Region-Based Convolutional Neural Nets for Localization of Glomeruli in Trichrome-Stained Whole Kidney Sections.

Background Histologic examination of fixed renal tissue is widely used to assess morphology and the progression of disease. Commonly reported metrics include glomerular number and injury. However, characterization of renal histology is a time-consuming and user-dependent process. To accelerate and improve the process, we have developed a glomerular localization pipeline for trichrome-stained kidney sections using a machine learning image classification algorithm.Methods We prepared 4-µm slices of kidneys from rats of various genetic backgrounds that were subjected to different experimental protocols and mounted the slices on glass slides. All sections used in this analysis were trichrome stained and imaged in bright field at a minimum resolution of 0.92 µm per pixel. The training and test datasets for the algorithm comprised 74 and 13 whole renal sections, respectively, totaling over 28,000 glomeruli manually localized. Additionally, because this localizer will be ultimately used for automated assessment of glomerular injury, we assessed bias of the localizer for preferentially identifying healthy or damaged glomeruli.Results Localizer performance achieved an average precision and recall of 96.94% and 96.79%, respectively, on whole kidney sections without evidence of bias for or against glomerular injury or the need for manual preprocessing.Conclusions This study presents a novel and robust application of convolutional neural nets for the localization of glomeruli in healthy and damaged trichrome-stained whole-renal section mounts and lays the groundwork for automated glomerular injury scoring.

Combating endometriosis by blocking proteasome and nuclear factor-kappaB pathways.

BACKGROUND: The objective of this study is to investigate the effect of pyrrolidine dithiocarbamate [PDTC; a nuclear factor-kappaB (NF-kappaB) inhibitor] and bortezomib (Velcade; a proteasome inhibitor) on the development of experimental endometriotic implants in rats. METHODS: Endometriosis was surgically induced in 30 rats using the method of Vernon and Wilson. Three weeks later the viability and volume of the implants were recorded and classified. Afterwards, rats were put into three groups with equal numbers. The groups were labelled as the control, the PDTC and the bortezomib groups. Seven days after treatment, a third laparotomy was done and the volume of implants was measured again. The animals were then sacrificed, and the implants were stained with Ki67, proliferating cell nuclear antigen (PCNA), CD34, CD31 and Masson's trichrome histochemical staining. RESULTS: In 80% of the implanted rats, vesicles at the suture region were observed, and the rats graded according to average vesicle diameter (D) as: Grade 1 (no vesicle, 20% of rats), Grade 2 (D < 2 mm, 33.3% of rats), Grade 3 (2 mm 4.5 mm, 26.7% of rats) and Grade 4 (D > 4.5 mm, 20% of rats). After treatment with PDTC or bortezomib, these percentages were decreased for Grades 3 and 4, and increased in Grade 1. The post-treatment implant volumes were decreased in the PDTC and bortezomib groups (P < 0.002 and P < 0.001), and slightly increased in the control group (P = 0.279). In the PDTC and bortezomib groups, CD34, CD31, PCNA and Ki67 expression levels were similar but were significantly reduced compared with the control group. CONCLUSIONS: PDTC and bortezomib may represent a novel therapeutic strategy for treatment of endometriosis.

Comparative effects of cationic triarylmethane, phenoxazine and phenothiazine dyes on horse serum butyrylcholinesterase.

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25( degrees )C in 50mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K(i) values of 4.5+/-0.50 microM, 0.41+/-0.007 microM, 0.44+/-0.086 microM and 0.050+/-0.0074 microM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K' values ( identical with[I](2)(0.5) extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20+/-0.096 microM, 0.0018+/-0.0015 microM, 0.92+/-0.23 microM and 0.23+/-0.08 microM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.

Antirepression as a second mechanism of transcriptional activation by a minor groove binding protein.

Competence for genetic transformation in the bacterium Bacillus subtilis is a bistable differentiation process governed by the minor groove DNA binding protein ComK. No detectable comK transcription occurs in the absence of an intact comK gene, indicating that ComK has auto-activating properties. ComK auto-stimulation, which is dependent on ComK binding to the comK promoter, is a critical step in competence development, ensuring quick and high-level expression of the late-competence genes. Auto-stimulation is also essential for the bistable expression pattern of competence. Here, we demonstrate that ComK acts as an activator at its own promoter by antagonizing the action of two repressors, Rok and CodY. Importantly, antirepression occurs without preventing binding of the repressing proteins, suggesting that ComK and the repressors might bind at distinct surfaces of the DNA helix. DegU, a DNA binding protein known to increase the affinity of ComK for its own promoter, potentiates the antirepression activity of ComK. We postulate that antirepression is primarily achieved through modulation of DNA topology. Although to our knowledge ComK is the only DNA binding protein shown to act in this novel fashion, other minor groove binding proteins may act similarly.

