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BACKGROUND: Whole-brain radiotherapy (WBRT) in patients with brain metastases (BM) is associated with neurocognitive decline. Given its crucial role in learning and memory, efforts to mitigate this toxicity have mostly focused on sparing radiation to the hippocampus. We hypothesized that BM are not evenly distributed across the brain and that several additional areas may be avoided in WBRT based on a low risk of developing BM. METHODS: We contoured 2757 lesions in a large, single-institution database of patients with newly diagnosed BM. BM centroids were mapped onto a standard brain atlas of 55 anatomic subunits and the observed percentage of BM was compared with what would be expected based on that region’s volume. A region of interest (ROI) analysis was performed in a validation cohort of patients from 2 independent institutions using equivalence and one-sample hypothesis tests. RESULTS: The brainstem and bilateral thalami, hippocampi, parahippocampal gyri, amygdala, and temporal poles had a cumulative risk of harboring a BM centroid of 4.83% in the initial cohort. This ROI was tested in 157 patients from the validation cohort and was found to have a 4.1% risk of developing BM, which was statistically equivalent between the 2 groups (P < 1 × 10(–6), upper bound). CONCLUSION: Several critical brain structures are at a low risk of developing BM. A risk-adapted approach to WBRT is worthy of further investigation and may mitigate the toxicities of conventional radiation.

Microbacterium foliorum is a naturally occurring bacteria in cruciferous vegetables and ripened cheese. The safety of M. foliorum SYG27B-MF has been assessed in both acute and subchronic studies and a battery of mutagenicity and clastogenicity tests. In a single dose acute study, the LD50 of M. foliorum SYG27B-MF was greater than 3 g/kg bw or 5.1 × 1016 colony forming unit (CFU)/kg bw, the highest dose tested. In a 90-day subchronic toxicity study in 80 Sprague-Dawley rats, no animals died and there were no treatment-related abnormalities at doses of 0, 500, 1000, or 2000 mg/kg bw. In a 90-day repeated toxicity test, the no-observed-adverse-effect level (NOAEL) M. foliorum SYG27B-MF was 2000 mg/kg/day or 3.4 × 1016 CFU/kg bw/day, the highest level tested. A mutagenicity study using reverse bacterial mutation tests and a genotoxicity study employing cultured hamster ovarian fibroblasts (CHO-K1) cell showed that M. foliorum SYG27B-MF was not mutagenic or clastogenic in the presence or absence metabolic activation. In an in vivo mouse micronucleus assay, M. foliorum SYG27B-MF did not induce did not induce micronuclei formation in the bone marrow cells of mice, indicating that it is non-clastogenic. The results from these studies support the safety of M. foliorum SYG27B-MF for use as a production organism for human food ingredients.

The safety of 3′-sialyllactose (3′-SL) sodium salt was evaluated by testing for gene mutations, in vivo and in vitro clastogenic activity, and animal toxicity in beagle dogs and rats. The results of all mutagenicity and genotoxicity tests were negative, indicating that 3′-SL does not have any mutagenic or clastogenic potential. The mean lethal dose (LD50) of 3′-SL sodium salt was well above 20 g/kg body weight (bw) in rats. A dose escalation acute toxicity study in Beagle dogs also indicated no treatment-related abnormalities. Subsequent 28-day and 90-day toxicity studies in Sprague- Dawley (SD) rats involved dietary exposure to 500, 1,000, and 2000 mg/kg bw of 3′-SL sodium salt and a water (vehicle) control. There were no treatment-related abnormalities on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, organ weights, relative organ weights, urinalysis parameters, or necropsy and histopathological findings. The No Observed Adverse Effect Level (NOAEL) of 3′-SL sodium salt was determined to be higher than 2000 mg/kg bw/day in an oral subchronic toxicity study in rats, indicating that the substance is an ordinary carbohydrate with the lowest toxicity rating. Results confirm that 3′-SL sodium salt has a toxicity profile similar to other non-digestible carbohydrates and naturally occurring human milk oligosaccharides (HMOs) and support its safety for human consumption in foods.

