonal antibody recognizing the extracellular domain of the 180 kDa bullous pemphigoid antigen (BPAG2) showed loss of fluorescent signal in the basal membrane zone of the skin. DNA sequencing revealed a homozygous C-to-G transversion at nucleotide position 899 in exon 11 of the COL17A1 gene, which encodes BPAG2. This mutation results in serine to cysteine at position 265, which is located in a highly conserved region of the intracellular domain of BPAG2. We showed that the proband's father was heterozygous for this mutation. In addition, we found a novel polymorphic substitution of C-to-G at nucleotide position 798 in exon 10 of the COL17A1 gene, which results in an I233M change in BPAG2 and is a common polymorphic allele in a limited Chinese population.

The paper described a new cDNA subtractive cloning strategy. This strategy was based on the 'cap-finder' method, 'long distance' polymerase chain reaction (PCR), streptavidin magnetic beads mediated subtraction, and spin column chromatography. When PCR products were resolved on agarose gel after three rounds of subtraction, the 'single gene difference' group displayed a predominantly enriched band, but the 'multiple gene difference' group did not display any apparent difference. Of 200 clones inserted with 0.7-2 kb fragments from the 'multiple gene difference' group, 50% were identified to be new sequences and 35% were known. Of 100 new sequences, 35% contained coding regions and 75% were confirmed by dot-blotting to be differentially expressed by genes of the target cell. The results suggested that this strategy might be very efficient for full-length cloning of differentially expressed genes of the cell.

Skeletal muscle fibers are specialized at the site of the neuromuscular synapse, and this polarity is acquired during development and maintained in the adult as a consequence of inductive interactions between nerve and muscle. This review summarizes recent experiments which demonstrate that two different signalling pathways, a transcriptional pathway and a post-translational pathway, have an important role in the formation and maintenance of the neuromuscular synapse and mediate the induction of polarity in skeletal muscle fibers.

The quantum Hall liquid-Wigner solid phase boundary as a function of filling factor, density (Landau-level mixing) and temperature displays a rich variety of phenomena. More importantly the density and temperature range where these phenomena could take place arc in an experimentally accessible regime. This is particularly true because of the availability of hole samples. We will present results of variational Monte Carlo calculations on the ground-state energy of the liquid and solid. Assuming the transition is first-order, we predict the density-dependent phase boundary at T = 0 for the basic filling factors ν = 1 3 , 1 5 , 1 7 , and 1 9 . At finite temperatures, treating the excitation spectrum as phonons for the s and magnetorotons for the liquid, we can calculate the low-temperature properties of the phase boundary. Because the entropy of the solid coming from the low-lying shear modes is large compared to the entropy of the liquid, i.e. there is a gap in the excitation spectrum, we find an interesting regime where the liquid freezes as the temperature is raised. This quantum freezing is analogous to the behavior near the minimum in the melting of the solid helium-superfluid helium boundary.