In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy

Whereas the morphogenesis of developing organisms is relatively well understood at the molecular level, the contribution of the mechanical properties of the cells to shape changes remains largely unknown, mainly because of the lack of quantified biophysical parameters at cellular or subcellular resolution. Here we designed an atomic force microscopy approach to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAMs). SAMs are highly organized structures that contain the plant stem cells, and generate all of the aerial organs of the plant. Building on modeling and experimental data, we designed a protocol that is able to measure very local properties, i.e. within 40-100 nm deep into the wall of living meristematic cells. We identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and even subcellular levels. Strikingly, we found that the outer cell wall was much stiffer at the tip of the meristem (5 ± 2 MPa on average), covering the stem cell pool, than on the flanks of the meristem (1.5 ± 0.7 MPa on average). Altogether, these results demonstrate the existence of a multiscale spatialization of the mechanical properties of the meristem surface, in addition to the previously established molecular and cytological zonation of the SAM, correlating with regional growth rate distribution.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献   

A computational study of atomic oxygen-doped silicon carbide nanotubes

We investigated the properties of atomic oxygen-doped (O-doped) models of representative (6,0) and (4,4) silicon carbide nanotubes (SiCNTs) by density functional theory (DFT) calculations of isotropic and anisotropic chemical shielding (CS) parameters of Si-29, O-17 and C-13 atoms for the optimized structures. The calculated parameters indicated the effects of O-doping on the electronic environments of the first neighboring atoms of the doped sites. Comparing the results of the zigzag and armchair models also indicated that the latter model detects more effects of the O-doping than the former one.     

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum‐free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.Download video file.^{(69M, mov)}     

Purification of immature neuronal cells from neural stem cell progeny

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.     

Age‐specific reference intervals for routine biochemical parameters in healthy neonates,infants, and young children in Iran

Age and sex need to be considered in the establishment of reference intervals (RIs), especially in early life when there are dynamic physiological changes. Since data for important biomarkers in healthy neonates and infants are limited, particularly in Iranian populations, we have determined age‐specific RIs for 7 laboratory biochemical parameters. This cross‐sectional study comprised a total of 344 paediatric participants (males: 158, females: 186) between the ages of 3 days and 30 months (mean age: 12.91 ± 7.15 months). Serum levels of creatinine, urea, uric acid, calcium, phosphate, vitamin D and high‐sensitivity C‐reactive protein (hs‐CRP) were measured using an Alpha classic‐AT plus auto‐analyser. We determined age‐specific RIs using CLSI Ep28‐A3 and C28‐A3 guidelines. No sex partitioning was required for any of the biomarkers. Age partitioning was required for kidney function tests and phosphate. The serum concentration of urea and creatinine increased with age, while phosphate and uric acid decreased with age. Age partitioning was not required for serum calcium, vitamin D, and hs‐CRP, which remained relatively constant throughout the age range. Age‐specific RIs for 7 routine biochemical markers were determined to address critical gaps in RIs in early life to help improve clinical interpretation of blood test results in young children, including neonates. Established age partitions demonstrate the biochemical changes that take place during child growth and development. These novel data will ultimately better disease management in the Iranian paediatric population and can be of value to clinical and hospital laboratories with similar populations.     

Fluid particle diffusion through high-hematocrit blood flow within a capillary tube

Fluid particle diffusion through blood flow within a capillary tube is an important phenomenon to understand, especially for studies in mass transport in the microcirculation as well as in solving technical issues involved in mixing in biomedical microdevices. In this paper, the spreading of tracer particles through up to 20% hematocrit blood, flowing in a capillary tube, was studied using a confocal micro-PTV system. We tracked hundreds of particles in high-hematocrit blood and measured the radial dispersion coefficient. Results yielded significant enhancement of the particle diffusion, due to a micron-scale flow-field generated by red blood cell motions. By increasing the flow rate, the particle dispersion increased almost linearly under constant hematocrit levels. The particle dispersion also showed near linear dependency on hematocrit up to 20%. A scaling analysis of the results, on the assumption that the tracer trajectories were unbiased random walks, was shown to capture the main features of the results. The dispersion of tracer particles was about 0.7 times that of RBCs. These findings provide good insight into transport phenomena in the microcirculation and in biomedical microdevices.     

Ultrastructural changes of sheep cumulus-oocyte complexes following different methods of vitrification

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.     

Association of Mycobacterium infections in patients with Mendelian susceptibility to mycobacterial disease with venous thromboembolism

An association between a hypercoagulable state and Mendelian susceptibility to mycobacterial disease (MSMD) has been established in a few studies; resultant thrombosis is considered rare. In a case‐control study, the prevalence of factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C mutations were investigated in mycobacterium‐infected patients. The study comprised 30 patients with mycobacterial infections (invasive, disseminated and/or recurrent infections with Bacille Calmette–Guerin or non‐tuberculosis mycobacteria and Mycobacterium Tuberculosis with positive results for acid‐fast bacilli and tuberculin skin tests) and 30 normal healthy controls. Forty female (66.7%) and 20 male subjects (33.3%) aged from 3 to 70 years were recruited into this study. Genotyping of targeted genes was performed by RT‐PCR and cytokine TNF‐α concentrations were quantified using a commercially available ELISA kit. Significant associations between mycobacterial infection and TNF‐α production after stimulating peripheral blood mononuclear cells with LPS alone and with IFN‐γ plus LPS were identified. Moreover, genotyping analysis in the studied population revealed a significant association between MTHFR c.677C>T (OR, 3.28; 95% CI, 1.35–7.92; P < 0.05), MTHFR c.1298A>C (OR, 2.33; 95% CI, 1.10–4.93; P < 0.05) and mycobacterial infection in affected patients, indicating susceptibility to venous thromboembolism according to previous studies. Additionally, mycobacterium‐infected patients had a significantly greater prevalence of MTHFR C677T and A1298C mutations than controls.     

Bioluminescence regenerative cycle (BRC) system: theoretical considerations for nucleic acid quantification assays

A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10(-18) mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).