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Viruses are obligate intracellular parasites that rely on host cell metabolism for successful replication. Thus, viruses rewire host cell pathways involved in central carbon metabolism to increase the availability of building blocks for successful propagation. However, the underlying mechanisms of virus-induced alterations to host metabolism are largely unknown. Noroviruses (NoVs) are highly prevalent pathogens that cause sporadic and epidemic viral gastroenteritis. In the present study, we uncovered several strain-specific and shared host cell metabolic requirements of three murine norovirus (MNV) strains, MNV-1, CR3, and CR6. While all three strains required glycolysis, glutaminolysis, and the pentose phosphate pathway for optimal infection of macrophages, only MNV-1 relied on host oxidative phosphorylation. Furthermore, the first metabolic flux analysis of NoV-infected cells revealed that both glycolysis and glutaminolysis are upregulated during MNV-1 infection of macrophages. Glutamine deprivation affected the viral lifecycle at the stage of genome replication, resulting in decreased non-structural and structural protein synthesis, viral assembly, and egress. Mechanistic studies further showed that MNV infection and overexpression of the non-structural protein NS1/2 increased the enzymatic activity of the rate-limiting enzyme glutaminase. In conclusion, the inaugural investigation of NoV-induced alterations to host glutaminolysis identified NS1/2 as the first viral molecule for RNA viruses that regulates glutaminolysis either directly or indirectly. This increases our fundamental understanding of virus-induced metabolic alterations and may lead to improvements in the cultivation of human NoVs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hafner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The development of new vaccines against the SARS-CoV-2 virus in response to the COVID-19 pandemic represents a milestone in the history of public health. However, due to the rapid development and short duration of these new vaccines, the full spectrum of side effects is not yet known. A 76-year-old man presented to the clinic for follow-up after being discharged from the emergency department for hyperglycemia. His medical history included well-controlled type 2 diabetes for two years, hypertension, and hyperlipidemia. He had recently noticed high home blood glucose readings over 400 mg/dL, and his hemoglobin A1c (mean 90-day glucose level) had increased from 6.5% to 12.6%. Notably, the patient reported having excellent health behaviors, including daily exercise, a closely monitored healthy diet, and regular blood glucose testing. After extensive endocrinology workup, the rapid change in blood glucose was thought to be due to his having recently received the COVID-19 messenger RNA (mRNA) vaccine. He was started on long- and short-acting insulin and a glucagon-like peptide-1 agonist (novel injectable type 2 diabetes medication), with improvement in blood glucose. He was tapered off all medications and remains on metformin 1,000 mg twice daily after one year.Whether the new COVID-19 mRNA vaccines directly incur hyperglycemia within certain groups of patients with diabetes is not known; thus, studies exploring the relationship between vaccine antigen binding and pancreatic function are needed., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Okon-Umoren et al.)

Objectives: Educational approaches for training physicians in clinical communications vary, and whether physicians apply the communication skills they learn or find them useful in the clinic is not well known. The aim of this study was to determine how first-year residents who received explicit instruction in 7 communication strategies would apply them in a simulation exercise and in clinical practice., Methods: First-year Internal Medicine residents at an urban teaching hospital received instruction in 7 systematic communication strategies: Ask-Tell-Ask, Teach-back, open-ended questioning, NURSE, open body language, pausing, and plain language. Residents were evaluated on their use of specific communication behaviors associated with the 7 strategies during a simulation exercise of disclosing a medical error to a standardized patient. Control group residents who did the simulation before attending the training program and training group residents who did the simulation after the training were compared. Residents were queried 6 months after the training program on their use of communication strategies during clinical practice., Results: A total of 27 residents participated ( n  = 13 control group; n  = 14 training group). The training group performed behaviors for "establishing patient understanding" significantly more often than the control group. Both groups used non-verbal communication and behaviors for addressing patient emotions at similar levels. Of the 24 residents who responded to the 6-month follow-up questionnaire, 24 (100%) reported using Ask-Tell-Ask, open-ended questioning, and Teach-back, and 22 (92%) reported using NURSE statements and non-verbal communication. Most respondents reported using the strategies in clinical practice often or very often (79%) and found the strategies useful or very useful (96%)., Conclusion: Providing explicit instruction in systematic communication strategies, particularly those focused on establishing patient understanding, may be an efficient approach for helping early career physicians develop effective communication skills that can be readily implemented during clinical training and practice., (© The Author(s) 2024.)

