ABSTRACTHistoplasmosis is an endemic mycosis in North America frequently reported along the Ohio and Mississippi River Valleys, although autochthonous cases occur in non-endemic areas. In the United States, the disease is provoked by two genetically distinct clades of Histoplasma capsulatum sensu lato, Histoplasma mississippiense (Nam1) and H. ohiense (Nam2). To bridge the molecular epidemiological gap, we genotyped 93 Histoplasma isolates (62 novel genomes) including clinical, environmental, and veterinarian samples from a broader geographical range by whole-genome sequencing, followed by evolutionary and species niche modelling analyses. We show that histoplasmosis is caused by two major lineages, H. ohiense and H. mississippiense; with sporadic cases caused by H. suramericanum in California and Texas. While H. ohiense is prevalent in eastern states, H. mississipiense was found to be prevalent in the central and western portions of the United States, but also geographically overlapping in some areas suggesting that these species might co-occur. Species Niche Modelling revealed that H. ohiense thrives in places with warmer and drier conditions, while H. mississippiense is endemic to areas with cooler temperatures and more precipitation. In addition, we predicted multiple areas of secondary contact zones where the two species co-occur, potentially facilitating gene exchange and hybridization. This study provides the most comprehensive understanding of the genomic epidemiology of histoplasmosis in the USA and lays a blueprint for the study of invasive fungal diseases.
Evolutionary constraints greatly favor compact genomes that efficiently encode proteins. However, several eukaryotic organisms, including apicomplexan parasites such as Toxoplasma gondii, Plasmodium falciparum and Babesia duncani, the causative agents of toxoplasmosis, malaria and babesiosis, respectively, encode very large proteins, exceeding 20 times their average protein size. Although these large proteins represent <1% of the total protein pool and are generally expressed at low levels, their persistence throughout evolution raises important questions about their functions and possible evolutionary pressures to maintain them. In this study, we examined the trends in gene and protein size, function and expression patterns within seven apicomplexan pathogens. Our analysis revealed that certain large proteins in apicomplexan parasites harbor domains potentially important for functions such as antigenic variation, erythrocyte invasion and immune evasion. However, these domains are not limited to or strictly conserved within large proteins. While some of these proteins are predicted to engage in conventional metabolic pathways within these parasites, others fulfill specialized functions for pathogen-host interactions, nutrient acquisition and overall survival.
Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.
CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.
Coccidioidomycosis, listed as a priority mycosis by the WHO, is endemic in the United States but often overlooked in Central and South America. Employing a multi-institutional approach, we investigate how disease characteristics, pathogen genetic variation, and environmental factors impact coccidioidomycosis epidemiology and outcomes in South America. We identified 292 cases (1978-2021) and 42 outbreaks in Piauí and Maranhão states, Brazil, the largest series outside the US/Mexico epidemic zone. The male-to-female ratio was 57.4:1 and the most common activity was armadillo hunting (91.1%) 4 to 30 days before symptom onset. Most patients (92.8%) exhibited typical acute pulmonary disease, with cough (93%), fever (90%), and chest pain (77%) as predominant symptoms. The case fatality rate was 8%. Our negative binomial regression model indicates that reduced precipitation levels in the current (p = 0.015) and preceding year (p = 0.001) predict heightened incidence. Unlike other hotspots, acidic soil characterizes this region. Brazilian strains differ genomically from other C. posadasii lineages. Northeastern Brazil presents a distinctive coccidioidomycosis profile, with armadillo hunters facing elevated risks. Low annual rainfall emerges as a key factor in increasing cases. A unique C. posadasii lineage in Brazil suggests potential differences in environmental, virulence, and/or pathogenesis traits compared to other Coccidioides genotypes.
The chemical and optical properties of secondary organic aerosols (SOA) have been widely studied through environmental chamber experiments, and some of the results have been parametrized in atmospheric models to help understand their radiative effects and climate influence. While most chamber studies investigate the aerosol formed from a single volatile organic compound (VOC), the potential interactions between reactive intermediates derived from VOC mixtures are not well understood. In this study, we investigated the SOA formed from pure and mixtures of anthropogenic (phenol and 1-methylnaphthalene) and/or biogenic (longifolene) VOCs using continuous-flow, high-NOx photooxidation chamber experiments to better mimic ambient conditions. SOA optical properties, including single scattering albedo (SSA), mass absorption coefficient (MAC), and refractive index (RI) at 375 nm, and chemical composition, including the formation of oxygenated organic compounds, organic-nitrogen compounds (including organonitrates and nitro-organics), and the molecular structure of the major chromophores, were explored. Additionally, the imaginary refractive index values of SOA in the multi-VOC system were predicted using a linear-combination assumption and compared with the measured values. When two VOCs were oxidized simultaneously, we found evidence for changes in SOA chemical composition compared to SOA formed from single-VOC systems, and this change led to nonlinear effects on SOA optical properties. The nonlinear effects were found to vary between different systems.
