The evolution of the type VI secretion system as a disintegration weapon.

The type VI secretion system (T6SS) is a nanomachine used by many bacteria to drive a toxin-laden needle into other bacterial cells. Although the potential to influence bacterial competition is clear, the fitness impacts of wielding a T6SS are not well understood. Here we present a new agent-based model that enables detailed study of the evolutionary costs and benefits of T6SS weaponry during competition with other bacteria. Our model identifies a key problem with the T6SS. Because of its short range, T6SS activity becomes self-limiting, as dead cells accumulate in its way, forming "corpse barriers" that block further attacks. However, further exploration with the model presented a solution to this problem: if injected toxins can quickly lyse target cells in addition to killing them, the T6SS becomes a much more effective weapon. We tested this prediction with single-cell analysis of combat between T6SS-wielding Acinetobacter baylyi and T6SS-sensitive Escherichia coli. As predicted, delivery of lytic toxins is highly effective, whereas nonlytic toxins leave large patches of E. coli alive. We then analyzed hundreds of bacterial species using published genomic data, which suggest that the great majority of T6SS-wielding species do indeed use lytic toxins, indicative of a general principle underlying weapon evolution. Our work suggests that, in the T6SS, bacteria have evolved a disintegration weapon whose effectiveness often rests upon the ability to break up competitors. Understanding the evolutionary function of bacterial weapons can help in the design of probiotics that can both establish well and eliminate problem species.

Incidental coronary artery calcification on non-gated CT thorax correlates with risk of cardiovascular events and death.

OBJECTIVES: To assess coronary artery calcification (CAC) on non-contrast non-ECG-gated CT thorax (NC-NECG-CTT) and to evaluate its correlation with short-term risk of cardiovascular disease (CVD) events and death. METHODS: Single-institution retrospective study including all patients 40-70 years old who underwent NC-NECG-CTT over a period of 6 months. Individuals with known CVD were excluded. The presence of CAC was assessed and quantified by the Agatston score (CACS). CAC severity was defined as mild (< 100), moderate (100-400), or severe (> 400). CVD events (including CVD death, myocardial infarction, revascularisation procedures, ischaemic stroke, acute peripheral atherosclerotic ischaemia), and all-cause mortality over a median of 3.5 years were recorded. Cox proportional-hazards regression modelling was performed including CACS, age, gender and CVD risk factors (smoking, hypertension, diabetes mellitus, dyslipidaemia, and family history of CVD). RESULTS: Of the total 717 eligible cases, 325 (45%) had CAC. In patients without CAC, there was only one CVD event, compared to 26 CVD events including 5 deaths in patients with CAC. The presence and severity of CAC correlated with CVD events (p < 0.001). A CACS > 100 was significantly associated with both CVD events, hazard ratio (HR) 5.74, 95% confidence interval: 2.19-15.02; p < 0.001, and all-cause mortality, HR 1.7, 95% CI: 1.08-2.66; p = 0.02. Ever-smokers with CAC had a significantly higher risk for all-cause mortality compared to never-smokers (p = 0.03), but smoking status was not an independent predictor for CVD events in any subgroup category of CAC severity. CONCLUSIONS: The presence and severity of CAC assessed on NC-NECG-CTT correlates with short-term cardiovascular events and death. KEY POINTS: • Patients aged 40-70 years old without known CVD but with CAC on NC-NECG-CTT have a higher risk of CVD events compared to those without CAC. • CAC (Agatston) score above 100 confers a 5.7-fold increase in the risk of short-term CVD events in these patients. • The presence and severity of CAC on NC-NECG-CTT may have prognostic and therapeutic implications.

Long noncoding RNA BHLHE40-AS1 promotes early breast cancer progression through modulating IL-6/STAT3 signaling.

Ductal carcinoma in situ (DCIS) is a nonobligate precursor to invasive breast cancer. Only a small percentage of DCIS cases are predicted to progress; however, there is no method to determine which DCIS lesions will remain innocuous from those that will become invasive disease. Therefore, DCIS is treated aggressively creating a current state of overdiagnosis and overtreatment. There is a critical need to identify functional determinants of progression of DCIS to invasive ductal carcinoma (IDC). Interrogating biopsies from five patients with contiguous DCIS and IDC lesions, we have shown that expression of the long noncoding RNA BHLHE40-AS1 increases with disease progression. BHLHE40-AS1 expression supports DCIS cell proliferation, motility, and invasive potential. Mechanistically, BHLHE40-AS1 modulates interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) activity and a proinflammatory cytokine signature, in part through interaction with interleukin enhancer-binding factor 3. These data suggest that BHLHE40-AS1 supports early breast cancer progression by engaging STAT3 signaling, creating an immune-permissive microenvironment.

