These biohydrogenation intermediates signal a coordinated downregulation of genes involved in mammary FA synthesis, transport, and esterification. We have previously reported decreased mammary expression of sterol response element‐binding protein 1 (SREBP1), SREBP1‐activating proteins, and thyroid hormone‐responsive spot 14 (S14) in the cow during diet‐induced milk fat depression (MFD), and treatment with trans‐10, cis‐12 CLA. Liver x receptors (LXR) and retinoid x receptors (RXR) regulate lipogenesis and are known to bind polyunsaturated FA and LXR agonist increases lipid synthesis in mammary epithelial cell culture. The current studies investigated if biohydrogenation products of rumen origin inhibit mammary lipogenesis through LXR and/or RXR. Expression of LXRs was not different in lactating compared to nonlactating bovine mammary tissue, and expression of LXRs, RXRα, and selected LXR and RXR target genes was not changed in mammary tissue during diet‐induced or CLA‐induced MFD in the cow. In bovine mammary epithelial cell culture, LXR agonist stimulated lipogenesis and expression of LXRß, ATP‐binding cassette 1 (ABCA1), SREBP1c, and S14, but LXR activation did not overcome CLA inhibition of lipogenesis and downregulation of LXRß, SREBP1c, and S14 expression. Lastly, expression of the LXR‐regulated carbohydrate‐responsive element‐binding protein (ChREBP) was higher in lactating than nonlactating tissue and was decreased during CLA‐induced MFD. We conclude that changes in mammary LXR expression in dairy cows are not involved in MFD and that trans‐10, cis‐12 CLA inhibition of lipogenesis and diet‐induced MFD appears independent of direct LXR signaling.Milk fat synthesis of ruminants can be inhibited by intermediates of ruminal fatty acid biohydrogenation including  Liver x receptors (LXR) and retinoid x receptors (RXR) regulate lipogenesis and are known to bind polyunsaturated FA, and LXR agonist increases lipid synthesis in mammary epithelial cell culture. However, expression of LXRs was not different in lactating compared to nonlactating bovine mammary tissue and do not appear to be involved in trans‐10, cis‐12 CLA inhibition of lipogenesis and diet‐induced milk fat depression in the cow. The CLA treatment involved a 3 day intravenous infusion of a CLA‐Intralipid emulsion  that supplied 10 g/d of trans‐10, cis‐12 CLA. The trans‐10, cis‐12 CLA methyl ester stock  contained 88.3% total CLA , 6.8% palmitic acid, 2.7% oleic acid, and 2.0% stearic acid. Methyl esters of CLA were selected as the convenience of the synthesis procedure make them more commonly available in larger quantities and ME of CLA are equally effective as free FA of CLA in reduction of milk fat in the cow  or tissue biopsies . Additionally, the effect of mammary physiological state was examined using mammary tissue collected from seven cows prior to lactation (approximately 30 days prepartum) and during established lactation  by needle biopsy according to Harvatine and Bauman . Second,μmol/L LXR agonist TO‐901317 , 10 μmol/L 9cRA (Sigma), or 75 μmol/L trans‐10, cis‐12 CLA alone or in combination. Treatments were performed in both basal media and media supplemented with serum and insulin as described in the figure legends. The CLA selected for cell culture was in free FA form to provide the natural form that does not require hydrolysis and contained 95.3% trans‐10, cis‐12 CLA and 2.2% linoleic acid . The CLA stock was complexed to BSA after formation of a potassium salt  were grown to 80–90% confluence in basal media  supplemented with 10% fetal calf serum  and 5 µg/mL bovine insulin . Cells were incubated in basal media in the absence of serum and insulin for 24 h before treatment. Cells were treated for 24 h with 5 n et al. .α  was performed according to Harvatine and Bauman  with min2 wells and transfected with 1 μg of luciferase plasmid and 0.067 μg of the control TK renilla plasmid  using Lipofectamine 2000 (Invitrogen). Luciferase plasmids included constructs containing approximately a 1.3 kb fragment of the murine SREBP1C promoter or its mutated version lacking the sterol response element (SRE), LXRE, or both  from Chen et al. (μL of Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activity was determined using the Dual‐Luciferase reporter assay (Promega) according to manufacturer's protocol.The MAC‐T cells were grown to 85% confluence in 9.6 cmn et al. . After tData were analyzed using the fit model procedure of JMP  using REML. The model for analysis of tissue expression profile included tissue and the geometric mean of three housekeeping genes [18S ribosomal subunit, ß‐actin, and ß2‐microglobulin  were not altered  and decreased by CLA when cultured in basal media or basal media supplemented with serum and insulin Fig. A,B. RXR Expression of direct targets of LXR was observed to determine functional LXR signaling mechanisms in bovine mammary epithelial cells. CLA decreased expression and TO9, 9cRA, and 9cRA+TO9 stimulated expression of FASN, SREBP1c, S14, and ABCA1 Fig. A–E. CLA The SREBP1c gene is regulated by both SREBP1 and LXR making interpretation of TO9 stimulation of SREBP1 expression more complicated. An SREBP1 reporter assay was used to determine the role of each independently in response to CLA. Mutation of the SRE and SRE & LXRE drastically decreased the activity of the SREBP1 promoter under basal conditions, but mutation of the LXRE alone had no effect Fig. A. CLA detrans‐10, cis‐12 CLA induces a similar response in many species including rats, mice, pigs, sheep, goats, and humans . Cell death was not observed in the current experiment with 5 μmol/L TO9 or in MAC‐T cells treated with 2.5 μmol/L TO9 . MAC‐T cells were derived from primary mammary epithelial cells from a lactating cow (Huynh et al. α was functionally important to S14 expression (Nagaya et al. The ability of CLA to reduce mammary expression of key lipogenic regulators, lipogenic enzymes, and LXR target gene in the presence of LXR and RXR agonists was investigated based on our working model Fig. . Signalitrans‐10, cis‐12 CLA was not expected to decrease expression of ABCA1, but ABCA1 is regulated by many factors including SREBP1a and SREBP2, and SREBP1a is highly expressed in many cell lines. However, mammary expression of ABCA1 was not decreased in the cow during MFD. Interestingly, trans‐9, trans‐11 CLA is an LXR agonist (El Roz et al. ATP‐binding cassette 1 is a LXR target gene, but is not regulated by SREBP1c (Liang et al. The SREBP1c promoter assay allowed dissection of the SREBP1c response to TO9 and CLA in bovine mammary epithelial cells. The drastic reduction in the activity of the SRE‐mutated promoter demonstrates a predominant role for SREBP1c in its own regulation and the lack of change in activity with mutation of the LXRE questions the importance of LXR in mammary SREBP1 regulation. Treatment with CLA caused the largest reductions in the activity of the wild‐type and SRE‐mutated promoter, although the magnitude of the decrease in the SRE promoter was small due to the low activity of the promoter. However, this does demonstrate the opportunity for a non‐SREBP1c mechanism. Additionally, CLA had no effect on the SRE & LXRE‐mutated promoter demonstrating a possible role of LXR in the CLA response. However, SREBP1 clearly has the predominant role in regulation of the SREBP1 promoter and the potential to explain the magnitude of responses observed during MFD in the cow.α or RXRα, but decreased expression of ChREBP in lactating mice. However, ChREBP protein was not detectable in adipose depleted mouse mammary cells (Rudolph et al. trans‐10, cis‐12 CLA treatment, but not during diet‐induced MFD in the current experiment (Fig. Glucose metabolism and fat synthesis are integrated by activation of ChREBP by xylulose 5‐phosphate, a metabolite of glucose, and activation of ChREBP simultaneously activates glycolysis and lipid synthesis through transcriptional regulation of target genes including l‐pyruvate kinase and FASN (Uyeda and Repa ent Fig. . Expresstrans‐10, cis‐12 CLA inhibition of lipogenesis does not support major CLA signaling through LXR. Recently, Ducheix et al. (In conclusion, the low expression of LXRs in lactating mammary tissue from the dairy cow raises questions about the importance of LXRs in milk fat synthesis. The lack of change in expression of specific LXR target genes and the failure of LXR and RXR agonists to overcome x et al.  reportedThe authors would like to specifically recognize the support and technical assistance of D.A. Dwyer. Additionally, the authors acknowledge the assistance of former Cornell students and employees Dr. S. Thorn, Dr. J.W. Perfield, Dr. M. Tanino, Dr. C. Tyburczy, Dr. E. Castañeda‐Gutiérrez, Dr. A.L. Lock, L. Furman, B. English, Dr. W. Waybright, and A. Garcia‐Ramirez.None declared.


