Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Distortionless, free-breathing, and respiratory resolved 3D diffusion weighted imaging of the abdomen.

PURPOSE: Abdominal imaging is frequently performed with breath holds or respiratory triggering to reduce the effects of respiratory motion. Diffusion weighted sequences provide a useful clinical contrast but have prolonged scan times due to low signal-to-noise ratio (SNR), and cannot be completed in a single breath hold. Echo-planar imaging (EPI) is the most commonly used trajectory for diffusion weighted imaging but it is susceptible to off-resonance artifacts. A respiratory resolved, three-dimensional (3D) diffusion prepared sequence that obtains distortionless diffusion weighted images during free-breathing is presented. Techniques to address the myriad of challenges including: 3D shot-to-shot phase correction, respiratory binning, diffusion encoding during free-breathing, and robustness to off-resonance are described. METHODS: A twice-refocused, M1-nulled diffusion preparation was combined with an RF-spoiled gradient echo readout and respiratory resolved reconstruction to obtain free-breathing diffusion weighted images in the abdomen. Cartesian sampling permits a sampling density that enables 3D shot-to-shot phase navigation and reduction of transient fat artifacts. Theoretical properties of a region-based shot rejection are described. The region-based shot rejection method was evaluated with free-breathing (normal and exaggerated breathing), and respiratory triggering. The proposed sequence was compared in vivo with multishot DW-EPI. RESULTS: The proposed sequence exhibits no evident distortion in vivo when compared to multishot DW-EPI, robustness to B0 and B1 field inhomogeneities, and robustness to motion from different respiratory patterns. CONCLUSION: Acquisition of distortionless, diffusion weighted images is feasible during free-breathing with a b-value of 500 s/mm2, scan time of 6 min, and a clinically viable reconstruction time.

MR-guided focused ultrasound therapy of extra-abdominal desmoid tumors: a multicenter retrospective study of 105 patients.

OBJECTIVE: To assess the safety and efficacy of magnetic resonance-guided focused ultrasound (MRgFUS) for the treatment extra-abdominal desmoids. METHODS: A total of 105 patients with desmoid fibromatosis (79 females, 26 males; 35 ± 14 years) were treated with MRgFUS between 2011 and 2021 in three centers. Total and viable tumors were evaluated per patient at last follow-up after treatment. Response and progression-free survival (PFS) were assessed with (modified) response evaluation criteria in solid tumors (RECIST v.1.1 and mRECIST). Change in Numerical Rating Scale (NRS) pain and 36-item Short Form Health Survey (SF-36) scores were compared. Treatment-related adverse events were recorded. RESULTS: The median initial tumor volume was 114 mL (IQR 314 mL). After MRgFUS, median total and viable tumor volume decreased to 51 mL (95% CI: 30-71 mL, n = 101, p < 0.0001) and 29 mL (95% CI: 17-57 mL, n = 88, p < 0.0001), respectively, at last follow-up (median: 15 months, 95% CI: 11-20 months). Based on total tumor measurements (RECIST), 86% (95% CI: 75-93%) had at least stable disease or better at last follow-up, but 50% (95% CI: 38-62%) of remaining viable nodules (mRECIST) progressed within the tumor. Median PFS was reached at 17 and 13 months for total and viable tumors, respectively. NRS decreased from 6 (IQR 3) to 3 (IQR 4) (p < 0.001). SF-36 scores improved (physical health (41 (IQR 15) to 46 (IQR 12); p = 0.05, and mental health (49 (IQR 17) to 53 (IQR 9); p = 0.02)). Complications occurred in 36%, most commonly 1st/2nd degree skin burns. CONCLUSION: MRgFUS reduced tumor volume, reduced pain, and improved quality of life in this series of 105 patients with extra-abdominal desmoid fibromatosis. CLINICAL RELEVANCE STATEMENT: Imaging-guided ablation is being increasingly used as an alternative to surgery, radiation, and medical therapy for the treatment of desmoid fibromatosis. MR-guided high-intensity focused ultrasound is an incisionless ablation technique that can be used to reduce tumor burden effectively and safely. KEY POINTS: • Desmoid fibromatosis was treated with MR-guided high-intensity focused ultrasound in 105 patients. • MR-guided focused ultrasound ablation reduced tumor volume and pain and improved quality of life. • MR-guided focused ultrasound is a treatment option for patients with extra-abdominal desmoid tumors.

