Studies have found that antipsychotics and antidepressants are associated with weight gain and obesity, particularly among women and some minority groups. Incarcerated populations  have a high prevalence of mental health problems and 15 % of offenders have been prescribed medications. Despite rates of antidepressant and antipsychotic use, investigations of weight gain and obesity in regard to these agents seldom have included offenders.This retrospective descriptive study (2005–2011) was conducted with a Department of Corrections in the east south central United States to investigate the relationship between antidepressant and antipsychotic agents, weight gain, obesity and race or gender differences. We sampled adult offenders who had an active record, at least two weight observations and height data. Offenders were classified into one of four mutually exclusive groups depending upon the type of medication they were prescribed: antidepressants, antipsychotics, other medications or no pharmacotherapy.p < .001 and p = .019, respectively. Women in the antidepressant group gained 6.4 kg compared to 2.0 kg for men, which was significant (p = .007). Although women in the antipsychotic group gained 8.8 kg compared to 1.6 kg for men, the finding was not significant (p = .122). Surprisingly, there were no significant differences in weight gain between African Americans and Whites in regard to antidepressants (p = .336) or antipsychotic agents (p = .335).The sample population for this study was 2728, which was 25.2 % of the total population. The population not on pharmacotherapy had the lowest baseline obesity rate (31.7 %) compared to offenders prescribed antipsychotics (43.6 %), antidepressants (43.6 %) or other medications (45.1 %). Offenders who were prescribed antidepressants or antipsychotics gained weight that was significantly different from zero, This study found that women and men offenders prescribed antidepressant or antipsychotic agents gained weight during their incarceration. Women prescribed antidepressants gained significantly more weight than men. However, there was no significant difference in weight gain between African Americans and Whites. Results suggest further investigation is needed to understand the effect of medication history, metabolic syndrome and to explain gender disparities. Obesity risk factors are complex and multifaceted, comprised of behaviors related to physical inactivity and excess caloric intake, as well as the environment where people live and social conditions, such as education and poverty CDC . Along wThe prevalence of obesity and weight gain are not distributed proportionately across populations, especially in regard to gender and race  also are at risk for disproportionate rates of obesity   gain more weight during their incarceration compared to the population that has no history of being prescribed these type of agents .Women who have ever been prescribed antidepressants or antipsychotics gain more weight than men.African Americans who have ever been prescribed antidepressants or antipsychotics gain more weight than Whites.There are gender and race differences in regard to weight gain for patients who have ever been prescribed either antidepressants or antipsychotics.This descriptive study investigated offenders in a state department of corrections to explain the relationship among changes in weight, psychotropic agents  and incarceration. The primary goal of this study was to investigate the effect that some psychotropic agents have on changes in weight. We hypothesized the following:This retrospective descriptive study was approved by an institutional review board (IRB), Protocol number: 10-0382F2L, at an academic health center and was conducted for 2005–2011 in collaboration with a Department of Corrections (DOC) in the east south central region of the United States.The DOC for this study had an electronic health record (EHR), from which all health data  were extracted. Clinic staff created an electronic record for all offenders upon their arrival at the DOC reception center, an assessment facility where everyone received a complete physical, mental health and dental examination. The DOC also used an offender management system (OMS), an electronic tracking and case management system, which collected demographic, social, criminal record (offense), sentence and parole data. The offender management record was created electronically and managed by correctional officers and case managers.The data extraction criterion for the EHR and OMS was active records  between June 1, 2005 and December 31, 2010. The date range was determined by the implementation of the DOC’s EHR, which occurred in 2005. All adult offenders (men and women) were eligible for inclusion. Offenders were excluded if they did not have at least two weight observations, a baseline and post baseline value (last recorded weight), and if they did not belong to a mutually exclusive medication group. Offenders also were excluded if there were no valid height data and their length of incarceration was not greater than zero. Offenders who met the inclusion criteria were assigned to a medication group based on pharmacy data identifying whether or not an offender had been prescribed an antidepressant or antipsychotic. These data allowed us to create four mutually exclusive groups: antidepressants, antipsychotics, other medications or no pharmacotherapy.Health record data for this study were primarily structured . Prescriptions were generated electronically using the National Drug Code (NDC). Diagnoses were entered using International Statistical Classification of Diseases (ICD-9) and supplemented by the Systematized Nomenclature of Medicine (SNOMED). The structured nature of prescriptions and diagnoses provided a degree of standardization. Clinic staff, primarily nurses, entered height and weight data. Data from the OMS were primarily entered by correctional officers into discreet fields. Race, ethnicity and gender were pre-populated based on court records.2). The calculated BMIs were classified using the World Health Organization’s (WHO) cut-off points , were not available in the extracted data; BMI was calculated as weight in kilograms (kg) divided by height in meters squared  was computed to evaluate whether or not populations have different proportions and odds ratios were computed to determine if the four mutually exclusive medication groups  differed in regard to exposure (medication use) and response (changes in weight). Differences in baseline and last weight observation (end of study) were examined using paired t-test to determine if the different groups on average gained weight that was statistically significant from zero. The paired t-test was performed for each group. Group comparisons for race and gender were made using the Wilcoxon rank-sum test. Comparisons for race were limited to African Americans and Whites, since there were too few other race and ethnic populations for meaningful analyses. The Kruskal-Wallis test was computed to evaluate differences among the four medication groups. To adjust for multiple comparisons and the increased probability of a Type I error , we performed the Bonferroni correction, which evaluates the significance of pairwise comparisons using an adjusted alpha .SAS® 9.4  was used to conduct statistical tests. Frequencies and percentages were conducted for nominal and categorical variables, such as race, gender, BMI classification, and medications. Means and standard deviations (SD) were calculated for continuous variables, including BMI, and percent weight change, length of time between weight observations. Chi-square (χThis study sampled 2728 offenders (25.2 %) out of a population of 10,841 who met the inclusion criteria . A large percent of the population was excluded for the following reasons: 1) medication groups were mutually exclusive; 2) offenders without chronic diseases did not report to clinics regularly and did not always have multiple weight observations; 3) patients primarily seen for mental health issues did not have vital statistics recorded in the health record on a consistent basis; 4) a segment of the population had not been incarcerated long enough to have multiple weight values.Despite excluding a large percent of the population, African Americans and offenders prescribed antidepressants or antipsychotics were overrepresented in the study sample; however, the percent of women offenders was less than the total population Table . Althougp = .353). The three groups also did not have significantly different last observed BMI (p = .913). Further, when antipsychotics (first generation and second generation) were compared, their baseline BMI was not significantly different (p = .860); last observed BMI also was not significantly different (p = .795) between the two subgroups. Table p = .204). When the length of time between baseline and last observed BMI for first generation antipsychotics (FGA) and second generation antipsychotics (SGA) were compared (Table p = .143).Table There were few offenders entering corrections who had a BMI classified as normal or underweight, as shown in Table As Table t-test was performed for all medications groups to indicate whether or not offenders in their respective group had changes in weight that was significantly different from zero. The test indicated that last observed weight was greater than baseline for the population prescribed antidepressants (p < .001), antipsychotics (p = .019), other medications (p = .018) and the no pharmacotherapy group (p < .001). In regard to the antipsychotic subgroups, changes in baseline and last observed weight was only significant for the population prescribed SGA (p = .033); FGA had a p-value = .209.The paired p < .001 and p = .001, respectively, as did African Americans and Whites, p = .030 and p < .001, respectively. Although women and men gained weight while taking antipsychotics, changes between baseline and last weight observed were not significant . African Americans and Whites, who were prescribed antipsychotics, also gained weight, but the increase was not significantly different from zero .Women and men, who were taking antidepressants, had weight gain significantly different from zero p < .001) in regard to medically significant weight gain. Larger proportions of patients taking antidepressants or antipsychotics  gained medically significant weight compared to those on other medications or no pharmacotherapy (22.6 % for both groups). There was no significant difference in proportions (p = .212) for medically significant weight gain between patients prescribed FGA or SGA.The four medication groups also had significantly different proportions  across the four medication groups. Weight change, percent weight change and change in BMI are highly collinear, given that all the variables are derived from weight observations and evidenced by the similarity in p-values; thus, subsequent analyses focus on weight change.Table Accounting for multiple comparisons , we performed a Bonferroni correction to identify which groups had significantly different weight changes from one another Table . The tesp = .448).When the antipsychotic subgroup was examined, offenders prescribed SGA gained 1.5 kg more weight compared to the FGA group Table , howeverp = .534). In regard to antipsychotics, African Americans compared to Whites gained more weight , but this differences was not significant (p = .157). There were no significant weight change differences between African Americans and Whites when the antipsychotic subgroup was examined. African Americans in the FGA group gained 2.1 kg compared to 1.3 kg for Whites (p = .313). In regard to SGA, African Americans also gained more weight than Whites , p = .355. While there were a few significant differences between African Americans and Whites in regard to length of time between baseline and last observed weight , the difference for the antidepressant and FGA groups were 4 and 6 months, respectively. There were no race differences for length of time between baseline and last observed weight for other medication groups.African Americans, who were prescribed antidepressants, on average, gained the same weight (2.6 kg) as Whites taking the same class of medication, which was not significant (p = .027), the difference was 6 months (Table The Wilcoxon two-sample test indicated that gender was significant (See Table hs Table .Table 8Ap = .423), even though women prescribed antipsychotics gained the most weight across groups. Further, women on FGA gained more weight than women taking SGA ; however, this differences was not significant (p = 1.0). There were no significant differences for weight change (p = .122) within men who were prescribed antidepressants (2.0 kg), antipsychotics (1.6 kg), other medications (0.6 kg) or no pharmacotherapy (1.3 kg). FGA and SGA differences for changes in weight for men also were not significant (p = .444), even though men gained more weight on SGA than men on FGA .Within the population of women offenders, there were no significant differences among women who were taking antidepressants (6.4 kg), antipsychotics (8.8 kg), other medications (4.3 kg) or no pharmacotherapy (3.7 kg) in regard to changes in weight (This study found that women and men offenders who were prescribed antidepressants or antipsychotics (FGA and SGA) gained weight during their incarceration over similar lengths of time, but the antidepressant group had weight gain that was significantly different from patients taking other medications. Weight gain differences between the antidepressant and no pharmacotherapy groups approached the significance level. Further, there was no significant difference in weight gain between SGA and FGA, even though SGA resulted in greater weight gain than FGA. There were gender differences in regard to antidepressant use where women gained significantly more weight compared to men; however, gender was not significant for antipsychotics (FGA and SGA). Differences in weight changes between African Americans and Whites were not significant for any of the psychotropic agents.Our results are consistent with previous studies, primarily outside of corrections, which found an association between weight gain and antidepressants  and weight gain in corrections. There were several limitations for this study, principally, the lack of medication history for offenders during their incarceration. Studies investigating antidepressant and antipsychotic use have found dose (ÜÇOk & Gaebel This study focused on descriptively investigating the effect of antidepressant and antipsychotic induced weight gain with a focus on race and gender differences. The findings of this study suggest further investigation is needed to understand the effect of mental health severity, medication history, metabolic syndrome and to explain gender disparities. Multivariable analyses will be needed to adjust for the effects from confounding factors with appropriate collinearity procedure (Yoo et al.


