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PubMed2026

Molecular and immune profiling of HER2-low, HER2 ultra-low, and HER2-null male breast cancer.

BACKGROUND: HER2 expression is described along a biological continuum from null to positive and serves as a critical biomarker for therapeutic guidance in breast cancer (BC). While HER2-low and ultra-low categories have emerged as actionable targets for antibody-drug conjugates (ADCs) in female BC, their molecular and immune characteristics remain largely unexplored in male breast cancer. METHODS: We profiled 214 male breast tumors using next-generation sequencing and whole-transcriptome sequencing to assess mutational, transcriptomic, and immune landscapes. Tumor mutational burden (TMB) was defined as high if > 10 mutations/Mb. Immune cell fractions were inferred using Quantiseq deconvolution. RESULTS: Among 214 samples, 66 (30.8%) were HER2-null, 53 (24.8%) HER2 ultra-low, 80 (37.4%) HER2-low, and 15 (7.0%) HER2-positive. HER2 ultra-low tumors exhibited a higher prevalence of PIK3CA mutations (39.2% vs 22.6%, p ≤ 0.05) compared to HER2-null. No significant differences were observed in TMB-high frequency or PD-L1 expression across subgroups. Immune composition differed primarily between HER2-null and HER2-expressing subgroups: HER2-ultra-low tumors showed higher B-cell infiltration, whereas HER2-null tumors were enriched in neutrophils. Transcriptomic analysis revealed upregulation of selected stemness-associated genes (NANOG, KLF4, POU5F1) and CEACAM1 in HER2-null tumors, while HER2-low and HER2-ultra-low tumors were largely similar across most molecular and immune readouts in this cohort. CONCLUSIONS: HER2-null male breast cancer appears to represent the most biologically divergent subgroup within the HER2-negative spectrum, whereas HER2-low and HER2-ultra-low tumors were largely similar in this cohort. These findings support further investigation of HER2-null disease as a distinct biological state and provide hypothesis-generating data for biomarker development in this rare population.

Male↗

PubMed2006

Polar positional information in Escherichia coli spherical cells.

Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.

Bacterial Proteins↗

PubMed2005

Visceral fat and beta cell function in non-diabetic humans.

AIMS/HYPOTHESIS: Preferential visceral adipose tissue (VAT) accumulation has been clearly associated with insulin resistance. In contrast, the impact of visceral obesity on beta cell function is controversial. METHODS: In 62 non-diabetic women and men (age 24-69 years, BMI 21-39 kg/m2), we measured VAT and subcutaneous adipose tissue (SAT) fat mass by magnetic resonance imaging. We also measured insulin secretion and beta cell function by C-peptide deconvolution and physiological modelling of data from a frequently sampled, 75-g, 3-h OGTT, respectively. RESULTS: VAT (range 0.1-3.1 kg) was strongly related to sex, age and BMI; SAT was related to sex and BMI. Controlling for sex, age, BMI and SAT by multivariate analysis, excess VAT was associated with a clinical phenotype comprising higher plasma glucose levels, BP, heart rate and serum transaminases. The corresponding metabolic phenotype consisted of insulin resistance (partial r=-0.38) and hyperinsulinaemia (partial r=0.29). The latter, however, was appropriate for the degree of insulin resistance regardless of obesity and abdominal fat distribution. Moreover, none of the model-derived parameters describing beta cell function (glucose sensitivity, rate sensitivity and potentiation) was independently associated with excess VAT. CONCLUSIONS/INTERPRETATION: In non-diabetic Caucasian adults of either sex, preferential visceral fat deposition in itself is part of an insulin-resistant phenotype. The insulin secretory response to a physiological challenge is increased to fully compensate for the insulin resistance, but the dynamics of beta cell function (glucose sensitivity, rate sensitivity and potentiation) are largely preserved.

Adipose Tissue↗

PubMed2002

Wound closure in sheared endothelial cells is enhanced by modulation of vascular endothelial-cadherin expression and localization.

