Shotgun metagenome sequencing of a Sudanese toombak snuff tobacco: genetic attributes of a high tobacco-specific nitrosamine containing smokeless tobacco product.

The most alarming aspect of the Sudanese toombak smokeless tobacco is that it contains high levels of highly toxic tobacco-specific nitrosamines (TSNAs). Understanding the microbiology of toombak is of relevance because TSNAs are an indirect result of microbial-mediated nitrate reductions. We conducted shotgun metagenomic sequencing on a toombak product for which relevant features are presented here. The microbiota was composed of over 99% Bacteria. The most abundant taxa included Actinobacteria, specifically the genera Enteractinococcus and Corynebacterium, while Firmicutes were represented by the family Bacillaceae and the genus Staphylococcus. Selected gene targets were nitrate reduction and transport, antimicrobial resistance, and other genetic transference mechanisms. Canonical nitrate reduction and transport genes (i.e. nar) were found for Enteractinococcus and Corynebacterium while various species of Staphylococcus exhibited a notable number of antimicrobial resistance and genetic transference genes. The nitrate reduction activity of the microbiota in toombak is suspected to be a contributing factor to its high levels of TSNAs. Additionally, the presence of antimicrobial resistance and transference genes could contribute to deleterious effects on oral and gastrointestinal health of the end user. Overall, the high toxicity and increased incidences of cancer and oral disease of toombak users warrants further investigation into the microbiology of toombak.

Microbial communities and gene contributions in smokeless tobacco products.

Smokeless tobacco products (STP) contain bacteria, mold, and fungi due to exposure from surrounding environments and tobacco processing. This has been a cause for concern since the presence of microorganisms has been linked to the formation of highly carcinogenic tobacco-specific nitrosamines. These communities have also been reported to produce toxins and other pro-inflammatory molecules that can cause mouth lesions and elicit inflammatory responses in STP users. Moreover, microbial species in these products could transfer to the mouth and gastrointestinal tract, potentially altering the established respective microbiotas of the consumer. Here, we present the first metagenomic analysis of select smokeless tobacco products, specifically US domestic moist and dry snuff. Bacterial, eukaryotic, and viral species were found in all tobacco products where 68% of the total species was comprised of Bacteria with 3 dominant phyla but also included 32% Eukarya and 1% share abundance for Archaea and Viruses. Furthermore, 693,318 genes were found to be present and included nitrate and nitrite reduction and transport enzymes, antibiotic resistance genes associated with resistance to vancomycin, ß-lactamases, their derivatives, and other antibiotics, as well as genes encoding multi-drug transporters and efflux pumps. Additional analyses showed the presence of endo- and exotoxin genes in addition to other molecules associated with inflammatory responses. Our results present a novel aspect of the smokeless tobacco microbiome and provide a better understanding of these products' microbiology. KEY POINTS: • The findings presented will help understand microbial contributions to overall STP chemistries. • Gene function categorization reveals harmful constituents outside canonical forms. • Pathway genes for TSNA precursor activity may occur at early stages of production. • Bacteria in STPs carry antibiotic resistance genes and gene transfer mechanisms.

Real-time dynamic modelling for the design of a cluster-randomized phase 3 Ebola vaccine trial in Sierra Leone.

BACKGROUND: Declining incidence and spatial heterogeneity complicated the design of phase 3 Ebola vaccine trials during the tail of the 2013-16 Ebola virus disease (EVD) epidemic in West Africa. Mathematical models can provide forecasts of expected incidence through time and can account for both vaccine efficacy in participants and effectiveness in populations. Determining expected disease incidence was critical to calculating power and determining trial sample size. METHODS: In real-time, we fitted, forecasted, and simulated a proposed phase 3 cluster-randomized vaccine trial for a prime-boost EVD vaccine in three candidate regions in Sierra Leone. The aim was to forecast trial feasibility in these areas through time and guide study design planning. RESULTS: EVD incidence was highly variable during the epidemic, especially in the declining phase. Delays in trial start date were expected to greatly reduce the ability to discern an effect, particularly as a trial with an effective vaccine would cause the epidemic to go extinct more quickly in the vaccine arm. Real-time updates of the model allowed decision-makers to determine how trial feasibility changed with time. CONCLUSIONS: This analysis was useful for vaccine trial planning because we simulated effectiveness as well as efficacy, which is possible with a dynamic transmission model. It contributed to decisions on choice of trial location and feasibility of the trial. Transmission models should be utilised as early as possible in the design process to provide mechanistic estimates of expected incidence, with which decisions about sample size, location, timing, and feasibility can be determined.

