Estimating misclassification error with small samples via bootstrap cross-validation

MOTIVATION: Estimation of misclassification error has received increasing attention in clinical diagnosis and bioinformatics studies, especially in small sample studies with microarray data. Current error estimation methods are not satisfactory because they either have large variability (such as leave-one-out cross-validation) or large bias (such as resubstitution and leave-one-out bootstrap). While small sample size remains one of the key features of costly clinical investigations or of microarray studies that have limited resources in funding, time and tissue materials, accurate and easy-to-implement error estimation methods for small samples are desirable and will be beneficial. RESULTS: A bootstrap cross-validation method is studied. It achieves accurate error estimation through a simple procedure with bootstrap resampling and only costs computer CPU time. Simulation studies and applications to microarray data demonstrate that it performs consistently better than its competitors. This method possesses several attractive properties: (1) it is implemented through a simple procedure; (2) it performs well for small samples with sample size, as small as 16; (3) it is not restricted to any particular classification rules and thus applies to many parametric or non-parametric methods.     

A Procedure for Staining Cartilage and Bone of Whole Vertebrate Larvae While Rendering All Other Tissues Transparent

A new procedure for clearing and differentially staining skeletons of vertebrate larvae is described. Body pigments of fixed specimens are bleached by treatment with alkaline H₂O₂. This process markedly enhances the transparency of alcian blue-alizarine red S stained whole animals without damaging the specimens. This procedure should be useful for analyzing skeletal structures in studies of embryology, comparative anatomy and teratology of small vertebrates.     

A multiple-tubes approach for accurate genotyping of very small DNA samples by using PCR: statistical considerations.

A multiple-tubes procedure is described for using PCR to determine the genotype of a very small DNA sample. The procedure involves dividing the sample among several tubes, then amplifying and typing the contents of each tube separately. The results are analyzed by a statistical procedure which determines whether a genotype can be conclusively assigned to the DNA sample. Simulation studies show that this procedure usually gives correct results even when the number of double-stranded fragments in the sample is as small as 30. The procedure remains effective even in the presence of small amounts of laboratory contamination. We find that the multiple-tubes procedure is superior to the standard one-tube procedure, either when the sample is small or when laboratory contamination is a potential problem; and we recommend its use in these situations. Because the procedure is statistical, it allows the degree of certainty in the result to be quantified and may be useful in other PCR applications as well.     

Detection of Mineralized Structures in Early Stages of Development of Marine Teleostei Using a Modified Alcian Blue-Alizarin Red Double Staining Technique for Bone and Cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 ixm in length can be clearly visualized. The method is suitable for both onto-genic studies during early stages of skeletal development in most marine fishes (e.g., Sporus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.     

Potential use of current X-ray contrast studies in the detection of small intestinal polyps in the Peutz-Jeghers syndrome

The paper presents the results of studies of the small bowel in 9 patients with the Peutz-Jeghers syndrome. The indications for the study were the clinical picture of gastrointestinal bleeding and the symptoms of ileus. The author's intubation enterographic procedure using the better composition of barium suspension and an infusion system for administering contract substances into the small bowel, an improved fractional contrasting procedure, and a procedure employing the agent Entero-view was applied for contrasting the small bowel. All the procedures revealed an obvious picture of polyposis of the small bowel. The minimum size of detected tumors was 0.3-0.4 cm in diameter. Emphasis is laid on the quantitative image of neoplasms with Entero-view and the therapeutic effect of intubation enterography in evolving small-small intestinal intussusception.     

Gluttonous predators: how to estimate prey size when there are too many prey

Prey size is an important factor in food consumption. In studies of feeding ecology, prey items are usually measured individually using calipers or ocular micrometers. Among amphibians and reptiles, there are species that feed on large numbers of small prey items (e.g. ants, termites). This high intake makes it difficult to estimate prey size consumed by these animals. We addressed this problem by developing and evaluating a procedure for subsampling the stomach contents of such predators in order to estimate prey size. Specifically, we developed a protocol based on a bootstrap procedure to obtain a subsample with a precision error of at the most 5%, with a confidence level of at least 95%. This guideline should reduce the sampling effort and facilitate future studies on the feeding habits of amphibians and reptiles, and also provide a means of obtaining precise estimates of prey size.     

