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With use of an electronic picture-scanning device and a digital computer, electron micrographs taken of a specimen along several different directions can be superimposed to form a montage that is more informative than the component images. Preliminary results indicate that one may thus study unstained, unshadowed biological material at high resolution.
The ability to separate biological macromolecules with good resolution on liquid chromatographic columns has depended on the development of suitable packing materials. In size exclusion chromatography, molecules are separated by size on the basis of differential permeation of the packing. Ion exchange, hydrophobic interaction (or reversed-phase), and affinity chromatography are all surface-mediated separation methods, although they depend on different retention mechanisms. High-performance liquid chromatographic columns designed for biopolymers offer major advantages over conventional columns in both speed and resolving power. The exponential growth of literature on the high-performance separation of peptides and proteins in particular indicates that the technique will become the dominant form of column liquid chromatography.
The biotin-binding site on avidin has been labeled with biotin conjugated to undecagold, an organometallic cluster compound containing 11 gold atoms in a core angestroms in diameter. Examination of unstained specimens by scanning transmission electron microscopy reveals the labeled sites directly, without computational averaging or filtering of the images. This approach should be widely applicable for determining the locations of subunits and functional site in biological macromolecules at a resolution at a resolution in range of 15 angstroms.
A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.
Methods are developed for the resolution of cultural and biological inheritance based on qualitative data on pairs of relatives. Analysis of data on schizophrenia concluded in favor of significant cultural inheritance, assortative mating and familial environment unique to twins, in addition to biological inheritance. Genetic and cultural heritabilities were estimated as 0.707 +/- 0.077 and 0.203 +/- 0.120, respectively. Familial environmental component unique to twins was estimated as 0.090 +/- 0.040.
PURPOSE: To investigate the clinical usefulness of the latency of the pupil light reflex by optimizing its measurement, characterizing its variability, and determining the sensitivity of pupil latency as a function of stimulus input in normal subjects. METHODS: Computerized binocular infrared pupillography was performed in 14 eyes of seven healthy subjects. Pupils were recorded simultaneously at 60 and 1000 Hz. Each eye was alternatively stimulated eight times for 50 ms every 2.5 seconds, increasing by 0.5 log units over a 2.0-log-unit range. To determine intersubject and intereye variability, 98 eyes of 49 healthy subjects were recorded at 60 Hz over a 3.0-log-unit range (15 degrees radius stimulation, four repetitions at each intensity). RESULTS: Accuracy and resolution of latency were limited by the number of light reflexes used to estimate the average latency and were significantly affected by sampling rate when the number of reflexes recorded was fewer than four. Binocular recording and interpolation of the 60-Hz recording to 300 Hz added resolution to the latency. Biological variability contributed more to interindividual variability than did measurement variability. The range of intereye afferent asymmetry of latency in normal subjects was only between 8.3 and 35 ms-less with brighter stimulus intensity. CONCLUSIONS: An optimal method for determination of the onset of the pupil light reflex was devised that consisted of filtering, interpolation of pupil recordings, and analysis of the first and second derivative of the pupil movement. Most of the variability in latency as a function of intensity in normal subjects was due to interindividual variation and latency was well matched between the two eyes of the subjects.
The usefulness of microdialysis was examined for the chronological determination of caffeine concentration in the brain and cerebrospinal fluids (CSF) following intravenous administration of caffeine in rats. The recovery percent of caffeine by microdialysis, the concentration ratio of caffeine in the dialysate against that in the brain tissue or CSF was determined. The recovery percent was proved to be constant at 5 different steady-state plasma concentrations of caffeine (0.1-280 nmol/ml) and in different collecting periods of dialysate ranging from 30 s to 10 min. The mean recovery percent in the brain and CSF were 10.9 and 13.1%, respectively. Thus, microdialysis was proved useful for determination of drug concentration in the tissue and biological fluids with time resolution of more than 30 s. The microdialysis method was then applied for the chronological determination of caffeine concentration in the brain and CSF following intravenous bolus administration. The estimated caffeine concentration in the brain and CSF was the same as those obtained by direct determination in isolated brain and CSF, respectively. Transfer of caffeine from plasma to brain and CSF were further pharmacokinetically analyzed using a modified 2-compartment model. In this kinetic model, the transfer of caffeine between the CSF and brain was neglected, since the mutual transfer of caffeine was not detected in in vivo experiments. Calculated curves were well fitted on observed caffeine concentrations in the plasma, brain and CSF.