Multiple myeloma, a plasma cell neoplasm, presents most commonly with anemia, hypercalcemia, renal failure, and bone pain. Only few cases of clinical aggressive presentation associated with bleeding were reported in the medical literature. The reported cases included gastrointestinal bleeding and cardiac tamponade. Spontaneous retroperitoneal haemorrhage as initial presentation has not been so far reported. We hereby report a case of a 64-year-old female who was found to have catastrophic hemorrhage in the retroperitoneal region that extended into intrathecal space causing cord compression. The case posed a significant diagnostic and management dilemma. This case emphasizes the need to think broadly and include multiple myeloma in the diagnosis of unexplained massive retroperitoneal bleeding. Multiple myeloma, a plasma cell neoplasm associated with production of monoclonal paraprotein, is second in prevalence amongst hematologic malignancies . The diaμL, haemoglobin of 9 g/dL, and platelets of 101 × 1000/μL. The peripheral smear showed rouleaux formation and plasma cells less than 10%. Earlier laboratory findings showed mild normocytic anemia, thrombocytopenia, and normal white blood count. Internal hemorrhage was greatly suspected given the clinical and laboratory findings. Other laboratory tests were significant for a creatinine of 2.5 mg/dL and a total calcium level of 9.0 mg/dL. Urinalysis was positive for blood and protein, 511 mg/dL. Prothrombin time was slightly elevated 14.2 sec and a normal activated partial thromboplastin time. Biochemical tests revealed a low total protein at 6.5 g/dL and serum albumin was low at 2.7 g/dL. Serum LDH was elevated at 240 U/L (60–160 U/L). Quantitative D-dimer was more than 0.5 mg/L . Fibrinogen was normal with a value of 194 mg/dL (193–423 mg/dL). Quantitative immunoglobulin showed an increased IgG at 2187 mg/dL and decreased IgA and IgM levels. Serum and urine immunofixation identified a monoclonal IgG lambda. CT abdomen revealed interval worsening of splenomegaly to 20 cm, multiple enlarged lymph nodes, and retroperitoneal haemorrhage extending from the left kidney to the left inguinal region. MRI of lumbar spine done emergently as the lower extremities showed progressive paralysis revealed critical long segment spinal cord compression extending from the T12 to L5 level secondary to medullary canal haemorrhage , PTT of 47 sec (25–35 sec), and fibrinogen of 75 mg/dL (193–423 mg/dL), worsening acute kidney injury with creatinine increasing from 2.5 mg/dL to 5.5 mg/dL, and acute hypoxic respiratory failure requiring endotracheal intubation due to severe refractory anemia leading to high output cardiac failure, and the chest X-ray revealed bilateral pulmonary vascular congestion and prominent left-sided pleural effusion. Finally, seven days after admission, her code status was changed to do-not-resuscitate per her family and healthcare proxy, given her refractory illness and poor prognosis. In the same day, she developed cardiac arrest and expired. Autopsy was obtained and gross evaluation revealed large blood clot in the left pelvis and retroperitoneal space measuring 40 cm in greatest dimension and weighing 1060 g indicating that the most likely cause of death was a haemorrhagic shock probably due to underlying bleeding diathesis secondary to platelets dysfunction. Microscopic evaluation confirmed the diagnosis of multiple myeloma with more than 60% CD138 and lambda light chain positive plasma cell in the bone marrow (Figures The patient was transferred to the ICU for developing severe haemorrhagic shock. Based on the clinical and laboratory data the diagnosis of multiple myeloma versus plasma cell leukaemia was considered. Plasma cell leukaemia was excluded as the peripheral blood showed less than 20% plasma cells which is the major criteria for diagnosing this condition. The cause of retroperitoneal bleeding in the absence of a bleeding disorder, vascular disease, and anticoagulant medications was considered to be related to the basic disease of multiple myeloma. Given the emergent and rapidly worsening condition, the patient was started on high dose corticosteroids and supportive measures including vasopressors and blood products including packed red blood cell, fresh frozen plasma, and platelets transfusion. The patient haematocrit and platelets count continued to drop precipitously. In 5 days, the hematocrit dropped from 9 g/dL to 5.6 g/dL and the platelets dropped from 101 × 1000/7 sec 25–5 sec, anThis case illustrates a rare and aggressive form of multiple myeloma presenting with retroperitoneal hemorrhage leading to cord compression. This type of presentation of multiple myeloma is reported for the first time in the medical literature. One case of retroperitoneal haemorrhage developed, however, during a well-established multiple myeloma and was reported in 1991 and ultimately light chain amyloidosis was detected which might have contributed to the bleeding complication in that case . In gene


Journal of Dairy Science from Oct., 2007 till May, 2010. The database consisted of a total of 442 observed means for MCY from 118 studies. The candidate factors that determine MCY were those which can be routinely measured in the field . Using a simple linear regression, the best equations for predicting milk fat yield(MFY) and milk protein yield (MPY) were MFY = 0.351 (±0.068) + 0.038 (±0.003) DMI (R2 = 0.27), and MPY = 0.552 (±0.071) + 0.031 (±0.002) DMI - 0.004 (±0.001) FpDM  (R2 = 0.38), respectively. The best equation for predicting milk fat content (%) explained only 12% of variations in milk fat content, and none of a single variable can explain more than 5% of variations in milk protein content. We concluded that among the tested variables, DMI was the only significant factor that affects MFY and both DMI and FpDM significantly affect MPY. However, predictability of linear equations was relatively low. Further studies are needed to identify other variables that can predict milk component yield more accurately.The objectives of this study were thus to identify most significant factors that determine milk component yield (MCY) using a meta-analysis and, if possible, to develop equations to predict MCY using variables that can be easily measured in the field. A literature database was constructed based on the research articles published in the  The manipulation of milk composition has been of interest to improve the nutritional value of milk and to increase the efficiency in manufacturing and processing of raw milk for dairy products . In add2 = 0.47) between DMI and MY using data set published in Journal of Dairy Science (Volumes 1 through 82). According to NRC[Many studies have tried to identify such factors affecting milk yield (MY) and MCY for past decades. DMI, dietary energy and protein or individual carbohydrate (CHO) and protein fractions might be important factors in controlling MY and milk protein yield (MPY) in dairy cows  explained only 12% of the variations in milk fat content, and none of a single variable could explain more than 5% of the variations in milk protein content. According to the review by Jenkins and McGuire indicateA further analysis was done to develop equations with variables that can be easily measured in the field for predicting MPY and MFY. DMI alone explained 27% of variations in MFY Figure  and 35% 2 = 0.27, root mean square error (RMSE) = 0.22), and MPY = 0.552 (±0.071) + 0.031 (±0.002) DMI - 0.004 (±0.001) FpDM , respectively.Using a random coefficient model with study as a random effect, we obtained 0.311 ±0.072) + 0.041 (±0.003) DMI  and 0.497 (±0.084) + 0.032 (±0.002) DMI - 0.003 (±0.001) FpDM  for predicting MFY and MPY, respectively . The detailed descriptive statistics of the database was described in Table The database consisted of a total of 442 treatment means from 118 studies published in the The independent variables used for explaining the variations in MCY  were DMI (kg/d), BW (kg), DMIpBW , FpDM , NDF (% DM), CP (% DM), starch (% DM), ADF (% DM), CP intake (kg/d), NDF intake (kg/d), starch intake (kg/d), and forage intake (kg/d). Among these variables, four to five predictive variables that sufficiently explained the variations in each dependent variable were selected using step-wise regression.The regression equations were developed in two phases. In the first phase, a random coefficients model was used using the MIXED procedure of SAS  with studies as a random variable to identify independent variables that were statistically significant for each equation.i,j; β the fixed effects parameter vector of which size is p; Z the designed N × (s × p) matrix that was blocked diagonally corresponding to each study (ni × p) to account for the random effect of each study; e the unknown vector of independent, identically and normally distributed random errors with mean 0 and variance σ2; N the total number of observations; s the number of studies; ni the number of observations that ith study; p the number of parameters, which is one (intercept) plus the number of variables used in the equation; and u and e are the normally distributed.where y is the vector of observed MCY of which size is N; X the n × p matrix of xAmong the acceptable regression models that had a linear combination of significant fixed effect variables, a model that had the lowest value of -2 restricted log likelihood, Akaike information criterion (AIC), the corrected Akaike information criterion (AICc) and Schwarz’s Bayesian criterion (SBC) was selected. The lowest value of those criteria above indicates a better model considering the number of observations, the number of parameters, and the maximum likelihood estimates. In the second phase, the parameters of the variables in the best model for each dependent variable, identified in the first phase, were estimated by fitting the prediction equations to a multiple regression model using GLM procedure of SAS .


Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of  L. kefiri strains were studied. None of the  L. kefiri strains tested caused α- or β-hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of  Kefir grains are composed of a complex community of yeasts, lactic acid, and acetic acid bacteria confined in a matrix of polysaccharides and proteins . The pro Lactobacillus, are extensively used as probiotics [As it is known, probiotics are “live microorganisms which, administered in adequate amounts, exert a beneficial effect to the health of the host” . Specifiobiotics , 8 sinceobiotics , 10 as wobiotics –13. Lactobacillus kefiri) are included in the QPS list of the European Union [ Lactobacillus strains have been associated with cases of sepsis, endocarditis, or bacteremia, mostly in association with a severe underlying disease [The study of the beneficial properties attributed to isolated microorganisms constitutes a field of great interest for the development of functional foods. Lactobacilli are generally regarded as safe (GRAS) and most of them  [Our workgroup has isolated and characterized numerous species of LAB and yeasts from kefir, including several strains ofs kefiri –24, one ls mL−1) . L. kefiri as a probiotic microorganism in vitro after verifying that secretion products and surface proteins from these heterofermentative lactobacilli exert a protective action against the invasion of Salmonella enterica serovar Enteritidis [ L. kefiri strains are able to preserve a high percentage of viability after both spray-drying [ L. kefiri's safety was ever evaluated. Since it is known that both the beneficial properties such as harmful characteristic are dependent on the strain, the individual study of the safety of potential probiotic microorganisms should be considered.We have already demonstrated the potential oferitidis  and thateritidis . On the y-drying , 29 and y-drying . However L. kefiri as a novel probiotic, we reported in this work some safety characteristics of L. kefiri strains, as well as the capacity of strains to produce antimicrobial compounds against some intestinal pathogens.Taking into account the potential of Lactobacillus kefiri strains CIDCA 8321, 8345, 8348, 83111, 83113, and 83115 [ Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 6538, Shigella flexneri ATCC 9199, Pseudomona aeruginosa ATCC 15442, a clinical isolate of Salmonella enterica serovar Enteritidis CIDCA 101 , enterohemorrhagic Escherichia coli EDL 933, Listeria monocytogenes ATCC 7644, and Bacillus cereus ATCC 10876. All mentioned strains, except B. cereus, were grown using brain heart infusion (BHI) broth  in agitation at 37°C for 16 h. B. cereus was grown in BHI growth supplemented with dextrose  1 g L−1 (BHIg) in agitation at 37°C for 16 h.Pure cultures used in this study comprisednd 83115 , 31. The−1; tryptone 10 g L−1; dextrose 10 g L−1; yeast extract 10 g L−1; Tween 80 1 g L−1; and bacteriological agar 15 g L−1) supplemented with 5% human blood (group O) and incubated for 48 h at 37°C under aerobic conditions. The appearance of clear zones around the bacterial colonies indicated the presence of β-hemolysis whereas green zones around the colonies suggested α-hemolysis. Enterococcus faecalis ATCC 29212 was included as a positive hemolytic control.Hemolysis was tested by growth of the strains on LAPTg agar (peptone 15 g Lμg mL−1. Stock solutions were diluted in LSM broth (90% IST plus 10% MRS) to obtain solutions with preliminary concentrations in the range of 0.25–128 μg mL−1. Bacterial inocula were prepared by suspending colonies from 48 h incubated in MRS medium to 5 mL 0.85% NaCl solution. Subsequently, inocula were adjusted to OD625 nm 0.18–0.24 and diluted 1 : 500 in LSM broth for inoculation of microdilution plates by adding 50 μL of diluted inoculum to each well containing 50 μL of an antibiotic solution. In these conditions, the bacterial inoculum was around 2-3 × 105 CFU mL−1 in the wells. After incubating plates under anaerobic conditions at 37°C for 48 hours, the MICs value was read as the lowest concentration of an antimicrobial agent in which visible growth was inhibited.The minimum inhibitory concentrations (MICs) of the antimicrobial agents tested  were detMICs results were compared with the recommended breakpoints for heterofermentative lactobacilli by the EFSA Panel on Additives and Products or Substances used in Animal Feed .Cat, chloramphenicol acetyltransferase gene, was assessed using the primers and PCR conditions described by Hummel et al. [ L. reuteri G4 was used as a positive control.l et al. . A plasmμL of a suspension OD625 nm 1 of L. kefiri strains was spotted into MRS agar and incubated for 24 h at 37°C. The following day, pathogens were seeded into soft BHI agar and plated over the spotted lactobacilli. After 18 h of incubation at 37°C, the inhibition halos were measured. The width of the clear zone (R) was calculated as follows: R = (dInhib − dSpot)/2, where dInhib is the diameter of the zone without pathogen growth and dSpot is the diameter of the spot. Inhibition scores are as follows: negative (−), R < 2 mm; low inhibition capacity (+), 2 mm < R < 5 mm; and high inhibition capacity (++), R > 6 mm. At least three independent experiments were performed.The agar spot test described by Schillinger and Lücke  was usedAll animal procedures were performed in strict accordance with the guidelines issued by the European Economic Community “86/609.”8 CFU of L. kefiri CIDCA 8348 (Lk group) or PBS (control group) daily for 21 days.Male 6-week-old Swiss albino mice  were quarantined 2 weeks after arrival and then randomized by body weight into experimental and control groups of 5–7 animals each. Mice were housed under standard laboratory conditions with free access to food and water. The temperature was kept at 22°C and a 12-hour light/dark schedule was maintained. Mice received by gavage 10Mice were weighted every two days; behavior and signs of pain were analyzed daily . At the Liver and spleen were removed and blood samples were collected aseptically. Liver and spleen were homogenized in 0.1% sterile PBS (0.1 g of organ per mL) and serially diluted. One hundred microliters of each organ homogenate or blood was plated on VRBG Agar  for enterobacteria and MRS agar for LAB. Plates were incubated under aerobic conditions for 24 h at 37°C for VRBG and for 48 h at 37°C for MRS before examination.Ileum content was washed with 1 mL sterile PBS and then serial dilutions were plated as indicated above.2-95% (v/v) air atmosphere [α, IFN-γ, and GM-CSF) measurements . All assays were performed according to the manufacturer's instructions.Explants were cultured in RPMI medium supplemented with 10% foetal bovine serum , 10 mg/L streptomycin and 10 IU/mL penicillin G, and 100 mg/L gentamicin  for 24 h at 37°C in a 5% (v/v) COmosphere . Superna L. kefiri strains isolated from kefir were studied in order to evaluate both their safety and antimicrobial properties.In the present work, six potentially probiotic in vitro was bacterial hemolytic activity. In this study, none of the L. kefiri strains tested caused α- or β-hemolysis (data not shown). In this genus, hemolytic activity has a very low frequency and only α-hemolysis has been reported for lactobacilli isolated from foods and dairy products [Since hemolysis is a common virulence factor among pathogens, the first safety parameter evaluatedproducts –41. L. kefiri strains are shown in  L. kefiri strains (data not shown). In this regard, Hummel et al. [ cat genes were susceptible to chloramphenicol; meanwhile, in other resistant strains cat genes could not be amplified. Further research, such as the study of the distribution of chloramphenicol MICs, could contribute to determine whether resistance is acquired (not acceptable strain) or intrinsic (acceptable strain) according to EFSA [Another important feature regarding safety is the sensitivity to antibiotics. The results obtained forl et al.  reported to EFSA . L. kefiri was evaluated just in two publications. Nawaz et al. [ L. kefiri strain isolated from a dairy product, which was resistant to kanamycin and tetracycline but sensitive to other antimicrobial agents tested in LSM medium. Chang et al. [ L. kefiri strains, among other lactobacilli, isolated from swine intestines were resistant to tetracycline, with MIC values higher than 256 μg mL−1, and that they possessed at least one resistance gene. Taking into account that tetracycline is the most widely used antimicrobial agent in swine production, its continuous administration might be selecting tetracycline resistant microorganisms on swine's microbiota. This feature and the different origin of our L. kefiri strains could contribute, at least in part, to the disagreement between our results and those from other authors.To our knowledge, antibiotic sensitivity ofz et al.  studied g et al.  observed L. kefiri strains studied. As observed in  L. kefiri strains than Gram negative bacteria. It is important to notice that the addition of MRS acidified with HCl or lactic acid to pH 4.3  was not able to produce inhibition of pathogens in our tests (data not shown). All the strains inhibited growth of Bacillus cereus and Staphylococcus aureus but none of them inhibited enterohemorrhagic Escherichia coli (EHEC). The strains L. kefiri CIDCA 8321, CIDCA 8348, and CIDCA 83111 were able to inhibit growth of the rest of the tested pathogens. Many mechanisms associated with bacterial inhibition have been described for Lactobacillus species [The secretion of molecules able to inhibit the growth of pathogens is a desirable characteristic, among others, for a potentially probiotic bacteria , and it  species . The pro species . L. kefiri CIDCA 8321, 8348, and 83111 demonstrated to be the most active strains against pathogens; however, CIDCA 8321 showed resistance to chloramphenicol. In consequence, among the other two strains, we selected CIDCA 8348 to perform in vivo studies in Swiss mice.Up to here,μL of a 109 CFU mL−1 suspension of L. kefiri CIDCA 8348 (Lk group) and mice receiving 100 μL of PBS (control group) daily for 21 days. Moreover, there were no differences in food and water intake between groups (data not shown). In accordance with these results, Lk group did not show any signs of pain, lethargy, dehydration, or diarrhea during treatment. No signs of inflammation or damage were observed in any organ during necropsy. Length of each mouse's colon was measured, since it has been reported that increasing levels of inflammation result in shortening of the colon [γ, TNF-α, and GM-CSF were observed in the small intestine and colon explants from Lk and control mice ( L. kefiri CIDCA 8348 administration [7 versus 4.8 ± 0.9 × 107) and LAB (1.1 ± 0.6 × 107 versus 2.6 ± 0.8 × 107) in the ileum were comparable between control and treated mice.As observed in he colon . No signhe colon  were obshe colon  who admirol mice . On the stration . Besides L. kefiri CIDCA 8348 isolated from a dairy product present a great potential as probiotic for human or animal use and can be used also for producing functional foods.Taking into account all these findings, we conclude that