Quantification of the Hemodynamic Changes of Cirrhosis with Free-Breathing Self-Navigated MRI.

BACKGROUND: Non-invasive assessment of the hemodynamic changes of cirrhosis might help guide management of patients with liver disease but are currently limited. PURPOSE: To determine whether free-breathing 4D flow MRI can be used to quantify the hemodynamic effects of cirrhosis and introduce hydraulic circuit indexes of severity. STUDY TYPE: Retrospective. POPULATION: Forty-seven patients including 26 with cirrhosis. FIELD STRENGTH/SEQUENCE: 3 T/free-breathing 4D flow MRI with soft gating and golden-angle view ordering. ASSESSMENT: Measurements of the supra-celiac abdominal aorta, supra-renal abdominal aorta (SRA), celiac trunk (CeT), superior mesenteric artery (SMA), splenic artery (SpA), common hepatic artery (CHA), portal vein (PV), and supra-renal inferior vena cava (IVC) were made by two radiologists. Measures of hepatic vascular resistance (hepatic arterial relative resistance [HARR]; portal resistive index [PRI]) were proposed and calculated. STATISTICAL ANALYSIS: Bland-Altman, Pearson's correlation, Tukey's multiple comparison, and Cohen's kappa. P < 0.05 was considered significant. RESULTS: Forty-four of 47 studies yielded adequate image quality for flow quantification (94%). Arterial structures showed high inter-reader concordance (range; ρ = 0.948-0.987) and the IVC (ρ = 0.972), with moderate concordance in the PV (ρ = 0.866). Conservation of mass analysis showed concordance between large vessels (SRA vs. IVC; ρ = 0.806), small vessels (celiac vs. CHA + SpA; ρ = 0.939), and across capillary beds (CeT + SMA vs. PV; ρ = 0.862). Splanchnic flow was increased in patients with portosystemic shunting (PSS) relative to control patients and patients with cirrhosis without PSS (P < 0.05, difference range 0.11-0.68 liter/m). HARR was elevated and PRI was decreased in patients with PSS (3.55 and 1.49, respectively) compared to both the control (2.11/3.18) and non-PSS (2.11/2.35) cohorts. DATA CONCLUSION: 4D flow MRI with self-navigation was technically feasible, showing promise in quantifying the hemodynamic effects of cirrhosis. Proposed quantitative metrics of hepatic vascular resistance correlated with PSS. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 2.

Data-driven self-calibration and reconstruction for non-cartesian wave-encoded single-shot fast spin echo using deep learning.

BACKGROUND: Current self-calibration and reconstruction methods for wave-encoded single-shot fast spin echo imaging (SSFSE) requires long computational time, especially when high accuracy is needed. PURPOSE: To develop and investigate the clinical feasibility of data-driven self-calibration and reconstruction of wave-encoded SSFSE imaging for computation time reduction and quality improvement. STUDY TYPE: Prospective controlled clinical trial. SUBJECTS: With Institutional Review Board approval, the proposed method was assessed on 29 consecutive adult patients (18 males, 11 females, range, 24-77 years). FIELD STRENGTH/SEQUENCE: A wave-encoded variable-density SSFSE sequence was developed for clinical 3.0T abdominal scans to enable 3.5× acceleration with full-Fourier acquisitions. Data-driven calibration of wave-encoding point-spread function (PSF) was developed using a trained deep neural network. Data-driven reconstruction was developed with another set of neural networks based on the calibrated wave-encoding PSF. Training of the calibration and reconstruction networks was performed on 15,783 2D wave-encoded SSFSE abdominal images. ASSESSMENT: Image quality of the proposed data-driven approach was compared independently and blindly with a conventional approach using iterative self-calibration and reconstruction with parallel imaging and compressed sensing by three radiologists on a scale from -2 to 2 for noise, contrast, sharpness, artifacts, and confidence. Computation time of these two approaches was also compared. STATISTICAL TESTS: Wilcoxon signed-rank tests were used to compare image quality and two-tailed t-tests were used to compare computation time with P values of under 0.05 considered statistically significant. RESULTS: An average 2.1-fold speedup in computation was achieved using the proposed method. The proposed data-driven self-calibration and reconstruction approach significantly reduced the perceived noise level (mean scores 0.82, P < 0.0001). DATA CONCLUSION: The proposed data-driven calibration and reconstruction achieved twice faster computation with reduced perceived noise, providing a fast and robust self-calibration and reconstruction for clinical abdominal SSFSE imaging. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:841-853.