We previously demonstrated that laminar shear stress enhances human coronary artery endothelial cell (HCAEC) wound closure via the mechanisms of cell spreading and migration. Because cell-cell junctional proteins such as vascular endothelial cell cadherin (VE-cadherin) are critical to cell-cell adhesion and motility, we tested the hypothesis that modulation of VE-cadherin expression under shear stress may be linked to this enhancement in wound closure. HCAEC monolayers were preconditioned to attain cellular alignment by shearing at 12 dynes/cm(2) for 18 hr in a parallel-plate flow chamber. Subsequently, they were divided into the following three groups: (i) control; (ii) treated with anti-cadherin-5 antibody; or (iii) treated with the calcium chelating agent EGTA. Next, the monolayers were wounded with a metal spatula and resheared at 20 dynes/cm(2) or left static. Time-lapse imaging was performed during the first 3 hr after imposition of these conditions. Immunocytochemistry or Western blot analyses for VE-cadherin expression were performed on all wounded monolayers. Deconvolution microscopy, three-dimensional cell-cell junctional reconstruction images, and histogram analyses of interendothelial junction signal intensities were performed on cells at the wound edge of a monolayer. Under shear, HCAEC demonstrated increased VE-cadherin immunofluorescence and protein expression despite an enhancement in wound closure compared with static conditions. In separate experiments, application with anti-cadherin-5 antibody or treatment with EGTA attenuated VE-cadherin expression and further enhanced wound closure compared with control shear and all static conditions. In addition, the pattern of VE-cadherin localization with these treatments became more intracellular and nuclear in appearance. These findings of changes in this junctional adhesion protein expression and localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.

Blotting, Western↗

PubMed2002

Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A.

Chromogranin A (CgA) is the index member of the chromogranin/secretogranin (or 'granin') family of regulated secretory proteins that are ubiquitously distributed in amine- and peptide-containing secretory granules of endocrine, neuroendocrine and neuronal cells. Because of their abundance and such widespread occurrence, granins have often been used as prototype proteins to elucidate mechanisms of protein targeting into dense-core secretory granules. In this study, we used a series of full-length, point mutant or truncated CgA-green fluorescent protein (GFP) chimeras to explore routing of CgA in neuroendocrine PC12 cells. Using sucrose gradient fractionation and 3D deconvolution microscopy to determine the subcellular localization of the GFP chimeras, as well as secretagogue-stimulated release, the present study establishes that a CgA-GFP fusion protein expressed in neuroendocrine PC12 cells is trafficked to the dense core secretory granule and thereby sorted to the regulated pathway for exocytosis. We show that information necessary for such trafficking is contained within the N-terminal but not the C-terminal region of CgA. We find that CgA's conserved N-terminal hydrophobic Cys(17)-Cys(38) loop structure may not be sufficient for sorting of CgA into dense-core secretory granules, nor is its stabilization by a disulfide bond necessary for such sorting. Moreover, our data reveal for the first time that the CgA(77-115) domain of the mature protein may be necessary (though perhaps not sufficient) for trafficking CgA into the regulated pathway of secretion.

Amino Acid Sequence↗

PubMed2004

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes.