P <0.05) increased the phosphorylation of the mTORC1 substrate ribosomal protein S6 kinase 1  and of insulin receptor substrate 1 , but suppressed insulin‐stimulated glucose uptake by 40% (P <0.01). These modifications were mTORC1‐dependent and were reversible. In vivo, leucine gavage reversibly increased S6K1 phosphorylation and IRS‐1 serine phosphorylation 5‐ to 12‐fold in skeletal muscle and impaired insulin tolerance of glucose (P <0.05) in lean rats. In insulin‐resistant rats, the impairment of whole blood glucose and AA metabolism induced by leucine gavage (0.001 < P <0.05) was more severe than that observed in lean rats; however, the impairment was reversible within 24 h of treatment. If these data are confirmed in long‐term studies, it would imply that the use of leucine/HPD in treating metabolic diseases is unlikely to have lasting negative effects on insulin sensitivity.High‐protein diets (HPDs) promote weight loss but other studies implicate these diets and their constituent amino acids (AAs) in insulin resistance. We hypothesized that AA‐induced insulin resistance is a temporal and reversible metabolic event. L6 myotubes were serum deprived for 4 h and then incubated in AA and/or insulin (100 nmol/L). Another group of cells was incubated overnight in AA + insulin, starved again, and then reincubated with AA and insulin. Mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) signaling and glucose uptake were then measured. Healthy or insulin‐resistant rats were gavaged with leucine (0.48 g/kg) and insulin sensitivity was examined. In myotubes, incubation with AA and insulin significantly ( Using in vitro and in vivo systems, we show that amino acid‐induced modulation of mTORC1/S6K1 signaling and serine phosphorylation of IRS‐1 in muscle cells/skeletal muscle are temporal and reversible. In addition, in both lean and obese rats, leucine‐induced impairment of whole body insulin sensitivity of glucose and amino acid is reversible within 24 h of treatment. Our data suggest that the use of leucine/amino acid in body weight management is unlikely to carry any lasting negative effect on insulin sensitivity. High‐protein diets (≥25% energy intake as proteins) , suppressor of cytokine signaling 1 (SOCS1) and SOCS3 are activated, leading to reduced phosphorylation of IRS1 Shepherd . In addiα‐Eagle's minimal essential medium (AMEM) and trypsin from Wisent ; and l‐leucine dehydrogenase from Calbiochem (EMD Millipore). Amino acid‐free medium (RPMI 1640) was obtained from US Biological . Glucose solutions and glucose/lactate buffers were obtained from Interscience .Fetal bovine serum and antibiotic–antimycotic preparations were purchased from Life Technologies . Immobilon Western Horseradish Peroxidase (HRP) chemiluminescence substrate was obtained from Millipore Corporation ; l‐leucine, phosphatase and protease inhibitor cocktails from Sigma‐Aldrich ; γ tubulin were obtained from Sigma‐Aldrich.Antibodies to phospho (ph)‐S6K1 (T389), total S6K1, ph‐IRS‐1 , total IRS‐1, ph‐Akt (T308), and HRP‐conjugated secondary antibodies (anti‐rabbit and anti‐mouse) were purchased from Cell Signaling Technology . Antibodies to μmol/L), for 30 min . In another experiment, myotubes were incubated in AA and insulin overnight. The next day, and to simulate a washout, the cells were starved for 4 h in serum‐free AMEM and then incubated in either AA or an AA‐free medium for 30 min, followed by incubation in insulin for an additional 30 min. Cells were then harvested and immunoblot analysis for the indicated antigens was carried out. In other experiments, after the incubation in AA and/insulin as described above, insulin‐stimulated glucose transport was conducted  were purchased from Charles River Laboratories Inc. . They were left to acclimatize for 1 week in the animal care facility at York University while being maintained at the standard 12:12‐h light–dark cycle at 22–23°C. They had free access to rat chow  and water. After acclimatization, the animals were handled 2–3 times per week in order to reduce stress due to handling on the day of the experiment. All experiments were approved by the York University Animal Care Committee and were performed in accordance with the Canadian Council for Animal Care guidelines.2O, 2.4 mL/100 g body weight). This amount is equivalent to 0.48 g leucine/kg, and represents ~40% of a rat's daily leucine consumption  injection was administered (0.75 U/kg body weight). Blood samples were collected at various times postinjection, and the resulting plasma analyzed for glucose , total BCAA . For rat studies, S6K1 and IRS‐1 data were analyzed by one‐way ANOVA, whereas glucose, BCAA, and insulin were analyzed by two‐way ANOVA and Tukey's multiple comparison test. Statistical significance was set at Incubation of L6 myotubes with a combination of AA and insulin increased T389 S6K1  and IRS‐1 serine phosphorylation  was conducted. Rats gavaged with leucine had significantly higher plasma blood glucose concentration only at 15 min from the initiation of ITT Fig. C.In young animals, insulin stimulates amino acid utilization for protein synthesis and suppresses proteolysis, leading to a decrease in plasma amino acid concentrations, especially those of the BCAA  phosphorylation was detected even in soleus muscles of fasted obese rats. Although these measures increased in response to leucine gavage, the values were not significantly different from basal Fig. A and B. Although plasma insulin was higher in obese relative to lean rats, leucine gavage had no significant effect on this hormone Fig. C.Fifteen minutes from the commencement of ITT in lean rats gavaged with water, plasma glucose was <50% of time zero value Fig. C. At equPlasma BCAA concentrations were higher in obese rats; in fact, the values in those gavaged with water were similar to the peak level seen in lean animals that were gavaged with leucine   can dephosphorylate IRS1 (Lazar and Saltiel In summary, we provide evidence that AA/leucine‐induced impairment of skeletal muscle insulin signaling and whole body insulin sensitivity of glucose in healthy rat is modest and reversible. In obese rats, although leucine administration further impaired insulin regulation of whole body glucose and amino acid metabolism, the effects were reversible. In studies that have attributed insulin resistance to high‐protein diet, leucine is thought to be the main mediator of this effect, therefore, our data have implications for human nutrition and management of metabolic diseases. While these data need to be confirmed in studies that examine the effects of long‐term high dietary protein intakes, our results suggest that the benefits derivable from the use of high‐protein diets by obese individuals are unlikely to be associated with worsening of insulin sensitivity.We thank Rolando Ceddia for guidance on glucose transport in muscle cells.None declared.


The purpose of this study was to examine whether the tendency to fear negative evaluation moderates the effect of fear emotion on the rejection of unfair offers in the ultimatum game (UG). A photograph of a fearful face or landscape was displayed subliminally  before the proposer’s offer in the UG was presented to participants. We used the Fear of Negative Evaluation Scale (FNES) to measure participants’ anxiety regarding negative evaluations from others. Results showed a significant interaction between FNES and condition (fearful face vs. landscape) in relation to the rejection of an unfair offer. Furthermore, the mean rejection rate of an unfair offer was significantly higher in the fearful face condition relative to that in the landscape condition among participants whose FNES scores were higher than the median; however, this difference was not observed in participants whose FNES scores were lower than the median. These results suggest that fear of negative evaluation moderates the effect of subliminal fear priming on the rejection of unfair offers in the UG, and that negative emotion induced by unconscious stimuli enhances rejection of these unfair offers. Factors leading to the rejection of unfair offers in the ultimatum game (UG)23479et al.31817Why do people reject unfair offers in the UG? Using the proximate factor explanation, many studies have shown that rejection is driven by responders feeling negative emotions411121314124351718Fear of negative evaluation from others has been examined in research on social anxiety conducted in the fields of psychiatry or clinical psychology24252728In this study, we used subliminal fear priming to evoke participants’ fear emotion by displaying a photograph of a fearful face for a duration that was too short to reach conscious awareness, and then compared rejection rates in this condition with that in the condition in which a landscape photograph was displayed. Furthermore, we used the Fear of Negative Evaluation ScaleWe predicted an interaction effect between FNES score and condition (fearful face vs. landscape) in relation to rejection of unfair offers in the UG, such that unfair offers would be more likely to be rejected in the fearful face condition relative to the landscape condition among people with a high FNES score.SD = 7.4). FNES scores did not differ significantly between men  and women , t (68) = 0.11, p = 0.911.The FNES demonstrated good reliability , and our participants’ mean score was 16.8  = 1.73, p = 0.088; ¥100/10% offer: female face: M = 0.73, SD = 0.45, male face: M = 0.80, SD = 0.40, t (69) = 1.52, p = 0.133. Therefore, we combined the rejection rates for female and male fearful faces to form an overall rejection rate for the fearful face condition.To examine the effect of the target’s sex on rejection rates, we compared mean rejection rates of unfair offers (out of ¥1000) for female and male fearful faces for each proposer’s offer. No effect of target’s sex was found: ¥200/20% offer: female face: F  = 0.044, p = 0.835) or order  = 1.363, p = 0.247), and the interaction between condition and order was nonsignificant  = 0.840, p = 0.363). Therefore, we concluded that the order of the proposers’ offers did not affect unfair offer rejection rates.We compared unfair offer rejection rates between the first and final six of the 12 trials completed, to examine the effect exerted by the order of proposers’ offers. A two-way analysis of variance revealed no significant main effects of condition  = 8.72, p = 0.004), condition  = 4.86, p = 0.031), and condition × FNES score  = 6.06, p = 0.016) were significant. However, the effects of FNES score  = 0.001, p = 0.977), offer × FNES score  = 0.68, p = 0.414), offer × condition  = 0.38, p = 0.543), and offer × condition × FNES score  = 0.38, p = 0.540) were nonsignificant.Mean rejection rates are shown in t test revealed that the mean rejection rate shown by participants whose total FNES scores were higher than the median (>17) in the fearful face condition  was significantly higher relative to that of the landscape condition , t (31) = 2.35, p = 0.025. However, this difference was not observed in participants whose total FNES scores were lower than the median (<17)  = 1.31, p = 0.198).As the interaction between condition and FNES score was significant, we combined rejection rates for the ¥100 (10%) and ¥200 (20%) offers and used this value as an indicator of the total mean rejection rate for unfair offers, before examining whether FNES scores differed according to the effect of subliminal fear priming on the rejection of unfair offers. A paired r = 0.286, p = 0.016; We then performed an additional analysis using the mean rejection rate for the fearful face condition minus that for the landscape condition as an indicator of the effect of subliminal fear priming on the rejection of unfair offers. Total FNES scores were positively correlated with the effect of subliminal fear priming on the rejection of unfair offers , who were recruited via posters distributed in classrooms; the monetary reward was emphasized at the point of recruitment. The methods were carried out in accordance with the approved guidelines. All experimental protocols were approved by the ethics committee at Tamagawa University and written informed consent was obtained from all participants.Participants were 70 undergraduate students .The timeline for each trial in the UG is shown in Subsequent to playing the UG, participants completed the FNES, which is a self-report scale that measures the tendency towards fear of social evaluation, and is used to assess social anxiety disorder in adults. The FNES contains 30 items, to which participants are required to respond ‘yes’ or ‘no’.How to cite this article: Takagishi, H. et al. Fear of Negative Evaluation Moderates the Effect of Subliminal Fear Priming on Rejection of Unfair Offers in the Ultimatum Game. Sci. Rep.6, 31446; doi: 10.1038/srep31446 (2016).