Optical contrast agents have been widely applied to enhance the sensitivity and specificity of optical imaging with near-infrared (NIR) light. However, because of the overwhelming scattering of light in biological tissues, the spatial resolution of traditional optical imaging degrades drastically as the imaging depth increases. Here, for the first time to our knowledge, we present noninvasive photoacoustic angiography of animal brains in vivo with NIR light and an optical contrast agent. When indocyanine green polyethylene glycol, a novel absorption dye with prolonged clearance, is injected into the circulatory system of a rat, it obviously enhances the absorption contrast between the blood vessels and the background tissues. Because NIR light can penetrate deep into the brain tissues through the skin and skull, we are able to successfully reconstruct the vascular distribution in the rat brain from the photoacoustic signals. On the basis of differential optical absorption with and without contrast enhancement, a photoacoustic angiograph of a rat brain is acquired that matches the anatomical photograph well and exhibits high spatial resolution and a much-reduced background. This new technology demonstrates the potential for dynamic and molecular biomedical imaging.
The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.
Gene expression profiling of single cells using single-cell and single-nucleus RNA sequencing (sxRNA-seq) enables researchers to characterize cellular heterogeneity and unraveling complex biological processes at unprecedented resolution. However, sxRNA-seq faces challenges due to the presence of ambient RNA, extraneous RNA molecules not originating from the cells of interest. Sample preparation is a major source of ambient RNA, where harsh conditions can lead to cell lysis and the release of intracellular RNA. This inescapable inclusion of ambient RNA can cause erroneous results and hinder downstream analyses. To address this issue, various methodologies have been developed to identify, quantify, and remove ambient RNA. Here, we rigorously evaluate 7 state-of-the-art methodologies for ambient RNA removal using simulated datasets, species-mixing experiments of varying complexities, and genotype-mixing experiments. We find that no single method performs the best across all datasets and metrics, but CellBender, DecontX and SoupX generally perform well.
Stress is often associated with water retention and its resolution with diuresis. The biological systems for the control of stress and water balance are very closely related. Corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP) are co-localised in the hypothalamus and often act synergistically. Atrial natriuretic peptide (ANP) can exert a feedback control on the hypothalamic/pituitary/adrenal axis. ANP has been shown to be anxiolytic, whereas AVP may be anxiogenic. AVP and ANP levels have been found to be abnormal in a range of stress disorders and psychiatric illnesses. Isatin is an endogenous anxiogenic factor which is also a potent inhibitor of the ANP receptor. It may provide a link between the function of monoamines during stress, and the control of water balance by ANP.
Histological correlations of the human lip with corresponding magnetic resonance (MR) images obtained ex vivo are presented. MR images were acquired with a dedicated low-field (0.1 T) MR system, with a resolution of 100 x 100 micron2 and a slice thickness of 1 mm using 3D-FLASH sequences. The outermost tissues visible on images are of high signal intensity, while the innermost connective tissues are of low signal intensity, both for skin and oral mucosa. It is possible to use a low-field system to obtain high resolution images of small biological samples.
The relationship between the developing embryo (both plant and animal) and a gravitational field has long been contemplated. The difficulty in designing critical experiments on the surface of the earth because of its background of 1 g, has been an obstacle to a resolution of the problem. Biological responses to gravity (particularly in plants) are obvious in many cases; however, the influence of gravity as an environmental input to the developing embryo is not as obvious and has proven to be extremely difficult to define. In spite of this, over the years numerous attempts have been made using a variety of embryonic materials to come to grips with the role of gravity in development. Three research tools are available: the centrifuge, the clinostat, and the orbiting spacecraft. Experimental results are now available from all three sources. Some tenuous conclusions are drawn, and an attempt at a unifying theory on gravitational influence on embryonic development is made.
