Impact of chronic hepatitis C on mortality in cirrhotic patients admitted to intensive-care unit

Background: Cirrhosis and severe sepsis are factors associated with increased mortality in intensive care unit (ICU), but chronic hepatitis C (CHC) has been less studied in ICU. The aim of this study was to analyze the impact of CHC on the mortality of cirrhotic patients admitted to ICU according to severe sepsis and decompensated cirrhosis. Methods: We carried out a retrospective study based on CHC-cirrhotic patients (CHC-group) admitted to ICU (n = 1138) and recorded in the Spanish Minimum Basic Data Set (2005-2010). A control-group (randomly selected cirrhotic patients without HIV, HBV, or HCV infections) was also included (n = 4127). The primary outcome variable was ICU mortality. The cumulative mortality rate on days 7, 30, and 90 in patients admitted to the ICUs was calculated by dividing the number of deaths by the number of patients admitted to the ICU. The adjusted hazard ratio (aHR) for death in the ICU was estimated through a semi-parametric Bayesian model of competing risk. Results: The CHC-group had a higher cumulative incidence of severe sepsis than the control-group in compensated cirrhosis (37.4 vs. 31.1 %; p = 0.024), but no differences between the CHC-group and the control-group in decompensated cirrhosis were found. Moreover, a higher cumulative incidence of severe sepsis was associated with decompensated cirrhosis compared to compensated cirrhosis in the control-group (40.1 vs. 31.1 %; p < 0.001) whereas this was not observed in the CHC group (38.1 vs. 37.4 %; p = 0.872). The CHC-group had higher cumulative mortality than the control-group by days 7 (47 vs. 41.3 %; p < 0.001), 30 (78.5 vs. 73.5 %; p < 0.001), and 90 (96.3 vs. 95.9 %; p < 0.001). In a competitive risk model, the CHC-group had a higher risk of dying if the ICU course was complicated by severe sepsis (adjusted hazard ratio (aHR) = 1.19; p = 0.003), but no significant values in patients with absence of severe sepsis were found (aHR = 1.09; p = 0.068). When patients were stratified by cirrhosis stage and severe sepsis, CHC patients with compensated cirrhosis had the higher risk of death if they had severe sepsis (aHR = 1.35; p = 0.002). Moreover, the survival was low in patients with decompensated cirrhosis and severe sepsis but we did not find significant differences between CHC-group and control-group. Conclusions: CHC was associated with an increased risk of death in cirrhotic patients admitted to ICUs, particularly in patients with compensated cirrhosis and severe sepsis.This research has been supported by Instituto de Salud Carlos III (grant numbers PI11/00245 & PI14CIII/00011 to SR and PI12/00019 to AAM). MAJS is supported by a contract of "Instituto de Salud Carlos III" (grant number CD13/00013)

