Labelled-replica techniques: post-shadow labelling of intramembrane particles in freeze-fracture replicas

Three methods are described for direct post-fracture, post-shadow labelling of individual classes of intramembrane particles (IMPs) in freeze-fracture replicas of biological membranes. The P-face IMPs corresponding to the acetylcholine receptor complexes (AChRs) of vertebrate neuroeffector junctions are identified by post-replication labelling with ferritin-antibody complexes and with neurotoxin-biotin-avidin-colloidal gold affinity ligands. (The freeze-etch nomenclature of Branton et al., 1975, is used in this report.) These post-shadow labelling techniques resemble conventional en bloc labelling techniques except that the labelling reagents must penetrate a thin but discontinuous layer of platinum superimposed on the molecules of interest. In the ‘sectioned labelled-replica technique’, the replicated and labelled tissues are stained, embedded in plastic and sectioned parallel to the replica-tissue interfaces. In the direct ‘labelled-replica techniques’, the replicated and labelled samples are freeze-dried or critical point dried, the labelled surfaces are stabilized by carbon coating, and the underlying tissues are dissolved, allowing the labelled-replicas to be examined as conventional freeze-fracture replicas. The unshadowed side of each AChR IMP is shown to retain sufficient biochemical information to permit both immunospecific and neurotoxin specific labelling despite formaldehyde fixation, freezing, fracturing, platinum shadowing, and thawing in aqueous media. A new mixed ferricyanide-osmium staining method reveals electron opaque structures spanning the membrane bilayer in the same size, number and distribution as the labelled IMPs. These experiments demonstrate the feasibility of identifying individual IMPs in freeze-fracture replicas and may allow the identification of specific membrane lesions in human disease.     

Accelerated image reconstruction using ordered subsets of projection data

The authors define ordered subset processing for standard algorithms (such as expectation maximization, EM) for image restoration from projections. Ordered subsets methods group projection data into an ordered sequence of subsets (or blocks). An iteration of ordered subsets EM is defined as a single pass through all the subsets, in each subset using the current estimate to initialize application of EM with that data subset. This approach is similar in concept to block-Kaczmarz methods introduced by Eggermont et al. (1981) for iterative reconstruction. Simultaneous iterative reconstruction (SIRT) and multiplicative algebraic reconstruction (MART) techniques are well known special cases. Ordered subsets EM (OS-EM) provides a restoration imposing a natural positivity condition and with close links to the EM algorithm. OS-EM is applicable in both single photon (SPECT) and positron emission tomography (PET). In simulation studies in SPECT, the OS-EM algorithm provides an order-of-magnitude acceleration over EM, with restoration quality maintained.     

Numerical calibration of donor systems for some GAP tests

Numerical models of various donor/attenuator systems utilized in gap tests were evaluated with the DYNA2D hydrocode. Three donor (pressed pentolite)/attenuator (PMMA) configurations were tested. Case I and Case 2 had a pentolite donor which was 2 cm × 2 cm right cylinder; the cell size was smaller in Case 1. Case 3 utilized a composite pentolite donor 2.07 cm long but having stepped diameters of one and two cm; the cell size was quite similar to that of Case 2. Peak (axial) pressure versus distance results from Case 1 were scaled and Compared to experimental data for both the NOL large scale gap test (LSGT) and the NSWC expanded large scale gap test (ELSGT). The comparison was found to be very good for the LSGT but less satisfactory for the ELSGT. Peak (axial) pressure versus distance results from Case 1 were the also compared to Case 2, a coarser zoned model similar to Case 1, and Case 3. This comparison showed that the composite donor produced peak (axial) pressures similar to those from the LSGT model. Normalized peak pressure versus radial distance and shock profiles were compared for the three cases and found to be quite similar. Additional normalized peak pressure versus radial distance and normalized pressure versus time information from Case 1 is plotted to show how the pentolite/PMMA rarefaction affects both the axial and radial pressure-time histories.     

