Salmonella isolates. The mean counts of AMB, TC, and S. aureus were log104.53, 3.97, and 3.88 log10cfu/g, respectively. Salmonella was isolated from 21 (70%) of the samples. Salmonella isolates in this study were highly susceptible to ciprofloxacin, gentamycin, and norfloxacin while they were resistant to erythromycin and tetracycline. High rate of multiple drug resistance was also noticed in Salmonella isolates. The microbial loads of meat were above the recommended microbial safety limits. Besides this, the isolation rate of Salmonella was high and high levels of drug resistance were documented for Salmonella isolates. Measures on handling and appropriate personal hygiene practices of workers in the retail shops are recommended to reduce the change of forborne disease outbreaks.The habit of raw meat consumption in addition to the poor hygienic standards and lack of knowledge contribute to food-borne diseases outbreaks. The objective of this research was to assess the bacterial quality and safety of fresh meat from retail Bahir Dar City, Ethiopia. A total of 30 fresh meat samples were collected from butcher shops. Standard bacteriological methods were used to isolate and enumerate bacteria. Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility testing of Meat is consumed by many people worldwide because of its nutritive composition. The protein profile of meat consists of amino acids that have been described as excellent due to the presence of all essential ones required by the body . It is c Salmonella, E. coli, and Shigella [In Ethiopia, minced or raw beef consumption is usually used for the preparation of a popular traditional Ethiopian dish known as locally “KITFO” and mostly it is consumed raw or partially cooked. This habit is a potential cause for food-borne illnesses in addition to, the common factors such as overcrowding, poverty, inadequate sanitary conditions, and poor general hygiene . The micShigella –10. Salmonella isolates from retail shops of Bahir Dar City, Ethiopia.The absence of organized slaughter house facility and the existence of small retail outlets have been the two biggest hurdles for hygienic production of meat . It is e2 and there are around 256,999 people living in there [This study was conducted in May 2015 in Bahir Dar town, which is the capital of Amhara National Regional State (ANRS) situated in the northern part of Ethiopia. Bahir Dar is located at 11°36′N latitude and 37°23′E longitude and has an elevation of 1840 m above sea level. The area of the town is about 160 kmin there . Salmonella. A total of 30 retail cut meat samples were collected from 30 purposively selected retail houses between 7:00 and 9:00 am. One kilogram of cut meat was aseptically collected with sterile glove and placed in a sterile glass beaker covered with aluminum foil. The samples were transported to the laboratory in ice box and bacteriological analysis was done within two hours of collection at Food Microbiology Laboratory of School of Chemical and Food Engineering, Bahir Dar University. The ambient temperature at the time of sample collection was 20°C.A cross-sectional study was conducted in retail meat shops to determine bacteriological quality and antibiogram of−2–10−4) were made from the homogenized sample and 1 ml from each sample of each dilution was taken and used for enumeration of aerobic mesophilic bacteria (AMB), total coliforms (TC), and S. aureus and the remaining homogenate was used for the isolation of Salmonella.Twenty-five grams of meat sample was mixed with 225 ml of 0.1% buffered peptone water  and homogenized for 2 minutes by using stomacher  . Tenfold Staphylococcus aureus count, samples were spread-plated in triplicate plates of Mannitol Salt Agar  and incubated at 30–37°C for 48 hrs and yellow colonies were counted [Enumeration of aerobic mesophilic bacteria was done using the pour plate techniques on plate count agar . One ml of homogenized sample was inoculated onto plate count agar, in triplicate and the plates were incubated aerobically at 32°C for a maximum of 48 hrs. After incubation, the plates having 30–300 colonies were counted using colony counter. Uninoculated media were incubated as negative control to check for sterility . Violate counted , 15. Salmonella (red colonies with or without black center) were picked and stored on Tryptic Soya Agar (TSA) slants for further purification and used for biochemical characterization and antimicrobial susceptibility tests [The homogenized sample was incubated at 37°C for 24 hrs and 1 ml of culture was transferred to 10 ml of selenite cysteine broth (SCB)  and incubated further at 37°C for 24 hrs. A loop full of culture from selenite cysteine broth culture was subcultured onto Xylose lysine deoxycholate (XLD) agar  plate and incubated aerobically at 37°C for 24 hrs. After incubation, 2-3 characteristic colonies ofty tests .μg), norfloxacin , amoxicillin , ampicillin , chloramphenicol , erythromycin , gentamicin , nalidixic acid , trimethoprim-sulfamethoxazole , cefoxitin , and tetracycline  . Morphologically identical 4–6 bacterial colonies from overnight culture were suspended in 5 ml nutrient broth and incubated for 4 hrs at 37°C. Turbidity of the broth culture was equilibrated to match 0.5 McFarland standards. The surface of Mueller-Hinton agar plate was evenly inoculated with the culture using a sterile cotton swab. The antibiotic discs were applied to the surface of the inoculated agar. After 18–24 hrs of incubation, the diameter of growth inhibition around the discs was measured and interpreted as sensitive, intermediate, or resistant according to Clinical and Laboratory Standards Institute [ E. coli ATCC 25922 was used as quality control for antimicrobial susceptibility tests.In vitro antimicrobial susceptibility tests were performed on Mueller-Hinton agar  using Kirby-Bauer disk disc diffusion technique . The ant Salmonella prevalence and antimicrobial susceptibility tests were expressed in terms of percentage.Data were analyzed using the statistical package for Social Science (SPSS) version 20 software by descriptive statistics. Results of bacterial counts were expressed in terms of mean log cfu/g and compared with Gulf Standards, 2002  Table 1Table 1. 10cfu/g having a mean value of 4.53 log10cfu/g  were resistant to two or more antibiotics. Five of the isolates were resistant to three or more antibiotics  were positive forxic acid . Among 2ibiotics . Salmonella isolates, all the Salmonella isolates showed high level of sensitivity (95–100%) to gentamycin, ciprofloxacin, norfloxacin, and chloramphenicol while high levels of resistance (66–90%) were documented against erythromycin and tetracycline. From a study done in Ethiopia, Reda et al. [. This could be due to the fact that ciprofloxacin and norfloxacin are relatively expensive and newly introduced, compared to the other common antibiotics. The routine practice of giving antimicrobial agents to domestic livestock as a means of preventing and treating diseases, as well as promoting growth, is an important factor in the emergence of antibiotic-resistant bacteria that are subsequently transferred to humans through the food chain [ Salmonella are acquired by eating contaminated foods of animal origin [With regard to the antimicrobial susceptibility profiles ofa et al.  and Farza et al.  reportedod chain , 32. Mosl origin , 34. S. aureus, and coliform counts which are above the recommended microbial safety limits. High bacterial loads and isolation of drug resistant Salmonella suggest a potential health risk to the consumers from the consumption of raw meat. These indicate poor handling and personal hygiene practices of workers in the retail shops and risk of food-borne disease. Investigation on antibiotic use in animal and animal feed is recommended.This study revealed high level of contamination in fresh meat as indicated by high aerobic mesophilic,


