Surface Coat Replacement Dynamics and Antigen Glycosylation in T. Brucei Influence Evasion of the Host Antibody Response

The protozoan parasite Trypanosoma brucei, a causative agent of human and animal trypanosomiasis, evades host immunity through antigenic variation of its variant surface glycoprotein (VSG) coat. During infection, the VSG coat elicits a robust antibody response, but the parasite escapes antibody-mediated clearance by repeatedly accessing its large genomic VSG repertoire and switching expression to antigenically distinct VSGs. Much of our knowledge of the process of VSG switching and the extent of VSG diversity has come from genetic analyses, but deep understanding of the T. brucei host-pathogen interface also requires examination of these topics at the protein level. In this thesis, I describe two protein-based studies of the VSG coat that reveal factors influencing trypanosome evasion of the host antibody response. First, I discuss my examination of the dynamics of VSG coat replacement following a genetic VSG switch, and the impact of this process on the parasite’s ability to escape the escalating host antibody response. Using a flow cytometry-based measurement strategy, I evaluated the rate of VSG replacement at the trypanosome surface, and showed that full coat replacement requires several days to complete. I then demonstrated through in vivo infection assays that parasites undergoing coat replacement are only vulnerable to clearance via early IgM antibodies for a limited time. Finally, IgM binding analyses and molecular modeling indicated that IgM loses its ability to mediate trypanosome clearance at unexpectedly early stages of coat replacement based on a critical density threshold of its cognate VSGs on the parasite surface. In the second part of this thesis, I describe a collaborative body of work examining the biochemical features of VSG3 (also termed MITat1.3 and VSG224) and their relevance to parasite interactions with host immunity. The crystal structure of VSG3 was solved, and analysis of the high-resolution structure revealed a glycan of a form previously unidentified in T. brucei, which was centrally located within a region likely to be involved in VSG-antibody interactions. Subsequent mouse infection and antibody binding analyses demonstrated that this VSG modification increases parasite virulence and facilitates evasion of host antibodies. Together, these studies increase our understanding of T. brucei infection dynamics and reveal that additional layers of VSG diversity can affect parasite immune evasion. The identification of an antigen density threshold for in vivo IgM functionality may also inform other systems in which IgM plays a crucial role. These results affirm that much remains to be learned about the interactions of the VSG coat with the host antibody response. Further examination of VSG biochemical diversity and the VSG-antibody interface may provide additional insights into T. brucei pathogenesis and potentially carry implications for other host-pathogen interactions or immune interactions in general

Hot-dip-zinc-coating of prefabricated structural steel components

This JRC-Scientific and Technical Report gives information from pre-normative research for methods to prevent liquid metal assisted cracking of prefabricated structural components during zinc-coating in the liquid zinc melt, that may impair the structural safety of structures in which the components are built in. This information provides a platform upon which further European design and product specifications can be developed. It may in particular affect the further developments of EN 1993, EN 1090, EN ISO 1461 and EN ISO 14713. This report gives the state-of-the-art in understanding the mechanism of liquid metal assisted cracking in the zinc bath and methods and models that may be used to avoid it. It could be a basis to propose rules for the design of steel components intended to be hot-dip-zinc-coated in such a way that the design is consistent with execution rules for hot-dip-zinc-coating. The workability of the rules proposed for all metal works and steel works that are fabricated under EN 1090 and galvanized according to the rules in this report is demonstrated by worked examples.JRC.DG.G.5-European laboratory for structural assessmen

Maintaining (locus of) control? : Assessing the impact of locus of control on education decisions and wages

This paper establishes that individuals with an internal locus of control, i.e., who believe that reinforcement in life comes from their own actions instead of being determined by luck or destiny, earn higher wages. However, this positive effect only translates into labor income via the channel of education. Factor structure models are implemented on an augmented data set coming from two different samples. By so doing, we are able to correct for potential biases that arise due to reverse causality and spurious correlation, and to investigate the impact of premarket locus of control on later outcomes

Patterns of alcohol consumption among individuals with alcohol use disorder during the COVID-19 pandemic and lockdowns in Germany