A non-iterative confidence interval estimating procedure for the intraclass kappa statistic with multinomial outcomes

We obtain the asymptotic sample variance of the intraclass kappa statistic for multinomial outcome data. A modified Wald type procedure based on this theory is then used for confidence interval construction. The results of a simulation study show that the proposed non-iterative approach performs very well in terms of confidence interval coverage and width for samples as small as 50. The procedure is illustrated with two examples from previously published medical studies.     

A SIMPLIFIED METHOD FOR THE URINARY EXCRETION TEST OF ABSORPTION OF COBALT 60 LABELLED VITAMIN B12

It is possible to do both radioiodine uptake studies and urinary excretion tests for absorption of Cobalt 60 vitamin B₁₂ with the same basic equipment. If a spectrometer is used and the samples properly prepared, the latter procedure can be performed quickly, accurately and with a minimum of laboratory equipment. This is a distinct advantage to a small laboratory with limited facilities, and should help to make both of these useful tests available for clinical use.     

Synergistic effect of TPA and T-cell mitogens in nonmammalian vertebrates

Summary The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was used as a comitogen with the plant lectins phytohemagglutinin (PHA) and concanavalin A (ConA) in short-term cultures of whole blood from nonmammalian vertebrates. Stimulation with TPA in addition to standard mitogens resulted in a synergistic effect, consistently yielding more metaphases than cultures stimulated with either PHA, ConA, or TPA alone and is successful with blood samples as small as 0.1 ml. The increased mitotic index makes it possible to use different banding procedures for systematic studies. Also, because the amount of blood needed is so small, this procedure, unlike other published techniques, does not require the destruction of smaller animals to do chromosome studies. This work was supported in part by National Institute of Environmental Health Science Grant 5-T32-ES07015-08 to the Environmental Toxicology Center at the University of Wisconsin-Madison.     

Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method

The bacterial mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance have functionally been reconstituted into giant unilamellar liposomes (GUVs) using an improved reconstitution method in the presence of sucrose. This method gives significant time savings (preparation times as little as 6 h) compared to the classical method of protein reconstitution which uses a dehydration/rehydration (D/R) procedure (minimum 2 days preparation time). Moreover, it represents the first highly reproducible method for functional reconstitution of MscS as well as MscS/MscL co-reconstitution. This novel procedure has the potential to be used for studies of other ion channels by liposome reconstitution.     

Automated microsequence analysis by use of radioactive phenylisothiocyanate.

The use of radioactive phenylisothiocyanate as a coupling reagent in conjunction with an automated protein sequenator permits N-terminal sequence analysis of 1.5 nmoles of protein. This procedure should facilitate chemical studies on several membrane proteins of current interest (e.g., histocompatability and Ir-associated alloantigens) which are available in only small quantities.     

Fluorescence-activated sorting of individual cells onto poly-L-lysine-coated slide areas

Cells sorted by a fluorescence-activated cell sorter are collected onto small areas of a glass slide. These collection areas have been coated with poly-L-lysine to attach the cells firmly to the glass surface. This simple procedure proved to be suitable to sort single cells and small cell populations with preservation of cytomorphology and viability without modifying the cell sorter. Additional studies on sorted cells may be performed, as shown by peroxidase-anti-peroxidase analysis of cellular antigens and by mRNA in situ hybridization.     

Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells

An extremely simple procedure for preparing cytoplasmic RNA from small numbers of cells is described. Cells are lysed with the detergent NP-40 and efficient extraction of protein from the postnuclear cytoplasmic lysate is ensured by denaturation with sodium dodecyl sulfate and urea. This procedure is suitable for preparing RNA from many cell types. All procedures have been scaled down to be performed in 1.5-ml microfuge tubes and thus RNA may be prepared from small numbers of cells. The procedure is extremely rapid and RNA is ready for Northern gel analysis in less than 30 min. Because so few steps are involved, RNA recovery is quantitative.     

