FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL.

NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Betaherpesvirus Virion Assembly and Egress.

Virions are the vehicle for cell-to-cell and host-to-host transmission of viruses. Virions need to be assembled reliably and efficiently, be released from infected cells, survive in the extracellular environment during transmission, recognize and then trigger entry of appropriate target cells, and disassemble in an orderly manner during initiation of a new infection. The betaherpesvirus subfamily includes four human herpesviruses (human cytomegalovirus and human herpesviruses 6A, 6B, and 7), as well as viruses that are the basis of important animal models of infection and immunity. Similar to other herpesviruses, betaherpesvirus virions consist of four main parts (in order from the inside): the genome, capsid, tegument, and envelope. Betaherpesvirus genomes are dsDNA and range in length from ~145 to 240 kb. Virion capsids (or nucleocapsids) are geometrically well-defined vessels that contain one copy of the dsDNA viral genome. The tegument is a collection of several thousand protein and RNA molecules packed into the space between the envelope and the capsid for delivery and immediate activity upon cellular entry at the initiation of an infection. Betaherpesvirus envelopes consist of lipid bilayers studded with virus-encoded glycoproteins; they protect the virion during transmission and mediate virion entry during initiation of new infections. Here, we summarize the mechanisms of betaherpesvirus virion assembly, including how infection modifies, reprograms, hijacks, and otherwise manipulates cellular processes and pathways to produce virion components, assemble the parts into infectious virions, and then transport the nascent virions to the extracellular environment for transmission.

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In†Vivo.

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.

Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species, including M. tuberculosis.

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 µg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.

Machine learning classification by fitting amplicon sequences to existing OTUs.

The ability to use 16S rRNA gene sequence data to train machine learning classification models offers the opportunity to diagnose patients based on the composition of their microbiome. In some applications, the taxonomic resolution that provides the best models may require the use of de novo operational taxonomic units (OTUs) whose composition changes when new data are added. We previously developed a new reference-based approach, OptiFit, that fits new sequence data to existing de novo OTUs without changing the composition of the original OTUs. While OptiFit produces OTUs that are as high quality as de novo OTUs, it is unclear whether this method for fitting new sequence data into existing OTUs will impact the performance of classification models relative to models trained and tested only using de novo OTUs. We used OptiFit to cluster sequences into existing OTUs and evaluated model performance in classifying a dataset containing samples from patients with and without colonic screen relevant neoplasia (SRN). We compared the performance of this model to standard methods including de novo and database-reference-based clustering. We found that using OptiFit performed as well or better in classifying SRNs. OptiFit can streamline the process of classifying new samples by avoiding the need to retrain models using reclustered sequences. IMPORTANCE There is great potential for using microbiome data to aid in diagnosis. A challenge with de novo operational taxonomic unit (OTU)-based classification models is that 16S rRNA gene sequences are often assigned to OTUs based on similarity to other sequences in the dataset. If data are generated from new patients, the old and new sequences must be reclustered to OTUs and the classification model retrained. Yet there is a desire to have a single, validated model that can be widely deployed. To overcome this obstacle, we applied the OptiFit clustering algorithm to fit new sequence data to existing OTUs allowing for reuse of the model. A random forest model implemented using OptiFit performed as well as the traditional reassign and retrain approach. This result shows that it is possible to train and apply machine learning models based on OTU relative abundance data that do not require retraining or the use of a reference database.

Pore structure controls stability and molecular flux in engineered protein cages.

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the Thermotoga maritima encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.

Cryo-EM structure and polymorphism of Aß amyloid fibrils purified from Alzheimer's brain tissue.

The formation of Aß amyloid fibrils is a neuropathological hallmark of Alzheimer's disease and cerebral amyloid angiopathy. However, the structure of Aß amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aß amyloid fibrils from meningeal Alzheimer's brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aß amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aß fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

Infection-Induced Changes Within the Endocytic Recycling Compartment Suggest a Roadmap of Human Cytomegalovirus Egress.

Human cytomegalovirus (HCMV) is an important pathogen in developing fetuses, neonates, and individuals with compromised immune systems. Gaps in our understanding of the mechanisms required for virion assembly stand in the way of development of antivirals targeting late stages of viral replication. During infection, HCMV causes a dramatic reorganization of the host endosecretory system, leading to the formation of the cytoplasmic virion assembly complex (cVAC), the site of virion assembly. As part of cVAC biogenesis, the composition and behavior of endosecretory organelles change. To gain more comprehensive understanding of the impact HCMV infection has on components of the cellular endocytic recycling compartment (ERC), we used previously published transcriptional and proteomic datasets to predict changes in the directionality of ERC trafficking. We identified infection-associated changes in gene expression that suggest shifts in the balance between endocytic and exocytic recycling pathways, leading to formation of a secretory trap within the cVAC. Conversely, there was a corresponding shift favoring outbound secretory vesicle trafficking, indicating a potential role in virion egress. These observations are consistent with previous studies describing sequestration of signaling molecules, such as IL-6, and the synaptic vesicle-like properties of mature HCMV virions. Our analysis enabled development of a refined model incorporating old and new information related to the behavior of the ERC during HCMV replication. While limited by the paucity of integrated systems-level data, the model provides an informed basis for development of experimentally testable hypotheses related to mechanisms involved in HCMV virion maturation and egress. Information from such experiments will provide a robust roadmap for rational development of novel antivirals for HCMV and related viruses.

Physical basis of amyloid fibril polymorphism.

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the ß-sheet twist, as well as peptide-peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-ß fibrils, and suggest that a small number of physical parameters control the observed fibril architectures.

Generation of a novel human cytomegalovirus bacterial artificial chromosome tailored for transduction of exogenous sequences.

The study of herpesviruses, including human cytomegalovirus (HCMV), is complicated by viral genome complexity and inefficient methods for genetic manipulation in tissue culture. Reverse genetics of herpesviruses has been facilitated by propagating their genomes in E. coli as bacterial artificial chromosomes (BACs), which enables complex and precise genetic manipulation using bacterial recombinational systems. Internal capsid volume imposes a strict limit on the length of genome that can be packaged efficiently. This necessitates deletion of presumably nonessential segments of the viral genome to allow for incorporation of the E. coli mini-F plasmid propagation sequence. To avoid deleting viral genes, several BACs utilize a Cre/LoxP system to self-excise the mini-F sequence upon reconstitution of virus in tissue culture. Here, we describe the adaptation of Cre/LoxP to modify the mini-F sequence of the HCMV TB40/E BAC, thus generating a new self-excisable BAC, TB40/E/Cre. After excision of the E. coli propagation sequence, a 2.7 kbp genome length deficit is created due to a preexisting deletion within the US2-US6 coding region. We exploited this deficit and an FKBP12 protein destabilization domain (ddFKBP) to create a novel gene transduction system for studying exogenous proteins during HCMV infection. Using TB40/E/Cre, we: i) found genome length-associated differences in growth and ii) demonstrated its utility as a system capable of efficient transduction of exogenous proteins and regulation of their accumulation over periods as short as 2h. TB40/E/Cre is a powerful tool of broad applicability that can be adapted to study HCMV replication and cell biology in a variety of contexts.