Understanding the interaction between climate and biotic evolution is crucial for deciphering the sensitivity of life. An enigmatic mass extinction occurred in the deep oceans during the Mid Pleistocene, with a loss of over 100 species (20%) of sea floor calcareous foraminifera. An evolutionarily conservative group, benthic foraminifera often comprise >50% of eukaryote biomass on the deep-ocean floor. Here we test extinction hypotheses  in the Tasman Sea, using geochemistry and micropalaeontology, and find evidence from several globally distributed sites that the extinction was caused by a change in phytoplankton food source. Coccolithophore evolution may have enhanced the seasonal ‘bloom' nature of primary productivity and fundamentally shifted it towards a more intra-annually variable state at ∼0.8 Ma. Our results highlight intra-annual variability as a potential new consideration for Mid Pleistocene global biogeochemical climate models, and imply that deep-sea biota may be sensitive to future changes in productivity. The cause of a 20% decline in sea floor calcareous foraminifera species during the Mid-Pleistocene remains enigmatic. Here, the authors present new geochemical evidence, from the Tasman Sea that supports a change in phytoplankton food source as the primary driver. The Mid Pleistocene transition MPT, ∼1.2–0.6 Ma) was characterized by global cooling, glacial stage lengthening, changing ocean circulation and evolution of terrestrial and marine biota.2–0.6 Ma245667910471132−], related to corrosiveness), sea-surface temperature (SST) and productivity. We also generated records of phytoplankton assemblages from two sites to compare with a published South Pacific recordGephyrocapsa occurred at ∼0.8 Ma, coinciding with the largest decline in benthic foraminifera. We conclude that small Gephyrocapsa evolution may have enhanced intra-annual variability of primary productivity and carbon flux reaching much of the mid-low latitude ocean floor where the extinction was centred, and that the extinction group may have been unable to cope with long periods of seasonally reduced organic carbon flux during parts of the year.In this study, we address these hypotheses by examining the first records of extinction group abundance generated together with proxies for bottom-water temperature, carbonate saturation  Site 593 over the period ∼0.75–1 Ma, covering the benthic extinction . Site 597Strictocostella scharbergana and Siphonodosaria lepidula, which decline abruptly at 0.85 and 0.8 Ma, respectively (Previous low-resolution (∼22 kyr) benthic foraminiferal data from Site 593 ref. . Our datectively . DiffereUvigerina peregrina (see Methods), ranges from ∼3 to 8 °C (modern temperature is 4.5 °C), with warmer interglacials . Bottom-water Δ[CO32−] at Site 593, reconstructed from B/Ca of epifaunal Cibicidoides wuellerstorfi (see Methods), shows relatively low values ranging from 10 to 25 μmol kg−1, with highest values recorded during cooler glacials and some potential negative outliers before 0.9 Ma , and the extinction group abundance does not co-vary with Δ[CO32−] during the pre-extinction interval , shows significant variability over the study interval, ranging from 10 to 18 °C, with cooler temperatures during glacials after ∼0.95 Ma. Since modern SSTs in the Tasman Sea are tightly coupled to the position of the productive Subtropical Front, we anticipate that shifts to Subtropical Front position might have influenced the organic matter flux. Sedimentary chlorin  occurred in the SE AtlanticGephyrocapsa could have been implicated in the benthic extinction, we paired records of nannoplankton and foraminiferal assemblage data in the Tasman Sea (Gephyrocapsa (>90%) during the extinction interval at ∼0.83 Ma. Interestingly, the first step in the extinction at ∼0.96 Ma at Site 593  than others , such that there is diachroneity in the onset of this event; however, the crucial point is that all global records with coccolith data show high abundances of small Gephyrocapsa at ∼0.8 Ma. In summary, evidence for a global peak in small Gephyrocapsa at ∼0.8 Ma comes from sites in the SE AtlanticGephyrocapsa event remains unknown; however, it may have been an evolutionary adaptation25Gephyrocapsa lineage is thought to be relatively independent of temperature and nutrients, but dependent on light intensity and day lengthG. caribbeanica at ∼0.6 Ma has been linked to increased blooms because of a sustained reduction in orbital eccentricity affecting day length and light intensityOur new nannoplankton data , togetheGephyrocapsa event, its correlation with the extinction at ∼0.8 Ma provides a new piece of evidence when evaluating the cause of the benthic extinction. Correlation itself does not prove causation; however, as phytoplankton provide a food supply for marine benthos, how might this change in phytoplankton assemblages have had an impact on benthic foraminifera? We propose that the most plausible extinction mechanism would be a change towards a more enhanced variation in annual export production and delivery of organic carbon to the deep ocean9107Gephyrocapsa was similar to its modern descendent, Emiliania huxleyi, a seasonal bloom species that dominates mid-low latitude nannoplankton assemblages during the Holocene25Gephyrocapsa may represent an early adaptation to more bloom-type ecology within nannoplankton, where intra-annual export production changed. Modern  global productivity is dominantly annually uneven outside the oligotrophic ocean gyres  was cored on the Challenger Plateau of the Tasman Sea in the SW Pacific Ocean. The upper 393 m of recovered sediment is foraminiferal-bearing nannofossil ooze, hosting very abundant and visually well-preserved benthic foraminiferaerstorfi . The tunerstorfi ) is cleaet al.2 kyr−1) was calculated by using sediment dry bulk density , sedimentation rate  and number of specimens per g  as follows:Benthic foraminiferal analyses were carried out at the British Geological Survey, Keyworth, UK, and at the University of Leicester, UK, using an Olympus SZX10 binocular microscope. Seventy-eight sediment samples of ∼10 cc were oven dried at 40 °C and washed with deionized water over a 63-μm sieve to remove clays (at Durham University). The >63-μm fraction was then dried, and individual species of foraminifera were counted under a binocular microscope and transferred into cardboard reference slides. Residues were sieved into size fractions, and picked separately, according to a procedure outline in ref. Calcareous nannofossil species were analysed at the British Geological Survey, Keyworth, UK. A total of 33 smear slides were prepared from the central part of fresh core sediment samples and were analysed using a Zeiss cross-polarizing light microscope at × 1,000 magnification. A counting phase was performed to obtain a qualitative and quantitative description of the assemblages based on percentage abundances of each recognized taxon . NannofoG. oceanica, G. caribbeanica, G. muellerae recognized during this interval as G. margereliG. omega and small Gephyrocapsa . Other taxa are present within the assemblages recognized at generic, specific and subspecific levels. At the generic level, minor taxa are represented by the genres Syracosphaera, Pontosphaera, Reticulofenestra and Rhabdosphaera. At the specific level, minor taxa are represented by the species Pseudoemiliania lacunosa, R. asanoi, Helicosphaera carteri, H. hyalina and Umbilicoshpaera sibogae. Finally, Coccolithus pelagicus and Calcidiscus leptoporus were distinguished at subspecific and submorphotype levels on the basis of their coccolith length. Refs 40,4140C. pelagicus subspecies: C. pelagicus pelagicus, the cold form, and C. pelagicus braarudii, the temperate and upwelling waters formC. leptoporus morphotypes have been distinguished: C. leptoporus type small ; C. lepμm; refs ); and C. μm refs ).Separate original samples were freeze-dried, homogenized, and alkenones and chlorins (diagenetic transformation products of chlorophyll)2424n-hexane (for hydrocarbons), dichloromethane (for ketones) and methanol (for polar compounds). Alkenones were quantified by Thermo Scientific Trace 1310 gas chromatograph fitted with a flame ionization detector. Separation was achieved with a fused silica column (30 m × 0.25 mm inner diameter) coated with 0.25 μm of 5% phenyl methyl siloxane (HP-5MS) and using He as the carrier gas. After injection, the following oven temperature programme was used: 60–200 °C at 20 °C min−1, 200–320 °C at 6 °C min−1, and then held at 320 °C for 35 min. SST was calculated using the UK37′ indexAlkenones were isolated from the second aliquot of the lipid extract using silica column chromatography, eluting with C. wuellerstorfi from the >250-μm fraction of each sample were analysed using an IsoPrime dual inlet mass spectrometer plus Multiprep device at the NERC Isotope Geosciences Laboratory. δ18O is reported as per mille (‰) deviations of the isotopic ratios (18O/16O), and is calculated on the V-PDB scale using an internal laboratory standard that is routinely calibrated against NBS-19 standards  for B/Ca. All trace element sample preparation and analysis occurred at the Godwin Laboratory for Palaeoclimate Research, Cambridge University (Uvigerina2−1), which has been shown to be suitable for this site for the interval from 0.4–0 Ma  per sample of benthic foraminifera iversity . All ana Ma ref. .32−] (defined as the difference between [CO32−]in situ and [CO32−]saturation) was calculated from the B/Ca ratio of epifaunal foraminifera C. wuellerstorfi (in μmol mol−1) using the following calibrationBottom-water Δ[COThe data that support the findings of this study are available within the article and its How to cite this article: Kender, S. et al. Mid Pleistocene foraminiferal mass extinction coupled with phytoplankton evolution. Nat. Commun. 7:11970 doi: 10.1038/ncomms11970 (2016).Supplementary Figures 1-5, Supplementary Table 1 and Supplementary References.Cibicidoides wuellerstorfi oxygen isotope data between 0.4 and 1.1 Ma for DSDP Site 593.Foraminiferal census data for DSDP Site 593.Calcareous nannofossil census data for DSDP Site 593 and ODP Site 980.Sediment UK37' and chlorin data for DSDP Site 593.Benthic foraminiferal trace element data for DSDP Site 593.


