Mitigation of a Coronavirus Disease 2019 Outbreak in a Nursing Home Through Serial Testing of Residents and Staff.

Nursing homes and long-term care facilities represent highly vulnerable environments for respiratory disease outbreaks, such as coronavirus disease 2019 (COVID-19). We describe a COVID-19 outbreak in a nursing home that was rapidly contained by using a universal testing strategy of all residents and nursing home staff.

A Case of Disseminated Histoplasmosis in a Patient with Rheumatoid Arthritis on Abatacept.

Biologic agents are effective treatments for rheumatoid arthritis but are associated with important risks, including severe infections. Tumor Necrosis Factor (TNF) α inhibitors are known to increase the risk of systemic fungal infections such as disseminated histoplasmosis. Abatacept is a biologic agent with a mechanism different from that of TNFα inhibitors: It suppresses cellular immunity by competing for the costimulatory signal on antigen-presenting cells. The risk of disseminated histoplasmosis for patients on abatacept is not known. We report a case of abatacept-associated disseminated histoplasmosis and review the known infectious complications of abatacept. While the safety of resuming biologic agents following treatment for disseminated histoplasmosis is also not known, abatacept is recommended over TNFα inhibitors for rheumatoid arthritis patients with a prior serious infection. We discuss the evidence supporting this recommendation and discuss alternative treatments for rheumatoid arthritis patients with a history of a serious infection.

Rasamsonia argillacea pulmonary and aortic graft infection in an immune-competent patient.

Rasamsonia argillacea (formerly known as Geosmithia argillacea) is a fungus recently recognized as a pathogen of immunocompromised patients. Here we report the first case of Rasamsonia infection in an immunocompetent host, presenting as a pulmonary and aortic graft infection. Its morphological similarity to nonpathogenic Penicillium species delayed the diagnosis and initiation of appropriate treatment.

Active Tuberculosis After Solid Organ Transplantation in Individuals With Negative Pretransplant QuantiFERON-TB Gold Testing: A Case Series.

Active tuberculosis (TB) in solid organ transplant (SOT) recipients most commonly occurs due to reactivation of latent infection and is associated with poor clinical outcomes, including allograft loss and death. National transplant societies, including the American Society of Transplantation, recommend screening for latent TB prior to transplant, with treatment in the peritransplant setting to reduce the subsequent risk of TB reactivation. Though screening is traditionally conducted using laboratory-based assays, such as the QuantiFERON-TB Gold, false negatives may occur in SOT candidates due to anergy from end-stage organ dysfunction, highlighting the need for a multimodal diagnostic approach. In this case series, we describe the clinical characteristics and outcomes of 3 SOT recipients at the University of Pennsylvania with negative pretransplant QuantiFERON-TB Gold testing who subsequently developed active TB in the posttransplant setting, contributing to a growing body of knowledge regarding this challenging population. Each patient experienced a complicated clinical course that arose in part from the lack of diagnosis of TB prior to transplant. Because all had epidemiologic risk factors for TB, the findings of our study highlight the need for more individualized approaches to pretransplant TB screening.

Identification of eukaryotic promoter regulatory elements using nonhomologous random recombination.

Understanding the regulatory logic of a eukaryotic promoter requires the elucidation of the regulatory elements within that promoter. Current experimental or computational methods to discover regulatory motifs within a promoter can be labor intensive and may miss redundant, unprecedented or weakly activating elements. We have developed an unbiased combinatorial approach to rapidly identify new upstream activating sequences (UASs) in a promoter. This approach couples nonhomologous random recombination with an in vivo screen to efficiently identify UASs and does not rely on preconceived hypotheses about promoter regulation or on similarity to known activating sequences. We validated this method using the unfolded protein response (UPR) in yeast and were able to identify both known and potentially novel UASs involved in the UPR. One of the new UASs discovered using this approach implicates Crz1 as a possible activator of Hac1, a transcription factor involved in the UPR. This method has several advantages over existing methods for UAS discovery including its speed, potential generality, sensitivity and lack of false positives and negatives.

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

Directed evolution and substrate specificity profile of homing endonuclease I-SceI.

The laboratory evolution of enzymes with tailor-made DNA cleavage specificities would represent new tools for manipulating genomes and may enhance our understanding of sequence-specific DNA recognition by nucleases. Below we describe the development and successful application of an efficient in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of nontarget sequences. We also applied a previously described in vitro selection method to reveal the comprehensive substrate specificity profile of the wild-type I-SceI homing endonuclease. Together these tools were used to successfully evolve mutant I-SceI homing endonucleases with altered DNA cleavage specificities. The most highly evolved enzyme cleaves the target mutant DNA sequence with a selectivity that is comparable to wild-type I-SceI's preference for its cognate substrate.

Highly sensitive in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity.

We have developed in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity. These selections require only generally accessible equipment, offer high degrees of enrichment of active molecules from mixtures of predominantly inactive species, can be applied to a variety of unrelated proteins, and require approximately 108-fold less material than existing synthetic molecule screening methods. Iterating these selections multiplies the net enrichment of active molecules, enabling enormous overall enrichment factors exceeding 106 to be achieved. Further, the selections can be adapted to select for binding specificity in addition to binding affinity. The application of methods described in this work may play a key role in the discovery of desired molecules from DNA-templated synthetic libraries.

DNA-templated organic synthesis and selection of a library of macrocycles.

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three "codons," into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.

Twisted amides inferred from QSAR analysis of hydrophobicity and electronic effects on the affinity of fluoroaromatic inhibitors of carbonic anhydrase.

QSAR has been used to elucidate the origin of the hydrophobicity and binding affinity of a small library of fluoroaromatic inhibitors of F131V carbonic anhydrase II. Our analysis predicted the presence of a twisted amide conformation for several bound inhibitors, which we confirmed crystallographically. We also determined that the hydrophobicity of the inhibitors as a whole results from the fragment hydrophobicities of their fluorobenzyl rings, corrected for field effects and the presence of an intramolecular F.H contact in solution. The loss of this interaction on binding to the enzyme makes the affinity sensitive to the same terms, but with the opposite dependence on the F.H contact. In the case of the four inhibitors bound as twisted amides, this F.H contact must be retained to some extent in the bound state in order for their affinities to be consistent with our QSAR analysis of the entire set of 17 molecules.