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Streptococcus pneumoniae is a major human pathogen and rising resistance to β-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Introduction: De-escalation of breast cancer treatment aims to reduce patient and financial toxicity without compromising outcomes. Level I evidence and National Comprehensive Cancer Network guidelines support omission of adjuvant radiation in patients aged >70 y with hormone-sensitive, pT1N0M0 invasive breast cancer treated with endocrine therapy. We evaluated radiation use in patients eligible for guideline concordant omission of radiation., Methods: Subgroup analysis of patients eligible for radiation omission from two pooled randomized controlled trials, which included stage 0-III breast cancer patients undergoing breast conserving surgery, was performed to evaluate factors associated with radiation use., Results: Of 631 patients, 47 (7.4%) met radiation omission criteria and were treated by 14 surgeons at eight institutions. The mean age was 75.3 (standard deviation + 4.4) y. Majority of patients identified as White (n = 46; 97.9%) and non-Hispanic (n = 44; 93.6%). The mean tumor size was 1.0 cm; 37 patients (88.1%) had ductal, 4 patients (9.5%) had lobular, and 17 patients (40.5%) had low-grade disease. Among patients eligible for radiation omission, 34 (72.3%) patients received adjuvant radiation. Those who received radiation were significantly younger than those who did not (74 y, interquartile range = 4 y, versus 78 y, interquartile range = 11 y, P = 0.03). There was no difference in radiation use based on size (P = 0.4), histology (P = 0.5), grade (P = 0.7), race (P = 1), ethnicity (P = 0.6), institution (P = 0.1), gender of the surgeon (P = 0.7), or surgeon (P = 0.1)., Conclusions: Fewer than 10% of patients undergoing breast conservation met criteria for radiation omission. Nearly three-quarters received radiation therapy with younger age being a driver of radiation use, suggesting ample opportunity for de-escalation, particularly among younger eligible patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: Factors contributing to the use of preoperative MRI remain poorly understood., Methods: Data from a randomized controlled trial of stage 0-3 breast cancer patients undergoing breast conserving surgery between 2016 and 2018 were analyzed., Results: Of the 396 patients in this trial, 32.6% had a preoperative MRI. Patient age, race, ethnicity, tumor histology, and use of neoadjuvant therapy were significant predictors of MRI use. On multivariate analysis, younger patients with invasive lobular tumors were more likely to have a preoperative MRI. Rates also varied significantly by individual surgeon (p < 0.001); in particular, female surgeons (39.9% vs. 24.0% for male surgeons, p = 0.001) and those in community practice (58.9% vs. 14.2% for academic, p < 0.001) were more likely to order preoperative MRI. Rates declined over the two years of the study, particularly among female surgeons., Conclusions: Preoperative MRI varies with patient age and tumor histology; however, there remains variability by individual surgeon., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Molecular subtype in invasive breast cancer guides systemic therapy. It is unknown whether molecular subtype should also be considered to tailor surgical therapy. The present investigation was designed to evaluate whether breast cancer subtype impacted surgical margins in patients with invasive breast cancer stage I through III undergoing breast-conserving therapy., Methods: Data from 2 randomized trials evaluating cavity shave margins (CSM) on margin status in patients undergoing partial mastectomy (PM) were used for this analysis. Patients were included if invasive carcinoma was present in the PM specimen and data for all 3 receptors (ER, PR, and HER2) were known. Patients were classified as luminal if they were ER and/or PR positive; HER2 enriched if they were ER and PR negative but HER2 positive; and TN if they were negative for all 3 receptors. The impact of subtype on the margin status was evaluated at completion of standard PM, prior to randomization to CSM versus no CSM. Non-parametric statistical analyses were performed using SPSS Version 26., Results: Molecular subtype was significantly correlated with race ( P = .011), palpability ( P = .007), and grade ( P < .001). Subtype did not correlate with Hispanic ethnicity ( P = .760) or lymphovascular invasion ( P = .756). In this cohort, the overall positive margin rate was 33.7%. This did not vary based on molecular subtype (positive margin rate 33.7% for patients with luminal tumors vs 36.4% for those with TN tumors, P = .425)., Discussion: Molecular subtype does not predict margin status. Therefore, molecular subtype should not, independent of other factors, influence surgical decision-making.

