Widespread perturbation of ETS factor binding sites in cancer.

Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer drivers. To predict driver non-coding variants (NCVs), we present a transcription factor (TF)-aware burden test based on a model of coherent TF function in promoters. We apply this test to NCVs from the Pan-Cancer Analysis of Whole Genomes cohort and predict 2555 driver NCVs in the promoters of 813 genes across 20 cancer types. These genes are enriched in cancer-related gene ontologies, essential genes, and genes associated with cancer prognosis. We find that 765 candidate driver NCVs alter transcriptional activity, 510 lead to differential binding of TF-cofactor regulatory complexes, and that they primarily impact the binding of ETS factors. Finally, we show that different NCVs within a promoter often affect transcriptional activity through shared mechanisms. Our integrated computational and experimental approach shows that cancer NCVs are widespread and that ETS factors are commonly disrupted.

Levels and drivers of urban black carbon and health risk assessment during pre- and COVID19 lockdown in Augsburg, Germany.

This study aimed to evaluate the levels and phenomenology of equivalent black carbon (eBC) at the city center of Augsburg, Germany (01/2018 to 12/2020). Furthermore, the potential health risk of eBC based on equivalent numbers of passively smoked cigarettes (PSC) was also evaluated, with special emphasis on the impact caused by the COVID19 lockdown restriction measures. As it could be expected, peak concentrations of eBC were commonly recorded in morning (06:00-8:00 LT) and night (19:00-22:00 LT) in all seasons, coinciding with traffic rush hours and atmospheric stagnation. The variability of eBC was highly influenced by diurnal variations in traffic and meteorology (air temperature (T), mixing-layer height (MLH), wind speed (WS)) across days and seasons. Furthermore, a marked "weekend effect" was evidenced, with an average eBC decrease of ∼35% due to lower traffic flow. During the COVID19 lockdown period, an average ∼60% reduction of the traffic flow resulted in ∼30% eBC decrease, as the health risks of eBC exposure was markedly reduced during this period. The implementation of a multilinear regression analysis allowed to explain for 53% of the variability in measured eBC, indicating that the several factors (e.g., traffic and meteorology) may contribute simultaneously to this proportion. Overall, this study will provide valuable input to the policy makers to mitigate eBC pollutant and its adverse effect on environment and human health.

Aerosol emissions from a marine diesel engine running on different fuels and effects of exhaust gas cleaning measures.

The emissions of marine diesel engines have gained both global and regional attentions because of their impact on human health and climate change. To reduce ship emissions, the International Maritime Organization capped the fuel sulfur content of marine fuels. Consequently, either low-sulfur fuels or additional exhaust gas cleaning devices for the reduction in sulfur dioxide (SO2) emissions became mandatory. Although a wet scrubber reduces the amount of SO2 significantly, there is still a need to consider the reduction in particle emissions directly. We present data on the particle removal efficiency of a scrubber regarding particle number and mass concentration with different marine fuel types, marine gas oil, and two heavy fuel oils (HFOs). An open-loop sulfur scrubber was installed in the exhaust line of a marine diesel test engine. Fine particulate matter was comprehensively characterized in terms of its physical and chemical properties. The wet scrubber led up to a 40% reduction in particle number, whereas a reduction in particle mass emissions was not generally determined. We observed a shift in the size distribution by the scrubber to larger particle diameters when the engine was operated on conventional HFOs. The reduction in particle number concentrations and shift in particle size were caused by the coagulation of soot particles and formation/growing of sulfur-containing particles. Combining the scrubber with a wet electrostatic precipitator as an additional abatement system showed a reduction in particle number and mass emission factors by >98%. Therefore, the application of a wet scrubber for the after-treatment of marine fuel oil combustion will reduce SO2 emissions, but it does not substantially affect the number and mass concentration of respirable particulate matters. To reduce particle emission, the scrubber should be combined with additional abatement systems.

Presbyopic LASIK using the Supracor algorithm and micromonovision in presbyopic myopic patients: 12-month visual and refractive outcomes.