Effect of charcoal-containing cigarette filters on gas phase volatile organic compounds in mainstream cigarette smoke.

Of the chemicals identified to date in mainstream cigarette smoke with known toxicological properties, the volatile organic compounds (VOCs) are considered the most hazardous group owing to their high abundance and toxicity. In this research we evaluate a recently introduced line of cigarettes that contain charcoal in their filters. The amount of charcoal in these filters ranged from 45 mg to 180 mg and were either dispersed among the filter material or contained in a small cavity in the filter segment. Charcoal has long been used for removing VOCs from both water and air. Our findings indicate that these cigarettes reduce machine generated mainstream smoke deliveries of a wide range of VOCs compared to a similar, non-charcoal filtered, cigarette. However, this reduction is dependent not only on the amount of charcoal present but also on the volume of smoke being drawn through the filter. While a brand with 45 mg charcoal reduces VOC delivery under ISO smoking conditions, charcoal saturation and breakthrough occur under more intense smoking conditions. Breakthrough is minimised for brands with the most charcoal. Overall, the brands with the most charcoal are effective at reducing VOC deliveries under even intense smoking conditions.

Analysis of toxic metals in commercial moist snuff and Alaskan iqmik.

The extent to which smokeless tobacco endangers human health is an ongoing subject of debate. Studies have shown that smokeless tobacco products contain high levels of biologically available nicotine and tobacco-specific nitrosamines. Toxic metals in smokeless tobacco products have been less extensively studied. In this study, concentrations of arsenic, barium, beryllium, cadmium, chromium, cobalt, lead, and nickel were measured in snuff products and iqmik tobacco, a product popular among some Alaska Natives. The average arsenic, cadmium, lead, and nickel concentrations in 17 commercially available brands were 0.23 +/- 0.06 microg/g, 1.40 +/- 0.31 microg/g, 0.45 +/- 0.13 microg/g and 2.28 +/- 0.36 microg/g, respectively. In 17 iqmik tobacco samples, the average arsenic, cadmium, lead, and nickel concentrations were 0.19 +/- 0.06 microg/g, 1.41 +/- 0.56 microg/g, 0.55 +/- 0.19 microg/g, and 2.32 +/- 1.63 microg/g, respectively. Using artificial saliva, the extractable levels of beryllium and lead were relatively low and consistent, whereas barium extracted from tobacco samples ranged from 2 to 21%. The group 1 and 2B carcinogens cadmium, cobalt, and nickel were more efficiently extracted by artificial saliva (30-65% of the cobalt, 20-46% of the nickel, and 21-47% of the cadmium).

Cadmium, lead, and thallium in smoke particulate from counterfeit cigarettes compared to authentic US brands.

Smoking remains the leading cause of preventable disease in the United States. Exposure to tobacco smoke leads to cancer, heart and lung disease, and addiction. The origin of the tobacco and cigarette manufacturing practices of counterfeit cigarettes are unknown. Because toxic metals are incorporated into the tobacco lamina during cultivation, the ambient metal content of the soil could produce significant differences in metal levels in both the tobacco and smoke of counterfeit cigarettes. We compared mainstream smoke cadmium, thallium, and lead deliveries from counterfeit and authentic brands. Mainstream smoke levels of all three metals were far greater for counterfeit than the authentic brands, in some cases by an order of magnitude. Significant differences still existed even after normalizing mainstream smoke metal levels with nicotine delivery; the counterfeits typically delivered much higher levels of all three analytes. Our findings, based on 21 different counterfeit samples, suggest that counterfeit cigarettes potentially result in a markedly greater exposure to toxic heavy metals than authentic brands, even after correcting for differences in nicotine intake. In view of the unknown health risks associated with inhaling higher levels of toxic metals, it is prudent to minimize exposure to toxic substances whenever possible.

Cadmium, lead, and thallium in mainstream tobacco smoke particulate.

