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As a reference laboratory for measles and rubella surveillance in Lombardy, we evaluated the association between SARS-CoV-2 infection and measles-like syndromes, providing preliminary evidence for undetected early circulation of SARS-CoV-2. Overall, 435 samples from 156 cases were investigated. RNA from oropharyngeal swabs (N = 148) and urine (N = 141) was screened with four hemi-nested PCRs and molecular evidence for SARS-CoV-2 infection was found in 13 subjects. Two of the positive patients were from the pandemic period (2/12, 16.7%, March 2020-March 2021) and 11 were from the pre-pandemic period (11/44, 25%, August 2019-February 2020). Sera (N = 146) were tested for anti-SARS-CoV-2 IgG, IgM, and IgA antibodies. Five of the RNA-positive individuals also had detectable anti-SARS-CoV-2 antibodies. No strong evidence of infection was found in samples collected between August 2018 and July 2019 from 100 patients. The earliest sample with evidence of SARS-CoV-2 RNA was from September 12, 2019, and the positive patient was also positive for anti-SARS-CoV-2 antibodies (IgG and IgM). Mutations typical of B.1 strains previously reported to have emerged in January 2020 (C3037T, C14408T, and A23403G), were identified in samples collected as early as October 2019 in Lombardy. One of these mutations (C14408T) was also identified among sequences downloaded from public databases that were obtained by others from samples collected in Brazil in November 2019. We conclude that a SARS-CoV-2 progenitor capable of producing a measles-like syndrome may have emerged in late June-late July 2019 and that viruses with mutations characterizing B.1 strain may have been spreading globally before the first Wuhan outbreak. Our findings should be complemented by high-throughput sequencing to obtain additional sequence information. We highlight the importance of retrospective surveillance studies in understanding the early dynamics of COVID-19 spread and we encourage other groups to perform retrospective investigations to seek confirmatory proofs of early SARS-CoV-2 circulation.

Background: There are known geographical differences in the epidemiology, clinical presentation, and management of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis. Currently, the pandemic of coronavirus disease 2019 (COVID-19) may provide additional uncertainty. In this retrospective observational study, we evaluated the phenotypes, treatments, and outcomes in Europe, Japan, and the United States (US) using contemporary cohorts of GPA and MPA. Methods: Patients with new-onset or severe relapse of GPA/MPA as classified by the Chapel Hill Consensus Conference classification between January 2020 and July 2020 were included in this study. Patients were enrolled from 7 sites in Europe (72 GPA, 65 MPA), 20 sites in Japan (19 GPA, 54 MPA), and 5 sites in the US (25 GPA, 19 MPA). We assessed baseline characteristics including demographics and clinical presentations, treatment regimens, and glucocorticoid doses in the three regions. End-stage renal disease (ESRD)-free survival at 1 year were compared using the adjusted survival curves. Results: There was no clear gender difference in GPA/MPA throughout the regions, and median age [interquartile range (IQR)] was 65 [57–73], 76 [71–82], and 70 [64–76] in Europe, Japan, and the US, respectively. For each region, patients were numerically older in MPA than GPA. Regarding atypical ANCA-status, that is, MPO-ANCA positivity in GPA (overall, 31 out of 116) was more prevalent in Japan (58%) and the US (44%) than in Europe (13%); PR3-ANCA positivity in MPA (overall, 16 out of 138) was more prevalent in Europe (15%) and the US (21%) than in Japan (3.7%). The frequency of lung involvement in GPA was consistent in each region, ranging from 52–60%, whereas those in MPA were more common in Japan (69%) than in Europe (39%) and the US (32%). The frequency of kidney involvement in GPA was inconsistent: 67% in Europe, 42% in Japan, and 88% in the US, whereas in MPA it was common in almost all patients in all regions. In Europe, Japan, and the US, the frequency of use of cyclophosphamide was 57%, 29%, and 34%, and that of rituximab was 63%, 40%, and 86%, the median oral prednisone dose (mg/day) was 40, 40, and 60 at the beginning and 5, 10, and 5 at 6 months, respectively. In Japan, 13 out of 73 patients (18%) were treated with glucocorticoid alone. The ESRD-free survival rates at 1 year in Europe, Japan, and the US were 78%, 80%, and 81%, respectively, calculated from the covariate-adjusted survival curves. Conclusions: Although the investigator's specialty should be considered, there are still regional differences in remission-induction regimens and glucocorticoid use, perhaps reflecting the diversity of clinical phenotypes of AAV. In the early stages of the COVID-19 pandemic, there was no apparent difference in ESRD-free survival among the three regions. Disclosures: TK received consulting fees from Chugai. AK received consulting fees from Otsuka, Vifor Pharma, UriSalt, Catalyst Biosciences, and Alexion. DG received consulting fees from ChemoCentryx and Aurinia. YK received research grants from Asahi-Kasei and Chugai, and consulting fees from Asahi-Kasei, Chugai, and Pfizer.