In this study, Beagle dogs were administered xylooligosaccharide (XOS, CAS # 87099-0) at doses of 0, 1250, 2500, and 5000 mg/kg/day by oral gavage for 26 weeks. A 4-week recovery period was added to observe delayed or reversible toxicity. Measurements included body weight, food consumption, clinical observations, temperature, electrocardiogram (ECG), urinalysis, blood chemistry, hematology, organ weight, gross necropsy, and histopathological examination. Except for transient diarrhea or vomiting, no treatment-related adverse effects were noted. In the mid-dose groups, transitional diarrhea was observed in the initial 1-2 weeks. In the high-dose groups, diarrhea and/or vomiting were observed episodically over the duration of treatment. However, they disappeared after XOS was withdrawn in the recovery period. Although there was a tendency toward less weight gain in the high-dose group animal group, this is typical in animals and humans fed non-digestible carbohydrates. This chronic toxicity study demonstrated that the no observed adverse effect level (NOAEL) of XOS is 2500 mg/kg body weight (BW)/day. Based on body surface area (conversion factor of 0.54 for dogs to human), this corresponds to daily doses of 1350 mg/kg BW or 81-108 g XOS in human adults weighing 60-80 kg.

Background: 20(S)-ginsenoside-Rg3 (C42H72O13), a natural triterpenoid saponin, is extracted from red ginseng. The increasing use of 20(S)-ginsenoside Rg3 has raised product safety concerns. Methods: In acute toxicity, 20(S)-ginsenoside Rg3 was singly and orally administrated to Kunming mice and Sprague–Dawley (SD) rats at the maximum doses of 1600 mg/kg and 800 mg/kg, respectively. In the 26-week toxicity study, we used repeated oral administration of 20(S)-ginsenoside Rg3 in SD rats over 26 weeks at doses of 0, 20, 60, or 180 mg/kg. Moreover, a 4-week recovery period was scheduled to observe the persistence, delayed occurrence, and reversibility of toxic effects. Results: The result of acute toxicity shows that oral administration of 20(S)-ginsenoside Rg3 to mice and rats did not induce mortality or toxicity up to 1600 and 800 mg/kg, respectively. During a 26-week administration period and a 4-week withdrawal period (recovery period), there were no significant differences in clinical signs, body weight, food consumption, urinalysis parameters, biochemical and hematological values, or histopathological findings. Conclusion: The mean oral lethal dose (LD50) of 20(S)-ginsenoside Rg3, in acute toxicity, is above 1600 mg/kg and 800 mg/kg in mice and rats, respectively. In a repeated-dose 26-week oral toxicity study, the no-observed-adverse-effect level for female and male SD rats was 180 mg/kg. Keywords: Oral toxicity study, Preclinical safety evaluation, Rats, Red ginseng, 20(S)-ginsenoside Rg3

We performed a series of toxicity studies on the safety of 6′-sialyllactose (6′-SL) sodium salt as a food ingredient. 6′-SL sodium salt, up to a maximum dose of 5000 μg/plate, did not increase the number of revertant colonies in five strains of Salmonella typhimurium in the presence or absence of S9 metabolic activation. A chromosomal aberration assay (using Chinese hamster lung cells) found no clastogenic effects at any concentration of 6′-SL sodium salt in the presence or absence of S9 metabolic activation. An in vivo bone marrow micronucleus test in Kunming mice showed no clastogenic activities with 6′-SL sodium salt doses up to 2000 mg/kg body weight (bw). In an acute toxicity study, the mean lethal dose of 6′-SL sodium salt was greater than 20 g/kg bw in rats. In a 13-week subchronic toxicity investigation, no effects were found at doses up to 5.0 g/kg bw of 6′-SL sodium salt in food consumption, body weight, clinical signs, blood biochemistry and hematology, urinalysis, or ophthalmic and histological macroscopic examination of organs. The no-observed-adverse-effect level (NOAEL) was 5.0 g/kg bw/day in rats.

Clinopodium chinense (Benth.) O. Ktze (Labiatae), known as 'Duanxueliu' in the Chinese Pharmacopoeia, has been widely used as a traditional Chinese medicine for the treatment of hemorrhagic disease. Total flavonoids from Clinopodium chinense (Benth.) O. Ktze (TFCC), the most active ingredient, possess a variety of properties, such as antioxygenation. Until now, evidence-based toxicity data on TFCC has been limited. This study evaluated the acute (in mice and rat) and the 28-day repeated-dose (in rat) toxicity study of TFCC, respectively. In acute study, oral administration of TFCC to rats and mice did not induce toxicity or mortality up to the maximum doses of 4000 and 5000 mg/kg, respectively. In subacute toxicity study, we administered TFCC at daily doses of 70, 210, and 630 mg/kg for 4 consecutive weeks to rats via gavage. We observed no changes in food consumption, water intake, body weight, chemistry and hematological parameters, organ weight, gross pathology or histopathology. No animals from any group died. These findings indicate that TFCC is relatively nontoxic, and provide practical guidance for selecting a safe dose for further investigation of TFCC in animal studies or clinical trials.