Hydrogen uptake of reduced graphene oxide and graphene sheets decorated with Fe nanoclusters

Graphene oxide (GO) has been prepared by employing modified Staudenmaier's method through thermal exfoliation of graphite oxide. High pressure hydrogen sorption isotherms up to 50 bar of GO, reduced by thermal reduction (TR-GO), chemical reduction (CR-GO) and graphene sheets decorated with Fe nanoclusters (Fe-GS) have been investigated. Thermal reduction of GO at 623 K under high vacuum yields TR-GO. Chemical reduction of GO using hydrazine forms CR-GO. Fe-GS was synthesized through arc-discharge between the ends of two graphite rods with one rod carrying Fe nanoparticles. The surface areas of these graphene samples were determined from the nitrogen adsorption isotherm employing Brunauer, Emmett and Teller (BET) method. Kelvin's equation was used to determine the pore size distribution of all graphene based samples. Hydrogen pressure-composition isotherms (PCI) were determined at 300 K and at 77 K, between 0.1 and 50 bar. Further, in this paper, we present a comparative adsorption isotherm analysis of hydrogen and helium on TR-GO. This reveals that the volume of hydrogen and helium adsorbed by TR-GO is nearly equal. The similar uptake volume determined for both hydrogen and helium indicates the possibility of monolayer adsorption of hydrogen and also nearly similar binding energy between TR-GO and H₂/He.     

Hydrogen energy in changing environmental scenario: Indian context

This paper deals with how the Hydrogen Energy may play a crucial role in taking care of the environmental scenario/climate change. The R&D efforts, at the Hydrogen Energy Center, Banaras Hindu University have been described and discussed to elucidate that hydrogen is the best option for taking care of the environmental/climate changes. All three important ingredients for hydrogen economy, i.e., production, storage and application of hydrogen have been dealt with. As regards hydrogen production, solar routes consisting of photoelectrochemical electrolysis of water have been described and discussed. Nanostructured TiO₂ films used as photoanodes have been synthesized through hydrolysis of Ti[OCH(CH₃)₂]₄. Modular designs of TiO₂ photoelectrode-based PEC cells have been fabricated to get high hydrogen production rate (10.35 lh⁻¹ m⁻²). However, hydrogen storage is a key issue in the success and realization of hydrogen technology and economy. Metal hydrides are the promising candidates due to their safety advantage with high volume efficient storage capacity for on-board applications. As regards storage, we have discussed the storage of hydrogen in intermetallics as well as lightweight complex hydride systems. For intermetallic systems, we have dealt with material tailoring of LaNi₅ through Fe substitution. The La(Ni_l − xFe_x)₅ (x = 0.16) has been found to yield a high storage capacity of 2.40 wt%. We have also discussed how CNT admixing helps to improve the hydrogen desorption rate of NaAlH₄. CNT (8 mol%) admixed NaAlH₄ is found to be optimum for faster desorption (3.3 wt% H₂ within 2 h). From an applications point of view, we have focused on the use of hydrogen (stored in intermetallic La–Ni–Fe system) as fuel for Internal Combustion (IC) engine-based vehicular transport, particularly two and three-wheelers. It is shown that hydrogen used as a fuel is the most effective alternative fuel for circumventing climate change.     

Five days of oral topotecan (Hycamtin), a phase I and pharmacological study in adult patients with solid tumours

Topotecan is a specific inhibitor to topoisomerase I. An oral formulation of topotecan is available with a bioavailability of 32-44% in humans. A phase I and pharmacological study of the oral formulation of topotecan administered daily for 5 days every 21 days was performed in adult patients with solid tumours to determine the maximum tolerated dose (MTD). Adult patients with a WHO performance status < or = 2 adequate haematological, hepatic and renal functions, with malignant solid tumours refractory to standard forms were entered into the study. Pharmacokinetics were performed on days 1 and 4 of the first course using a validated high performance liquid chromatographic assay. 29 patients entered the study, all patients were evaluable for toxicity and response. The doses studied in the 29 patients were 1.2, 1.8, 2.3, 2.7 mg/m2/day and a fixed dose of 4 mg/day without surface area adjustment. A total of 109 courses were given. Dose limiting toxicity (DLT) was reached at a dose of 2.7 mg/m2/day and consisted of CTC (NCI-Common Toxicity Criteria) grade IV granulocytopenia. The regimen was well tolerated. Non-haematological toxicities were mild, including fatigue, anorexia, nausea, vomiting and diarrhoea. A significant correlation was observed between the percentage decrease in white blood cells versus the area under the curve (AUC(t)) of topotecan lactone (R = 0.76 P < 0.01) which was modelled by a sigmoidal Emax function. The correlation coefficient between the absolute topotecan dose administered and the AUC(t) was R = 0.52 (P = 0.04). Pharmacokinetics of the fixed dose of 4 mg/day were comparable to the 2.3 mg/m2/day dose. DLT in this phase I study of five daily doses of oral topotecan every 21 days was granulocytopenia. The recommended dose for phase II studies is 2.3 mg/m2/day or alternatively, a fixed dose of 4 mg/day.     