The deliveries of cadmium, thallium, and lead in mainstream smoke particulate from cigarettes with different smoke delivery designs were determined by inductively coupled plasma-mass spectrometry in order to investigate their impact on the delivery of these known toxic compounds. Analyses showed that the levels of all three metals in smoke particulate were associated with their tar delivery category. After normalizing the metal concentrations to tar, there were no longer any statistically significant delivery differences between full-flavor, light or ultra-light cigarettes. When the concentrations were normalized to nicotine, the mean levels from the three delivery groups were much smaller than before normalization. But unlike the case using tar to normalize, in some of the cases, there were still some statistically significant differences in the nicotine-normalized results. These findings suggest that if smokers compensate for differences in nicotine intake, they receive exposures to toxic heavy metals from ultra-light, light and full-flavor cigarettes that are more similar than results would suggest from using the Federal Trade Commission method alone.

The development of a quantitative flexibility test for body armour and comparison with wearer trials.

In this paper a mechanical flexibility test is developed which can be used to assess multi layer body armour systems. This is compared with a subjective manual test, and then with the results of wearer trials conducted using the recently approved ISO body armour standard ISO 14876-1 (2002). A series of trials was conducted on six different ballistic and/or stab resistant body armour types with a variety of protection levels and constructions. These were tested using the mechanical test system in which the armour was forced through a 200 mm hole by a 100 mm hemispherical plunger. The results of this test were then compared to a second set of trials in which flexibility of the same armour was assessed by manual handling and flexing of the armour. Finally an ergonomic wearer trial was conducted with four armours according to ISO 14876-1 (2002) each armour being assessed by four volunteers and the results compared to flexibility data collected in the first two trials. It was shown that the mechanical flexibility test produced results which were in good agreement with a purely subjective flexibility assessment. These results in turn showed reasonable but not exact correlation with the wearer trials. The ISO wearer trials addressed other factors such as overall comfort and fit of the systems and so the results were not purely a function of flexibility.

Determination of tar, nicotine, and carbon monoxide yields in the mainstream smoke of selected international cigarettes.

OBJECTIVE: Survey of nicotine, tar, and carbon monoxide (CO) smoke deliveries from 77 cigarette brands purchased in 35 countries was conducted using a standardised machine smoking method. The goal of this study was to determine regional variations and differences in the tar, nicotine, and CO smoke yields of a cigarette brand manufactured by a leading transnational corporation and of non-US locally popular cigarette brands. DESIGN: The majority of the cigarettes were purchased in each of the participating countries by delegate members of the World Health Organization and forwarded to the Centers for Disease Control and Prevention for analysis. Smoke deliveries were determined using a standardised smoking machine method and subsequent gravimetric and gas chromatography analysis. RESULTS: The smoke deliveries varied widely. Mainstream smoke deliveries varied from 6.8 to 21.6 mg tar/cigarette, 0.5 to 1.6 mg nicotine/cigarette, and 5.9 to 17.4 mg CO/cigarette. In addition to the smoke deliveries, the cigarettes were examined to determine physical parameters such as filter composition, length, and ventilation levels. CONCLUSION: Analysis of the smoke deliveries suggested that cigarettes from the Eastern Mediterranean, Southeast Asia, and Western Pacific WHO regions tended to have higher tar, nicotine, and CO smoke deliveries than did brands from the European, American, or African WHO regions surveyed.

Concentrations of nine alkenylbenzenes, coumarin, piperonal and pulegone in Indian bidi cigarette tobacco.

Indian-made bidi cigarettes sold in the United States are available in a variety of exotic (e.g. clove, mango) and candy-like (e.g. chocolate, raspberry) flavors. Because certain tobacco flavorings contain alkenylbenzenes and other toxic or carcinogenic chemicals, we measured the concentration of flavor-related compounds in bidi tobacco using a previously developed method. Twenty-three brands of bidis were sampled using automated headspace solid-phase microextraction and subsequently analyzed for 12 compounds by gas chromatography-mass spectrometry. Two alkenylbenzene compounds, trans-anethole and eugenol, were found in greater than 90% of the brands analyzed. Methyleugenol, pulegone and estragole were each detected in 30% or more of the brands, whereas safrole and elemicin were not detected in any of the brands. The flavor-related compounds with the highest tobacco concentrations were eugenol (12,000 microg/g tobacco) and trans-anethole (2200 microg/g tobacco). The highest eugenol and trans-anethole concentrations found in bidi tobacco were about 70,000 and 7500 times greater, respectively, than the highest levels previously found in US cigarette brands. Measurement of these compounds is crucial to evaluation of potential risks associated with inhaling highly concentrated flavor-related compounds from bidis or other tobacco products.

