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Objectives: This study tested the near-term reproducibility of (18)fluorodeoxyglucose positron emission tomography (FDG-PET) imaging of atherosclerosis. Background: It is known that FDG-PET can measure inflammation within the aorta, carotid, and vertebral arteries with histologic validation in humans and animal models of disease. By tracking changes in inflammation over time, PET could be used as a surrogate marker of antiatheroma drug efficacy. However, the short-term variability and reproducibility of the technique are unknown. Methods: We imaged the carotid arteries and aorta in 11 subjects with FDG-PET/computed tomography twice, 14 days apart. We assessed interobserver and intraobserver agreement and interscan variability. Results: Interscan plaque FDG variability over 2 weeks was very low; intraclass correlation coefficients (ICC) ranged between 0.79 and 0.92. Interobserver agreement was high across all territories imaged except aortic arch (ICC values from 0.90 to 0.97, arch 0.71). Intraobserver agreement was high, with ICC values between 0.93 and 0.98. Conclusions: Spontaneous change in plaque FDG uptake is low over 2 weeks, with favorable inter- and intraobserver agreement. Power calculations suggest that drug studies using FDG-PET imaging would require few subjects compared with other imaging modalities. This study strengthens the case for FDG-PET as a noninvasive plaque imaging technique. [ABSTRACT FROM AUTHOR]

The transcriptomes of a 2-pyrone-4,6-dicarboxylic acid-producing strain of Novosphingobium aromaticivorans DSM12444 were determined when grown in minimal medium containing glucose alone or glucose plus vanillin, ferulic acid, or the β-5-linked aromatic dimer dehydrodiconiferyl alcohol as carbon sources. Here, we present the RNA-sequencing data we obtained., Competing Interests: The authors declare no conflict of interest.

Objective: To characterize the patient population using weekend and evening appointments for screening mammography versus standard appointment times across four outpatient facilities in our academic health system., Methods: In this institutional review board-approved retrospective cohort study, there were 203,101 screening mammograms from 67,323 patients who had a screening mammogram performed at outpatient centers at a multisite academic institution from January 1, 2015, to December 31, 2022. Screening appointments were defined as "standard appointment time" (between 8 am and 5 pm on Monday through Friday) or "weekend or evening appointment time" (scheduled after 5 pm on Monday through Friday or at any time on a Saturday or Sunday). Associations between appointment group and patient characteristics were analyzed using univariate and multivariate logistic regression., Results: Most screening mammograms (n = 185,436, 91.3%) were performed at standard times. The remainder (n = 17,665, 8.7%) were performed during weekends or evenings. As we created additional weekend and evening appointments after the coronavirus disease 2019 pandemic, the annual percentage of all screening mammograms performed on evenings and weekends increased. On multivariate analysis, when compared with standard appointment times, we found that patients who were younger than age 50 (P < .001), a race other than non-Hispanic White (P < .001), non-English speakers (P < .001), and from less advantaged zip codes (P < .03) were more likely to use weekend and evening appointment times compared with those aged 70 and above, non-Hispanic White patients, English speakers, and those from the most advantaged zip codes., Conclusions: Weekend and evening appointment availability for screening mammograms might improve screening access for all patients, particularly for those younger than age 50, those of races other than non-Hispanic White, and those from less advantaged zip codes., (Copyright © 2024 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Introduction: MRI-guided biopsy is the standard of care for breast imaging findings seen only by MRI. Although a non-zero false-negative rate of MRI-guided breast biopsy has been reported by multiple studies, there are varied practice patterns for imaging follow-up after a benign concordant MRI guided biopsy. This study assessed the outcomes of benign concordant MRI-guided biopsies at a single institution., Patients and Methods: This IRB-approved, retrospective study included patients with MRI-guided biopsies of breast lesions from November 1, 2014, to August 31, 2020. Only image-concordant breast lesions with benign histopathology and those follow up with MRI imaging or excision were included in the study., Results: Out of 275 lesions in 216 patients that met the inclusion criteria, 274 lesions were followed with MRI (range, 5-79 months; average, 25.5 months) and showed benign or stable features upon follow-up. One out of 275 lesions (0.4%), a 6 mm focal nonmass enhancement, was ultimately found to represent malignancy after initial MRI-guided biopsy yielded fibrocystic changes. The lesion was stable at a 6-month follow-up MRI but increased in size at 18 months. Repeat biopsy by ultrasound guidance yielded invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS)., Conclusion: Breast MRI-guided biopsy has a low false-negative rate. Our single malignancy from a total of 275 lesions gives a false negative rate of 0.4%. This data also supports a longer follow-up interval than the commonly performed 6-month follow-up, in order to assess for interval change., Competing Interests: Disclosures The authors have stated that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This work aims to perform a cross-site validation of automated segmentation for breast cancers in MRI and to compare the performance to radiologists. A three-dimensional (3D) U-Net was trained to segment cancers in dynamic contrast-enhanced axial MRIs using a large dataset from Site 1 (n = 15,266; 449 malignant and 14,817 benign). Performance was validated on site-specific test data from this and two additional sites, and common publicly available testing data. Four radiologists from each of the three clinical sites provided two-dimensional (2D) segmentations as ground truth. Segmentation performance did not differ between the network and radiologists on the test data from Sites 1 and 2 or the common public data (median Dice score Site 1, network 0.86 vs. radiologist 0.85, n = 114; Site 2, 0.91 vs. 0.91, n = 50; common: 0.93 vs. 0.90). For Site 3, an affine input layer was fine-tuned using segmentation labels, resulting in comparable performance between the network and radiologist (0.88 vs. 0.89, n = 42). Radiologist performance differed on the common test data, and the network numerically outperformed 11 of the 12 radiologists (median Dice: 0.85-0.94, n = 20). In conclusion, a deep network with a novel supervised harmonization technique matches radiologists' performance in MRI tumor segmentation across clinical sites. We make code and weights publicly available to promote reproducible AI in radiology., (© 2024. The Author(s) under exclusive licence to Society for Imaging Informatics in Medicine.)

