Whole Genome Assembly of a Hybrid Trypanosoma cruzi Strain Assembled with Nanopore Sequencing Alone.

Trypanosoma cruzi is the causative agent of Chagas disease, which causes 10,000 deaths per year. Despite the high mortality associated with Chagas, relatively few parasite genomes have been assembled to date, with genome assemblies unavailable even for some commonly used laboratory strains. This is at least partially due to T. cruzi's highly complex and highly repetitive genome, which defies investigation using traditional short read sequencing methods. Here, we have generated a high-quality whole genome assembly of the hybrid Tulahuen strain, a commercially available Type VI strain, using long read Nanopore sequencing without short read scaffolding. The assembled genome contains 25% repeat regions, 17% variable multigene family members, and 27% transposable elements and is of comparable quality to T. cruzi genome assemblies that utilized both long and short read data. Notably, we find that regions with transposable elements are significantly enriched for multicopy surface proteins, and that surface proteins are, on average, closer to transposable elements than other coding regions. This finding suggests that mobile genetic elements such as transposons may drive recombination within surface protein gene families. This work demonstrates the feasibility of nanopore sequencing to resolve complex regions of T. cruzi genomes, and with these resolved regions, provides support for a possible mechanism for genomic diversification.

Whole Genome Assembly of a Hybrid Trypanosoma cruzi Strain Assembled with Nanopore Sequencing Alone.

Trypanosoma cruzi is the causative agent of Chagas disease, which causes 10,000 deaths per year. Despite the high mortality caused by the pathogen, relatively few parasite genomes have been assembled to date; even some commonly used laboratory strains do not have publicly available genome assemblies. This is at least partially due to T. cruzi's highly complex and highly repetitive genome: while describing the variation in genome content and structure is critical to better understanding T. cruzi biology and the mechanisms that underlie Chagas disease, the complexity of the genome defies investigation using traditional short read sequencing methods. Here, we have generated a high-quality whole genome assembly of the hybrid Tulahuen strain, a commercially available Type VI strain, using long read Nanopore sequencing without short read scaffolding. Using automated tools and manual curation for annotation, we report a genome with 25% repeat regions, 17% variable multigene family members, and 27% transposable elements. Notably, we find that regions with transposable elements are significantly enriched for surface proteins, and that on average surface proteins are closer to transposable elements compared to other coding regions. This finding supports a possible mechanism for diversification of surface proteins in which mobile genetic elements such as transposons facilitate recombination within the gene family. This work demonstrates the feasibility of nanopore sequencing to resolve complex regions of T. cruzi genomes, and with these resolved regions, provides support for a possible mechanism for genomic diversification.

Amplicon Sequencing Reveals Complex Infection in Infants Congenitally Infected With Trypanosoma Cruzi and Informs the Dynamics of Parasite Transmission.

Congenital transmission of Trypanosoma cruzi is an important source of new Chagas infections worldwide. The mechanisms of congenital transmission remain poorly understood, but there is evidence that parasite factors are involved. Investigating changes in parasite strain diversity during transmission could provide insight into the parasite factors that influence the process. Here we use amplicon sequencing of a single copy T. cruzi gene to evaluate the diversity of infection in clinical samples from Chagas positive mothers and their infected infants. Several infants and mothers were infected with multiple parasite strains, mostly of the same TcV lineage, and parasite strain diversity was higher in infants than mothers. Two parasite haplotypes were detected exclusively in infant samples, while one haplotype was never found in infants. Together, these data suggest multiple parasites initiate a congenital infection and that parasite factors influence the probability of vertical transmission.

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood.

Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3-11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area.

We report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seropositivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors. We additionally describe the longitudinal dynamics of immunoglobulin-G, immunoglobulin-M, and in vitro neutralizing antibody titers in COVID-19 patients. Neutralizing antibodies rise in tandem with immunoglobulin levels following symptom onset, exhibiting median time to seroconversion within one day of each other, and there is >93% positive percent agreement between detection of immunoglobulin-G and neutralizing titers.

Predicted Structural Variability of Mycobacterium tuberculosis PPE18 Protein With Immunological Implications Among Clinical Strains.