Background: We sought to determine factors affecting time to surgery (TTS) to identify potential modifiable factors to improve timeliness of care., Methods: Patients with clinical stage 0-3 breast cancer undergoing partial mastectomy in 2 clinical trials, conducted in ten centers across the US, were analyzed. No preoperative workup was mandated by the study; those receiving neoadjuvant therapy were excluded., Results: The median TTS among the 583 patients in this cohort was 34 days (range: 1-289). Patient age, race, tumor palpability, and genomic subtype did not influence timeliness of care defined as TTS ≤30 days. Hispanic patients less likely to have a TTS ≤30 days ( P = .001). There was significant variation in TTS by surgeon ( P < .001); those practicing in an academic center more likely to have TTS ≤30 days than those in a community setting (55.1% vs 19.3%, P < .001). Patients who had a preoperative ultrasound had a similar TTS to those who did not (TTS ≤30 days 41.9% vs 51.9%, respectively, P = .109), but those who had a preoperative MRI had a significantly increased TTS (TTS ≤30 days 25.0% vs 50.9%, P < .001). On multivariate analysis, patient ethnicity was no longer significantly associated with TTS ≤30 ( P = .150). Rather, use of MRI (OR: .438; 95% CI: .287-.668, P < .001) and community practice type (OR: .324; 95% CI: .194-.541, P < .001) remained independent predictors of lower likelihood of TTS ≤30 days., Conclusions: Preoperative MRI significantly increases time to surgery; surgeons should consider this in deciding on its use.

Canada's current non-legislated oversight system for animal-based science not only fails to adequately incentivize the replacement of sentient animals as best scientific practice in any meaningful way, but also fails to adequately protect those animals bred, harmed, and killed in the name of science. In this paper, we outline the various shortcomings of the Canadian Council on Animal Care, and we highlight the need for Canada to move towards national legislation akin to that seen in other jurisdictions like the U.K. We conclude that while legislation alone cannot ensure the replacement of sentient animals in science, it appears to be a precondition for significant progress in animal protection and for the development and adoption of non-animal methods.

Modern breast cancer treatment is multidisciplinary. Comprehensive breast centers are uniquely positioned to treat patients in a multidisciplinary fashion, providing timely diagnoses, state-of-the-art treatment options, and survivorship care. Important ancillary services can improve patients' emotional, financial, physical, and sexual distress. Patient navigators are the link between these provided services and the patient., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In advanced age, there is an accelerated decline in skeletal muscle mass that appears to be secondary to repeated cycles of denervation-reinnervation and eventually, failed reinnervation. However, whether variation in reinnervation capacity explains why some muscles are less vulnerable to age-related atrophy has not been addressed. In this study we examined changes in neuromuscular junction (NMJ) morphology, fiber cross-sectional area (CSA) and fiber type, accumulation of severely atrophied myofibers, and expression of a marker of denervation in four muscles that exhibit differences in the degree of age-related atrophy and which span the extremes of fiber type composition in 8 mo old (8 M) and 34 mo old (34 M) male Fischer 344 Brown Norway F1 hybrid rats. Aging muscle atrophy was most pronounced in the fast twitch gastrocnemius (Gas; 25%) and similar between extensor digitorum longus (EDL) and slow-twitch soleus (Sol) muscle (14-15%), whereas the slow-twitch adductor longus (AL) increased in mass by 21% between 8 M and 34 M (P < 0.05 for all). Only the Sol exhibited significant alterations in fiber type with aging, and there was a decrease in fiber CSA in the Gas, EDL, and Sol (P < 0.05) with aging that was not seen in the AL. Muscles that atrophied had an increased fraction of severely atrophic myofibers (P < 0.05), but this was not observed in the AL. The Gas and EDL both demonstrated a similar degree of age-related remodeling of pre- and post-synaptic NMJ components. On the other hand, pre- and post-synaptic morphology underwent greater changes with aging in the AL, and many of these same morphological variables were already greater in the Sol vs AL at 8 M, suggesting the Sol had already undergone substantial remodeling and may be nearing its adaptive limits. Consistent with this idea, analysis of NMJ morphology in Sol from 3 M rats exhibited similar values as 8 M AL, and the Sol demonstrated greater expression of the denervation marker neural cell adhesion molecule (NCAM) compared to the AL at 34 M. Collectively, our results are consistent with NMJ remodeling capacity being finite with aging and that maintained remodeling potential confers atrophy protection in aging skeletal muscle by reducing the degree of persistent denervation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