Influence of Neotyphodium coenophialum on copper concentration in tall fescue

Poor performance of livestock that graze tall fescue (Festuca arundinacea Schreb.) has been associated with the endophyte fungus Neotyphodium coenophialum [Morgan-Jones and Gams] Glenn, Bacon, and Hanlin). Recent evidence suggests lowered Cu status and a depression of Cu-related immune function in steers that graze endophyte-infected (E+) tall fescue. Greenhouse and field studies investigated relationships between the endophyte and Cu concentrations in tall fescue. Seventeen infected 'Kenhy' clones were divided, and one plant of each pair was treated three times with Benomyl to remove the endophyte (E-). Plants were watered with nutrient solution in a greenhouse for 6 mo before sampling. Copper concentrations were greater (P < .001) in E- than in E+ clones (3.4 vs 2.8 microg/g; SE, .06). In the second greenhouse experiment, genetically similar E+ and E- 'Kentucky'-31 (KY-31) and 'Georgia Jessup' were grown from seed and fertilized with nutrient solution to produce mature plants. Copper concentrations were higher (P < .05) in E- than in E+ tall fescue (8.6 vs 7.6 microg/g; SE, .3). In a field plot experiment in Texas, E+ and E- KY-31 were grown with 0, 50, and 100% replacement of potential evapotranspiration. By September, Cu concentrations were higher (P < .05) in E- than in E+ tall fescue (7.3 vs 6.6 microg/g; SE, .2). In pasture experiments, KY-31 E+ (> 70% infection level) and E- (< 5% infection level) tall fescue were grown in Virginia at two locations with three rates of N fertilizer. Copper concentrations were higher (P < .05) in E- than in E+ tall fescue (4.8 vs 4.5 microg/g; SE, .1) and increased (P < .01) linearly in response to N. Our data demonstrate that the presence of the endophyte is associated with lower Cu concentrations in tall fescue, which may contribute to lowered Cu status in animals and thus contribute to the etiology of fescue toxicity.     

Prenatal diagnosis of partial monosomy 13q associated with occipital encephalocoele in a fetus

The pre- and postnatal findings of a fetus with a de novo del(13)(pter-->q21:) and an occipital encephalocoele are described. Maternal serum alpha-fetoprotein (AFP) screening at 19 weeks' gestation demonstrated a high level of 2.5 multiples of the median (MOM) and ultrasonography at 27 weeks' gestation showed severe intrauterine growth retardation, cardiomegaly, an occipital encephalocoele, and a calvarial defect. Genetic amniocentesis revealed a karyotype of 46,XX,del(13)(pter-->q21:). The proband postnatally displayed additional abnormalities such as microphthalmia, hypertelorism, large low-set ears, and micrognathia. We discuss the association of central nervous system (CNS) malformations with 13q deletions and emphasize that pregnancies with neural tube defects warrant cytogenetic analysis, especially when additional fetal abnormalities and neonatal dysmorphism are observed.     

Abnormal gastro-oesophageal reflux in Chinese with atypical chest pain

Although atypical chest pain has been well described in the Western population, its frequency in Chinese is unknown. Over a period of 42 months, we studied 521 Chinese patients with chest pain and identified 108 patients (20.7%) whose pain was not related to cardiac causes, as determined by exercise ECG or cardiac catheterization. Using 24 h ambulatory pH monitoring and baseline oesophageal manometry, 28.7, 19.4 and 5.6% of these patients were found to have abnormal reflux parameters, abnormal manometric findings or both, respectively. There were significantly more patients complaining of chest pain during the study in the gastro-oesophageal reflux disease (GERD) group than in the non-GERD group (16/31 vs 20/77; P < 0.001). The lower oesophageal sphincter pressure was lower in those with abnormal reflex parameters than in those with normal reflux parameters (12.7 +/- 5.4 vs 17.8 +/- 5.8 mmHg; P < 0.05). There was no significant difference in symptoms, such as heartburn (54.8 vs 42.9%), regurgitation (38.7 vs 35.1%) and dysphagia (19.4 vs 24.7%), among the two groups. Non-specific changes were the most frequent baseline motility pattern. In conclusion, atypical chest pain and gastro-oesophageal reflux disease are not uncommon in Chinese and this deserves special emphasis as the continuation of anti-anginal drugs may aggravate their condition.     

Validation of the Applied Biosystems Prism 377 automated sequencer for the forensic short tandem repeat analysis

The Applied Biosystems (ABI) Prism 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well-to-read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51 degrees C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software. Complete resolution of 1 base differences was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2-fold higher than the 373A and the sensitivity was increased by one third.