Immobilized Sample Amplification for Quantitative Determination of Retroviruses

Immobilized sample amplification (ISA) is a novel method for amplification, detection, monitoring, and quantitative determination of nucleic acids from a minute amount of sample. We present here a novel quantitative ISA assay for retroviruses using a replication-defective recombinant retrovirus as a model retrovirus. Samples, as small as 5 to 10 microl or as large as 1 ml or more in volume, are readily immobilized on a nylon or polyester matrix. Retroviral RNA is directly amplified following the rehydration of the immobilized samples, thus eliminating the needs for retroviral RNA extraction. An ISA assay of a 10-microl viral sample generates results equal to or better than that of RT-PCR on equivalent amount RNA isolated from larger sample volumes. Recovery of RNA from small volumes, such as 10 microl, is almost impossible, whereas ISA assay detects retroviruses from as small as 1 to 5 microl of viral samples containing 10(4) cfu/ml determined by colony-forming assay. Extraction of RNA from a small amount of infectious viral samples not only is a difficult, biohazardous procedure, but also introduces random errors which contribute to variability in viral quantitation. Since the ISA method eliminates the isolation/extraction of the nucleic acids, it significantly shortens the handling time for the biohazardous materials and simplifies the procedure for analyzing small quantities of biological samples. This method detects less than 10 infectious retroviral particles as determined by both colony-forming assay and electron microscope studies. The format and protocol of this quantitative ISA assay can be easily automated to fit into numerous platforms, thus making it attractive for laboratory automation.     

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.     

Synthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with labeled acyl groups

A facile procedure for the small scale chemical synthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPL) is reported. Under optimal conditions, 36% of the sn-glycero-3-phosphocholine was converted to DPL. This procedure is suitable for small scale preparation of DPL with very high specific radioactivity from labeled palmitic acid. Furthermore, the labeled DPL obtained will serve as precursor for the chemical or enzymatic synthesis of other labeled phospholipids.     

A technique for retrograde intubation in mice

Endrotracheal intubation is critical for some experimental studies in mice, but the animals' small size makes the procedure difficult. The authors describe a new, easily learned retrograde intubation method using angioplasty guide wire. They twice intubated anesthetized mice successfully with no airway complications caused by puncture of the trachea.     

Synthesis of l,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine with Labeled Acyl Groups

A facile procedure for the small scale chemical synthesis of l,2-dipalmitoyl-Sn -glycero-3-phosphocholine (DPL) is reported. Under optimal conditions, 36% of the sn-glycero-3-phosphocholine was converted to DPL. This procedure is suitable for small scale preparation of DPL with very high specific radioactivity from labeled palmitic acid. Furthermore, the labeled DPL obtained will serve as precursor for the chemical or enzymatic synthesis of other labeled phospholipids.     

Expression,purification and preliminary crystallization study of RpaC protein from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803

State transitions in cyanobacteria are physiological adaptation mechanisms that change the interaction of the phycobilisomes with the photosystem I and photosystem II core complexes. This mechanism is essential for cyanobacteria at low light intensities. Previous studies of cyanobacteria have identified a gene named rpaC, which appears to be specifically required for state transitions. The gene product of rpaC is very probably a transmembrane protein that is a structural component of the phycobilisome-photosystem II supercomplex. However, the physiological role of RpaC protein is unclear. Here we report the construction of an expression system that enables high production of fusion protein TrxHisTagSTag-RpaC, and describe suitable conditions for purification of this insoluble protein at a yield of 3 mg per 1 dm³ of bacterial culture. Cleavage with HRV 3C protease to remove the TrxHisTagSTag portion resulted in low yields of RpaC-protein (∼ 30 μg/dm³ of bacterial culture), therefore the applicability to structural studies was tested for the fusion protein only. Several preliminary conditions for crystallization of TrxHisTagSTag-RpaC were set up under which microcrystals were obtained. This set of conditions will be a good starting point for optimization in future crystallization experiments. TrxHisTagSTag-RpaC protein may prove useful in biochemical studies where the small size of RpaC protein is limiting the investigation of interactions with significantly larger parts of the photosynthetic apparatus. Furthermore, the purification procedure described here might also be applied to the production and purification of other small membrane proteins for biochemical and structural studies.