"Animal-based research should be held to the highest ethical standards" is becoming an increasingly common refrain. Though I think such a commitment is what we should expect of those using animals in science, much as we would if the participants were humans, some key insights of discussions in applied ethics and moral philosophy only seem to slowly impact what reasonably qualifies as the highest standards in animal research ethics. Early in my paper, I will explain some of these insights and loosely tie them to animal research ethics. Two emergent practices in laboratory animal science, positive reinforcement training and "rehoming," will then be discussed, and I will defend the view that both should be mandatory on no more ethical grounds than what is outlined in the first section. I will also provide reasons for foregrounding the moral significance of dissent and why, most of the time, an animal research subject's sustained dissent should be respected. Taken together, what I will defend promises to change how at least some animals are used in science and what happens to them afterwards. But I will also show how an objective ethics requires nothing less. Ignoring these constraints in the scientific use of animals comes at the cost of abandoning any claim to adhering to our highest ethical standards and, arguably, any claim to the moral legitimacy of such scientific use., (© The Author(s) 2020. Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Single-center studies have demonstrated that resection of cavity shave margins (CSM) halves the rate of positive margins and re-excision in breast cancer patients undergoing partial mastectomy (PM). We sought to determine if these findings were externally generalizable across practice settings., Methods: In this multicenter randomized controlled trial occurring in 9 centers across the United States, stage 0-III breast cancer patients undergoing PM were randomly assigned to either have resection of CSM ("shave" group) or not ("no shave" group). Randomization occurred intraoperatively, after the surgeon had completed their standard PM. Primary outcome measures were positive margin and re-excision rates., Results: Between July 28, 2016 and April 13, 2018, 400 patients were enrolled in this trial. Four patients (2 in each arm) did not meet inclusion criteria after randomization, leaving 396 patients for analysis: 196 in the "shave" group and 200 to the "no shave" group. Median patient age was 65 years (range; 29-94). Groups were well matched at baseline for demographic and clinicopathologic factors. Prior to randomization, positive margin rates were similar in the "shave" and "no shave" groups (76/196 (38.8%) vs. 72/200 (36.0%), respectively, P = 0.604). After randomization, those in the "shave" group were significantly less likely than those in the "no shave" group to have positive margins (19/196 (9.7%) vs. 72/200 (36.0%), P < 0.001), and to require re-excision or mastectomy for margin clearance (17/196 (8.7%) vs. 47/200 (23.5%), P < 0.001)., Conclusion: Resection of CSM significantly reduces positive margin and re-excision rates in patients undergoing PM., Competing Interests: The authors report no conflicts of interest., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Microbiology is at a turning point in its 120-year history. Widespread next-generation sequencing has revealed genetic complexity among bacteria that could hardly have been imagined by pioneers such as Pasteur, Escherich and Koch. This data cascade brings enormous potential to improve our understanding of individual bacterial cells and the genetic basis of phenotype variation. However, this revolution in data science cannot replace established microbiology practices, presenting the challenge of how to integrate these new techniques. Contrasting comparative and functional genomic approaches, we evoke molecular microbiology theory and established practice to present a conceptual framework and practical roadmap for next-generation microbiology.

The predatory bacterium B. bacteriovorus grows and divides inside the periplasm of Gram-negative bacteria, forming a structure known as a bdelloplast. Cell division of predators inside the dead prey cell is not by binary fission but instead by synchronous division of a single elongated filamentous cell into odd or even numbers of progeny cells. Bdellovibrio replication and cell division processes are dependent on the finite level of nutrients available from inside the prey bacterium. The filamentous growth and division process of the predator maximizes the number of progeny produced by the finite nutrients in a way that binary fission could not. To learn more about such an unusual growth profile, we studied the role of DivIVA in the growing Bdellovibrio cell. This protein is well known for its link to polar cell growth and spore formation in Gram-positive bacteria, but little is known about its function in a predatory growth context. We show that DivIVA is expressed in the growing B. bacteriovorus cell and controls cell morphology during filamentous cell division, but not the number of progeny produced. Bacterial Two Hybrid (BTH) analysis shows DivIVA may interact with proteins that respond to metabolic indicators of amino-acid biosynthesis or changes in redox state. Such changes may be relevant signals to the predator, indicating the consumption of prey nutrients within the sealed bdelloplast environment. ParA, a chromosome segregation protein, also contributes to bacterial septation in many species. The B. bacteriovorus genome contains three ParA homologs; we identify a canonical ParAB pair required for predatory cell division and show a BTH interaction between a gene product encoded from the same operon as DivIVA with the canonical ParA. The remaining ParA proteins are both expressed in Bdellovibrio but are not required for predator cell division. Instead, one of these ParA proteins coordinates gliding motility, changing the frequency at which the cells reverse direction. Our work will prime further studies into how one bacterium can co-ordinate its cell division with the destruction of another bacterium that it dwells within., (Copyright © 2020 Milner, Ray, Saxon, Lambert, Till, Fenton and Sockett.)