The interaction between the behavioral and physiological immune systems provides fertile ground for research. Here, we examine the interactions between fear of disease, collectivism/individualism, disgust, visual perception and salivary IgA. First, we parsed collectivism/individualism into ancestry and psychological processes and examined their relationships to fear of disease. Both ancestral and psychological collectivists scored higher on a test of hypochondria than individualists. Additionally, in two studies we exposed participants to slides of diseased, injured or healthy individuals. Diseased and injured stimuli were rated as equally disgusting, while diseased stimuli were rated as more disgusting than healthy stimuli. We measured salivary IgA in participants before and after they viewed the stimuli. Participants provided information on their ancestral collectivism or individualism. Salivary IgA levels increased after participants viewed images of diseased or injured individuals. Participants with collectivist ancestry tended to react to the diseased and injured images with an increase in IgA, while levels of IgA remained the same or decreased in individualists in one study but we failed to replicate the effect in the second study. An increased salivary IgA response to potentially diseased individuals is adaptive, because salivary IgA plays an important role in protecting individuals from contracting an infection. The response may be related to increased preoccupation with disease states. One of the most potent agents for natural selection is infectious disease , the disgust response and collectivism/individualism. As we gathered data on collectivism/individualism in Hawaii it became clear that the concept was multi-faceted. On the one hand, we theorized that an individual could be influenced by her close relatives through both a heritable tendency and through socialization processes resulting in collectivist or individualist behavior . On the other hand, an individual in a multi-cultural society like Hawaii, that combines elements from both the east and the west, will be confronted with both collectivist and individualist ideals through work, school and other social activities thereby being in a position to combine collectivist and individualist ideas .In the current series of studies, we tested hypotheses associated with pathogen avoidance. Specifically, in Study 1 we examined ancestral collectivism/individualism, whether an individual's maternal and paternal ancestors were from a collectivist or individualist society, versus psychological collectivism and individualism measured with Triandis’  collectiWe explored the relationship between ancestral and psychological collectivism/individualism and health behavior by examining their relationships to perceived susceptibility for disease and hypochondria.The sample consisted of 110 students attending University of Hawaii at Hilo (UHH) and Hawaii Community College (HCC). Participants completed a questionnaire that assessed age, ethnicity of both maternal and paternal grandparents, parents and participant's identified ethnicity. UHH boasts an exceptionally diverse student body, with many individuals of mixed ancestry and diverse ages. Due to this heterogeneity, participants were also assigned an ethnicity percentage. For example, if a participant reported her mother as Japanese, and her father as Caucasian, but identified herself as Japanese, she was coded as Asian, collectivist, and given an ethnicity value of 50%. If the above individual identified herself as Caucasian, however, she would have been coded as Caucasian, individualistic and assigned an ethnicity value of 50%. For certain analyses, we used only individuals of greater than 50% ethnicity. We felt that by concentrating on individuals with greater than 50% ethnicity, we could decrease the variance between individuals thereby increasing our statistical power to reveal differences between collectivists and individualists .Table 1N = 37), Asian (N = 39), Pacific Islander (N = 10), Hispanic (N = 4) and African-American (N = 2). Africans  and N = 107 for the individualism sub-scale (α = .74).Cronbach's alpha was used to compute internal consistency of all scales. The psychological collectivism/individualism scale was obtained from Triandis . The 32 α = .74). This was a different scale than the one used by Faulkner et al. . The scale that measured HB was obtained from an online site, Know Your Own Mind (www.tran4mind.u-net.com/hypo.htm). Participants answered 25 questions about their perceptions of their own health in a ‘Yes, Maybe, No’ format. A response of ‘maybe’ was always scored as ‘0’. ‘Yes’ and ‘no’ answers were scored so that the final score represented increased HB . The original internal consistency analysis was low (α = .58), so we performed a principal component factor analysis on the 25 questions in the HB scale and reduced the number of questions to the 11 shown in α = .62).The PVD scale was obtained from Duncan, Schaller, and Park . The 15-r et al.  and Navar et al.  but was r et al.  revealedF = 10.47; p = .002) but not on the PVD scale  = 1.86; p = .18) or either of its factors. Similarly, psychological collectivism was significantly correlated with the HB scale , the PVD scale and to a lesser extent the PVD scales' factors . On the other hand, neither HB (r = .04) nor PVD (r = −.06) was significantly correlated with psychological individualism. The scatter plots for the correlations of psychological collectivism and individualism with HB are displayed in N = 54), psychological collectivism was related to HB  but psychological individualism was not . The same pattern was found for ancestral individualists (N = 37); psychological collectivism was correlated with HB  but not psychological individualism . Age was significantly correlated with PVD  and its infect factor  but even when age was used as a covariate in the ancestral analysis the relationship remained non-significant  = 2.12; p = .14). HB and PVD were significantly correlated . Women scored significantly higher on the PVD (MN = 56.6 (SE = 1.44)) than men (50.8 (SE = 2.05); F = 6.86; p = .01)) and on its infect factor  = 7.58; p = .007) but not on the germ factor or psychological collectivism, individualism, HB or age.We used one-way ANOVAs to examine the effects of ancestral collectivism/individualism and gender on the disease vulnerability scales (HB and PVD), and simple correlations to examine the relationships between psychological collectivism and individualism on the scales. Participants with high ancestral collectivism scored significantly higher on the HB scale (MN = 12.56 (SE = 0.47)) than those with high ancestral individualism (MN = 10.47 (SE = 0.53); Study 2 examined whether exposure to a series of images of diseased versus physically injured individuals would result in increased levels of salivary IgA. Stimuli were rated by independent observers as equally disgusting. Additionally, we examined whether the responses of individuals from predominantly collectivist ancestry differed from individuals from predominantly individualist ancestry. We hypothesized that individuals with more collectivist ancestry should have heightened salivary IgA responses.n = 18), Asian (n = 14) or Pacific Islander (n = 6). Caucasians were classified as individualists, while Asians and Pacific Islanders were classified as collectivists  on a 7-point Likert scale, with 7 being ‘very disgusting’ and 1 being ‘not disgusting at all’ (MInjury = 3.5 (SD = 1.56); MDisease = 3.4 (SD = 1.19); F = 0.07; p = .80).Participants were randomly assigned to one of two conditions: the experimental condition, in which participants watched a slideshow of individuals afflicted with visually perceptible diseases . For IgA, the sensitivity was 2.5 µg/mL, the intra-assay coefficients of variation ranged from 4.49 to 6.99 and the inter-assay coefficients of variation ranged from 8.65 to 8.93. For cortisol, the sensitivity was less than 0.003 µg/mL, the intra-assay coefficients of variation ranged from 3.35 to 3.65 and the inter-assay coefficients of variation ranged from 3.75 to 6.41.Saliva was collected before and after each slideshow. Participants were asked to rinse their mouths with water prior to collection and then collect saliva through the passive drool method, allowing the saliva to collect on the bottom of their mouths and then easing the saliva into a tube. Participants sat on a futon behind a folding screen to collect saliva and were provided with paper towels to minimize embarrassment. Saliva was stored at −20°C until IgA and cortisol levels could be analyzed through enzyme-linked immunosorbent assay (ELISA). ELISAs were conducted with kits and procedures obtained from r = .52; p = .001). The other correlations were not significant (ps > .31). A repeated measures ANOVA (Ss/Group × Time) with age as a covariate was used to analyze the data on IgA levels. Time of measurement was statistically significant  = 14.2; p = .001) but Group (MNInjury = 64.1 (7.4); MNDisease = 62.2 (7.4)) was not  = 0.03; p = .86). Participants' salivary IgA rose significantly after they viewed either type of stimuli (MN = 65.8 (6.8)) from baseline levels (MN = 60.6 (5.4)). The interaction between Group and Time was not significant (Injury: MNBaseline = 60.8 (7.8), MNPostView = 67.5 (9.7); Disease: MNBaseline = 60.4 (7.8), MNPostView = 64.1 (9.7); F = 0.05; p = .82).Repeated measures ANOVAs (SPSS 20) were used to examine differences between Groups (Injury versus Disease) and to examine Collectivism/Individualism across the pre- and post-measurements. Simple correlations were conducted between potentially confounding variables. The simple correlations between possible confounding variables found one significant positive correlation between age and experimental IgA levels  = 2.23; p = .14). When only participants with greater than 50% ancestry were examined, there was a trend for the Time × Ancestry interaction  = 3.63; p = .07). Collectivists displayed increased IgA levels after exposure to the stimuli, while individualists displayed decreased IgA levels  = 47.6; p < .0001) but not the individualists' scores  = 1.28; p = .28). Cortisol levels did not differ across the time periods, across the groups or across collectivism/individualism.To examine collectivism/individualism and their relationship to change in IgA levels, we performed two analyses. The first analysis used data from all participants' perceived cultural identity and the second analysis used only participants who reported greater than 50% ancestral collectivism/individualism . We collA levels . After tIn Study 2, we controlled the disgust levels in the two sets of stimuli and found no difference between the disease and injury slideshows in eliciting a salivary IgA response. For Study 3, we partially replicated Study 2 but also wanted to use a set of control stimuli that were similar to the disease stimuli but not disgusting. Another difference between the two studies was that Study 2 was conducted in the autumn (September–November) and Study 3 in the winter/spring (February–April).N = 33), Asian (N = 47) or Pacific Islander (N = 14). One participant was Iraqi; she was classified as collectivistic, along with the Asians and Pacific Islanders. Caucasians were classified as individualists  rated the control stimuli (MControl = 1.3 (SD = 0.22)) as significantly less disgusting than the disease stimuli (MDisease = 3.4 (SD = 1.42); F = 35.4; p < .0001). Saliva samples were collected before and after participants viewed the slideshow. Samples were treated and analyzed similar to those in Study 2.Participants were randomly assigned to either the experimental or control condition. The experimental (disease) slideshow was not changed from Study 2; however, the control slideshow was replaced with images of visually healthy individuals in settings similar to those in the disease slideshow. Slides were shown in a random order, for five seconds each. In contrast to Study 2, an independent group of observers ; therefore, we did not use age as a covariate in the analyses. We conducted the same analyses as in Study 2. Time of measurement was not significant (MNBaseline = 37.3 (2.3); MNPostView = 38.05 (2.4); F = 0.15; p = .70) nor was the Group effect  = 0.42; p = .52) or interaction (Control: MNBaseline = 36.3 (3.3), MNPostView = 36.2 (3.5); Disease: MNBaseline = 38.3 (3.3), MNPostView = 39.9 (3.4); F = 0.17; p = .68). Additionally none of the main effects or interactions was significant when we examined participants from collectivist versus individualist backgrounds, regardless of whether we used all participants or only participants with >50% ancestral collectivism/individualism backgrounds (ps > .20).Repeated measures ANOVAs (SPSS 20) were used to examine the differences between Groups (Control versus Disease) and to examine Collectivism/Individualism across the pre- and post-measurements. Simple correlations were conducted between potentially confounding variables. Age was not correlated with IgA levels in Study 3 (et al. (People with collectivist ancestry could differ from those with individualist ancestries in their reactions to signs of possible disease in a number of ways. For example, they might display a sensitized disgust response, a larger stress reaction or heightened vigilance. Interestingly, Schaller et al.  did not We found that collectivists scored higher on a questionnaire measuring HB than individualists. This relationship was found whether we examined ancestral or psychological collectivism. Hypochondria is associated with an acute awareness and preoccupation with bodily functions; therefore, this attitude may predispose people to be more aware of, and reactive to, illness symptoms in themselves and others. It could be that hypochondria is simply an extreme form of an adaptive behavioral response. The relationship between collectivism, hypochondria and the immune system is deserving of further study.Individualists live in areas of the world that host fewer pathogens than the tropics (Fincher et al., In Study 2, people with collectivist ancestry displayed increased salivary IgA to a slideshow of diseased persons while people with individualist ancestry actually showed the opposite response, although the decrease was not significant. Although the simple effects for people from collectivist ancestry differed, there was only a trend for the interaction to be significant so these findings are equivocal. Additionally, we failed to replicate the findings in Study 3. At this point, the conservative conclusion is that there is no relationship between collectivism/individualism and the salivary IgA response. It has been hypothesized that collectivism was initially adaptive for persons living in environments with historically high parasitism rates (Fincher et al., The reported studies have several limitations but indicate the direction of future research. First, a more refined way of defining ancestral and psychological collectivism versus individualism would improve the research. One way to accomplish this would be to replicate the research in countries in which collectivists or individualists evolved and compare their responses to second or third generation immigrants of the same ancestry. Additionally, it would be interesting to follow individuals from collectivist and individualist backgrounds after they begin living in multi-cultural settings to examine whether adopting different attitudes toward family or egoism results in behavioral and/or physiological shifts. Second, we did not try to recruit equal numbers of collectivists/individualists because we were using students at UHH and HCC as participants. At this point in time, more women are attending university than men and, in Hawaii, especially at UHH, Asian students outnumber Caucasians. Future research could recruit from a more diverse pool of participants with the aim of achieving more balanced samples. Third, we examined participants at the height of the flu season in Study 3; it would be interesting to examine IgA responses across different time periods and seasons.