Congenital prosopagnosia, the innate impairment in recognizing faces, is a very heterogeneous disorder with different phenotypical manifestations. To investigate the nature of prosopagnosia in more detail, we tested 16 prosopagnosics and 21 controls with an extended test battery addressing various aspects of face recognition. Our results show that prosopagnosics exhibited significant impairments in several face recognition tasks: impaired holistic processing (they were tested amongst others with the Cambridge Face Memory Test (CFMT)) as well as reduced processing of configural information of faces. This test battery also revealed some new findings. While controls recognized moving faces better than static faces, prosopagnosics did not exhibit this effect. Furthermore, prosopagnosics had significantly impaired gender recognition—which is shown on a groupwise level for the first time in our study. There was no difference between groups in the automatic extraction of face identity information or in object recognition as tested with the Cambridge Car Memory Test. In addition, a methodological analysis of the tests revealed reduced reliability for holistic face processing tests in prosopagnosics. To our knowledge, this is the first study to show that prosopagnosics showed a significantly reduced reliability coefficient  in the CFMT compared to the controls. We suggest that compensatory strategies employed by the prosopagnosics might be the cause for the vast variety of response patterns revealed by the reduced test reliability. This finding raises the question whether classical face tests measure the same perceptual processes in controls and prosopagnosics. Congenital prosopagnosia refers to the lifelong, innate impairment in identifying someone by his or her face (first case description by Two main aspects of face perception put faces apart from most other objects: (1). Faces are recognized at the individual level (identification); (2). They are processed holistically. While identification is a clear concept, what exactly is meant with the term “holistic processing” is not well defined and there are numerous controversies about the use of the terms holistic and configural processing e.g., . Here wepart-whole test  for prosopagnosics and 20.3 months (SD = 1.6) for controls. During the first session, participants performed the CFMT, test number 1, a surprise recognition test (number 3), and a similarity rating test (5). The second session included the CCMT, 2, the composite face test (4), a gender recognition test (6), and a facial motion advantage test (7). In both sessions, participants could take self-paced breaks between the experiments.The experiments were conducted in two sessions lying about 2 years apart: On average, 24.6 months  and Psychtoolbox . ParticiWe tested 16 congenital prosopagnosic participants (from now on referred to as “prosopagnosics”) and 21 control participants (“controls”) matched as closely as possible to the prosopagnosic participants in terms of age and sex see .Table 1All participants provided informed consent. All participants had normal or corrected-to-normal visual acuity, but we did not formally assess color perception, contrast sensitivity, stereoscopic vision or other neuropsychological measures or personality traits . As no brain imaging was available to exclude the presence of brain lesions, we relied on participants’ self-testimonies or parents’ testimonies. To provide an objective measure of face processing abilities and to maintain comparability with other studies, we tested all participants with the CFMT. Individual results and z-scores are given in The prosopagnosics were diagnosed by a semi-structured interview , approveAll controls were compensated with 8 Euro per hour. The controls did not participate in the full diagnostic interview but in a questionnaire and reported to have no problems in recognizing faces of their friends and family members.η2) and linear regressions were calculated with IBM SPSS statistics Version 20. T tests and their effect sizes Cohen’s d (d), Mann–Whitney U tests and tests’ internal consistency reliability coefficients were calculated with Matlab2011b.The description of the dependent variables is given for each test individually. All analyses were conducted with Matlab2011b (The MathWorks Inc.) and IBM SPSS statistics Version 20 (IBM Corp. Released 1 Formula (2)). Statistical difference between prosopagnosics’ and controls’ split-half reliability coefficients was calculated as statistical difference between correlation coefficients  of the test faces, while prosopagnosics scored 54.8%, (SD = 5.9). The difference between groups was significant  = 94.7, p < .001, η2 = .73), with prosopagnosics performing worse than controls.We calculated the overall recognition performance as the percentage of correctly recognized faces per participant. Prosopagnosics showed a significantly reduced face recognition ability compared to controls. This result reflects the impaired holistic face processing and face memory of prosopagnosic participants and replicates findings of many previous studies e.g., .The CCMT  is a tesDennett and colleagues found a significant correlation between the scores of their CCMT and participants’ general interest in cars and knowledge of car makes and models. Therefore, we ran an additional test for car expertise after completing the CCMT, to be able to account for this possible influence and correct the CCMT scores for car expertise.As a detailed description is given in the original study , we giveSixteen cars from the CCMT (four target and twelve distractor cars) were presented one after the other to the participants along with three answer choices of possible car makes and models see . ParticiThe car images in both tests had a visual angle of 5.7° horizontally and 11.4° vertically.SD = 12.9) of the cars, and prosopagnosics scored 75.1% (SD = 12.7). For the car expertise test, controls correctly identified 64.6% (SD = 16.1) of the car makes, and prosopagnosics scored 52.0% (SD = 19.9).The performance measure in both tasks was the percentage of correctly recognized cars per participant. F = 0.31, p = .58, η2 = .01). For the car expertise, test the control group exhibited significantly greater expertise in car models than the prosopagnosics  = 4.57, p = .04, η2 = .12). Therefore, we compared both groups’ CCMT scores while controlling for the car expertise. For this, we ran a linear regression with car expertise scores as predictor. The residuals of the regression did not differ significantly between groups  = 0.64, p = .43, η2 = .02), indicating that the CCMT scores do not differ between groups after controlling for car expertise.  = –0.33, p = .75, d = –0.11).)For the CCMT, there was no significant difference in scores between prosopagnosics and controls . In each test phase, we used 16 static images of the target actors  were depicted, each showing four different facial expressions. Both the exhibited expressions and the actors’ identities differed in both sets. The videos had a mean length of 2.7 s , each performing four different facial expressions that participants had to name. The order of the videos was pseudorandom such that no actor was seen twice in a row. Participants had to start each video per key press and could watch it only once. After each video, they typed in their interpretation of the facial expression (maximum 80 characters). No feedback was given. After this implicit learning phase, participants performed a surprise old–new recognition task. For this, the participants saw 32 different images: Four images from each of the four target actors and four images from four new distractor actors. Participants had to decide for each image whether the actor had been seen during the learning phase or not by pressing the relevant keys on the keyboard. Stimuli were presented for 2 s or until key press, whichever came first. The next image appeared as soon as an answer was entered. The order of the pictures was pseudorandom, such that no actor was seen twice in a row. No feedback was given. All participants reported that they had not anticipated the surprise recognition task after the expression naming.The second part was conducted to control for the effect of surprise. The design was similar, with the difference that participants knew that an old–new recognition task would follow the explicit learning phase. Again, the participants watched 16 videos of four different actors. This time they did not need to name the facial expressions but could concentrate on remembering the appearance of the actors. Afterwards they once more had to recognize the actors among the distractors.d′-scores as Z(hits)—Z. d′-score of 2.09 (SD = 0.88) in the first, surprise part and 2.66 (SD = 0.91) in the second part. Prosopagnosics achieved a mean d′-score of 1.03 (SD = 0.64) in the first part and 1.48 (SD = 0.87) in the second part. A two-way repeated measures ANOVA of the factors participant group  and test part  was conducted on the d′-scores. Recognition performance was significantly higher in the second part compared to the first, surprise part  = 7.1 p = .012, η= .17) and controls performed significantly better than prosopagnosics  = 29.9, p < .001, η= .46). The interaction between parts and participant groups was not significant  = 0.11, p = .75, η2 < .01).For each participant, we calculated the t(15) > 6.4, p < .001, d > 1.61; controls for both parts t(20) > 10.8, p < .001, d > 2.48). However, ceiling effects were present for the controls in the second part, as 33% of the controls scored above 95% accuracy , 52.4% scored above 90% accuracy ), see Prosopagnosics and controls performed significantly above chance level . The ceiling performance of the controls reinforces this observation: It may have led to underestimate the improvement between test parts for controls, yet the improvement for prosopagnosics between test parts was still not bigger than for the controls. Because prosopagnosics’ performance was significantly above chance level in the first, surprise part, we conclude that they extracted and stored identity-relevant information even when not paying attention to that information. We suggest two equally possible explanations. First, contrary to our hypothesis, prosopagnosics had engaged their strategies not only during the explicit but also during the implicit learning phase. They were thus able to extract and store featural characteristics even without conscious effort. The second possible explanation is that prosopagnosics’ recognition system does not differ fundamentally from that of the controls in so far that in both groups the mechanisms of holistic processing and extracting identification-relevant information seem to occur automatically in explicit as well as in implicit learning conditions. These automatic mechanisms are exhibited by prosopagnosics, yet are reduced compared to controls. In our next experiment, we investigate whether indeed holistic processing abilities are still present, though in reduced form in prosopagnosics.Several studies state that the key to a well-functioning face recognition system lies in holistic face processing. Holistic processing is defined as the integration of all facial information, for