PURPOSE: To evaluate the visual and refractive outcomes for presbyopia and myopia treatment using the Teneo 317 M2 platform and the myopic Supracor algorithm. SETTING: Percy Military Hospital and Private Laser Victor Hugo Center, Paris, France. DESIGN: Observational retrospective nonrandomized study. METHODS: 50 eyes (25 patients) treated with bilateral myopic Supracor and micromonovision using the Teneo 317 M2 platform and followed up for 12 months. Study outcomes included binocular and monocular visual acuities (without correction for distance and near vision), the spherical equivalent, predictability, stability, safety, optical aberrations, and complications. RESULTS: The mean age was 50.6 ± 2.7 years, and the mean preoperative spherical equivalent was -2.6 ± 1.4 diopters. At 12 months postoperatively, the mean binocular uncorrected distance visual acuity was 0.02 ± 0.03 logMAR, and 24 patients (96%) achieved an acuity of 20/25 or better. The binocular uncorrected near visual acuity was equal to Jaeger 1 in 18 patients (72%) and Jaeger 2 or better in 23 patients (92%). 12 eyes (24%) had lost 1 Snellen line, and 1 eye (2%) had lost 2 Snellen lines of monocular corrected distance visual acuity. No cases required retreatment; however, 1 eye (2%) underwent revision surgery because of diffuse lamellar keratitis. CONCLUSIONS: This study suggests that Supracor using the Teneo 317 M2 platform is a safe and effective technique for myopia and presbyopia treatment. Supracor is therefore a viable alternative to monovision for presbyopia and myopia. However, a careful patient selection is essential to satisfy realistic expectations.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia.

Activated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1ß release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

Effect of Atmospheric Aging on Soot Particle Toxicity in Lung Cell Models at the Air-Liquid Interface: Differential Toxicological Impacts of Biogenic and Anthropogenic Secondary Organic Aerosols (SOAs).

BACKGROUND: Secondary organic aerosols (SOAs) formed from anthropogenic or biogenic gaseous precursors in the atmosphere substantially contribute to the ambient fine particulate matter [PM ≤2.5µm in aerodynamic diameter (PM2.5)] burden, which has been associated with adverse human health effects. However, there is only limited evidence on their differential toxicological impact. OBJECTIVES: We aimed to discriminate toxicological effects of aerosols generated by atmospheric aging on combustion soot particles (SPs) of gaseous biogenic (ß-pinene) or anthropogenic (naphthalene) precursors in two different lung cell models exposed at the air-liquid interface (ALI). METHODS: Mono- or cocultures of lung epithelial cells (A549) and endothelial cells (EA.hy926) were exposed at the ALI for 4 h to different aerosol concentrations of a photochemically aged mixture of primary combustion SP and ß-pinene (SOAßPIN-SP) or naphthalene (SOANAP-SP). The internally mixed soot/SOA particles were comprehensively characterized in terms of their physical and chemical properties. We conducted toxicity tests to determine cytotoxicity, intracellular oxidative stress, primary and secondary genotoxicity, as well as inflammatory and angiogenic effects. RESULTS: We observed considerable toxicity-related outcomes in cells treated with either SOA type. Greater adverse effects were measured for SOANAP-SP compared with SOAßPIN-SP in both cell models, whereas the nano-sized soot cores alone showed only minor effects. At the functional level, we found that SOANAP-SP augmented the secretion of malondialdehyde and interleukin-8 and may have induced the activation of endothelial cells in the coculture system. This activation was confirmed by comet assay, suggesting secondary genotoxicity and greater angiogenic potential. Chemical characterization of PM revealed distinct qualitative differences in the composition of the two secondary aerosol types. DISCUSSION: In this study using A549 and EA.hy926 cells exposed at ALI, SOA compounds had greater toxicity than primary SPs. Photochemical aging of naphthalene was associated with the formation of more oxidized, more aromatic SOAs with a higher oxidative potential and toxicity compared with ß-pinene. Thus, we conclude that the influence of atmospheric chemistry on the chemical PM composition plays a crucial role for the adverse health outcome of emissions. https://doi.org/10.1289/EHP9413.