BACKGROUND: The technique of Fluorescence In-Situ Hybridization (FISH), a hybrid of cytogenetics and molecular biology has increased the resolution and application of cytogenetics in various neoplastic processes. In various types of leukemias, primary investigation by conventional cytogenetic [CC] technique followed by FISH has increased our understanding of the abnormal clonal formation involving different gene region. AIMS: Present study is aimed to use different kinds of in-house FISH probes in various hematological malignancies and its correlation with conventional cytogenetic finding. MATERIAL AND METHODS: Cytogenetic study was carried out in 360 patients either from peripheral blood or from bone marrow cells suspected for various types of leukemias. Four of 360 cases were further selected for FISH study by using different types of in-house probes, such as BAC [Bacterial Artificial Chromosome], PAC [Phague Artificial Chromosome], alphoid, PCP [Partial Chromosome Paint] and WCP [Whole Chromosome paint]. RESULTS: The results confirmed breakpoints of inversion 16 and del 16 in case 2 and 3 respectively. Whereas, case 1 did not confirm the cytogenetic findings of t(15;17) by PML/RARa fusion signals as multiple cell lines were involved in the patients. PCP and WCP were helpful in the identification of the marker chromosome in case 1. Telomeric and centromeric probes confirmed the cytogenetic findings of t(5;7) in case 4. CONCLUSION: We observe from this study that, in addition to the conventional cytogenetic study, FISH study provide further confirmation of chromosomal rearrangements. This facilitates our understanding of the neoplastic process more precisely for the better prognostication of the patient.
The effectiveness of an optical coherence tomography (OCT) system depends largely on the light source chosen. Published data on the optical properties of tissues are used to quantify the exponential attenuation of broadband light on transport through tissue. The effective attenuation coefficient is taken to be the sum of the absorption and scattering coefficients. This is used to demonstrate the effect on the spectra of a wide range of published OCT sources and the change in system resolution induced, and hence to comment on the suitability of different sources for OCT. The tissues studied include skin dermis, liver, and gallbladder. Sources at higher wavelengths are shown to be capable of high-resolution OCT imaging at greater depths. Titanium:sapphire lasers would be most suited for high-resolution OCT over comparatively shallow depths into tissue. For lower-resolution applications of OCT, a semiconductor optical amplifier and ytterbium fiber sources have better powers and bandwidths than superluminescent diodes. The resolution of OCT systems is not reduced significantly with imaging depth.
Technical advances on several frontiers have expanded the applicability of existing methods in structural biology and helped close the resolution gaps between them. As a result, we are now poised to integrate structural information gathered at multiple levels of the biological hierarchy - from atoms to cells - into a common framework. The goal is a comprehensive description of the multitude of interactions between molecular entities, which in turn is a prerequisite for the discovery of general structural principles that underlie all cellular processes.
Identifying the genes underlying genetically complex traits is of fundamental importance for medicine, agriculture, and evolutionary biology. However, the level of resolution offered by traditional quantitative trait locus (QTL) mapping is usually coarse. We analyze here a trait closely related to fitness, ovariole number. Our initial interspecific mapping between Drosophila sechellia (8 ovarioles/ovary) and D. simulans (15 ovarioles/ovary) identified a major QTL on chromosome 3 and a minor QTL on chromosome 2. To refine the position of the major QTL, we selected 1038 additional recombinants in the region of interest using flanking morphological markers (selective phenotyping). This effort generated approximately one recombination event per gene and increased the mapping resolution by approximately seven times. Our study thus shows that using visible markers to select for recombinants can efficiently increase the resolution of QTL mapping. We resolved the major QTL into two epistatic QTL, QTL3a and QTL3b. QTL3a shows sign epistasis: it has opposite effects in two different genetic backgrounds, the presence vs. the absence of the QTL3b D. sechellia allele. This property of QTL3a allows us to reconstruct the probable order of fixation of the QTL alleles during evolution.
Jet-based technologies are increasingly being explored as potential high-throughput and high-resolution methods for the manipulation of biological materials. Previously shown to be of use in generating scaffolds from biocompatible materials, we were interested to explore the possibility of using electrospinning technology for the generation of scaffolds comprised of living cells. For this, it was necessary to identify appropriate parameters under which viable threads containing living cells could be produced. Here, we describe a method of electrospinning that can be used to deposit active biological threads and scaffolds. This has been achieved by use of a coaxial needle arrangement where a concentrated living biosuspension flows through the inner needle and a medical-grade poly(dimethylsiloxane) (PDMS) medium with high viscosity (12,500 mPa s) and low electrical conductivity (10-15 S m-1) flows through the outer needle. Using this technique, we have identified the operational conditions under which the finest cell-bearing composite microthreads are formed. Collected cells that have been cultured, postelectrospinning, have been viable and show no evidence of having incurred any cellular damage during the bionanofabrication process. This study demonstrates the feasibility of using coaxial electrospinning technology for biological and biomedical applications requiring the deposition of living cells as composite microthreads for forming active biological scaffolds.