example, shape of nose, mouth, and eyes (features) and their spatial distances (configuration). This information is combined into a whole gestalt, making it harder to process the information individually . A classWe used the “complete design” version of this experiment . In the complete design, holistic processing is indexed by an interdependence of congruency and alignment: Performance is better in congruent than in incongruent trials . Misalignment reduces the congruency effect, as it disrupts holistic processing. We use this version of the experiment because it has been suggested that it may better separate face-specific from non-face-specific effects than the “classic” design  of the first composite face was the same as the top half of the second face within the same trial. In the “different” condition, the two top halves differed. In the congruent condition, the bottom halves were same if the top halves were same or they were different if the top halves were different. In the incongruent condition, the bottom halves were different if the top halves were the same and vice versa. In the aligned condition, top and bottom halves were placed exactly on top of each other. For the misaligned condition, the top half was displaced to the right, while the bottom part was displaced to the left such that the middle of one half was placed adjacent to the edge of the other half. All face images were presented upright for the upright condition or rotated by 180° for the inverted condition.The combination of upright or inverted condition with aligned or misaligned conditions was tested in four separate blocks. The block order was balanced across participants. Each of the four blocks contained 120 trials: 30 trials of each combination of same and different trials, and congruent and incongruent trials. The order of trials was randomized.In each trial, participants had to indicate whether the two face halves comprising the eyes were the same or not. Participants responded during the inter-trial interval of 2 s by pressing the relevant keys on the keyboard. No feedback was given. After every 20 trials and also between blocks participants were able to take a self-paced break. Before testing, there were 10 training trials for each of the four different blocks. Blocks were trained in the same order as they would appear during the actual testing.d′-scores as Z–Z. The congruency effect was calculated by subtracting d′-scores of incongruent from congruent conditions. SD = 1.27) for aligned and 0.1 (SD = 0.8) for misaligned trials, while prosopagnosics obtained a mean congruency effect of 0.65 (SD = 0.61) for aligned and 0.31 (SD = 0.48) for misaligned trials. In the inverted condition controls obtained a mean congruency effect of 0.36 (SD = 0.70) for aligned and 0.14 (SD = 0.52) for misaligned trials, while prosopagnosics obtained a mean congruency effect of –0.11 (SD = 0.46) for aligned and 0.27 (SD = 0.45) for misaligned trials.For each participant we calculated the F = 23.54, p < .001, η= .40) and there was no significant difference between participant groups  = 0.93, p = .34, η2 = .03). The interaction between alignment and participant group was significant, indicating that the congruency effect was more affected by misalignment in the control group than for prosopagnosics  = 7.71, p = .009, η= .18). A post hoc analysis for prosopagnosics revealed that their congruency effect was significantly smaller for the misaligned than aligned condition  = 12.90, p = .003, η= .34). This indicates that controls and prosopagnosics exhibit evidence of holistic processing for upright faces.As misalignment and inversion are both control conditions for the measurement of holistic processing, we consider these two factors separately. First, we looked at the congruency effect for the upright condition only, using misalignment as control condition. A two-way repeated measures ANOVA on participant group  and alignment  was conducted. The congruency effect was larger for the aligned than the misaligned conditions  = 16.0, p < .001, η= .31) and controls showed overall a larger congruency effect than prosopagnosics  = 10.11, p = .003, η= .22). The interaction between orientation and group was non-significant, indicating that the inversion factor did not affect prosopagnosics and controls differently  = 0.23, p = .64, η= .01).Second, we looked at the congruency effect for the upright-aligned versus the inverted-aligned conditions only, using inversion as control condition. A two-way repeated measures ANOVA for the aligned condition on orientation  and participant group  was conducted. As expected, the congruency effect was larger for upright than inverted conditions  = 7.29, p = .016, η= .16). This was not the case for controls, who showed no difference in congruency effects  = 1.27, p = .27, η= .03).Additionally, we investigated more closely the negative congruency effect observed for prosopagnosics in the inverted-aligned condition see . The con2 Importantly, the difference in congruency effect for aligned versus misaligned trials is significant for prosopagnosics, which indicates that their holistic processing ability is still present yet impaired.The congruency effect in interdependence with (a) alignment or (b) orientation serves as a measure of holistic processing. For the upright condition, using (a) misalignment as control condition, we found that controls showed a larger difference in congruency effect for aligned versus misaligned trials compared to prosopagnosics. These results suggest that holistic processing is impaired or utilized to a smaller extent by prosopagnosics in this task. This replicates the results of previous reports of decreased holistic processing for prosopagnosics compared to controls .2 ImportWhen we used (b) the inversion effect  to assess holistic processing, no significant difference between groups appeared (non-significant interaction). Thus, we did not find differences in holistic processing between groups, which is contrary to the expectations given by our design. Furthermore, our results also imply that prosopagnosics show more holistic processing for misaligned face halves than aligned halves when seen inverted. Similar “inversion superiority effects” for prosopagnosics have been described before . HoweverIn sum, if we concentrate on the upright condition that can be clearly interpreted, prosopagnosics, compared to controls, show a smaller difference between the congruency effects obtained in the aligned and misaligned condition. This indicates that holistic processing  is present but impaired for prosopagnosics.The composite task is generally regarded to be the best method to assess holistic face processing. There are many other methods  might be involved as well and in what way  or their configuration, but they shared the same skin texture and outer shape see . Skin teThe stimuli had a visual angle of 5.7° horizontally and 8.6° vertically. To prevent pixel matching, the faces were presented at different random positions on the screen within a viewing angle of 7.6° horizontally and 10.5° vertically.Participants rated the perceived pairwise similarity of the faces within each set on a Likert scale from 1 (very little similarity) to 7 . They were advised to use the whole range of ratings during the experiment. In each trial, the first face was displayed for 2 s, followed by a pixelated face mask for 0.8 s, and then the second face for another 2 s. Afterwards, the Likert scale was displayed and participants marked their rating by moving a slider on the scale via the arrow keys and confirmed their choice by pressing the relevant key on the keyboard. The start position of the slider was randomized. The next trial started as soon as the rating was confirmed. There were no time restrictions, but participants were told to answer without too long considerations. After every 20 trials, participants could have a self-paced break.The faces of each set were compared with each other and with themselves. We were only interested in trials comparing faces manipulated along the same dimension . Similarity ratings were close to seven (high similarity) for identical faces and dropped with decreasing similarity. For each participant, we fitted a linear regression to the mean similarity ratings, again separately for featural and for configural ratings. The similarity levels served as predictors. The steepness of the slopes was then used as measure of sensitivity: The steeper the slopes, the stronger the participant perceived the configural or featural changes.SD = 0.23) for featural and 0.70 (SD = 0.15) for configural changes. Prosopagnosics obtained a mean sensitivity score of 1.09 (SD = 0.20) for featural and 0.51 (SD = 0.23) for configural changes. We analyzed the sensitivity scores with a two-way repeated-measures ANOVA with the factors change type  and participant group . Participants exhibited a higher sensitivity towards featural than configural changes  = 172.76, p < .001, η= .83), and prosopagnosics showed an overall reduced sensitivity compared to controls  = 9.34, p = .004, η= .21). The interaction between change type and participant group was non-significant  = 0.41, p = .53, η= .01).The use of parametric stimuli in our tests allowed a more fine-grained quantification of potential configural and especially of featural processing impairment in prosopagnosics than most other studies. We morphed facial features  between two faces in several steps in each set in terms of their color and shape, thus creating faces differing gradually for these features. In contrast, most other studies only exchanged features between faces. Furthermore, we took care to use faces without make-up and all our stimuli had been controlled for their naturalness  from our in-house 3D face database. As visible in Participants had to judge the gender of each face. The faces were shown one at a time and stayed on screen until a response was given by pressing the relevant keys on the keyboard. The next image appeared as soon as a response was entered. The order of trials was randomized. No feedback was given. Participants were instructed to answer as correctly and as quickly as possible.SD = 4.8), while prosopagnosics scored very well too at 84.4% (SD = 5.9). Nevertheless, prosopagnosics performed significantly worse than controls as revealed by a one-way ANOVA  = 16.62, p < .001, η= .32).For each participant, percent correct accuracy was calculated. Prosopagnosics exhibited a significantly lower gender recognition ability compared to controls. This differs from the self-reports of prosopagnosics  and alsomotion advantage). Again, this advantage may potentially be even stronger for prosopagnosics than for controls who do not need to rely on this additional information for recognition. Therefore, we wanted to investigate the influence of appearance  and motion on face recognition for prosopagnosics compared to controls, by using dynamic stimuli in which the same people changed their look between learning and test. We also included static stimuli and faces that did not change their appearance  as control conditions. We expected that, for the identical stimuli, the prosopagnosics would retain their usual compensatory strategies, while for the changed stimuli they