Deep residual network for off-resonance artifact correction with application to pediatric body MRA with 3D cones.

PURPOSE: To enable rapid imaging with a scan time-efficient 3D cones trajectory with a deep-learning off-resonance artifact correction technique. METHODS: A residual convolutional neural network to correct off-resonance artifacts (Off-ResNet) was trained with a prospective study of pediatric MRA exams. Each exam acquired a short readout scan (1.18 ms ± 0.38) and a long readout scan (3.35 ms ± 0.74) at 3 T. Short readout scans, with longer scan times but negligible off-resonance blurring, were used as reference images and augmented with additional off-resonance for supervised training examples. Long readout scans, with greater off-resonance artifacts but shorter scan time, were corrected by autofocus and Off-ResNet and compared with short readout scans by normalized RMS error, structural similarity index, and peak SNR. Scans were also compared by scoring on 8 anatomical features by two radiologists, using analysis of variance with post hoc Tukey's test and two one-sided t-tests. Reader agreement was determined with intraclass correlation. RESULTS: The total scan time for long readout scans was on average 59.3% shorter than short readout scans. Images from Off-ResNet had superior normalized RMS error, structural similarity index, and peak SNR compared with uncorrected images across ±1 kHz off-resonance (P < .01). The proposed method had superior normalized RMS error over -677 Hz to +1 kHz and superior structural similarity index and peak SNR over ±1 kHz compared with autofocus (P < .01). Radiologic scoring demonstrated that long readout scans corrected with Off-ResNet were noninferior to short readout scans (P < .05). CONCLUSION: The proposed method can correct off-resonance artifacts from rapid long-readout 3D cones scans to a noninferior image quality compared with diagnostically standard short readout scans.

Restriction spectrum imaging: An evolving imaging biomarker in prostate MRI.

Restriction spectrum imaging (RSI) is a novel diffusion-weighted MRI technique that uses the mathematically distinct behavior of water diffusion in separable microscopic tissue compartments to highlight key aspects of the tissue microarchitecture with high conspicuity. RSI can be acquired in less than 5 min on modern scanners using a surface coil. Multiple field gradients and high b-values in combination with postprocessing techniques allow the simultaneous resolution of length-scale and geometric information, as well as compartmental and nuclear volume fraction filtering. RSI also uses a distortion correction technique and can thus be fused to high resolution T2-weighted images for detailed localization, which improves delineation of disease extension into critical anatomic structures. In this review, we discuss the acquisition, postprocessing, and interpretation of RSI for prostate MRI. We also summarize existing data demonstrating the applicability of RSI for prostate cancer detection, in vivo characterization, localization, and targeting. LEVEL OF EVIDENCE: 5 J. Magn. Reson. Imaging 2017;45:323-336.

Spatial transcriptomic analysis drives PET imaging of tight junction protein expression in pancreatic cancer theranostics.