Wounding patterns and human performance in knife attacks: optimising the protection provided by knife-resistant body armour.

BACKGROUND: Stab attacks generate high loads, and to defeat them, armour needs to be of a certain thickness and stiffness. Slash attacks produce much lower loads and armour designed to defeat them can be far lighter and more flexible. METHODS AND SUBJECTS: Phase 1: Human performance in slash attacks: 87 randomly selected students at the Royal Military College of Science were asked to make one slash attack with an instrumented blade on a vertically mounted target. No instructions on how to slash the target were given. The direction, contact forces and velocity of each attack were recorded. Phase 2: Clinical experience with edged weapon attacks: The location and severity of all penetrating injuries in patients attending the Glasgow Royal Infirmary between 1993 and 1996 were charted on anatomical figures. RESULTS: Phase 1: Two types of human slash behaviour were evident: a 'chop and drag' blow and a 'sweep motion' type of attack. 'Chop and drag' attacks had higher peak forces and velocities than sweep attacks. Shoulder to waist blows (diagonal) accounted for 82% of attacks, 71% of attackers used a long diagonal slash with an average cut length of 34 cm and 11% used short diagonal attacks with an average cut length of 25 cm. Only 18% of attackers slashed across the body (short horizontal); the average measured cut length of this type was 28 cm. The maximum peak force for the total sample population was 212 N; the maximum velocity was 14.88 m s(-1). The 95 percentile force for the total sample population was 181 N and the velocity was 9.89 m s(-1). Phase 2: 431 of the 500 patients had been wounded with edged weapons. The average number of wounds sustained by victims in knife assaults was 2.4. The distribution of wounds by frequency and severity are presented. CONCLUSIONS: Anti-slash protection is required for the arms, neck, shoulders, and thighs. The clinical experience of knife-attack victims provides information on the relative vulnerabilities of different regions of the body. It is anticipated that designing a tunic-type of Police uniform that is inherently stab and slash resistant will eventually replace the current obvious and often bulky extra protective vest. Attempts at making a combined garment will need to be guided by ergonomic considerations and field testing. A similar anatomical regional risk model might also be appropriate in the design of anti-ballistic armour and combined anti-ballistic and knife-resistant armour.

Reliability of individual differences in initial sensitivity and acute tolerance to nitrous oxide hypothermia.

On average, the hypothermia exhibited by rats receiving 60% nitrous oxide (N2O) eventually abates despite the continued inhalation of the drug (i.e., acute tolerance develops). However, large individual differences occur in both the magnitude of hypothermia achieved and the degree of acute tolerance that develops. To determine whether the degree of temperature loss and subsequent recovery during N2O administration are reliable characteristics of an individual, we measured intraperitoneal temperature via telemetry in 77 Long-Evans rats that each received 60% N2O for 5 h during two sessions separated by 14 days. Good intersession reliability (Pearson's r) was observed for simple change and adjusted change scores for both initial N2O temperature sensitivity (.61 < or = r < or = .62), and acute tolerance development (.46 < or = r < or = .52). In a separate experiment, three groups of rats were selected based on their individual body temperature patterns during an initial N2O administration: (1) insensitive to N2O hypothermia (n = 8); (2) marked hypothermia followed by acute tolerance development (n = 6); and (3) marked hypothermia followed by little acute tolerance development (n = 6). When retested 10 days later, each group exhibited a body temperature profile similar to that observed during the initial N2O exposure. Thus, the temperature profile observed during a rat's initial exposure to 60% N2O reflects a reproducible response for that animal.

Amiloride-sensitive signals and NaCl preference and appetite: a lick-rate analysis.

Rats prefer hypotonic and isotonic NaCl solutions to water in long-access drinking paradigms. To focus on the role of taste signals in NaCl preference, licking patterns of rats with 30-s exposure to NaCl solutions (0-0.5 M) were examined when they were either water deprived, sodium depleted, or not deprived (NaCl mixed in dilute sucrose). In all three conditions, rats displayed a preference for NaCl. The addition of 100 microM amiloride, a sodium channel blocker, to NaCl did not change rats' licking when they were sodium replete but dramatically reduced licking when they were deplete. Transection of the chorda tympani (CT) nerve, an afferent pathway for amiloride-sensitive Na(+) signals, had no effect on NaCl preference in nondeprived rats and only a modest effect on those that were Na(+) deplete. Amiloride was found to exert significant suppression of NaCl intake in Na(+)-depleted rats with transection of the CT, supporting the existence of other afferent pathways for transmission of amiloride-sensitive Na(+) signalling. Together, these studies argue for the involvement of different neural signalling mechanisms in NaCl preference in the presence and absence of explicit Na(+) need.

