Do senescence markers correlate in vitro and in situ within individual human donors?

Little is known on how well senescence markers in vitro and in situ correlate within individual donors. We studied correlations between the same and different in vitro markers. Furthermore, we tested correlations between in vitro markers with in situ p16INK4a positivity.From 100 donors (20-91 years), cultured dermal fibroblasts were assessed for reactive oxygen species (ROS), telomere-associated foci (TAF), p16INK4a and senescence-associated ß-gal (SAß-gal), with/ without 0.6 µM rotenone for 3 days (short-term). In fibroblasts from 40 donors, telomere shortening, ROS and SAß-gal were additionally assessed, with/ without 20 nM rotenone for 7 weeks (long-term). In skin from 52 donors, the number of p16INK4a positive dermal cells was assessed in situ.More than half of the correlations of the same senescence markers in vitro between duplicate experiments and between short-term versus long-term experiments were significant. Half of the different senescence marker correlations were significant within the short-term and within the long-term experiments. The different senescence markers in vitro were not significantly correlated intra-individually with in situ p16INK4a positivity.In conclusion, the same and different senescence markers are frequently correlated significantly within and between in vitro experiments, but in vitro senescence markers are not correlated with p16INK4a positivity in situ.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

Highly Sensitive Laser Scanning of Photon-Upconverting Nanoparticles on a Macroscopic Scale.

An upconversion laser scanner has been optimized to exploit the advantages of photon-upconverting nanoparticles (UCNPs) for background-free imaging on a macroscopic scale. A collimated 980 nm laser beam afforded high local excitation densities to account for the nonlinear luminescence response of UCNPs. As few as 2000 nanoparticles were detectable, and the linear dynamic range covered more than 5 orders of magnitude, which is essentially impossible by using conventional fluorescent dyes. UCNPs covered by a dye-doped silica shell were separated by agarose gel electrophoresis and scanned by a conventional fluorescence scanner as well as the upconversion scanner. Both optical labels could be detected independently. Finally, upconversion images of lateral flow test strips were recorded to facilitate the sensitive and quantitative detection of disease markers. A marker for the parasitic worm Schistosoma was used in this study.

Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.

Feasibility of a lateral flow test for neurocysticercosis using novel up-converting nanomaterials and a lightweight strip analyzer.

UNLABELLED: Neurocysticercosis is a frequent parasitic infection of the human brain, occurring in most of the world, and requires imaging of the brain to diagnose. To determine the burden of disease and to simplify diagnosis, a field-friendly rapid lateral flow (LF) based antibody screening test was developed. The assay utilizes novel nano-sized up-converting phosphor (UCP) reporter particles in combination with a portable lightweight analyzer and detects antibodies in serum samples reactive with bacterial-expressed recombinant (r) T24H, a marker for detecting neurocysticercosis cases. Three sequential flow steps allow enrichment of antibodies on the Test (T) line and consecutive binding of protein-A coated UCP reporter particles. Antibody binding was determined by measuring 550 nm emission after excitation of the UCP label with a 980 nm infrared (IR) diode. Clinical sensitivity and specificity of the assay to detect cases of human neurocysticercosis with 2 or more viable brain cysts were 96% and 98%, respectively, using a sample set comprised of sera from 63 confirmed cases and 170 healthy parasite-naïve non-endemic controls. IN CONCLUSION: Proof-of-principle, of a rapid UCP-LF screening assay for neurocysticercosis was demonstrated. The assay utilized bacterial-expressed rT24H as a potential alternative for baculovirus-expressed rT24H. Performance of the UCP-LF assay was excellent, although further studies need to confirm that bacterial expressed antigen can entirely replace previously used baculovirus antigen. In addition, the increasing availability of commercial sources for UCP reporter materials as well as the accessibility of affordable semi-handheld scanners may allow UCP-based bioanalytical systems for point-of-care to evolve at an even faster pace.

Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels.

The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.

Novel Ex Vivo Culture Method for the Study of Dupuytren's Disease: Effects of TGFß Type 1 Receptor Modulation by Antisense Oligonucleotides.