Multivalent peptides and proteins for collagen imaging

Collagens are abundant proteins present in the extracellular matrix where they provide tensile stiffness to organs and tissues. They are an important marker for several physiological processes including tissue remodeling and a variety of diseases. Collagens form a superfamily of proteins that all have highly repetitive primary sequences necessary to form triple helices. In biology the affinity and specificity of binding are often enhanced by combining multiple weak interactions, an effect that is known as multivalency. Multivalency plays a major role in cellsurface interactions, in the immune system, and in protein-protein interactions. Since collagens are highly repetitive they are attractive, but complex targets to study using multivalent probes. The focus of the research in this thesis was the development and study of new collagen targeting strategies based on multivalent display of peptides and proteins. In Chapter 2 a new synthetic method is described for making well-defined proteinfunctionalized micelles and liposomes via native chemical ligation (NCL). A cysteinefunctionalized phospholipid was synthesized to allow native chemical ligation on micelles and liposomes. Using expressed protein ligation enhanced yellow fluorescent protein (EYFP) was prepared with a C-terminal thioester and successfully ligated to micelles consisting of cysteinefunctionalized phospholipids. Next, EYFP-liposomes were prepared in a similar manner and fluorescence spectroscopy showed that the proteins retained their fluorescent properties. The general applicability of NCL was further tested using CNA35, a collagen-binding protein that has been applied for fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approx 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and to bind collagen with a 150-fold higher affinity compared to the monovalent protein. This proof of principle study showed that NCL allows the direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipidbased assemblies. In Chapter 3 the efficiency of the NCL reaction was further improved, which allowed a more detailed study of the affinity and specificity of multivalent protein micelles for collagen. Micelles were prepared with 2 to 20 CNA35 proteins on the surface and Surface Plasmon resonance (SPR) and solid-phase binding assays were used to study the thermodynamics and kinetics of micelle binding. SPR measurements revealed that multivalent protein micelles are remarkably stable, showing no dissociation from the collagen surface after extensive washing, a finding that has fundamental implications for the applications of micelles in molecular imaging and targeted drug delivery. The collagen affinity of monovalent CNA35 was attenuated by at least 900-fold by introducing a single point mutation (Y175K). Remarkably, multivalent CNA35 (Y175K)-micelles were able to bind collagen depending on the valency. Micelles with only one protein were unable to bind to collagen, while micelles containing 5 proteins showed some binding and micelles with on average 17 proteins showed full ‘restoration’ of the collagen binding. This increase in affinity was independent of the micelle as scaffold, since liposomal display showed a similar increase in affinity depending on the protein density on the surface. Furthermore, these CNA35(Y175K)-micelles were successful in ex vivo collagen imaging in kidney tissue. The collagen specificity did not change upon multivalent display of CNA35(Y175K). This could be due to the dynamic nature of the micelle resulting in adaptation to the binding site architecture. Interestingly, a difference in collagen specificity was observed using both types of micelles when imaging collagen type IV in rat kidney tissue, with the CNA35(Y175K)-micelles apparently detecting only a subset of the type IV collagen architectures. Since targeted micelles and liposomes are popular in vivo drug delivery and molecular imaging vehicles, detailed understanding of the exchange of lipid-functionalized proteins in these lipid-based assemblies is essential. In Chapter 4 the fluorescent proteins ECFP and EYFP were used as a FRET pair to investigate the lipid exchange behavior of protein-functionalized liposomes and micelles. No exchange was observed between protein-functionalized phospholipids in the liposomes that are typically used for molecular imaging. However, protein-functionalized lipids were found to exchange between micelles showing a half-time of exchange of almost 2 hours at room temperature, but only 4 min at 37 °C. FRET was also used to show that protein-functionalized micelles remained intact at least until a lipid concentration as low as 0.15 µM, indicating that they are even more stable than previously assumed. Although CNA35 micelles and liposomes can be used for collagen imaging their applicability is hampered by their size and the fact that CNA35 lacks collagen type specificity. In Chapter 5 peptide-based collagen targeting was pursued. A collagen type I binding peptide with low affinity was multimerized on a dendritic scaffold to mimic the 5-fold presentation on the phage. This new approach yielded a high-affinity binder with a collagen affinity that is comparable to that of CNA35. SPR measurements and ex vivo collagen imaging of pericardial tissue showed that the peptide probe outperformed CNA35 in specificity. Furthermore, the general applicability of this new approach was investigated by screening phage display libraries to find peptide binders for collagen types II, IV and V. Several collagen type V binding peptides were discovered with low affinity but high specificity. Remarkably, the scrambled peptide sequences also specifically bound collagen type V indicating that the order of amino acids does not matter much. Again, multimerization of these peptides increased the affinity by at least 500-fold compared to the monovalent peptides. In vitro and ex vivo collagen binding experiments showed that these pentavalent peptides were very specific for collagen type V. Most current targeted drug delivery and molecular imaging applications use antibodies or antibody domains as targeting ligands. To extend the applicability of the approaches described in chapter 2 and 3, a general strategy to obtain antibody fragments with reactive Cterminal thioesters was developed in Chapter 6. Antibodies and antibody fragments contain essential disulfide bonds to stabilize their structure and maintain binding activity, which complicates the recombinant production in E. coli. In this chapter we showed that singledomain antibodies can be targeted to the periplasm as intein-fusion proteins to allow disulfide bond formation. After protein extraction and purification the single-domain antibodies were isolated with a reactive C-terminal thioester. Single-domain antibodies were coupled via NCL to micelles and shown to be functional. This approach can be extended to other antibody formats and shows that NCL can be used to functionalize a variety of nanoparticles without compromising activity. Chapter 7 elaborates on the possible applications of the collagen probes described in this thesis. The CNA35-functionalized micelles and liposomes can be prepared with imaging moieties to use them as targeted molecular imaging probes. Also, drugs can be easily incorporated in micelles and liposomes to allow targeted drug delivery. Furthermore, multivalent display of mutant CNA35 on micelles and liposomes can be envisioned to discriminate tissues with high and low collagen content. In contrast to CNA35-functionalized particles the peptide-functionalized dendritic wedges allow for specific collagen imaging. Also promising is the use of peptide wedges as targeted molecular imaging probes by connecting them with wedges containing imaging moieties. Furthermore, the possibilities of peptidefunctionalized micelles as collagen probes are discussed, since they combine the specificity of the peptides with the ease of preparation and biocompatibility of the micelles. In conclusion, the work described in this thesis has provided more insight into the importance of using multivalent interactions for collagen targeting and imaging. The multivalent display of CNA35 on micelles and liposomes resulted in high affinity collagen probes that were able to target collagen in vitro and ex vivo. Furthermore, multivalent display of collagen binding peptides resulted in high affinity probes that are collagen type specific. These results demonstrate the potential of multivalent peptide and protein interactions for imaging of repetitive targets in general

