Genomically recoded with optimized functional phenotypes.

Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, C321.∆A.

Universal Genetic Testing for Newly Diagnosed Invasive Breast Cancer.

Importance: Between 5% and 10% of breast cancer cases are associated with an inherited germline pathogenic or likely pathogenic variant (GPV) in a breast cancer susceptibility gene (BCSG), which could alter local and systemic therapy recommendations. Traditional genetic testing criteria misses a proportion of these cases.

Objective: To evaluate the prevalence and clinicopathological associations of GPVs in 2 groups of BCSGs among an ethnically diverse cohort of women with newly diagnosed breast cancer.

Elastocapillary effects determine early matrix deformation by glioblastoma cell spheroids.

During cancer pathogenesis, cell-generated mechanical stresses lead to dramatic alterations in the mechanical and organizational properties of the extracellular matrix (ECM). To date, contraction of the ECM is largely attributed to local mechanical stresses generated during cell invasion, but the impact of "elastocapillary" effects from surface tension on the tumor periphery has not been examined. Here, we embed glioblastoma cell spheroids within collagen gels, as a model of tumors within the ECM.

SPAK-dependent cotransporter activity mediates capillary adhesion and pressure during glioblastoma migration in confined spaces.

The invasive potential of glioblastoma cells is attributed to large changes in pressure and volume, driven by diverse elements, including the cytoskeleton and ion cotransporters.  However, how the cell actuates changes in pressure and volume in confinement, and how these changes contribute to invasive motion is unclear. Here, we inhibited SPAK activity, with known impacts on the cytoskeleton and cotransporter activity and explored its role on the migration of glioblastoma cells in confining microchannels to model invasive spread through brain tissue.

System-wide optimization of an orthogonal translation system with enhanced biological tolerance.

Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site-specifically incorporate biologically relevant non-standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post-translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems-level biology of OTS development and utilization remains underexplored.

Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine.

Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production.

A multi-purpose, regenerable, proteome-scale, human phosphoserine resource for phosphoproteomics.

Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation.

Cell type-independent profiling of interactions between intracellular pathogens and the human phosphoproteome.

Interactions between proteins from intracellular pathogens and host proteins in an infected cell are often mediated by post-translational modifications encoded in the host proteome. Identifying protein modifications, such as phosphorylation, that dictate these interactions remains a defining challenge in unraveling the molecular mechanisms of pathogenesis. We have developed a platform in engineered bacteria that displays over 110,000 phosphorylated human proteins coupled to a fluorescent reporter system capable of identifying the host-pathogen interactome of phosphoproteins (H-PIP).

An Unusual Enteric Yolk Sac Tumor: First Report of an Ovarian Germ Cell Tumor Associated With a Germline Pathogenic Variant in DICER1.

A variety of unusual tumors are associated with both germline and somatic DICER1 pathogenic variants (PVs), including, in the female genital tract, embryonal rhabdomyosarcoma at various sites and ovarian Sertoli-Leydig cell tumor. There have been occasional reported cases of ovarian germ cell tumors [mainly yolk sac tumor (YST)] harboring DICER1 PVs but, as far as we are aware, none of these has been proven to have a germline provenance. We report an unusual enteric variant of ovarian YST in a 28-yr-old woman associated with a germline PV c.

Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria.

Alcohol as friend or foe in autoimmune diseases: a role for gut microbiome?

Alcohol is well known for promoting systemic inflammation and aggravating multiple chronic health conditions. Thus, alcohol may also be expected to serve as a risk factor in autoimmune diseases. However, emerging data from human and animal studies suggest that alcohol may in fact be protective in autoimmune diseases.

Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response.

Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence.

Ultrafast electron diffraction from nanophotonic waveforms via dynamical Aharonov-Bohm phases.

Electron interferometry via phase-contrast microscopy, holography, or picodiffraction can provide a direct visualization of the static electric and magnetic fields inside or around a material at subatomic precision, but understanding the electromagnetic origin of light-matter interaction requires time resolution as well. Here, we demonstrate that pump-probe electron diffraction with all-optically compressed electron pulses can capture dynamic electromagnetic potentials in a nanophotonic material with sub-light-cycle time resolution via centrosymmetry-violating Bragg spot dynamics. The origin of this effect is a sizable quantum mechanical phase shift that the electron de Broglie wave obtains from the oscillating electromagnetic potentials within less than 1 fs.

The mechanism of β--methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

β--methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking.

Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome.

Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated.

Alcohol shifts gut microbial networks and ameliorates a murine model of neuroinflammation in a sex-specific pattern.

Alcohol is a widely consumed dietary component by patients with autoimmune neuroinflammatory diseases, but current evidence on the effects of alcohol in these conditions is confounding. Epidemiological studies suggest moderate consumption of alcohol may be protective in some autoimmune diseases; however, this correlation has not been directly investigated. Here, we characterize the effects of moderate-dose alcohol in a model system of autoimmune neuroinflammation, experimental autoimmune encephalomyelitis (EAE).

Expression of authentic post-translationally modified proteins in organisms with expanded genetic codes.

Cellular signaling and regulatory cascades often rely on post-translational modification of proteins, particularly phosphorylation, to quickly and effectively relay signals from a variety of inputs. Numerous kinases, the effectors of phosphorylation, and kinase networks have been implicated in human diseases. Until recently, an inability to produce high yields of physiologically phosphorylated proteins has proven to be a substantial barrier toward our understanding of many enzymatic processes.

Francisella tularensis Transmission by Solid Organ Transplantation, 2017.

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure.

Combined in-depth, 3D, en face imaging of the optic disc, optic disc pits and optic disc pit maculopathy using swept-source megahertz OCT at 1050 nm.

Purpose: To demonstrate papillary imaging of eyes with optic disc pits (ODP) or optic disc pit associated maculopathy (ODP-M) with ultrahigh-speed swept-source optical coherence tomography (SS-OCT) at 1.68 million A-scans/s. To generate 3D-renderings of the papillary area with 3D volume-reconstructions of the ODP and highly resolved en face images from a single densely-sampled megahertz-OCT (MHz-OCT) dataset for investigation of ODP-characteristics.

CE: Original Research: Primary Care Providers and Screening for Military Service and PTSD.

Unlabelled: : Background: Most veterans have the option of receiving their health care from the Veterans Health Administration or through primary care providers in the private sector. However, there is some evidence that fewer than half of community-based, private sector primary care and mental health providers screen their patients for military service, particularly in rural areas, leaving these veterans less likely to be screened for posttraumatic stress disorder (PTSD) and other military service-related conditions.

Purpose: To determine whether primary care providers in the private sector are screening patients for military service and subsequent PTSD.

Aminoacyl-tRNA quality control is required for efficient activation of the TOR pathway regulator Gln3p.

The aminoacylation status of the cellular tRNA pool regulates both general amino acid control (GAAC) and target of rapamycin (TOR) stress response pathways in yeast. Consequently, fidelity of translation at the level of aminoacyl-tRNA synthesis plays a central role in determining accuracy and sensitivity of stress responses. To investigate effects of translational quality control (QC) on cell physiology under stress conditions, phenotypic microarray analyses were used to identify changes in QC deficient cells.

Translational fidelity and mistranslation in the cellular response to stress.

Faithful translation of mRNA into the corresponding polypeptide is a complex multistep process, requiring accurate amino acid selection, transfer RNA (tRNA) charging and mRNA decoding on the ribosome. Key players in this process are aminoacyl-tRNA synthetases (aaRSs), which not only catalyse the attachment of cognate amino acids to their respective tRNAs, but also selectively hydrolyse incorrectly activated non-cognate amino acids and/or misaminoacylated tRNAs. This aaRS proofreading provides quality control checkpoints that exclude non-cognate amino acids during translation, and in so doing helps to prevent the formation of an aberrant proteome.

Fully human CD19-specific chimeric antigen receptors for T-cell therapy.

Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA libraries.

Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae.

Amino acid starvation activates the protein kinase Gcn2p, leading to changes in gene expression and translation. Gcn2p is activated by deacylated tRNA, which accumulates when tRNA aminoacylation is limited by lack of substrates or inhibition of synthesis. Pairing of amino acids and deacylated tRNAs is catalyzed by aminoacyl-tRNA synthetases, which use quality control pathways to maintain substrate specificity.

MS-READ: Quantitative measurement of amino acid incorporation.

Ribosomal protein synthesis results in the genetically programmed incorporation of amino acids into a growing polypeptide chain. Faithful amino acid incorporation that accurately reflects the genetic code is critical to the structure and function of proteins as well as overall proteome integrity. Errors in protein synthesis are generally detrimental to cellular processes yet emerging evidence suggest that proteome diversity generated through mistranslation may be beneficial under certain conditions.