CRISPRi functional genomics in bacteria and its application to medical and industrial research.

SUMMARYFunctional genomics is the use of systematic gene perturbation approaches to determine the contributions of genes under conditions of interest. Although functional genomic strategies have been used in bacteria for decades, recent studies have taken advantage of CRISPR (clustered regularly interspaced short palindromic repeats) technologies, such as CRISPRi (CRISPR interference), that are capable of precisely modulating expression of all genes in the genome. Here, we discuss and review the use of CRISPRi and related technologies for bacterial functional genomics. We discuss the strengths and weaknesses of CRISPRi as well as design considerations for CRISPRi genetic screens. We also review examples of how CRISPRi screens have defined relevant genetic targets for medical and industrial applications. Finally, we outline a few of the many possible directions that could be pursued using CRISPR-based functional genomics in bacteria. Our view is that the most exciting screens and discoveries are yet to come.

A thematic analysis of MDMA-related harm and harm reduction experiences and knowledge in Aotearoa New Zealand.

BACKGROUND: Methylenedioxymethamphetamine (MDMA) is a popular drug worldwide and use is prevalent in Aotearoa New Zealand. Although associated with some significant harms, including fatalities, MDMA is ultimately less harmful than other commonly consumed drugs. We aimed to expand the understanding of MDMA harm and harm reduction strategies from a consumer perspective so that national harm reduction efforts can be better informed. METHODS: We conducted 14 semi-structured focus group discussions including 60 people (aged 18-67, median = 21) who use MDMA in the Southern region of Aotearoa New Zealand to explore their thoughts and experiences regarding MDMA associated harm and harm reduction. Reflexive thematic analysis was conducted from a critical realist perspective. RESULTS: Five themes were generated; (1) Mindset and setting matters; (2) Looking after your body and mind, not overdoing it; (3) Other substances increase risk and harm; (4) Trusted friends and peers are protective; and (5) Valid information is key for healthy self-determination; and one subtheme 5.1) Drug checking is essential harm reduction. CONCLUSIONS: We discuss the implications for MDMA consumers and aim to inform national drug policy and the harm reduction practices of consumers and organisations, for the ultimate purpose of reducing MDMA-related harm in Aotearoa New Zealand.

Basal forebrain and prelimbic cortex connectivity is related to behavioral response in an attention task.

The basal forebrain (BF) is critical for the motivational recruitment of attention in response to reward-related cues. This finding is consistent with a role for the BF in encoding and transmitting motivational salience and readying prefrontal circuits for further attentional processing. We recorded local field potentials to determine connectivity between prelimbic cortex (PrL) and BF during the modulation of attention by reward-related cues. We find that theta and gamma power are robustly associated with behavior. Power in both bands is significantly lower during trials in which an incorrect behavioral response is made. We find strong coherence during responses that are significantly stronger when a correct response is made. We show that information flow is largely monodirectional from BF to and is strongest when correct responses are made. These experiments demonstrate that connectivity between BF and the PrL increases during periods of increased motivational recruitment of attentional resources.

Harm reduction behaviours and harm experiences of people who use 3,4-methylenedioxymethamphetamine (MDMA) in Aotearoa New Zealand.