Dupuytren's disease (DD) is a benign fibroproliferative disease of the hand. It is characterized by the excessive production of extracellular matrix (ECM) proteins, which form a strong fibrous tissue between the handpalm and fingers, permanently disrupting the fine movement ability. The major contractile element in DD is the myofibroblast (MFB). This cell has both fibroblast and smooth muscle cell-type characteristics and causes pathological collagen deposition. MFBs generate contractile forces that are transmitted to the surrounding collagen matrix. Μajor profibrotic factors are members of the transforming growth factor-ß (TGFß) pathway which directly regulate the expression levels of several fibrous proteins such as collagen type 1, type 3, and α-smooth muscle actin. Molecular modulation of this signaling pathway could serve as a therapeutic approach. We, therefore, have developed an ex vivo "clinical trial" system to study the properties of intact, patient-derived resection specimens. In these culture conditions, Dupuytren's tissue retains its three-dimensional (3D) structure and viability. As a novel antifibrotic therapeutic approach, we targeted TGFß type 1 receptor (also termed activin receptor-like kinase 5) expression in cultured Dupuytren's specimens by antisense oligonucleotide-mediated exon skipping. Antisense oligonucleotides targeting activin receptor-like kinase 5 showed specific reduction of ECM and potential for clinical application.Molecular Therapy-Nucleic Acids (2014) 3, e142; doi:10.1038/mtna.2013.69; published online 21 January 2014.

MAVIS: an integrated system for live microscopy and vitrification.

Cryo-electron microscopy of vitrified biological samples can provide three-dimensional reconstructions of macromolecules and organelles within bacteria and cells at nanometer scale resolution, even in native conditions. Localization of specific structures and imaging of cellular dynamics in cellular cryo-electron microscopy is limited by (i) the use of cryo-fixation to preserve cellular structures, (ii) the restricted availability of electron dense markers to label molecules inside cells and (iii) the inherent low contrast of cryo electron microscopy. These limitations can be mitigated to a large extend by correlative light and electron microscopy, where the sample is imaged by both light and electron microscopy. Here we present a Microscopy and Vitrification Integrated System (MAVIS) that combines a light microscope with a plunger to vitrify thin specimens. MAVIS provides the capability for fluorescence light microscopic imaging of living cells and bacteria that are adhered to an electron microscopy grid and subsequent vitrification within a time frame of seconds. The instrument allows targeting of dynamic biological events in time and space by fluorescence microscopy for subsequent cryo light and electron microscopy. Here we describe the design and performance of the MAVIS, illustrated with biological examples.

A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study.

Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal antibody infliximab (IFX). The applied chromatographic method utilizes sequential flows of diluted serum, wash buffer, and an immunoglobulin generic label on LF strips with a Test line comprised of TNF-α. The successive flows permitted enrichment of IFX at the Test line before the label was applied. The label, luminescent upconverting phosphor (UCP) particles coated with protein-A, emits a 550-nm visible light upon excitation with 980-nm infrared light. IFX concentrations were determined through measurement of UCP fluorescence at the Test line. The assay was optimized to detect IFX levels as low as 0.17 µg/mL in serum. For patients with IBD, this limit is appropriate to detect levels associated with loss of response (0.5 µg IFX/mL). The assay was evaluated with clinical samples from patients with Crohn's disease and correlated well within the physiologically relevant range from 0.17 to 10 µg/mL with an IFX-specific ELISA. Performance of the assay was further successfully validated with samples from blood donors, IFX negative IBD patients, and rheumatoid arthritis patients that had developed anti-IFX antibodies. Because of its generic nature, the assay is suited for detecting most therapeutic anti-TNF-α monoclonal antibodies.

A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen.

UNLABELLED: An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA-ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP-LF test indicates a better performance than the ELISA. The UCP-LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings. CONCLUSION: The dry format UCP-LF assay was shown to provide a robust and easy to use format for rapid testing of CAA antigen in serum. It performed at least as good as the ELISA with respect to sensitivity and specificity, and was found to be superior with respect to speed and simplicity of use. Worldwide shipping at ambient temperature of the assay reagents, and the availability of small scanners to analyze the CAA UCP-LF strip, are two major steps towards point-of-care (POC) applications in remote and resource poor environments to accurately identify low (30 pg CAA/mL serum; equivalent to about 10 worm pairs) to heavy Schistosoma infections.