Multiplex, randomized CRISPR interference sequencing (MuRCiS) allows for the simultaneous identification of multiple gene knockouts that together influence microbial processes. Here, we report on an updated analysis tool called Auto-MuRCiS that utilizes Docker to make the analysis of these data rapid and more user-friendly., Competing Interests: The authors declare no conflict of interest.

Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans . We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans . Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans . A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales., Importance: In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products., Competing Interests: The authors declare no conflict of interest.

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our integrated systems approach allowed us to propose a model for how RNA G4s can present barriers to E. coli growth and that reducing the rate of translation can compensate for G4-related stress., Competing Interests: Competing Interest Statement: There are no conflicts of interest with this study.

To advance knowledge of microbial communities capable of fermenting agro-industrial residues into value-added products, we report metagenomes of microbial communities from six anaerobic bioreactors that were fed a mixture of ultra-filtered milk permeate and cottage cheese acid whey. These metagenomes produced 122 metagenome-assembled genomes that represent 34 distinct taxa., Competing Interests: The authors declare no conflict of interest.

The Gram-negative pathogen Acinetobacter baumannii is considered an "urgent threat" to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF "SigAb;" however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE. We combine promoter mutagenesis, motif scanning, and ChIP-seq to define the direct SigAb regulon, which consists of sigAb itself, the stringent response mediator, relA , and the uncharacterized small RNA, " sabS ." However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper induced SigAb-dependent transcription. Further, we found that two uncharacterized genes in the sigAb operon, " aabA " and " aabB ", have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb , aabA , and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii ., Competing Interests: Competing Interest None.

Members of the " Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 " Ca . Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 ( ppk1 ) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most " Ca . Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the " Ca . Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 " Ca . Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1 , and genome-scale phylogenetic analyses do not belong to any of the currently recognized " Ca . Accumulibacter" species for which we propose the new species designation " Ca . Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, " Ca . A. necessarius" and " Ca . A. propinquus" accounted for more than 40% of the " Ca . Accumulibacter" community, whereas the newly proposed " Ca . A. jenkinsii" accounted for about 5% of the " Ca . Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the " Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the " Ca . Accumulibacter" species enriched under these conditions, including one novel species for which we propose " Ca . Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional " Ca . Accumulibacter" species further refine the phylogeny of the " Ca . Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems., Competing Interests: The authors declare no conflict of interest.