Problem: Systematically investigating annual Accreditation Council for Graduate Medical Education (ACGME) Resident/Fellow Survey results by directly gathering trainee feedback could uncover training program problems and clarify misunderstandings as they arise, leading to faster corrective actions and program improvement., Approach: The Focus Group Forum (FGF) was created based on the utilization-focused evaluation approach to systematically gather comprehensive, high-quality, actionable trainee feedback on specific annual ACGME survey results and involve trainees in program improvement (Henry Ford Hospital, 2021). Trainees from programs with survey results indicating <80% compliance within several content areas were invited to attend FGF sessions. During FGF sessions, neutral moderators experienced in conducting focus groups and creating psychologically safe spaces and neutral scribes gathered trainee feedback on survey results through structured, iterative discussions and an anonymous electronic polling system. Summaries of FGF findings were created, combined with actual annual ACGME survey data, and used to develop recommended corrective actions and monitoring plans., Outcomes: In 2021, 6 training programs had survey results below the institution's compliance threshold for 4-8 of the 9 content areas. Of the 180 trainees (from the 6 programs) invited to attend an FGF session, 79 (44%) participated. Five key issues were identified: misinterpretation of several survey questions, lack of knowledge of institutional policies and procedures, perceived inability to share feedback with faculty, feelings of being overwhelmed with administrative duties, and lack of sufficient protected time for educational activities and requirements., Next Steps: The authors are developing an FGF process for faculty so that all stakeholders have a voice regarding annual ACGME survey results. They are also improving scheduling processes so that feedback from experienced trainees who are leaving the institution will not be missed and developing longer-term processes for tracking outcomes since time for implementing corrective actions before the next ACGME survey is limited., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Association of American Medical Colleges.)

Background: Research has explored the problems that women encounter during a medical career; however, the advice that experienced women physicians would give to women who have not yet entered the field is needed to reveal how the medical work landscape is evolving and to provide real-world narratives to help career seekers make informed choices., Objective: By eliciting women's perspectives on their medical careers by asking them what advice they would give to aspiring women physicians, we aimed to reveal areas for improving career satisfaction of women physicians and to inform those who advise women considering a medical career., Methods: In this qualitative study, we used a phenomenological approach to conduct semi-structured one-on-one interviews with 24 women physicians to query the advice they would give to women contemplating a career in medicine., Results: Thematic analysis of interview transcriptions revealed 10 themes that women physicians communicated as being important to consider before deciding to become a physician. Although some advice had a cautionary tone, encouraging and practical advice was also conveyed. The most abundant themes concerned the centrality of patient care, a passion for practicing medicine, and the importance of planning. Other key topics included family and friends, self-reflection, life balance, finances, ethics, maintaining presence, and two overt cautionary statements., Conclusion: Interviews revealed that meaning and purpose derived from a medical career and maintaining work-life balance are valued by some women physicians. Participants were encouraging in recommending medicine as a career choice for women, while highlighting some challenges.

The Henry Ford Health System provides patients with a safe, improved system of continuous kidney replacement therapy using a proprietary, 24-hour sustained low-efficiency dialysis (SLED). The SLED system utilizes regional citrate anticoagulation (RCA) in conventional hemodialysis machines that have been configured to provide slow dialytic therapy. Within our hospital complex, SLED-RCA systems are deployed in intensive care units distributed over 4 floors in 2 buildings. This widespread footprint represents a spatial challenge for hemodialysis technicians. Fifteen SLED-RCA machines may be running at one time, and each deployed unit may signal an alarm for multiple reasons. Previously, audible alarms prompted intensive care unit nurses to identify the alarming machine and manually notify technicians by telephone. Technicians would then travel to resolve the alarm. To improve the process of addressing SLED-RCA machine alarms, we developed a remote alert alarm system that wirelessly notifies hemodialysis technicians of specific machine alarms. A quality improvement analysis of nearly 1,000 SLED-RCA alarms over a 1-week period revealed that the average time for alarm correction with a remote alert alarm system was approximately 5 minutes. Reducing alarm resolution time may free technicians and nurses for other critical duties., (© 2022 The Author(s).)

Health systems were abruptly plunged into a crisis as SARS-CoV-2 exploded into a pandemic in spring 2020. In March-April 2020, Metropolitan Detroit was a US "hotspot." As a large health system with five hospitals and two behavioural health inpatient facilities, a health insurance company, a medical group and physician network, and 41 ambulatory clinics normally hosting over 10,000 daily patient encounters, the Henry Ford Health System deployed numerous strategies in the management of this upheaval. As hospitals and Emergency Departments were inundated with COVID-19 patients, other services and activities needed to shut down as state-mandated policies were promulgated, new internal and external communication networks established, and management of employees and resources such as ventilators, ICU beds, personal protective equipment, and laboratory supplies became critical challenges. We describe herein the system-wide strategies implemented and lessons learned in the operation of a health system in the initial throes of a global pandemic., (© 2021 The Authors. The International Journal of Health Planning and Management published by John Wiley & Sons Ltd.)