Aquaporin-4 and brain edema.

Aquaporin-4 (AQP4) is a water-channel protein expressed strongly in the brain, predominantly in astrocyte foot processes at the borders between the brain parenchyma and major fluid compartments, including cerebrospinal fluid (CSF) and blood. This distribution suggests that AQP4 controls water fluxes into and out of the brain parenchyma. Experiments using AQP4-null mice provide strong evidence for AQP4 involvement in cerebral water balance. AQP4-null mice are protected from cellular (cytotoxic) brain edema produced by water intoxication, brain ischemia, or meningitis. However, AQP4 deletion aggravates vasogenic (fluid leak) brain edema produced by tumor, cortical freeze, intraparenchymal fluid infusion, or brain abscess. In cytotoxic edema, AQP4 deletion slows the rate of water entry into brain, whereas in vasogenic edema, AQP4 deletion reduces the rate of water outflow from brain parenchyma. AQP4 deletion also worsens obstructive hydrocephalus. Recently, AQP4 was also found to play a major role in processes unrelated to brain edema, including astrocyte migration and neuronal excitability. These findings suggest that modulation of AQP4 expression or function may be beneficial in several cerebral disorders, including hyponatremic brain edema, hydrocephalus, stroke, tumor, infection, epilepsy, and traumatic brain injury

Unimpaired attentional disengagement in toddlers with autism spectrum disorder

A prominent hypothesis holds that ‘sticky’ attention early in life in children with autism spectrum disorder (ASD) limits their ability to explore and learn about the world. Under this hypothesis, the core clinical symptoms of ASD – restricted interests, repetitive behaviors and impaired social/communication abilities – could all result from impaired attentional disengagement during development. However, the existence of disengagement deficits in children with ASD is controversial, and a recent study found no deficit in 5- to 12-year-olds with ASD. Nonetheless, the possibility remains that disengagement is impaired earlier in development in children with ASD, altering their developmental trajectory even if the attentional deficit itself is remediated or compensated for by the time children with ASD reach school age. Here, we tested this possibility by characterizing attentional disengagement in a group of toddlers just diagnosed with ASD (age 21 to 37 months). We found strikingly similar performance between the ASD and age-matched typically developing (TD) toddlers, and no evidence of impaired attentional disengagement. These results show that even at a young age when the clinical symptoms of ASD are first emerging, disengagement abilities are intact. Sticky attention is not a fundamental characteristic of ASD, and probably does not play a causal role in its etiology.Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (Award F32-HD075427