Recent advancements in vaccinology have led to the development of the M72/AS01E subunit vaccine, of which the major component is the Mycobacterium tuberculosis (MTB) PPE18 protein. Previous studies have demonstrated the genetic variability of the gene encoding PPE18 protein and the resulting peptide changes in diverse clinical strains of MTB; however, none have modeled the structural changes resulting from these peptide changes and their immunological implications. In this study, we investigated the structural predictions of 29 variant PPE18 proteins previously reported. We found evidence that PPE18 is at least a two-domain protein, with a highly conserved first domain and a largely variable second domain that has different coevolutionary clusters. Further, we investigated putative epitope sites in the clinical variants of PPE18 using prediction software. We found a negative relationship between T-cell epitope number and residue variability, while B-cell epitope likelihood was positively correlated with residue variability. Moreover, we found far more residues in the second domain predicted to be B-cell epitopes compared with the first domain. These results suggest an important functional role of the first domain and a role in immune evasion for the second, which extends our knowledge base of the basic biology of the PPE18 protein and indicates the need for further study into non-traditional immunological responses to TB.

Sexual Dimorphism in Response to an NRF2 Inducer in a Model for Pachyonychia Congenita.

Sex is an influential factor regarding pathophysiology and therapeutic response in human disease. Pachyonychia congenita is caused by mutations in keratin genes and typified by dystrophic lesions affecting nails, glands, oral mucosa, and palmar-plantar epidermis. Painful palmar-plantar keratoderma (PPK) severely impairs mobility in pachyonychia congenita. Mice genetically null for keratin 16 (Krt16), one of the genes mutated in pachyonychia congenita, develop pachyonychia congenita-like PPK. In male Krt16-/- mice, oxidative stress associated with impaired glutathione synthesis and nuclear factor erythroid-derived 2 related factor 2 (NRF2)-dependent gene expression precedes PPK onset, which can be prevented by topical sulforaphane-mediated activation of NRF2. We report here that sulforaphane treatment fails to activate NRF2 and prevent PPK in female Krt16-/- mice despite a similar set of molecular circumstances. Follow-up studies reveal a temporal shift in PPK onset in Krt16-/- females, coinciding with sex-specific fluctuations in footpad skin glutathione levels. Dual treatment with sulforaphane and diarylpropionitrile, an estrogen receptor beta selective agonist, results in NRF2 activation, normalization of glutathione levels, and prevention of PPK in female Krt16-/- mice. These findings point to a sex difference in NRF2 responsiveness that needs be considered when exploring NRF2 as a therapeutic target in skin disorders.

Randomized, split-body, single-blinded clinical trial of topical broccoli sprout extract: Assessing the feasibility of its use in keratin-based disorders.

BACKGROUND: Epidermolysis bullosa simplex is a skin-blistering disorder caused by mutations in keratin (K)14 or K5. Treatment with nuclear factor (erythroid-derived 2)-like 2 inducer sulforaphane ameliorated skin blistering in Krt14-null mice, correlating with induction of K17. To be therapeutically useful for epidermolysis bullosa simplex, topical broccoli sprout extract (BSE), enriched for sulforaphane, would ideally induce the expression of homologous keratins (eg, K6, K17, K16) in the basal layer of human epidermis without impacting expression of defective keratins (K5/K14). OBJECTIVE: The purpose of this 1-week, randomized, split-body, single-blinded, placebo-controlled trial was to assess the impact of BSE on keratin expression. METHODS: Five subjects (34-71 years old) applied BSE (500 nmol of sulforaphane/mL) or vehicle alone to the inner aspect of the arm daily. Expression of keratin, nuclear factor (erythroid-derived 2)-like 2, and other markers was assessed using reverse transcription-polymerase chain reaction and indirect immunofluorescence. RESULTS: One subject (age 71 years) was excluded a posteriori because of poor tissue quality. Topical BSE activated nuclear factor (erythroid-derived 2)-like 2 and up-regulated K17 in the epidermis of all subjects, had variable effects on K16 and K6 expression, and did not alter expression of K14 or K5. LIMITATIONS: Small sample size is a limitation. CONCLUSION: BSE represents an attractive therapeutic candidate for K14-associated epidermolysis bullosa simplex.

Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes.

Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16-/- mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid-derived 2 related factor 2-dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16-/- mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16-/- footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16-/- mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment.