WIS‐neuromath enables versatile high throughput analyses of neuronal processes

Automated analyses of neuronal morphology are important for quantifying connectivity and circuitry in vivo, as well as in high content imaging of primary neuron cultures. The currently available tools for quantification of neuronal morphology either are highly expensive commercial packages or cannot provide automated image quantifications at single cell resolution. Here, we describe a new software package called WIS‐NeuroMath, which fills this gap and provides solutions for automated measurement of neuronal processes in both in vivo and in vitro preparations. Diverse image types can be analyzed without any preprocessing, enabling automated and accurate detection of neurites followed by their quantification in a number of application modules. A cell morphology module detects cell bodies and attached neurites, providing information on neurite length, number of branches, cell body area, and other parameters for each cell. A neurite length module provides a solution for images lacking cell bodies, such as tissue sections. Finally, a ganglion explant module quantifies outgrowth by identifying neurites at different distances from the ganglion. Quantification of a diverse series of preparations with WIS‐NeuroMath provided data that were well matched with parallel analyses of the same preparations in established software packages such as MetaXpress or NeuronJ. The capabilities of WIS‐NeuroMath are demonstrated in a range of applications, including in dissociated and explant cultures and histological analyses on thin and whole‐mount sections. WIS‐NeuroMath is freely available to academic users, providing a versatile and cost‐effective range of solutions for quantifying neurite growth, branching, regeneration, or degeneration under different experimental paradigms. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish¹, plants^{2, 3}, arthropods^{4, 5}, fungi⁶, and bacteria⁷. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested^8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears¹¹, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments.We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously^{9, 10}, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)¹². This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies'' group (Queens University)¹⁰. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs^{5, 13}. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations^{14, 15}. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber¹³. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere¹⁶.

The Cold Stage System

The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 μm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).     

Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13^NEGSSEA4^POSTra-1-60^POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.     

Di-alkylated paromomycin derivatives: Targeting the membranes of Gram positive pathogens that cause skin infections

A collection of paromomycin-based di-alkylated cationic amphiphiles differing in the lengths of their aliphatic chain residues were designed, synthesized, and evaluated against 14 Gram positive pathogens that are known to cause skin infections. Paromomycin derivatives that were di-alkylated with C₇ and C₈ linear aliphatic chains had improved antimicrobial activities relative to the parent aminoglycoside as well as to the clinically used membrane-targeting antibiotic gramicidin D; several novel derivatives were at least 16-fold more potent than the parent aminoglycoside paromomycin. Comparison between a di-alkylated and a mono-alkylated paromomycin indicated that the di-alkylation strategy leads to both an improvement in antimicrobial activity and to a dramatic reduction in undesired red blood cell hemolysis caused by many aminoglycoside-based cationic amphiphiles. Scanning electron microscopy provided evidence for cell surface damage by the reported di-alkylated paromomycins.     

Hemicellulose modification promotes cadmium hyperaccumulation by decreasing its retention on roots in Sedum alfredii

Plant and Soil - Plant growth in the Arctic is often nutrient limited due to temperature constraints on decomposition and low atmospheric input of nitrogen (N). Local hotspots of nutrient...     

Association between carbonyl sulfide uptake and (18)Δ during gas exchange in C(3) and C(4) leaves

Carbonyl sulfide (COS) and C(18)OO exchange by leaves provide potentially powerful tracers of biosphere-atmosphere CO(2) exchange, and both are assumed to depend on carbonic anhydrase (CA) activity and conductance along the diffusive pathway in leaves. We investigated these links using C(3) and C(4) plants, hypothesizing that the rates of COS and C(18)OO exchange by leaves respond in parallel to environmental and biological drivers. Using CA-deficient antisense lines of C(4) and C(3) plants, COS uptake was essentially eliminated and discrimination against C(18)OO exchange ((18)Δ) greatly reduced, demonstrating CA's key role in both processes. (18)Δ showed a positive linear correlation with leaf relative uptake (LRU; ratio of COS to CO(2) assimilation rates, A(s)/A(c), normalized to their respective ambient concentrations), which reflected the effects of stomatal conductance on both COS and C(18)OO exchange. Unexpectedly, a decoupling between A(s) and (18)Δ was observed in comparing C(4) and C(3) plants, with a large decrease in (18)Δ but no parallel reduction in A(s) in the former. This could be explained by C(4) plants having higher COS concentrations at the CA site (maintaining high A(s) with reduced CA) and a high phosphoenolpyruvate carboxylase/CA activity ratio (reducing (18)O exchange efficiency between CO(2) and water, but not A(s)). Similar A(s) but higher A(c) in C(4) versus C(3) plants resulted in lower LRU values in the former (1.16 ± 0.20 and 1.82 ± 0.18 for C(4) and C(3), respectively). LRU was, however, relatively constant in both plant types across a wide range of conditions, except low light (<191 μmol photon m(-2) s(-1)).     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Light-Induced Changes within Photosystem II Protects Microcoleus sp. in Biological Desert Sand Crusts against Excess Light

The filamentous cyanobacterium Microcoleus vaginatus, a major primary producer in desert biological sand crusts, is exposed to frequent hydration (by early morning dew) followed by desiccation during potentially damaging excess light conditions. Nevertheless, its photosynthetic machinery is hardly affected by high light, unlike “model” organisms whereby light-induced oxidative stress leads to photoinactivation of the oxygen-evolving photosystem II (PSII). Field experiments showed a dramatic decline in the fluorescence yield with rising light intensity in both drying and artificially maintained wet plots. Laboratory experiments showed that, contrary to “model” organisms, photosynthesis persists in Microcoleus sp. even at light intensities 2–3 times higher than required to saturate oxygen evolution. This is despite an extensive loss (85–90%) of variable fluorescence and thermoluminescence, representing radiative PSII charge recombination that promotes the generation of damaging singlet oxygen. Light induced loss of variable fluorescence is not inhibited by the electron transfer inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), nor the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that reduction of plastoquinone or O₂, or lumen acidification essential for non-photochemical quenching (NPQ) are not involved. The rate of Q_A ⁻ re-oxidation in the presence of DCMU is enhanced with time and intensity of illumination. The difference in temperatures required for maximal thermoluminescence emissions from S₂/Q_A ⁻ (Q band, 22°C) and S_2,3/Q_B ⁻ (B band, 25°C) charge recombinations is considerably smaller in Microcoleus as compared to “model” photosynthetic organisms, thus indicating a significant alteration of the S₂/Q_A ⁻ redox potential. We propose that enhancement of non-radiative charge recombination with rising light intensity may reduce harmful radiative recombination events thereby lowering ¹O₂ generation and oxidative photodamage under excess illumination. This effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacteria to the higher plant chloroplast.     

Novel oxime and oxime ether derivatives of 12,14-dichlorodehydroabietic acid: design, synthesis, and BK channel-opening activity

Oxime ether derivatives of the benzylic ketone of 12,14-dichlorodehydroabietic acid (diCl-DHAA, 4b) were synthesised, and their BK channel-opening activity was evaluated in an assay system of CHO-K1 cells expressing hBKalpha channels. Oxime ether structure on the B ring of diCl-DHAA significantly increased the BK channel-opening activity.