We apply spatial transcriptomics and proteomics to select pancreatic cancer surface receptor targets for molecular imaging and theranostics using an approach that can be applied to many cancers. Selected cancer surfaceome epithelial markers were spatially correlated and provided specific cancer localization, whereas the spatial correlation between cancer markers and immune- cell or fibroblast markers was low. While molecular imaging of cancer-associated fibroblasts and integrins has been proposed for pancreatic cancer, our data point to the tight junction protein claudin-4 as a theranostic target. Claudin-4 expression increased ∼16 fold in cancer as compared with normal pancreas, and the tight junction localization conferred low background for imaging in normal tissue. We developed a peptide-based molecular imaging agent targeted to claudin-4 with accumulation to ∼25% injected activity per cc (IA/cc) in metastases and ∼18% IA/cc in tumors. Our work motivates a new approach for data-driven selection of molecular targets.

Induction of interleukin-10 in the T helper type 17 effector population by the G protein coupled estrogen receptor (GPER) agonist G-1.

Interleukin-10 (IL-10) is a potent suppressor of the immune system, commonly produced by CD4(+) T cells to limit ongoing inflammatory responses minimizing host damage. Many autoimmune diseases are marked by large populations of activated CD4(+) T cells within the setting of chronic inflammation; therefore, drugs capable of inducing IL-10 production in CD4(+) T cells would be of great therapeutic value. Previous reports have shown that the small molecule G-1, an agonist of the membrane-bound G-protein-coupled estrogen receptor GPER, attenuates disease in an animal model of autoimmune encephalomyelitis. However, the direct effects of G-1 on CD4(+) T-cell populations remain unknown. Using ex vivo cultures of purified CD4(+) T cells, we show that G-1 elicits IL-10 expression in T helper type 17 (Th17) -polarized cells, increasing the number of IL-10(+) and IL-10(+) IL-17A(+) cells via de novo induction of IL-10. T-cell cultures differentiated in the presence of G-1 secreted threefold more IL-10, with no change in IL-17A, tumour necrosis factor-α, or interferon-γ. Moreover, inhibition of extracellular signal-regulated kinase (but not p38 or Jun N-terminal kinase) signalling blocked the response, while analysis of Foxp3 and RORγt expression demonstrated increased numbers of IL-10(+) cells in both the Th17 (RORγt(+)) and Foxp3(+) RORγt(+) hybrid T-cell compartments. Our findings translated in vivo as systemic treatment of male mice with G-1 led to increased IL-10 secretion from splenocytes following T-cell receptor cross-linking. These results demonstrate that G-1 acts directly on CD4(+) T cells, and to our knowledge provide the first example of a synthetic small molecule capable of eliciting IL-10 expression in Th17 or hybrid T-cell populations.

Alternative approach of hepatocellular carcinoma surveillance: abbreviated MRI.

This review focuses on emerging abbreviated magnetic resonance imaging (AMRI) surveillance of patients with chronic liver disease for hepatocellular carcinoma (HCC). This surveillance strategy has been proposed as a high-sensitivity alternative to ultrasound for identification of patients with early-stage HCC, particularly in patients with cirrhosis or obesity, in whom sonographic visualization of small tumors may be compromised. Three general AMRI approaches have been developed and studied in the literature - non-contrast AMRI, dynamic contrast-enhanced AMRI, and hepatobiliary phase contrast-enhanced AMRI - each comprising a small number of selected sequences specifically tailored for HCC detection. The rationale, general technique, advantages and disadvantages, and diagnostic performance of each AMRI approach is explained. Additionally, current gaps in knowledge and future directions are discussed. Based on emerging evidence, we cautiously recommend the use of AMRI for HCC surveillance in situations where ultrasound is compromised.

Convolutional neural network-automated hepatobiliary phase adequacy evaluation may optimize examination time.

