Response of fibrinolytic activity to venous occlusion.

Resting fibrinolytic activity and the response of the fibrinolytic system to venous occlusion were studied in a group of healthy middle-aged men. All subjects showed increased fibrinolytic activity but of varying degrees. There was a linear relationship between resting and occluded levels of fibrinolytic activity but many subjects with lower levels of fibrinolytic activity showed an anomalous response. Responses over the expected level were more common than unexpectedly low levels of response. Fibrinogen and plasminogen concentrations were inversely correlated with fibrinolytic activity.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

ACSL4-dependent ferroptosis does not represent a tumor-suppressive mechanism but ACSL4 rather promotes liver cancer progression

Ferroptosis is a novel type of programmed cell death that differs from apoptosis in that it involves iron-dependent peroxidation of membrane phospholipids. Its role in a variety of human disorders, including cancer has been hypothesized in recent years. While it may function as an endogenous tumor suppressor in a variety of cancers, its role during initiation and progression of liver cancer, particularly hepatocellular carcinoma (HCC), is yet unknown. Because HCC is most commonly found in chronically injured livers, we utilized two well-established mouse models of chronic injury-dependent HCC formation: Treatment with streptozotocin and high-fat diet as metabolic injury model, as well as treatment with diethylnitrosamine and carbon tetrachloride as toxic injury model. We used mice with hepatocyte-specific deletion of Acsl4, a key mediator of ferroptosis, to explore the significance of ferroptotic cell death in hepatocytes, the cell type of origin for HCC. Surprisingly, preventing ferroptotic cell death in hepatocytes by deleting Acsl4 does not increase the formation of HCC. Furthermore, Acsl4-deficient livers display less fibrosis and proliferation, especially in the HCC model of toxic damage. Intriguingly, in this model, the absence of ACSL4-dependent processes such as ferroptosis significantly slow down the growth of HCC. These findings suggest that during HCC formation in a chronically injured liver, ferroptotic cell death is not an endogenous tumor-suppressive mechanism. Instead, we find that ACSL4-dependent processes have an unanticipated cancer-promoting effect during HCC formation, which is most likely due to aggravated liver damage as demonstrated by increased hepatic fibrosis. Previous studies suggested that ferroptosis might have beneficial effects for patients during HCC therapy. As a result, during HCC progression and therapy, ferroptosis may have both cancer-promoting and cancer-inhibitory effects, respectively.Subject terms: Cancer models, Cancer genetics, Cell death     

Identification of a linear B-cell epitope on the Schistosoma japonicum saposin protein,SjSAP4: Potential as a component of a multi-epitope diagnostic assay

BackgroundSchistosoma japonicum is one of three major species of blood flukes causing schistosomiasis, a disease, which continues to be a major public health issue in the Philippines. SjSAP4, a member of a multigene family of saposin-like proteins, is a recognized immunodiagnostic biomarker for schistosomiasis japonica. This study aimed to identify linear B-cell epitopes on SjSAP4 and to validate their potential as components of a multi-epitope assay for the serological diagnosis of schistosomiasis japonica.MethodologySjSAP4-derived peptides were expressed as GST-peptide-fused proteins and these were Western blot probed with human serum samples from S. japonicum Kato-Katz (KK)-positive individuals and uninfected controls. A core epitope was further identified by Western blotting through probing a series of truncated peptides with the schistosomiasis patient sera. The diagnostic performance of the core epitope-containing peptides and the full-length SjSAP4 was evaluated by enzyme-linked immunosorbent assay (ELISA) using a panel of sera collected from subjects resident in a schistosomiasis-endemic area of the Philippines.Main findingsAs a result of the peptide mapping, one peptide (P15) was found to be highly immunogenic in the KK-positive individuals. We subsequently showed that -S¹⁶³QCSLVGDIFVDKYLD¹⁷⁸- is a core B-cell epitope of P15. Subsequent ELISAs incorporating SjSAP4, SjSAP4-Peptide and SjSP-13V2-Peptide showed a sensitivity of 94.0%, 46.0% and 74.0%, respectively, and a specificity of 97.1%, 100% and 100%, respectively. Notably, complementary recognition of the B-cell epitopes (SjSAP4-Peptide and SjSP-13V2-Peptide) was observed in a subset of the KK-positive individuals. A dual epitope-ELISA (SjSAP4-Peptide + SjSP-13V2-Peptide-ELISA) showed a diagnostic sensitivity of 84.0% and a specificity of 100%.Conclusions/SignificanceIn this study, -S¹⁶³QCSLVGDIFVDKYLD¹⁷⁸- was identified as a dominant linear B-cell epitope on SjSAP4. This peptide and the complementary recognition of other B-cell epitopes using sera from different KK-positive individuals can provide the basis of developing a multi-epitope assay for the serological diagnosis of schistosomiasis.     