Quantitative analysis of acetates in cigarette tobacco using solid-phase microextraction and gas chromatography-mass spectrometry.

A method incorporating solid-phase microextraction (SPME) and gas chromatography-mass spectrometry for the headspace analysis of selected volatile organic compounds present in cigarette tobacco is developed and evaluated. Quantitative information on methyl, ethyl, n-propyl, isopropyl, isopropenyl, vinyl, and butyl acetates present in 29 different flavor variants (full, light, and ultra-light) of the top ten selling brands in the United States is presented. The concentrations of the various acetate analytes range from the low nanaogram to microgram levels per cigarette. Clear differences are observed in the concentrations of various acetates when comparing the levels in brands from different manufacturers. The SPME technique provides a method that allows high sample throughput, requires little sample preparation, and yields useful analytical information. High precision is obtained on multiple measurements of cigarettes from an individual pack, but lower precision levels are observed in general when comparing results obtained on the analysis of cigarettes from different packs of the same brand. The higher pack-to-pack variations may be due in part to product aging with a proportionate amount of evaporative loss of the relatively volatile acetates.

An assessment of human performance in stabbing.

Stab-resistant body armour is now becoming a standard item of equipment for police officers in the United Kingdom. In the UK these are usually required to have a stab resistance as specified by the Police Scientific Development Branch KR42 standard [G. Parker, PSDB Stab Resistant Body Armour Test Procedure, Police Scientific Development Branch, Publication No 10/93, 1993]. There are several other test standards, all of which specify that body armour must resist penetration by a specific blade type delivered at a specific energy level or range of levels. However, the actual range of energy levels specified varies over almost an order of magnitude and the basis for these levels is not clearly defined. This paper describes tests to determine the energy range and characteristics of stabbing actions that might be directed against stab resistant body armour by an assailant. The energy and velocity that can be achieved in stabbing actions has been determined for a number of sample populations. Volunteers were asked to stab a target using an instrumented knife that measured the axial force and acceleration during the stabbing. The maximum energy obtained in underarm stabbing actions was 64 J whilst overarm stabbing actions could produce 115 J. The loads produced on contact with the target often approached 1000 N.

Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry.

A model experiment for the 'on-line' screening of substrate libraries by enzymes using combinatorial libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed. The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and component of a H-gamma-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored. It shows the feasibility of 'two-dimensional' screening of substrate libraries by ESI-FTICR mass spectrometry.

Analysis of combinatorial libraries using electrospray Fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization coupled with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to provide information about complete combinatorial libraries of small peptides containing 10(3)-10(4) components. The fidelity of attempted synthesis steps can be ascertained rapidly, and, when the extremely high resolution FTICR mass spectra are combined with appropriate computer simulation, both diversity and degeneracy of the libraries as synthesized can be assessed.

Pulsed-gas glow discharge for ultrahigh mass resolution measurements with fourier transform ion cyclotron resonance mass spectrometry.

A new pulsed-gas glow discharge (GD) source has been developed for use with an external ion source Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. With pulsed argon gas introduction into the GD source, the gas load and pressure in the mass analyzer region were greatly reduced; this resulted in improved mass resolution. Mass resolution of greater than 1 450 000 (fwhm) has been achieved for Cu(+) ions from a brass sample, the highest reported for any type of GD mass spectrometer. The pulsed-gas GD source promises analytical usefulness for ultrahigh resolution measurements in GD mass spectrometry.

Internal glow discharge-fourier transform ion cyclotron resonance mass spectrometry.

A glow discharge (GD) ion source has been developed to work within the high magnetic field of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Characterization of this source revealed that the optimum operating voltage, pressure, and current are significantly lower than those for normal glow discharges. The sputter rate was lowered to 1/30th of that found with a normal glow discharge source operated external to the high magnetic field region. Operation of the GD source closer to the FTICR analyzer cell than with previous experimental designs resulted in improved ion transport efficiency. Preliminary results from this internal GD source have established detection limits in the low parts per million range for selected elemental species.