Objective: The goal of this study was to determine patient factors associated with the use of self-scheduling of screening mammograms (SMs) using an online portal., Methods: All SMs scheduled at our multisite academic institution from January 1, 2015, to December 31, 2022, were included. The frequency of self-scheduling via an online portal was calculated per year. Univariate and multivariate logistic regression models with generalized estimating equation were used to estimate associations between patient characteristics and scheduling format after accounting for correlations between mammograms performed on the same woman., Results: During the study period, 250,369 SMs were performed in 74,860 unique patients (mean age 59 ± 12 years). Of these, 36,200 (14.5%) were self-scheduled via the online portal. Self-scheduling increased each year, from 3.7% in 2015 to 36.9% in 2022. Younger age, non-Black race, being an English speaker, and being from a nondisadvantaged zip code were significant predictors of self-scheduling on univariate and multivariate logistic regression. Age <50 years versus age ≥70 years was the patient characteristic that most strongly predicted the likelihood of self-scheduling (adjusted odds ratio 5.4, 95% confidence interval 5.2-5.6)., Conclusions: Over 8 years (2015-2022), utilization of self-scheduling for screening mammography using an online patient portal increased from 3.7% to 36.9%. Age < 50 years was the patient characteristic that most strongly predicted likelihood of self-scheduling., (Copyright © 2023 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Background: Supplemental screening with breast MRI is recommended annually for patients who have greater than 20% lifetime risk for breast cancer. While there is robust data regarding features of mammographic screen-detected breast cancers, there is limited data regarding MRI-screen-detected cancers., Patients and Methods: Screening breast MRIs performed between August 1, 2016 and July 30, 2022 identified 50 screen-detected breast cancers in 47 patients. Clinical and imaging features of all eligible cancers were recorded., Results: During the study period, 50 MRI-screen detected cancers were identified in 47 patients. The majority of MRI-screen detected cancers (32/50, 64%) were invasive. Pathology revealed ductal carcinoma in situ (DCIS) in 36% (18/50), invasive ductal carcinoma (IDC) in 52% (26/50), invasive lobular carcinoma in 10% (5/50), and angiosarcoma in 2% (1/50). The majority of patients (43/47, 91%) were stage 0 or 1 at diagnosis and there were no breast cancer-related deaths during the follow-up periods. Cancers presented as masses in 50% (25/50), nonmass enhancement in 48% (25/50), and a focus in 2% (1/50). DCIS was more likely to present as nonmass enhancement (94.4%, 17/18), whereas invasive cancers were more likely to present as masses (75%, 24/32) (P < .001). All cancers that were stage 2 at diagnosis were detected either on a baseline exam or more than 4 years since the prior MRI exam., Conclusion: MRI screen-detected breast cancers were most often invasive cancers. Cancers detected by MRI screening had an excellent prognosis in our study population. Invasive cancers most commonly presented as a mass., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Importance: DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria., Competing Interests: The authors declare no conflict of interest.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms., Competing Interests: NE, KM, JT, AD, KJ, KB, TD, MM No competing interests declared

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Purpose: For patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, it is standard of care to perform pre- and post-NAC imaging to evaluate response to therapy prior to surgery. In this study we assess outcome metrics of magnetic resonance imaging (MRI) following NAC., Methods: We conducted a retrospective analysis of patients with invasive breast cancer who underwent a breast MRI before and after NAC between 2016 and 2021 at a single, multisite academic institution. All breast MRI studies were characterized as either radiologic complete response (rCR) or non-rCR. Corresponding surgical pathology reports were reviewed and categorized as pathologic complete response (pCR) or non-pCR. We defined a positive test as having residual enhancement on MRI (non-rCR) and a positive outcome as having residual disease on final surgical pathology (non-pCR)., Results: There were 225 patients included in the study (mean age 52 ± 12 years). Breast cancer receptor distribution was HR+/HER2- (n = 71, 32%), HR+/HER2+ (n = 51, 23%), HR-/HER2- (n = 72, 32%), and HR-/HER2+ (n = 31, 14%). In total, 78 (35%) had rCR and 77 (34%) had pCR; 43 (19%) had both rCR and pCR. The overall accuracy rate was 69% (156/225), sensitivity 76% (113/148), specificity 56% (43/77), positive predictive value 77% (113/147), and negative predictive value 55% (43/78). The PPV was significantly associated with receptor status (p = 0.004). No patient or imaging characteristics were associated with sensitivity., Conclusion: Breast MRI only moderately predicts pathologic response for invasive breast cancer treated with NAC (overall accuracy 69%). PPV is significantly associated with receptor status., Competing Interests: Declaration of competing interest The authors have no declarations of competing interests to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Management of circumscribed breast masses seen on MRI is largely extrapolated from mammography and US data with limited MRI-specific data available. This study aimed to assess clinical and MRI imaging features of malignant circumscribed breast masses., Methods: In this IRB-approved retrospective study, breast MRIs performed between April 1, 2008, and August 30, 2020, containing circumscribed masses, excluding multiple bilateral circumscribed masses, were reviewed. Clinical and imaging features of all eligible masses were recorded, and associations with malignant outcomes were assessed using Fisher's exact test and Wilcoxon rank sum test, with P < 0.05 considered significant., Results: For the 165 masses that met study criteria in 158 women, the mean age was 48 years (SD 12.0 years). Nine of 165 masses were malignant (5.5%). Round masses were significantly more likely to be malignant (7/37, 18.9%) compared to oval masses (2/128, 1.7%) (P < 0.001). Among masses with available dynamic contrast kinetics data, the malignancy rate was 0/84 (0%) for persistent kinetics, 2/23 (8.7%) for plateau kinetics, and 4/24 (16.7%) for washout kinetics (P = 0.002). The malignancy rate for oval masses without washout kinetics was 0% (0/92). T2 hyperintense masses had a malignancy rate of 7/104 (6.7%), and homogeneously enhancing masses had a malignancy rate of 5/91 (5.5%)., Conclusion: These data support the use of mass shape and dynamic contrast enhancement kinetics to guide management of circumscribed breast masses seen by MRI, with oval masses without washout kinetics and any circumscribed mass with persistent kinetics showing no malignancies in this study., (© Society of Breast Imaging 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Walters, Mohan, Myers, Ingle, Donohue and Noguera.)