Human and animal research both operate within established standards. In the United States, criticism of the human research environment and recorded abuses of human research subjects served as the impetus for the establishment of the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, and the resulting Belmont Report. The Belmont Report established key ethical principles to which human research should adhere: respect for autonomy, obligations to beneficence and justice, and special protections for vulnerable individuals and populations. While current guidelines appropriately aim to protect the individual interests of human participants in research, no similar, comprehensive, and principled effort has addressed the use of (nonhuman) animals in research. Although published policies regarding animal research provide relevant regulatory guidance, the lack of a fundamental effort to explore the ethical issues and principles that should guide decisions about the potential use of animals in research has led to unclear and disparate policies. Here, we explore how the ethical principles outlined in the Belmont Report could be applied consistently to animals. We describe how concepts such as respect for autonomy and obligations to beneficence and justice could be applied to animals, as well as how animals are entitled to special protections as a result of their vulnerability.

Though there is a burgeoning interest in applied Buddhist ethics, Buddhist animal research ethics remains an underdeveloped area. In this paper I will explore how some central Buddhist ethical considerations can usefully engage our use of other animals (henceforth, animals) in science. As the scientific use of animals is broad, I will narrow my focus to laboratory science. I will show that, though a Buddhist abolitionism would not be unmotivated, it is possible to reject it. While doing so, it will be important to resist emphasizing elements of Buddhist thought that merely provide reasons to adopt the dominant ethical framework governing laboratory animal research ethics, known as the 3Rs. Though I will suggest how a Buddhist animal research ethics can sometimes permit the use of animals in harmful research, it will also require ethical constraints that resonate with some of the more progressive elements in 'Western' bioethics., (© 2019 John Wiley & Sons Ltd.)

Penicillin and related antibiotics disrupt cell wall synthesis to induce bacteriolysis. Lysis in response to these drugs requires the activity of cell wall hydrolases called autolysins, but how penicillins misactivate these deadly enzymes has long remained unclear. Here, we show that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae ( Sp ). We find that during exponential growth, Sp cells primarily produce lipid-anchored TAs called lipoteichoic acids (LTAs) that bind and sequester the major autolysin LytA. However, penicillin-treatment or prolonged stationary phase growth triggers the degradation of a key LTA synthase, causing a switch to the production of wall-anchored TAs (WTAs). This change allows LytA to associate with and degrade its cell wall substrate, thus promoting osmotic lysis. Similar changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase-induced lysis for other important Gram-positive pathogens., Competing Interests: JF, GD, AF, DR, TB No competing interests declared, (© 2019, Flores-Kim et al.)

Purpose: To compare the accuracy and reliability of a natural language processing (NLP) algorithm with manual coding by radiologists, and the combination of the two methods, for the identification of patients whose computed tomography (CT) reports raised the concern for lung cancer., Methods: An NLP algorithm was developed using Clinical Text Analysis and Knowledge Extraction System (cTAKES) with the Yale cTAKES Extensions and trained to differentiate between language indicating benign lesions and lesions concerning for lung cancer. A random sample of 450 chest CT reports performed at Veterans Affairs Connecticut Healthcare System between January 2014 and July 2015 was selected. A reference standard was created by the manual review of reports to determine if the text stated that follow-up was needed for concern for cancer. The NLP algorithm was applied to all reports and compared with case identification using the manual coding by the radiologists., Results: A total of 450 reports representing 428 patients were analyzed. NLP had higher sensitivity and lower specificity than manual coding (77.3% v 51.5% and 72.5% v 82.5%, respectively). NLP and manual coding had similar positive predictive values (88.4% v 88.9%), and NLP had a higher negative predictive value than manual coding (54% v 38.5%). When NLP and manual coding were combined, sensitivity increased to 92.3%, with a decrease in specificity to 62.85%. Combined NLP and manual coding had a positive predictive value of 87.0% and a negative predictive value of 75.2%., Conclusion: Our NLP algorithm was more sensitive than manual coding of CT chest reports for the identification of patients who required follow-up for suspicion of lung cancer. The combination of NLP and manual coding is a sensitive way to identify patients who need further workup for lung cancer.