How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP-effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of dual-purpose peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP-effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP-effector interface.
The total oxidizable precursor (TOP) assay has been extensively used for detecting PFAS pollutants that do not have analytical standards. It uses hydroxyl radicals (HO•) from the heat activation of persulfate under alkaline pH to convert H-containing precursors to perfluoroalkyl carboxylates (PFCAs) for target analysis. However, the current TOP assay oxidation method does not apply to emerging PFAS because (i) many structures do not contain C-H bonds for HO• attack and (ii) the transformation products are not necessarily PFCAs. In this study, we explored the use of classic acidic persulfate digestion, which generates sulfate radicals (SO4-•), to extend the capability of the TOP assay. We examined the oxidation of Nafion-related ether sulfonates that contain C-H or -COO-, characterized the oxidation products, and quantified the F atom balance. The SO4-• oxidation greatly expanded the scope of oxidizable precursors. The transformation was initiated by decarboxylation, followed by various spontaneous steps, such as HF elimination and ester hydrolysis. We further compared the oxidation of legacy fluorotelomers using SO4-• versus HO•. The results suggest novel product distribution patterns, depending on the functional group and oxidant dose. The general trends and strategies were also validated by analyzing a mixture of 100000- or 10000-fold diluted aqueous film-forming foam (containing various fluorotelomer surfactants and organics) and a spiked Nafion precursor. Therefore, (1) the combined use of SO4-• and HO• oxidation, (2) the expanded list of standard chemicals, and (3) further elucidation of SO4-• oxidation mechanisms will provide more critical information to probe emerging PFAS pollutants.
Isomerized amino acid residues have been identified in many peptides extracted from tissues or excretions of humans and animals. These isomerized residues can play key roles by affecting biological activity or by exerting an influence on the process of aging. Isomerization occurs spontaneously and does not result in a mass shift. Thus, identifying and localizing isomerized residues in biological samples is challenging. Herein, we introduce a fast and efficient method using tandem mass spectrometry (MS) to locate isomerized residues in peptides. Although MS2 spectra are useful for identifying peptides that contain an isomerized residue, they cannot reliably localize isomerization sites. We show that this limitation can be overcome by utilizing MS3 experiments to further evaluate each fragment ion from the MS2 stage. Comparison at the MS3 level, utilizing statistical analyses, reveals which MS2 fragments differ between samples and, therefore, must contain the isomerized sites. The approach is similar to previous work relying on ion mobility to discriminate MS2 product ions by collision cross-section. The MS3 approach can be implemented using either ion-trap or beam-type collisional activation and is compatible with the quantification of isomer mixtures when coupled to a calibration curve. The method can also be implemented in combination with liquid chromatography in a targeted approach. Enabling the identification and localization of isomerized residues in peptides with an MS-only methodology will expand accessibility to this important information.
Connected and Autonomous Vehicles (CAV) require positioning that is consistently reliable and accurate. This is achieved through the choice of sensors and the real-time selection of high-quality measurements. Global Navigation Satellite Systems (GNSS) are the foundation to achieve accurate absolute positioning. GNSS Common-mode Errors (CME)mitigation can be realized with Differential GNSS (DGNSS) approach and Precise Point Positioning (PPP) techniques. With the evolution of the International GNSS Service (IGS) Multi-GNSS Experiment (MGEX), Real-time PPP (RT-PPP) corrections for multi-GNSS have only recently become accessible.
GNSS measurements are prone to outliers. This results in an inherent performance versus risk trade-off in CAV state estimation applications. Recently proposed Risk-Averse Performance Specified (RAPS) methods address this trade-off by optimally selecting a subset of measurements to minimize risk while achieving a target performance. The existing RAPS literature presents cases where the performance specification is stated for the full information matrix. However, those methods are not computationally efficient as required for real-time and do not address situations where that specification is infeasible.
This dissertation focuses on the Diagonal Performance-Specified RAPS (DiagRAPS) formulation. This dissertation begins with a review of GNSS measurement models and real-time CME mitigation techniques, such as DGNSS, PPP, and Virtual Network DGNSS (VN-DGNSS). It then develops the theory of DiagRAPS for both binary and non-binary measurement selection variables. Algorithms suitable for real-time applications are proposed within Linear Programming (LP) and Mixed-Integer Linear Programming (ILP) optimization frameworks, achieving polynomial time complexity. The convergence and computation costs of these algorithms are discussed. For binary DiagRAPS, a novel convex reformulation is derived, leading to a globally optimal solution that can be solved using existing tools. Additionally, a soft constraint optimization approach is proposed for situations when the specified performance is unfeasible. Finally, this dissertation evaluates DiagRAPS state estimation approaches using real-world multi-GNSS data from challenging environments for both DGNSS and RT-PPP applications. The results reveal that the locally optimal approach achieves state estimation performance comparable to the global solution. Both binary and non-binary DiagRAPS outperform traditional methods. Notably, the non-binary approach yielded the lowest computation cost and the best overall performance.