MBL polymorphisms and TB has been studied in various populations, the results are controversial. The aim of this study was to investigate mannose-binding lectin (MBL) gene polymorphisms with susceptibility to pulmonary tuberculosis (PTB) in a Lur population of Iran.Tuberculosis (TB) is caused by infection of Mycobacterium tuberculosis. Host genetic variability is an important determinant of the risk of developing TB in humans. Although the association between MBL gene polymorphisms  were genotyped by using PCR Single Strand Conformation Polymorphism (SSCP) technique in a Lur population living in Lorestan Province, consisting of 100 patients with pulmonary tuberculosis (PTB) age and sex matched 100 healthy controls (HCs). Association analyses were performed with the SPSS 21 statistical software.In this case-control study, four functional MBL (HH) genotype polymorphism significantly was associated with increased susceptibility to TB . Additionally, H allele showed a significant association with increased risk of TB . Also, the distribution of L allele in patients was significantly lower frequency in TB patients compared to controls . However, the allelic and genotypic frequencies of AB, XY and PQ polymorphisms were not significantly different between the patients and the controls. We couldn't detect any significant differences between haplotypes among TB patients and healthy controls.We found that HH genotype and H allele may increase the susceptibility to pulmonary TB in the Lur population of Iran, although L allele may decrease the susceptibility to pulmonary TB in this population. We suggest that it is necessary to further more studies with larger sample size and other ethnic population.Our findings demonstrated that  Furthermore, Host genetic variability is an important determinant of the risk of developing TB in humans MBL polymorphisms and susceptibility to active TB.Mannose-Binding Lectin (MBL) is a member of the collectin family that recognizes pathogens by its carbohydrate-recognition domains MBL2 gene (10q11.2-q21) encodes for the mannose binding lectin and presents several polymorphisms, four of which are known for their functional effect AB (rs1800450)), and in the promoter and 5′-untranslated regions (nt − 550 HL (rs11003125), nt − 221 XY (rs7096206), and nt + 4 PQ (rs7095891)) of the MBL2 gene are associated with serum levels and/or functions of MBL. Many studies showed these genetic variations are associated with a wide variety of diseases, including respiratory tract infections MBL polymorphisms may contribute to pulmonary tuberculosis susceptibility.MBL gene polymorphisms and the susceptibility to PTB. However, in previously reported studies MBL2 polymorphisms have conflicting results showing protection from or susceptibility to TB MBL2 sequence variants and TB was observed AB were associated with increased susceptibility to TB in Chinese origin YX were associated with increased susceptibility to TB among Chinese HL, but not YX, PQ or AB, were associated with decreased susceptibility to TB among Chinese MBL polymorphisms to PTB susceptibility, but the findings have been contradictory.Previous studies have not reached a consensus regarding the association between the MBL polymorphisms and TB.Conflicting results are not unexpected in association studies for several reasons, including small sample size, marginal statistical significance, detection of genotypes, or ethnic heterogeneity. Therefore, here we conducted a genetic association case-control study in a Lur population of Lorestan origin, to define the association between the 22.1The study was approved by the Ethical Committee of Lorestan University of Medical Sciences and Informed consent was taken from all subjects before blood sampling and questionnaire investigation. All subjects agreed to take part in the study. The patients included in this study were newly diagnosed PTB patients registered in the health center of Khorramabad city of Lorestan Province from January 2016 to January 2017. Cases were 100 unrelated Lur individuals selected with newly diagnosed pulmonary tuberculosis, positive sputum smear and/or culture and significant symptoms of typical PTB, chest radiography consistent with active disease. Then, patients with any autoimmune disease, chronic renal failure and any chronic inflammatory disease were excluded. The control group composed of healthy individuals who matched in age and gender. Controls were 100 unrelated Iranian individuals of the same race and geographic region. None of the controls showed any clinical manifestations of PTB at the time of blood sample collection, and no evidence of prior PTB noted on chest radiography. The healthy control group composed of healthy individuals who matched in age and gender with the patient group. Additionally, all study subjects had parents of the same race.2.2MBL genetic polymorphisms in the Iranian population, α and β errors. We used a value of at least 10% for the prevalence of polymorphisms, an α error of 0.05, a β error of 0.2, a 10% expected difference, and match ratio of 1:1. The minimum sample size was estimated to be 100 for the PTB group and 100 for the HC group.The sample size was determined by the following factors: the known prevalence 2.3MBL gene polymorphisms, that previously suggested by Wu et al. MBL gene, the AB, HL, PQ and XY polymorphisms were detected in patients and controls using their genomic DNA. PCR amplifications were performed using purified DNA Mastercycler  in 25 μl reaction volumes. Thermocycling parameters were as follows: 94 °C for 4 min and 35 cycles of 94 °C for 30 s, 59 °C for 60 s and 72 °C for 45 s, with a final extension at 72 °C for 5 min. PCR amplification products were analyzed on a polyacrylamide gel to detect alterations in PCR-amplified products. The primer sequences were previously reported A case-control study was conducted in 100 pulmonary tuberculosis (PTB) patients and 100 healthy controls (HCs). Blood samples were collected from all subjects. We extracted genomic DNA from 5 ml Venous blood samples using the QIAmp kit  according to the manufacturer's protocol. Polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) technique were used to study the frequencies of 2.4AB, HL, PQ and XY polymorphisms were ascertained by direct counting in the PTB patients and control group. Departure from Hardy-Weinberg Equilibrium (HWE) of All polymorphisms frequency was assessed by an exact test in both patient and control groups. Data were managed and analyzed using SPSS 21 program. We calculated differences in the genotypic and allelic frequencies of AB, HL, PQ and XY polymorphisms between the cases group and control group by Chi-square test and Fisher's exact test The genotypic and allelic frequencies of 3A total of 100 cases with smear-positive PTB and 100 healthy controls were recruited to the study. The mean age of cases was  and the mean age of controls was . 