Most bacterial cells are surrounded by an essential cell wall composed of the net-like heteropolymer peptidoglycan (PG). Growth and division of bacteria are intimately linked to the expansion of the PG meshwork and the construction of a cell wall septum that separates the nascent daughter cells. Class A penicillin-binding proteins (aPBPs) are a major family of PG synthases that build the wall matrix. Given their central role in cell wall assembly and importance as drug targets, surprisingly little is known about how the activity of aPBPs is controlled to properly coordinate cell growth and division. Here, we report the identification of MacP (SPD&#95;0876) as a membrane-anchored cofactor of PBP2a, an aPBP synthase of the Gram-positive pathogen Streptococcus pneumoniae We show that MacP localizes to the division site of S. pneumoniae , forms a complex with PBP2a, and is required for the in vivo activity of the synthase. Importantly, MacP was also found to be a substrate for the kinase StkP, a global cell cycle regulator. Although StkP has been implicated in controlling the balance between the elongation and septation modes of cell wall synthesis, none of its substrates are known to modulate PG synthetic activity. Here we show that a phosphoablative substitution in MacP that blocks StkP-mediated phosphorylation prevents PBP2a activity without affecting the MacP-PBP2a interaction. Our results thus reveal a direct connection between PG synthase function and the control of cell morphogenesis by the StkP regulatory network., Competing Interests: The authors declare no conflict of interest.

Most bacterial cells are surrounded by a peptidoglycan cell wall that is essential for their integrity. The major synthases of this exoskeleton are called penicillin-binding proteins (PBPs) 1,2 . Surprisingly little is known about how cells control these enzymes, given their importance as drug targets. In the model Gram-negative bacterium Escherichia coli, outer membrane lipoproteins are critical activators of the class A PBPs (aPBPs) 3,4 , bifunctional synthases capable of polymerizing and crosslinking peptidoglycan to build the exoskeletal matrix 1 . Regulators of PBP activity in Gram-positive bacteria have yet to be discovered but are likely to be distinct due to the absence of an outer membrane. To uncover Gram-positive PBP regulatory factors, we used transposon-sequencing (Tn-Seq) 5 to screen for mutations affecting the growth of Streptococcus pneumoniae cells when the aPBP synthase PBP1a was inactivated. Our analysis revealed a set of genes that were essential for growth in wild-type cells yet dispensable when pbp1a was deleted. The proteins encoded by these genes include the conserved cell wall elongation factors MreC and MreD 2,6,7 , as well as a membrane protein of unknown function (SPD&#95;0768) that we have named CozE (coordinator of zonal elongation). Our results indicate that CozE is a member of the MreCD complex of S. pneumoniae that directs the activity of PBP1a to the midcell plane where it promotes zonal cell elongation and normal morphology. CozE homologues are broadly distributed among bacteria, suggesting that they represent a widespread family of morphogenic proteins controlling cell wall biogenesis by the PBPs.

Background: Prior traumatic experiences have been associated with poorer coping strategies, greater distress, and more posttraumatic stress disorder (PTSD) symptoms following a subsequent cancer diagnosis affecting their survival. However, the impact of prior physical traumatic injury on cancer survival has not been examined., Methods: The present study matched patients from the same Level 1 Trauma center who appeared in both the trauma and cancer registries. A total of 498 patients met the criteria between 1998 and 2014 who have experienced both a diagnosis of cancer and a physical traumatic injury. The survival between the patients who had physical trauma before cancer (TBC) versus those that had physical trauma after the cancer diagnosis (TAC) were compared., Results: The TBC group had a higher percentage of males (48 % vs 33 % p = 0.001) and motor vehicle collisions (18 % vs 7 %, p < 0.001), than the TAC group. TBC patients were also significantly younger than TAC patients at the time of the physical traumatic event (68.7 ± 14.6 vs 76.2 ± 12.0 years, p < 0.001), and longer length of time between the cancer diagnosis and physical traumatic injury (2.9 ± 2.9 vs 1.7 ± 2.6 years, p < 0.001). The overall probability of survival for the entire sample was 68 %. Percent survival for the TBC (n = 251) and TAC (n = 247) groups was 56 and 80 % respectively (p < 0.001). Results were consistent regardless of stage of cancer at diagnosis (hazard ratio (HR (Standard Error)). After adjusting for comorbidities Charlson comorbidity index (CCI) (HR = 1.2 (0.06), p = 0.009)), cancer stage (HR = 2.8 (0.12), p < 0.001)), lung cancer (HR = 1.7 (0.25), p < 0.001) and bladder cancer (HR = 3.5 (0.55), p = 0.02), experiencing a prior physical traumatic injury was associated with an increased HR for mortality of 4.6 (0.93), p < 0.001)., Conclusions: A physical traumatic episode before cancer diagnosis (TBC) increased the risk of death 4.6 fold compared to the TAC group even after adjusting for CCI, stage of cancer at diagnosis, lung cancer, and bladder cancer. These findings suggest considering a history of physical traumatic injury in cancer patients as a possible risk factor for faster cancer progression and mortality.

