More Than Results: The Clinical and Research Relationship in the Evolving Detection and Surveillance of SARS-CoV-2.

INTRODUCTION: During the coronavirus disease 2019 (COVID-19) pandemic, real-time reverse transcription polymerase chain reaction (RT-PCR) became an essential tool for laboratories to provide high-sensitivity qualitative diagnostic testing for patients and real-time data to public health officials. Here we explore the predictive value of quantitative data from RT-PCR cycle threshold (Ct) values in epidemiological measures, symptom presentation, and variant transition. METHODS: To examine the association with hospitalizations and deaths, data from 74,479 patients referred to the Avera Institute for Human Genetics (AIHG) for COVID-19 testing in 2020 were matched by calendar week to epidemiological data reported by the South Dakota Department of Health. We explored the association between symptom data, patient age, and Ct values for 101 patients. We also explored changes in Ct values during variant transition detected by genomic surveillance sequencing of the AIHG testing population during 2021. RESULTS: Measures from AIHG diagnostic testing strongly explain variance in the South Dakota state positivity percentage (R2 = 0.758), a two-week delay in hospitalizations (R2 = 0.856), and a four-week delay in deaths (R2 = 0.854). Based on factor analysis of patient symptoms, three groups could be distinguished which had different presentations of age, Ct value, and time from collection. Additionally, conflicting Ct value results among SARSCoV- 2 variants during variant transition may reflect the community transmission dynamics. CONCLUSIONS: Measures of Ct value in RT-PCR diagnostic assays combined with routine screening have valuable applications in monitoring the dynamics of SARS-CoV-2 within communities.

Pathogenesis of Tobacco-Associated Lung Adenocarcinoma Is Closely Coupled with Changes in the Gut and Lung Microbiomes.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a-/- mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a-/- mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

B cell-dependent subtypes and treatment-based immune correlates to survival in stage 3 and 4 lung adenocarcinomas.

Lung cancer is the leading cause of cancer-related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non-small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late-stage NSCLC. The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA-Seq) with deconvolution of RNA-Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B-cell rich patient group had increased expression of CXCL13 and greater abundance of PD1+ CD8 T cells. The presence of B cells and PD1+ CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS). We then assessed the predictive and prognostic utility of these cell types and TLS within publicly available stage 3 and 4 lung adenocarcinoma (LUAD) RNA-Seq datasets. As previously described by others, pre-treatment expression of intratumoral 12-chemokine TLS gene signature is associated with progression free survival (PFS) in patients who receive treatment with immune checkpoint inhibitors (ICI). Notably and unexpectedly pre-treatment percentages of intratumoral B cells are associated with PFS in patients who receive surgery, chemotherapy, or radiation. Further studies to confirm these findings would allow for more effective patient selection for both ICI and non-ICI treatments.

VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P(2) in yeast and mouse.

The signalling lipid PI(3,5)P(2) is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P(2) levels. Mutations that lower PI(3,5)P(2) cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P(2) was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P(2) regulatory complex by direct contact with the known regulators of PI(3,5)P(2): Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P(2) regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P(2). Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.

Critical role of 7,8-didemethyl-8-hydroxy-5-deazariboflavin for photoreactivation in Chlamydomonas reinhardtii.

DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.

Design and Development of a Behaviorally Active Recombinant Neurotrophic Factor.

INTRODUCTION: Carbamoylated erythropoietin (CEPO) is a chemically engineered, nonhematopoietic derivative of erythropoietin (EPO) that retains its antidepressant and pro-cognitive effects, which are attributed to the increased expression of neurotrophic factors like brain derived neurotrophic factor (BDNF), in the central nervous system. However, the chemical modification process which produces CEPO from erythropoietin (EPO) requires pure EPO as raw material, is challenging to scale-up and can also cause batch-to-batch variability. To address these key limitations while retaining its behavioral effects, we designed, expressed and analyzed a triple, glutamine, substitution recombinant mimetic of CEPO, named QPO. METHODS AND MATERIALS: We employ a combination of computational structural biology, molecular, cellular and behavioral assays to design, produce, purify and test QPO. RESULTS: QPO was shown to be a nonhematopoietic polypeptide with significant antidepressant-like and pro-cognitive behavioral effects in rodent assays while significantly upregulating BDNF expression in-vitro and in-vivo. The in-silico binding affinity analysis of QPO bound to the EPOR/EPOR homodimer receptor shows significantly decreased binding to Active Site 2, but not Active Site 1, of EPOR. DISCUSSION: The results of the behavioral and gene expression analysis imply that QPO is a successful CEPO mimetic protein and potentially acts via a similar neurotrophic mechanism, making it a drug development target for psychiatric disorders. The decreased binding to Active Site 2 could imply that this active site is not involved in neuroactive signaling and could allow the development of a functional innate repair receptor (IRR) model. Substituting the three glutamine substitution residues with arginine (RPO) resulted in the loss of behavioral activity, indicating the importance of glutamine residues at those positions.