Cell morphology drives spatial patterning in microbial communities.

The clearest phenotypic characteristic of microbial cells is their shape, but we do not understand how cell shape affects the dense communities, known as biofilms, where many microbes live. Here, we use individual-based modeling to systematically vary cell shape and study its impact in simulated communities. We compete cells with different cell morphologies under a range of conditions and ask how shape affects the patterning and evolutionary fitness of cells within a community. Our models predict that cell shape will strongly influence the fate of a cell lineage: we describe a mechanism through which coccal (round) cells rise to the upper surface of a community, leading to a strong spatial structuring that can be critical for fitness. We test our predictions experimentally using strains of Escherichia coli that grow at a similar rate but differ in cell shape due to single amino acid changes in the actin homolog MreB. As predicted by our model, cell types strongly sort by shape, with round cells at the top of the colony and rod cells dominating the basal surface and edges. Our work suggests that cell morphology has a strong impact within microbial communities and may offer new ways to engineer the structure of synthetic communities.

Nationwide randomised trial evaluating elective neck dissection for early stage oral cancer (SEND study) with meta-analysis and concurrent real-world cohort.

BACKGROUND: Guidelines remain unclear over whether patients with early stage oral cancer without overt neck disease benefit from upfront elective neck dissection (END), particularly those with the smallest tumours. METHODS: We conducted a randomised trial of patients with stage T1/T2 N0 disease, who had their mouth tumour resected either with or without END. Data were also collected from a concurrent cohort of patients who had their preferred surgery. Endpoints included overall survival (OS) and disease-free survival (DFS). We conducted a meta-analysis of all six randomised trials. RESULTS: Two hundred fifty randomised and 346 observational cohort patients were studied (27 hospitals). Occult neck disease was found in 19.1% (T1) and 34.7% (T2) patients respectively. Five-year intention-to-treat hazard ratios (HR) were: OS HR = 0.71 (p = 0.18), and DFS HR = 0.66 (p = 0.04). Corresponding per-protocol results were: OS HR = 0.59 (p = 0.054), and DFS HR = 0.56 (p = 0.007). END was effective for small tumours. END patients experienced more facial/neck nerve damage; QoL was largely unaffected. The observational cohort supported the randomised findings. The meta-analysis produced HR OS 0.64 and DFS 0.54 (p < 0.001). CONCLUSION: SEND and the cumulative evidence show that within a generalisable setting oral cancer patients who have an upfront END have a lower risk of death/recurrence, even with small tumours. CLINICAL TRIAL REGISTRATION: NIHR UK Clinical Research Network database ID number: UKCRN 2069 (registered on 17/02/2006), ISCRTN number: 65018995, ClinicalTrials.gov Identifier: NCT00571883.

Close Resection Margins Do Not Influence Local Recurrence in Patients With Oral Squamous Cell Carcinoma: A Prospective Cohort Study.

PURPOSE: This study investigated the clinical relevance of the distance between the resection margin and tumor cells of the primary sites for oral squamous cell carcinoma, with particular attention to local recurrence rate. PATIENTS AND METHODS: All patients diagnosed with oral squamous cell carcinoma from 1995 to 2006 and treated primarily with surgery formed the initial cohort of the study. Patient with various degrees of dysplasia in the margin, patients who received radiotherapy, and patients who died of causes other than oral cancer were excluded. Margins 1 to 5 mm were considered close. A margin of at least 5 mm was considered free of disease (clear). Local recurrence was defined as tumor development at the site of the primary tumor during the follow-up period (≥5 yr). The Fisher exact test was used to determine the relevance of the differences between the studied groups (free vs close margins) in relation to local recurrence. RESULTS: Histologic analysis of the specimens was performed. Of the 53 patients, 32 had free margins and 3 of the 32 had a local recurrence. In addition, 21 patients had close margins and 3 of the 21 had a local recurrence. The difference between the 2 groups was not statistically relevant. CONCLUSIONS: The authors advocate that the strategy of using close resection margins as a generic indicator for local recurrence and adverse prognosis might have to be reassessed. The histopathologic evidence of tumor cells within a distance less than 0.5 cm from the surgical margins does not necessarily seem to offer a certain indication for additional treatment. Other prognostic factors, such as involvement of cervical lymph nodes and tumor depth, must be considered in the decision making for further treatment.