Introduction: The exact prevalence of obstructive sleep apnea (OSA) is unknown, and primary care providers are left with conflicting guidance on screening criteria from various institutions. The purpose of this study was to identify health care gaps in OSA diagnosis for patients at high risk of OSA., Methods: A retrospective medical record review was performed assessing adult patients (≥ 18 years) who had outpatient visits in family medicine clinics, located in the cities of Detroit, Troy, and Commerce, Michigan in 2018. The primary outcome was the number of patients assessed for OSA. Patients determined as high risk for OSA had at least 3 of the following criteria: (1) hypertension, (2) age 50 years and older, (3) male gender, and (4) body mass index > 35 kg/m 2 . Statistical approach included univariate and logistic regression analysis. Manual chart review of 200 randomly selected records was performed to determine the most common reasons for OSA screening., Results: Out of 30,022 patients, 4,911 (16.4%) were at high risk for OSA, of which 1,524 (31.0%) were assessed for OSA. Logistic regression analysis of high-risk patients revealed that male sex (odds ratio, 1.84; 95% CI, 1.51-2.26; P < .001 ) and body mass index > 35 kg/m 2 (odds ratio, 4.96; 95% CI, 4.04-6.09; P < .001 ) were significantly associated with OSA evaluation. Race was not associated with OSA assessment., Conclusion: Because many individuals at high risk for OSA are not referred for evaluation, improved guidance on OSA screening based on objective risk factors is needed., Competing Interests: Competing and conflicting interests: None declared., (© Copyright 2022 by the American Board of Family Medicine.)

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Developing as a physician requires an enormous amount of complex training, and quality of instruction greatly affects training outcomes. But while physicians are expected to teach trainees within the clinic, they often do not receive formal training in effective instructional practices. Providing faculty development programs is one way that institutions can help physicians develop teaching skills, but these programs often are developed without the input of educational specialists and not based in educational theory. In this methodology paper, we describe a 5-module curriculum that was developed in a cross-disciplinary collaboration between instructional designers and physician faculty. By merging educational and medical expertise and using adult learning theory with the Charlotte-Danielson educational framework, an essentials for clinical teaching educational endorsement program (ECTEEP) was created as a feature of the institutional curriculum within a large, urban teaching hospital. Here we describe how the program was developed through a physician-educator partnership, outline the program's key content, and highlight essential aspects of successful implementation. The ECTEEP incorporates active learning approaches within an abbreviated format, distilling 5 critical aspects of effective teaching that are relevant to the clinical environment: cultural humility and safe learning environments, instruction practices for engaging learners, instruction and assessment strategies, receiving and giving feedback, and mentorship and coaching. A central feature of the program is that facilitators actively model the teaching behaviors they are conveying, which underscores the critical importance of facilitator preparation and skill. Our curriculum is offered here as a basic template for institutions that may want to establish a program for enhancing physician teaching skill., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Introduction: Several overdoses of the antiepileptic drug perampanel have been reported in adults, but very few have been reported in children. We report the case of an observed exploratory ingestion of perampanel in a 2-year-old child that resulted in ataxia and prolonged coma., Case Report: A previously healthy 2-year-old female patient presented to the emergency department (ED) 30 minutes after the witnessed ingestion of 30 mg of perampanel (2 mg/kg). Within minutes of ingestion, she displayed ataxia and inability to walk. Upon ED presentation, she had normal vital signs but was minimally responsive with physical stimulation. Naloxone was given without response. She was intubated because of profound central nervous system depression and transferred to a pediatric tertiary care facility. She remained intubated with no pharmacological sedation. Physical exam showed a horizontal nystagmus. Detailed neurologic examination of ataxia and coordination was not possible, and she did not demonstrate hyperreflexia, clonus, or rigidity. Her mental status gradually improved, and she was extubated approximately 72 hours after exposure. After extubation, the patient still exhibited truncal ataxia and did not return to baseline until 96 hours post ingestion. Serum drawn approximately 16 hours after exposure showed 870 ng/mL perampanel (ref < 20 ng/mL). She remained hemodynamically stable throughout her hospital course, despite protracted depressed mental status., Discussion: Given the severity of our patient's presentation, pediatric patients showing symptoms of perampanel overdose should be triaged to the ED for evaluation in anticipation of a prolonged clinical course with decreased consciousness and hypoventilation.

