An inexpensive,accurate method for measuring leaf area and defoliation through digital image analysis

We report a protocol using a common desk-top scanner and public domain software for measuring existing leaf area and leaf area removed as a result of herbivory. We compared the accuracy and precision of this method to that of a standard leaf area meter. Both methods were used to measure metal disks of a known area, the area of soybean (Glycine max L.) leaves, and the area removed by simulating leaf feeding with a hole-punch. We varied the amount of injury across a low, medium, and high degree of simulated feeding. The mean area of 10 cm2 and 50 cm2 metal disks was more accurately estimated with the leaf area meter than the desk-top scanner. Leaf area estimates from both methods were highly correlated. The desk-top scanner accurately estimated the leaf area removed from the low, medium, or high degree of simulated leaf feeding. However, the leaf area meter overestimated low levels of simulated feeding injury. Though measuring a leaf's surface area with a desk-top scanner requires two steps (creating a digital image file and calculating the area represented by that image), the overall time required to measure leaf injury is shorter than with a leaf area meter. This relatively simple and inexpensive method of estimating leaf area and feeding damage has advantages in certain experimental situations where a prefeeding measurement of the leaf is impossible or undesirable, or when small amounts of feeding occur.     

Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping

Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.     

A more cost-effective method of preoperative computerized imaging

Stimulated by the explosive expansion of the computerized desk top publishing industry during the past few years, microcomputer hardware and software are evolving at a staggering rate. Memory is rapidly increasing, and prices are declining. I have found that with the hardware and software described in this paper, I was able to obtain, in a much more cost-effective manner, as useful preoperative information for my practice as I could obtain with more expensive "turnkey" (only one use) computerized imaging systems. This type of microcomputer, of course, is not limited to just the imaging system, but can be used for a variety of other programs as well, such as word processing, slide labeling and production, spreadsheet functions, billing and filing, and numerous business and other applications. The ease of use with readily available 35-mm slides of my patients has greatly enhanced the appeal of this system. Computerized imaging, when used as an educational tool, can be very helpful in preoperative planning, resident teaching, and for illustration and discussion of a patient's proposed surgery. The electronic imaging disclaimer compiled by the American Society of Plastic and Reconstructive Surgeons has been extremely helpful in clarifying the limits of computerized imaging and reducing any false expectations that my patients might have. All of us are experiencing the dawn of a very exciting evolution.     

Enzymatic switching for efficient and accurate translesion DNA replication

When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) δ or ε, a switch occurs to allow translesion synthesis by DNA polymerase η, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol δ, Pol ε and Pol η and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis–syn thymine–thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol η and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.     

A more accurate method for measurement of tuberculocidal activity of disinfectants

The current Association of Official Analytical Chemists method for testing tuberculocidal activity of disinfectants has been shown to be inaccurate and to have a high degree of variability. An alternate test method is proposed which is more accurate, more precise, and quantitative. A suspension of Mycobacterium bovis BCG was exposed to a variety of disinfectant chemicals and a kill curve was constructed from quantitative data. Data are presented that show the discrepancy between current claims, determined by the Association of Official Analytical Chemists method, of selected commercially available products and claims generated by the proposed method. The effects of different recovery media were examined. The data indicated that Mycobacteria 7H11 and Middlebrook 7H10 agars were equal in recovery of the different chemically treated cells, with Lowenstein-Jensen agar having approximately the same recovery rate but requiring incubation for up to 3 weeks longer for countability. The kill curves generated for several different chemicals were reproducible, as indicated by the standard deviations of the slopes and intercepts of the linear regression curves.     

CARNOY: A new digital measurement tool for palynology

Quantitative data play an important role in palynological research. With the advent of digital imaging in light and electron microscopy, palynologists now have the opportunity to perform measurements faster and more precisely than ever before. Several image analysis software packages already exist for these tasks, but they are often expensive, difficult to use or not adapted to the specific needs of palynologists. After studying the daily workflow of a palynologist, we designed CARNOY, an image analysis application written from the ground up for use in palynology and morphology. CARNOY offers an easy-to-use interface and several features to make measuring easier and faster. The program can export measurements to almost every other software package for further analysis and is available for free on the Internet.     

A more accurate method for measurement of tuberculocidal activity of disinfectants.