Objective: To examine whether lockdown measures are associated with AC and consumption-related temporal and psychological within-person mechanisms. Design, setting, and participants: This quantitative, intensive, longitudinal cohort study recruited 1743 participants from 3 sites from February 20, 2020, to February 28, 2021. Data were provided before and within the second lockdown of the COVID-19 pandemic in Germany: before lockdown (October 2 to November 1, 2020); light lockdown (November 2 to December 15, 2020); and hard lockdown (December 16, 2020, to February 28, 2021). Main outcomes and measures: Daily ratings of AC (main outcome) captured during 3 lockdown phases (main variable) and temporal (weekends and holidays) and psychological (social isolation and drinking intention) correlates. Results: Of the 1743 screened participants, 189 (119 [63.0%] male; median [IQR] age, 37 [27.5-52.0] years) with at least 2 alcohol use disorder (AUD) criteria according to the Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) yet without the need for medically supervised alcohol withdrawal were included. These individuals provided 14 694 smartphone ratings from October 2020 through February 2021. Multilevel modeling revealed significantly higher AC (grams of alcohol per day) on weekend days vs weekdays (β = 11.39; 95% CI, 10.00-12.77; P < .001). Alcohol consumption was above the overall average on Christmas (β = 26.82; 95% CI, 21.87-31.77; P < .001) and New Year's Eve (β = 66.88; 95% CI, 59.22-74.54; P < .001). During the hard lockdown, perceived social isolation was significantly higher (β = 0.12; 95% CI, 0.06-0.15; P < .001), but AC was significantly lower (β = -5.45; 95% CI, -8.00 to -2.90; P = .001). Independent of lockdown, intention to drink less alcohol was associated with lower AC (β = -11.10; 95% CI, -13.63 to -8.58; P < .001). Notably, differences in AC between weekend and weekdays decreased both during the hard lockdown (β = -6.14; 95% CI, -9.96 to -2.31; P = .002) and in participants with severe AUD (β = -6.26; 95% CI, -10.18 to -2.34; P = .002). Conclusions and relevance: This 5-month cohort study found no immediate negative associations of lockdown measures with overall AC. Rather, weekend-weekday and holiday AC patterns exceeded lockdown effects. Differences in AC between weekend days and weekdays evinced that weekend drinking cycles decreased as a function of AUD severity and lockdown measures, indicating a potential mechanism of losing and regaining control. This finding suggests that temporal patterns and drinking intention constitute promising targets for prevention and intervention, even in high-risk individuals

Measuring self-regulation in everyday life: reliability and validity of smartphone-based experiments in alcohol use disorder

Self-regulation, the ability to guide behavior according to one’s goals, plays an integral role in understanding loss of control over unwanted behaviors, for example in alcohol use disorder (AUD). Yet, experimental tasks that measure processes underlying self-regulation are not easy to deploy in contexts where such behaviors usually occur, namely outside the laboratory, and in clinical populations such as people with AUD. Moreover, lab-based tasks have been criticized for poor test–retest reliability and lack of construct validity. Smartphones can be used to deploy tasks in the field, but often require shorter versions of tasks, which may further decrease reliability. Here, we show that combining smartphone-based tasks with joint hierarchical modeling of longitudinal data can overcome at least some of these shortcomings. We test four short smartphone-based tasks outside the laboratory in a large sample (N = 488) of participants with AUD. Although task measures indeed have low reliability when data are analyzed traditionally by modeling each session separately, joint modeling of longitudinal data increases reliability to good and oftentimes excellent levels. We next test the measures’ construct validity and show that extracted latent factors are indeed in line with theoretical accounts of cognitive control and decision-making. Finally, we demonstrate that a resulting cognitive control factor relates to a real-life measure of drinking behavior and yields stronger correlations than single measures based on traditional analyses. Our findings demonstrate how short, smartphone-based task measures, when analyzed with joint hierarchical modeling and latent factor analysis, can overcome frequently reported shortcomings of experimental tasks

Measuring self-regulation in everyday life: Reliability and validity of smartphone-based experiments in alcohol use disorder