46 individuals of cases were males and 54 individuals were females, and 58 individuals of controls were males and 42 individuals were females.MBL polymorphisms in all subjects did not deviate from the Hardy-Weinberg equilibrium (P > 0.05). The results of the association between the MBL AB (+ 230) (rs1800450), HL (− 550) (rs11003125), PQ (+ 4) (rs7095891) and XY (− 221) (rs7096206) genotypic and allelic frequencies and the risk of PTB were listed in The genotype distribution of HH, HL, LL genotype frequencies of the MBL gene were 35%, 43% and 22%, while these were 22%, 48% and 30% in healthy individuals respectively. The genotypic frequencies of MBL AB, PQ and XY polymorphisms did not have significant difference between the PTB and the controls. Based on the analysis of loci, we found that MBL (HH) genotype polymorphism significantly was associated with increased susceptibility to TB . Additionally, H allele showed a significant association with increased risk of TB . Also, the distribution of L allele in patients was significantly lower frequency in TB patients compared to controls . For MBL-HL (rs11003125) polymorphism, the genotype (HH) and H allele increased PTB risk by 1.909-fold and 1.525-fold respectively among patients compared with controls. Also, the L allele decreased PTB risk by 0.656-fold among patients compared with controls  (rs1800450), HL (− 550) (rs11003125), PQ (+ 4) (rs7095891) and XY (− 221) (rs7096206) and PTB susceptibility in this population. In our study we showed that MBL (HH) genotype and the H allele are involved in the susceptibility to TB development in a Lur population of Iran. A significant association was found for L allele to resistant against the pulmonary tuberculosis. The genotypic and allelic frequencies of MBL AB, XY and PQ polymorphism showed no correlations with PTB susceptibility By contrast, our study did not replicate previous studies of associations between MBL polymorphisms and susceptibility to PTB.Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. However, a relatively small proportion of people infected with Mycobacterium tuberculosis will develop TB MBL gene polymorphism and pulmonary TB infection MBL-AB polymorphism, affect the serum level as well as configuration and function of MBL more significantly than other MBL-AB gene polymorphism is more common than the other BB genotype may be associated with susceptibility to TB, since they observed a significant increase in the frequency of BB genotype in TB patients than controls. Additionally, Capparelli et al. BB genotype among patients (22.3% vs. 3.5%in controls) in an Italian population. Our results are inconsistent with some previous studies, a possible explanation could be their ethnic differences.The relation between MBL gene polymorphisms and susceptibility to TB has been studied in different populations. The effect of low MBL levels on TB has been controversial. Some studies reported low MBL levels as associated with protection against the disease, whereas others estimated a relation with increased susceptibility MBL2 variants, either structural alleles (codons 54) in Gambian children MBL genotype in Caucasian patients compared to control subjects. The same tendency was also observed in patients of other ethnic origin. The authors hypothesized that heterozygosity for MBL2 variant alleles, responsible for low serum MBL levels, was associated with protection against clinical TB. Studies in Danish patients MBL2 polymorphisms at the codons 54 and susceptibility to TB. A large study in India showed that a significantly increased genotype frequency of MBL mutant homozygotes was seen in pulmonary TB patients as compared with that in control subjects On other hand, some studies have shown that MBL deficiency protects against disease caused by M. tuberculosis MBL (HH) and (HL) genotype was associated with susceptibility to TB, although in our study MBL (HL) genotype were not associated with susceptibility to pulmonary tuberculosis Our results showed similar outcome as a study that carried out in PTB patients of Chines. In Wu et al. study, the BB genotype frequency distribution was similar between patients and controls, but the B allele, as well as the AB genotype, was significantly more frequent in TB patients than healthy controls; the homozygous AA genotype, responsible for higher levels of circulating MBL proteins, was more frequent in controls than in TB patients AB MBL2 variant in TB patients and healthy controls in a population from the Amazon region (Northern of Brazil), without finding any evidence of association between this polymorphism and TB AB gene polymorphism, significant association was found in the dominant model , homozygote comparison , and B vs. A . The results suggested that individuals with B allele may have an increased risk of TB as compared with wild type AA homozygotes in Chinese population. In their study no significant association was found between MBL2 + 4 PQ gene polymorphism and the risk of TB. They suggests that individuals carrying the MBL2 codon 54 B allele may have an increased risk of TB as compared with AA homozygous, whereas MBL2 + 4 PQ gene polymorphism is possibly not associated with TB in Chinese population In daCruz et al. study, the TB is a multifactorial disease and its susceptibility depends not only upon host genetic aspects, but also on mycobacteria characteristics and on interactions with environmental factors. In addition, different genetic backgrounds and environmental factors may also influence the results. There is no doubt about the difficulty in comparing results from studies conducted in different populations, even when the same allele or haplotype are analyzed and the same study design is used. These paradoxical findings could be due to the Ethnic difference, different between genotyping technique, criteria for sample inclusion and exclusion and the small sample size of this study and could be caused by gene-environment interaction, gene-gene interaction, and gene-agent interactions 5MBL polymorphisms suggests that MBL (HH) genotype, H and L allele associate with PTB risk in the Lur population of Iran. For better understanding of the association between these polymorphisms and the risk of TB, larger sample size and more studies, especially those investigating haplotypes as well as gene-gene and gene-environment interactions, are required in the future. Further studies are required to determine how MBL polymorphisms influence susceptibility to Mycobacterium tuberculosis infection.Our comprehensive analysis of The authors declare no conflict of interest.


