RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

Discovery and Verification of Osteopontin and Beta-2-microglobulin as Promising Markers for Staging Human African Trypanosomiasis

Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and β-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and β-2-microglobulin in CSF of S2 patients (μg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.Human African trypanosomiasis (HAT), or sleeping sickness, is caused by an extracellular protozoan parasite of the genus Trypanosoma, which is transmitted through the bite of a tsetse fly (genus Glossina). Two morphologically identical subspecies of the parasite, are responsible for the two geographically and clinically different forms of HAT: a chronic form, widespread in West and Central Africa, caused by T. b. gambiense, and an acute form, endemic in eastern Africa, caused by T. b. rhodesiense (1). In both forms of the disease, parasites are initially localized in the blood stream, lymph, and peripheral tissues; this is the first or hemolymphatic stage (S1). During this stage, patients present generic clinical features that are common to other infectious diseases such as human immunodeficiency virus (HIV), malaria, and tuberculosis (TB), which can coexist with HAT, thus making its early diagnosis difficult (2). If treatment is not carried out, the disease progresses to the second or meningoencephalitic stage (S2) after trypanosomes cross the blood-brain barrier (BBB) and invade the central nervous system (CNS). This phase is characterized by a broad range of neurological signs that are indicative of CNS involvement (1). Diagnosis of HAT is based on parasitological demonstration of parasites in blood or lymph-node aspirate (3). All positive or suspect patients have to undergo a lumbar puncture and cerebrospinal fluid (CSF)1 examination, to determine whether they have second stage disease (4). According to the World Health Organization (WHO) guidelines, the meningoencephalitic stage is defined by the presence of parasites in CSF and/or a white blood cell (WBC) count of more than 5 cells per μl (5). Other parameters, such as intrathecal IgM production could also provide additional information to determine whether the CNS is involved (6, 7).Treatment of HAT patients varies depending on the infecting parasite and the stage of disease (5, 8). S2 drugs in current use, including melarsoprol, eflornithine, and a combination of nifurtimox and eflornithine have several limitations, such as a high rate of toxicity (melarsoprol causes death to 5% of treated patients) (9), complex logistics, and mode of administration (6, 10). Consequently, staging is a vital step in the diagnosis and treatment of HAT. However, the poor specificity or sensitivity of WBC counting and of parasitological techniques for demonstration of parasites in CSF, highlight the need for discovery of better tools for staging the disease.Several attempts have been made during the last decade to identify potential biomarkers able to discriminate between the two stages of sleeping sickness. Most of the efforts focused on cytokines and chemokines, because the patient''s immune system plays a crucial role in the brain pathology (11–14).Proteomic approaches are increasingly being applied in biomedical research and clinical medicine to investigate body fluids as a source of biomarkers (15), including the diagnosis of neurological disorders such as Alzheimer''s disease (16), Parkinson''s disease (17), and multiple sclerosis (18, 19). The protein composition of CSF is strictly regulated and can reflect the physiological or pathological state of the CNS (15). Thus in the present study, we addressed the challenge of staging HAT by analyzing CSF from T. b. gambiense patients using two complementary proteomic strategies: a classical approach based on two-dimensional gel electrophoresis (2-DE), and quantitative mass spectrometry (MS) using isobaric tandem mass tag (TMT) technology (sixplex TMT® MS/MS) (20).     

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods

Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results

CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion

This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.     

Acute insulin infusion decreases plasma ghrelin levels in uncomplicated obesity

BACKGROUND: Plasma ghrelin levels have been shown to decrease after insulin infusion in lean subjects. Nevertheless, the mechanism of the suggested inhibitory effect of insulin on ghrelin is still unclear and no data about the effect of acute insulin infusion on plasma ghrelin concentration in obese subjects are available. OBJECTIVE: We sight to evaluate plasma ghrelin concentration during an hyperinsulinemic euglycemic clamp in uncomplicated obese subjects. METHODS: 35 uncomplicated obese subjects, body mass index (BMI) 43.3+/-10.1 kg/m(2), 33 women and 2 men, mean age 34.9+/-10, with a history of excess fat of at least 10 years underwent euglycemic hyperinsulinemic clamp. Blood samples for ghrelin were performed at baseline and steady state of euglycemic insulin clamp. RESULTS: Ghrelin concentrations decreased over time to 10.6+/-15% (range 2-39%) of baseline, from a mean of 205.53+/-93.79 pg/ml to 179.03+/-70.43 pg/ml during the clamp (95% CI, 10.69 to 36.44, P<0.01). In a univariate linear regression analysis baseline plasma ghrelin levels were inversely correlated to BMI (r=-0.564, P=0.04). A linear positive trend between whole body glucose utilization (M(FFMkg) index) and ghrelin reduction during the clamp was found (chi(2) 3.05, p=0.05). CONCLUSIONS: Our data seem to suggest that hyperinsulinemia during a euglycemic clamp is able to suppress plasma ghrelin concentrations in uncomplicated obesity. This effect appears to be positively related to insulin sensitivity.     

Characterization of a natural variant of human NDP52 and its functional consequences on mitophagy

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing–remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.Subject terms: Autophagy, Molecular modelling, Immunological disorders     

Molecular dynamics of mesophilic-like mutants of a cold-adapted enzyme: insights into distal effects induced by the mutations

Networks and clusters of intramolecular interactions, as well as their "communication" across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted α-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted α-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme.     

Ecological impact of introduced fish in high altitude lakes: a case of study from the European Alps

Fish introduction is a major threat to alpine lake biota leading to the loss of native species and to the degeneration of natural food-webs. This study provides an extensive investigation on the impact of the introduced fish Salvelinus fontinalis on the native communities of alpine lakes in the Gran Paradiso National Park. We compared the macroinvertebrate and zooplankton communities of six stocked and nine fishless lakes with a repeated sampling approach during the summers 2006–2009. The impact of fish presence on alpine lake fauna is often mediated by the strong seasonality governing these ecosystems, and it dramatically affects the faunal assemblage of littoral macroinvertebrates and the size, structure, and composition of the pelagic zooplankton community with a strong selective predation of the more visible taxa. Direct ecological impacts include a decrease or extinction of non-burrower macroinvertebrates and of large zooplankton species, while small zooplankton species and burrower macroinvertebrates were indirectly advantaged by fish presence. Due to the existence of a compensation between rotifers and crustaceans, fish presence does not affect total zooplankton biomass and diversity even if fish are a factor of ecological exclusion for large crustaceans. These compensatory mechanisms are a key process surrounding the impact of introduced fish in alpine lakes.