Background: Legalization of medical and recreational cannabis is a major contributor to pediatric cannabis exposures. The trends and magnitude of pediatric cannabis exposures in Michigan after medical cannabis legalization in 2008 have not been assessed., Objective: To describe the temporal trends of pediatric cannabis exposures reported to the Michigan Poison Center (MiPC) after medical cannabis was legalized in 2008 and 1 year after legalization of recreational cannabis in 2018., Methods: Retrospective electronic chart review of pediatric (<18 years old) single-substance cannabis exposures reported to the MiPC from January 1, 2008 to December 31, 2019. Routes of cannabis exposure were reported as ingestion, inhalation, and unknown. Types of ingested cannabis products were also documented., Results: Between 2008 and 2019, 426 pediatric cannabis single exposures were reported. The median patient age was 6.0 years (interquartile range 2-15 years). Age distribution was bimodal. A total of 327 (76.8%) exposures were from cannabis ingestion, 79 (18.5%) from inhalation, 2 (0.5%) from both ingestion and inhalation, and 18 (4.2%) from unknown route. The doubling time for number of cases was 2.1 years, and the total number of annual reported cases increased after 2016. Teenagers (13-17 years) had the highest number of inhalational exposures, whereas young children (0-5 years) had the highest number of ingestions., Conclusion: Single-substance pediatric cannabis exposures reported to the Michigan Poison Center increased after medical cannabis was legalized in 2008 through recreational legalization in 2018., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A long-standing paradigm in virology was that non-enveloped viruses induce cell lysis to release progeny virions. However, emerging evidence indicates that some non-enveloped viruses exit cells without inducing cell lysis, while others engage both lytic and non-lytic egress mechanisms. Enteric viruses are transmitted via the faecal-oral route and are important causes of a wide range of human infections, both gastrointestinal and extra-intestinal. Virus cellular egress, when fully understood, may be a relevant target for antiviral therapies, which could minimize the public health impact of these infections. In this review, we outline lytic and non-lytic cell egress mechanisms of non-enveloped enteric RNA viruses belonging to five families: Picornaviridae , Reoviridae , Caliciviridae , Astroviridae and Hepeviridae . We discuss factors that contribute to egress mechanisms and the relevance of these mechanisms to virion stability, infectivity and transmission. Since most data were obtained in traditional two-dimensional cell cultures, we will further attempt to place them into the context of polarized cultures and in vivo pathogenesis. Throughout the review, we highlight numerous knowledge gaps to stimulate future research into the egress mechanisms of these highly prevalent but largely understudied viruses.

The mitochondrial network plays a critical role in the regulation of innate immune signaling and subsequent production of proinflammatory cytokines such as IFN-β and IL-1β. Dynamin-related protein 1 (DRP1) promotes mitochondrial fission and quality control to maintain cellular homeostasis during infection. However, mechanisms by which DRP1 and mitochondrial dynamics control innate immune signaling and the proinflammatory response are incompletely understood. Here we show that macrophage DRP1 is a positive regulator of TNF-α production during sterile inflammation or bacterial infection. Silencing macrophage DRP1 decreased mitochondrial fragmentation and TNF-α production upon stimulation with lipopolysaccharide (LPS) or methicillin-resistant Staphylococcus aureus (MRSA) infection. The defect in TNF-α induction could not be attributed to changes in gene expression. Instead, DRP1 was required for post-transcriptional control of TNF-α. In contrast, silencing DRP1 enhanced IL-6 and IL-1β production, indicating a distinct mechanism for DRP1-dependent TNF-α regulation. Our results highlight DRP1 as a key player in the macrophage pro-inflammatory response and point to its involvement in post-transcriptional control of TNF-α production., 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 © 2021 Gao, Reynolds, Passalacqua, Sexton, Abuaita and O’Riordan.)

Performic acid (PFA) is an emerging disinfectant to inactivate bacterial and viral microorganisms in wastewater. In this study, the inactivation kinetics of murine norovirus (MNV) by PFA, in phosphate buffer and municipal secondary effluent wastewater, are reported for the first time. PFA decay followed first-order kinetics and the inactivation of MNV was governed by the exposure of microorganisms to PFA, i.e., the integral of the PFA concentration over time (integral CT or ICT). The extension of the Chick-Watson model, in the ICT domain, described well the reduction of MNV by PFA, with determined ICT-based inactivation rate constants, k d , of 1.024 ± 0.038 L/(mg·min) and 0.482 ± 0.022 L/(mg·min) in phosphate buffer and wastewater, respectively, at pH 7.2. Furthermore, the simultaneous PFA inactivation of MNV and fecal indicators indigenously present in wastewater such as fecal coliforms and enterococci showed that 1-log reduction could be achieved with ICT of 2, 1.5, and 3.5 mg·min/L, respectively. When compared with the most commonly used peracid disinfectant of municipal wastewater, peracetic acid (PAA), the ICT requirements determined using the fitted ICT-based kinetic models were ∼20 times higher for PAA than PFA, indicating a much stronger inactivation power of the PFA molecule.