The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Myers, Ingle, Walters, Fortney, Scarborough, Donohue and Noguera.)

Rationale and Objectives: A solitary dilated duct (SDD) is a single asymmetrically dilated breast duct with diameter more than 2 mm. The Breast Imaging Reporting and Data System (BI-RADS) fifth edition recommends additional imaging and biopsy for SDDs without demonstrated benign etiology, however management of this rare entity remains controversial. This study describes practice patterns, malignancy rate, and features associated with high-risk/malignant SDDs to better stratify patients requiring biopsy versus follow-up., Materials and Methods: This IRB-approved retrospective study identified mammographic, sonographic and MRI exams utilizing the term "solitary dilated duct" at a multisite academic institution between 1/1/2010 and 12/31/2020. Clinical and imaging features, BI-RADS assessments, and outcomes were analyzed. Univariate and multivariate analyses identified predictors of high-risk/malignant histology., Results: SDDs identified in 49 women (mean age 56.1 years) were assessed as BI-RADS 4/5 (31/49, 63%), BI-RADS 3 (9/49, 18%), or BI-RADS 2 (9/49, 18%). Most sampled lesions were benign (16/31, 52%) and the remaining were high-risk (15/31, 48%, all papillary lesions). The only papilloma with atypia on core biopsy upgraded to grade 2 DCIS on excision (malignancy rate 1/49, 2%). All anechoic SDDs were benign (n=13), and all benign SDDs lacked internal vascularity. SDDs with associated masses were associated with malignant/high-risk outcomes on multivariate analysis (p < .001)., Conclusion: The BI-RADS fifth edition recommends biopsy for SDDs without demonstrated benign etiology. In our 11-year study period, practice patterns were variable with a low malignancy rate of 2%. Our findings suggest that anechoic SDDs may be followed, and SDDs with associated masses or internal vascularity require biopsy., (Copyright © 2022 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

BACKGROUND. Studies have shown improved targeting and sampling of noncalcified lesions (asymmetries, masses, and architectural distortion) with digital breast tomosynthesis (DBT)-guided biopsy in comparison with digital mammography (DM)-guided stereotactic biopsy. Literature that compares the two techniques specifically for sampling calcifications has been scarce. OBJECTIVE. The purpose of this study was to compare the performance and outcomes of DM- and DBT-guided biopsy of suspicious calcifications. METHODS. This retrospective study included 1310 patients (mean age, 58 ± 12 [SD] years) who underwent a total of 1354 9-gauge vacuum-assisted core biopsies of suspicious calcifications performed at a single institution from May 22, 2017, to December 31, 2021. The decision to use a DM-guided or DBT-guided technique was made at the discretion of the radiologist performing the biopsy. Procedure time, the number of exposures during the procedure, and the histopathologic outcomes were recorded. The two techniques were compared using a two-sample t test for continuous variables and a chi-square test for categoric variables. Additional tests were performed using generalized estimating equations to control for the effect of the individual radiologist performing the biopsy. RESULTS. A total of 348 (26%) biopsies used DM guidance, and 1006 (74%) used DBT guidance. The mean procedure time was significantly lower for DBT-guided biopsy (14.9 ± 8.0 [SD] minutes) than for DM-guided biopsy (24.7 ± 14.3 minutes) ( p < .001). The mean number of exposures was significantly lower for DBT-guided biopsy (4.1 ± 1.0 [SD] exposures) than for DM-guided biopsy (9.1 ± 3.3 exposures) ( p < .001). The differences in procedure time and number of exposures remained significant (both p < .001) when controlling for the effect of the radiologist performing the biopsy. There were no significant differences (all p > .05) between DM-guided and DBT-guided biopsy in terms of the malignancy rate on initial biopsy (20% vs 19%), the rate of high-risk lesion upgrading (14% vs 22%), or the final malignancy rate (23% vs 22%). CONCLUSION. DBT-guided biopsy of suspicious calcifications can be performed with shorter procedure time and fewer exposures compared with DM-guided biopsy, without a significant difference in rates of malignancy or high-risk lesion upgrading. CLINICAL IMPACT. The use of a DBT-guided, rather than a DM-guided, biopsy technique for suspicious calcifications can potentially reduce patient discomfort and radiation exposure without affecting clinical outcomes.