Development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids.

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive "sample-to-result" diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

Molecular diagnosis of minimal residual disease in head and neck cancer patients.

AIM: Locoregional recurrences and distant metastases in adequately treated head and neck squamous cell carcinoma (HNSCC) patients have a dismal effect on survival. Tumor cells that escape histopathological detection might be the prime cause of this effect. We evaluated whether minimal residual cancer (MRC) in deep surgical margins and disseminated tumor cells (DTCs) in bone marrow aspirates are associated with clinicohistopathological parameters and outcome. METHODS: Submucosal samples of deep resection margins of 105 HNSCC patients with histopathologically tumor-free surgical margins were analysed for the presence of MRC using hLy-6D qRT-PCR. Bone-marrow aspirates of 76 of these patients were analysed for DTCs by immunocytochemical staining. Presence of molecular-positive deep surgical margins, presence of DTC in bone marrow aspirates, and clinicohistopathological parameters were tested for associations with survival parameters by univariate and multivariate analyses. RESULTS: In addition to lymph node stage, it appeared that vasoinvasive growth and particularly infiltrative growth pattern are significant predictors for locoregional recurrence (p = 0.041 and p = 0.006, respectively) and disease-free survival (p = 0.014 and p = 0.008, respectively). Remarkably, neither the presence of molecular-positive deep surgical margins nor that of DTC in bone marrow aspirates were significantly related to outcome. CONCLUSIONS: The presence of vasoinvasive and infiltrative growth in HNSCC tumor specimens are significant risk-factors for locoregional recurrence and disease-free survival. At present there seems no role for molecular analysis of deep surgical margins and bone marrow aspirates in predicting outcome with the methods used.

Microarray-based identification of age-dependent differences in gene expression of human dermal fibroblasts.

Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated ß-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/ß-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects.

Human in vivo longevity is reflected in vitro by differential metabolism as measured by (1)H-NMR profiling of cell culture supernatants.

The offspring of nonagenarian siblings suffer less from age related conditions and have a lower risk of mortality compared to their partners. Fibroblast strains derived from such offspring in middle age show different in vitro responses to stress, more stress-induced apoptosis and less senescence when compared to strains of their partners. Aiming to find differences in cellular metabolism in vitro between these fibroblast strains, cell culture supernatants collected at 24 hours and five days were analysed using (1)H nuclear magnetic resonance (NMR)-based metabolic footprinting. Between 24 hours and five days of incubation, supernatants of all fibroblast strains showed decreased levels of glucose, pyruvate, alanine-glutamine (ala-gln), valine, leucine, isoleucine, serine and lysine and increased levels of glutamine, alanine, lactate and pyroglutamic acid. Strains from offspring and their partners were compared using a partial least squares-discriminant analysis (PLS-DA) model based on the data of the five-day time point. The ala-gln and glucose consumption were higher for fibroblast strains derived from offspring when compared to strains of their partners. Also, production of glutamine, alanine, lactate and pyroglutamic acid was found to be higher for fibroblast strains derived from offspring. In conclusion, differences in NMR-based metabolic profiles of human cells in vitro reflect the propensity for human longevity of the subjects from whom these were derived.

Fully automated attenuation measurement and motion correction in FLIP image sequences.

Fluorescence loss in photobleaching (FLIP) is a method to study compartment connectivity in living cells. A FLIP sequence is obtained by alternatively bleaching a spot in a cell and acquiring an image of the complete cell. Connectivity is estimated by comparing fluorescence signal attenuation in different cell parts. The measurements of the fluorescence attenuation are hampered by the low signal to noise ratio of the FLIP sequences, by sudden sample shifts and by sample drift. This paper describes a method that estimates the attenuation by modeling photobleaching as exponentially decaying signals. Sudden motion artifacts are minimized by registering the frames of a FLIP sequence to target frames based on the estimated model and by removing frames that contain deformations. Linear motion (sample drift) is reduced by minimizing the entropy of the estimated attenuation coefficients. Experiments on 16 in vivo FLIP sequences of muscle cells in Drosophila show that the proposed method results in fluorescence attenuations similar to the manually identified gold standard, but with standard deviations of approximately 50 times smaller. As a result of this higher precision, cell compartment edges and details such as cell nuclei become clearly discernible. The main value of this method is that it uses a model of the bleaching process to correct motion and that the model based fluorescence intensity and attenuation estimates can be interpreted easily. The proposed method is fully automatic, and runs in approximately one minute per sequence, making it suitable for unsupervised batch processing of large data series.