Helianthus annuus L.) cultivars interacting with three alternating temperature regimes and to select the most salt tolerant cultivars to plant in the saline region. Seeds were germinated in the isotonic NaCl and polyethylene glycol (PEG) solutions of −0.45, −0.90, −1.34, −1.79, and −2.24 MPa at 10 : 20, 15 : 25, and 20 : 30°C temperature regimes. Both NaCl and PEG inhibited germination, but the effects of NaCl were less as compared to that of PEG, which means that adverse effects of PEG on germination were due to osmotic effect rather than specific ion accumulation. For the three cultivars, higher germination occurred at 10 : 20°C in NaCl treatments and at 20 : 30°C in the isotonic PEG treatments. Among the three cultivars, Sandaomei (SDM) is the most tolerant to salt and PEG stress.Salinization has severe influences on agriculture in the whole world. The main aims of this work were to evaluate osmotic effect and ion effect of NaCl on seed germination of three sunflower ( Seed germination is a crucial phase in the life cycle of plants , which d Atriplex prostrata and Atriplex patula [ Aristida adscensionis and Artemisia ordosica [+ had positive effect that promoted seed germination of barley in previous study [Seeds of plant often germinate best under nonsaline conditions and their germination decreases with increase in salinity level , 6, 7. Sx patula  and Arisordosica . Howeverus study . A betteSunflower belonging to the family Asteraceae is the world's fourth largest oilseed crop , 17. And Polygonum aviculare [ Sarcobatus vermiculatus [ Allenrolfea occidentalis [ Aeluropus lagopoides [ Urochondra setulosa [ Sporobolus ioclados [Temperature can interact with salinity to affect seed germination. Although, higher salinity may inhibit germination, the detrimental effect of salinity is generally reduced at optimal germination temperatures. For example, inviculare  and Sarciculatus , decreasdentalis  and Aelugopoides . In othesetulosa  or Sporoioclados . Salinitioclados .Thus, the aim of the present study was as follows: (a) to determine the germination response of sunflower seeds to NaCl solutions; (b) to differentiate the osmotic effect from ion toxic effect through comparison of NaCl with the metabolically inactive osmotic agent PEG; (c) to evaluate the effects of salinity/PEG, temperature, cultivars, and in particular the interactions between these factors on germination percentage and germination rate in sunflower seeds; and (d) to determine the most tolerant sunflower cultivar that will adapt to the arid or semiarid regions.Helianthus annuus L.) cultivars Sandaomei (SDM), Daxin (DX), and Longkui (LK) were used as seed materials in this study. They are all widely planted cultivars in the study region. Seeds of SDM and DX were obtained from a saline soil in Jilin Province , northeast of China in October 2012, and seeds of LK were obtained from Institute of Crop Breeding, Department of Agricultural Science, Heilongjiang Province, in September 2012. Seeds were decorticated and achenes were stored at room temperature (23 ± 2°C) before use.Three sunflower  with temperatures of 10 : 20, 15 : 25, and 20 : 30°C (dark : light). Seeds were germinated under different osmotic potentials induced by two osmotica, NaCl, and polyethylene glycol (PEG) at −0.45, −0.90, −1.34, −1.79, and −2.24 MPa. Distilled water was used as control. Seeds were germinated in twofold of filter paper placed in Petri dishes (diameter 12 cm) with 15 mL of test solution. The dishes were sealed with parafilm to prevent evaporation. Four replicates of 25 seeds were used for each treatment. Germination was recorded every day for 7 days. Seeds were considered to have germinated when the radicle emerged (1 mm).G/t, where G is the percentage of seed germinated at 1 day interval and t is the total germination period [The rate of germination was calculated using a modified Timson's index of germination velocity (IGV) = ∑n period . The maxP < 0.05). These data were analyzed using SPSS . The regression analysis between germination rate and osmotic potential was carried out using Sigma Plot .Germination data was transformed (arcsine) before statistical analysis. A four-way analysis of variance (ANOVA) was carried out to test effects of main factors  and their interactions on the final germination percentage and rate of germination. A LSD test was used to determine least significant range between means (P < 0.05) individual effects of temperature, cultivar, water potential, osmoticum (NaCl/PEG), and their interactions on germination percentage and rate of germination . For cultivar LK, rate of germination in PEG treatments at 15 : 25°C was slightly higher than that at 20 : 30°C  NaCl solutions at 20 : 30°C. Flowers [Seed germination of cultivar SDM was 100% or nearly at above −1.34 MPa NaCl solutions at all three temperatures and 50% in −2.24 MPa (500 mM) NaCl solutions at 10 : 20°C Figures . A halop0 : 30°C  and only Flowers  also poi Flowers .NaCl and PEG both affected seed germination of sunflower severely; PEG had a greater inhibitory effect than NaCl for the three cultivars, especially in lower water potentials (−1.79 and −2.24 MPa). These results agree with the statements that NaCl had a less effect on the germination and seedling growth of cowpea than PEG  and seedP < 0.05) between the two osmotica, which means the osmotic effect plays the main role. In water potentials between −0.9 and −2.24 MPa, ions of NaCl have a positive role: the ions in the cells could be used as osmolytes to maintain cellular osmotic potential lower than that of the ambient environment, allowing water entry into the cells [+ and Cl− ions by the seed, maintaining a water potential gradient allowing water uptake during seed germination [+ concentration results of SDM can provide part of the evidence . As the water potential decreased beyond −2.24 MPa, the toxic effect appeared, because the germination decreased to zero. The physiological regulatory mechanism of sunflower in salinity conditions would be studied in further experiments.In this study, in higher water potentials , the germination was similar to the control and there were no significant differences  effects of temperature, cultivar, water potential, and osmoticum and their interactions on germination percentage and rate of germination  or lower (10 : 20°C) temperature and the opposite was observed at average temperature (15 : 25°C). LK also appeared to germinate more rapidly at 15 : 25°C than the other two alternating temperatures. This may be because cultivar LK is from Heilongjiang Province, which is in the north of Jilin Province and the annual temperature there is a little lower than Jilin Province. Thus, the optimum temperature for cultivar LK is lower.Western Jilin Province  in China belongs to the Northeast Farming-Grazing Interlocking District and arid and semiarid region, which was once a productive region but now has serious problems of salinization due to human activity frequently destroying the ecosystem equilibrium . The ave+ concentrations of the early seedling in the isotonic PEG and NaCl solutions were similar, while the Na+ concentrations of the early seedling in NaCl solutions were dramatically greater than the PEG solutions with the same water potentials. This implies that Na+ can enter the seed and act positive roles in seed germination.Supplementary Table: The K


The mean concentration of mercury was found maximum in the blood samples of older age group (62.8 µg/L) in dentists and (44.3 µg/L) in dental assistants. The comparison of mercury concentration among dentists, dental assistants, and controls (pairing based on their ages) revealed that the concentration increased with the age and experience among the dentists and dental assistants. Moreover, the mercury concentration in all the studied dental wastewater samples, collected from twenty-two dental clinics, was found to be exceeding the recommended discharge limit of 0.01 mg/L. Therefore, we recommend that immediate steps must be taken to ensure appropriate preventive measures to avoid mercury vapors in order to prevent potential health hazards to dentistry personnel. Strong regulatory and administrative measures are needed to deal with mercury pollution on emergency basis.The objective of this study was to assess the occupational exposure to mercury in dentistry and associated environmental emission in wastewater of Lahore, Pakistan. A total of ninety-eight blood samples were collected comprising 37 dentists, 31 dental assistants, and 30 controls. Results demonstrate that the dentistry personnel contained significantly higher mean concentration of mercury in their blood samples (dentists: 29.835  Mercury has been used in dental silver amalgam for the last two centuries , as a deThe dentist and their assistants have been occupationally exposed to different forms of mercury across the world , 9. MoreIn addition to this, studies have also reported that the use of mercury in dentistry is consequently associated with 10–70% of the total daily mercury load in the wastewater collection system . This coA total of 98 individuals comprising 37 dentists, 31 dental clinic assistants, and thirty control group individuals were sampled randomly, covering the diverse environment of Lahore City. Sampling took place in March and April 2015. Each individual was questioned and interviewed to get the information about their daily routine on the following variables, that is, age, gender, working hours, years of experience, nutrition habits , smoking, and amalgam filling per week . A basic+2 in HNO3 to other blood plasma samples to roughly double the original mercury concentration. All the measurements were assessed in µg/L and expressed in terms of total blood mercury, considering the plasma and erythrocyte ratio, 2 : 3 [Five milliliters of venous blood was collected in metal-free vacutainers. The blood samples were centrifuged at 1500 ×g for 20 minutes at 5°C. The packed erythrocytes and plasma were separated by means of a serum separator while plasma samples were subjected to mercury determination for further analysis . The samµg/L (<10 µg/L), individuals with mild exposure such as dentists as 10–15 µg/L, individuals with high exposure such as patients as 15–50 µg/L, and individuals with significantly higher exposure when the whole blood mercury level is >50 µg/mL (test ID: HG-8618).Finally, the whole population was analyzed against three levels of mercury as suggested by Mayo Medical Laboratories according to the exposure and effects. These levels are normal as 0–9 The wastewater samples were obtained from twenty-two dental clinics at the end of the working day having no mercury separation technique. The sampling was performed at two points, that is, the discharge point of dental wastewater into municipal wastewater collection system (grab samples) and the side-holding tank attached to dental chairs (mostly composite samples). Three replicate samples were collected from each sampling point on three consecutive working days. All the wastewater samples were collected and preserved in accordance with the standard methods of the American Public Health Association . The wasThe accuracy of mercury analysis was assessed using advanced mercury analyzer by running samples in triplicate. Recovery varied between 92.3 and 101.4%. A good agreement was found between the obtained mean and the certified value. Furthermore, 15% of the randomly selected samples were analyzed thrice in order to evaluate the reproducibility.p < 0.05) was performed. Among questionnaire variables, age, sex, working hours, years of experience, and number of amalgam filling were considered as independent variables, while mercury concentration in blood samples was considered as a dependent variable. The data was tested for the assumption of normality using the Kolmogorov-Smirnov test. In the end, the Correspondence Analysis (CA) was performed to describe the relationships of age groups with different levels of mercury as risk/exposure factor among dentists and dental assistants.The STATISTICA 7.0 software  was employed to perform the statistical analysis. The descriptive statistical parameters such as arithmetic mean, standard deviation, and the respective confidence limits were calculated for the blood mercury content and one-way ANOVA (p < 0.05).The descriptive statistics of mean mercury concentration in the blood samples of dentists, dental assistants, and controls is summarized in µg/L) was recorded in group 4 (51–60 years)  . The invfindings , 23, 24 findings –27.