Self-regulation, the ability to guide behavior according to one's goals, plays an integral role in understanding loss of control over unwanted behaviors, for example in alcohol use disorder (AUD). Yet, experimental tasks that measure processes underlying self-regulation are not easy to deploy in contexts where such behaviors usually occur, namely outside the laboratory, and in clinical populations such as people with AUD. Moreover, lab-based tasks have been criticized for poor test-retest reliability and lack of construct validity. Smartphones can be used to deploy tasks in the field, but often require shorter versions of tasks, which may further decrease reliability. Here, we show that combining smartphone-based tasks with joint hierarchical modeling of longitudinal data can overcome at least some of these shortcomings. We test four short smartphone-based tasks outside the laboratory in a large sample (N = 488) of participants with AUD. Although task measures indeed have low reliability when data are analyzed traditionally by modeling each session separately, joint modeling of longitudinal data increases reliability to good and oftentimes excellent levels. We next test the measures' construct validity and show that extracted latent factors are indeed in line with theoretical accounts of cognitive control and decision-making. Finally, we demonstrate that a resulting cognitive control factor relates to a real-life measure of drinking behavior and yields stronger correlations than single measures based on traditional analyses. Our findings demonstrate how short, smartphone-based task measures, when analyzed with joint hierarchical modeling and latent factor analysis, can overcome frequently reported shortcomings of experimental tasks

Patterns of Alcohol Consumption Among Individuals With Alcohol Use Disorder During the COVID-19 Pandemic and Lockdowns in Germany

Importance Alcohol consumption (AC) leads to death and disability worldwide. Ongoing discussions on potential negative effects of the COVID-19 pandemic on AC need to be informed by real-world evidence. Objective To examine whether lockdown measures are associated with AC and consumption-related temporal and psychological within-person mechanisms. Design, Setting, and Participants This quantitative, intensive, longitudinal cohort study recruited 1743 participants from 3 sites from February 20, 2020, to February 28, 2021. Data were provided before and within the second lockdown of the COVID-19 pandemic in Germany: before lockdown (October 2 to November 1, 2020); light lockdown (November 2 to December 15, 2020); and hard lockdown (December 16, 2020, to February 28, 2021). Main Outcomes and Measures Daily ratings of AC (main outcome) captured during 3 lockdown phases (main variable) and temporal (weekends and holidays) and psychological (social isolation and drinking intention) correlates. Results Of the 1743 screened participants, 189 (119 [63.0%] male; median [IQR] age, 37 [27.5-52.0] years) with at least 2 alcohol use disorder (AUD) criteria according to the Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) yet without the need for medically supervised alcohol withdrawal were included. These individuals provided 14 694 smartphone ratings from October 2020 through February 2021. Multilevel modeling revealed significantly higher AC (grams of alcohol per day) on weekend days vs weekdays (β = 11.39; 95% CI, 10.00-12.77; P < .001). Alcohol consumption was above the overall average on Christmas (β = 26.82; 95% CI, 21.87-31.77; P < .001) and New Year’s Eve (β = 66.88; 95% CI, 59.22-74.54; P < .001). During the hard lockdown, perceived social isolation was significantly higher (β = 0.12; 95% CI, 0.06-0.15; P < .001), but AC was significantly lower (β = −5.45; 95% CI, −8.00 to −2.90; P = .001). Independent of lockdown, intention to drink less alcohol was associated with lower AC (β = −11.10; 95% CI, −13.63 to −8.58; P < .001). Notably, differences in AC between weekend and weekdays decreased both during the hard lockdown (β = −6.14; 95% CI, −9.96 to −2.31; P = .002) and in participants with severe AUD (β = −6.26; 95% CI, −10.18 to −2.34; P = .002). Conclusions and Relevance This 5-month cohort study found no immediate negative associations of lockdown measures with overall AC. Rather, weekend-weekday and holiday AC patterns exceeded lockdown effects. Differences in AC between weekend days and weekdays evinced that weekend drinking cycles decreased as a function of AUD severity and lockdown measures, indicating a potential mechanism of losing and regaining control. This finding suggests that temporal patterns and drinking intention constitute promising targets for prevention and intervention, even in high-risk individuals

African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat

The African trypanosome, Trypanosoma brucei spp., is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its Variant Surface Glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and “switching” to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4Å resolution crystal structure of variant VSG3 manifests heretofore unappreciated divergence in the tertiary fold and oligomeric state. The structure also reveals an O linked carbohydrate on the top surface of VSG3, a modification previously unknown in African trypanosomes. Mass spectrometric analysis indicates that this O -glycosylation site is heterogeneously occupied in VSG3 by 0 to 3 hexose residues and is also present in other VSGs. We demonstrate that this O -glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino acid sequence to include surface posttranslational modifications with immunomodulatory impact