The Clinical Importance of Preoperative Rectal Swabs in Patients after Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

Background: Surgical site infections are among the most common healthcare-associated infections, especially in patients undergoing cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). The aim of this retrospective study was to examine postoperative infectious complications according to preoperative screening findings of nasal and rectal swabs. Methods: Two hundred four consecutive patients received nasal and rectal swab examination for multidrug-resistant (MDR) bacteria within 30 days before the operation in patients where CRS and HIPEC were planned. Inclusion criteria were as follows: confirmed peritoneal metastases (histologically and/or cytologically); age under 85 years; adequate renal, liver, and bone marrow function; no sign of infection preoperatively; resectable disease; and CRS and HIPEC procedure. If surgical site infection occurred, the microbial spectrum of the site was assessed. One hundred twenty-one patients (63 female [52.1%] and 58 male [47.9%]) met the criteria and were further analyzed retrospectively. Statistical correlations between postoperative complications and risk factors were investigated by univariate and multivariate analysis. Results: Postoperative complications in total were observed in 57 patients (47.1%) with major complications (Clavien-Dindo grades 3-4) in 15 patients (12.4%) and infectious complications in 37 (30.6%) patients. The overall prevalence of nasal MRSA carriage was 3.28%, and the overall prevalence of rectal MDR bacteria carriage was 10.7%. In propensity score analysis, colonized patients compared to noncolonized patients showed increased total complications (CD1-5, p = 0.025), infectious complications (p = 0.028), surgical site infections (p = 0.022) as well as pneumonia (p = 0.016). Multivariate analysis showed that in addition to preoperative rectal colonization, American Society of Anesthesiologists score was a risk factor for postoperative complications. Conclusions: Preoperative 3-MRGN and vancomycin-resistant enterococcus colonization were associated with increased complications and surgical site infections. Special antimicrobial treatment pathways are necessary for these patients to reduce postoperative complications due to colonization.

Puff-Resolved Analysis and Selected Quantification of Chemicals in the Gas Phase of E-Cigarettes, Heat-Not-Burn Devices, and Conventional Cigarettes Using Single-Photon Ionization Time-of-Flight Mass Spectrometry (SPI-TOFMS): A Comparative Study.

INTRODUCTION: A wide array of alternative nicotine delivery devices (ANDD) has been developed and they are often described as less harmful than combustible cigarettes. This work compares the chemical emissions of three ANDD in comparison to cigarette smoke. All the tested ANDD are characterized by not involving combustion of tobacco. AIMS AND METHODS: Single-photon ionization time-of-flight mass spectrometry (SPI-TOFMS) is coupled to a linear smoking machine, which allows a comprehensive, online analysis of the gaseous phase of the ANDD aerosol and the conventional cigarette (CC) smoke. The following devices were investigated in this study: a tobacco cigarette with a glowing piece of coal as a heating source, an electric device for heating tobacco, and a first-generation electronic cigarette. Data obtained from a standard 2R4F research cigarette are taken as a reference. RESULTS: The puff-by-puff profile of all products was recorded. The ANDD show a substantial reduction or complete absence of known harmful and potentially harmful substances compared with the CC. In addition, tar substances (i.e. semivolatile and low volatile aromatic and phenolic compounds) are formed to a much lower extent. Nicotine, however, is supplied in comparable amounts except for the investigated electronic cigarette. CONCLUSIONS: The data show that consumers switching from CC to ANDD are exposed to lower concentrations of harmful and potentially harmful substances. However, toxicological and epidemiological studies must deliver conclusive results if these reduced exposures are beneficial for users. IMPLICATIONS: The comparison of puff-resolved profiles of emissions from different tobacco products, traditional and alternative, may help users switch to lower emission products. Puff-resolved comparison overcomes technical changes, use modes between products and may help in their regulation.

Bacterobilia in pancreatic surgery-conclusions for perioperative antibiotic prophylaxis.

