Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background

Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.

Methods

The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.

Results

We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.

Conclusion

In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.     

Congestive heart failure with preserved ejection fraction is associated with severely impaired dynamic Starling mechanism

Sedentary aging leads to increased cardiovascular stiffening, which can be ameliorated by sufficient amounts of lifelong exercise training. An even more extreme form of cardiovascular stiffening can be seen in heart failure with preserved ejection fraction (HFpEF), which comprises ~40~50% of elderly patients diagnosed with congestive heart failure. There are two major interrelated hypotheses proposed to explain heart failure in these patients: 1) increased left ventricular (LV) diastolic stiffness and 2) increased arterial stiffening. The beat-to-beat dynamic Starling mechanism, which is impaired with healthy human aging, reflects the interaction between ventricular and arterial stiffness and thus may provide a link between these two mechanisms underlying HFpEF. Spectral transfer function analysis was applied between beat-to-beat changes in LV end-diastolic pressure (LVEDP; estimated from pulmonary artery diastolic pressure with a right heart catheter) and stroke volume (SV) index. The dynamic Starling mechanism (transfer function gain between LVEDP and the SV index) was impaired in HFpEF patients (n = 10) compared with healthy age-matched controls (n = 12) (HFpEF: 0.23 ± 0.10 ml·m?2·mmHg?1 and control: 0.37 ± 0.11 ml·m?2·mmHg?1, means ± SD, P = 0.008). There was also a markedly increased (3-fold) fluctuation of LV filling pressures (power spectral density of LVEDP) in HFpEF patients, which may predispose to pulmonary edema due to intermittent exposure to higher pulmonary capillary pressure (HFpEF: 12.2 ± 10.4 mmHg2 and control: 3.8 ± 2.9 mmHg2, P = 0.014). An impaired dynamic Starling mechanism, even more extreme than that observed with healthy aging, is associated with marked breath-by-breath LVEDP variability and may reflect advanced ventricular and arterial stiffness in HFpEF, possibly contributing to reduced forward output and pulmonary congestion.     

Serological and parasitological prevalence of Toxoplasma gondii in wild birds from Colorado

Ground-feeding birds are considered important in the epidemiology of Toxoplasma gondii because they serve as indicators of soil contamination by oocysts, and birds of prey are indicators of T. gondii prevalence in rodents and other small mammals. Cats excrete environmentally resistant oocysts after consuming tissues of T. gondii -infected birds. In the present study, sera and tissues from 382 wild birds from Colorado were tested for T. gondii infection. Antibodies to T. gondii were found in 38 birds with the use of the modified agglutination test (MAT, 1∶25 titer). Tissues (brains, hearts) of 84 birds were bioassayed in mice. Viable T. gondii was isolated from 1 of 1 barn owl (Tyto alba), 1 of 5 American kestrels (Falco sparverius), 1 of 7 ferruginous hawks (Buteo regalis), 1 of 4 rough-legged hawks (Buteo lagopus), 2 of 13 Swainson's hawks (Buteo swainsoni), and 1 of 25 red-tailed hawks (Buteo jamaicensis). This is the first time T. gondii has been isolated from the barn owl, ferruginous hawk, rough-legged hawk, and Swainson's hawk.     

ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi-derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero-heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.     

