Optimal molecular profiling of tissue and tissue components

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.     

Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells

Purpose

To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear.

Methods

An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre.

Results

Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype.

Conclusions

Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.     

Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay

Hepatocyte growth factor (HGF) is a mesenchymal-derived cytokine. It exerts in vitro a motogenic effect on various target cells, which is displayed either by cell scattering, locomotion, and migration during the wound repair process of cultured cells, or invasiveness through the extracellular matrix. Although it is known that HGF influences the motogenic effect of endothelial cells, the precise effects of HGF during angiogenesis are still poorly understood. To identify genes regulated via HGF signaling in HUVECs, we used the differential display polymerase chain reaction. In this study, thymosin beta4 was found to be differentially expressed in HGF-treated HUVECs compared with control. Data from HPLC profile and induction of MMPs indicate that HGF may affect the biological behavior of HUVECs through a combination of the direct effects of HGF itself and indirect effects mediated via induction of thymosin beta4 in vitro.     

Net dissolved inorganic nitrogen production in hyporheic mesocosms with contrasting sediment size distributions

The interstitial spaces within streambeds are recognized as an important location of dissolved inorganic nitrogen (DIN) transformations in streams. However, it remains uncertain how physical characteristics of streambeds affect the magnitude and net outcome of subsurface nitrogen transformations. We tested whether the size distribution of streambed sediments, in isolation from the influence of streambed topography and groundwater upwelling, could affect net DIN uptake or production along interstitial flow paths. Mesocosms constructed from PVC pipe (15 cm diameter × 1 m long) were filled with either coarse gravel/cobble or gravel/cobble mixed with finer sediments (5 mesocosms per sediment treatment). Mesocosms were submerged in a stream and oriented, so that surface water flowed through the sediments. After 2 months incubation, we measured DIN in interstitial water at 20 cm intervals and dissolved oxygen at 10 cm intervals along mesocosm flow paths. In both sediment types, DIN concentrations increased longitudinally along mesocosm flow paths in the direction of interstitial flow, indicating net DIN production. Although DIN increased to higher concentrations in mesocosms with fine sediments, greater exchange flow through coarse sediments resulted in similar rates of net DIN production and delivery to surface water. Production of DIN in both sediment types was concentrated within the first 10 cm of interstitial flow paths, with no significant production further along the flow paths. Coarse sediments had higher rates of oxygen consumption per unit sediment volume than the coarse–fine sediment mix, suggesting interstitial water velocity may be an important factor affecting hyporheic microbial metabolism.     

Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross.     

Indigogenic substrates for detection and localization of enzymes

Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic histochemistry. Indoxyl substrates also are used to localize enzyme activities in non-indigogenic techniques. In indoxyl-azo methods, the released indoxyl couples with a diazonium salt to form an azo dye. In indoxyl-tetrazolium methods, the oxidizing agent is a tetrazolium salt, which is reduced by the indoxyl to an insoluble coloured formazan. Indoxyl-tetrazolium methods operate only at high pH; the method for alkaline phosphatase is used extensively to detect this enzyme as a label in immunohistochemistry and in Western blots. The insolubility of indigoid dyes in water limits the use of indigogenic substrates in biochemical assays for enzymes, but the intermediate indoxyl and leucoindigo compounds are strongly fluorescent, and this property is exploited in a variety of sensitive assays for hydrolases. The most commonly used substrates for this purpose are glycosides and carboxylic and phosphate esters of N-methylindoxyl. Indigogenic enzyme substrates are among many chromogenic reagents used to facilitate the identification of cultured bacteria. An indoxyl substrate must be transported into the organisms by a permease to detect intracellular enzymes, as in the blue/white test for recognizing E. coli colonies that do or do not express the lac-z gene. Secreted enzymes are detected by substrate-impregnated disks or strips applied to the surfaces of cultures. Such devices often include several reagents, including indigogenic substrates for esterases, glycosidases and DNAse.     

Quantitative mass spectrometric immunoassay of insulin like growth factor 1

Reported in this work are the development of mass spectrometric immunoassay (MSIA) devices and methods for the qualitative analysis of IGF-1 and -2, and the rigorous quantification of IGF-1 from human plasma. A method involving addition of SDS in moderate concentration to unfractionated plasma for disrupting IGF/IGFBP complexes was initially developed. The method is suitable for the direct extraction of the IGFs and subsequent mass spectrometric analysis. Rat plasma, containing IGF-1 that is mass shifted from human IGF-1, was used as an internal reference standard (IRS) for the quantification of IGF-1 directly from human plasma. A standard curve with linear dynamic range of at least 2 orders of magnitude was constructed from serially diluted IGF-1 standards containing equal amounts of rat plasma. Using the standard curve, IGF-1 levels in plasma samples from eight individuals were determined. The limit of detection for the IGF-1 MSIA was also evaluated and established to be approximately 15 pM. The assay is rapid and can be performed in parallel via high-throughput robotics processing. Furthermore, the mass spectrometry aspect of the developed IGF-1 immunoassay offers a new dimension in the ongoing study of IGF-1 and related diseases.     

What effect does classroom separation have on twins' behavior, progress at school, and reading abilities?

We investigated the effects of classroom separation on twins' behavior, progress at school, and reading abilities. This investigation was part of a longitudinal study of a nationally-representative sample of twins (the E-risk Study) who were assessed at the start of school (age 5) and followed up (age 7). We examined three groups of twins: pairs who were in the same class at both ages; pairs who were in separate classes at both ages; and pairs who were in the same class at age 5, but separated by age 7. When compared to those not separated, those separated early had significantly more teacher-rated internalizing problems and those separated later showed more internalizing problems and lower reading scores. Monozygotic (MZ) twins showed more problems as a result of separation than dizygotic (DZ) twins. No group differences emerged for externalizing problems, ADHD or prosocial behaviors. The implications of the findings for parents and teachers of twins, and for school practices about separating twins, are discussed.     

Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.