Neotomodon alstoni is fed a regular rodent chow, some individuals develop obesity, representing an opportunity to compare the effects of spontaneous obesity upon the circadian organization in this species with that observed in other rodents with induced obesity. We report differences in the free running circadian locomotor activity rhythm and in the effects of light pulses between lean and obese mice. Also, the photo-induced expression of the c-Fos protein and vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus (SCN) were examined at circadian time (CT) 14 and 22. We show that obese mice have a larger dispersion of the period of circadian locomotor rhythm in constant darkness. Photic induced phase shifts are nearly 50% shorter at CT 14, and 50% larger at CT 22 than in lean mice. The photoinduction of VIP in the SCN at CT 22 was larger in obese mice, which may be related to the differences observed in photic phase shifting. Our work indicates that the obesity in Neotomodon has effects on the neural mechanisms that regulate the circadian system.Obesity is a growing problem worldwide with a clear impact on health status. It is also a condition that negatively affects circadian rhythms. When the mouse  In natural conditions, entrainment of the clock by external periodic signals is an evolutionary advantage, because it allows rhythms to adapt and anticipate natural periodic changes 23. Light2thalamus , 6. Undethalamus . These bthalamus .Interactions among central pacemaker, peripheral oscillators, and the external environment converge in entrained output rhythms . Recent 91011131416171819ob/ob mice, genetically unable to produce leptin, displayed a larger photic phase shifting than wild-type mice [clock mutant mice that show altered metabolic responses [db/db mice, lacking the long isoform of the leptin receptor, display longer free-running period and increased photoinduction of c-Fos during the late night than db/+ mice do [Obesity has received particular attention from researchers, mainly because this condition has greatly increased worldwide and has been linked to an environment that offers an abundance of calorie-rich foods and few opportunities for physical activities , 22. Divype mice , while iype mice . Finallyesponses  also shoesponses . Genetic mice do .In mammals, glutamate is the main neurotransmitter involved in light transduction of the circadian response ; howeverNeotomodon alstoni, is an advantageous model for a variety of behavioral and physiological studies, including circadian rhythms and its relation to obesity [40N. alstoni offers a different perspective, since some of these mice develop obesity with standard rodent diets, while others remain lean. As they grow, almost half of the population – either wild-captured or F1 offspring – becomes progressively overweight and eventually obese after 4–6 months. The body weight of these obese specimens is almost twice that of lean mice, indicating a probable biological predisposition for overweight in some sector of the population [N. alstoni born in vivarium conditions and we address the mechanisms of such differences by studying the photoinduction of c-Fos synthesis in the SCN, as observed by others [Diverse animal models offer unique and powerful experimental approaches. They allow us to understand the underlying mechanisms that relate metabolic disorders to circadian disruption and to develop therapeutic strategies , 36, 37. obesity 4041 and besity 40. In thispulation , 40. In y others , 23 as wy others , 34.Neotomodon alstoni born and raised in captivity were used. All animals were held under a 12 h: 12 h light-dark (LD) cycle . Mice were individually housed in cages containing wood chips. Food chow  and tap water were provided ad libitum. For 6 months after weaning, body weight  was followed in male offspring (F1), and the animals were then separated into two groups: lean (BW = 45 ± 3 g) and obese (BW = 76 ± 4 g)  Figure . Mice be) Figure . All proAt the end of the study, mice were deeply anesthetized with sodium pentobarbital (50 mg/kg) and perfused transcardially with 50 ml of saline solution (0.9%) followed immediately by 50 ml of 4% paraformaldehyde in 0.1 M phosphate buffer .Activity in a non-wheel running environment was continuously monitored using a light beam detection system and collected with data acquisition software  as described elsewhere . Each beThe phase shifts were tested at circadian times  according to a previous work  considerCircadian changes of locomotor activity rhythm were analyzed in double-plotted actograms employing Actiview software , and significant circadian periods (p < 0.05) were obtained with the Periodogram analysis provided by CHRONOSFIT® Software . Phase s2O2 . After this, sections were mounted on poly-D-lysine  slides and coverslipped with CC/mount .Lean and obese mice were treated as in the phase-shifting protocol described above at CT 14 or CT 22. A control group was euthanized without receiving light pulse  while mice in the experimental group, were exposed to a 1-h pulse of fluorescent (200 lx) white light . After perfusion, brains were removed and post-fixed in paraformaldehyde solution for 24 h, then transferred successively to 10, 20, and 30% sucrose solutions in 0.1 M PB. Frozen coronal sections of 40 μm were cut with a cryostat. There are no stereotaxic studies of the brain of this rodent species; therefore, as an approximation to the medial SCN, we examined slides above the optic chiasm used as a reference. Sections were serially collected; one set of slides was processed with the c-Fos antibody  and other series with VIP antibody . Slides were incubated for 72 h at 4°C in PB saline (PBS), 1% of Goat Serum, and 0.3% Triton X-100 (PBSGT). Tissues were rinsed three times for 10 min in PBS and then incubated for 2 h with biotinylated secondary antibody  1: 5000 in PBSGT. Slices were rinsed three times for 10 min and incubated for 2 h with the Vectastain Elite ABC Kit  and washed three times again for 10 min in PBS. The tissue was incubated with diaminobenzidine  and H2. VIP staining was also evaluated in each slide as the average optical density of 25 consecutive 100-µm radius fields, distributed in the SCN area, by using Image-J software . Background optical density was established in the optic chiasm and subtracted from each of the samples; average of all fields is reported as optical density.After IHC, slides from each experimental condition were inspected and photographed with a microscope  coupled to a digital camera (Axiocam Erc53), and the software Axiovision 4.8  was used for further analysis. The c-Fos immunoreactive cells in the SCN were evaluated by visually counting stained nuclei on each side of the SCN in a gridded area of about 90,000 µmNeotomodon offspring had a body weight increase similar to that of the wild mice observed before [th month. Figure th month. At this age, significant differences were noted in epididymal fat . However, a larger dispersion of τ was observed in constant darkness in obese than in lean mice . Day-to-day onset stability in DD in free run (absolute difference in minutes) revealed a more stable onset in lean than in obese mice , but the difference was not statistically significant.In order to address mechanisms of photic resetting, we tested the magnitude of phase shift at CT 06, CT 14 and CT 22. Representative examples of actograms are shown Figure . Gray sqNeotomodon mice show different photic induction of c-Fos in the SCN, we used immunohistochemistry to explore differences between obese and lean mice after a light pulse at CT 14 and CT 22. Figure To determine if the obese Figure Neotomodon alstoni mice, excluding any influence of wheel running on circadian rhythms [ad