Nicotinergic agents can act as both chemokines and chemoattractants for cell migration. Epidermal keratinocytes both synthesize acetylcholine and use it as a paracrine and autocrine regulator of cell motility. To gain a mechanistic insight into nicotinergic control of keratinocyte motility, we determined types of nicotinic acetylcholine receptors and signaling pathways regulating keratinocyte chemokinesis and chemotaxis, using respective modifications of the agarose gel keratinocyte outgrowth assay. Random migration of keratinocytes was significantly (P<0.05) inhibited by hemicholinum-3, a metabolic inhibitor of acetylcholine synthesis, as well as by the alpha-conotoxins MII and AuIB, preferentially blocking alpha3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the alpha3beta2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of alpha3beta2 included activation of the protein-kinase-C isoform delta and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of alpha7 nicotinic acetylcholine receptor. The alpha7-preferring antagonist alpha-bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the alpha7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-alpha7 antisense oligonucleotides and alpha7-knockout mice. The signaling pathway mediating alpha7-dependent keratinocyte chemotaxis included intracellular calcium, activation of calcium/calmodulin-dependent protein-kinase II, conventional isoforms of protein-kinase C, phosphatidylinositol-3-kinase and engagement of Rac/Cdc42. Redistribution of alpha7 immunoreactivity to the leading edge of keratinocytes upon exposure to a chemoattractant preceded crescent shape formation and directional migration. Application of high-resolution deconvolution microscopy demonstrated that, on the cell membrane of keratinocytes, the nicotinic acetylcholine receptor subunits localize with the integrin beta1. The obtained results demonstrate for the first time that alpha3 and alpha7 nicotinic acetylcholine receptors regulate keratinocyte chemokinesis and chemotaxis, respectively, and identify signaling pathways mediating these functions, which has clinical implications for wound healing and control of cancer metastases.

Acetylcholine↗

PubMed2005

Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells.

The mechanism of target cell lysis in cytotoxic lymphocyte-mediated death is not well understood, and the role of granzymes in this process is unclear. Chemical functional probes were thus prepared for the major granzymes A and B to deconvolute their role in natural killer cell-mediated lysis of target cells. These biotinylated and substrate specificity-based diphenyl phosphonates allowed facile evaluation of selectivity through activity-based profiling in cell lysates and intact cells. Both inhibitors were found to be extremely selective in vitro and in cells. Use of these inhibitors in cell-based assays revealed granzyme A to be a minor effector and granzyme B to be a major effector of target cell lysis by natural killer cells. These studies indicate that the proapoptotic granzyme B functions also as a pronecrotic effector of target cell death.

Apoptosis↗

PubMed2005

Intracellular localization and trafficking of fluorescein-labeled cisplatin in human ovarian carcinoma cells.

PURPOSE: We sought to identify the subcellular compartments in which cisplatin [cis-diamminedichloroplatinum (DDP)] accumulates in human ovarian carcinoma cells and define its export pathways. EXPERIMENTAL DESIGN: Deconvoluting digital microscopy was used to identify the subcellular location of fluorescein-labeled DDP (F-DDP) in 2008 ovarian carcinoma cells stained with organelle-specific markers. Drugs that block vesicle movement were used to map the traffic pattern. RESULTS: F-DDP accumulated in vesicles and were not detectable in the cytoplasm. F-DDP accumulated in the Golgi, in vesicles belonging to the secretory export pathway, and in lysosomes but not in early endosomes. F-DDP extensively colocalized with vesicles expressing the copper efflux protein, ATP7A, whose expression modulates the cellular pharmacology of DDP. Inhibition of vesicle trafficking with brefeldin A, wortmannin, or H89 increased the F-DDP content of vesicles associated with the pre-Golgi compartments and blocked the loading of F-DDP into vesicles of the secretory pathway. The importance of the secretory pathway was confirmed by showing that wortmannin and H89 increased whole cell accumulation of native DDP. CONCLUSIONS: F-DDP is extensively sequestered into vesicular structures of the lysosomal, Golgi, and secretory compartments. Much of the distribution to other compartments occurs via vesicle trafficking. F-DDP detection in the vesicles of the secretory pathway is consistent with a major role for this pathway in the efflux of F-DDP and DDP from the cell.

Adenosine Triphosphatases↗

PubMed2003

Sequestration of connexin43 in the early endosomes: an early event of Leydig cell tumor progression.