BACKGROUND: Jaundice or preoperative cholestasis (PC) are typical symptoms of pancreatic masses. Approximately 50% of patients undergo preoperative biliary drainage (PBD) placement. PBD is a common cause of bacterobilia (BB) and is a known surgical site infection risk factor. An adjustment of preoperative antibiotic prophylaxis (PAP) may be reasonable according to the profile of BB. For this, we examined the microbiological findings in routine series of patients. AIM: To investigate the incidence and profile of biliary bacterial colonization in patients undergoing pancreatic head resections. METHODS: In the period from January 2009 to December 2015, 285 consecutive pancreatic head resections were performed. Indications for surgery were malignancy (71%), chronic pancreatitis (18%), and others (11%). A PBD was in 51% and PC was in 42%. The standard PAP was ampicillin/sulbactam. Intraoperatively, a smear was taken from the hepatic duct. An analysis of the isolated species and resistograms was performed. Patients were categorized according to the presence or absence of PC (PC+/PC-) and PBD (PBD+/PBD-) into four groups. Antibiotic efficiency was analyzed for standard PAP and possible alternatives. RESULTS: BB was present in 150 patients (53%). BB was significantly more frequent in PBD+ (n =120) than in PBD- (n = 30), P < 0.01. BB was present both in patients with PC and without PC: (PBD-/PC-: 18%, PBD-/PC+: 30%, PBD+/PC-: 88%, PBD+/PC+: 80%). BB was more frequent in malignancy (56%) than in chronic pancreatitis (45%). PBD, however, was the only independent risk factor in multivariate analysis. In total, 357 pathogens (342 bacteria and 15 fungi) were detected. The five most common groups (n = 256, 74.8%) were Enterococcus spp. (28.4%), Streptococcus spp. (16.9%), Klebsiella spp. (12.6%), Escherichia coli (10.5%), and Enterobacter spp. (6.4%). A polymicrobial BB (PBD+: 77% vs PBD-: 40%, P < 0.01) and a more frequent detection of Enterococcus (P < 0.05) was significantly associated with PBD+. In PBD+, the efficiency of imipenem and piperacillin/tazobactam was significantly higher than that of the standard PAP (P < 0.01). CONCLUSION: PBD-/PC- and PBD-/PC+ were associated with a low rate of BB, while PBD+ was always associated with a high rate of BB. In PBD+ patients, BB was polymicrobial and more often associated with Enterococcus. In PBD+, the spectrum of potential bacteria may not be covered by standard PAP. A more potent alternative for prophylactic application, however, was not found.

In Vivo Immunological Effects of CD73 Deficiency.

BACKGROUND/AIMS: The extracellular ecto-5'-nucleotidase (CD73) is involved in the production of immunosuppressive adenosin (Ado), which can influence different immune cells through the specific adenosine receptors. The main aim of this work was to characterize immune cell populations as well as serum cytokine level in healthy CD73-deficient mice compared to healthy wild-type animals. METHODS: Profound immnophenotyping of splenocytes from healthy CD73-deficient and wild-type mice was done using flow cytometry (FACS analysis). Cytokine measurement in the serum of the animals was carried out with a Bio-Plex assay. RESULTS: The CD73-deficience leads to an increase in a percentage of NK cells and pDC, as well as influences expression of the costimulatory molecules CD80 and CD86. The knockout mice in opposite to wild-type animals show high amount of effector CD4+ T-cells in the spleens. No changes have been found in the subpopulations of CD8+ T-cells. Besides, CD73-deficience leads to a decrease in the percentage of regulatory T cells. Compared with the wild-type animals we found that CD73 knockout mice possess low serum concentration of IL-6. CONCLUSION: This in vivo study clear demonstrated certain immunological changes in the CD73-deficient mice and thus immunoregulatory potential of CD73 molecule. This makes this extracellular enzyme to a real immune check point molecule, attractive for further investigations and clinical studies.

How to do it: setting up a PrEP service in an integrated sexual reproductive health service setting.