libitum as it occurs in wild mice (Figure Metabolic dysfunction has recently been related to the circadian timing system, prompting intense interest in the linkage between these two areas of physiology research 1718. Alt17 rhythms , 47. VivN. alstoni mice show different changes in the photic response, reducing delays at CT 14 and increasing the advances at CT 22, which are different from those observed in other rodent models, such as mice fed high fat diet that present a decrease in the advance zone [ob/ob mice in which phase shifts of locomotor activity are larger in the delay zone [db/db mice at CT 22 [Neotomodon or the way in which overweight occurs. Some individuals of Neotomodon mice become obese after six months of regular chow diet; therefore, obesity developed by consuming a high fat diet in few weeks may have a different impact on the circadian system than obesity achieved by regular diet.Research in rodent models that closely mimic the changes in humans are essential . Obesitynce zone  or in thlay zone . Howeverat CT 22 . Therefoat CT 22 ; we shouin vitro of the rat’s SCN indicate that leptin changes the membrane potential and the spike shape, inducing phase advances in the firing rate [Per transcription in mice, suggesting that leptin also exerts a positive feedback effect on the circadian pacemaker [Neotomodon, since previous observations indicate high levels of leptin at late subjective night [Neotomodon [Neotomodon mice, to see if there is some relationship with the differences in phase shift induced by light.Obese mice present hyperleptinemia and some other metabolic and neural changes related to food intake 4041. Exp40ing rate , 11. Furacemaker . It is ive night . It is pThe number of cells expressing c-Fos in lean mice is different between CT 14 and CT 22 Figure  because 5051in vivo and in vitro [Neotomodon SCN may show changes along the day (Figure Neotomodon should be tested. In a previous study, we observed differences in VPAC2 expression [Neotomodon [At postnatal ages, a circadian rhythm in VIP expression has been observed in the SCN neurons of rats in vitro –54, but in vitro . Our resy Figure . Obese mpression  as well otomodon . In the otomodon . Hence, otomodon , and it Neotomodon alstoni affects the way in which the circadian pacemaker oscillates in constant conditions and how it responds to photic stimulation. Our results contrast with other models of obese rodent tested. The advantage of studying the mechanisms affected in a species where obesity is somehow “spontaneous” is the comparison with other animal models in which obesity is induced by genetic selection or nutritional manipulations. It would also be important to consider if the changes observed are present in other rodent models where some individuals, even within the same strain, show resistance to protocols of diet-induced obesity [60Together, the results presented here suggest that the obesity condition in  obesity 6061. Stu


Philaenus spumarius, Aphrophoridae) and leafhoppers  taking off from smooth (glass) and rough  surfaces. On glass, the propulsive hind legs of Philaenus froghoppers slipped, resulting in uncontrolled jumps with a fast forward spin, a steeper angle and only a quarter of the velocity compared with jumps from rough surfaces. By contrast, Aphrodes leafhoppers took off without their propulsive hind legs slipping, and reached low take-off angles and high velocities on both substrates. This difference in jumping ability from smooth surfaces can be explained not only by the lower acceleration of the long-legged leafhoppers, but also by the presence of 2–9 soft pad-like structures (platellae) on their hind tarsi, which are absent in froghoppers. High-speed videos of jumping showed that platellae contact the surface briefly (approx. 3 ms) during the acceleration phase. Friction force measurements on individual hind tarsi on glass revealed that at low sliding speeds, both pushing and pulling forces were small, and insufficient to explain the recorded jumps. Only when the tarsi were pushed with higher velocities did the contact area of the platellae increase markedly, and high friction forces were produced, consistent with the observed jumps. Our findings show that leafhoppers have special adhesive footpads for jumping from smooth surfaces, which achieve firm grip and rapid control of attachment/detachment by combining anisotropic friction with velocity dependence.Many hemipteran bugs can jump explosively from plant substrates, which can be very smooth. We therefore analysed the jumping performance of froghoppers ( Some of the most proficient jumping insects are found among plant-sucking bugs in the hemipteran sub-order Auchenorrhyncha, including froghoppers –3 and leα = tan−1 = tan−1(1/μ) is limited by the friction coefficient μ . Friction coefficients μ for rigid, dry surfaces are typically less than 1. Assuming μ = 0.35 , which could be achieved by adhesive structures that strengthen the surface contact when they accelerate before take-off. However, the use of adhesive structures for jumping comes with several biomechanical challenges. Firstly, if the feet of the propulsive legs adhered too well to the surface, they would slow down the jump. As insect jumps are very brief, an extremely rapid control of surface adhesion would be required.Many jumping hemipteran and orthopteran insects are generalist herbivores that live and feed on multiple species of plants ,16, whicon glass ), insectSecondly, most adhesive devices used by climbing insects are directional, i.e. they stick when legs are pulled towards the body but detach when pushed, thereby allowing easy detachment during locomotion  and Aphrodes bicinctus/makarovi leafhoppers (Cicadellidae) can jump from smooth surfaces. Philaenus frog-hoppers have relatively short hind legs (66% of the body length), are able to accelerate in less than 1 ms to take-off velocities of up to 4.7 m s−1, when jumping from high-density foam [Aphrodes leafhoppers have longer hind legs (84% of the body length), take longer (4.4 ms) to accelerate and achieve take-off velocities of up to 2.9 m s−1 on high-density foam [Insect jumping performance has been studied mostly by allowing insects to jump from rough substrates such as twigs, high-density foam or sandpaper ,5,31,32.Aphrodes leafhoppers and Philaenus froghoppers able to jump from smooth surfaces? (ii) Does jumping performance differ between smooth and rough surfaces? (iii) What structures come into surface contact during take-off and how are they adapted? (iv) How is attachment and detachment controlled in these structures?The mechanisms and potential adaptations of insects for jumping from smooth surfaces have not been studied. Here, we address the following questions: (i) are 2.2.1.Aphrodes of the bicinctus Schrank 1776/makarovi Zachvatkin 1948 group (Cicadellidae) and the froghopper Philaenus spumarius Linnaeus 1758 (Aphrophoridae). We collected 31 adult A. bicinctus/makarovi  and 43 adult P. spumarius (13.3 ± 0.4 mg) on meadows around Cambridge (UK) from several species of plants. Both insects have been recorded to live on diverse host plants [We studied two hemipterans of similar size, the leafhopper t plants ,34. Obse2.2.The tarsal morphology of leafhoppers and froghoppers was examined in a Leica MZ 16 stereo microscope . For scanning electron microscopy (SEM), legs were fixed in 4% glutaraldehyde in 0.1 M PIPES buffer at pH 7.4 for 48 h at 4°C. They were then washed with de-ionized water and dehydrated in increasing concentrations of ethanol (up to 96% ethanol). The legs were air-dried and mounted on stubs, sputter coated with a 20 nm layer of gold, and examined in a FEI XL 30-FEG SEM  at 10 kV.2.3.To compare jumping performance on smooth