Loss of function SMAD4 nonstop mutations in human cancer.

BACKGROUND: SMAD4 is a tumour suppressor gene that is mutated in a variety of cancers. SMAD4 nonstop mutations, which convert stop codons to sense codons that extend transcription, have been identified in genomic databases but have not been characterised in human pathology samples. The frequency of SMAD4 nonstop mutations and the consequences of nonstop mutations on SMAD4 protein expression are unknown. METHODS: We retrospectively analysed our cancer sequencing database of 38,002 tumour specimens and evaluated the spectrum of SMAD4 mutations. SMAD4 protein expression was evaluated by immunohistochemistry in tumours with SMAD4 nonstop mutations. RESULTS: In total, 1956 SMAD4 mutations were identified in 1822 tumours. SMAD4 mutations were most common in tumours of the gastrointestinal tract and included nonsense variants (n = 344), frameshift indels (n = 258), splice site variants (n = 104), and missense variants at codon R361 (n = 245). In a subset of cases with immunohistochemistry, SMAD4 expression was lost in 23 of 25 tumours (92%) with protein truncating variants and in 7 of 27 tumours (26%) with missense variants. Four cases harboured SMAD4 nonstop mutations. SMAD4 nonstop mutations were identified in two pancreatic adenocarcinomas, one colonic adenocarcinoma, and one non-small cell lung carcinoma. Immunohistochemistry demonstrated loss of SMAD4 protein expression in each of the four tumours with SMAD4 nonstop mutations. CONCLUSION: SMAD4 nonstop mutations are associated with loss of SMAD4 protein expression in multiple tumour types. SMAD4 nonstop mutations should be clinically interpreted as pathogenic loss of function alterations when identified in cancer sequencing panels.

Heat-shock proteases promote survival of Pseudomonas aeruginosa during growth arrest.

When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.

Hidden in plain sight: urinary Cryptococcus neoformans missed by routine diagnostics in a patient with acute leukemia.

Cryptococcuria is a rare manifestation of localized cryptococcal disease. We present a case of Cryptococcus neoformans urinary tract infection in an immunocompromised host missed by routine laboratory workup. The patient had negative blood cultures, a negative serum cryptococcal antigen (CrAg), and "non-Candida yeast" growing in urine culture that was initially dismissed as non-pathogenic. The diagnosis was ultimately made by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) from a repeat urine culture after transfer to a tertiary care center. Cryptococcus should be considered in the differential of refractory urinary tract infections growing non-Candida yeast.

Inducible transposon mutagenesis for genome-scale forward genetics.

Transposon insertion sequencing (Tn-seq) is a powerful method for genome-scale functional genetics in bacteria. However, its effectiveness is often limited by a lack of mutant diversity, caused by either inefficient transposon delivery or stochastic loss of mutants due to population bottlenecks. Here, we introduce "InducTn-seq", which leverages inducible mutagenesis for temporal control of transposition. InducTn-seq generates millions of transposon mutants from a single colony, enabling the sensitive detection of subtle fitness defects and transforming binary classifications of gene essentiality into a quantitative fitness measurement across both essential and non-essential genes. Using a mouse model of infectious colitis, we show that InducTn-seq bypasses a highly restrictive host bottleneck to generate a diverse transposon mutant population from the few cells that initiate infection, revealing the role of oxygen-related metabolic plasticity in pathogenesis. Overall, InducTn-seq overcomes the limitations of traditional Tn-seq, unlocking new possibilities for genome-scale forward genetic screens in bacteria.

Characterization of the ospZ promoter in Shigella flexneri and its regulation by VirB and H-NS.

OspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter and investigated its regulation by H-NS and VirB in Shigella flexneri. We show that H-NS represses and VirB partially derepresses the ospZ promoter. H-NS-mediated repression requires sequences located between -731 and -412 relative to the beginning of the ospZ gene. Notably, the VirB-dependent derepression of ospZ requires the same VirB binding sites as are required for the VirB-dependent derepression of the divergent icsP gene. These sites are centered 425 bp upstream of the ospZ gene but over 1 kb upstream of the icsP transcription start site. Although these VirB binding sites lie closer to ospZ than icsP, the VirB-dependent increase in ospZ promoter activity is lower than that observed at the icsP promoter. This indicates that the proximity of VirB binding sites to Shigella promoters does not necessarily correlate with the level of VirB-dependent derepression. These findings have implications for virulence gene regulation in Shigella and other pathogens that control gene expression using mechanisms of transcriptional repression and derepression.

A20 Restricts NOS2 Expression and Intestinal Tumorigenesis in a Mouse Model of Colitis-Associated Cancer.