µg/L), followed by dental assistants (22.798 µg/L) and controls (3.276 µg/L). These high levels of mercury in dental personnel indicate the chronic accumulation of mercury in the blood of dentists and dental assistants due to their occupational exposure of elemental mercury vapors. A study from Pakistan revealed that 100% of the studied private dental clinics have significantly higher levels of mercury vapors in indoor air than ATSDR limit [Among the three working classes, that is, dentists, dental assistants, and controls , the maxDR limit . The inhDR limit , 29. ConDR limit , the merDR limit , the higDR limit . So the µg/L; however, the concentration was significantly higher in the remaining population, that is, 32 dentists (86%). Among these dentists, five individuals (16%) were found to have mercury concentration ranging within 10–15 µg/L (mild exposure); 20 individuals (62%) had mercury concentration within 15–50 µg/L (high exposure); and 7 individuals (22%) were found to have mercury concentration significantly higher than 50 µg/mL (significantly high exposure). The complete illustration is provided in According to the Mayo-derived standards, as explained previously, only five dentists (14%) were found to have mercury concentration lower than the devised limit, that is, 10 µg/L (mild exposure); 22 individuals (71%) ranged within 15–50 µg/L (high exposure); and 1 individual (3%) was found to have mercury concentration higher than the 50 µg/L (significantly high exposure)  had mercury concentration within the range, whereas 6 individuals (19%) ranged within 10–15 xposure) . Howeverxposure) .µg/L) was found in personnel with more than 10 years' work experience followed by those with 5–10 years' work experience (18.154 µg/L), and the lowest was found in those with less than five years' work experience (11.749 µg/L). Karahalil et al. [Higher blood mercury content was also significantly associated with dental personnel's daily working hours  which isl et al.  and Baell et al.  have alsµg/L) was recorded in personnel with greater than 10 fillings performed per week, followed by 5–10 fillings per week (32.156 µg/L) and less than 5 fillings per week (16.781 µg/L). Ritchie et al. [The number of amalgam fillings per week performed by dental personnel has shown an increasing trend for mercury concentration in blood samples . The hige et al.  found sie et al. , 32, 33.µg/L, which is relatively higher than the subjects having no amalgam fillings (µg/L), followed by those with two to five amalgam fillings (26.440 µg/L), and the lowest being among those with less than two amalgam fillings (19.463 µg/L).The subjects with own amalgam filling are found to have mean mercury concentration of 22.902 fillings . The resfillings , the higOverwhelmingly, the mercury concentrations in all the groups of dentists and dental assistants were significantly higher than in the control group. A similar trend was observed by different studies, whereby the mercury concentration in dentists was higher than in those individuals not occupationally exposed , 19, 26.The mercury concentrations released into the environment through wastewater discharges from dental clinics are depicted in Mercury concentrations in wastewater discharge point samples are of a similar magnitude as those reported by Welland  and wereµg/L in dental wastewater [This high release of mercury into the environment in the present study is mainly associated with residual noncontact amalgam and waste amalgam from filling removal, with no mercury recycling and/or separation activities involved. The residual noncontact amalgam can be easily recycled with generation potential of 211 mg of mercury/day/chair , which wstewater . The metstewater . The higstewater . Thus, tThe mercury concentrations in dental personnel are found to be significantly elevated than in controls, with the highest mean concentrations recorded in older and experienced dentists. The total mercury concentrations in all the dental wastewater samples were also exceeding limits in all samples. Thus, based on the present study, instantaneous steps shall be taken to safeguard the health of the dentists and dental assistants through appropriate preventive measures for mercury vapors by utilization of alternative filling material. As far as wastewater discharges are concerned, it is recommended to ensure the implementation of particular legislation and to deal with mercury in wastewater in Pakistan.


Few studies on spatial patterns or secular trends in human leishmanias have been conducted in Morocco. This study aimed to examine spatial patterns and trends associated with the human leishmaniasis incidence rate (HLIR) at the province/prefecture level between 2003 and 2013 in Morocco.Only the available published country data on the HLIR between 2003 and 2013, from the open access files of the Ministry of Health, were used. Secular trends were examined using Kendall’s rank correlation. An exploratory spatial data analysis was also conducted to examine the spatial autocorrelation , and spatial diffusion at the province/prefecture level. The influence of various covariates  on the HLIR was tested via spatial regression (ordinary least squares regression).CI = 12.9–13.1) for cutaneous leishmaniasis (CL) and 0.4 per 100 000 population (95 % CI = 0.4–0.5) for visceral leishmaniasis (VL). The available data on HLIR were based on combined CL and VL cases, however, as the CL cases totally outnumbered the VL ones, HLIR may be considered as CL incidence rate. At the provincial level, a secular increase in the incidence rate was observed in Al Hoceima (P = 0.008), Taounate (P = 0.04), Larache (P = 0.002), Tétouan (P = 0.0003), Khenifra (P = 0.008), Meknes (P = 0.03), and El Kelaa (P = 0.0007), whereas a secular decrease was observed only in the Chichaoua province (P = 0.006). Even though increased or decreased rate was evident in these provinces, none of them showed clustering of leishmaniasis incidence. Significant spatial clusters of high leishmaniasis incidence were located in the northeastern part of Morocco, while spatial clusters of low leishmaniasis incidence were seen in some northwestern and southern parts of Morocco; there was spatial randomness in the remaining parts of the country. Significant clustering was seen from 2005 to 2013, during which time the Errachidia province was a permanent ‘hot spot’. Global Moran’s I increased from 0.2844 (P = 0.006) in 2005 to 0.5886 (P = 0.001) in 2011, and decreased to 0.2491 (P = 0.004) in 2013. It was found that only poverty had an effect on the HLIR (P = 0.0003), contributing only 23 % to this (Adjusted R-squared = 0.226).At the country level, no secular variation was observed. Poisson annual incidence rate estimates were 13 per 100 000 population  contains supplementary material, which is available to authorized users. Please see Additional file Arrêté ministériel N° 683–95 (31 mars 1995)), a leishmaniasis control program  for CL and 0.4 per 100 000 population (95 % CI = 0.4–0.5) for VL. It is well known that CL cases significantly outnumber VL cases [Leishmania tropica, and to interrupt transmission of cutaneous leishmaniasis due to L. major in concerned foci, by the end of 2016” [Leishmaniasis is a vector borne disease, which has a wide clinical and epidemiological diversity with respect to leishmania parasite types, vector species, and reservoirs. In Morocco, leishmaniasis is a public health concern . Parasit in 1997 , and the in 1997 . Between in 1997 . PoissonP-values > 0.05)  and 1) urbanization, considering ‘province’ and ‘prefecture’ as the two degrees of urbanization; 2) population density; and 3) social factors, including ‘poverty rate’ and ‘vulnerability rate’ as covariates, were examined.2 [In this study, all of Morocco was considered see Fig. . The cou2 . It is sMorocco’s population was 30 079 000 in 2003, increasing to 32 951 000 in 2013 .Since the beginning of 2015, Morocco has been administratively subdivided into 12 regions. However, as the current study is interested only in the period of 2003–2013, the previous subdivisions , which cHuman leishmaniasis is an endemic disease that falls within the framework of a national Ministry of Health program related to vector-borne diseases . The SSHCovariates were chosen based on World Health Organization (WHO) guidelines  and dataHaut-Commissariat du Plan,” which is the Moroccan Census Bureau, and available for the years 2004 and 2007 [Poverty rate data and vulnerability rate data, by province and prefecture, were freely accessible from “and 2007 . Because8. For VL, a total of 1 480 events were observed [8. Variations in human leishmaniasis incidence over time by region .A free-license shapefile map of Morocco, comprising only 47 provinces/prefectures, was downloaded  and modiAccording to the Moroccan Census Bureau, during the 11-study period (2003–2013), 15 of the 59 provinces/prefectures have been split each into two provinces, which led the SSHHI-Ministry of Health to provide HLIR for more than 59 provinces . The incTo examine spatial patterns and spatial diffusion, the exploratory spatial data analysis approach –17 was aTo test the covariates’ influence on leishmaniasis incidence, a spatial regression analysis (ordinary least squares) was carried out (see Table P = 0.01), a secular time increase was observed in R15 (P = 0.008) and R16 (P = 0.02), and a secular time decrease was observed in R7 (P = 0.04). No secular time variation was observed in the remaining administrative regions (P ≥ 0.05)  was observed in region 13 (R13) in 2010  and the Taounate province (P = 0.04). In R16, the rate increase was evident in the Larache province (P = 0.002) and in the Tétouan province (P = 0.0003). In R13, it was evident in the Khenifra province (P = 0.008) and the Meknes province (P = 0.03). The rate decrease in R7 was evident in the Chichaoua province (P = 0.006), even though an increase was observed in the El Kelaa province (P = 0.0007). No secular time variation was observed in the uncited provinces (P ≥ 0.05). The yellow color in Fig. The increased incidence rate in R15 was evident in the Al Hoceima province . Significant clustering was seen from 2005 to 2013 (see Table P = 0.001). It seemed to be the main location from where leishmaniasis spread: from 2003 to 2007, high incidence clustering was observed in the north neighboring provinces of the Errachidia province, including in Figuig, Jrada, and Boulemane; in 2008, high incidence clustering disappeared in the north neighboring provinces and appeared in south neighboring provinces, such as Ouarzazate and Zagora , but further spreading, from 2010 to 2013, in the aforementioned south neighboring provinces all the way to southwestern neighboring provinces, such as Azilal and El Haouz .A total