Astilbin is an active flavonoid compound isolated from Rhizoma Smilacis Glabrae. It has been widely used as an anti-hepatic, anti-arthritic, and anti-renal injury agent. However, its safety has not yet been established. The objective of this study was to evaluate 4-week repeated oral toxicity and genotoxicity of astilbin. We examined oral toxicity in Sprague-Dawley rats after daily oral administration of astilbin at 50, 150, and 500 mg/kg for 4 weeks. Negative control animals received the same volume of the solvent. Astilbin administration did not lead to death, body weight gain, food consumption, or adverse events. There were no significant differences in toxicity between the astilbin and control group; we observed no toxic effects on hematological or urinalysis parameters, biochemical values, organ weight, or histopathological findings. We assessed the genotoxicity of astilbin with the Ames test (TA97a, TA98, TA100, TA102, and TA1535), chromosomal aberration assay (using Chinese hamster ovary cells), and mammalian micronucleus test (in mice). We found no genotoxicity in any tested strains. The no-observed-adverse-effect level (NOAEL) for astilbin in the 4-week repeated oral toxicity study in rats was greater than 500 mg/kg body weight/day, regardless of gender. Results also suggested that astilbin does not have genotoxicity potential.

Intakes of ready-to-eat cereal (RTEC) have been inversely associated with risk factors of chronic diseases such as cardiovascular disease (CVD), type 2 diabetes, and certain cancers; however, their relations with total and cause-specific mortality remain unclear.To prospectively assess the associations of RTEC intakes with all causes and disease-specific mortality risk.The study included 367,442 participants from the prospective National Institutes of Health (NIH)-AARP Diet and Health Study. Intakes of RTEC were assessed at baseline.Over an average of 14 years of follow-up, 46,067 deaths were documented. Consumption of RTEC was significantly associated with reduced risk of mortality from all-cause mortality and death from CVD, diabetes, all cancer, and digestive cancer (all p for trend0.05). In multivariate models, compared to nonconsumers of RTEC, those in the highest intake of RTEC had a 15% lower risk of all-cause mortality and 10%-30% lower risk of disease-specific mortality. Within RTEC consumers, total fiber intakes were associated with reduced risk of mortality from all-cause mortality and deaths from CVD, all cancer, digestive cancer, and respiratory disease (all p for trend0.005).Consumption of RTEC was associated with reduced risk of all-cause mortality and mortality from specific diseases such as CVD, diabetes, and cancer. This association may be mediated via greater fiber intake.

Intakes of whole grains and cereal fiber have been inversely associated with the risk of chronic diseases; however, their relation with total and disease-specific mortality remain unclear. We aimed to prospectively assess the association of whole grains and cereal fiber intake with all causes and cause-specific mortality.The study included 367,442 participants from the prospective NIH-AARP Diet and Health Study (enrolled in 1995 and followed through 2009). Participants with cancer, heart disease, stroke, diabetes, and self-reported end-stage renal disease at baseline were excluded.Over an average of 14 years of follow-up, a total of 46,067 deaths were documented. Consumption of whole grains were inversely associated with risk of all-cause mortality and death from cancer, cardiovascular disease (CVD), diabetes, respiratory disease, infections, and other causes. In multivariable models, as compared with individuals with the lowest intakes, those in the highest intake of whole grains had a 17% (95% CI, 14-19%) lower risk of all-cause mortality and 11-48% lower risk of disease-specific mortality (all P for trend0.023); those in the highest intake of cereal fiber had a 19% (95% CI, 16-21%) lower risk of all-cause mortality and 15-34% lower risk of disease-specific mortality (all P for trend0.005). When cereal fiber was further adjusted, the associations of whole grains with death from CVD, respiratory disease and infections became not significant; the associations with all-cause mortality and death from cancer and diabetes were attenuated but remained significant (P for trend0.029).Consumption of whole grains and cereal fiber was inversely associated with reduced total and cause-specific mortality. Our data suggest cereal fiber is one potentially protective component.