Objectives: Multiple exposures to gadolinium-based contrast agents (GBCAs) is known to be associated with gadolinium deposition in the brain in certain patients. Such deposition has been correlated with specific brain MRI findings, although most available data is in patients with underlying neurologic disorders. We aim to prospectively evaluate brain MRI signal changes as well as neurologic and neuropsychologic testing results in women undergoing screening breast MRI., Methods: In this IRB-approved, HIPAA-compliant prospective study, 9 women with 5 or more exposures to linear and/or macrocyclic GBCA due to screening breast MRI underwent noncontrast brain MRI, neurologic exam and neuropsychologic testing. Women with underlying neurologic, psychologic, hepatic or renal disorders were excluded., Results: The mean total number of GBCA exposures was 8 (standard deviation 2.7), with 63/72 (87%) of the exposures being linear agents. There was no association between brain MRI signal changes and abnormalities on neurologic or neuropsychologic examination. There was no association between total number of GBCA exposures and abnormalities on neurologic or neuropsychologic examination., Conclusion: In this prospective exploratory study of 9 women with 5 or more GBCA exposures due to screening breast MRI, there was no association between brain MRI signal changes and clinical abnormalities on neurologic or neuropsychologic examination. While larger studies are needed in this patient population, the lack of clinical impact of multiple GBCA exposures in this study is reassuring., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: This study assessed the upgrade rates of high-risk lesions (HRLs) in the breast diagnosed by MRI-guided core biopsy and evaluated imaging and clinical features associated with upgrade to malignancy., Methods: This IRB-approved, retrospective study included MRI-guided breast biopsy exams yielding HRLs from August 1, 2011, to August 31, 2020. HRLs included atypical ductal hyperplasia (ADH), lobular carcinoma in situ (LCIS), atypical lobular hyperplasia (ALH), radial scar, and papilloma. Only lesions that underwent excision or at least 2 years of MRI imaging follow-up were included. For each HRL, patient history, imaging features, and outcomes were recorded., Results: Seventy-two lesions in 65 patients were included in the study, with 8/72 (11.1%) of the lesions upgraded to malignancy. Upgrade rates were 16.7% (2/12) for ADH, 100% (1/1) for pleomorphic LCIS, 40% (2/5) for other LCIS, 0% (0/19) for ALH, 0% (0/18) for papilloma, and 0% (0/7) for radial scar/complex sclerosing lesion. Additionally, two cases of marked ADH bordering on DCIS and one case of marked ALH bordering on LCIS, were upgraded. Lesions were more likely to be upgraded if they presented as T2 hypointense (versus isotense, OR 6.46, 95% CI 1.27-32.92) or as linear or segmental non-mass enhancement (NME, versus focal or regional, p = 0.008)., Conclusion: Our data support the recommendation that ADH and LCIS on MRI-guided biopsy warrant surgical excision due to high upgrade rates. HRLs that present as T2 hypointense, or as linear or segmental NME, should be viewed with suspicion as these were associated with higher upgrade rates to malignancy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To assess the relationship between sociodemographic factors and adherence rates in patients with a BI-RADS 3 assessment., Methods: This retrospective cohort study reviewed data from all patients with a BI-RADS 3 assessment on mammography and ultrasound examinations at a single, multisite academic institution, which serves a diverse urban-suburban population, from January 1, 2015, to December 13, 2017. Appropriate follow-up was defined as returning for the first follow-up examination 3 to 9 months after the index examination. Associations between BI-RADS 3 adherence rates and patient sociodemographic characteristics were evaluated using logistic regression., Results: There were 4,038 patients in our study period; 2,437 patients (60%) had appropriate follow-up, 765 (19%) patients had delayed follow-up, and 836 patients (21%) were lost to follow-up. The overall malignancy rate was 1.4% (46 of 3,202). Older age, retired employment status, and Medicare insurance status were associated with increased adherence to BI-RADS 3 follow-up recommendations. Black race, single relationship status, Medicaid and self-pay insurance status, and living in a top 15% disadvantaged zip code were associated with decreased adherence. On multivariate analysis, older age remained associated with increased adherence and Medicaid insurance status with decreased adherence. Time between index examination and cancer diagnosis was shorter in patients who had timely follow-up (202 days [interquartile range 183-358] versus 392 days [interquartile range 365-563], P ≤ .001), although there was not a significant difference in stage at diagnosis (P = .46)., Discussion: Multiple sociodemographic factors are associated with low adherence to BI-RADS 3 follow-up recommendations suggesting that more frequent and targeted interventions are needed to close disparity gaps., (Copyright © 2022. Published by Elsevier Inc.)