Partial Superficial, Superficial, and Total Parotidectomy in the Management of Benign Parotid Gland Tumors: A 10-Year Prospective Study of 205 Patients.

PURPOSE: The aim of this report is to present an overview of the authors' experience in treating parotid gland tumors for a period of 10 years. This report describes patients' demographics, surgical outcomes, and complications and discusses the management of benign disease with particular emphasis on the importance of facial nerve dissection. PATIENTS AND METHODS: A total of 205 consecutive patients with different parotid gland tumors underwent surgery at Northampton General Hospital (Northampton, UK) from October 2000 to November 2010. Data were prospectively collected and entered into an electronic database. Patients' demographics, clinical tumor size, type of operation, fine-needle aspiration result, facial nerve status, final histopathologic report, and intraoperative and postoperative complications were recorded and analyzed. RESULTS: This study confirmed that good results in low recurrence rate and minimal risk of facial nerve weakness can be achieved with operations less aggressive than traditional superficial parotidectomy, such as partial superficial parotidectomy. Transient facial nerve palsy was significantly more frequent after total (40%; P < .001) and superficial (28%; P < .05) parotidectomy, respectively, than after partial superficial parotidectomy (9.6%). CONCLUSION: Because the risk or recurrence is higher when surgery is performed by inexperienced surgeons, the authors advocate that parotid gland surgery should be performed by adequately trained operators and the surgical specimen ideally should be examined by a histopathologist experienced in the diagnosis of salivary gland tumors. Recurrence rate for these tumors increases with time; therefore, long-term follow-up is required for these patients.

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion.

INTRODUCTION: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated ß-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized. METHODS: Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers. RESULTS: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification. CONCLUSION: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

Coarse-graining DNA for simulations of DNA nanotechnology.

To simulate long time and length scale processes involving DNA it is necessary to use a coarse-grained description. Here we provide an overview of different approaches to such coarse-graining, focussing on those at the nucleotide level that allow the self-assembly processes associated with DNA nanotechnology to be studied. OxDNA, our recently-developed coarse-grained DNA model, is particularly suited to this task, and has opened up this field to systematic study by simulations. We illustrate some of the range of DNA nanotechnology systems to which the model is being applied, as well as the insights it can provide into fundamental biophysical properties of DNA.

Bringing bike share to a low-income community: lessons learned through community engagement, Minneapolis, Minnesota, 2011.

BACKGROUND: High prevalence of physical inactivity contributes to adverse health outcomes. Active transportation (cycling or walking) is associated with better health outcomes, and bike-sharing programs can help communities increase use of active transportation. COMMUNITY CONTEXT: The Minneapolis Health Department funded the Nice Ride Minnesota bike share system to expand to the Near North community in Minneapolis, Minnesota. Near North is a diverse, low-income area of the city where residents experience health disparities, including disparities in physical activity levels. METHODS: The installation of new bike share kiosks in Near North resulted in an environmental change to support physical activity. Community engagement was conducted pre-intervention only and consisted of focus groups, community meetings, and interviews. Postintervention data on bike share trips and subscribers were collected to assess intervention effectiveness. OUTCOME: Focus group participants offered insights on facilitators and barriers to bike share and suggested system improvements. Community engagement efforts showed that Near North residents were positive about Nice Ride and wanted to use the system; however, the numbers of trips and subscriptions in Near North were low. INTERPRETATION: Results show that the first season of the expansion was moderately successful in improving outreach efforts and adapting bike share to meet the needs of low-income populations. However, environmental change without adequate, ongoing community engagement may not be sufficient to result in behavior change.

Collective protection against the type VI secretion system in bacteria.