Pre-exposure prophylaxis for HIV (PrEP) has been shown to reduce transmission of HIV in a number of trials; however, there is limited evidence regarding the optimal way to deliver PrEP through pre-existing UK services, particularly through fully integrated drop-in sexual health service models. PrEP in the form of Truvada was launched in Wales in July 2017. We set up a PrEP service to be delivered via our drop-in integrated sexual reproductive health service. In the first 5 months of PrEP service provision, we found unforeseen levels of comorbidity, polypharmacy and renal impairment in our cohort of PrEP patients. As a result, we have altered our service model and all patients are now followed up in booked appointment PrEP clinics run by members of the HIV team. Those patients with estimated glomerular filtration rate (eGFR) of 60-70 mL/min or with eGFR of 60-80 mL/min and with comorbidities impacting on renal function are monitored every 4-6 weeks initially, and PrEP has been incorporated into our pre-existing virtual HIV renal clinic for discussion with a renal physician. The PrEP team clinicians report that monitoring and managing the PrEP cohort is now easier in its appointment-only format, although some patients have reported that they would prefer a drop-in system.

A case report of small bowel obstruction from previously undiagnosed lobular breast carcinoma: First in Australian literature.

INTRODUCTION: This case report is the first in the Australian literature of a patient, without prior diagnosis, presenting with a bowel obstruction secondary to lobular breast cancer. This highlights a relatively rare cause of bowel obstruction, but also the importance of breast self-examination as a compliment to the current BreastScreen Australia program. PRESENTATION OF CASE: A 67-year-old female presented to the Emergency Department with a 48-h history of sharp, constant epigastric pain, vomiting and constipation. The patient proceeded to emergency laparotomy for presumed large bowel obstruction, which revealed a stricture in the distal terminal ileum causing a distal small bowel obstruction. A right hemicolectomy was performed. Histopathology revealed the terminal ileum stricture to be metastatic lobular breast carcinoma. Clinical examination of the patient's right breast revealed a lesion suggestive of the primary malignancy despite a normal ultrasound and mammogram in 2014. After failing to progress, a CT scan was performed which revealed progressive small and large bowel distension. A repeat laparotomy was performed revealing dilated large bowel without obstructing pathology and an intact anastomosis. A loop ileostomy was performed. Following a further febrile episode, the patient decided to withdraw care and the patient passed away three weeks into her admission from suspected intra-abdominal sepsis. DISCUSSION: Breast cancer is becoming the third most common cancer amongst Australian women with a significant burden of disease and mortality. CONCLUSION: Despite the rare presentation, this case reminds the medical community and general population of the importance of breast self-examination and the BreastScreen Australia program.

The impact of temporal artery biopsy on surgical practice.

BACKGROUND: Giant cell arteritis (GCA) has the potential to cause irreversible blindness and stroke in affected patients [1-4]. Temporal artery biopsy (TAB) remains the gold standard test for GCA [6-8]. Recent literature suggests that TAB does not change management of patients with suspected GCA and that ultrasound scan (USS) may be sufficient enough alone to confirm the diagnosis [9-11,13]. The aim of this study is to therefore determine the impact of TAB on current surgical practice and emergency theatre services. MATERIALS AND METHODS: A retrospective clinical study was performed of patients who had undergone TAB at the Caboolture Hospital from January 2010 to September 2015. Demographic and clinical data was collected from patient's medical records in regards to GCA. RESULTS: A total of 55 TAB were performed on 50 patients. Only two TAB were positive for GCA. Thirty-eight (76%) patients had a pre-TAB ACR criteria score of ≥3. Pre-operative corticosteroids were administered in forty-five (90%) patients, on average 4 ± 10 days pre-TAB. Mean time to TAB was 1.6 ± 1.6 days following their booking. Ninety-one percent of TAB were performed by surgical registrars. All TAB were performed using local anaesthesia alone. CONCLUSIONS: TAB is an expensive procedure with a low positive yield. Recent evidence suggests promising results with USS in diagnosing GCA. With the exceedingly low positive TAB results found in this study, patients with suspected GCA should be investigated in accordance with the above algorithm. The routine use of USS will reduce the number of negative TAB performed.