Ferrate(VI) (Fe VI O 4 2 , Fe(VI)) is an emerging oxidant/disinfectant to treat a wide range of contaminants and microbial pollutants in wastewater. This study describes the inactivation of murine norovirus (MNV) by Fe(VI) in phosphate buffer (PB) and secondary effluent wastewater (SEW). The decay of Fe(VI) had second-order kinetics in PB while Fe(VI) underwent an initial demand followed by first-order decay kinetics in SEW. The Chick-Watson inactivation kinetic model, based on integral CT (ICT) dose, well fitted the inactivation of MNV in both PB and SEW. In PB, the values of the inactivation rate constant ( k d ) decreased with an increase in pH, which was related to the reaction of protonated Fe(VI) species (HFeO 4 - ) with MNV. Higher k d was observed in SEW than in PB. The inactivation of indigenous fecal coliforms (FC) in SEW was also measured. A two-population double-exponential model that accounted for both dispersed and particle-associated FC well fitted the inactivation data with determined k d and particle-associated inactivation rate constant ( k p ). Results show that Fe(VI) was more effective in inactivating dispersed FC than MNV. The MNV inactivation results obtained herein, coupled with the detailed modeling, provide important information in designing an Fe(VI) wastewater disinfection process.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

The innate immune system senses microbial ligands through pattern recognition and triggers downstream signaling cascades to promote inflammation and immune defense mechanisms. Emerging evidence suggests that cells also recognize alterations in host processes induced by infection as triggers. Protein ubiquitination and deubiquitination are post-translational modification processes essential for signaling and maintenance of cellular homeostasis, and infections can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the impact of ubiquitin homeostasis alteration on macrophage function. Perturbation of ubiquitin homeostasis led to a rapid and transient burst of reactive oxygen species (ROS) that promoted macrophage inflammatory and anti-infective capacity. Moreover, we found that ROS production was dependent on the NOX2 phagocyte NADPH oxidase. Global alteration of the ubiquitin proteome also enhanced proinflammatory cytokine production in mice stimulated with a sub-lethal dose of LPS. Collectively, our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.

The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics and energetic profiling of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus (MNV) infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in macrophages revealed that glycolysis is an important factor for optimal MNV infection, while inhibiting the PPP and OXPHOS showed a relatively minor impact of these pathways on MNV infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased viral protein production and viral RNA. The requirement of glycolysis was specific for MNV (but not astrovirus) infection, independent of the type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMP-activated protein kinase (AMPK), two master regulators of cellular metabolism, implicating Akt signaling in upregulating host metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates glycolysis as a virulence determinant. They further point to cellular metabolism as a novel therapeutic target for norovirus infections and improvements in current human norovirus culture systems. IMPORTANCE Viruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase glycolysis upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high-efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies., (Copyright © 2019 Passalacqua et al.)

In Nature, Gerlach et al. (Nature 2018;563:705-709) report that methicillin-resistant Staphylococcus aureus camouflages its surface by displaying a 'stealth' wall teichoic acid (WTA) isomer. WTA can act as a cloak to limit exposure of surface antigens to the immune system, but this report indicates that even the cloak can become immunologically silent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Bacterial pathogens have evolved to exploit humans as a rich source of nutrients to support survival and replication. The pathways of bacterial metabolism that permit successful colonization are surprisingly varied and highlight remarkable metabolic flexibility. The constraints and immune pressures of distinct niches within the human body set the stage for understanding the mechanisms by which bacteria acquire critical nutrients. In this article we discuss how different bacterial pathogens carry out carbon and energy metabolism in the host and how they obtain or use key nutrients for replication and immune evasion.