BACKGROUND: 3,4-Methylenedioxymethamphetamine (MDMA) is drug of high prevalence in Aotearoa New Zealand and is the primary drug analysed by legal drug checking services. We aimed to address the gap in literature pertaining to MDMA-related harm reduction behaviour and harm experiences within the country. METHODS: An online survey was used to assess the harm reduction behaviours (e.g., limiting consumption, planning use, seeking information) of people who use MDMA, in addition to their use of reagent testing and the major national drug checking and harm reduction service, KnowYourStuffNZ. RESULTS: In total, 915 people completed the survey (60.7% females, aged 18-65, median = 24, IQR = 20-28). Frequency of various MDMA-related harm reduction behaviours differed, although these were carried out relatively frequently by most participants. Those who reported experiencing harm (physical, psychological, spiritual, social) from MDMA, or another drug presumed to be MDMA, reported less frequent harm reduction behaviours than non-harmed consumers. Reagent testing of MDMA had been conducted by 42.3% of the sample. Approximately 27% of the sample had used KnowYourStuffNZ services. Of KnowYourStuffNZ clients, 95.9% reported learning about harm reduction, and 53.3% reported changing their behaviour because of the service. Reasons for not using the KnowYourStuffNZ service were primarily lack of availability in local area (32.8%) or at relevant events (51.8%), and lack of concern with substance quality (29.8%). MDMA harm was reported by 14.4% of the sample, whilst reported harm was more common from consumption of presumably non-MDMA substances, self-reported as being mistaken for MDMA. Harm was primarily physical or psychological. Potential MDMA dependence was apparent in 6.9% of the sample. CONCLUSIONS: The findings highlight potential targets for harm reduction education and interventions and emphasize the need for greater availability of readily accessible drug checking services in Aotearoa New Zealand.

A Targeted Genome-scale Overexpression Platform for Proteobacteria.

Targeted, genome-scale gene perturbation screens using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale overexpression face key limitations. Here, we develop a facile approach for genome-scale gene overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We create a platform for comprehensive gene targeting using CRISPR-associated transposition (CAST) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome-scale in Escherichia coli, recovering known antibiotic targets and genes with unexplored roles in antibiotic function. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.

Rolling through TikTok: An analysis of 3,4-methylenedioxymethamphetamine-related content.

INTRODUCTION: TikTok has quickly gained popularity through its platforming of large amounts of short video content. Given its widespread popularity, unrestricted access and poor content monitoring may allow 3,4-methylenedioxymethamphetamine (MDMA)-related content to influence perception of MDMA use. We aimed to investigate how MDMA-related videos are portrayed on TikTok and explore MDMA-related harm reduction content. METHODS: MDMA-related hashtags and sounds were utilised to collect data from TikTok (n = 498). Video views, likes, comments and shares were recorded and quantified, and videos were coded for depiction/sentiment towards MDMA and thematic content. RESULTS: The total sample view count was 82,413,781. Videos had a median view count of 28,900 (SD = ±561,645), median like count of 2269 (SD = ±102,904), median comment count of 52 (SD = ±755), and median share count of 34 (SD = ±3292). Most videos depicted MDMA neutrally (40.6%), while 34.9% were positive. MDMA intoxication was presumed in 40.2% of videos. The analysis produced seven themes, of which humour was the most common (80.5%). Harm reduction content was present in nine videos, viewed 999,700 times, and consisted of mixed subject matter. DISCUSSION AND CONCLUSIONS: Similar themes and issues surrounding drug-related content on TikTok are relevant to MDMA, and intoxication was present in a significant portion of the sample. Better monitoring or regulation of content could potentially offset harm that may arise from consumption of such content. Promotion of harm reduction content could also be trialled to minimise harm.

Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii.

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing, they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize A. baumannii essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that may explain synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.IMPORTANCEAcinetobacter baumannii is a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistant A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical for A. baumannii viability. We show that genes involved in aerobic metabolism are central to A. baumannii physiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii.

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing (Tn-seq), they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize A. baumannii essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that underpin synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.

Impact of Prostate Urethral Lift Device on Prostate Magnetic Resonance Image Quality.

