Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent target list. This global survey of HBV integration events, together with recently published whole-genome sequencing analyses, furthered our understanding of the HBV–related HCC.

To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. Spy is unlike the structure of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors.

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The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.The thioredoxin fold is the core scaffold of numerous proteins that control disulfide redox activity in the cell (1-3). These redox proteins share very little sequence homology, but all of them incorporate the four-stranded ␤-sheet, three flanking ␣-helices, and the redox-active CXXC motif of the TRX 5 fold (Fig. 1A). The archetype of the family is thioredoxin (4), a disulfide reductase that maintains a reducing cytosolic environment. Other TRX fold redox proteins include the Dsb proteins (1), which regulate the formation of disulfide bonds in prokaryotes, and protein-disulfide isomerase (5), which catalyzes the oxidation and shuffling of disulfides in the endoplasmic reticulum of eukaryotic cells.This wide range of redox activities of TRX fold proteins is thought to be a consequence of modifications to the common scaffold, which result in different redox properties. Thus, the redox potential of Escherichia coli thioredoxin is very reducing, at Ϫ271 mV (6, 7), whereas that of the oxidizing periplasmic protein E. coli DsbA is Ϫ120 mV (8). Thioredoxin fold proteins that participate in a wide range of thiol disulfide exchange reactions, such as the eukaryotic protein-disulfide isomerases, have intermediate redox potentials (around Ϫ160 mV (9)).Thioredoxin-related proteins provide an attractive model for the study of how protein function is dictated by sequence and three-dimensional structure; this is because their functions are, in part, determined by their redox properties, which in turn, are easy to quantify. For example, mutations in thioredoxin that make its redox potential more oxidative complement null mutations in the oxidase DsbA (10, 11). A detailed understanding of how thioredoxin fold sequence affects redox properties provides an excellent opportunity to relate sequence and function. Previous work has focused on the role of the CXXC "redox rheostat" active site in determining the properties of thioredoxin-related proteins (3,12,13). Experiments that exchange the X-X dipeptide of one thiol-disulfide oxidoreductase with that of another generally result in an oxidoreductase with a redox potential partially shifted in the direction of the oxidoreductase protein that served as the source of the dipepti...