Cysteine Oxidation in Proteins: Structure, Biophysics, and Simulation.

Cysteine side chains can exist in distinct oxidation states depending on the pH and redox potential of the environment, and cysteine oxidation plays important yet complex regulatory roles. Compared with the effects of post-translational modifications such as phosphorylation, the effects of oxidation of cysteine to sulfenic, sulfinic, and sulfonic acid on protein structure and function remain relatively poorly characterized. We present an analysis of the role of cysteine reactivity as a regulatory factor in proteins, emphasizing the interplay between electrostatics and redox potential as key determinants of the resulting oxidation state. A review of current computational approaches suggests underdeveloped areas of research for studying cysteine reactivity through molecular simulations.

The endosymbiont Wolbachia rebounds following antibiotic treatment.

Antibiotic treatment has emerged as a promising strategy to sterilize and kill filarial nematodes due to their dependence on their endosymbiotic bacteria, Wolbachia. Several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity. However, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. Concerns about resistance arising from mass drug administration necessitate a careful exploration of potential Wolbachia recrudescence. In the present study, we investigated the long-term effects of the FDA-approved antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Genome sequence analyses of Wolbachia revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of densely packed Wolbachia within the worm's ovarian tissues were observed by confocal microscopy and remained in worms treated with rifampicin, suggesting that they may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. To our knowledge, these clusters have not been previously described and may be the source of the Wolbachia rebound.

Comparative Performance of Latest-Generation and FDA-Cleared Serology Tests for the Diagnosis of Chagas Disease.

Confirmed diagnosis of chronic Chagas disease (CD) requires positive results by two different IgG serology tests. Variable sensitivity has been reported among tests and in different geographic regions. Inadequate specificity presents a particular challenge in low-prevalence settings such as the United States. This study provides a direct comparison of the latest-generation IgG serology assays with four previously assessed FDA-cleared tests. Seven hundred ten blood donor plasma specimens were evaluated by Wiener Lisado and Wiener v.4.0 enzyme-linked immunosorbent assays (ELISAs) and Abbott PRISM Chagas chemiluminescent assay (ChLIA). Sensitivity and specificity were assessed relative to infection status as determined by the original blood donation testing algorithm. All three latest-generation assays demonstrated 100% specificity (95% confidence interval [CI], 98.6 to 100.0). Wiener Lisado, Wiener v.4.0, and Abbott PRISM had sensitivities of 97.1% (95% CI, 95.1 to 98.4), 98.9% (95% CI, 97.4 to 99.6), and 95.5% (95% CI, 93.2 to 97.3), respectively. As with previously evaluated FDA-cleared tests, all three assays had the highest reactivity and sensitivity in samples from donors born in South America and lowest reactivity and sensitivity in specimens from those born in Mexico, with intermediate results in specimens from Central American donors. Wiener v.4.0 had the highest diagnostic sensitivity in all comparisons. Our findings suggest that the latest-generation CD serology tests could improve diagnostic sensitivity without affecting specificity.

Chagas Disease Serological Test Performance in U.S. Blood Donor Specimens.

Chagas disease affects an estimated 300,000 individuals in the United States. Diagnosis in the chronic phase requires positive results from two different IgG serological tests. Three enzyme-linked immunosorbent assays (ELISAs) (Hemagen, Ortho, and Wiener) and one rapid test (InBios) are FDA cleared, but comparative data in U.S. populations are sparse. We evaluated 500 seropositive and 300 seronegative blood donor plasma samples. Country of birth was known for 255 seropositive specimens, which were grouped into regions as follows: Mexico (n = 94), Central America (n = 88), and South America (n = 73). Specimens were tested by the four FDA-cleared IgG serological assays. Test performance was evaluated by two comparators and latent class analysis. InBios had the highest sensitivity (97.4% to 99.3%) but the lowest specificity (87.5% to 92.3%). Hemagen had the lowest sensitivity (88.0% to 92.0%) but high specificity (99.0% to 100.0%). The level of sensitivity was intermediate for Ortho (92.4% to 96.5%) and Wiener (94.0% to 97.1%); both had high specificity (98.8% to 100.0% and 96.7% to 99.3%, respectively). The levels of antibody reactivity and clinical sensitivity were lowest in donors from Mexico, intermediate in those from Central America, and highest in those from South America. Our findings provide an initial evidence base to improve laboratory diagnosis of Chagas disease in the United States. The best current testing algorithm would employ a high-sensitivity screening test followed by a high-specificity confirmatory test.

Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases.

BACKGROUND: Autosomal dominant mutations in α-synuclein, TDP-43 and tau are thought to predispose to neurodegeneration by enhancing protein aggregation. While a subset of α-synuclein, TDP-43 and tau mutations has been shown to increase the structural propensity of these proteins toward self-association, rates of aggregation are also highly dependent on protein steady state concentrations, which are in large part regulated by their rates of lysosomal degradation. Previous studies have shown that lysosomal proteases operate precisely and not indiscriminately, cleaving their substrates at very specific linear amino acid sequences. With this knowledge, we hypothesized that certain coding mutations in α-synuclein, TDP-43 and tau may lead to increased protein steady state concentrations and eventual aggregation by an alternative mechanism, that is, through disrupting lysosomal protease cleavage recognition motifs and subsequently conferring protease resistance to these proteins. RESULTS: To test this possibility, we first generated comprehensive proteolysis maps containing all of the potential lysosomal protease cleavage sites for α-synuclein, TDP-43 and tau. In silico analyses of these maps indicated that certain mutations would diminish cathepsin cleavage, a prediction we confirmed utilizing in vitro protease assays. We then validated these findings in cell models and induced neurons, demonstrating that mutant forms of α-synuclein, TDP-43 and tau are degraded less efficiently than wild type despite being imported into lysosomes at similar rates. CONCLUSIONS: Together, this study provides evidence that pathogenic mutations in the N-terminal domain of α-synuclein (G51D, A53T), low complexity domain of TDP-43 (A315T, Q331K, M337V) and R1 and R2 domains of tau (K257T, N279K, S305N) directly impair their own lysosomal degradation, altering protein homeostasis and increasing cellular protein concentrations by extending the degradation half-lives of these proteins. These results also point to novel, shared, alternative mechanism by which different forms of neurodegeneration, including synucleinopathies, TDP-43 proteinopathies and tauopathies, may arise. Importantly, they also provide a roadmap for how the upregulation of particular lysosomal proteases could be targeted as potential therapeutics for human neurodegenerative disease.

Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads.

Onchocerciasis and lymphatic filariasis are neglected tropical diseases caused by infection with filarial worms. Annual or biannual mass drug administration with microfilaricidal drugs that kill the microfilarial stages of the parasites has helped reduce infection rates and thus prevent transmission of both infections. However, success depends on high population coverage that is maintained for the duration of the adult worm's lifespan. Given that these filarial worms can live up to 14 years in their human hosts, a macrofilaricidal drug would vastly accelerate elimination efforts. Here, we have evaluated the repurposed drug pyrvinium pamoate as well as newly synthesized analogs of pyrvinium for their efficacy against filarial worms in vitro and in vivo. We found that pyrvinium pamoate, tetrahydropyrvinium and one of the analogs were highly potent in inhibiting worms in in vitro whole-worm screening assays, and that all three compounds reduced female worm fecundity and inhibited embryogenesis in the Brugia pahangi-gerbil in vivo model of infection.