Extracting information from imaging cytometry: a review

The extraction of statistically meaningful quantitative information from microscopy images is increasingly important for modern biological research. Obtaining accurate, quantitative information from biological specimens, however, is a complex process that requires optimization of several parameters. One must consider the number of probes, fluorescent channels required, type of plate to be used, number of fields to be acquired and optimal resolution for image acquisition. The extraction of information from images is dependent on and can be aided greatly by careful consideration of the factors involved in the image acquisition process. I summarize here the general principles behind the imaging and software technology that is used to quantify images and highlight particular issues of concern for critically applying image quantitation techniques for research.     

Gene expression and activity of digestive proteases in <Emphasis Type="Italic">Daphnia</Emphasis>: effects of cyanobacterial protease inhibitors

Background  

The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia 's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels.     

Redox regulation of carbonic anhydrases via thioredoxin in chloroplast of the marine diatom Phaeodactylum tricornutum

Thioredoxins (Trxs) are important regulators of photosynthetic fixation of CO(2) and nitrogen in plant chloroplasts. To date, they have been considered to play a minor role in controlling the Calvin cycle in marine diatoms, aquatic primary producers, although diatoms possess a set of plastidic Trxs. In this study we examined the influences of the redox state and the involvement of Trxs in the enzymatic activities of pyrenoidal carbonic anhydrases, PtCA1 and PtCA2, in the marine diatom Phaeodactylum tricornutum. The recombinant mature PtCA1 and -2 (mPtCA1 and -2) were completely inactivated following oxidation by 50 μm CuCl(2), whereas DTT activated CAs in a concentration-dependent manner. The maximum activity of mPtCAs in the presence of 6 mm reduced DTT increased significantly by addition of 10 μm Trxs from Arabidopsis thaliana (AtTrx-f2 and -m2) and 5 μm Trxs from P. tricornutum (PtTrxF and -M). Analyses of mPtCA activation by Trxs in the presence of DTT revealed that the maximum mPtCA1 activity was enhanced ～3-fold in the presence of Trx, whereas mPtCA2 was only weakly activated by Trxs, and that PtTrxs activate PtCAs more efficiently compared with AtTrxs. Site-directed mutagenesis of potential disulfide-forming cysteines in mPtCA1 and mPtCA2 resulted in a lack of oxidative inactivation of both mPtCAs. These results reveal the first direct evidence of a target of plastidic Trxs in diatoms, indicating that Trxs may participate in the redox control of inorganic carbon flow in the pyrenoid, a focal point of the CO(2)-concentrating mechanism.     

Historical trends in frog populations in New Zealand based on public perceptions

Surveys were distributed to New Zealand land users in 1998 and 2008 to acquire information about New Zealand frogs with the aim of compiling and mapping their distribution and inferred population trends without costly and time-consuming field surveys. The overall frog population trend was reported as declining, with possible causes reported as an increase in agriculture, an increase in the distribution of predatory fish and disease. The resultant maps could be used for four main purposes: 1) to identify regions where Litoria populations are known to occur, which can be eliminated when considering suitable regions for translocation of Leiopelma; 2) to identify growing or stable populations of Litoria species, which may assist future disease surveys, population monitoring and to identify sources of genetic material that may serve as an Ark for declining Australian populations; 3) to highlight populations that are in decline to enable effective targeting of detailed disease studies; and 4) to approximate the stability of amphibian populations in the absence of more accurate, but costly, scientific monitoring.     

Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum

Photosynthetic organisms in nature often experience light fluctuations. While low light conditions limit the energy uptake by algae, light absorption exceeding the maximal rate of photosynthesis may go along with enhanced formation of potentially toxic reactive oxygen species. To preempt high light-induced photodamage, photosynthetic organisms evolved numerous photoprotective mechanisms. Among these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to dissipate thermally the excessively absorbed energy. Diatoms thrive in all aquatic environments and thus belong to the most important primary producers on earth. qE in diatoms is provided by a concerted action of Lhcx proteins and the xanthophyll cycle pigment diatoxanthin. While the exact Lhcx activation mechanism of diatom qE is unknown, two lumen-exposed acidic amino acids within Lhcx proteins were proposed to function as regulatory switches upon light-induced lumenal acidification. By introducing a modified Lhcx1 lacking these amino acids into a Phaeodactylum tricornutum Lhcx1-null qE knockout line, we demonstrate that qE is unaffected by these two amino acids. Based on sequence comparisons with Lhcx4, being incapable of providing qE, we perform domain swap experiments of Lhcx4 with Lhcx1 and identify two peptide motifs involved in conferring qE. Within one of these motifs, we identify a tryptophan residue with a major influence on qE establishment. This tryptophan residue is located in close proximity to the diadinoxanthin/diatoxanthin-binding site based on the recently revealed diatom Lhc crystal structure. Our findings provide a structural explanation for the intimate link of Lhcx and diatoxanthin in providing qE in diatoms.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.