PURPOSE: To develop and evaluate the performance of a fully-automated convolutional neural network (CNN)-based algorithm to evaluate hepatobiliary phase (HBP) adequacy of gadoxetate disodium (EOB)-enhanced MRI. Secondarily, we explored the potential of the proposed CNN algorithm to reduce examination length by applying it to EOB-MRI examinations. METHODS: We retrospectively identified EOB-enhanced MRI-HBP series from examinations performed 2011-2018 (internal and external datasets). Our algorithm, comprising a liver segmentation and classification CNN, produces an adequacy score. Two abdominal radiologists independently classified series as adequate or suboptimal. The consensus determination of HBP adequacy was used as ground truth for CNN model training and validation. Reader agreement was evaluated with Cohen's kappa. Performance of the algorithm was assessed by receiver operating characteristics (ROC) analysis and computation of the area under the ROC curve (AUC). Potential examination duration reduction was evaluated descriptively. RESULTS: 1408 HBP series from 484 patients were included. Reader kappa agreement was 0.67 (internal dataset) and 0.80 (external dataset). AUCs were 0.97 (0.96-0.99) for internal and 0.95 (0.92-96) for external and were not significantly different from each other (p = 0.24). 48 % (50/105) examinations could have been shorter by applying the algorithm. CONCLUSION: A proposed CNN-based algorithm achieves higher than 95 % AUC for classifying HBP images as adequate versus suboptimal. The application of this algorithm could potentially shorten examination time and aid radiologists in recognizing technically suboptimal images, avoiding diagnostic pitfalls.

Gadoxetate-enhanced Abbreviated MRI for Hepatocellular Carcinoma Surveillance: Preliminary Experience.

Purpose: To describe a single-center preliminary experience with gadoxetate disodium-enhanced abbreviated MRI for hepatocellular carcinoma (HCC) screening and surveillance in patients with cirrhosis or chronic hepatitis B virus (cHBV). Materials and Methods: This was a retrospective study of consecutive patients aged 18 years and older with cirrhosis or cHBV who underwent at least one gadoxetate-enhanced abbreviated MRI examination for HCC surveillance from 2014 through 2016. Examinations were interpreted prospectively by one of six abdominal radiologists for clinical care. Clinical, imaging, and other data were extracted from electronic medical records. Diagnostic adequacy was assessed in all patients. Diagnostic accuracy was assessed in the subset of patients who could be classified as having HCC or not having HCC on the basis of a composite reference standard. Results: In this study, 330 patients (93% with cirrhosis; 45% women; mean age, 59 years) underwent gadoxetate-enhanced abbreviated MRI. In the 330 patients, 311 (94.2%) baseline gadoxetate-enhanced abbreviated MRI examinations were diagnostically adequate. Of 141 (43%) of the 330 patients, 91.4% (129 of 141) could be classified as not having HCC and 8.6% (12 of 141) could be classified as having HCC. Baseline gadoxetate-enhanced abbreviated MRI had 0.92 sensitivity (95% confidence interval [CI]: 0.62, 1.00) and 0.91 specificity (95% CI: 0.84, 0.95) for detection of HCC. Of the 330 patients who underwent baseline gadoxetate-enhanced abbreviated MRI, 187 (57%) were lost to follow-up. Conclusion: Gadoxetate-enhanced abbreviated MRI is feasible clinically, has a high diagnostic adequacy rate, and, on the basis of our preliminary experience, accurately depicts HCC in high-risk patients. Strategies to enhance follow-up compliance are needed.© RSNA, 2019Keywords: Abdomen/GI, Cirrhosis, Liver, MR-Imaging, Oncology, ScreeningSupplemental material is available for this article.

Fully automated convolutional neural network-based affine algorithm improves liver registration and lesion co-localization on hepatobiliary phase T1-weighted MR images.