Glycosylation is a post-translational modification of proteins in which carbohydrate chains, known asglycans, are linked to lipids or proteins. Many biological proteins are glycosylated, and these glycans are important in the necessary biological functionality of proteins. In this dissertation, the structure/function relationship of highly glycosylated proteins are explored using molecular dynamics simulations and experiment in concert, specifically: 1) the structural & mechanistic explanation of preventing COVID-19 infection with a novel inhaled antiviral compound, 2) the dynamics of the SARS-CoV-2 spike glycoprotein interacting with its cofactors on the host-cell and exploitation of said interaction to develop a rapid antigen test, 3) the mechanism of airborne transmission of the SARS-CoV-2 virion in a respiratory aerosol, and 4) the relationship between glycosylation and functional dynamics of T-cell immunoglobulin and mucin domain containing proteins.

Case investigation and contact tracing (CICT) is a strategy for preventing transmission of infectious diseases, deployed during the COVID-19 pandemic, to identify individuals before becoming symptomatic and/or infectious so they could test and quarantine in order to break chains of transmission. Since May 11, 2020, the UC San Diego Return to Learn program conducted CICT for UCSD students, faculty, and staff. Data collected through contact tracing efforts provides an estimate for adherence to social distancing efforts. Our study aimed to identify the number of close contacts per case as well as factors associated with changes in the number of close contacts. Using available data, cases investigated between July 2020 and April 2021, we examined case characteristics and determined the number of close contacts reported by cases over time. Trends in the number of contacts per case over time were examined using linear regression. Of the 968 cases investigated during this period, 33.9% were White/Middle Eastern, 51.1% were female, and 63.2% were 18-29 years old. Cases were stratified based on university affiliation, in which 56.4% were students (including student employees) and 43.6% were employees of UC San Diego. Findings indicated that the number of close contacts per case had a statistically significant association with age group and student status. Analyses showed an increase in the mean number of close contacts per case over time. Furthermore, we observed that the number of contacts per case increased after stay-at-home orders ended in San Diego county (p<0.001), but did not decrease after business closures and mask mandates were implemented (p=0.76). These findings suggest that changes in time and mitigation protocols (i.e., quarantine) can impact social distancing adherence by proxy of close contacts, which provide insights for future outbreak mitigation efforts and policy planning.

Microbe-host interactions are mediated by protein-carbohydrate binding processes. The microbial adherence to cellular targets is a crucial step in pathogenesis. N-Glycans are oligosaccharides attached to the polypeptide of proteins and can be found on the surface of mammalian cells. Within the respiratory and intestinal tracts, highly glycosylated epithelial cells represent the primary boundary separating embedded host tissues from pathogens. Currently, there are limited methods to comprehensively explore the roles of glycans involved in these infections, therefore both virus and bacterium were employed in this study to investigate this topic. SARS‑CoV‑2, the causative agent for the COVID-19 pandemic, reaches into the respiratory tract, and Salmonella. Typhi can infect the intestinal tract and cause typhoid fever.The cell surface glycome was manipulated via metabolic engineering. N-Glycans on the cell underwent cell membrane extraction, enzymatic release, enrichment and was eventually analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC micro-fluidic chip. Peptides and glycopeptides from host cells were obtained using cell membrane extraction and trypsin digestion. Peptides were desalted by solid-phase extraction with C18 cartridges, whereas glycopeptides were enriched with HILIC. They were then both analyzed using an Orbitrap Fusion Lumos LC-MS/MS system. Viral infection assay and confocal microscopy were employed to explore the effect of glycome on binding with SARS-Cov-2. Bacterial adherence and invasion assays were used to assess bacterial infection capacity with different types of N-glycans dominating the host cell surfaces. We established a cell-based model that enabled us to perform reliable structure-phenotype correlative experiments and compare the effect of individual N-glycan types in microbial infection. Using specific inhibitors, we created host cell surfaces that were primarily fucosylated, sialylated, undecorated, or contained mainly oligomannose structures. All the glycomic cell profiles here were confirmed via Q-TOF LC/MS. Confocal microscopy helped to reinforce the notion that HMOs, such as sialylated structures, can function as decoys to prevent SARS-Cov2 infection. After modifying the host cell glycosylation, binding assays showed the spike protein had a strong affinity towards sialylated N-Glycans. Combined with molecular dynamics simulations, our data further demonstrated that the spike protein, which recognized with host receptors to initiate viral entry, preferentially bound to sialic acids in α2-3 linkage. Bacterial adherence assay illustrated that fucosylated N-glycans on HCT116 cell surface increased significantly in the number of adhered Salmonella. In HCT116 cells, fucosylation was tunable, providing variable amounts of exogenous fucose. We then found that adherence of Salmonella is associated with the abundance of host fucosylated N-glycans. Furthermore, adherence of Salmonella to cells could be blocked by co-incubation with fucose or pretreatment of cells with fucosidase. The results proved that fucose residues on host cells bind with Salmonella. We performed qualitative and quantitative analyses of membrane proteins from host cells. The proteomic analysis showed that the metabolic engineering did not change the abundance of membrane proteins, which indicated that host protein expression did not contribute to the altered adherence. Meanwhile, glycoproteomic analysis yielded site-specific glycopeptide information. The glycopeptides were identified and quantified using a standard glycoproteomic workflow. We found that the attached N-glycans rather than glycosylation sites were manipulated in host cells. These results highlighted the importance of glycans in host-microbe interactions. This study also developed a lectin proximity oxidative labeling (Lectin PROXL) method to identify lectin-binding glycoproteins. The lectin was modified with a probe to produce hydroxide radicals. Thus, the lectin-recognized glycoproteins are oxidized and identified using a conventional proteomic process. All the lectin probes oxidized around 70% of glycoproteins. The specificity and sensitivity of each lectin were assessed utilizing glycomic and glycoproteomic data. Furthermore, the sialic acid- and fucose-binding lectins have higher specificity and sensitivity than other lectins. This approach provides an unprecedented perspective of lectin- glycoprotein interactions and protein networks mediated by distinct glycan types on cell membranes.