Whole-genome sequencing was used to compare longitudinal isolates of Staphylococcus aureus that developed resistance to oxacillin (MIC up to 16 μg/ml). The mecA gene was absent. A novel 5-bp TATCC frameshift insertion in a gene encoding an ABC transporter similar to that of the teichoic acid translocation ATP-binding protein TagH and a 3-bp GCT nonframeshift insertion in the pdhA pyruvate dehydrogenase gene were detected in the oxacillin-resistant isolates., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: The genetic network involved in the bacterial cell cycle is poorly understood even though it underpins the remarkable ability of bacteria to proliferate. How such network evolves is even less clear. The major aims of this work were to identify and examine the genes and pathways that are differentially expressed during the Caulobacter crescentus cell cycle, and to analyze the evolutionary features of the cell cycle network., Results: We used deep RNA sequencing to obtain high coverage RNA-Seq data of five C. crescentus cell cycle stages, each with three biological replicates. We found that 1,586 genes (over a third of the genome) display significant differential expression between stages. This gene list, which contains many genes previously unknown for their cell cycle regulation, includes almost half of the genes involved in primary metabolism, suggesting that these "house-keeping" genes are not constitutively transcribed during the cell cycle, as often assumed. Gene and module co-expression clustering reveal co-regulated pathways and suggest functionally coupled genes. In addition, an evolutionary analysis of the cell cycle network shows a high correlation between co-expression and co-evolution. Most co-expression modules have strong phylogenetic signals, with broadly conserved genes and clade-specific genes predominating different substructures of the cell cycle co-expression network. We also found that conserved genes tend to determine the expression profile of their module., Conclusion: We describe the first phylogenetic and single-nucleotide-resolution transcriptomic analysis of a bacterial cell cycle network. In addition, the study suggests how evolution has shaped this network and provides direct biological network support that selective pressure is not on individual genes but rather on the relationship between genes, which highlights the importance of integrating phylogenetic analysis into biological network studies.

Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediate S. aureus (VISA). In this study, we characterized two isogenic USA300 S. aureus isolates collected sequentially from a single patient with endocarditis where the S. aureus isolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein of S. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII, sarT, and saeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquired in vivo. Creation of de novo mutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype.

Background: Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated., Results: In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF., Conclusions: To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.

Background: Although genome-wide transcriptional analysis has been used for many years to study bacterial gene expression, many aspects of the bacterial transcriptome remain undefined. One example is antisense transcription, which has been observed in a number of bacteria, though the function of antisense transcripts, and their distribution across the bacterial genome, is still unclear., Methodology/principal Findings: Single-stranded RNA-seq results revealed a widespread and non-random pattern of antisense transcription covering more than two thirds of the B. anthracis genome. Our analysis revealed a variety of antisense structural patterns, suggesting multiple mechanisms of antisense transcription. The data revealed several instances of sense and antisense expression changes in different growth conditions, suggesting that antisense transcription may play a role in the ways in which B. anthracis responds to its environment. Significantly, genome-wide antisense expression occurred at consistently higher levels on the lagging strand, while the leading strand showed very little antisense activity. Intrasample gene expression comparisons revealed a gene dosage effect in all growth conditions, where genes farthest from the origin showed the lowest overall range of expression for both sense and antisense directed transcription. Additionally, transcription from both strands was verified using a novel strand-specific assay. The variety of structural patterns we observed in antisense transcription suggests multiple mechanisms for this phenomenon, suggesting that some antisense transcription may play a role in regulating the expression of key genes, while some may be due to chromosome replication dynamics and transcriptional noise., Conclusions/significance: Although the variety of structural patterns we observed in antisense transcription suggest multiple mechanisms for antisense expression, our data also clearly indicate that antisense transcription may play a genome-wide role in regulating the expression of key genes in Bacillus species. This study illustrates the surprising complexity of prokaryotic RNA abundance for both strands of a bacterial chromosome.

Background: Bacillus anthracis produces two catecholate siderophores, petrobactin and bacillibactin, under iron-limited conditions. Here, we investigate how variable iron and oxygen concentrations influence the biosynthetic output of both siderophores in B. anthracis. In addition, we describe the differential levels of transcription of select genes within the B. anthracis siderophore biosynthetic operons that are responsible for synthesis of petrobactin and bacillibactin, during variable growth conditions., Methodology/principal Findings: Accumulation of bacillibactin in B. anthracis Sterne (34F(2)) and in a mutant lacking the major superoxide dismutase (ΔsodA1) was almost completely repressed by the addition of 20 µM of iron. In contrast, petrobactin synthesis in both strains continued up to 20 µM of iron. Accumulation of petrobactin and bacillibactin showed a slight increase with addition of low levels of paraquat-induced oxidative stress in wild type B. anthracis Sterne. Cultures grown with high aeration resulted in greater accumulation of petrobactin relative to low aeration cultures, and delayed the repressive effect of added iron. Conversely, iron-depleted cultures grown with low aeration resulted in increased levels of bacillibactin. No difference was found in overall superoxide dismutase (SOD) activity or transcriptional levels of the sodA1 and sodA2 genes between iron-depleted and iron-replete conditions at high or low aeration, suggesting that SOD regulation and iron metabolism are separate in B. anthracis. The highest transcription of the gene asbB, part of the petrobactin biosynthetic operon, occurred under iron-limitation with high aeration, but transcription was readily detectable even under iron-replete conditions and in low aeration. The gene dhbC, a member of the bacillibactin biosynthetic operon, was only transcribed under conditions of iron-depletion, regardless of growth aeration., Conclusion: These data suggest that bacillibactin regulation is highly sensitive to iron-concentration. In contrast, although regulation of petrobactin is less dependent on iron, it is likely subject to additional levels of regulation that may contribute to virulence of B. anthracis.

Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity.

While the mRNA expression patterns of homeotic genes have been examined in numerous arthropod species, data on their protein accumulation is extremely limited. To address this gap, we analyzed the protein expression pattern of the hox gene Sex combs reduced (Scr) in six hemimetabolous insects from four divergent orders (Thysanura, Orthoptera, Dictyoptera and Hemiptera). Our comparative analysis reveals that the original domain of SCR expression was likely confined to the head and then subsequently moved into the prothorax (T1) in winged insect lineages. The data also show a trend toward the posteriorization of the anterior boundary of SCR expression in the head, which starts in the mandibles (Thysanura) and then gradually shifts to the maxillary (Orthoptera) and labial segments (Dictyoptera and Hemiptera), respectively. In Thermobia (firebrat) and Oncopeltus (milkweed bug) we also identify instances where SCR protein is not detected in regions where mRNA is expressed. This finding suggests the presence of a post-transcriptional regulatory mechanism of Scr in these species. Finally, we show that SCR expression in insect T1 legs is highly variable and exhibits divergent patterning even among related species. In addition, signal in the prothoracic legs of more basal insect lineages cannot be associated with any T1 specific features, indicating that the acquisition of SCR in this region preceded any apparent gain of function. Overall, our results show that Scr expression has diverged considerably among hemimetabolous lineages and establish a framework for subsequent analyses to determine its role in the evolution of the insect head and prothorax.

Although gene expression has been studied in bacteria for decades, many aspects of the bacterial transcriptome remain poorly understood. Transcript structure, operon linkages, and information on absolute abundance all provide valuable insights into gene function and regulation, but none has ever been determined on a genome-wide scale for any bacterium. Indeed, these aspects of the prokaryotic transcriptome have been explored on a large scale in only a few instances, and consequently little is known about the absolute composition of the mRNA population within a bacterial cell. Here we report the use of a high-throughput sequencing-based approach in assembling the first comprehensive, single-nucleotide resolution view of a bacterial transcriptome. We sampled the Bacillus anthracis transcriptome under a variety of growth conditions and showed that the data provide an accurate and high-resolution map of transcript start sites and operon structure throughout the genome. Further, the sequence data identified previously nonannotated regions with significant transcriptional activity and enhanced the accuracy of existing genome annotations. Finally, our data provide estimates of absolute transcript abundance and suggest that there is significant transcriptional heterogeneity within a clonal, synchronized bacterial population. Overall, our results offer an unprecedented view of gene expression and regulation in a bacterial cell.

Background: Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains., Methodology/principal Findings: Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241), an attenuated strain of B. anthracis (Sterne 34F(2)), and an avirulent B. cereus strain (10987)--during exponential growth in two distinct atmospheric environments: 14% CO(2)/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2) environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment., Conclusions/significance: We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and highlights the importance of looking beyond differences in gene content in comparative genomics studies.

Unlabelled: Here, we report the development of SOCS (short oligonucleotide color space), a program designed for efficient and flexible mapping of Applied Biosystems SOLiD sequence data onto a reference genome. SOCS performs its mapping within the context of 'color space', and it maximizes usable data by allowing a user-specified number of mismatches. Sequence census functions facilitate a variety of functional genomics applications, including transcriptome mapping and profiling, as well as ChIP-Seq., Availability: Executables, source code, and sample data are available at http://socs.biology.gatech.edu/

Microarray analyses were conducted to evaluate the paraquat-induced global transcriptional response of Bacillus anthracis Sterne (34F(2)) to varying levels of endogenous superoxide stress. Data revealed that the transcription of genes putatively involved in metal/ion transport, bacillibactin siderophore biosynthesis, the glyoxalase pathway, and oxidoreductase activity was perturbed most significantly. A B. anthracis mutant lacking the superoxide dismutase gene sodA1 (Delta sodA1) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the Delta sodA1 mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and Delta sodA1 cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The Delta sodA1 mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in B. anthracis. A double mutant lacking both the sodA1 and sodA2 genes (Delta sodA1 Delta sodA2) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between sodA1 and sodA2, and a role for the sod genes in manganese homeostasis.