and rough surfaces, insects were placed on a smooth glass coverslip (18 × 18 × 0.17 mm) or on sandpaper (glued onto an 18 × 18 mm plate) with 30 μm nominal asperity size. If the insects did not jump spontaneously, they were carefully prompted using a fine natural hair paintbrush. Jumps were recorded with two high-speed video cameras positioned to achieve dorsal and lateral views, either at 1000 frames per second (fps) using two Redlake PCI 1000 B/W  or at 4700 fps using two Phantom v. 7.1 .atlab script  by digitising the insects' body position in both camera views; take-off angle and velocity were calculated trigonometrically from the flight trajectory over the first 2 ms in which the insect was airborne.Each recorded jump was analysed using a custom-written M2.4.Aphrodes leafhoppers and Philaenus froghoppers were placed on glass coverslips on an inverted Leica DM IRE2 microscope. Contact areas were visualized using a 5× lens and bright field epi-illumination from a 100 W mercury arc lamp. This illumination produces high-contrast images of the pad contact areas [Aphrodes using a FASTCAM 1024 PCI high-speed camera  at 5000 fps. Contact areas and a simultaneous close-up side view of the insect were recorded in Aphrodes and Philaenus using two Phantom v. 7.1 high-speed cameras  at 4700 fps. In total, we obtained high-speed close-up recordings of the hind tarsi for 12 jumps of five Aphrodes and nine jumps of eight Philaenus.To record the contact areas of hind tarsi during the acceleration phase of a jump (defined as the time between the first visible hind leg movement and take-off), ct areas . Contactatlab. In jumps of Aphrodes, velocities along the sliding trajectory of platellae in contact with the glass coverslip were measured by manually digitising the proximal end of the platella contact area three times (to reduce the measurement error) using ImageJ. We used the average of the three measured values for each frame. As we digitised the proximal end of the platella contact zone, it is possible that we slightly underestimated the sliding distance . The first frame without visible surface contact of the hind tarsi was defined as ‘take-off’ and the time as 0 ms .In most of the recordings, contact areas of only one hind leg was visible. For each jump, the contact area was measured using a threshold algorithm in M2.5.Aphrodes and Philaenus on glass were measured using a two-dimensional force transducer mounted on a three-dimensional motor positioning stage. A custom LabVIEW  program collected data and controlled movements of the force transducer and video trigger signals . The hiPhilaenus, mean forces of 34 mN were found in the direction of the jump with a mean take-off angle of 46.8° [Aphrodes, mean forces of 11 mN were found with a mean take-off angle of 37.1° [−1 in random order and at different positions on the glass coverslip to avoid accumulation of adhesive secretion [−1 could not be tested with our set-up. For each velocity, slides were performed in both the pushing and pulling direction  in random order. After each slide, the foot was left in contact for 2 s before a 0.5 mm s−1 pull-off in the normal direction. The noise level of the measurement set-up for both adhesion and friction was less than 0.2 mN.In previously recorded jumps of of 37.1° , giving ecretion ,36. Thesnlme [crank [Statistical analysis of the data was performed in R v. 3.0.2 . Data arnlme  and Pagee [crank .3.3.1.Philaenus froghoppers and Aphrodes leafhoppers possess rows of conical, sclerotized spines ventrally at the distal end of their hind tibia. In Philaenus, these spines do not articulate with the tibia, but in Aphrodes they are hinged. Philaenus has similar rows of sclerotized spines also on the first and second tarsomeres but Aphrodes has only individual small spines.The tarsus of leafhoppers and froghoppers consists of three segments and a distal pretarsus containing claws and a bi-lobed adhesive pad (arolium) . The hinPhilaenus, a single flexible hair , protrudes from the dorsal side of each spine (b). By contrast, the tarsus of Aphrodes bears many conspicuous, rounded cuticular outgrowths  (d–f). They are 65–100 μm long  and less sclerotized than the surrounding cuticle with a pale, yellowish colour (e). In lateral view, they appear approximately straight on the dorsal side, but convex on the ventral side (f).In cutella’ ), 60 to ch spine b. By contellae’, –42), whianimals) d–f. Theyh colour e. In latral side f.3.2.Philaenus jumped from smooth glass their hind legs always slipped . The slipping of the propulsive hind legs on glass resulted in significantly steeper jumps  and a rapid forward spin (clockwise in N = 10 insects), whereas Philaenus jumping from sandpaper showed a weak backspin . Both the steeper take-off angle and the forward spin result from the near-complete loss of forward thrust when the hind legs slip. In normal jumps without slipping, the hind legs push both backward  and downward (perpendicular to the surface), but they can only push downward when they slip, thereby accelerating the insect's rear end upward. This produces a forward rotation of the insect's body around the front feet (while these are still in contact), accelerating the body centre of mass upward , indicating that most of the energy is dissipated by the slipping and kicking hind legs [When s upward . Once thind legs .Figure Philaenus, we never observed Aphrodes slip on any of the substrates . Consistently, their take-off velocity did not depend significantly on the substrate  and they jumped with similar take-off angles . The jumps of Aphrodes showed only minimal backspin on both substrates . The total kinetic energy was similar for jumps on both surfaces .In contrast with 3.3.Philaenus were observed in contact with the glass surface . Within the first two frames of visible hind leg movements, however, these hairs were already detached and remained out of contact while the leg was slipping. In eight out of nine jumps the hind leg arolium was in contact before the jump, but also detached at the start of the acceleration phase.Before take-off from glass, fine hairs on the hind tarsi of Aphrodes  showed that two to nine platellae on the first and second tarsomere came into surface contact at the start of the acceleration phase (when the first hind leg movements became visible) and remained in contact until take-off ; the mean contact area was 2484 ± 258 μm2 . The platellae were in surface contact for 3.1 ± 0.1 ms. Contact area increased rapidly at the beginning of the contact phase, both by expansion of the contact area of individual platellae  and by additional platellae coming into contact . Contact areas decreased rapidly 0.6 to 0.2 ms before take-off. In three jumps , hind leg arolia were in surface contact before the jump, but their contact area strongly decreased or they detached at the beginning of the acceleration phase.Contact area recordings for the hind legs of jumping N = 12 jumps of five leafhoppers), corresponding to up to 5.3 times the maximal length of the contact area of one platella . Platellae slid fastest at the start of the contact phase, with peak velocities ranging from 15 to 154 mm s−1, and then slowed down or completely stopped before take-off. In some jumps, platellae even moved slightly in the direction of the jump just before detachment . Higher velocities of greater than 160 mm s−1 were sometimes observed when the platellae came into initial contact, but the contact areas were blurred, suggesting incomplete surface contact.During the brief contact phase, the platellae slid backwards (against the direction of the jump) over a short distance . The sliding distance ranged from 7 to 358 μm , the total contact area of the platellae was small