The objective of this study was to estimate the independent associations between intake of phosphorus (P) and bone health parameters such as bone mineral content (BMC) and bone mineral density (BMD). It provides odds ratio (OR) of osteoporosis with quartiles of P intake adjusted for covariates (i.e., age, gender, BMI, and consumption of calcium (Ca), protein, total dairy foods, and vitamin D as well as intakes of supplemental Ca, vitamin D, and multivitamins/minerals). Data came from males and females aged 13–99 years who participated in the 2005–2010 National Health and Nutrition Examination Survey (NHANES). Analyses showed that higher P intake was associated with higher Ca intake, and that dietary Ca:P ratios (0.51-0.62, with a mean of 0.60 for adults) were adequate in all age/gender groups. High intake of P was positively associated with BMC in female teenagers (Q4 vs. Q1: BMC, 30.9 ± 1.1 vs. 29.0 ± 0.5 g, P = 0.001). It was also positively associated with BMC and BMD as well as reduced risk of osteoporosis in adults >20 years of age (Q4 vs. Q1: OR of osteoporosis, 0.55; 95% confidence interval [CI], 0.39- 0.79; P = 0.001; BMC, 37.5 ± 0.4 vs. 36.70 ± 0.3 g, P < 0.01; BMD, 0.986 ± 0.004 vs. 0.966 ± 0.005 g/cm2, P < 0.05). The data suggest that high intake of P has no adverse effect on bone metabolism in populations with adequate Ca intake, and that it is also associated with positive bone parameters in some age/gender groups.

To evaluate the potential toxicity of refined arachidonic acid-rich oil (RAO) derived from Mortierella alpina (M. alpina) XM027, we performed a 90-day subchronic study in F1 Sprague Dawley (SD) rats. This study was preceded by a 4-week pretreatment period of parental (F0) rats and exposure of the F0 dams throughout mating, gestation, and lactation. The results indicated that RAO, at dose levels of 0.5%, 1.5%, and 5%, did not affect either reproductive performance of the parental rats, or any characteristics of the pups. In the subchronic study with the offspring (F1) rats, no treatment related abnormalities were observed. In summary, no observable adverse effect level (NOAEL) in this study was placed at 5% RAO, the highest level tested. This level corresponds to approximately 3750 mg/kg in F0 females, 2850 mg/kg in F0 males, 4850 mg/kg in F1 females, and 4480 mg/kg in F1 males.

Some new sufficient conditions for determining global asymptotic stability of an equilibrium point under control saturation are presented. For an open-loop stable system, we show that there exists a class of linear time invariant state feedback matrices which globally stablizes an equilibrium under control saturation. In addition, we show that for open-loop unstable plants, an equilibrium can never be globally stabilized using a certain class of saturated linear time invariant state feedback. Finally, we extend the stability results to nonlinear systems whose nonlinearities are globally Lipschitz.

DNA vaccines are cost-effective and versatile, though intracellular delivery has been challenging in humans. Alternative delivery modalities such as electroporation have demonstrated improved immune responses, but are painful. In this single-center, double-blind, medical device trial, we evaluated the safety and tolerability of Easy Vax™ dermal electroporation system, alone (without DNA) in healthy adults. Three randomized protocol doses were administered to 10 subjects (80% white, 60% female, mean age: 32.1 years) in each of two areas (total of six doses). Two subjects complained of shooting pain, burning and/or tingling when doses were administered to the forearm region, but not the lateral deltoid regions. Subsequent doses for the remaining eight subjects were restricted to the deltoid regions only. Tolerability pain scores never exceeded 3 of 10 in the 11-Point Pain Rating scale, and 12 of 100 in the Visual Analog Scale (VAS), and lower in follow-up evaluations (P < 0.0001), with no significant difference between the three dosing protocols. Electrical properties of the skin, measured automatically by the device, showed no correlation between pain intensity and skin conductance. In conclusion, the Easy Vax™ electroporation device is safe and well tolerated when administered over the lateral deltoid skin regions in healthy volunteers.