BACKGROUND AND AIMS: Colon cancer can occur sporadically or in the setting of chronic inflammation, such as in patients with inflammatory bowel disease. We previously showed that A20, a critical negative regulator of tumor necrosis factor signal transduction, could regulate sporadic colon cancer development. In this report, we investigate whether A20 also acts as a tumor suppressor in a model of colitis-associated cancer. METHODS: Colitis and colitis-associated tumors were induced in wild-type and A20 intestinal epithelial cell-specific knockout (A20dIEC) mice using dextran sodium sulfate and azoxymethane. Clinicopathologic markers of inflammation were assessed in conjunction with colonic tumor burden. Gene expression analyses and immunohistochemistry were performed on colonic tissue and intestinal enteroids. Nitric oxide (NO) production and activity were assessed in whole colonic lysates and mouse embryonic fibroblasts. RESULTS: A20dIEC mice develop larger tumors after treatment with dextran sodium sulfate and azoxymethane than wild-type mice. In addition to elevated markers of inflammation, A20dIEC mice have significantly enhanced expression of inducible nitric oxide synthase (iNOS), a well-known driver of neoplasia. Enhanced iNOS expression is associated with the formation of reactive nitrogen species and DNA damage. Loss of A20 also enhances NO-dependent cell death directly. CONCLUSION: Mechanistically, we propose that A20 normally restricts tumor necrosis factor-induced nuclear factor kappa B-dependent production of iNOS in intestinal epithelial cells, thereby protecting against colitis-associated tumorigenesis. We also propose that A20 plays a direct role in regulating NO-dependent cell death.

A nationwide assessment of perceptions of research-intense academic careers among predoctoral MD and MD-PhD trainees.

INTRODUCTION: While previous studies have described career outcomes of physician-scientist trainees after graduation, trainee perceptions of research-intensive career pathways remain unclear. This study sought to identify the perceived interests, factors, and challenges associated with academic and research careers among predoctoral MD trainees, MD trainees with research-intense (>50%) career intentions (MD-RI), and MD-PhD trainees. METHODS: A 70-question survey was administered to 16,418 trainees at 32 academic medical centers from September 2012 to December 2014. MD vs. MD-RI (>50% research intentions) vs. MD-PhD trainee responses were compared by chi-square tests. Multivariate logistic regression analyses were performed to identify variables associated with academic and research career intentions. RESULTS: There were 4433 respondents (27% response rate), including 2625 MD (64%), 653 MD-RI (15%), and 856 MD-PhD (21%) trainees. MD-PhDs were most interested in pursuing academia (85.8%), followed by MD-RIs (57.3%) and MDs (31.2%). Translational research was the primary career intention for MD-PhD trainees (42.9%). Clinical duties were the primary career intention for MD-RIs (51.9%) and MDs (84.2%). While 39.8% of MD-PhD respondents identified opportunities for research as the most important career selection factor, only 12.9% of MD-RI and 0.5% of MD respondents shared this perspective. Interest in basic research, translational research, clinical research, education, and the ability to identify a mentor were each independently associated with academic career intentions by multivariate regression. CONCLUSIONS: Predoctoral MD, MD-RI, and MD-PhD trainees are unique cohorts with different perceptions and interests toward academic and research careers. Understanding these differences may help to guide efforts to mentor the next generation of physician-scientists.

Identification of Fitness Determinants during Energy-Limited Growth Arrest in Pseudomonas aeruginosa.

Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are poorly defined. In this study, we exploited the fact that Pseudomonas aeruginosa can remain viable over several weeks when limited for organic carbon (pyruvate) as an electron donor or oxygen as an electron acceptor. ATP values were reduced under both types of limitation, yet more severely in the absence of oxygen. Using transposon-insertion sequencing (Tn-seq), we identified fitness determinants in these two energy-limited states. Multiple genes encoding general functions like transcriptional regulation and energy generation were required for fitness during carbon or oxygen limitation, yet many specific genes, and thus specific activities, differed in their relevance between these states. For instance, the global regulator RpoS was required during both types of energy limitation, while other global regulators such as DksA and LasR were required only during carbon or oxygen limitation, respectively. Similarly, certain ribosomal and tRNA modifications were specifically required during oxygen limitation. We validated fitness defects during energy limitation using independently generated mutants of genes detected in our screen. Mutants in distinct functional categories exhibited different fitness dynamics: regulatory genes generally manifested a phenotype early, whereas genes involved in cell wall metabolism were required later. Together, these results provide a new window into how P. aeruginosa survives growth arrest.IMPORTANCE Growth-arrested bacteria are ubiquitous in nature and disease yet understudied at the molecular level. For example, growth-arrested cells constitute a major subpopulation of mature biofilms, serving as an antibiotic-tolerant reservoir in chronic infections. Identification of the genes required for survival of growth arrest (encompassing entry, maintenance, and exit) is an important first step toward understanding the physiology of bacteria in this state. Using Tn-seq, we identified and validated genes required for fitness of Pseudomonas aeruginosa when energy limited for organic carbon or oxygen, which represent two common causes of growth arrest for P. aeruginosa in diverse habitats. This unbiased, genome-wide survey is the first to reveal essential activities for a pathogen experiencing different types of energy limitation, finding both shared and divergent activities that are relevant at different survival stages. Future efforts can now be directed toward understanding how the biomolecules responsible for these activities contribute to fitness under these conditions.

The physiology of growth arrest: uniting molecular and environmental microbiology.

Most bacteria spend the majority of their time in prolonged states of very low metabolic activity and little or no growth, in which electron donors, electron acceptors and/or nutrients are limited, but cells are poised to undergo rapid division cycles when resources become available. These non-growing states are far less studied than other growth states, which leaves many questions regarding basic bacterial physiology unanswered. In this Review, we discuss findings from a small but diverse set of systems that have been used to investigate how growth-arrested bacteria adjust metabolism, regulate transcription and translation, and maintain their chromosomes. We highlight major questions that remain to be addressed, and suggest that progress in answering them will be aided by recent methodological advances and by dialectic between environmental and molecular microbiology perspectives.