The amino-terminus of mouse DNA methyltransferase 1 forms an independent domain and binds to DNA with the sequence involving PCNA binding motif.

DNA methylation patterns in genome are maintained during replication by a DNA methyltransferase Dnmt1. Mouse Dnmt1 is a 180 kDa protein comprising the N-terminal regulatory domain, which covers 2/3 of the molecule, and the rest C-terminal catalytic domain. In the present study, we demonstrated that the limited digestion of full-length Dnmt1 with different proteases produced a common N-terminal fragment, which migrated along with Dnmt1 (1-248) in SDS-polyacrylamide gel electrophoresis. Digestion of the N-terminal domains larger than Dnmt1 (1-248) with chymotrypsin again produced the fragment identical to the size of Dnmt1 (1-248). These results indicate that the N-terminal domain of 1-248 forms an independent domain. This N-terminal domain showed DNA binding activity, and the responsible sequence was narrowed to the 79 amino acid residues involving the proliferating cell nuclear antigen (PCNA) binding motif. The DNA binding activity did not distinguish between DNA methylated and non-methylated states, but preferred to bind to the minor groove of AT-rich sequence. The DNA binding activity of the N-terminal domain competed with the PCNA binding. We propose that DNA binding activity of the N-terminal domain contributes to the localization of Dnmt1 to AT-rich sequence such as Line 1, satellite, and the promoter of tissue-specific silent genes.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

BACKGROUND: Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. RESULTS: The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. CONCLUSION: RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications.

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.

DNA binding and gene activation properties of the Nmp4 nuclear matrix transcription factors.

Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To date, these are the only Cys(2)His(2) zinc finger proteins known to bind within the minor groove of homopolymeric (dA.dT) DNA. Nmp4 isoforms contain from 5 to 8 Cys(2)His(2) zinc fingers, an SH3-binding domain that overlaps with a putative AT-hook and a polyglutamine-alanine repeat (poly(QA)). To determine the mechanistic significance of Cys(2)His(2) zinc finger association with this unusual consensus DNA binding element, we identified the Nmp4 DNA-binding and transcriptional activation domains. Zinc fingers 2, 3, and 6 mediated association with the homopolymeric (dA.dT) COL1A1/MMP DNA consensus element. The N terminus of the Nmp4 protein exhibited a strong trans-activation capacity when fused to the GAL4 DNA-binding domain, but this activity was masked within the context of the full-length Nmp4-GAL4 DNA-binding domain chimera. However, upon binding to the COL1A1/MMP homopolymeric (dA.dT) element, the native Nmp4 protein up-regulated transcription, and the poly(QA) domain acquired a significant role in trans-activation. We propose that allosteric effects induced upon zinc finger association with the homopolymeric (dA.dT) minor groove confer context-specific functionality to this unusual family of Cys(2)His(2) transcription factors.

Purification and characterization of a putative vitellogenin from the ovary of amphioxus (Branchiostoma belcheri tsingtaunese).

An oocyte-yolk protein was purified by double-step chromatography from amphioxus ovaries. The purified protein appeared to exist as a homodimer of approximately 320 kDa in native polyacrylamide gel electrophoresis (PAGE), and was reduced to a single monomer of approximately 160 kDa in sodium dodecyl sulfate-PAGE (SDS-PAGE). The protein was characterized as a phospholipoglycoprotein by native PAGE and staining of gels for phosphorus with methyl green, for lipids with oil red O and Sudan black B, and for carbohydrates using periodic acid/Schiff reagent. In addition, the amino acid composition of the oocyte-yolk protein was generally similar to that of vitellogenins (Vgs) isolated from different phyla of animals including both vertebrates and invertebrates. The purified phospholipoglycoprotein is thus considered as putative amphioxus Vg.

Intermediate filaments reconstituted from vimentin, desmin, and glial fibrillary acidic protein selectively bind repetitive and mobile DNA sequences from a mixture of mouse genomic DNA fragments.