BACKGROUND: Liver alignment between series/exams is challenged by dynamic morphology or variability in patient positioning or motion. Image registration can improve image interpretation and lesion co-localization. We assessed the performance of a convolutional neural network algorithm to register cross-sectional liver imaging series and compared its performance to manual image registration. METHODS: Three hundred fourteen patients, including internal and external datasets, who underwent gadoxetate disodium-enhanced magnetic resonance imaging for clinical care from 2011 to 2018, were retrospectively selected. Automated registration was applied to all 2,663 within-patient series pairs derived from these datasets. Additionally, 100 within-patient series pairs from the internal dataset were independently manually registered by expert readers. Liver overlap, image correlation, and intra-observation distances for manual versus automated registrations were compared using paired t tests. Influence of patient demographics, imaging characteristics, and liver uptake function was evaluated using univariate and multivariate mixed models. RESULTS: Compared to the manual, automated registration produced significantly lower intra-observation distance (p < 0.001) and higher liver overlap and image correlation (p < 0.001). Intra-exam automated registration achieved 0.88 mean liver overlap and 0.44 mean image correlation for the internal dataset and 0.91 and 0.41, respectively, for the external dataset. For inter-exam registration, mean overlap was 0.81 and image correlation 0.41. Older age, female sex, greater inter-series time interval, differing uptake, and greater voxel size differences independently reduced automated registration performance (p ≤ 0.020). CONCLUSION: A fully automated algorithm accurately registered the liver within and between examinations, yielding better liver and focal observation co-localization compared to manual registration.

The incidence of pulmonary embolism and associated FDG-PET findings in IV contrast-enhanced PET/CT.

RATIONALE AND OBJECTIVES: Most fluorine-18 fluorodeoxyglucose (FDG)-positron emission tomography with computed tomography (PET/CT) studies are performed on cancer patients. These patients are at increased risk of pulmonary embolism (PE). In this retrospective review, we determined the rate of PE, and the prevalence of associated FDG-PET findings on intravenous (IV) contrast-enhanced PET/CT. MATERIALS AND METHODS: We identified all PET/CT studies performed at our institution with a reported finding of PE between January 2005 and October 2012. The medical record was reviewed for symptoms, which were identified after the diagnosis of PE, and whether the patients received treatment. The prevalence of associated FDG-PET findings was determined. RESULTS: A total of 65 total cases of PE (of 182,72 total PET/CT examinations) were identified of which 59 were previously unknown. This gives an incidental PE (IPE) rate of 0.32%. Of the patients where sufficient clinical information was available, 34 of 36 (94%) were treated either with therapeutic anticoagulation or inferior vena cava filter, and 30 of 36 (83%) were asymptomatic in retrospect. Of the patients with IPE, we found nine (15.2%) with associated focal pulmonary artery hypermetabolism, three (5.1%) with hypermetabolic pulmonary infarction, and one with increased isolated right ventricular FDG uptake (1.7%). One case of chronic PE demonstrated a focal hypometabolic filling defect in a pulmonary artery on PET. CONCLUSIONS: We found IPE in 0.32% of PET/CT scans. Focal pulmonary artery hypermetabolism or hypometabolism, and hypermetabolic pulmonary artery infarction with the "rim sign" were uncommonly associated with PE. These findings could raise the possibility of IPE in non-IV contrast-enhanced PET/CT studies.

The G protein-coupled estrogen receptor (GPER) agonist G-1 expands the regulatory T-cell population under TH17-polarizing conditions.

The transcription factor Foxp3 is critical to the suppressive phenotype of CD4+ regulatory T cells. Studies have clearly shown that numerous autoimmune diseases are marked by the presence of activated CD4+ T cells within the setting of chronic inflammation. Therefore, drugs capable of inducing Foxp3 expression in activated CD4+ T cells could be of great therapeutic interest. We have previously shown that the small molecule G-1, an agonist directed against the membrane-bound G protein-coupled estrogen receptor, can induce IL10 expression in naive CD4+ T cells. In addition, we and others have demonstrated that G-1 attenuates disease in an animal model of experimental autoimmune encephalomyelitis. Using ex vivo cultures of purified CD4+ T cells, we show that G-1 can elicit Foxp3 expression under TH17-polarizing conditions, which mimic the in situ inflammatory milieu of several autoimmune diseases. These findings build upon previous results demonstrating the immunosuppressive properties of the novel estrogenic small molecule G-1.

Characterization of sporulation histidine kinases of Bacillus anthracis.

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.