Smoking increases the risk of developing lung cancer. Genetic loci have been identified which could form the basis of a lung cancer susceptibility test; but little is known whether such a test would interest or motivate those trying to quit smoking. To address this, we investigated the attitudes of people trying to quit smoking towards genetic susceptibility testing for lung cancer. Participant's attitudes to topics associated with lung cancer susceptibility testing were assessed; were they interested in genetic testing? What impact would a hypothetical high- or low- risk result have on smoking cessation? 680 self-completion questionnaires were given to individuals attending National Health Service stop smoking clinics in three different areas of the United Kingdom between 2011 and 2012. 139 questionnaires were returned, giving a 20 % response rate. Participants expressed an interest in a genetic susceptibility test for lung cancer and almost all reported that a high-risk result would increase their motivation to stop smoking. However, many participants had a neutral attitude towards a low-risk result. Most participants agreed their smoking habit could lead to lung cancer. Lung cancer susceptibility testing may be a useful incentive to help people quit smoking. This study suggests the need for genetic services to work with smoking cessation teams if routine testing becomes available in the future.

Among the "hard cases" of captive animal research is the continued use of chimpanzees in harmful experimental science. In a recent article I contend that contemporary animal welfare science and chimpanzee behavioral studies permit, if not require, a reappraisal of the moral significance of chimpanzee dissent from participation in certain experiments. In what follows, I outline my earlier argument, provide a brief survey of some central concepts in pediatric research ethics, and use these to enrich an understanding of chimpanzee dissent useful for research ethics.

Bacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss of zapE results in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation of zapE leads to elongation of Escherichia coli and affects the dynamics of the Z-ring during division. In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner. IMPORTANCE Bacterial cell division has mainly been characterized in vitro. In this report, we could identify ZapE as a novel cell division protein which is not essential in vitro but is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. As zapE is not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future.

Inherited microbial symbionts can modulate host susceptibility to natural enemy attack. A wider range of symbionts influence host population demography without altering individual susceptibility, and it has been suggested that these may modify host disease risk through altering the rate of exposure to natural enemies. We present the first test of this thesis, specifically testing whether male-killing symbionts alter the epidemiology of a sexually transmitted infection (STI) carried by its host. STIs are typically expected to show female-biased epidemics, and we first present a simple model which indicates that male-biased STI epidemics may occur where symbionts create female-biased population sex ratios. We then examined the dynamics of a STI in the ladybird beetle Adalia bipunctata, which is also host to a male-killing bacterium. We present evidence that male-biased epidemics of the STI are observed in natural populations when the male-killer is common. Laboratory experiments did not support a role for differential susceptibility of male and female hosts to the STI, nor a protective role for the symbiont, in creating this bias. We conclude that the range of symbionts likely to alter parasite epidemiology will be much wider than previously envisaged, because it will additionally include those that impact host demography alone.

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin-MreB while cell division is governed by tubulin-FtsZ. A ring-like structure containing FtsZ (the Z ring) at mid-cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid-cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB-FtsZ interaction is required for transfer of cell-wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.

Background: Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as "obligate predators" because only by mutations, often in gene bd0108, are 1 in ~1x10(7) of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome., Results: To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired., Conclusions: The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.

Autism, typically described as a spectrum neurodevelopmental disorder characterized by impairments in verbal ability and social reciprocity as well as obsessive or repetitious behaviours, is currently thought to markedly affect more males than females. Not surprisingly, this encourages a gendered understanding of the Autism Spectrum. Simon Baron-Cohen, a prominent authority in the field of autism research, characterizes the male brain type as biased toward systemizing. In contrast, the female brain type is understood to be biased toward empathizing. Since persons with autism are characterized as hyper-systemizers and hypo-empathizers, Baron-Cohen suggests that, whether they are male or female, most possess an "extreme male brain profile." We argue that Baron-Cohen is misled by an unpersuasive gendering of certain capacities or aptitudes in the human population. Moreover, we suggest that this may inadvertently favour boys in diagnosing children with Autism Spectrum Disorders. If this is correct, it could also have rather serious consequences for treatment and services for girls (and women) on the Autism Spectrum.

Wolbachia is one of the most common symbionts of arthropods. Its establishment requires lateral transfer to and successful transmission within novel host species. However, Wolbachia performs poorly when introduced into new host species, and models predict that Wolbachia should seldom be able to establish from low initial frequencies. Recently, various symbionts, including Wolbachia, have been shown to protect their hosts from natural enemies. Hence, Wolbachia invasion may be facilitated by the dynamic interaction between it, its host, and a natural enemy. We model such an interaction whereby Wolbachia induces either complete resistance, partial resistance, or tolerance to a host-specific pathogen and also induces the common manipulation phenotype of cytoplasmic incompatibility (CI). We show that the presence of the pathogen greatly facilitates Wolbachia invasion from rare and widens the parameter space in which "imperfect" Wolbachia strains can invade. Furthermore, positive frequency-dependent selection through CI can drive Wolbachia to very high frequencies, potentially excluding the pathogen. These results may explain a poorly understood aspect of Wolbachia biology: it is widespread, despite performing poorly after transfer to new host species. They also support the intriguing possibility that Wolbachia strains that encode both CI and natural-enemy resistance could potentially rid insects, including human disease vectors, of important pathogens.

