Background: Weeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called Multisystem Inflammatory Syndrome in Children (MIS-C).Gastrointestinal symptoms are common in MIS-C patients and severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not previously been identified.Methods: Here, we analyzed biospecimens from 100 children: 19 children with MIS-C, 26 with acute COVID-19, and 55 controls. Stool was assessed for SARS-CoV-2 by RT-PCR and plasma was assessed for markers of breakdown of mucosal barrier integrity, including zonulin.Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As proof of concept, we treated a MIS-C patient with larazotide, a zonulin antagonist, and monitored impact on antigenemia and clinical response. Results:We showed that in MIS-C, prolonged presence of SARS-CoV-2 in the GI tract leads to release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The MIS-C patient treated with larazotide displayed a coinciding decrease in plasma SARS-CoV-2 Spike antigen levels, inflammatory markers, and a resultant clinical improvement above that achieved with currently available treatments. Conclusion:These mechanistic data of MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

Here we describe TRACE (T7 polymerase-driven continuous editing), a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase. TRACE induces high rates of mutagenesis over multiple cell generations in genes Reprints and permissions information is available at www.nature.com/reprints.

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease-of-readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a parallelized manner. Here, we report a multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesis to light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with video game music, which is equivalent to 84 trits or 110 bits of data.

Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals.U sing Single Molecule Arrays( Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibodymediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in ab iosafety level 2l aboratory within two hours.W eu sed this assayt oa ssess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for as hort period of time and showthat neutralization and antibody levels increase over time.Wealso adapted the assayfor SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy,C OVID-19 infected, and vaccinated individuals.T his assayi sh ighly adaptable for clinical applications,s uch as vaccine development and epidemiological studies.

Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals.U sing Single Molecule Arrays( Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibodymediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in ab iosafety level 2l aboratory within two hours.W eu sed this assayt oa ssess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for as hort period of time and showthat neutralization and antibody levels increase over time.Wealso adapted the assayfor SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy,C OVID-19 infected, and vaccinated individuals.T his assayi sh ighly adaptable for clinical applications,s uch as vaccine development and epidemiological studies.

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease of readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a highly parallelized manner. Here, we report a novel multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co 2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesisto light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with music from the 1985 Nintendo video game Super Mario Brothers TM , which is equivalent to 84 trits or 110 bits of data.In the era of Big Data, molecular DNA has become an increasingly attractive medium for the storage and archiving of digital data 1-6 . This is primarily attributed to its ultra-high storage density, which is currently estimated to be in the hundreds of petabytes per gram DNA 7 . The attractiveness of DNA as a data storage medium is additionally bolstered by its durability, longevity and energy efficiency compared to counterpart storage mediums; both analog and digital 8,9 . However, for storing a meaningful volume of digital data, the synthesis of many unique DNA sequences at long lengths is required. While advances in array-based Oligonucleotide Library Synthesis (OLS) technology have enabled highly multiplexed DNA oligonucleotide synthesis, production in this format still relies on decades old phosphoramidite chemical synthesis methods 10 . Many time-consuming steps of expensive and harsh reactions with the accumulation of toxic by-products greatly limit chemical synthesis as the demand for longer and larger quantities of DNA oligonucleotides increases 11 . 3 Recently, the use of terminal deoxynucleotidyl transferase (TdT), a template-independent polymerase, to synthesize DNA oligonucleotides was shown to be a promising alternative to chemical synthesis [12][13][14] . Because synthesis reactions are performed under aqueous conditions, many limiting aspects of the phosphoramidite chemistry can be circumvented or improved upon. This would be ideal for digital data storage in DNA; however, due to the natural promiscuity of TdT, controlling the enzyme in a sequencespecific manner is challenging [15][16][17] . In order to overcome this challenge, a recent study showed controlled TdT extension activity with apyrase by degrading free nucleotides needed for synthesis 14 .Incubation with optimized ratios of apyrase, TdT and nucleotide over multiple cycles resulted in the successful synthesis of several DNA oligonucleot...