would make more use of the dynamic information.Most studies testing holistic face recognition abilities of prosopagnosics use only static face stimuli. Furthermore, those face images often are identical for training and testing. Such tasks do not reflect the everyday challenges encountered by prosopagnosics, as people move, speak, and might alter their look on a day-to-day basis. On the one hand, the different looks of people might complicate recognition for prosopagnosics maybe even more than for controls who do not rely on these non-facial attributes for recognition. On the other hand, the additional dynamic information might give additional cues for prosopagnosics, thus facilitating recognition  while for the other half of the targets the second recording was presented at test (changed stimuli). Participants were informed that the look might change between learning and testing. The order of trials was randomized during learning and testing. Which target actresses were tested in the identical or changed condition was counterbalanced across participants. During learning and testing participants could see each stimulus only once. During testing, the stimuli were presented as long as described above or until key press, whichever came first. The next stimulus started as soon as an answer was entered by pressing the relevant keys on the keyboard. No feedback was given. Between blocks participants were able to take a self-paced break.d′-scores for each participant. d′-score of 2.79 (SD = 0.53) for dynamic stimuli and 2.25 (SD = 0.65) for static stimuli. Prosopagnosics achieved a mean d′-score of 1.87 (SD = 0.74) for dynamic stimuli and 1.85 (SD = 0.61) for static stimuli. For the changed condition, controls achieved a mean d′-score of 1.72 (SD = 0.71) for dynamic stimuli and 1.40 (SD = 0.71) for static stimuli. Prosopagnosics achieved a mean d′-score of 1.09 (SD = 0.76) for dynamic stimuli and 1.19 (SD = 0.48) for static stimuli.We calculated F = 117.32, p < .001, η2 = .77) and a significantly better performance for controls  = 15.46, p < .001, η= .31). The interaction was non-significant  = 2.42, p = .13, η2 = .07), indicating that there was no difference between controls and prosopagnosics in how the changed appearance of the faces influenced their performance.We ran a two-way repeated measures ANOVA for the identical and changed condition versus participant group . We found a significantly better performance in the identical condition  = 4.99, p = .032, η= .13) and a statistically better performance for controls than prosopagnosics  = 15.62, p < .001, η= .31). Interaction between stimulus type and participant group was significant  = 4.14, p = .0496, η= .11). Post hoc analysis of simple effects of stimulus type  revealed that controls performed better for dynamic than static stimuli in the identical condition  = 8.65, p = .005, η= .18), while there was no difference between dynamic and static stimuli for prosopagnosics .We also conducted two-way repeated measures ANOVAs on stimulus type  and participant group  for the identical and for the changed condition separately. For the identical condition, we found a statistically better performance for dynamic than static stimuli  = 0.44, p = .51, η= .01). We found a better performance for controls than prosopagnosics  = 8.03, p = .008, η= .19). The interaction between stimulus type and participant group was non-significant  = 1.54, p = .22, η= .04).For the changed condition, there was no performance difference for static versus dynamic stimuli , our study also brings to light some fundamental difference in the quality of the obtained data. It reveals that classical tests engaging holistic processing might not be adequate for prosopagnosic participants although they are well adapted for fine-grained investigations of face recognition in neurotypical populations.Prosopagnosics in this study displayed reduced face recognition (CFMT) and normal object recognition (CCMT). More interestingly, we found that prosopagnosics showed automatic retrieval of identity information (surprise recognition test), indicating that their unconscious mechanisms for extracting identification-relevant information are still active whenever they view faces. Furthermore, we could replicate the finding that prosopagnosics show limited holistic processing (composite face test), supporting the assumption that holistic processing is decisive for efficient identification . We furtThe tests’ reliability coefficients obtained for the controls and prosopagnosics reached good levels most of the time. However, for tests of holistic face processing employing static stimuli exclusively, in particular for the CFMT, prosopagnosics obtained only a fraction of the reliability coefficients of controls. We suggest that prosopagnosics use varying strategies  to compensate for their face recognition impairment, which leads to their inconsistent answering behavior in those tests. While we admit of course that this suggestion is a speculation from our part, the use of strategies by prosopagnosics has been widely reported in the literature . Future That last finding, reduced test reliability when testing prosopagnosics, has important implications for our current study in particular and for research on prosopagnosia at large. An additional unsuccessful goal of our current study had been to assess a large group of prosopagnosics with a variety of tests with the goal of finding subgroups. In hindsight, after completion of our study, the general opinion is now that a much larger number of prosopagnosic participants is needed for finding clear subgroups, owing to various potential factors introducing noise in the test data, two of them being genetic diversity  and co-mWith our extended battery of existing and newly created tests and our large sample size of prosopagnosic and control participants, we were able to refine our knowledge about face perception processes in general and for congenital prosopagnosia in particular. Furthermore, we are the first to reveal that the response behavior of prosopagnosics in tests for holistic processing differs from controls, as indicated by their noticeably reduced test reliability. Future work will need to examine the robustness and cause of this phenomenon. Additionally, better tests need to be designed, with higher reliabilities for prosopagnosics. Such tests would provide more robust results allowing to obtain a more accurate picture and better classification of the impairment.


Castor fiber) in Poland. Cd concentrations in liver (0.21 ± 0.44 μg/g) and in kidney (2.81 ± 4.52 μg/g) were lower in juvenile than in adult beavers. Pb concentrations in liver (0.08 ± 0.03 μg/g) and kidney (0.08 ± 0.03 μg/g) were similar among all individuals, while both Cu and Zn levels were higher in liver  than in kidney . Cu levels also differed between juveniles and adults. We reviewed the literature reporting metal concentrations in beavers. Our results indicate metal contamination in beavers away from important industrial emission sources and suggest the natural environment should be regularly monitored to ensure their levels are below recommended, legal values.Heavy metals are persistent environmental contaminants, and wild animals are increasingly exposed to the harmful effects of compounds of anthropogenic origin, even in areas distant from industrial centers. We used atomic absorption spectrometry to determine levels of cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn) in liver and kidney of wild Eurasian beavers ( Wild animals are exposed to environmental contaminants of both natural and anthropogenic origin and are suitable bioindicators of environmental pollution  and lead (Pb), as well as essential elements in which toxicity is determined by the dose, as copper (Cu) and zinc (Zn). Although these four elements occur naturally in the environment, they are supplied in large quantities through domestic and industrial pollution  and aspen (Populus tremuloides), as well as on non-woody terrestrial and aquatic plants (Haarberg and Rosell Castor fiber) as a “partially protected” species  tissues obtained from industrial areas, generally regarded as polluted  to assess trace element contents in a region away from major industrial centers and (ii) to evaluate the degree of contamination against legal Cd and Pb levels recommended by the European Commission in farm animal edible tissues , Warmia and Mazury Region, northeastern Poland, in Summer 2012. Warmia and Mazury is a traditional agricultural region with extensive forests and numerous lakes. The region is an important recreational center for local and international tourists who seek activities in a natural environment. The sampling site was away from a major industrial complex, which could putatively be a source of pollutants in their vicinity. Animals were collected upon approval of the Regional Nature Conservation Authority (Opinion No. RDOŚ-28-OOP-6631-0007-638/09/10/pj 2010) and the Local Ethics Committee (Opinion No. 11/2010).All beavers were collected during daytime with a net and transported to the laboratory in cages specially adapted for this purpose . Each working solution was analyzed in duplicate and calibration curves were periodically verified. Recoveries of all four elements were within the range 86–109 % and relative standard deviations were below 10 %.Analysis for each element was performed based on calibration curves plotted from working standards. For calibration, we used commercial stock solutions (1000 mg/l) of analytical grade for all four elements (J.T.Baker®). Working standards were prepared by dissolving stock solutions with 0.1 M HNOt test. Concentrations in beaver livers and kidneys were compared between age or sex classes using a non-parametric Mann-Whitney U test. Pair-wise comparisons between element concentrations in each organ were performed using a Spearman rank correlation analysis. We report arithmetic means, standard deviations (SD), median, and range. Where necessary, data were transformed to meet statistical assumptions. Significance level was set at p < 0.05, and all statistical analyses were run using R version 3.0.0  and Zn . Additionally, Zn levels were significantly correlated to Cd levels in kidney .We captured six female  and four male  beavers Table . Mean con = 6) and male (n = 4) beavers for any of the four metal concentrations in liver or kidney  than in juveniles (n = 4) in both liver  and kidney . In liver, Cu levels were significantly lower in adults than in juveniles , and in kidney, Cu levels were significantly higher in adults than in juveniles .We found no differences between female  and stone marten (Martes foina): Bilandžić et al. Tyto capensis): Ansara-Ross et al. Pygoscelis spp.): Jerez et al. Contrastingly, some metal concentrations were affected by the animals’ age. Higher liver and kidney Cd concentrations were found in adult beavers than in juveniles, indicating that this metal bioaccumulates over time (Parker and Hamr Castor canadensis) in habitats located 175 km from an ore smelter in Ontario, Canada  and willow , trees that accumulate high levels of Cd particularly on acidic soils that increase the mobility of heavy metals  as well as in Canada and Russia (Jankowska et al.


Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of  The use of complementary and alternative medicine is becoming increasingly popular worldwide1356910Regulatory bodies such as the US Food and Drug Administration (FDA), the UK Medicines and Healthcare Products Regulatory Agency (MHRA) and the Therapeutic Goods Administration (TGA) in Australia are entrusted with regulating herbal products, including TCMs, prepared or sold within their borders. Medicines that are thought to contain low-risk ingredients often only require the manufacturer to declare what is contained within their products, a form of regulation referred to as ‘light-touch’The field of complementary and alternative medicine polarises the public, health care professionals, regulators and researchers, but efficacy arguments aside, most would agree that herbal CAMs should be safe, unadulterated, legal and honestly labelled. In this study, we set out to evaluate the content of 26 legally purchased TCMs, using a combination of molecular screening methods. This study is the first of its kind, and aims to provide much-needed pharmacovigilance strategies for pre- and post-market auditing of herbal CAMs.The plant and animal composition of the 26 TCMs were genetically analysed using both 454 (Roche) GS Junior and Ion Torrent PGM NGS platforms . MultiplThe taxonomic assignments from the mtDNA and plastid DNA data are listed in Each of the 26 TCM samples was extensively tested for potential adulterants using LC-MS analysis. Each of the compounds was carefully assessed relative to known standards, and a limit of quantitation and limit of detection was constructed for each compound. A total of 18 different toxicological compounds were detected in 13 TCMs listed in The amounts of the adulterants and/or undeclared substances detected were variable between samples as indicated in Twenty-five out of the 26 TCM samples were screened for heavy metal contamination using SF-ICP-MS. The aqueous state of TCM17 prevented it from being analysed using the above method. Three potentially toxic metals to humans, arsenic, cadmium and lead were the focus of this analysis, many of which are present in quantities that exceed recommended daily intakes see . Eleven This study describes the first detailed combined DNA, toxicological, and heavy metal audit of herbal medicine employing both next generation DNA sequencing technologies and mass spectrometry-based detection. The focus of this study was on 26 TCMs purchased “over the counter” within Australia, however many of these medicines are also for sale on the international market. Of the 26 TCM samples, 12 were classified as “Listed” by the TGA, with the remainder having no listing meaning that they should not have been commercially available. In Australia, and elsewhere, herbal products such as TCMs are regarded as low-risk and therefore undergo less stringent regulation. In some jurisdictions TCMs are simply classified as ‘dietary supplements’. Regardless of how they are categorised, the regulatory framework relies heavily, or exclusively on the assumption that manufacturers are making accurate declarations regarding the composition of their products. In the case of the TCMs studied here, it is clear that such declarations are not always correct; i.e. there were major discrepancies between what was declared, either to the regulatory agency, or to the consumer in the form of an ingredient list.et al. (2012) had demonstrated that a DNA-based NGS approach is a powerful way to scrutinise TCM for plant and animal componentsCoghlan Panthera. This sample contained DNA from snow leopard (Panthera uncia), and possibly tiger (Panthera tigris), both of which are listed within the Convention on the International Trade in Endangered Species of flora and fauna (CITES) Appendix I. Appendix I affords the highest level of trade restriction. This result was checked using independent DNA isolations from TCM8 and sequencing them on 454, Ion Torrent and Illumina NGS platforms. Finally, the presence of snow leopard was confirmed by successful amplification of P. uncia using a species-specific PCR assay1617Panthera DNA in a TGA ‘Listed’ medicine is therefore of great concern. On the basis of this identification alone, TCM8 is illegal to import or sell and an incorrect declaration has been made to the regulatory agency.The TCM that most clearly breaches international law is TCM8 (used to treat “arthritis” and “pain”) where two mtDNA signatures (differing by one polymorphism) were recovered that provided a 100% match to species within the genus Panthera (TCM8), pit viper (TCM 11) and frogs (TCM 19) are likely to have been deliberately added as a primary ingredient. However the reproducible detection of DNA from ‘domestic’ animals  is of concern due to the clear carcinogenic risks associated with aristolochic acid519Ephedra genus in this same TCM makes the product illegal to sell in most jurisdictions. Four TCM samples contained DNA that could be assigned to the genus Apocynum, which has very restricted use and is on the registry of poisonous (FDA), restricted or prohibited plants (MHRA and TGA). Apocynum species are known to contain cardiac glycosides (e.g. cymarin), which are cardiotoxins that can cause arrhythmias.The presence of DNA assigning to plant species such as Up to 24% of TCM preparations have been previously reported to be adulterated with pharmaceuticalsFrom a toxicological perspective, TCM2  was perhaps the most concerning as a ‘cocktail’ of six undeclared compounds were detected: ephedrine (1097 μg/g), chlorpheniramine (1025 μg/g), salicylic acid (25.2 μg/g), amoxicillin (18 μg/g), methylephedrine (10 μg/g) and small quantities of warfarin (4 μg/g). The interactions of the analgesic, antibiotic, stimulant and antihistamine drugs in this single TCM preparation are difficult to predict. This may be even more significant if the preparation is used by children or pregnant women.Ephedra DNA. In other instances Ephedra DNA could not be detected when ephedrine was present, and this may be because a synthetic adulterant was added to the preparation or, alternatively, the DNA was too highly degraded for detection. In another example, there was no obvious genetic sequence that could account for the brucine (3405 μg/g), and strychnine (1509 μg/g) detected in TCM1, although species outside of the Strychnos genus may also generate these alkaloids.In some instances there was a clear correlation between the genetic and toxicological analyses. The ephedrine detected in TCM1 correlates with the detection of Morus , including whether the product is categorised as a listed medicine by the regulatory body (the TGA), can be found in the et al. (2012)2 (Sigma), 20 mM dithiothreitol solution , 1% sodium dodecyl sulfate (Invitrogen), and Milli-Q water. 200 μL of digest was mixed with 1 mL of Qiagen  PB buffer and purified and washed on a silica spin column and finally eluted with 50 μL of EB buffer .DNA characterisation of the TCM samples followed Coghlan, Haile trnL g/h, rbcL, and 16S rRNA25262 , 1 × Taq polymerase buffer , 0.4 μM dNTPs , 0.1 mg bovine serum albumin  when required to counteract inhibition, 0.4 μM of each primer, 0.2 μL of AmpliTaq Gold DNA polymerase  and SYBR-Green dye. The qPCR conditions were: 50 cycles of 95 °C for 30 seconds, annealing at primer specific temperature  tags were designed for the plant and mammal primer sets used for qPCR above. Amplicon sequencing was carried out on both a 454  GS Junior instrument as described previouslyde novo method32http://dx.doi.org/10.5061/dryad.ps04c.The sequencing output files were retrieved, filtered, and processed using Geneious (v6.1)4 × g and diluted 1:10 with LC-MS water for analysis. The final internal standard concentration was 5 ng/mL.TCM samples were crushed in a clean mortar and pestle, and 5 mg of each was extracted with methanol containing 0.05 mg/mL diphenhydramine , for 1 minute at 6500 rpm in a Precellys tissue lyser . Extracts were centrifuged for 5 minutes at 1.3 × 1018 (100 mm × 2·1 mm × 3 μm) , with mobile phase A being LC-MS water and mobile phase B being 0.1% formic acid in acetonitrile. Analysis was repeated with mobile phase B as methanol to expand coverage of possible adulterants. The nebuliser gas (air) was set at 70 psi, the vortex gas at 30 psi and 300°C and the drying gas at 25 psi and 200°C (both N2). Mobile phase was held at 95% A for 2 minutes before ramping to 100% B by 9 minutes, held for 5 minutes, back to 95% A by 15 minutes, held for 5 minutes.Samples were analysed using a Varian 212 HPLC system coupled to a 325-MS triple quadrupole mass spectrometer, using electrospray ionisation . The column was a Restek Ultra Aqueous CEighteen likely adulterants were selected and, to establish optimal MS/MS conditions, standards of each were infused into the MS at 1 μg/mL. A minimum of two transitions per compound were selected and the dwell time for each transition was 0.025 s for a total scan time of 0.85 s, with the detector set at 1100 V. Standard curves were generated for each compound over the range 0.1 to 100 ng/ml. Data was analysed using Varian MS Workstation v6.9.3 (Agilent Technologies), with peak areas normalised to the internal standard.3) to screen for bioavailable toxic metals. The “acid leachable” method does not determine total metal concentrations but is representative of metals released from the TCM samples in-vitro. For reporting purposes, the concentrations determined by the method should be considered an underestimate of the total metal concentrations contained within TCM. Powdered TCM samples (~0.3 g) were added to acid-cleaned polypropylene vials containing 5 mL of ultrapure nitric acid  and leached for 24 hours at room temperature. Following acid leaching samples were made up to 10 mL with ultrapure water (>18 mΩ), filtered (acid-cleaned 0.2 μm PTFE filter membrane) and diluted 1: 75 with 3% ultrapure HNO3.TCM samples were leached in concentrated nitric acid (HNO3), replicates and quality control standards  were determined at regular intervals throughout the analysis. Internal standard normalised ions intensities were quantified using “matrix matched” external standards prepared by serial dilution from commercial primary standards. The results were blank subtracted and corrected for dilution. TCMs were deemed non-compliant if the concentration of the heavy metals lead, cadmium and arsenic were above the threshold daily intake levels set by the TGA for a 60kg person.Sixteen heavy metals were determined under ultra-trace clean conditions using a sector-field inductively coupled plasma mass spectrometer  housed in the Curtin University ultra-clean TRACE facility. Samples were mixed with an internal standard (Indium) and introduced to the SF-ICP-MS using an auto-sampler  enclosed in a metal free hood. Instrumental and full procedural blanks . Sci. Rep. 5, 17475; doi: 10.1038/srep17475 (2015).


Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2–3 mm tubes, which were placed in pairs, separated by a small gap, on semi‐permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum‐free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80%  had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin+ neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies. ©2015. The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, LtdThe ability to grow embryonic organs  In addition, ex vivo organ culture can be used to assess the effects on organogenesis of physical manipulations or the addition of exogenous chemicals, such as growth factors. With respect to the mammalian small intestine, previous studies have begun to explore these aspects using, as examples, organ culture of intact embryonic gut rudiments /F12 (Gibco BRL), insulin (10 mg/l), sodium selenite (5 mg/l) and transferrin (5.5 mg/l). For some experiments, this medium was supplemented with R‐spondin 1 , a factor that promotes canonical WNT/β‐catenin signalling and stimulates intestinal epithelial proliferation in postnatal mice , an epithelial cell–cell adhesion protein , a visceral muscle cytoskeletal protein , and the mouse anti E‐cadherin antibody described above. Endogenous mouse immunoglobulins were blocked using the Mouse on Mouse Kit . After washing, appropriate secondary antibodies were applied for 1 h. Finally, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining was undertaken to detect cell nuclei. Dextrans were visualized through an Olympus BX51 microscope and images captured with a Coolsnap ES camera (Photometrics) through MetaVue Software (Molecular Devices). Immunofluorescence was visualized with a Fluoview FV1000 confocal microscope (Olympus) and images were acquired using Fluoview v. 3.1b software (Olympus). The images were then processed and analysed using ImageJ (http://rsb.info.nih.gov/ij) and Adobe Photoshop CS5. Specific band pass filter sets were used to prevent bleed‐through between channels.For whole‐mount immunofluorescence, rudiments were washed in 1% bovine serum albumin (BSA) in 1% PBS and 0.1% Triton X‐100 for 2 h, then blocked with 1% BSA in 1% PBS, 0.1% Triton X‐100 and 5% heaet al., αSMA+  demonstrated that the rudiments curved as they grew, with the presumed mesenteric side in the concavity, so that the proximate ends of adjacent explants became poorly aligned. Therefore, in an attempt to maintain orientation, upon being explanted the rudiment pairs were linked with a single suture threaded through their lumens  vs non‐aligned  pairs .By contrast, on day 3 of culture, nine (26%) of 34 rudiment pairs either still had a gap between them or, if their ends touched, the epithelial zones of the two organs were not well aligned Figure F, G. In αSMA+ zone was present  from explants fed basal medium alone . The average starting distance between adjacent rudiment pairs in basal medium alone vs pairs in medium supplemented with R‐spondin 1 was not significantly different .Finally, we tested whether addition of 100 ng/ml of R‐spondin 1 affected growth or fusion. The ends of rudiments exposed to this exogenous growth factor displayed sheet‐like extensions by day 3 of culture and these were not found in rudiments grown in basal medium alone (see supporting information, Figure et al., et al., ex vivo is beginning to provide a source of gut tissues that can be used to complement the refashioning of aberrant gut by surgery (Sala et al., et al., We believe that insights from experiments to study the healing and growth of intestines in organ culture will ultimately inform the design of novel therapies for human disease. An example of the latter is short bowel syndrome, a potentially fatal condition arising from a variety of developmental and acquired insults, such as congenital atresia and necrotizing enterocolitis (Khalil et al., et al., et al. (et al. (et al., et al., The current results confirm reports (Abud , et al.  used thi (et al.  showed t et al., , might iThe authors declare no conflicts of interest.Video S1. Spontaneous peristalsis in healed jejunal neo‐organVideo S2. Spontaneous peristalsis in an embryonic day 17 jejunumFigure S1. Histology of embryonic jejunumFigure S2. Distances between rudiment pairs at the time of being explantedFigure S3. A neural network in the wall of embryonic day 17 jejunumFigure S4. Effects of R‐spondin 1 on jejunal explantSupporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.