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Bdellovibrio bacteriovorus is a famously fast, flagellate predatory bacterium, preying upon Gram-negative bacteria in liquids; how it interacts with prey on surfaces such as in medical biofilms is unknown. Here we report that Bdellovibrio bacteria "scout" for prey bacteria on solid surfaces, using slow gliding motility that is present in flagellum-negative and pilus-negative strains.

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.

Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation. Although the divergence observed at some host-resistance and parasite-infectivity genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Phi2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.

We studied the two mreB genes, encoding actinlike cytoskeletal elements, in the predatory bacterium Bdellovibrio bacteriovorus. This bacterium enters and replicates within other Gram-negative bacteria by attack-phase Bdellovibrio squeezing through prey outer membrane, residing and growing filamentously in the prey periplasm forming an infective "bdelloplast," and septating after 4 h, once the prey contents are consumed. This lifestyle brings challenges to the Bdellovibrio cytoskeleton. Both mreB genes were essential for viable predatory growth, but C-terminal green fluorescent protein tagging each separately with monomeric teal-fluorescent protein (mTFP) gave two strains with phenotypic changes at different stages in predatory growth and development. MreB1-mTFP cells arrested growth early in bdelloplast formation, despite successful degradation of prey nucleoid. A large population of stalled bdelloplasts formed in predatory cultures and predation proceeded very slowly. A small proportion of bdelloplasts lysed after several days, liberating MreB1-mTFP attack-phase cells of wild-type morphology; this process was aided by subinhibitory concentrations of an MreB-specific inhibitor, A22. MreB2-mTFP, in contrast, was predatory at an almost wild-type rate but yielded attack-phase cells with diverse morphologies, including spherical, elongated, and branched, the first time such phenotypes have been described. Wild-type predatory rates were seen for all but spherical morphotypes, and septation of elongated morphotypes was achieved by the addition of A22.

Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5.

The genetic basis of infection in host-parasite interactions has traditionally been considered within the framework of either gene-for-gene (GFG) or matching-allele models. We present an alternative model, termed inverse-gene-for-gene (IGFG), where pathogen infectiousness is determined by parasite recognition of host signals and/or receptors or where there is active host searching by parasites. We show that coevolutionary dynamics under IGFG are both qualitatively and quantitatively different from those of the GFG model, and we suggest that this new approach may be applicable to a range of important host-parasite systems that are not currently catered for by the existing frameworks.

This paper integrates some Buddhist moral values, attitudes and self-cultivation techniques into a discussion of the ethics of cognitive enhancement technologies - in particular, pharmaceutical enhancements. Many Buddhists utilize meditation techniques that are both integral to their practice and are believed to enhance the cognitive and affective states of experienced practitioners. Additionally, Mahāyāna Buddhism's teaching on skillful means permits a liberal use of methods or techniques in Buddhist practice that yield insight into our selfnature or aid in alleviating or eliminating duhkha (i.e. dissatisfaction). These features of many, if not most, Buddhist traditions will inform much of the Buddhist assessment of pharmaceutical enhancements offered in this paper. Some Buddhist concerns about the effects and context of the use of pharmaceutical enhancements will be canvassed in the discussion. Also, the author will consider Buddhist views of the possible harms that may befall human and nonhuman research subjects, interference with a recipient's karma, the artificiality of pharmaceutical enhancements, and the possible motivations or intentions of healthy individuals pursuing pharmacological enhancement. Perhaps surprisingly, none of these concerns will adequately ground a reflective Buddhist opposition to the further development and continued use of pharmaceutical enhancements, either in principle or in practice. The author argues that Buddhists, from at least certain traditions - particularly Mahāyāna Buddhist traditions - should advocate the development or use of pharmaceutical enhancements if a consequence of their use is further insight into our self-nature or the reduction or alleviation of duhkha.