Connexins form gap junction channels that allow intercellular communication between neighboring cells. Compelling evidence has revealed that Cx are tumor-suppressor genes and reduced Cx expression has been related with uncontrolled cell growth in tumors and transformed cells. In the present study, we addressed Cx transcriptional and posttranscriptional regulations during the earlier stage of testicular tumors confined to Leydig cells in a transgenic mice model. In situ hybridization indicated that connexin43 (Cx43) mRNA was highly expressed either at early tumorogenesis (3 m) characterized by intense proliferation of Leydig cells, or at advanced tumorogenesis (6-7 m) when tumor cells completely invaded the testis. In contrast, Cx43 protein analyzed by Western blotting or classic immunohistochemical analyses was present at the beginning of tumor progression, but was dramatically reduced as tumor advanced. Application of high-resolution deconvolution microscopy to testis sections demonstrates that cells that proliferate exhibited an aberrant cytoplasmic Cx43 localization, in contrast to the expected plasma membrane Cx43 localization in normal Leydig cells. Dual immunofluorescence labeling with specific markers of cellular compartments shows that cytoplasmic Cx43 signal was mainly sequestered within early endosomes. Altogether, this study provides the first evidence that impaired Cx43 trafficking in endosomes is an early event associated with uncontrolled cell proliferation that could serve as a neoplastic marker.

Animals↗

PubMed1983

Long-term storage of samples for flow cytometric DNA analysis.

A simple procedure for long-term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine-needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were stored for up to one year by freezing at -80 degrees C. Statistical analysis of the results showed no change in the fractions of cells in the cell cycle phases as determined by deconvolution of the DNA-histograms. It was found that in addition to the intrinsic sample variation from the parameter estimation by deconvolution, there was significant intraday and interday variation. Hence the most accurate results are obtained if different aliquots of a sample are measured on different days rather than on the same day. Use of the storage method thus has the potential of increasing the accuracy of the analysis. The storage method makes sample collection independent of immediate subsequent analysis. This has enabled us to perform large internally controlled experiments, involving more samples than can be analyzed in one day, to examine tumor samples from different hospitals and to utilize fully the capacity of our flow cytometer. The method was a prerequisite for developing an accurate standardization procedure for DNA content determination.

Analysis of Variance↗

PubMed2004

Approaches to determine the enthalpy of crystallisation, and amorphous content, of lactose from isothermal calorimetric data.

Amorphous lactose will crystallise rapidly if its glass transition temperature is reduced below its storage temperature. This is readily achieved by storing samples at ambient temperature and a relative humidity (RH) of greater than 50%. If the sample is monitored in an isothermal microcalorimeter as it crystallises, the heat changes associated with the event can be measured; indeed this is one of the methods used to quantify the amorphous content of powders and formulations. However, variations in the calculation methods used to determine these heat changes have led to discrepancies in the values reported in the literature and frequently make comparison of data from different sources difficult. Data analysis and peak integration software allow the selection and integration of specific areas of complex traces with great reproducibility; this has led to the observation that previously ignored artefacts are in fact of sufficient magnitude to affect calculated enthalpies. In this work a number of integration methodologies have been applied to the analysis of amorphous spray-dried lactose, crystallised under 53 or 75% RH at 25 degrees C. The data allowed the selection of a standard methodology from which reproducible heat changes could be determined. The method was subsequently applied to the analysis of partially amorphous lactose samples (containing 1-100% (w/w) amorphous content) allowing the quantification limit of the technique to be established. It was found that the best approach for obtaining reproducible results was (i) to crystallise under an RH of 53%, because this slowed the crystallisation response allowing better experimental measurement and (ii) to integrate all the events occurring in the ampoule, rather than trying to select only that region corresponding to crystallisation, since it became clear that the processes occurring in the cell overlapped and could not be deconvoluted. The technique was able to detect amorphous contents as low as 1% (w/w), using this integration strategy, although it was observed that the calibration plot constructed showed a negative deviation from linearity. It is suggested that such non-ideal behaviour results from the formation of varying ratios of alpha-lactose monohydrate, anhydrous alpha-lactose and anhydrous beta-lactose.

Calorimetry↗

PubMed2026

A refined MASH-HCC model identifies macrophage Gadd45b as a key orchestrator of inflammation-driven neoplastic progression.