Bacteria commonly face attacks from other strains using the type VI secretion system (T6SS), which acts like a molecular speargun to stab and intoxicate competitors. Here we show how bacteria can work together to collectively defend themselves against these attacks. This project began with an outreach activity: while developing an online computer game of bacterial warfare, we noticed that one strategist ("Slimy") that made extracellular polymeric substances (EPS) was able to resist attacks from another strategist that employed the T6SS ("Stabby"). This observation motivated us to model this scenario more formally, using dedicated agent-based simulations. The model predicts that EPS production can serve as a collective defence mechanism, which protects both producing cells and neighbouring cells that do not make EPS. We then tested our model with a synthetic community that contains a T6SS-wielding attacker (Acinetobacter baylyi), and two T6SS-sensitive target strains (Escherichia coli) that either secrete EPS, or not. As predicted by our modelling, we find that the production of EPS leads to collective protection against T6SS attacks, where EPS producers protect each other and nearby non-producers. We identify two processes that explain this protection: EPS sharing between cells and a second general mechanism whereby groups of resistant cells shield susceptible cells, which we call "flank protection". Our work shows how EPS-producing bacteria can work together to defend themselves from the type VI secretion system.

The evolution of short- and long-range weapons for bacterial competition.

Bacteria possess a diverse range of mechanisms for inhibiting competitors, including bacteriocins, tailocins, type VI secretion systems and contact-dependent inhibition (CDI). Why bacteria have evolved such a wide array of weapon systems remains a mystery. Here we develop an agent-based model to compare short-range weapons that require cell-cell contact, with long-range weapons that rely on diffusion. Our model predicts that contact weapons are useful when an attacking strain is outnumbered, facilitating invasion and establishment. By contrast, ranged weapons tend to be effective only when attackers are abundant. We test our predictions with the opportunistic pathogen Pseudomonas aeruginosa, which naturally carries multiple weapons, including CDI and diffusing tailocins. As predicted, short-range CDI can function at low and high frequencies, while long-range tailocins require high frequency and cell density to function effectively. Head-to-head competition experiments with the two weapon types further support our predictions: a tailocin attacker defeats CDI only when it is numerically dominant, but then we find it can be devastating. Finally, we show that the two weapons work well together when one strain employs both. We conclude that short- and long-range weapons serve different functions and allow bacteria to fight both as individuals and as a group.

Bacterial defences: mechanisms, evolution and antimicrobial resistance.

Throughout their evolutionary history, bacteria have faced diverse threats from other microorganisms, including competing bacteria, bacteriophages and predators. In response to these threats, they have evolved sophisticated defence mechanisms that today also protect bacteria against antibiotics and other therapies. In this Review, we explore the protective strategies of bacteria, including the mechanisms, evolution and clinical implications of these ancient defences. We also review the countermeasures that attackers have evolved to overcome bacterial defences. We argue that understanding how bacteria defend themselves in nature is important for the development of new therapies and for minimizing resistance evolution.

The impact of lip-split mandibulotomy on patients treated for pT2 oral tongue squamous cell carcinoma: a study of 224 patients.

BACKGROUND: Head and neck surgeons often face a challenge in order to achieve adequate three-dimensional resection of tumours in the oral cavity, especially in the dentate patient. METHODS: We compared the outcomes of lip-split mandibulotomy and trans-oral access, respectively, in patients treated for primary pT2 oral tongue SCC with regard to the status of the resection margins and the incidence of tumour recurrence. RESULTS: Multivariate analysis showed a non-significant effect of the surgical technique used to the reported recurrence, F(1, 224) = 0.350, p = .555 and a significant effect on the margins achieved F(1, 224) = 11.381, p = .001. CONCLUSIONS: Defects after excision of larger and more posterior tumours that are going to be reconstructed with free flaps represent a more probable indication for using an osteotomy access technique. Lip-split mandibulotomy is a low-morbidity technique which can deliver a sound oncological outcome and can be relatively easily taught to less experienced surgeons.

Cryopreservation of Human Adipose Tissues and Adipose-Derived Stem Cells with DMSO and/or Trehalose: A Systematic Review.

Adipose tissue senescence is implicated as a major player in obesity- and ageing-related disorders. There is a growing body of research studying relevant mechanisms in age-related diseases, as well as the use of adipose-derived stem cells in regenerative medicine. The cell banking of tissue by utilising cryopreservation would allow for much greater flexibility of use. Dimethyl sulfoxide (DMSO) is the most commonly used cryopreservative agent but is toxic to cells. Trehalose is a sugar synthesised by lower organisms to withstand extreme cold and drought that has been trialled as a cryopreservative agent. To examine the efficacy of trehalose in the cryopreservation of human adipose tissue, we conducted a systematic review of studies that used trehalose for the cryopreservation of human adipose tissues and adipose-derived stem cells. Thirteen articles, including fourteen studies, were included in the final review. All seven studies that examined DMSO and trehalose showed that they could be combined effectively to cryopreserve adipocytes. Although studies that compared nonpermeable trehalose with DMSO found trehalose to be inferior, studies that devised methods to deliver nonpermeable trehalose into the cell found it comparable to DMSO. Trehalose is only comparable to DMSO when methods are devised to introduce it into the cell. There is some evidence to support using trehalose instead of using no cryopreservative agent.