of 59 provinces and prefectures were considered for the spatial analysis. Moran’s I statistics over this period were examined. Dispersed high and low values were shown in 2003 , whereas random clustering was seen in 2004 . To further examine the effect of this covariate, a LISA cluster map of poverty rates . In 2013, the incidence rate was 3.2 per 100,000 population [In a previous study , Merione to 2012 . The pre to 2012 ) were obpulation  in the EMeriones shawi is not allowed [Algeria is a neighboring country of Morocco and the second largest focus of CL in the world after Afghanistan . Looking allowed  in Alger allowed . They we allowed , to the Clusters of low leishmaniasis incidence were seen in the southern part of Morocco, including in the Tan Tan, Tarfaya, Laayoun, Boujdour, and Essmara provinces, where incidence rates were as low as zero, which is consistent with findings of a study carried out in the 1990s that reported a scarcity of vector species and an absence of leishmaniasis cases . AnthropL. tropica reported to be transmitted by the sandfly P. sergenti (see Table L. tropica [L. tropica. However, if an unknown reservoir host was predominatly present in Moroccan provinces where CL due to L. tropica were reported [In Morocco, CL, due to  tropica , 33 has reported , an evidreported , howeverTo determine other potential factors that may affect leishmaniasis incidence in Morocco, the poverty rate, the vulnerability rate, population density, and urbanization at the province/prefecture level were examined. Only poverty was found to have a significant effect on leishmaniasis incidence, contributing only 23 % to this (see Table This study had limitations. Data related to human CL and VL cases, separately published by province/prefecture or by region, are not available. However, the distribution of different forms and species of leishmaniasis throughout the country is documented elsewhere , which hThe strength of this study is the description of the spatial patterns of human leishmaniasis incidence over an 11-year period in the whole of Morocco, including in the south of it where related information was lacking, the identification of provinces/prefectures showing either secular time increase or significant clustering, and the examination of some covariates that may influence human leishmaniasis incidence. All this was done by means of not-well-known scientific approaches in Morocco, such as exploratory spatial data analysis. Findings indicate the need for different stakeholders to address leishmaniasis in Morocco.New information on leishmaniasis incidence in Morocco was provided. This study highlights the importance of conducting spatial analysis as to determine where conducting studies on leishmaniasis and implementing vector and rodent control measures should be a priority. Further studies and new scientific approaches including risk assessment and GIS tools would identify underlying risk factors related to leishmaniasis; studying small areas and relying on data at the level of the individual, provided necessary data are available, would yield more information on risk factors that may affect leishmaniasis incidence in Morocco. This may be considered in future studies related to leishmaniasis. Poverty contributes to leishmaniasis in Morocco, which requires joint efforts of many stakeholders.Ethical approval for this study is not applicable as the data are publicly available.


Hardcore smokers are smokers who have smoked for many years and who do not intend to quit smoking. The “hardening hypothesis” states that light smokers are more likely to quit smoking than heavy smokers (such as hardcore smokers). Therefore, the prevalence of hardcore smoking among smokers would increase over time. If this is true, the smoking population would become harder to reach with tobacco control measures. In this study we tested the hardening hypothesis.We calculated the prevalence of hardcore smoking in the Netherlands from 2001 to 2012. Smokers were ‘hardcore’ if they a) smoked every day, b) smoked on average 15 cigarettes per day or more, c) had not attempted to quit in the past 12 months, and d) had no intention to quit within 6 months. We used logistic regression models to test whether the prevalence changed over time. We also investigated whether trends differed between educational levels.Among smokers, the prevalence of hardcore smoking decreased from 40.8 % in 2001 to 32.2 % in 2012. In the general population, it decreased from 12.2 to 8.2 %. Hardcore smokers were significantly lower educated than non-hardcore smokers. Among the general population, the prevalence of hardcore smoking decreased more among higher educated people than among lower educated people.We found no support for the hardening hypothesis in the Netherlands between 2001 and 2012. Instead, the decrease of hardcore smoking among smokers suggests a ‘softening’ of the smoking population. In the past decades, smoking prevalence has declined globally , and in hardening hypothesis [According to the pothesis , light spothesis , 5. In tIf the hardening hypothesis is supported, the portion of so-called ‘hardcore smokers’ in the population of smokers would have increased over the last years. Generally, hardcore smokers are smokers who have smoked for many years and do not intent to quit . CompareOn consumption, most studies agree that smokers can be classified as hardcore smokers if they smoke daily , 8–16 anIn this study, we chose a definition that was most similar to most of the wide variety of definitions that exist in the field. This way, the results from our study could be compared the findings of others. In addition, our criteria have been shown to be related to a lower likelihood of quitting . As a rePrevious studies suggest that smokers have hardened in some countries or within subgroups. Some found hardening among English adults from 2000 to 2010  and Norwhardcore smoker as well [Educational inequalities in smoking are widening in both the Netherlands and other European countries , 22. Als as well , 11. Pre as well . As a re as well , it woulThe current study is performed with data from 2001 to 2012 from the Netherlands. In the current study, we investigated whether the smoking population hardened in the Netherlands during the period 2001-2012. We also investigated whether differences in hardcore smoking existed between educational levels and whether these differences have changed over time. To identify such population trends, we used repeated cross-sectional survey data from a large nationally representative sample of the general population in the Netherlands.As smoking is predicted by education , the disWe used data from the Dutch Continuous Survey of Smoking Habits: a cross-sectional web survey that monitors the smoking habits of the Dutch population . RespondThe Central Committee on Research Involving Human Subjects in the Netherlands required no ethical approval for this non-medical survey research.We categorized respondents as non-smoker, non-hardcore smoker or hardcore smoker. We determined smoking status by asking: ‘Do you ever smoke or do you not smoke at all?’ Smokers were ‘hardcore’ if they a) smoked every day , 14, b) We assessed age, sex, employment, number of cigarettes per day and whether participants used roll-your-own cigarettes or factory-made cigarettes. We assessed highest attained education and categorized participants in three groups (Dutch names in brackets). Lower educated people either received primary education, lower secondary education (MAVO) or lower vocational education (LBO). Intermediate educated people received intermediate vocational education (MBO) or higher secondary education . Higher educated people had attained tertiary education .2-tests).First, we tested for groups differences between hardcore smokers and non-hardcore smokers on age (t-test) or any other characteristics . In the second step, we added a three-level ordinal variable for education. In the final step, we added interaction variables to test whether the prevalence of hardcore smoking had developed differently between educational levels. We controlled for age and sex, because age and sex are known predictors of hardcore smoking . As the In a secondary analysis, we investigated whether the trend in hardcore smoking would have been different if we had used another definition of hardcore smoking. Some studies did not use consumption to define hardcore smokers , 25. Theχ2  = 4.50, p = .034, φ = .007, and higher educated participants, χ2  = 4011.91, p < .001, φ = .189. Our weighted dataset of hardcore smokers, also included more women, χ2  = 4.48, p < .034, φ = .031, and higher educated participants over time, χ2  = 219.20, p < .001, φ = .169Table t(2873.61) = 3.23, p = .002, d = .104, and more likely to be lower or intermediate educated, χ2  = 108.50, p < .001, φ = .165. They were also less likely to be student and more likely to be unemployed or unable to work, χ2  = 78.23, p < .001, φ = .141. Finally, hardcore smokers were more likely than non-hardcore smokers to smoke roll-your-own cigarettes, χ2  = 333.53, p < .001, φ = .290. We found no significant differences in sex in 2012, χ2  = 3.78, p = .053, φ = .031.Table p < .001  as well. Second, the softening may be a result of changing social norms. Societal norms about smoking may have changed over time and this process might have increased quitting throughout the whole smoking population.Both explanations are in line with Rose’s theory, which states that tobacco control measures and social norms do not only influence light smokers, but the population as a whole . TherefoIn line with previous research , 11, 13,In line with other studies , 12, we A strength of our study is that we used repeated cross-sectional data from a large representative sample of the general population. This allowed us to examine trends in hardcore smoking among both smokers and among the general population. Because we had a large sample, we were also able to identify differences in trends between educational levels.trends in hardcore smoking. These trends may be more informative about future characteristics of the smoking populations than prevalence rates.A potential concern is the definition of hardcore smokers. Although several studies investigated the prevalence of hardcore smoking before, no clear definition of hardcore smokers currently exist. In our study, we therefore used a definition that is most comparable to other studies. As many studies have used different definitions, it is difficult to compare the prevalence of hardcore smoking between studies . By usinFuture research may focus on the use of e-cigarettes among hardcore smokers. A recent study showed that Dutch tobacco smokers are increasingly aware of e-cigarettes and that many have started to use them . If futuDespite the softening of the smoking population, about 8.2 % of the Dutch population is still a hardcore smoker. This group remains particularly vulnerable to death, disease, and lower quality of life. Therefore, interventions targeting hardcore smokers are still needed to further decrease the prevalence of hardcore smoking in the Netherlands . PreviouIn line with previous studies , our studecreased between 2001 and 2012. This suggests that the population of smokers has softened, instead of hardened. There was no support for the hardening hypothesis in the Netherlands. Among the general population, hardcore smoking decreased at a higher rate among higher educated people than among lower educated people. This may be explained by increasing educational differences in smoking among the general population.The prevalence of hardcore smoking among smokers