Predatory Bdellovibrio bacteriovorus bacteria are remarkable in that they attach to, penetrate and digest other Gram-negative bacteria, living and replicating within them until all resources are exhausted, when they escape the prey ghost to invade fresh prey. Remarkable remodeling of both predator and prey cell occurs during this process to allow the Bdellovibrio to exploit the intracellular niche they have worked so hard to enter, keeping the prey "bdelloplast" intact until the end of predatory growth. If one views motile non-predatory bacteria in a light microscope, one is immediately struck by how rare it is for bacteria to collide. This highlights how the cell surface of Bdellovibrio must be specialized and adapted to allow productive collisions and further to allow entry into the prey periplasm and subsequent secretion of hydrolytic enzymes to digest it. Bdellovibrio can, however, also be made to grow artificially without prey; thus, they have a large genome containing both predatory genes and genes for saprophytic heterotrophic growth. Thus, the membrane and outer surface layers are a patchwork of proteins encompassing not only those that have a sole purpose in heterotrophic growth but also many more that are specialized or employed to attach to, enter, remodel, kill and ultimately digest prey cells. There is much that is as yet not understood, but molecular genetic and post-genomic approaches to microbial physiology have enhanced the pioneering biochemical work of four decades ago in characterizing some of the key events and surface protein requirements for prey attack.

In this article, we critically examine some of the ethical challenges and interpretive difficulties with possible future non-clinical applications of pediatric fMRI with a particular focus on applications in the classroom and the courtroom - two domains in which children come directly in contact with the state. We begin with a general overview of anticipated clinical and non-clinical applications of pediatric fMRI. This is followed by a detailed analysis of a range of ethical challenges and interpretive difficulties that trouble the use of fMRI and are likely to be especially acute with non-clinical uses of the technology. We conclude that knowledge of these challenges and difficulties should influence policy decisions regarding the non-clinical uses of fMRI. Our aim is to encourage the development of future policies prescribing the responsible use of this neuroimaging technology as it develops both within and outside the clinical setting.

Interspecific pathogen interactions can profoundly affect pathogen population dynamics and the efficacy of control strategies. However, many pathogens exhibit cyclic abundance patterns (e.g., seasonality), and temporal asynchrony between interacting pathogens could reduce the impact of those interactions. Here we use an extension of our previously published model to investigate the effects of cycles on pathogen interaction. We demonstrate that host immune memory can maintain the impact of an interaction, even when the effector pathogen abundance is low or the pathogen is absent. Paradoxically, immune memory can result in pathogens interacting more strongly when temporally out of phase. We find that interactions between species can result in changes to the temporal pattern of the affected species. We further demonstrate that this may be observed in a natural host-pathogen system. Given the continuing debate regarding the relevance of pathogen interactions in natural systems and increasing concern about treatment strategies for coinfections, both the discovery of a shift in cycle in empirical data and the mechanism by which we identified it are important. Finally, because the model structure used here is analogous to models of a simple predator-prey system, we also consider the consequences of these findings in the context of that system.

Background: Many parasites of medical and veterinary importance are transmitted by cold-blooded intermediate hosts or vectors, the abundance of which will vary with ambient temperatures, potentially altering disease prevalence. In particular, if global climate change will increase mean ambient temperature in a region endemic with a human pathogen then it is possible that the incidence of disease will similarly increase. Here we examine this possibility by using a mathematical model to explore the effects of increasing long-term mean ambient temperature on the prevalence and abundance of the parasite Schistosoma mansoni, the causative agent of schistosomiasis in humans., Principal Findings: The model showed that the impact of temperature on disease prevalence and abundance is not straightforward; the mean infection burden in humans increases up to 30 degrees C, but then crashes at 35 degrees C, primarily due to increased mortalities of the snail intermediate host. In addition, increased temperatures changed the dynamics of disease from stable, endemic infection to unstable, epidemic cycles at 35 degrees C. However, the prevalence of infection was largely unchanged by increasing temperatures. Temperature increases also affected the response of the model to changes in each parameter, indicating certain control strategies may become less effective with local temperature changes. At lower temperatures, the most effective single control strategy is to target the adult parasites through chemotherapy. However, as temperatures increase, targeting the snail intermediate hosts, for example through molluscicide use, becomes more effective., Conclusions: These results show that S. mansoni will not respond to increased temperatures in a linear fashion, and the optimal control strategy is likely to change as temperatures change. It is only through a mechanistic approach, incorporating the combined effects of temperature on all stages of the life-cycle, that we can begin to predict the consequences of climate change on the incidence and severity of such diseases.

Parasites are ubiquitous in biological systems and antagonistic coevolution between hosts and parasites is thought be a major ecological and evolutionary force. Recent experiments using laboratory populations of bacteria and their parasitic viruses, phage, have provided the first direct empirical evidence of antagonistic coevolution in action. In this article we describe this model system and synthesise recent findings that address the causes and consequences of antagonistic coevolution.