Droplet printing reveals the importance of micron-scale structure for bacterial ecology.

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.

The evolution of tit-for-tat in bacteria via the type VI secretion system.

Tit-for-tat is a familiar principle from animal behavior: individuals respond in kind to being helped or harmed by others. Remarkably some bacteria appear to display tit-for-tat behavior, but how this evolved is not understood. Here we combine evolutionary game theory with agent-based modelling of bacterial tit-for-tat, whereby cells stab rivals with poisoned needles (the type VI secretion system) after being stabbed themselves. Our modelling shows tit-for-tat retaliation is a surprisingly poor evolutionary strategy, because tit-for-tat cells lack the first-strike advantage of preemptive attackers. However, if cells retaliate strongly and fire back multiple times, we find that reciprocation is highly effective. We test our predictions by competing Pseudomonas aeruginosa (a tit-for-tat species) with Vibrio cholerae (random-firing), revealing that P. aeruginosa does indeed fire multiple times per incoming attack. Our work suggests bacterial competition has led to a particular form of reciprocation, where the principle is that of strong retaliation, or 'tits-for-tat'.

Intestinal parasites in six Islamic medieval period latrines from 10th-11th century Córdoba (Spain) and 12th-13th century Mértola (Portugal).

OBJECTIVE: To investigate the types of intestinal parasites that infected people living in Islamic period southern Iberia (al-Andalus), and compare with other regions of Europe. MATERIALS: Four cesspits from 10th-11th century CE Córdoba (Spain), and two from 12th-13th century Mértola (Portugal). METHODS: Sediment from each cesspit was analyzed using digital light microscopy and enzyme-linked immunosorbent assay (ELISA). RESULTS: Analysis revealed eggs of roundworm (Ascaris lumbricoides) in every cesspit analyzed, but no evidence of other species of helminth or protozoal parasites. CONCLUSION: Differences were noted between parasite species found in Mediterranean Europe and northern Europe, where a range of zoonotic parasites were endemic alongside sanitation-related parasites. We suggest that the scarcity of zoonotic parasites in southern Europe in the medieval period may reflect contrasts in climate between northern and southern Europe. SIGNIFICANCE: The repeated identification of roundworm eggs suggests that al-Andalus was less hygienic than historically depicted. We did not note a difference between parasites found in Muslim and Christian regions of Iberia, and the predominance of parasites spread by fecal contamination of food is consistent with past research. LIMITATIONS: The eggs of some species of parasite are fragile, so may theoretically have been present in the population but did not survive for us to identify them. SUGGESTIONS FOR FURTHER RESEARCH: To further investigate the role of climate upon the parasites that affected past human populations.

Cooperation, competition and antibiotic resistance in bacterial colonies.

Bacteria commonly live in dense and genetically diverse communities associated with surfaces. In these communities, competition for resources and space is intense, and yet we understand little of how this affects the spread of antibiotic-resistant strains. Here, we study interactions between antibiotic-resistant and susceptible strains using in vitro competition experiments in the opportunistic pathogen Pseudomonas aeruginosa and in silico simulations. Selection for intracellular resistance to streptomycin is very strong in colonies, such that resistance is favoured at very low antibiotic doses. In contrast, selection for extracellular resistance to carbenicillin is weak in colonies, and high doses of antibiotic are required to select for resistance. Manipulating the density and spatial structure of colonies reveals that this difference is partly explained by the fact that the local degradation of carbenicillin by ß-lactamase-secreting cells protects neighbouring sensitive cells from carbenicillin. In addition, we discover a second unexpected effect: the inducible elongation of cells in response to carbenicillin allows sensitive cells to better compete for the rapidly growing colony edge. These combined effects mean that antibiotic treatment can select against antibiotic-resistant strains, raising the possibility of treatment regimes that suppress sensitive strains while limiting the rise of antibiotic resistance. We argue that the detailed study of bacterial interactions will be fundamental to understanding and overcoming antibiotic resistance.