Metabolic dysfunction-associated steatohepatitis (MASH) is emerging as a leading driver of hepatocellular carcinoma (HCC), yet the molecular mechanisms linking metabolic stress, chronic inflammation and tumorigenesis remain poorly understood. Here we established a metabolically relevant, time-efficient MASH-to-HCC model in C57BL/6N mice by combining a MASH diet with controlled CCl4 administration, enabling stepwise recapitulation of MASH-associated neoplastic progression. Using this model, we identified growth arrest and DNA damage 45b (Gadd45b) as a novel MASH-derived protumorigenic regulator selectively activated under metabolic stress. Integrated analyses of human bulk and single-cell transcriptomic datasets and mouse transcriptomic deconvolution revealed concordant macrophage remodeling and GADD45B/Gadd45b expression dynamics during MASH-to-HCC progression. Mechanistically, fatty acids and TNFα preferentially induced Gadd45b in macrophages, where it amplified TNFα-NF-κB signaling. Macrophage-derived inflammatory signals subsequently induced Gadd45b and NF-κB activation in hepatocytes, establishing a feed-forward inflammatory loop that promoted fibrogenic and partial EMT-like programs and tumor spheroid formation. Importantly, temporal profiling during spheroid formation and progression revealed transient induction of Gadd45b during early spheroid establishment, but not during later progression, indicating that Gadd45b-mediated inflammatory signaling primarily promotes tumor initiation rather than subsequent growth. Consistent with human data, Gadd45b expression increased with disease severity and positively correlated with inflammatory factors in the MASH-HCC model, whereas pharmacological inhibition attenuated the Gadd45b-inflammation signaling axis. Collectively, our findings establish macrophage Gadd45b as a key orchestrator linking metabolic stress, chronic inflammation, and neoplastic transformation during MASH-to-HCC progression. Our refined MASH-HCC model provides a robust platform for mechanistic studies and preclinical evaluation of inflammation-targeted therapies.

Journal Article↗

PubMed2026

Integrative multi-omics profiling of insomnia-related molecular features reveals microbiome, immune, and therapy-relevant heterogeneity in colorectal cancer.

Emerging evidence implicates insomnia as a potential risk factor in carcinogenesis, potentially involving systemic inflammation, circadian disruption, and microbiome alterations. However, the molecular associations linking insomnia-related features to colorectal cancer (CRC), particularly with respect to tumor biology, immune microenvironmental states, and therapy-relevant phenotypes, remain largely unexplored. Multi-omics integration of genomic, transcriptomic, and microbiome data from 3,026 CRC patients across seven independent cohorts, including a large, well-annotated Clinical Omics study of Colorectal Cancer in China (COCC) cohort, enabled insomnia-based molecular classification through unsupervised non-negative matrix factorization (NMF) clustering. The insomnia subtype (IS) was biologically characterized via pathway enrichment, immune deconvolution, microbial profiling, and single-cell transcriptomics. Furthermore, an insomnia score (ISscore) was developed and validated in multiple cohorts for risk stratification and assessment of treatment-response-related indicators in CRC. Unsupervised clustering revealed two distinct molecular subtypes (IS1/IS2), with IS2 demonstrating significantly poorer survival. IS2 exhibited marked activation of EMT/angiogenesis pathways versus cell cycle activation in IS1. The IS2 microenvironment showed increased immunosuppression-related infiltration and exhausted T cell signatures, together with intratumoral microbiome variation characterized by depletion of Ruminococcaceae UCG-002 and enrichment of Hungatella/Selenomonas. The ISscore system stratified survival risk and was associated with computational indicators of immunotherapy response. Single-cell analysis nominated PPIA-BSG as a potential cell-cell communication signal involving high-ISscore tumor cells, CXCL12+ endothelial cells, and CLEC9A+ dendritic cell subsets. This multi-omics characterization of insomnia-CRC interplay suggests that insomnia-related molecular features are associated with an immunologically distinct and microbiome-altered tumor ecosystem. The ISscore provides a reproducible framework for capturing insomnia-related molecular heterogeneity, supporting risk stratification and future evaluation of therapy-relevant phenotypes.IMPORTANCEChronic insomnia affects millions, but it is not typically considered a cancer risk factor. Our study, analyzing vast biological data from over 3,000 colorectal cancer patients, uncovers a potential link between a person's predisposition to insomnia and their risk of developing this disease. This suggests that the biological pathways related to sleep may play a role in cancer development. Understanding this connection opens up new avenues for identifying individuals at higher risk and developing novel prevention strategies for colorectal cancer.

colorectal cancer↗

PubMed2026

Malignant epithelial states drive immune dysfunction in ampulla of Vater carcinoma.