The hippocampus has been implicated in anxiety disorders and post-traumatic stress disorder (PTSD); human studies suggest that a dysfunctional hippocampus may be a vulnerability factor for the development of PTSD. In the current study, we examined the effect of hippocampal damage in avoidance learning, as avoidance is a core symptom of all anxiety disorders. First, the effect of hippocampal damage on avoidance learning was investigated in outbred Sprague Dawley (SD) rats. Second, the function of the hippocampus in Wistar-Kyoto (WKY) rats was compared to SD rats. The WKY rat is an animal model of behavioral inhibition, a risk factor for anxiety, and demonstrates abnormal avoidance learning, marked by facilitated avoidance acquisition and resistance to extinction. The results of the current study indicate that hippocampal damage in SD rats leads to impaired extinction of avoidance learning similar to WKY rats. Furthermore, WKY rats have reduced hippocampal volume and impaired hippocampal synaptic plasticity as compared to SD rats. These results suggest that hippocampal dysfunction enhances the development of persistent avoidance responding and, thus, may confer vulnerability to the development of anxiety disorders and PTSD. The development of post-traumatic stress disorder (PTSD) and anxiety disorders is a function of an individual’s experience and inherent vulnerability. While much research effort has been devoted to the effects of traumatic stress on individuals, less effort has been devoted to the study of vulnerability factors. Vulnerability or risk factors may be inherited  or due to prior experiences .The hippocampus is a brain region implicated in PTSD. Patients with PTSD have reduced hippocampal volume  rat is an animal model of behavioral inhibition and displays many characteristics related to anxiety disorders. Trait behavioral inhibition is a vulnerability factor for the development of anxiety disorders, as behaviorally inhibited children are more likely to develop anxiety disorders  were 300–350 g and male Wistar-Kyoto  rats were 200–250 g at the start of the behavioral study. Thirty-five SD rats underwent surgery for lesions or sham procedures. Eight SD and eight WKY rats were behaviorally tested without surgery. All rats were housed individually on a 12-h light/dark cycle with lights turning on at 7:00 a.m. Training and testing were performed during the light phase of the light/dark cycle. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the Veterans Affairs Medical Center at East Orange, New Jersey.Male SD rats (Rats were anesthetized with isoflurane (2%). Burr holes were drilled into the skull overlying the hippocampus or entorhinal cortex. The coordinates (in mm) for the entorhinal cortex lesion sites in relation to bregma were as follows (four sites per hemisphere): AP -5.3, ML ±6.5, DV -5.0; AP -6.0, ML ±6.5, DV -5.0; AP -6.7, ML ±5.0, DV -6.5; AP -7.4, ML ± 5.0, DV -6.5. The coordinates for the hippocampal lesion sites in relation to bregma were as follows (five sites per hemisphere): AP -2.5, ML ±1.6, DV -3.8; AP -4.2, ML ±2.6, DV -3.1; AP -5.3, ML ±4.4, DV -3.4; AP -5.8, ML ±5.6, DV -4.1; AP -6.0, ML ±5.6, DV -4.1. Injections were made bilaterally. The needle of a Hamilton syringe was inserted into the desired location to infuse saline for sham surgery or ibotenic acid (10 μg/μl) to damage hippocampus or entorhinal cortex. Infusions occurred at a rate of 0.1 μl/min with a volume of 0.5 μl dispensed per site. Rats were allowed at least 10 days to recover from surgery. Extent and location of lesions are depicted in Figure Rats were trained in an operant box with a lever (10.5 cm above the floor), a cue light (20.5 cm above the grid floor), and a speaker (26 cm above the grid floor) on one wall and a light (14 W) on the opposite wall that was constantly lit during the session. Scrambled footshocks were delivered through the grid floor . The operant box was enclosed in a sound-attenuating box.Avoidance learning occurred as described previously  was performed to determine whether significant effects in extinction remained after covarying performance on the last acquisition session. Interactions were evaluated by F-test.Data are expressed as mean ± standard error of the mean. Performance was assessed by calculating the proportion of trials in each session with an avoidance response. A mixed design ANOVA with session as the within subject factor and lesion/strain as the between subjects factor was performed. All lesion groups and unoperated SD and WKY groups were included in this overall ANOVA and are captured in the lesion/strain factor. Separate analyses were performed for the acquisition and the extinction phases. To determine whether non-specific responding might be increased by lesion or strain, lever presses during each minute of the ITI were analyzed. Mean lever presses per trial per minute was determined for the ITI and assessed statistically using a mixed design ANOVA with session and ITI minute as within subject factors and lesion or strain as a between subjects factor. Statistical analysis was performed with α = 0.05 using SPSS for Windows . Mixed design analysis of variance (ANOVA) was used to compare groups. Mauchly’s test was used to determine violations in the assumptions of sphericity for repeated measure factors and Greenhouse–Geisser correction was used in the appropriate situations to correct for violations  at three months of age. All rats were given at least one week to acclimate to the new surroundings prior to recordings. Recordings were performed during the light phase of the light/dark cycle. Procedures were as described previously  response curves were generated to monitor changes in slope of field EPSP (fEPSP) and population spike amplitude  before and after high frequency stimulation (HFS). The i/o curves were generated using 100–1000 μA stimulation. Average waveforms were generated from five evoked responses at each stimulus intensity, and slopes of fEPSP and population spike were measured from these averaged waveforms. Two i/o curves were used to establish baseline. HFS to induce LTP was based on parameters established previously  × 2 (time) × 7 (stimulus Intensity) repeated measures ANOVA. Statistical analysis was performed with SPSS similar to that described for the behavioral studies.At the end of behavioral testing or recording, all animals were perfused intracardially with saline followed by formalin. Brains were extracted and submerged overnight in formalin followed by 30% sucrose. Brains were sectioned (50 μm) through the hippocampus and entorhinal cortex with a sliding microtome. Sections were stained with cresyl violet. For the lesion study, location and extent of brain damage were assessed Figure . For then = 5 for each strain) were sacrificed, perfused, and brains extracted. Brains were sectioned at 50 μm thickness and every fifth section was collected and stained with cresyl violet. Volume of the hippocampus, neocortex, corpus callosum, and striatum was estimated using the Cavalieri method  = 30.55, p < 0.001]. Acquisition of avoidance did not differ between lesion groups nor between strains   = 6.02, p < 0.001]. ITI responding was greater in the first minute of the ITI as compared to the second or third minutes . Whereas the main effect of lesion/strain  was not significant, the lesion/strain × session × ITI interaction  did reach significance. In post hoc analysis of each ITI minute, lesion/strain affected the first minute ITI response   ≤ 1.61, p > 0.171; lesion/strain × session interaction, F ≤ 1.17, p > 0.196]. Comparisons made between sham and lesions and between unoperated SD and WKY rats for first minute ITI responses revealed that hippocampal but not entorhinal lesions facilitated responding , and strain differences trended toward significance with WKY tending to make more ITI responses than SD rats . Thus, hippocampal but not entorhinal cortex lesions increased ITI responding during the first, but not second or third, minutes of the ITI  = 13.73, p < 0.001]. Lesion/strain differed  but session did not interact with lesion/strain . Post hoc analysis demonstrated that rats with hippocampal lesions and WKY rats extinguished their avoidant responding slower than the other groups (p < 0.05)  = 5.71, p < 0.001], demonstrating persistent avoidant responding in rats with hippocampal lesions and WKY rats but not rats with entorhinal cortex lesions.Hippocampal lesion and WKY rats were impaired in extinction of avoidance responding Figures A,B. OverF5,25 = 13.7F = 306.47, p < 0.001]. ITI responses were most numerous during early extinction sessions and gradually decreased with extinction training   = 2.53, p = 0.01], but did not interact with session, . The triple interaction did not reach significance, . Upon further analysis, it was determined that lesion/strain was significantly different during the first minute of ITI,   = 2.44, p = 0.048]; however, post hoc analysis found no significant group differences. Thus, hippocampal lesions increased ITI responding during the first, but not second or third, minute of the ITI  = 6.348, p < 0.05]. WKY rats had significantly smaller hippocampus  and cortex  compared to SD rats  = 5.229 p = 0.028] and the phase × stimulus intensity interaction  were significant  = 13.139, p < 0.001]. In contrast to SD rats, LTP of the fEPSP was not observed in WKY rats  = 1.913, p = 0.198] nor the phase × stimulus intensity interaction  were significant. The main effect of stimulus intensity was significant, [F = 22.234 p < 0.001].Long-term potentiation (LTP) of the mPP to DG synapse was impaired in WKY rats. Evoked field potentials had similar waveforms in SD and WKY rats Figure . LTP of ion  and the phase × stimulus intensity interaction [F = 7.014 p < 0.001] were significant. The main effect of stimulus intensity was also significant, . LTP of the population spike was not observed in WKY rats  = 4.291; corrected p = 0.085] nor the phase × stimulus intensity interaction  were significant, although the main effect of stimulus intensity was significant, .Similar to fEPSP, LTP of the population spike was observed in SD but not in WKY rats Figures  and 7A,BAn abnormal hippocampus may provide risk for developing PTSD. Smaller hippocampal