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asb mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system.

Various computational approaches have been proposed for operon prediction, but most algorithms rely on experimental or functional data that are only available for a small subset of sequenced genomes. In this study, we explored the possibility of using phylogenetic information to aid in operon prediction, and we constructed a Bayesian hidden Markov model that incorporates comparative genomic data with traditional predictors, such as intergenic distances. The prediction algorithm performs as well as the best previously reported method, with several significant advantages. It uses fewer data sources and so it is easier to implement, and the method is more broadly applicable than previous methods--it can be applied to essentially every gene in any sequenced bacterial genome. Furthermore, we show that near-optimal performance is easily reached with a generic set of comparative genomes and does not depend on a specific relationship between the subject genome and the comparative set. We applied the algorithm to the Bacillus anthracis genome and found that it successfully predicted all previously verified B. anthracis operons. To further test its performance, we chose a predicted operon (BA1489-92) containing several genes with little apparent functional relatedness and tested their cotranscriptional nature. Experimental evidence shows that these genes are cotranscribed, and the data have interesting implications for B. anthracis biology. Overall, our findings show that this algorithm is capable of highly sensitive and accurate operon prediction in a wide range of bacterial genomes and that these predictions can lead to the rapid discovery of new functional relationships among genes.

Due to its potential as a bioweapon, Bacillus anthracis has received a great deal of attention in recent years, and a significant effort has been devoted to understanding how this organism causes anthrax. There has been a particular focus on the inhalational form of the disease, and studies over the past several years have painted an increasingly complex picture of how B. anthracis enters the mammalian host, survives the host's defense mechanisms, disseminates throughout the body and causes death. This article reviews recent advances in these areas, with a focus on how the bacterium interacts with its host in establishing infection and causing anthrax.

The Bacillus anthracis chromosome encodes four unique, putative superoxide dismutase (sod) genes. During exponential growth and sporulation, sodA1, sodA2, and sodC are transcribed constitutively throughout the growth cycle as individual genes. In contrast, the transcription of sod15 occurs mainly during late exponential and sporulation phases as part of a four-gene operon that may be involved in spore formation. Vegetative cell and spore lysates of wild-type Sterne and superoxide dismutase deletion (Deltasod) mutants show detectable SOD activity for SODA1 and SODA2, and protein analysis suggests that these two proteins form active homodimers and heterodimers. A comparison of the growth of parental versus Deltasod mutants under various chemical oxidative stresses indicates that DeltasodA1 mutants are particularly sensitive to endogenously produced superoxide, whereas DeltasodA2, Deltasod15, and DeltasodC mutants remain as resistant to this stress as the parental strain. In addition, in mouse survival assays, Deltasod15 and DeltasodA1 were responsible for less end-point death, but the level of decreased virulence does not fall within a statistically significant range. Collectively, these data show that sodA1 acts as a major protectant from intracellular superoxide stress, that sod15 is transcribed as part of an operon that may play a role in cell morphology, and that sodA2 and sodC may have minor roles that are not apparent in the conditions tested here.

Interactions between Bacillus anthracis and host macrophages represent critical early events in anthrax pathogenesis, but their details are not clearly understood. Here we report the first genomewide characterization of the transcriptional changes within macrophages infected with B. anthracis and the identification of several hundred host genes that were differentially expressed during this intracellular stage of infection. These loci included both genes that are known to be regulated differentially in response to many other bacterial pathogens and those that appear to be differentially regulated in response to B. anthracis but not other bacterial species that have been tested. These data provide a transcriptional basis for a variety of physiological changes observed during infection, including the induction of apoptosis caused by the infecting bacteria. The expression patterns underlying B. anthracis-induced apoptosis led us to test further the importance of one very highly induced macrophage gene, that for ornithine decarboxylase. Our data show that this enzyme plays an important and previously unrecognized role in suppressing apoptosis in B. anthracis-infected cells. We have also characterized the transcriptional response to anthrax lethal toxin in activated macrophages and found that, following toxin treatment, many of the host inflammatory response pathways are dampened. These data provide insights into B. anthracis pathogenesis as well as potential leads for the development of new diagnostic and therapeutic options.