An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels.

Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2 Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

Relative Telomere Repeat Mass in Buccal and Leukocyte-Derived DNA.

Telomere length has garnered interest due to the potential role it may play as a biomarker for the cellular aging process. Telomere measurements obtained from blood-derived DNA are often used in epidemiological studies. However, the invasive nature of blood draws severely limits sample collection, particularly with children. Buccal cells are commonly sampled for DNA isolation and thus may present a non-invasive alternative for telomere measurement. Buccal and leukocyte derived DNA obtained from samples collected at the same time period were analyzed for telomere repeat mass (TRM). TRM was measured in buccal-derived DNA samples from individuals for whom previous TRM data from blood samples existed. TRM measurement was performed by qPCR and was normalized to the single copy 36B4 gene relative to a reference DNA sample (K562). Correlations between TRM from blood and buccal DNA were obtained and also between the same blood DNA samples measured in separate laboratories. Using the classical twin design, TRM heritability was estimated (N = 1892, MZ = 1044, DZ = 775). Buccal samples measured for TRM showed a significant correlation with the blood-1 (R = 0.39, p < 0.01) and blood-2 (R = 0.36, p < 0.01) samples. Sex and age effects were observed within the buccal samples as is the norm within blood-derived DNA. The buccal, blood-1, and blood-2 measurements generated heritability estimates of 23.3%, 47.6% and 22.2%, respectively. Buccal derived DNA provides a valid source for the determination of TRM, paving the way for non-invasive projects, such as longitudinal studies in children.

The UVS9 gene of Chlamydomonas encodes an XPG homolog with a new conserved domain.

Nucleotide excision repair (NER) is a key pathway for removing DNA damage that destabilizes the DNA double helix. During NER a protein complex coordinates to cleave the damaged DNA strand on both sides of the damage. The resulting lesion-containing oligonucleotide is displaced from the DNA and a replacement strand is synthesized using the undamaged strand as template. Ultraviolet (UV) light is known to induce two primary forms of DNA damage, the cyclobutane pyrimidine dimer and the 6-4 photoproduct, both of which destabilize the DNA double helix. The uvs9 strain of Chlamydomonas reinhardtii was isolated based on its sensitivity to UV light and was subsequently shown to have a defect in NER. In this work, the UVS9 gene was cloned through molecular mapping and shown to encode a homolog of XPG, the structure-specific nuclease responsible for cleaving damaged DNA strands 3' to sites of damage during NER. 3' RACE revealed that the UVS9 transcript is alternatively polyadenylated. The predicted UVS9 protein is nearly twice as long as other XPG homologs, primarily due to an unusually long spacer region. Despite this difference, amino acid sequence alignment of UVS9p with XPG homologs revealed a new conserved domain involved in TFIIH interaction.

Association of intracellular proteins with folded major histocompatibility complex class I molecules.

The major histocompatibility complex (MHC) class I molecule is responsible for presenting peptide antigens at the cell surface for recognition by cytotoxic T lymphocytes. Several chaperone molecules interact with the MHC class I heavy chain and release when the MHC groove folds around peptide. Two additional proteins, invariant chain and amyloid precursor-like protein 2 (APLP2), interact specifically and stably with MHC class I molecules that have folded peptide-binding grooves. Invariant chain and APLP2 also affect MHC class I cell-surface expression, and so may play a part in MHC class I trafficking. Association of APLP2 with the MHC class I molecule appears to be regulated by a viral protein, the adenovirus E3/19K protein. Analysis of the interactions of these proteins with each other and with MHC class I will clarify how presentation of antigens by MHC class I is controlled by events that occur subsequent to MHC class I folding.

Effect of linkage of transduction domain sequences to a lymphoma idiotype DNA vaccine on vaccine effectiveness.