Employing the whole-genome PCR technique, intermediate filaments (IFs) reconstituted from vimentin, desmin, and glial fibrillary acidic protein were shown to select repetitive and mobile DNA sequence elements from a mixture of mouse genomic DNA fragments. The bound fragments included major and minor satellite DNA, telomere DNA, minisatellites, microsatellites, short and long interspersed nucleotide elements (SINEs and LINEs), A-type particle elements, members of the mammalian retrotransposon-like (MaLR) family, and a series of repeats not assignable to major repetitive DNA families. The latter sequences were either similar to flanking regions of genes; possessed recombinogenic elements such as polypurine/polypyrimidine stretches, GT-rich arrays, or GGNNGG signals; or were characterized by the distribution of oligopurine and pyrimidine motifs whose sequential and vertical alignment resulted in patterns indicative of high recombination potentials of the respective sequences. The different IF species exhibited distinct quantitative differences in DNA selectivities. Complexes consisting of vimentin IFs and DNA fragments containing LINE, (GT)(n) microsatellite, and major satellite DNA sequences were saturable and dynamic and were formed with high efficiency only when the DNAs were partially denatured. The major-groove binder methyl green exerted a stronger inhibitory effect on the binding reaction than did the minor-groove binder distamycin A; the effects of the two compounds were additive. In addition, DNA footprinting studies revealed significant configurational changes in the DNA fragments on interaction with vimentin IFs. In the case of major satellite DNA, vimentin IFs provided protection of the T-rich strand from cleavage by DNase I, whereas the A-rich strand was totally degraded. Taken together, these observations suggest that IF protein(s) bind to double-stranded DNAs at existing single-stranded sites and, taking advantage of their helix-destabilizing potential, further unwind them via a cooperative effort of their N-terminal DNA-binding regions. A comparison of the present results with literature data, as well as a search in the NCBI database, showed that IF proteins are related to nuclear matrix attachment region (MAR)-binding proteins, and the DNA sequences they interact with are very similar or even identical to those involved in a plethora of DNA recombination and related repair events. On the basis of these comparisons, IF proteins are proposed to contribute in a global fashion, not only to genetic diversity, but also to genomic integrity, in addition to their role in gene expression.

Distamycin-induced inhibition of formation of a nucleoprotein complex between the terminase small subunit G1P and the non-encapsidated end (pacL site) of Bacillus subtilis bacteriophage SPP1.

The small subunit of the Bacillus subtilis bacteriophage SPP1 terminase (G1P) forms a sequence-specific nucleoprotein complex with the SPP1 non-encapsidated end (pacL site) during initiation of DNA encapsidation. Gel mobility shift assay was used to study the G1P-pacL interaction. Distamycin, a minor groove binder that induces local distortion of the DNA, inhibits G1P-pacL complex formation. The competition of G1P with distamycin for DNA binding at the pacL site is independent of the order of addition of the reactants. Other minor groove binders, such as spermine or Hoechst 33258, which do not distort DNA, failed to compete with G1P for pacL DNA binding. Cationic metals, which generate a repertoire of DNA structures different from that caused by the minor groove binders, can partially reverse the distamycin-induced inhibition of G1P binding to pacL DNA. The major groove binder methyl green, which does not distort sequence-directed bending of pacL DNA, competes with G1P for binding at the pacL site. Our data suggest that the natural sequence-directed bend that exists within the pacL site is the architectural element that facilitates assembly of a nucleoprotein complex and hence initiation of DNA encapsidation by bacteriophage SPP1.

On the structure of active chromatin. A flow linear dichroism study of chromatin fractionated by nuclease digestion.

Nuclei from Ehrlich ascites cells were treated with micrococcal nuclease or DNase I and extracted with 1 mM EDTA. The chromatin fraction released by this procedure showed positive flow linear dichroism (LD) at low salt (2 mM NaCl) while the non-released fraction had negative LD. Furthermore, the chromatin structure responsible for the positive LD was found to be labile: The LD was reduced by heat (37 degrees C) or RNase treatment and inverted to a negative LD by electric fields (10 kV/cm) and by the presence of DNA binding dyes.

Detection of bacterial phosphatase activity by means of an original and simple test.

A new test for the detection of bacterial phosphatase activity has been devised. The test is performed using agar media containing both methyl green (MG) and phenolphthalein diphosphate (PDP); in these media phosphatase-producing strains grow deep-green-stained colonies whereas non-producing strains do not. A total of 739 different strains were tested, including 593 staphylococci, 95 micrococci, 11 streptococci, 10 corynebacteria, 14 enterobacteria, and 16 candidae. All strains found phosphatase-positive according to the conventional phosphatase test displayed deep-green-stained colonies on MG-PDP media, whereas all phosphatase-negative strains showed unstained colonies on the same media. The main advantages of the present phosphatase test as compared with other conventional ones are that it is more simple to perform, it can reveal the phosphatase activity of colonies grown in deep agar, and can be incorporated into commercial multitest kits.

The action of some triphenylmethane dyes on yeast and erythrocyte membranes.

The action of a series of triphenylmethane dyes on the membranes of two different cell types, yeast cells and erythrocytes, have been studied. The action of the dyes on yeast cells resembles the action of other positively charged bactericides. The sequence of the activity in the dye series is completely different for yeast cells and erythrocytes. Pararosaniline and crystal violet are much more active than malachite green and brilliant green on yeast cells, whereas the reverse sequence of activities applies in erythrocytes. The carbinol form of the dyes plays an important role as regards dye interaction with erythrocytes. Transition of the dye into the carbinol form is in water extremely slow, but is greatly accelerated in the presence of an organic phase, at least for malachite green and brilliant green, but not for crystal violet and pararosaniline. This explains the different action of the two categories of dyes on erythrocytes. Experiments with pure carbinol also support the hypothesis.