PURPOSE: Prostatic urethral lift with UroLift is a minimally invasive approach to treat symptomatic benign prostatic hypertrophy. This device causes artifacts on prostate magnetic resonance images. Our aim was to evaluate the impact of artifact on prostate magnetic resonance image quality. MATERIALS AND METHODS: This was a single-center retrospective review of patients with UroLift who subsequently had prostate magnetic resonance imaging. Two readers graded UroLift artifact on each pulse sequence using a 5-point scale (1-nondiagnostic; 5-no artifact). Prostate Imaging Quality scores were assigned for the whole data set. The volume of gland obscured by artifact was measured. Linear and logistic regression models were used to identify predictors of poor image quality. RESULTS: Thirty-seven patients were included. Poor image quality occurs more in the transition zone than the peripheral zone (15% vs 3%), at base/mid regions vs the apex (13%, 9%, and 5%, respectively) and on diffusion-weighted images vs T2-weighted and dynamic contrast-enhanced sequences (27%, 0.3%, 0%, respectively; P < .001). Suboptimal image quality (ie, Prostate Imaging Quality score <2) was found in 16%-24% of exams. The percentage of gland obscured by the UroLift artifact was higher on diffusion-weighted images and dynamic contrast-enhanced sequences than T2-weighted (32%, 9%, and 6%, respectively; P < .001). CONCLUSIONS: UroLift artifact negatively affects prostate magnetic resonance image quality with greater impact in the mid-basal transition zone, obscuring a third of the gland on diffusion-weighted images. Patients considering this procedure should be counseled on the impact of this device on image quality and its potential implications for any image-guided prostate cancer workup.

Cognitive and arginine metabolic correlates of temporal dysfunction in the MIA rat model of schizophrenia risk.

As a hallmark characteristic of schizophrenia, abnormal perception of time is thought to arise from cognitive impairment; however, the absence of translational models indexing this pathological relationship creates barriers to understanding the functional and biological bases of timing impairments. Here, we investigate the relationship between timing and cognition using the maternal immune activation (MIA) rat model of schizophrenia. We additionally investigate the role of prefrontal cortex L-arginine metabolism in these processes via high-performance liquid chromatography and liquid chromatography/mass spectrometry. Results revealed that MIA rats exhibit greater underestimation of interval durations (2-8 s); greater underestimation corresponded with declines in sustained attention capacity. Working memory impairments were not found to contribute to timing deficits. These findings represent the first direct identification of a timing-attention relationship within rodents and are discussed with respect to the dopamine hypothesis of temporal pace. We also found that MIA exposure altered aspects of arginine metabolism as observed in schizophrenia, and we present preliminary evidence suggesting that these changes have functional consequences for cognition. These findings support the MIA rat model as a valuable tool for future investigations exploring the biological instantiation of interrelated timing and cognitive deficits in schizophrenia. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

The instrumental role of operant paradigms in translational psychiatric research: Insights from a maternal immune activation model of schizophrenia risk.

Rigorous behavioral analysis is essential to the translation of research conducted using animal models of neuropsychiatric disease. Here we discuss the use of operant paradigms within our lab as a powerful approach for exploring the biobehavioral bases of disease in the maternal immune activation rat model of schizophrenia. We have investigated a range of disease features in schizophrenia including abnormal perception of time, cognition, learning, motivation, and internal state (psychosis), providing complex insights into brain and behavior. Beyond simple phenotyping, implementing sophisticated operant procedures has been effective in delineating aspects of pathological behavior, identifying interacting pathologies, and isolating contributing mechanisms of disease. We provide comment on the strengths of operant techniques to support high-quality behavioral investigations in fundamental neuropsychiatric research.

Influence of Enema and Dietary Restrictions on Prostate MR Image Quality: A Multireader Study.

PURPOSE: To evaluate the effect of enema and dietary restrictions on prostate MR image quality metrics and to assess inter-reader agreement for these metrics. METHODS: This retrospective study included 195 men divided into groups based on their compliance with preparation instructions before prostate MRI (Enema + Diet, n = 98; Enema, n = 42; Diet, n = 35; Control [no compliance], n = 20). Four readers independently assessed six image quality metrics on a 5-point scale. Between-group comparisons were made using Wilcoxon rank sum test. Inter-reader agreement was calculated using Fleiss' kappa. RESULTS: Compared with the Control group, image quality with respect to rectal stool/gas, distortion of diffusion-weighted images, overall image quality, and confidence in assessment was higher in the Enema + Diet, Enema, and Diet groups (p  < 0.05 for all comparisons). The Enema + Diet and Enema groups had significantly higher scores than the Diet group for rectal stool/gas (p < 0.001 and 0.005, respectively). The Enema + Diet and Diet groups had higher scores than the Control group for rectal peristalsis (p = 0.027 and 0.009, respectively), but there were no significant differences in motion artifacts on T2-weighted images. Agreement among readers was fair, with kappa values ranging from 0.25 to 0.37. CONCLUSION: Enema and dietary restriction can improve the quality of prostate MRI by decreasing rectal distension and distortion of diffusion-weighted images and by increasing reader confidence in image assessment. Inter-reader agreement using subjective criteria for analysis of MRI quality is fair.