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Despite technological advancements focused on reducing breast cancer mortality through early detection, there have been reported disparities in the access to these imaging services with underserved patient populations (including racial minority groups and patients of low socioeconomic status) showing underutilization compared to other patient groups. These underserved populations tend to have more advanced breast cancer presentations, in part due to delays in diagnosis resulting in later stage of disease presentation. To make matters worse, the COVID-19 pandemic declared in March 2020 has resulted in significant healthcare disruptions leading to extensive delays in breast imaging services which are expected to negatively impact breast cancer mortality long-term. Given the worsening disparity in breast cancer mortality among racial/ethnic minorities and financially disadvantaged groups, it is vital to address these disparity gaps with the goal of reducing the barriers to timely breast cancer diagnosis and addressing breast cancer mortality differences among breast cancer patients. Therefore, this review aims to provide a discussion highlighting the disparities related to breast imaging access, the effects of the COVID-19 pandemic on these disparities, current targeted interventions implemented in breast imaging practices to reduce these disparities, and future directions on the journey to reducing disparity gaps for breast imaging patients. Tackling the root cause factors of the persistent breast cancer-related disparities is critical to meeting the needs of patients who are disadvantaged and can lead to continued improvement in the quality of individualized care for patients who have higher breast cancer morbidity and mortality risks., (Copyright © 2022 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

BACKGROUND. Recall rates are lower for digital breast tomosynthesis (DBT) than for full-field digital mammography (FFDM). This difference could have important implications with respect to one-view asymmetries given that missed cancers are often visible on one view. OBJECTIVE. The purpose of this study is to compare the outcomes of one-view asymmetries recalled from DBT versus FFDM screening examinations and to determine predictors of malignancy among recalled asymmetries. METHODS. This retrospective study first determined recall rates associated with one-view asymmetries for screening mammography performed using DBT and FFDM from July 14, 2016, through July 14, 2020. Further analyses included patients recalled for a one-view asymmetry who completed subsequent diagnostic workup and all recommended follow-up. Patient and cancer characteristics were extracted from the electronic health record. RESULTS. The recall rate associated with asymmetries was lower for DBT screening (2.5% [3169/128,755]) than for FFDM screening (3.4% [815/23,898]) ( p < .001). Further analyses of patients who completed diagnostic workup and subsequent follow-up included 3119 patients (mean age, 57 years) for DBT screening and 811 patients (mean age, 56 years) for FFDM screening. Distribution of final BI-RADS categories from subsequent diagnostic workup was not different between the two modalities ( p > .99). The frequency of malignancy was not different between asymmetries recalled from DBT (1.7% [54/3119]) and FFDM (1.7% [14/811]) ( p > .99). Malignant asymmetries identified on FFDM versus DBT were more frequently associated with architectural distortion on diagnostic workup (35.7% [5/14] vs 9.3% [5/54]) ( p < .001) and were more commonly invasive ductal carcinoma (92.9% vs 57.4%) and less commonly invasive lobular carcinoma (0.0% vs 24.1%) ( p = .05). In multivariable analysis, independent predictors of malignancy among recalled asymmetries from DBT were age (for 55-69 years, odds ratio [OR] = 2.40 [ p = .04]; for ≥ 70 years, OR = 7.93 [ p < .001]; reference, < 55 years) and breast density (not dense, OR = 2.47 [ p = .001]; reference, dense breasts). CONCLUSION. Recalled asymmetries were less frequent for DBT than for FFDM. The malignancy rate was low for both modalities (1.7%). Age 55 years old and older and lower breast density predicted malignancy for DBT-recalled asymmetries. CLINICAL IMPACT. Our results support the use of DBT to reduce unnecessary recalls without altering PPV for asymmetry-associated malignancies. Patient factors should be considered when assessing whether a potential asymmetry on DBT screening represents overlapping fibroglandular tissue or a suspicious finding that requires diagnostic workup.

The transcriptomes of Zymomonas mobilis 2032 were captured during the fermentation of ammonia fiber expansion (AFEX)-pretreated corn stover and switchgrass hydrolysates containing different concentrations of glucose and xylose. RNA samples were collected when Z. mobilis was fermenting glucose or xylose. Here, we present transcriptome sequencing (RNA-Seq) data obtained during separate phases of glucose or xylose consumption.