Chronic inhibition of the respiratory chain in human fibroblast cultures: differential responses related to subject chronological and biological age.

Respiratory chain function becomes less efficient with age resulting in increased levels of damaging reactive oxygen species. We compared rotenone-exposed fibroblast strains from young and old subjects and from offspring of nonagenarian siblings and the partners of the offspring. Rotenone increased reactive oxygen species levels, inhibited growth rate, and increased telomere shortening (all p < .05). Non-stressed strains from young subjects showed lower reactive oxygen species levels (p = .031) and higher growth rates (p = .002) than strains from old subjects. Stressed strains from young subjects showed smaller increases in reactive oxygen species levels (p = .014) and larger decreases in growth rate (p < .001) than strains from old subjects. Telomere-shortening rates were not different between groups. Stress-induced decreases in growth rate were larger in strains from offspring than from partners (p = .05). Strains from young and old subjects are differentially affected by chronic inhibition of the respiratory chain. Changed growth rates in strains from offspring resemble those from strains from young subjects.

mRNA cycles through hypoxia-induced stress granules in live Drosophila embryonic muscles.

In some myopathies, hypoxia can be the result of pathologic effects like muscle necrosis and abnormal blood flow. At the molecular level, the consequence of hypoxic conditions is not yet fully understood. Under stress conditions, many housekeeping gene mRNAs are translationally silenced, while translation of other mRNAs increases. Alterations to the pool of mRNAs available for translation lead to the formation of so-called stress granules containing both mRNAs and proteins. Stress granule formation and dynamics have been investigated using cells in culture, but have not yet been examined in vivo. In Drosophila embryonic muscles, we found that hypoxia induces the formation of sarcoplasmic granules containing the established stress granule markers RIN and dFMR1. Upon restoration of normoxia, the observed granules were decreased in size, indicating that their formation might be reversible. Employing photobleaching approaches, we found that a cytoplasmic reporter mRNA rapidly shuttles in and out of the granules. Hence, stress granules are highly dynamic complexes and not simple temporary storage sites. Although mRNA rapidly cycles through the granules, its movement throughout the muscle is, remarkably, spatially restricted by the presence of yet undefined myofiber domains. Our results suggest that in hypoxic muscles mRNA remains highly mobile; however, its movement throughout the muscle is restricted by certain boundaries. The development of this Drosophila hypoxia model makes it possible to study the formation and dynamics of stress granules and their associated mRNAs and proteins in a living organism.

Reversible aggregation of PABPN1 pre-inclusion structures.

Increased aggregation of misfolded proteins is associated with aging, and characterizes a number of neurodegenerative disorders caused by homopolymeric amino acid expansion mutations. PABPN1 is an aggregation-prone nuclear protein. Natural aggregation of wild-type (WT) PABPN1 is not known to be disease-associated, but alanine-expanded PABPN1 (expPABPN1) accumulates in insoluble intranuclear inclusions in muscle of patients with oculopharyngeal muscular dystrophy (OPMD). We applied microscopic image quantification to study PABPN1 aggregation process in living cells. We identified transitional pre-inclusion foci and demonstrate that these structures significantly differ between WT- and expPABPN1-expressing cells, while inclusions of these proteins are indistinguishable. In addition to the immobile PABPN1 in inclusions, in the nucleoplasm of expPABPN1 expressing cells we also found a fraction of immobile proteins, representing pre-aggregated species. We found that pre-aggregated and pre-inclusion structures are reverted by a PABPN1 specific affinity binder while inclusion structures are not. Together our results demonstrate that the aggregation process of WT- and expPABPN1 differs in steps preceding inclusion formation, suggesting that pre-aggregated protein species could represent the cytotoxic structures.