The Rab11-family interacting proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner

After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma, suggesting that specific parasite PV proteins may be involved in these processes.     

Effect of clinician information sessions on diagnostic testing for Chagas disease

BackgroundChagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened.Methodology/Principal findingsWe conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11).Conclusion/SignificanceIn this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.     

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1 ^−/−‐ and Tlr8 ^−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.     

Persistence of Water within Perched Basins of the Peace-Athabasca Delta, Northern Canada

The Peace-Athabasca Delta (PAD) is one of the world’s largest freshwater deltas. Many of its shallow (<1.5 m) lakes and wetlands are perched above surrounding waterways. The delta has experienced a number of wetting and drying intervals. The latest drying trend ended in 1996 when high waters, generated under ice-jam and open-water conditions, recharged a number of the perched basins. The objective of this study was to determine the relative importance of hydroclimatic components on the persistence of water. A water-balance model was developed to simulate water-level responses following a flood event. Basin response was tested against a range of historical hydroclimatic conditions that have occurred in the delta during the 20th century. Ponded water duration in a 0.8 m deep perched basin was 5 years for the cool-dry period of the 1920s, slightly longer for the post-1974 flood era, and up to 9 years for the wet conditions of the 1940s and 1950s. Water drawdown occurred in almost every year and was almost exclusively due to evaporation exceeding precipitation. Net groundwater flux was minimal. Given the overlying importance of the floodwater component in the water balance of perched basins, the next step is to investigate the causes, spatial sources and frequency of flooding. This water balance model presented in this paper offers a useful tool for the management of the duration of water in perched wetlands of the PAD, which can help preserve essential habitat for wildlife.     

Shifts in plant functional community composition under hydrological stress strongly decelerate litter decomposition

Litter decomposition is a key process of nutrient and carbon cycling in terrestrial ecosystems. The decomposition process will likely be altered under ongoing climate change, both through direct effects on decomposer activity and through indirect effects caused by changes in litter quality. We studied how hydrological change indirectly affects decomposition via plant functional community restructuring caused by changes in plant species’ relative abundances (community‐weighted mean (CWM) traits and functional diversity). We further assessed how those indirect litter quality effects compare to direct effects. We set up a mesocosm experiment, in which sown grassland communities and natural turf pieces were subjected to different hydrological conditions (dryness and waterlogging) for two growing seasons. Species‐level mean traits were obtained from trait databases and combined with species’ relative abundances to assess functional community restructuring. We studied decomposition of mixed litter from these communities in a common “litterbed.” These indirect effects were compared to effects of different hydrological conditions on soil respiration and on decomposition of standard litter (direct effects). Dryness reduced biomass production in sown communities and natural turf pieces, while waterlogging only reduced biomass in sown communities. Hydrological stress caused profound shifts in species’ abundances and consequently in plant functional community composition. Hydrologically stressed communities had higher CMW leaf dry matter content, lower CMW leaf nitrogen content, and lower functional diversity. Lower CWM leaf N content and functional diversity were strongly related to slower decomposition. These indirect effects paralleled direct effects, but were larger and longer‐lasting. Species mean traits from trait databases had therefore considerable predictive power for decomposition. Our results show that stressful soil moisture conditions, that are likely to occur more frequently in the future, quickly shift species’ abundances. The resulting functional community restructuring will decelerate decomposition under hydrological stress.