Inhibition of Isoleucyl-tRNA Synthetase by the Hybrid Antibiotic Thiomarinol.

Hybrid antibiotics are an emerging antimicrobial strategy to overcome antibiotic resistance. The natural product thiomarinol A is a hybrid of two antibiotics: holothin, a dithiolopyrrolone (DTP), and marinolic acid, a close analogue of the drug mupirocin that is used to treat methicillin-resistant Staphylococcus aureus (MRSA). DTPs disrupt metal homeostasis by chelating metal ions in cells, whereas mupirocin targets the essential enzyme isoleucyl-tRNA synthetase (IleRS). Thiomarinol A is over 100-fold more potent than mupirocin against mupirocin-sensitive MRSA; however, its mode of action has been unknown. We show that thiomarinol A targets IleRS. A knockdown of the IleRS-encoding gene, ileS, exhibited sensitivity to a synthetic analogue of thiomarinol A in a chemical genomics screen. Thiomarinol A inhibits MRSA IleRS with a picomolar Ki and binds to IleRS with low femtomolar affinity, 1600 times more tightly than mupirocin. We find that thiomarinol A remains effective against high-level mupirocin-resistant MRSA and provide evidence to support a dual mode of action for thiomarinol A that may include both IleRS inhibition and metal chelation. We demonstrate that MRSA develops resistance to thiomarinol A to a substantially lesser degree than mupirocin and the potent activity of thiomarinol A requires hybridity between DTP and mupirocin. Our findings identify a mode of action of a natural hybrid antibiotic and demonstrate the potential of hybrid antibiotics to combat antibiotic resistance.

Impaired discrimination of a subanesthetic dose of ketamine in a maternal immune activation model of schizophrenia risk.

BACKGROUND: Animal models of psychiatric diseases suffer from a lack of reliable methods for accurate assessment of subjective internal states in nonhumans. This gap makes translation of results from animal models to patients particularly challenging. AIMS/METHODS: Here, we used the drug-discrimination paradigm to allow rats that model a risk factor for schizophrenia (maternal immune activation, MIA) to report on the subjective internal state produced by a subanesthetic dose of the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. RESULTS/OUTCOMES: The MIA rats' discrimination of ketamine was impaired relative to controls, both in the total number of rats that acquired and the asymptotic level of discrimination accuracy. This deficit was not due to a general inability to learn to discriminate an internal drug cue or internal state generally, as MIA rats were unimpaired in the learning and acquisition of a morphine drug discrimination and were as sensitive to the internal state of satiety as controls. Furthermore, the deficit was not due to a decreased sensitivity to the physiological effects of ketamine, as MIA rats showed increased ketamine-induced locomotor activity. Finally, impaired discrimination of ketamine was only seen at subanesthetic doses which functionally correspond to psychotomimetic doses in humans. CONCLUSION: These data link changes in NMDA responses to the MIA model. Furthermore, they confirm the utility of the drug-discrimination paradigm for future inquiries into the subjective internal state produced in models of schizophrenia and other developmental diseases.

Metagenome Sequencing to Explore Phylogenomics of Terrestrial Cyanobacteria.

Cyanobacteria are ubiquitous microorganisms with crucial ecosystem functions, yet most knowledge of their biology relates to aquatic taxa. We have constructed metagenomes for 50 taxonomically well-characterized terrestrial cyanobacterial cultures. These data will support phylogenomic studies of evolutionary relationships and gene content among these unique algae and their aquatic relatives.