A surgeon-led model to improve operating theatre change-over time and overall efficiency: A randomised controlled trial.

BACKGROUND: The non-operative time during the process of patient change-over between operating theatre cases is a significant source of delay and overall theatre inefficiency. The aim of this study was to integrate and trial a working strategy to improve this change-over time. METHOD: This was a single-blinded, randomised controlled intervention study comparing a surgeon-led, team-based model of strategies versus routine patient change-over. This model was trialled by a single surgeon, and the primary outcome was the difference in change-over times compared with 4 other surgeons who were blinded and served as controls. Secondary outcome measures included overall differences in complications between the groups, and the number and differences in operative case cancellations due to inadequate theatre time. RESULTS: 1265 patients were randomised into 5 general surgical lists, and included all major and minor cases. Median number of operative cases were 214 per surgeon, with an overall median change over time of 17.9 ± 3.7 min. Surgeon A in the intervention group had a median change-over time of 12.1 ± 5.4 min (p < 0.001), with a median difference of 8.5 min ± 21.4 min (p < 0.0001), translating to a 58% reduction in median change-over time between the intervention and control groups. There were no differences in complication rates amongst the groups. The intervention group had no cancellations due to lack of time, compared with 37 cancellations in the control group. CONCLUSION: This study demonstrates a statistically significant improvement in median change-over times using this model. This re-design can be implemented without incurring extra costs, staff, or operating theatres.

Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.

Impaired peripheral Th1 CD4+ T cell response to Escherichia coli proteins in patients with Crohn's disease and ankylosing spondylitis.

BACKGROUND: To clarify the impact of T cell responses towards enteric antigens for chronic intestinal inflammation, we determined T helper 1 reactivity towards conserved Escherichia coli proteins in patients with Crohn's disease (CD) and healthy individuals and patients with ankylosing spondylitis (AS), who also often show microscopic inflammatory lesions within the gut or even develop overt inflammatory bowel disease. METHODS: We determined the frequency of IFNγ+CD40L+ cells/CD4+ T cells after stimulation of whole blood with pools of E. coli proteins. RESULTS: The E. coli-specific Th1 response was significantly reduced in CD patients and to a lower extent also in AS patients. CONCLUSIONS: E. coli is a target for polyclonal Th1 responses in healthy individuals. The impairment of these responses in CD and AS patients might be due to recruitment of enterobacteria-specific Th1 cells to the gut or might reflect inadequate priming of adaptive immune response.

Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin.

Earlier, we have shown that spontaneously isolated non-pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin-like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep-2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli-carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep-2 cells. Moreover, internalization of non-invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.

Influence of filter ventilation on the chemical composition of cigarette mainstream smoke.

Total yields of cigarette smoke constituents are greatly influenced by smoking behaviour, the tobacco blend as well as a variety of cigarette design parameters. Thereby, filter ventilation, i.e. diluting the smoke by providing a zone of microscopic holes around the circumference of the filter is one method to reduce the yield of 'tar' and other smoke compounds. However, little is known how these design variations influence the combustion conditions, and therefore, the overall chemical pattern of the smoke. In this paper single photon ionization-time-of-flight mass spectrometry (SPI-TOFMS) is used to characterize and compare cigarettes on a puff-by-puff basis, which differ only in filter ventilation magnitude. The research cigarettes investigated were made from Virginia tobacco and featured filter ventilations of 0% (no ventilation), 35%, and 70%. The cigarettes were smoked under two different puffing regimes, one using the puffing parameters of the conventional International Organization for Standardization (ISO) smoking regime and a more intense smoking condition. Results show that every variation entails a change of the chemical pattern, whereby, in general, cigarettes with 0% filter ventilation as well as the intense smoking regime lead to a more complete combustion compared to the ISO smoking conditions and the high ventilated cigarettes. Changes in the overall patterns can also be observed during the smoking for individual puffs. Some substances dominate the first puff, some species are more pronounced in the middle puffs, whereas others are preferably formed in the last puffs. This demonstrates the high complexity of the occurring processes. Results might help to understand the formation and decomposition reactions taking place when a cigarette is smoked and offer scope for targeted reduction strategies for specific toxicants or groups of toxicants in the smoke.