EndALL-Virus by Suriya Sundaram In the midst of the real-life coronavirus ordeal, you are in a world where a virus has put humanity on the brink of extinction. The virus has taken everything you love from you. You have nothing left to lose. You are humanity's last hope... will you make the right decisions to save man-kind?

Cells have evolved proteins that detect foreign DNA, RNA, or proteins which then activate cellular pathways to combat bacterial or viral infection.1–4 Apolipoprotein B editing complex 3 (APOBEC3 or A3) is a host cytidine deaminase that deaminates cytidine to uridine in viral DNA in the cytoplasm causing hyper G→A mutation leading to destabilization and degradation of the viral genome.5–8 HIV viral infectivity factor (vif) has evolved to regulate A3 degradation of viral DNA.9 Vif hijacks host ubiquitination machinery to degrade A3s and prevent packaging of A3 into the viral particle.10,11 Vif hijacks the cotranscription factor core factor binding unit beta (CBF-beta), Elongin B (ELOB) and Elongin C (ELOC) to form the VCBC complex.12–15 VCBC binds A3s and Cullin 5 (Cul5) for ubiquitination and degradation of A3 thereby preventing packaging.12,16 Antigen binding fragments (Fabs) were generated against VCBC using a naïve B-cell Fab phage display library to isolate biological tools that are specific for the host-virus interaction.17 Two high affinity Fabs were found to bind at distinct epitopes on VCBC and produced distinct phenotypes when expressed in cells as single chain variable fragments (scFvs).17 The Fab 3C9 shields A3F from ubiquitination and restores packaging of A3F into the viral particle. The Fab 1D1 blocks binding of Cul5 and ubiquitination in vitro. The scFv 1D1 prevented ubiquitination of A3F but did not restore packaging. Affinity purification mass spectroscopy (AP-MS) in HEK293Ts with 3C9 scFv and 1D1 scFv showed different interactomes in the presence of vif. AP-MS with 1D1 scFv did not interact with CBF-beta, an important component of the VCBC complex. Spatial and temporal elucidation of proteins interacting with the complex will further determine events effecting viral infectivity.

Antibodies and antibody-like molecules are broadly used as biochemical reagents and therapeutics. They are highly valued as probes that can distinguish between protein targets with extraordinary molecular detail. A deep understanding of natural antibody structure and function has enabled the development of fully synthetic human antibody libraries for in vitro display. These synthetic libraries recapitulate the sophistication of molecular recognition in antibody complementarity determining regions (CDRs) and in vitro display permits exquisite control over selection conditions. Thus, protein engineers can tailor-make antibodies to bind challenging and non-conventional targets.Here, we describe three examples of protein engineering that leverage synthetic antibody libraries and in vitro display technologies to generate binders to novel epitopes. In Chapter 1, we utilize recombinant antibody pairs to target the post-translational modification (PTM) phosphotyrosine (pY) in folded protein epitopes. In Chapter 2, we develop and deploy a single-domain antibody (sdAb) library to rapidly identify inhibitors to SARS-CoV-2 viral entry. In Chapter 3, we utilize chemo-epitope specific sdAbs to create small-molecule-dependent switches and further engineer them to control cellular therapies.

One of the factors which perpetuates gender inequality is the inequitable division of household labor, and particularly the division of child caretaking labor. Even when women are employed outside the home, many remain primarily responsible for household duties and child caretaking. This research utilizes individual interviews (N=40) with heterosexual, married or cohabitating parents of children 5 and under where fathers do the majority of child caretaking. I explore how lead-dad households are similar to and different from those with a traditional lead-mother arrangement, as well as what motivates lead-father families to choose this arrangement, and how they explain it to others. This project also considers the impact of the COVID-19 pandemic on lead-dad households and whether it has disrupted household roles in these families. This project uses atypical or negative cases to better understand the issue of gender identity of both mothers and fathers in atypical households.