The landmark paper recently published by Croucher et al. (2011) reports the draft genome sequencing of 240 Streptococcus pneumoniae isolates and demonstrates the power of comparative genomics in the epidemiological and phylogenetic analysis of bacterial pathogens. Until recently, the focus of bacterial genome projects has been individual strains or small numbers of clinical isolates. However, with the advent of next generation sequencing platforms such as Roche 454 and Illumina, bacterial genomes can be inexpensively and rapidly sequenced. The first large-scale genomic sequencing project to utilize these technologies for a similar purpose (also by the Bentley group) analyzed 63 clonally related methicillin-resistant Staphylococcus aureus isolates (Harris et al., 2010). Croucher et al. (2011) analyzed temporally and geographically diverse isolates of a single S. pneumoniae lineage, the multidrug resistant Spain 23F ST81 (PMEN1) pandemic clone, to provide insights into the microevolution of this important pathogen. Streptococcus pneumoniae is a major worldwide public health concern. It is responsible for an estimated 1.6 million deaths each year, half of which are children under the age of five (World Health Organization, 2003). Efforts to limit pneumococcal disease have focused primarily on antibiotic intervention and vaccination. However, after initial, and often dramatic, successes the effectiveness of these measures has consistently been overcome by the extremely adaptable and resilient pneumococcus. S. pneumoniae is naturally competent and readily incorporates DNA fragments derived from pneumococcal and non-pneumococcal bacterial species into its genome by homologous recombination. High levels of recombination have resulted in extreme diversity within pneumococcal populations from which variant genotypes can rapidly emerge in the presence of changing selective pressures. The primary pressures outside of natural immunity are antibiotics and pneumococcal conjugate polysaccharide vaccines. Resistant and multidrug resistant pneumococci have emerged since the late 1960s and now severely limit treatment options for pneumococcal diseases (Reinert, 2009). The incidences of invasive pneumococcal disease (IPD) and antibiotic resistant pneumococci decreased dramatically following the introduction of the heptavalent conjugate vaccine PCV7 in 2000 (Dagan, 2009). However, the incidence of IPD and resistance is again rising due to the emergence of multidrug resistant, non-vaccine “replacement” serotypes. Croucher et al. (2011) documented the “escape” of the PMEN1 clone from PCV7 by indentifying several independent horizontal transfer events that resulted in serotype switching of PMEN1 strains from vaccine serotype 23F to non-vaccine serotypes. This suggested strong selective pressures for capsule switch variants exerted by the PCV7-immunized human immune system. Evidence for the selective pressures exerted by antibiotics was also reported. Analysis of integrative and conjugative elements revealed rapid degeneration of such elements but with consistent retention of determinants of resistance to antibiotics commonly used in treatment of upper respiratory infections (e.g., β-lactams and macrolides). It should be expected that the pneumococcus will adapt quickly to new antibiotic pressures and vaccines such as the 13-valent PCV vaccine (PCV13) introduced in the United States early 2010. This emphasizes the importance of surveillance and epidemiology to track the emergence of new clones and variations. In addition to overcoming clinical interventions, the plasticity of the pneumococcal genome has confounded epidemiological analyses. Multilocus sequence typing (MLST) is currently the preferred method for comparing relatedness of isolates within a given bacterial species, and relies on sequence comparison of seven highly conserved alleles. However, the high rate of recombination in pneumococci greatly reduces the resolution of MLST-derived pneumococcal phylogenies due to horizontal acquisition of single nucleotide polymorphisms (SNPs) by recombination events involving MLST alleles. Croucher et al. (2011) avoided this problem by removing horizontally inherited sequences from the phylogenetic analyses. Recombination events were identified by comparison of each genome to a Spain 23F ST81 reference genome (ATCC 700669; Croucher et al., 2009). The phylogeny constructed using only vertically inherited SNPs proved to be a superior estimation of the evolution of the clone based on the correlation between the distance from the root of the tree and the date of isolation of each strain. By comparing the more accurate phylogeny to the dates and locations of isolation, the authors were able to pinpoint the likely origins of different clades and to discern information about the geographic spread of the clone. Future studies utilizing large-scale genomic comparison in S. pneumoniae will likely focus on the influence of antibiotic and vaccine pressures and on the evolution of pneumococcal populations. Whereas Croucher et al. (2011) utilized “deep” sequencing into a single lineage, “wide” genomic analysis of isolates that encompass entire pneumococcal populations will provide insights into the evolution and interactions of multiple lineages. In addition, it will elucidate the effects of localized factors such as host population genetic composition and confounding health and socioeconomic factors. In particular, it will clarify the effects of vaccine and antibiotic pressures on the population and identify candidate lineages for deep sequencing. Sequencing technologies have also enabled analysis of entire bacterial populations in individual niches, such as the Human Microbiome Project (HMP). The HMP attempts to elucidate the composition and role of the microbiota that inhabit the human body. Such studies in the nasopharynx will provide a better understanding of the role of pneumococcal carriage in invasive disease as well as inter- and intra-species horizontal gene transfer in S. pneumoniae. Similarly, study of the evolution of pneumococcal isolates within a single host will help define risks of acquired antibiotic resistance in the presence of antibiotic therapy and other interventions. Current and next generation technologies will be invaluable for epidemiologists and microbiologists studying non-pneumococcal pathogens, and commensals. Large-scale genomic analyses may lead to identification of novel therapeutic agents and new vaccine targets. Croucher et al. (2011) are one of the first groups to realize the full potential of the developments in DNA sequencing technologies that powered these large scale comparative genomic projects. This report signals the beginning of an era of discovery that will lead to comprehensive understanding of bacterial evolution, will revolutionize epidemiology, and allow the development of more effective and longer lasting strategies to treat infectious diseases.