BACKGROUND: Ampulla of Vater (AoV) carcinoma is a rare malignancy arising at the junction of intestinal and pancreatobiliary epithelium. Its heterogeneous clinical behavior and histological diversity have hindered therapeutic advances, and the cellular basis of this heterogeneity remains unclear. We aimed to construct a single-cell transcriptomic atlas of AoV carcinoma, with a focus on identifying epithelial subtypes and their interactions with the tumor microenvironment (TME). METHODS: We performed single-cell RNA sequencing on eight primary AoV tumors and four matched normal tissues. Comprehensive clustering and transcriptomic analyses identified cell-type composition, epithelial heterogeneity, and tumor-immune interactions. Findings were validated using deconvolution of bulk RNA-seq data from 62 AoV carcinoma patients. Results Malignant epithelial cells were categorized into four distinct subtypes: Int-Wnt, PB-KRAS, Int-Hypoxia, and Cycling stage. PB-KRAS cells exhibited stem-like transcriptional programs and high genomic instability. Deconvolution analysis of bulk RNA-seq data from the independent AoV cohort revealed that enrichment of the PB-KRAS subtype correlated with tumor recurrence and poor survival. Our immune profiling analysis discovered a significant association between PB-KRAS subtype and GZMK+ CD8+ T cells, which are in a pre-dysfunctional state, alongside SPP1+ macrophages exhibiting immunosuppressive traits. Spatial transcriptome data further supports the immunosuppressive natures of TME around PB-KRAS subtype malignant epithelial cells in AoV carcinoma. CONCLUSIONS: Our study presents a single-cell atlas of AoV carcinoma, highlighting the molecular diversity of malignant epithelium and its association with the immune microenvironment. The PB-KRAS subtype emerges as a stem-like, immunosuppressive tumor state associated with poor prognosis, providing insights for future therapeutic targeting.

Ampulla of Vater carcinoma↗

PubMed2004

S-cone connections of the diffuse bipolar cell type DB6 in macaque monkey retina.

Previous studies of primate retinae have shown that diffuse bipolar (DB) cells contact all the cones in their dendritic field, suggesting there is no spectral selectivity in the functional input to DB cells. However, since short-wavelength sensitive (S) cones make up less than 10% of the total cone population, specialized connectivity with S-cones is difficult to detect. In the present study, the S-cone connectivity of a subtype of DB cells, the DB6 cell, was studied in macaque monkey retina. Pieces of macaque retina were processed with antibodies to CD15 to stain DB6 cells and antibodies to the S-cone opsin to identify S-cones. Immunoreactivity was visualized using immunoperoxidase or immunofluorescence. Some preparations were additionally processed with peanut agglutinin coupled to fluorescein to reveal medium- and long-wavelength sensitive (M/L) cones. The preparations were analyzed using conventional and deconvolution light microscopy. The majority of DB6 cells had one or two S-cones in their dendritic field and the majority of S-cones were located in the dendritic field of DB6 cells. On average, 80% of the S-cones and 81% of the M/L cones contacted DB6 cells. The average number of dendritic terminals at cone pedicles did not differ between the cone types. However, the total number of DB6 dendritic terminals receiving input from M/L-cone pedicles was about eight times higher than the total number of dendritic terminals at S-cone pedicles. In conclusion, DB6 cells make indiscriminate contact with all cone types, but receive their major input from M/L-cones and thus carry a "Yellow-ON" spectral signal.

Animals↗

PubMed2006

Resampling and deconvolution of linear time-of-flight records for enhanced protein profiling.