Infiltrative Renal Malignancies: Imaging Features, Prognostic Implications, and Mimics.

Infiltrative renal malignancies are a subset of renal masses that are morphologically characterized by a poorly defined interface with the renal parenchyma. Infiltrative renal malignancies are less common but more aggressive than more typical renal malignancies and carry an overall worse prognosis. Although an infiltrative renal process often represents a malignant neoplasm, infiltrative masses include a wide spectrum of diseases including primary renal cortical, medullary, and pelvic tumors; lymphoproliferative processes; metastases; and various infectious, inflammatory, immune-mediated, and vascular mimics. The imaging features of these masses are often nonspecific, but with the appropriate history, laboratory results, and clinical context, the radiologist can help narrow the diagnosis and guide further treatment. An invited commentary by Lee is available online.Online supplemental material is available for this article. ©RSNA, 2021.

Infiltrative Renal Masses: Clinical Significance and Fidelity of Documentation.

BACKGROUND: The prevalence of infiltrative renal masses (IRMs) and fidelity of documentation of infiltrative features remain unclear. OBJECTIVE: To investigate the prevalence/significance of IRMs and assess whether infiltrative features were documented preoperatively. DESIGN, SETTING, AND PARTICIPANTS: A total of 522 patients with renal tumors managed with partial/radical nephrectomy (2012-2014) whose pathology demonstrated locally advanced and/or aggressive histology were analyzed. Preoperative computed tomography/magnetic resonance imaging was retrospectively/independently reviewed by two radiologists. IRMs were required to have a poorly defined interface with parenchyma and nonelliptical shape in one or more distinct/unequivocal areas. Infiltrative features were defined as extensive or focal. INTERVENTION: Partial/radical nephrectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cancer-specific mortality (CSM) was estimated using cumulative-incidence analysis. Significant and independent predictors of CSM were evaluated using Cox proportional hazard analysis. RESULTS AND LIMITATIONS: Median tumor size was 6.9cm; renal cell carcinomas (RCCs) predominated (92%). Image review confirmed 133 IRMs (25%), including 103 RCCs; 59 had sarcomatoid or poorly differentiated features. IRMs were larger and more often symptomatic compared than non-IRMs, and disseminated disease was also more common for IRMs (all p<0.001). Overall, 109 IRMs were imaged at our center; 42 were documented as IRMs in preoperative radiology reports, while infiltrative features were not documented in 67 (61%). Only four (6%) of these 67 were documented as infiltrative by the surgical team. Infiltrative features were more often focal in undocumented IRMs. On multivariable analysis, infiltrative features, disseminated disease, and non-RCC histology were independent predictors of CSM (hazard ratio or HR [95% confidence interval {CI}]=1.73 [1.21-2.47], 2.98 [2.10-4.23], and 2.79 [1.86-4.62], respectively). Among IRMs, extensive infiltrative features and disseminated disease were associated with CSM (HR [95% CI]=1.98 [1.27-3.07] and 2.35 [1.52-3.63], respectively), while documentation status failed to show an association. Excluding patients with disseminated disease or residual cancer after surgery, recurrence rates were 62% for IRMs versus 22% for non-IRMs (p<0.001), and there was again no significant difference between documented and undocumented IRMs (p=0.36). Limitations include a retrospective design. CONCLUSIONS: Twenty-five percent of locally advanced/histologically aggressive renal tumors exhibited infiltrative features, although many were not documented as IRMs. Among this high-risk surgical population, infiltrative features were independent predictors of CSM, irrespective of whether they were documented or not. Our data suggest that infiltrative features should be assessed and documented routinely during evaluation of renal masses. PATIENT SUMMARY: Infiltrative renal masses may be more common than previously appreciated, although many were not documented as infiltrative during preoperative evaluation. Our data suggest that infiltrative features have a strong impact on prognosis and should be assessed and documented routinely during radiologic and clinical evaluation of renal masses.