Anaerobic microbiomes can be used to recover the chemical energy in agroindustrial and municipal wastes as useful products. Here, we report a total of 109 draft metagenome-assembled genomes from a bioreactor-fed carbohydrate-rich dairy manure hydrolysate. Studying these genomes will aid us in deciphering the metabolic networks in anaerobic microbiomes.

Here, we report the metagenomes from five anaerobic bioreactors, operated under different conditions, that were fed carbohydrate-rich thin stillage from a corn starch ethanol plant. The putative functions of the abundant taxa identified here will inform future studies of microbial communities involved in valorizing this and other low-value agroindustrial residues.

Fermentative microbial communities can be utilized for the conversion of various agroindustrial residues into valuable chemicals. Here, we report 34 metagenomes from anaerobic bioreactors fed lactose-rich ultrafiltered milk permeate and 278 metagenome-assembled genomes (MAGs). These MAGs can inform future studies aimed at generating renewable chemicals from dairy and other agroindustrial residues.

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria., Competing Interests: The authors have declared that no competing interests exist.

Unlike mammography and US, limited data exist to establish well-defined criteria for MRI findings that have a ≤2% likelihood of malignancy. Therefore, determining which findings are appropriate for a BI-RADS 3 assessment on MRI remains challenging and variable among breast radiologists. Emerging data suggest that BI-RADS 3 should be limited to baseline MRI examinations (or examinations with less than two years of prior comparisons) performed for high-risk screening and only used for masses with all of the typical morphological and kinetic features suggestive of a fibroadenoma or dominant enhancing T2 hypointense foci that is distinct from background parenchymal enhancement and without suspicious kinetics. This article presents an updated discussion of BI-RADS 3 assessment (probably benign) for breast MRI using current evidence., (© Society of Breast Imaging 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Architectural distortion without a sonographic correlate is an indication for digital breast tomosynthesis-guided vacuum-assisted biopsy (DBT-VAB). However, when the finding is not visualized on the day of biopsy, the procedure is canceled. This study reports the outcomes of canceled DBT-VAB of architectural distortion due to nonvisualization., Methods: In this IRB-approved retrospective study, chart review was performed to identify DBT-VABs of architectural distortion at our institution between June 1, 2017, and November 1, 2020, that were canceled because of nonvisualization at the time of biopsy. Cases without follow-up imaging were excluded. Statistical analysis, including the frequency of cases yielding malignancy by the end of the study period, was performed., Results: In total, 7.2% (39/544) of architectural distortions recommended for biopsy during the study period were canceled because of nonvisualization, 30 of which had follow-up imaging and were included in the study. Mean patient age was 56 years (standard deviation [SD], 9.6 years) and mean follow-up time was 26.7 months (SD, 11.2 months; range, 8.4-50.9 months). During the follow-up period, 16.7% (5/30) underwent repeat biopsy attempt, with one malignant result (1/30, 3.3%; SD, 18%; 95% confidence interval: 0.6%-16.7%). In total, 86.7% (26/30) of cases were declared benign during the follow-up period and 10% (3/30) remained stable with a BI-RADS 3 assessment category., Conclusion: During available follow-up, there was a low likelihood that distortions not visualized at the time of DBT-VAB represented malignancy (3.3%, 1/30). While this low malignancy rate is reassuring, imaging follow-up is warranted., (© Society of Breast Imaging 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbial communities using anammox bacteria to remove nitrogen are increasingly important in wastewater treatment. We report on 25 metagenome-assembled genomes of low-abundance microbes from a partial nitritation anammox bioreactor system that have not been described previously. These data add to the body of information about this important wastewater treatment system.

This study describes 94 patients who presented with suspected COVID-19 vaccine-related axillary adenopathy on breast imaging. All biopsies recommended within 12 weeks of the second vaccine dose were benign. Among women not recommended for biopsy, the median interval between the second vaccine dose and ultrasound follow-up was 15.9 weeks. Three biopsies yielding malignant diagnoses were recommended 12.0-13.1 weeks after the second vaccine dose. Lengthening imaging follow-up to 12-16 weeks after the second dose may reduce unnecessary biopsy recommendations.

There is growing interest in producing beneficial products from wastes using microbiomes. We previously performed multiomic analyses of a bioreactor microbiome that converted carbohydrate-rich lignocellulosic residues to medium-chain carboxylic acids. Here, we present draft metagenome-assembled genomes from this microbiome, obtained from reactors in which xylose was the primary carbon source.