Patient idiotype-specific vaccines for treatment of non-Hodgkin's lymphoma have shown promise in clinical trials, encouraging efforts to enhance the effectiveness of idiotype vaccines further. It has previously been found that for some other types of experimental vaccines, the addition of transduction domains has improved vaccine immunogenicity. Transduction domains are short amino acid sequences that are capable of increasing transport through cellular membranes. In this study, we tested murine B cell 38C13 lymphoma idiotype DNA vaccines with human immunodeficiency virus (HIV) Tat-derived transduction sequences for efficacy against 38C13 challenge. The rate of tumor onset was similar for the idiotype and transduction domain-conjugated idiotype vaccine groups. At days 22-23 postchallenge, the number of surviving mice was significantly higher in the group that had received a DNA vaccine consisting of the 38C13 idiotype sequence plus modified Tat transduction sequence, in comparison with the group that received idiotype-only vaccines. Although the overall survival difference was not statistically significant following day 24, a trend toward an increased survival rate for mice receiving idiotype plus Tat-derived transduction domains was maintained through day 106 postchallenge. Thus, the addition to idiotype vaccines of specific sequences that facilitate intracellular transport may have potential to improve the effectiveness of such vaccines.

A charged amino acid residue in the transmembrane/cytoplasmic region of tapasin influences MHC class I assembly and maturation.

Tapasin influences the quantity and quality of MHC/peptide complexes at the cell surface; however, little is understood about the structural features that underlie its effects. Because tapasin, MHC class I, and TAP are transmembrane proteins, the tapasin transmembrane/cytoplasmic region has the potential to affect interactions at the endoplasmic reticulum membrane. In this study, we have assessed the influence of a conserved lysine at position 408, which lies in the tapasin transmembrane/cytoplasmic domain. We found that substitutions at position K408 in tapasin affected the expression of MHC class I molecules at the cell surface, and down-regulated tapasin stabilization of TAP. In addition to affecting TAP interaction with tapasin, the substitution of alanine, but not tryptophan, for the lysine at tapasin position 408 increased the amount of tapasin found in association with the open, peptide-free form of the HLA-B8 H chain. Tapasin K408A was also associated with more folded, beta(2)-microglobulin-assembled HLA-B8 molecules than wild-type tapasin. Consistent with our observation of a large pool of tapasin K408A-associated HLA-B8 molecules, the rate at which HLA-B8 migrated from the endoplasmic reticulum was slower in tapasin K408A-expressing cells than in wild-type tapasin-expressing cells. Thus, the alanine substitution at position 408 in tapasin may interfere with the stable acquisition by MHC class I molecules of peptides that are sufficiently optimal to allow MHC class I release from tapasin.

REX1, a novel gene required for DNA repair.

Nucleotide excision repair is a major pathway for repairing UV light-induced DNA damage in most organisms. Using insertional mutagenesis, we isolated a UV-sensitive mutant of Chlamydomonas reinhardtii that is blocked in the excision of cyclobutane pyrimidine dimers. The mutant is also sensitive to the alkylating agent, methyl methanesulphonate. We have cloned REX1, a novel gene that rescues the mutant. The gene is unusual in a eukaryotic organism in that it is predicted to encode two different proteins, a small protein (8.9 kDa) and a larger protein (31.8 kDa). Neither protein is homologous to known DNA repair proteins. Partial complementation is achieved with subclones of the gene encoding only the 8.9-kDa protein. The 8.9-kDa protein has homologues in many organisms including Saccharomyces cerevisiae, Arabidopsis, and humans. The 31.8-kDa protein appears to be less conserved. These findings may be of general importance for DNA repair in other organisms.

The amyloid precursor-like protein 2 and the adenoviral E3/19K protein both bind to a conformational site on H-2Kd and regulate H-2Kd expression.

A protein of unknown physiological function, called amyloid precursor-like protein 2 (APLP2), forms an association with the murine class I molecule K(d) that is up-regulated by the presence of the adenoviral protein E3/19K. We have extended these findings to show that APLP2 and E3/19K associate preferentially with folded K(d) and not with the open form. APLP2 was detectable at the cell surface, but its surface expression was not up-regulated by the concurrent expression of K(d). Experimental down-regulation of APLP2 expression caused a consistent increase in the surface expression of K(d), indicating that APLP2 normally reduces K(d) surface expression. These data suggest a role for APLP2 in controlling the maturation of major histocompatibility complex class I molecules.

The Ig-like domain of tapasin influences intermolecular interactions.

Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L(d)/tapasin association can be segregated from reconstitution of folded L(d) surface expression. This finding suggests that peptide acquisition by L(d) is influenced by tapasin functions that are independent of L(d) binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L(d), and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L(d). These data are consistent with a higher overall affinity between L(d) and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin's stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin's electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.