The current Association of Official Analytical Chemists method for testing tuberculocidal activity of disinfectants has been shown to be inaccurate and to have a high degree of variability. An alternate test method is proposed which is more accurate, more precise, and quantitative. A suspension of Mycobacterium bovis BCG was exposed to a variety of disinfectant chemicals and a kill curve was constructed from quantitative data. Data are presented that show the discrepancy between current claims, determined by the Association of Official Analytical Chemists method, of selected commercially available products and claims generated by the proposed method. The effects of different recovery media were examined. The data indicated that Mycobacteria 7H11 and Middlebrook 7H10 agars were equal in recovery of the different chemically treated cells, with Lowenstein-Jensen agar having approximately the same recovery rate but requiring incubation for up to 3 weeks longer for countability. The kill curves generated for several different chemicals were reproducible, as indicated by the standard deviations of the slopes and intercepts of the linear regression curves.     

CD45-assisted PanLeucogating for accurate,cost-effective dual-platform CD4+ T-cell enumeration

BACKGROUND: North American and European guidelines for dual-platform (DP) flow cytometry recommend absolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtained on a hematology analyzer and the percentages of CD4+ cells among lymphocytes (CD4%/lympho) obtained by flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match between these common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocyte counts (white cell counts [WCC]) and CD4% among the gated CD45+ leucocytes (CD4%/leuco) can be determined with greater accuracy. METHODS: We introduced "PanLeucogating," i.e., we used total leucocytes as the common denominator for improving the precision of DP absolute CD4 counting. Correlations and Bland-Altman tests were used for statistical analysis. RESULTS: First, 22 stabilized blood product samples were provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy and precision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next, 183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly high correlation and comparable precision of absolute CD4 counts were observed between the SP volumetric method and DP PanLeucogating (R(2) = 0.990; bias 6 +/- SD 17%). The SP volumetric method showed lower levels of agreement with the DP lymphocyte gating (R(2) = 0.758; bias 14 +/- SD 51%) and with the SP bead-based method (R(2) = 0.923; bias 4 +/-SD 31%). CONCLUSIONS: These observations show that DP leucocyte counts (WCC) should replace lymphocyte counts as the "common denominator" although CD4%/lympho values can, as an extra step, be also provided readily if requested. When coupled with quality control for WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cell assay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4, antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.     

An accurate method for measurement of aldosterone production

A method is described for isolation of the acid-hydrolysable metabolite of aldosterone in sufficient purity to measure accurately the daily production rate. Values obtained with six hospital patients were 84-131mug./day on a daily intake of 100m-equiv. of Na(+) and 227-464mug./day on a daily intake of 10m-equiv. of Na(+). Corresponding values for aldosterone excretion were also recorded, but these are a poor index of production rate since they represent from 1.6 to 9.8% of the total daily output of aldosterone.     

A simple and accurate method for the measurement of the growth of cell monolayers

Summary A procedure has been developed for the accurate, quick, and simple indexing of the growth of insect cell monolayers with a reticule in the eyepiece of an inverted phasecontrast microscope. A magnification of ×500 is desirable for accuracy and ease of counting. Cell nuclei whose circumferences include any of the 25 points in the reticule are enumerated in each microscopic field. Counts of as few as five fields gave an error of 18.8% per flask. Ten fields gave an error of 11.5% and provided a sufficiently accurate comparison for many purposes. Forty counts allowed the measurement of differences between treatments with an error of 5.85%. A large number of treatments can be handled simultaneously, due to the small number of replicates necessary and the small amount of time required per treatment. When the capacity of different batches of lactalbumin hydrolysate to support growth was tested in our insect tissue culture medium, some of them were found to be suitable and others unsuitable. Doubling time of cell populations was measured, after the ratio of the average area of the nuclei at time 0 to that at timeX was used to multiply the number of nuclei counted at timeX. Average nuclear areas were satisfactorily measured by simple measurements of nuclear diameter on an ocular micrometer. The cell nuclei tended to decrease in area when cell monolayers reached confluence or became crowded. The number of replicates required was reduced, because the same flask could be used several times without disturbing the cell monolayer. The method of counting nuclei in a monolayer by means of reticule points and a phase-contrast microscope can also be adapted to the estimation of the absolute number of cells growing on the bottom of a flask. Portion of a thesis by the senior author submitted for the Ph.D. degree to the Graduate College of the University of Illinois. This research was supported in part by grant GB 20915 from the National Science Foundation and United States Public Health Service grant AI 6392 from the National Institutes of Health.     

An efficient and cost-effective isotope labeling protocol for proteins expressed in shape Escherichia coli

A cost-effective protocol for uniform ¹⁵N and/or¹³ C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.     

A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D

Background  

To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge.     

Practical do-it-yourself device for accurate volume measurement of breast

A simple and accurate method of measuring differences in breast volume based on Archimedes' principle is described. In this method, a plastic container is placed on the breast of the patient who is lying in supine position. While the breast occupies part of the container, the remaining part is filled with water and the volume is measured. This method allows the measurement of the volume differences of asymmetric breasts and also helps the surgeon to estimate the size of the prosthesis to be used in augmentation mammaplasty.     

A cost-effective method for reducing soil disturbance-induced errors in static chamber measurement of wetland methane emissions

Static chambers used for sampling methane (CH₄) in wetlands are highly sensitive to soil disturbance. Temporary compression around chambers during sampling can inflate the initial chamber CH₄ headspace concentration and/or lead to generation of non-linear, unreliable flux estimates that must be discarded. In this study, we tested an often-used rubber gasket (RG)-sealed static chamber against a water-filled gutter (WFG) seal design that could be set up and sampled from a distance of 2 m with a newly designed remote rod sampling system to reduce soil disturbance. Compared to conventional RG design, our remotely sampled static chambers reduced the chance of detecting inflated initial CH₄ concentrations (>3.6 ppm) from 66 to 6 % and nearly doubled the proportion of robust linear regressions (r ² > 0.9) from 45 to 86 %. Importantly, the remote rod sampling system allows for more accurate and reliable CH₄ sampling without costly boardwalk construction. This paper presents results demonstrating that the remote rod sampling system combined with WFG static chambers improves CH₄ data reliability by reducing initial gas measurement variability due to chamber disturbance when tested on a mineral soil-restored wetland in Charles City County, Virginia, USA.     

HaploRec: efficient and accurate large-scale reconstruction of haplotypes

Background  

Haplotypes extracted from human DNA can be used for gene mapping and other analysis of genetic patterns within and across populations. A fundamental problem is, however, that current practical laboratory methods do not give haplotype information. Estimation of phased haplotypes of unrelated individuals given their unphased genotypes is known as the haplotype reconstruction or phasing problem.     

A catchment-oriented and cost-effective policy for water protection

Governmental programmes and international agreements to counteract eutrophication have largely not attained agreed objectives (e.g. reduction by half of the anthropogenic nitrogen load on Swedish coastal waters). Important components of such programmes are improved removal of nitrogen in municipal treatment plants and changed agricultural practices. In addition, increased N-removal during runoff, i.e. restoration of ponds and wetlands, is an important strategy. One explanation of the fact that the objectives have yet not been achieved might be that the most effective step to counteract diffuse pollution has not been fully implemented. It is therefore important to stress the potential of effective measures and find ways to fully implement them at the watershed level. It is important to avoid excessive applications of fertilizers because this leads to an exponential increase in leaching. Field experiments indicate that the use of winter crops or an undersown catch crop outside the main cropping season has reduced nitrate losses by up to 75% in single years, and by nearly 50% over successive years. In southern Sweden, the area of wetlands has been reduced considerably (more than 90%) by melioration activities. In a recent project, budget studies with restored ponds verified the importance of ponds and wetlands in nitrogen retention. Per unit area, increased nitrogen loading implied increased nitrogen retention, but often a decrease in the percent retained. Ponds with depths of 0.4–2.0 m and hydrological loads of 0.14–5.2 m³ m⁻² day⁻¹ were created. One hundred and fifty to seven thousand kg N ha⁻¹ year⁻¹ was removed in ponds loaded by streams dominated by agricultural run off. A pond receiving pre-treated municipal wastewater removed 8000 kg N ha⁻¹ year⁻¹. The upper limit for N-removal is set by the hydrological conditions. Sedimentation of organic material must be favoured in order to obtain adequate conditions for denitrification. To achieve the governmental objective in nitrogen load reduction changed cultivation practices within the agricultural sector must be combined with restoration of ponds/wetlands.