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.Motoneurons are extremely extended neurons that mediate the control of all muscle types by the central nervous system. Therefore, diseases involving progressive motoneuron degeneration such as amyotrophic lateral sclerosis (ALS)¹ (OMIM: 105400) or spinal muscle atrophy (OMIM: 253300) are particularly devastating and generally fatal disorders. Today, ALS is believed to form a phenotypic continuum with the disease entity frontotemporal lobe degeneration (OMIM: 600274) (1, 2). About 10% of ALS cases are known to be inherited, but the vast majority are considered sporadic. The number of inherited cases might be underestimated because of incomplete family histories, non-paternity, early death of family members, or incomplete penetrance (3).Mutations in several genes have been reported for the familial form, including in Sod1 (4), Als2 (5), Setx (6), Vapb (7), Tardbp (8, 9), Fus/Tls (10, 11), Vcp (12), Pfn1 (13), and several others (reviewed in Ref. 14). The most frequent genetic cause of inherited ALS was recently shown to be a hexanucleotide repeat expansion in an intron of a gene of unknown function called C9orf72 (15–17). Based on the spectrum of known mutations, several disease mechanisms for ALS have been proposed, including dysfunction of protein folding, axonal transport, RNA splicing, and metabolism (reviewed in Refs. 14, 18, and 19). Despite intensive research, it is still unclear whether a main common molecular pathway or mechanism underlies motoneuron degeneration in ALS and frontotemporal lobe degeneration. Spinal muscle atrophy is caused by homozygous mutations or deletions in the survival of motor neuron gene (Smn1) that presumably impair the RNA metabolism through diminished functionality of the Smn1 gene product (20). Over recent decades several model systems have been established to investigate ALS (21). These include transgenic animal models such as mouse (22), drosophila (23), and zebrafish (24). In cell-based studies, primary motoneurons cultured from rodent embryos (25) or motoneuron-like cell lines are employed. Primary cells are considered to more closely mimic the in vivo situation, but they are more challenging to establish and maintain. In contrast, the degree of functional relevance of cell lines can be difficult to establish, but they can be propagated without limitation and are well suited for high-throughput analysis. In particular, the spinal cord neuron–neuroblastoma hybrid cell line NSC-34 (26) and the mouse neuroblastoma cell line N2a (27) are widely used not only to assess motoneuron function, but also to study disease mechanisms in motoneurons (28, 29).As proteins are the functional actors in cells, proteomics should be able to make important contributions to the characterization and evaluation of cellular models. In particular, by identifying and quantifying the expressed proteins and bioinformatically interpreting the results, one can obtain enough information to infer functional differences. Our laboratory has previously shown proof of concept of such an approach by comparing the expression levels of about 4,000 proteins between primary hepatocytes and a hepatoma cell line (30). Very recently, mass-spectrometry-based proteomics has achieved sufficient depth and accuracy to quantify almost the entire proteome of mammalian cell lines (31–33). Furthermore, new instrumentation and algorithms now make it possible to perform label-free quantification between multiple cellular systems and with an accuracy previously associated only with stable isotope labeling techniques (34, 35).To evaluate the suitability of motoneuron-like cell lines as cellular model systems for research on ALS and related disorders, we characterized the proteomes of two widely used cell lines, NSC-34 and N2a, and compared them with the proteomes of mouse primary motoneurons and non-neuronal control cell lines. To generate primary motoneurons, we employed a recently described culturing system that makes it possible to isolate highly enriched motoneuron populations in less than 8 h (25). We identified more than 10,000 proteins and investigated differences in quantitative levels of individual neuron-associated proteins and pathways related to motoneuron function and disease mechanisms.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.     

Sequentially Deposited versus Conventional Nonfullerene Organic Solar Cells: Interfacial Trap States,Vertical Stratification,and Exciton Dissociation

Bulk heterojunction (BHJ) nonfullerene organic solar cells prepared from sequentially deposited donor and acceptor layers (sq‐BHJ) have recently been shown to be highly efficient, environmentally friendly, and compatible with large area and roll‐to‐roll fabrication. However, the related photophysics at donor‐acceptor interface and the vertical heterogeneity of donor‐acceptor distribution, critical for exciton dissociation and device performance, have been largely unexplored. Herein, steady‐state and time‐resolved optical and electrical techniques are employed to characterize the interfacial trap states. Correlating with the luminescent efficiency of interfacial states and its nonradiative recombination, interfacial trap states are characterized to be about 40% more populated in the sq‐BHJ devices than the as‐cast BHJ (c‐BHJ), which probably limits the device voltage output. Cross‐sectional energy‐dispersive X‐ray spectroscopy and ultraviolet photoemission spectroscopy depth profiling directly visualize the donor–acceptor vertical stratification with a precision of 1–2 nm. From the proposed “needle” model, the high exciton dissociation efficiency is rationalized. This study highlights the promise of sequential deposition to fabricate efficient solar cells, and points toward improving the voltage output and overall device performance via eliminating interfacial trap states.