and only slightly larger in the pushing direction , much smaller than the areas observed during natural jumps . The friction forces were also small in both the pushing and pulling direction : when the tarsi were pushed, the contact area of each platella expanded dramatically , so that their size was consistent with that observed during natural jumps. This contact area expansion was associated with a strongly increased friction force . The friction coefficient for 5 mm s−1 velocity at 5 mN normal force was μ = 1.00, which would allow forward jumps with take-off angles as low as 44.9°. By contrast, the contact area during pulling decreased slightly with velocity (figure 6b). Nevertheless, pulling friction increased with sliding velocity , but forces were much lower than for pushing .However, a very different behaviour occurred for higher sliding velocities . Our highest experimentally applied velocity of 5 mm s−1 was still lower than the naturally observed sliding velocities (see above); it is therefore likely that even higher friction coefficients are reached during natural jumps, allowing even lower take-off angles without slipping.Thus, the contact area and friction force of platellae changed both with direction and velocity. Most of the increase in contact area and friction occurred for velocities changing from 0.1 mm s figures  and 6b. c; electronic supplementary material, tables S1–S3). For a pushing velocity of 5 mm s−1 and normal forces increasing from 1 to 5 mN, the friction coefficient for Aphrodes decreased from 2.86 to 1.00.Variation of the normal force had only a small effect on the frictional behaviour of the platellae. At lower normal forces of 3 and 1 mN, platellae showed a similar direction and velocity dependence. However, the friction coefficient decreased with increasing normal forces . Shear stresses in the pulling direction appeared to be larger than in the pushing direction . However, it is likely that this result is an artefact, because as a result of the bending of the platellae, more hard tarsal spines could come into contact during pulling, thereby increasing the friction forces and leading to an overestimate of the platella shear stress.Friction force per contact area (shear stress) increased approximately linearly with velocity for both sliding directions .Adhesion forces of the platellae were very small; when hind leg tarsi were pulled off perpendicularly with a speed of 0.5 mm sPhilaenus froghoppers, we observed that some of the thin, long acutellae protruding from behind the tarsal spines also made close contact with the substrate , they were generally low, and for higher velocities in the pushing direction they were much lower than those of Aphrodes . At 5 mm s−1 and 5 mN normal force, the mean peak shear force in the pushing direction was 1.3 mN (N = 5), resulting in a friction coefficient of μ = 0.26, which would only allow steep upward jumps . Similar to the results for leafhoppers, the friction coefficient of Philaenus tarsi decreased from 0.71 to 0.26 when normal forces increased from 1 to 5 mN at a pushing velocity of 5 mm s−1 .Although platellae are absent in  figures a and 7. 4.Philaenus froghoppers and Aphrodes leafhoppers differ in their ability to jump from smooth surfaces. Froghoppers performed more powerful jumps than leafhoppers on the rough sandpaper surface, where the tibial and tarsal spines on their hind legs could interlock with asperities. However, they slipped on glass, resulting in jumps that were slower (four-times lower take-off velocity), had less kinetic energy (nine-times less than on sandpaper), were directed steeper upward, and had a rapid forward spin. By contrast, leafhoppers never slipped and jumped with similar take-off velocities and angles from both glass and sandpaper.Our results show that c). By contrast, friction forces for the tarsal hairs of Philaenus froghoppers at 5 mN normal force were very small, allowing only steep upward jumps with take-off angles more than 75.6°, close to the observed mean take-off angle from glass of 71.3°. Our results indicate that higher normal forces closer to the expected normal force of 12.4 mN per leg for Philaenus froghoppers [figure 6f), and therefore lead to even larger (steeper) minimum take-off angles.This difference in jumping ability from smooth surfaces can be explained not only by the different pushing forces produced by both insects, but also by the presence of specialized structures called platellae on the hind tarsi of leafhoppers, which are absent in froghoppers. Apart from the correlation of jumping performance with tarsal morphology, four other lines of evidence support this conclusion: first, our high-speed contact area recordings revealed that the platellae came into contact with the surface during the acceleration phase of a jump, but almost never when standing or walking. Second, platellae produced much larger contact areas than the acutellae of froghoppers, and they remained in contact during the acceleration phase (unlike the acutellae). Third, the platellae slid backward during the acceleration phase of the jump, speaking against firm interlocking. The reduction in sliding speed of the platellae without a change in contact area during the acceleration phase could reflect a variation of the insect's acceleration, but it is more likely based on the depletion of fluid from the adhesive contact zone of the platellae, similar to observations for lubricated rubber  or smootfigure 6Aphrodes explain their jump performance on glass? Assuming a jump acceleration of 613 m s−2 , a body mass of 18.4 mg and a take-off angle of 47.7°, the shear force can be estimated as 7.6 mN, or 3.8 mN per hind leg. With the observed mean contact area of 2484 μm2, a shear stress of 1530 kPa would be required. This shear stress is slightly higher than the mean shear stress measured experimentally for platellae at 5 mm s−1 velocity and 5 mN normal force (1111 kPa). However, as the platellae slid with peak velocities of 15 to 154 mm s−1 during natural jumps, and as shear stresses increase with sliding velocity, it is likely that these high shear stresses were produced by the platellae. This extrapolation to higher velocities  is justified, as the shear stress of rubbery pads steadily increases with velocity up to the onset of stick-slip, which we never observed for sliding platellae [Can the contact area of platellae observed in natural jumps of Platellae have been described as ‘transparent, fleshy, relatively short setae with thick and even slightly swollen, blunt, rounded tips’ , and werOur results show that platellae combine two mechanisms to control friction rapidly and reliably for jumping. The surface contact of platellae is both direction and velocity dependent: contact areas and forces were large only when the hind leg was pushed rapidly, and remained small at low pushing speeds or when pulled. This mechanism ensures good grip during the acceleration phase when the legs are pushed and easy detachment when the legs are pulled at take-off. The engagement of platellae only during the contact phase of a jump may also lessen damage and wear, which would reduce attachment performance .A similar direction dependence has been reported for many insect adhesive pads –25,27,49The difference in function between the platellae and the adhesive arolium in leafhoppers provides a clear example of the widespread division of labour between proximal and distal attachment organs on the same foot ,27,29,53The combined direction and velocity dependence of platellae may be an adaptation for the extremely rapid control of attachment required for jumping. Platellae detached in 0.2 to 0.6 ms, much faster than vertically climbing geckos  or antsThe