GA-SmaAt-GNet: Generative Adversarial Small Attention GNet for Extreme Precipitation Nowcasting

In recent years, data-driven modeling approaches have gained significant attention across various meteorological applications, particularly in weather forecasting. However, these methods often face challenges in handling extreme weather conditions. In response, we present the GA-SmaAt-GNet model, a novel generative adversarial framework for extreme precipitation nowcasting. This model features a unique SmaAt-GNet generator, an extension of the successful SmaAt-UNet architecture, capable of integrating precipitation masks (binarized precipitation maps) to enhance predictive accuracy. Additionally, GA-SmaAt-GNet incorporates an attention-augmented discriminator inspired by the Pix2Pix architecture. This innovative framework paves the way for generative precipitation nowcasting using multiple data sources. We evaluate the performance of SmaAt-GNet and GA-SmaAt-GNet using real-life precipitation data from the Netherlands, revealing notable improvements in overall performance and for extreme precipitation events compared to other models. Specifically, our proposed architecture demonstrates its main performance gain in summer and autumn, when precipitation intensity is typically at its peak. Furthermore, we conduct uncertainty analysis on the GA-SmaAt-GNet model and the precipitation dataset, providing insights into its predictive capabilities. Finally, we employ Grad-CAM to offer visual explanations of our model's predictions, generating activation heatmaps that highlight areas of input activation throughout the network.Comment: 18 pages, 11 figur

The use of the SF-36 questionnaire in adult survivors of childhood cancer: evaluation of data quality, score reliability, and scaling assumptions

BACKGROUND: The SF-36 has been used in a number of previous studies that have investigated the health status of childhood cancer survivors, but it never has been evaluated regarding data quality, scaling assumptions, and reliability in this population. As health status among childhood cancer survivors is being increasingly investigated, it is important that the measurement instruments are reliable, validated and appropriate for use in this population. The aim of this paper was to determine whether the SF-36 questionnaire is a valid and reliable instrument in assessing self-perceived health status of adult survivors of childhood cancer. METHODS: We examined the SF-36 to see how it performed with respect to (1) data completeness, (2) distribution of the scale scores, (3) item-internal consistency, (4) item-discriminant validity, (5) internal consistency, and (6) scaling assumptions. For this investigation we used SF-36 data from a population-based study of 10,189 adult survivors of childhood cancer. RESULTS: Overall, missing values ranged per item from 0.5 to 2.9 percent. Ceiling effects were found to be highest in the role limitation-physical (76.7%) and role limitation-emotional (76.5%) scales. All correlations between items and their hypothesised scales exceeded the suggested standard of 0.40 for satisfactory item-consistency. Across all scales, the Cronbach's alpha coefficient of reliability was found to be higher than the suggested value of 0.70. Consistent across all cancer groups, the physical health related scale scores correlated strongly with the Physical Component Summary (PCS) scale scores and weakly with the Mental Component Summary (MCS) scale scores. Also, the mental health and role limitation-emotional scales correlated strongly with the MCS scale score and weakly with the PCS scale score. Moderate to strong correlations with both summary scores were found for the general health perception, energy/vitality, and social functioning scales. CONCLUSION: The findings presented in this paper provide support for the validity and reliability of the SF-36 when used in long-term survivors of childhood cancer. These findings should encourage other researchers and health care practitioners to use the SF-36 when assessing health status in this population, although it should be recognised that ceiling effects can occur