Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ.

14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH2, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3ß phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.

Nanobody assemblies with fully flexible topology enabled by genetically encoded tetrazine amino acids.

Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups-site-specifically inserted by genetic code expansion at Nb surface sites-are compatible with Nb folding and function, enabling Nb assembly at any desired point. Using two anti-SARS-CoV-2 Nbs with viral neutralization ability, we created Nb homo- and heterodimers with improved properties compared with conventionally linked Nb homodimers, which, in the case of our tetrazine-conjugated trimer, translated into enhanced viral neutralization. Thus, this tetrazine-based approach is a generally applicable strategy that greatly increases the accessible range of Nb assembly topologies, and thereby adds the optimization of topology as an effective avenue to generate Nb assemblies with improved efficacy.

Severe Acute Respiratory Syndrome Coronavirus 2 Transmission in a Georgia School District-United States, December 2020-January 2021.

BACKGROUND: To inform prevention strategies, we assessed the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and settings in which transmission occurred in a Georgia public school district. METHODS: During 1 December 2020-22 January 2021, SARS-CoV-2-infected index cases and their close contacts in schools were identified by school and public health officials. For in-school contacts, we assessed symptoms and offered SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) testing; performed epidemiologic investigations and whole-genome sequencing to identify in-school transmission; and calculated secondary attack rate (SAR) by school setting (eg, sports, elementary school classroom), index case role (ie, staff, student), and index case symptomatic status. RESULTS: We identified 86 index cases and 1119 contacts, 688 (61.5%) of whom received testing. Fifty-nine of 679 (8.7%) contacts tested positive; 15 of 86 (17.4%) index cases resulted in ≥2 positive contacts. Among 55 persons testing positive with available symptom data, 31 (56.4%) were asymptomatic. Highest SARs were in indoor, high-contact sports settings (23.8% [95% confidence interval {CI}, 12.7%-33.3%]), staff meetings/lunches (18.2% [95% CI, 4.5%-31.8%]), and elementary school classrooms (9.5% [95% CI, 6.5%-12.5%]). The SAR was higher for staff (13.1% [95% CI, 9.0%-17.2%]) vs student index cases (5.8% [95% CI, 3.6%-8.0%]) and for symptomatic (10.9% [95% CI, 8.1%-13.9%]) vs asymptomatic index cases (3.0% [95% CI, 1.0%-5.5%]). CONCLUSIONS: Indoor sports may pose a risk to the safe operation of in-person learning. Preventing infection in staff members, through measures that include coronavirus disease 2019 vaccination, is critical to reducing in-school transmission. Because many positive contacts were asymptomatic, contact tracing should be paired with testing, regardless of symptoms.

Is Symptom Screening Useful for Identifying COVID-19 Infection in School Settings? Georgia, USA.

This study's goal was to characterize the utility of symptom screening in staff and students for COVID-19 identification and control of transmission in a school setting. We conducted a secondary analysis of cross-sectional data for staff, students and associated household members in a Georgia school district exposed to COVID-19 cases who received RT-PCR testing and symptom monitoring. Among positive contacts, 30/49 (61%) of students and 1/6 (17%) of staff reported no symptoms consistent with COVID-19. Symptom sensitivity was 30% in elementary students and 42% in middle/high students. Fifty-three percent (10/19) of symptomatic positive contacts had at least one household member test positive for SARS-CoV-2 compared with 50% (10/20) of asymptomatic positive contacts. The absence of symptoms in children is not indicative of a lack of SARS-CoV-2 infection or reduced risk of infection for associated household members. Testing all close contacts of people with COVID-19 in schools is needed to interrupt transmission networks.

Clusters of SARS-CoV-2 Infection Among Elementary School Educators and Students in One School District - Georgia, December 2020-January 2021.

In-person learning benefits children and communities (1). Understanding the context in which transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), occurs in schools is critical to improving the safety of in-person learning. During December 1, 2020-January 22, 2021, Cobb and Douglas Public Health (CDPH), the Georgia Department of Public Health (GDPH), and CDC investigated SARS-CoV-2 transmission in eight public elementary schools in a single school district. COVID-19 cases* among educators and students were either self-reported or identified by local public health officials. Close contacts (contacts)† of persons with a COVID-19 case received testing. Among contacts who received positive test results, public health investigators assessed epidemiologic links, probable transmission directionality, and the likelihood of in-school transmission.§ Nine clusters of three or more epidemiologically linked COVID-19 cases were identified involving 13 educators and 32 students at six of the eight elementary schools. Two clusters involved probable educator-to-educator transmission that was followed by educator-to-student transmission and resulted in approximately one half (15 of 31) of school-associated cases. Sixty-nine household members of persons with school-associated cases were tested, and 18 (26%) received positive results. All nine transmission clusters involved less than ideal physical distancing, and five involved inadequate mask use by students. Educators were central to in-school transmission networks. Multifaceted mitigation measures in schools, including promotion of COVID-19 precautions outside of school, minimizing in-person adult interactions at school, and ensuring universal and correct mask use and physical distancing among educators and students when in-person interaction is unavoidable, are important in preventing in-school transmission of SARS-CoV-2. Although not required for reopening schools, COVID-19 vaccination should be considered as an additional mitigation measure to be added when available.