Background Digital breast tomosynthesis (DBT) has improved the accuracy of mammography, including resolving many breast asymmetries as overlapping breast tissue. The pathologic outcomes of persistent developing asymmetries visualized at DBT are not well established. Purpose To characterize the outcomes and the predictors of malignancy for developing asymmetries visualized at DBT without a sonographic correlate. Materials and Methods This retrospective study included all tomosynthesis-guided biopsies of developing asymmetries performed at a single institution from May 2017 through January 2020. A reader study including three breast imaging radiologists determined interrater agreement and inclusion into the study. Electronic medical records were used to extract patient characteristics, imaging characteristics, and pathologic diagnoses. The Wilcoxon rank sum test, Fisher exact test, and χ 2 test were used to analyze correlations of patient and imaging characteristics with likelihood of malignancy. Results The reader study included 95 DBT examinations with moderate interrater reliability (Fleiss κ = 0.45). There was majority reader agreement in 85 of the 95 DBT examinations (89%) of 83 women (median age, 56 years; interquartile range, 47-69 years), and this finalized the study data set. At pathologic examination, most asymmetries (68 of 85, 80%) were benign, with common diagnoses being fibrocystic change ( n = 20), stromal fibrosis ( n = 10), and fat necrosis ( n = 10). The overall malignancy rate was 20% (17 of 85 asymmetries; 95% CI: 12, 29); 15 of the 17 malignancies (88%) were invasive cancers. Malignancies were more common in women with a personal history of breast cancer (35% vs 10%, P = .02). Conclusion In 85 developing asymmetries visualized at digital breast tomosynthesis without a sonographic correlate, there was a 20% (95% CI: 12, 29) malignancy rate, which was higher than the rates of malignancy for a developing asymmetry detected at digital mammography. © RSNA, 2021 See also the editorial by Skaane in this issue.

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221&#95;0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Identification of essential genes is key to understanding the required processes and gene products of organisms under one or more conditions. Transposon sequencing (Tn-seq) has been used to predict essential genes or ones that conditionally impact fitness in a wide variety of organisms. Here, we describe the generation of genome-scale mutant libraries and the analysis of Tn-seq data to identify essential genes from cultures grown in a single condition as well as those that are conditionally important by analyzing the behavior of these mutant libraries in different growth environments. While we illustrate the approach using data derived from Tn-seq analysis of the α-proteobacteria Rhodobacter sphaeroides and Zymomonas mobilis, the protocols and systems we describe should be generally applicable to a variety of organisms., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Lignin is a potential source of valuable chemicals, but its chemical depolymerization results in a heterogeneous mixture of aromatics and other products. Microbes could valorize depolymerized lignin by converting multiple substrates into one or a small number of products. In this study, we describe the ability of Novosphingobium aromaticivorans to metabolize 1-(4-hydroxy-3-methoxyphenyl)propane-1,2-dione (G-diketone), an aromatic Hibbert diketone that is produced during formic acid-catalyzed lignin depolymerization. By assaying genome-wide transcript levels from N. aromaticivorans during growth on G-diketone and other chemically-related aromatics, we hypothesized that the Lig dehydrogenases, previously characterized as oxidizing β-O-4 linkages in aromatic dimers, were involved in G-diketone metabolism by N. aromaticivorans. Using purified N. aromaticivorans Lig dehydrogenases, we found that LigL, LigN, and LigD each reduced the Cα ketone of G-diketone in vitro but with different substrate specificities and rates. Furthermore, LigL, but not LigN or LigD, also reduced the Cα ketone of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one (GP-1) in vitro , a derivative of G-diketone with the Cβ ketone reduced, when GP-1 was provided as a substrate. The newly identified activity of these Lig dehydrogenases expands the potential range of substrates utilized by N. aromaticivorans beyond what has been previously recognized. This is beneficial both for metabolizing a wide range of natural and non-native depolymerized lignin substrates and for engineering microbes and enzymes that are active with a broader range of aromatic compounds. IMPORTANCE Lignin is a major plant polymer composed of aromatic units that have value as chemicals. However, the structure and composition of lignin have made it difficult to use this polymer as a renewable source of industrial chemicals. Bacteria like Novosphingobium aromaticivorans have the potential to make chemicals from lignin not only because of their natural ability to metabolize a variety of aromatics but also because there are established protocols to engineer N. aromaticivorans strains to funnel lignin-derived aromatics into valuable products. In this work, we report a newly discovered activity of previously characterized dehydrogenase enzymes with a chemically modified by-product of lignin depolymerization. We propose that the activity of N. aromaticivorans enzymes with both native lignin aromatics and those produced by chemical depolymerization will expand opportunities for producing industrial chemicals from the heterogenous components of this abundant plant polymer.