volume and associated poorer learning were observed in soldiers with PTSD and their non-combat, non-PTSD twin siblings  but not in WKY rats. One explanation for persistent avoidance responding during extinction training is that hippocampal lesions increase general activity, including lever-press responding. However, the lack of group differences during minutes 2 and 3 of the ITI suggests that this is not the case. The increase in ITI responding in WKY rats during the acquisition phase has been previously reported (Beck et al., Depression and anxiety are commonly comorbid (Kessler et al., In summary, previous human studies with PTSD patients have suggested that an abnormal hippocampus may be a risk factor for developing PTSD. Here, we present evidence that hippocampal damage facilitates the development of persistent avoidance responding, similar to symptoms of anxiety disorders in humans. Moreover, we provide support that an animal model of behavioral inhibition, a risk factor for anxiety disorders (Kagan et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lactobacilli reduced the surface tension of phosphate buffer saline (PBS) from 72.0 to 39.5 mNm−1 pH 7.2 and its critical micelle concentration (CMC) was found to be 2.5 mg mL−1. Low cost production of Lactobacilli derived biosurfactant was carried out at lab scale fermenter which yields 0.8 mg mL−1 biosurfactant. The biosurfactant was found least phytotoxic and cytotoxic as compared to the rhamnolipid and sodium dodecyl sulphate (SDS) at different concentration. Structural attributes of biosurfactant were determined by FTIR, NMR (1H and 13C), UPLC-MS, and fatty acid analysis by GCMS which confirmed the presence of glycolipid type of biosurfactant closely similar to xylolipids. Biosurfactant is mainly constituted by lipid and sugar fractions. The present study outcomes provide valuable information on structural characterization of the biosurfactant produced by L. helveticus MRTL91. These findings are encouraging for the application of Lactobacilli derived biosurfactant as nontoxic surface active agents in the emerging field of biomedical applications.A probiotic strain of  Bacillus, Pseudomonas, and other genus of soil inhabitant microorganisms are commonly reported for the biosurfactant production but, due to pathogenic nature, their application is limited to only environmental applications /100%.The phytotoxicity assay of the biosurfactant was determined in a static seed germination and root elongation of theslightly . Solutio2 atmosphere. A standardized quantity of cells (1 × 104) was inoculated in 100 μL of DMEM in 96-well culture plates and incubated for stabilization for 24 h before the treatment. The stock solution of biosurfactant was prepared in DMSO at concentration of 10 μg/1 μL. The final quantities of biosurfactant were added 25 μg, 12.5 μg, and 6.25 μg in the cytotoxicity assay and incubated for 24 h at 37°C in 5% CO2 atmosphere. After 24 h, 15 μL dye solution from the CellTiter 96 nonradioactivity cell proliferation assay kit  was added into the wells and kept for 4 h incubation as per the recommendation. Afterward, the 100 μL stopping solution was added in all the wells and incubated overnight to dissolve formazan product to get uniform readings. The absorbance was recorded at 570 nm in microplate spectrophotometer . The DMSO used as solvent was taken as negative control in the assay. To estimate the cytotoxicity of biosurfactant, biologically originated rhamnolipid and Sodium dodecyl sulfate (SDS) were used as positive controls.The cytotoxicity of biosurfactant was checked on mouse fibroblast (ATCC L929) cell line . The cel−1). The decrease in surface tension was compared with the surface tension of production medium, that is, whey (53.5 mNm−1). Samples were withdrawn at regular intervals and experimental data of biomass, lactose consumed, and measurement of surface tension plotted in −1 and the surface tension was reduced down to 39.5 mNm−1. Biosurfactant concentration was found to be approximately 0.80 gL−1. Increase in initial lactose concentration yields higher biomass and biosurfactant produced by different lactobacilli [ Lactobacillus paracasei subsp. paracasei was also found to be growth-associated. Biosurfactant concentration production was reported to be maximal at stationary growth phase. The similar pattern of biosurfactant production and lactose utilization was also reported [−1). The results obtained by L. helveticus MRTL91 confirmed that the strain is a significant biosurfactant producer. And whey based medium can be used as an alternative substrate for large scale production of biosurfactant.Strain MRTL91 was found putative biosurfactant producer. The lowest value of surface tension was achieved after 10 h of fermentation in the stationary phase . The compound showed the C–H stretching vibrations in the transmittance range 2932 cm−1 indicating the aliphatic chain. 1720 cm−1 (for the C=O ester bond) and 1273 cm−1 were found to be ether and C–O stretching vibration in sugars, 1041 cm−1 (polysaccharides), 702 cm−1, and 648 cm−1 (for CH2 group) confirming the presence of glycolipid moieties. Biosurfactant produced by L. helveticus has been chemically characterized. Results of TLC, FTIR, 1H NMR, 13C NMR, and GCMS spectra suggest that it consists of several compounds such as octadecanoic acid as main lipid consisting of long aliphatic chain and polysaccharides. Proton and carbon NMR analysis confirmed the presence of –CH3 (0.896 ppm), –(CH2)n– (1.286 ppm), –(CH2–COO)– (2.324 ppm), –O–CH– (4.386 ppm), and –CH2=CH– (7.535 ppm) , and GCMS confirmed the presence of octadecanoic acid containing glycolipid with a cumulative molecular weight of 391.32 m/z. GCMS analysis of biosurfactant showed major peaks for octadecanoic acid, a fatty acid at a retention time of 8.46 min  was 250°C determined from TGA curve. The weight of polymer was drastically lost around and above 290°C and continued gradually to decrease [Thermal stability of BS is a significant property for its commercial application at extreme temperature. Thermal degradation of BS was carried out by TG analysis . Approxi50–400°C . Moisturosa MA01 . The degdecrease . BS isol L. helveticus was found noncytotoxic and nonphytotoxic. Microbial surface active agents are generally regarded as less toxic and biodegradable biomaterial [Biosurfactant produced bymaterial . But duematerial . The litmaterial . Brassica nigra L. and Triticum aestivum L. Different concentration of biosurfactant was prepared at concentration equal to the half of critical micelle concentration (CMC) value, equal to CMC, and twice the CMC. In present study, about 100% seed germination was observed in both types of seeds. But seed germination was declined in the treatment of seeds with SDS   Tables . Biosurf−1, 0.125 mg mL−1, and 0.0625 mg mL−1. Cell viability was found maximum about 43.3% at 6.25 mg mL−1 in case of biosurfactant produced by strain MRTL91 while positive control rhamnolipid showed 35.3% viability quiet close to SDS, that is, 35.99%. But increase in the concentration of BS also declined the cellular viability. At concentration of 25 mg mL−1, cell viability was found 30.9% as compared to rhamnolipid which showed 32.87% of cell viability. While DMSO used as diluent did not show any significant cytotoxicity, the highest biosurfactant concentration studied showed a significant decrease of the total number of viable cells, probably due to a prevalence of a detergent-like effect leading to cell membrane disruption [Cytotoxicity of BS was evaluated using mouse fibroblast (ATCC L929) cell line. The mouse fibroblasts cells were selected and generally regarded suitable for cytotoxicity assessment. Mouse fibroblast cells are recommended for in vitro evaluation of medical devices by the International Organization for Standardization (2009). During cytotoxicity determination, different concentrations of cell bound BS and purified rhamnolipids  were prepared in DMSO . Whereassruption . It is iμg mL−1. Biosurfactant produced by Sphingobacterium detergens was studied for its cytotoxicity and antiproliferative effects on different cell lines. When comparing cytotoxicity values (IC50) of the two fractions in fibroblast and keratinocyte cell cultures, fraction B was found less cytotoxic, showing lower toxicity than the reference compound SDS, indicating low skin irritability [ L. helveticus would be ideal for potential application in cleaning/coating material for several biomedical equipment and cosmetic formulations.Various studies on evaluation of cytotoxicity of biosurfactant reported in literature, the lack of cytotoxicity is anticipated when you wish to formulate ecofriendly and safe antiadhesive suspension directly to be used for human health. Typically, the cytotoxicity seems linked to its interactions with the phospholipids of cell membrane and therefore cell lysis. Cochis et al.  have reptability . Accordi L. helveticus MRTL 91 was isolated and structurally characterized as being similar to xylolipid. The FTIR and NMR analysis of biosurfactant revealed the presence of sugar and lipid fractions. Structurally the BS is characterized as a glycolipid with hexadecanoic fatty acid (C16) chain. The minimum surface tension and the CMC were found similar to the previous reports of biosurfactant produced by other lactobacilli. Their potential application in products for human consumption such as cosmetics and pharmaceuticals or food additives requires an accurate characterization of possible toxic side effects. Biosurfactant was confirmed as nonphytotoxic and noncytotoxic compound as compared with other microbial and chemically synthesized surface active agents. This is the first compilation of the information on L. helveticus derived biosurfactant, structural elucidation, and toxicity assessment. Biosurfactant from LAB, that is, GRAS status organism, is safe for oral consumption and biomedical applications. Structural elucidation opens new horizon for biosurfactants applications in pharmaceuticals/cosmetics and suitable alternative to conventional antimicrobials and antimicrobial resistance.The identification and structural characterization of new biosurfactant is gaining interest from the commercial point of view. The BS produced by