We have developed a peak deconvolution strategy that is applicable to the full mass range of a time-of-flight (TOF) spectrum. This strategy involves resampling a spectrum to create a time series that has equal peak widths (in time) across the entire spectrum, and then using the deconvolution filters we have previously described. We use this technique to deconvolve the protein mass spectra for blood serum and cell lysates acquired on three separate TOF instruments. Following deconvolution, we resolve spectral structures consistent with expected events such as multiply charged ions, matrix adducts and post-translational protein modifications. The deconvolution procedure produces a 40% improvement in the resolution and enhanced experimental sensitivity over the full length of the linear TOF record, up to m/z 150 000. This approach is particularly appropriate for automated data analysis and peak detection in dense TOF spectra.

Algorithms↗

PubMed2005

Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.

Ischemia causes kidney tubular cell damage and abnormal renal function. The kidney is capable of morphological restoration of tubules and recovery of function. Recently, it has been suggested that cells repopulating the ischemically injured tubule derive from bone marrow stem cells. We studied kidney repair in chimeric mice expressing GFP or bacterial beta-gal or harboring the male Y chromosome exclusively in bone marrow-derived cells. In GFP chimeras, some interstitial cells but not tubular cells expressed GFP after ischemic injury. More than 99% of those GFP interstitial cells were leukocytes. In female mice with male bone marrow, occasional tubular cells (0.06%) appeared to be positive for the Y chromosome, but deconvolution microscopy revealed these to be artifactual. In beta-gal chimeras, some tubular cells also appeared to express beta-gal as assessed by X-gal staining, but following suppression of endogenous (mammalian) beta-gal, no tubular cells could be found that stained with X-gal after ischemic injury. Whereas there was an absence of bone marrow-derived tubular cells, many tubular cells expressed proliferating cell nuclear antigen, which is reflective of a high proliferative rate of endogenous surviving tubular cells. Upon i.v. injection of bone marrow mesenchymal stromal cells, postischemic functional renal impairment was reduced, but there was no evidence of differentiation of these cells into tubular cells of the kidney. Thus, our data indicate that bone marrow-derived cells do not make a significant contribution to the restoration of epithelial integrity after an ischemic insult. It is likely that intrinsic tubular cell proliferation accounts for functionally significant replenishment of the tubular epithelium after ischemia.

Animals↗

PubMed2005

Deconvolution microscopy.

Since its introduction in 1983, deconvolution microscopy has become a key image-processing tool for visualizing the cellular structures of fixed and living specimens in three dimensions and at subresolution scale. The last 20 years have seen the development of many different applications based on deconvolution microscopy, including a wide variety of optical setup and deconvolution algorithms. This chapter aims to summarize and to describe in detail the major features of this technology, from theoretical aspects to practical solutions. It will begin by explaining the principle of image formation in three-dimensional optical sectioning microscopy. As deconvolution microscopy provides, in essence, a means of overcoming the limits of optical microscopy, the second part of this chapter is dedicated to the theoretical and experimental description of image generation through a microscope. Methods will be detailed for the determination of point spread function, as a crucial step for the characterization of any optical system and a key preliminary step for image deconvolution. The challenges faced and the various possibilities for determining this function precisely will be discussed. All possible sources of aberrations and image degradation processes will be discussed. In the third part of this chapter, we will introduce the acquisition setup and requirements for compliance between acquisition and deconvolution processes. Typical setups for fixed and living cell observation will be detailed, with key features for optimizing speed and reducing artifacts. In the fourth and last part of this chapter, we will describe, in theoretical terms, the various restoration algorithms commonly used in the field of optical microscopy and will provide results obtained with some of the commercially available packages. We shall conclude by considering the prospects for future solutions (currently under development) aiming to handle more easily the huge amounts of data generated by rapid multi-dimensional living cell microscopy. Designed for use by standard cell biologists and hardware and software engineers and developers, this chapter has been written to provide a clear explanation of the wide-reaching and powerful domain of deconvolution microscopy.

Algorithms↗