Simultaneous on-line size and chemical analysis of gas phase and particulate phase of cigarette mainstream smoke.

This paper describes the combined set-up of on-line chemical analysis of gas phase by single-photon ionisation/resonance enhanced multiphoton ionisation-time-of-flight mass spectrometry (SPI/REMPI-TOFMS) and on-line particle size analysis by differential electrical mobility particle spectrometry (DMS 500) for the investigation of fresh cigarette mainstream smoke. SPI is well suited for the investigation of a great variety of organic species, whereas REMPI is highly sensitive for aromatic compounds. Gas phase measurements of filtered and unfiltered smoke are possible with the SPI/REMPI-TOFMS in order to determine the influence of the presence of particles on the chemical composition of the gas phase. Initial results are shown for the characterisation and comparison of three pure Virginia tobacco research cigarettes having filter ventilations of 0%, i.e. no filter ventilation, 35% and 70% ventilation. The three cigarette types are smoked under two different smoking regimes, a standard regime using puff parameters equivalent to the conventional International Standard Organisation regime and a more intense smoking regime. For the gas phase, qualitative puff-by-puff resolved yields of three selected compounds (acetaldehyde, phenol and styrene) are shown and compared. For particulate matter, particle number, count median diameter and total surface area are illustrated on a puff-by-puff basis. Yields of the chemicals analysed, puff number and surface area are in good agreement with the intensity of the smoking regime and the dilution of smoke by filter ventilation. However, gaseous compounds are influenced differently, depending whether an absolute particle filter is present or not, i.e. they can be totally removed (phenol), partially removed (styrene) or not affected (acetaldehyde). For particle analysis, the count median diameter decreases from puff to puff and is strongly dependent on the smoking regime and ventilation rate. Thereby, 0% ventilated cigarettes smoked under the intense regime result in the smallest count median diameters of ca. 180 nm, whereas 70% ventilated cigarettes smoked with a standard regime lead to the largest values of up to 280 nm. As particle diameter increases, particle number decreases as a consequence of increasing time for particle coagulation.

Characterization of puff-by-puff resolved cigarette mainstream smoke by single photon ionization-time-of-flight mass spectrometry and principal component analysis.

Soft single photon ionization-time-of-flight mass spectrometry (SPI-TOFMS) and principal component analysis (PCA) were applied for the characterization and discrimination of the chemical patterns of all individual cigarette puffs from the 2R4F University of Kentucky research reference cigarette. The SPI-TOFMS was connected to a smoking machine, and 10 cigarettes were smoked under defined smoking conditions. A total of 41 detected mass signals could be clearly assigned to smoke constituents (e.g., unsaturated hydrocarbons, aromatic species, sulfurous compounds, and nitrogen-containing substances). For further analysis, the on-line recorded mass signals were added up for each cigarette puff resulting in a single summed mass spectrum for each puff. The so-achieved puff-by-puff resolved yields were additionally normalized by the corresponding total ion signal, which eliminated the influences of varying amounts of burnt tobacco. These values were incorporated into a PCA to find differences and similarities in the chemical patterns of the individual cigarette puffs. In addition, absolute (without normalization) and normalized puff resolved yields were used to clarify occurring trends. Thereby, it was shown that the chemical pattern of the first cigarette puff was very unique, whereby extraordinary high yields of unsaturated hydrocarbons are mainly responsible for this. Depending on the smoking procedure, the chemical pattern of the second puff can also be separated from the first and the third puff. In this case, nitrogen-containing substances play an important role. Puffs three to eight show only small but observable differences. These changes are greater influenced by oxygen-containing and sulfurous smoke constituents. The findings reveal that the overall chemical patterns of machine-smoked cigarette puffs vary quite a lot during the smoking process. This lets us assume that the burden of hazardous compounds for the human smoker also differs from puff to puff.