mechanical control of surface contact may be based on the morphology and orientation of the platellae: when the foot is pushed (with the spines in surface contact), a torque will develop around the base of each platella, pressing more of its soft convex side into contact and deforming it. When pulled, this torque is reversed and will help to raise the platellae away from the surface, resulting in smaller contact areas.Philaenus also contact the surface during the acceleration phase of jumps. The similar anatomical position of platellae and acutellae  suggests that both structures are homologous [Philaenus slipped when trying to jump from glass. However, we observed that some froghopper species with more acutellae can also perform successful jumps from glass , suggesting that even acutellae can provide grip if present in sufficient numbers.Our results show that the tarsal hairs (acutellae) of mologous . In contPhilaenus to jump from smooth glass affects their jumping performance on plants under natural conditions. The surfaces of most plant leaves and stems are not only more curved, but also rougher and softer than glass [Philaenus avoid plants with very smooth surfaces, while Aphrodes can cope with a wider range, but more detailed ecological data are needed to test this hypothesis.It is still unclear to what extent the inability of an glass ,57, so tPhilaenus are shorter than those of Aphrodes. As longer hind legs increase the time to accelerate and reduce the forces acting on the feet, Burrows & Sutton [Aphrodes 4.4 mN versus Philaenus 11.6 mN; [Philaenus produces 2.6 times more shear force while accelerating. Assuming that the shear stress of tarsal pads is approximately constant (i.e. ignoring the effect of sliding velocity), Philaenus would require an approximately 2.6 times larger contact area of its tarsal pads than Aphrodes to avoid slipping. While such larger pad areas might be anatomically possible, this difference suggests that jumping pads such as platellae are easier to realize in combination with longer legs and lower accelerations. Smaller forces for longer limbs may also expose the pads to less damage and wear, allowing the use of softer adhesive structures for attachment. However, even short-legged leafhoppers jumping with high accelerations such as Ulopa reticulata [The hind legs of & Sutton  hypothes11.6 mN; ,5) showsticulata .The adaptations for jumping from smooth surfaces reported here provide an extreme example for rapid control of surface attachment. The study of such attachment systems may help to uncover general mechanisms for switchable adhesion and inspire biomimetic synthetic devices.


In addition to testosterone (T), the emerging role of sex hormone-binding globulin (SHBG) in pathogenesis of metabolic syndrome (MetS) has been noted recently. However, reports of associations with serum adipocytokine levels are still limited. Therefore, we conducted this study to evaluate whether serum T and SHBG levels are independent predictors for the risk of MetS that are associated with adiponectin and leptin levels in 614 Taiwanese men over 40 years old collected from a free health screening. Subjects in the lowest quartile of TT and SHBG levels are exposed to a 1.58 and 3.22 times risk of developing MetS, as compared to those in the highest quartile of TT and SHBG levels. However, SHBG retains its significance independent of TT as a MetS risk predictor, but not vice versa. In addition, SHBG was significantly correlated with both adiponectin and leptin levels even after adjusting for TT levels. In conclusion, SHBG served as a major predictor for the risk of MetS and was correlated with serum adiponectin and leptin levels that are independent of T. Further studies are needed to elucidate the true role of SHBG in the pathogenesis of MetS and possible mechanisms associated with serum adiponectin and leptin levels. It has become a major public health concern in the twenty-first century due to its rapidly increasing prevalence in recent years and its utility as a predictor for the risk of type 2 diabetes mellitus (DM) and cardiovascular disease3. Patients with MetS have a 5-fold increase in risk for type 2 DM and twofold increase in risk for cardiovascular disease over the subsequent 5 to 10 years3. Increased adipose tissue with dysregulation of adipocytokines is thought to play a major role in the pathogenesis of MetS4. Adiponectin and leptin are the two most important adipocytokines that can modulate insulin sensitivity, body weight and inflammatory status5.Metabolic syndrome (MetS) is a collection of cardio-metabolic risk factors, including obesity, insulin resistance, hypertension, and dyslipidemia, appearing to affect approximately 10–40% of adult populations worldwide8. Testosterone (T) is the principal sex hormone in men with both androgenic and anabolic effects that decline gradually, especially after the age of 409. It consists of three components, including T bound to sex hormone‑binding globulin (SHBG), T bound to albumin, and free testosterone (FT)11. Traditionally, FT is considered the main component with hormonal activity, and serum SHBG is considered a transport protein for T to target tissues, regulating the circulating concentration of FT11. However, recent research has demonstrated that serum SHBG can directly affect cells by binding to their receptors and acting as a hormone12. In addition, polymorphisms in the SHBG gene have been associated with the risk of type 2 DM, suggesting a causal role for SHBG in metabolic disease risk14. A large number of epidemiological studies have also reported that both low T and SHBG levels can increase the risk of MetS in men15. Some studies even suggested that serum SHBG levels may play a more dominant role than T levels in determining the risk of MetS18.In East Asia, the prevalence of MetS has been reported higher in men and increased with ageAlthough many studies have investigated the relationships of serum T and SHBG levels with the risk of MetS in men, only a few have focused on their relationships with serum adipocytokines, especially adiponectin and leptin levels. Therefore, we conducted this study to evaluate whether serum T and SHBG levels are independent predictors for the risk of MetS that are associated with adiponectin and leptin levels in a large sample of middle- to old-aged Taiwanese men.Of the 656 men participating in the health screening, 42 subjects were excluded due to current malignancies 13 cases), current use of medications that would interfere with the measurement of natural testosterone levels (17 cases) and incomplete evaluation (12 cases), leaving 614 subjects with a mean age of 55.8 ± 5.7 years (range: 43–83 years). The baseline characteristics of the study population are summarized in Table 3 cases, 2, p < 0.001), lower education levels , higher prevalence of current smoking  and alcohol drinking , lower serum TT , SHBG  and adiponectin  levels, and higher leptin levels  than those without MetS (Table Table TT was significantly correlated with waist circumference (WC) , diastolic blood pressures , fasting blood glucose (FBG)  and triglyceride (TG) . SHBG was significantly correlated with WC , FBG , TG , and high density lipoprotein (HDL)  . Subjects in the lowest quartile of TT and SHBG levels were exposed to 1.58 and 3.22 times the risk of having MetS respectively, when compared to those in the highest quartile of TT and SHBG levels Table . When adIn multiple linear regression analyses, both TT and SHBG levels were significantly positively correlated with adiponectin levels  and negatively correlated with leptin levels  after adjusting for age, educational level, alcohol drinking, and cigarette smoking Tables  and 6. HIn a large sample of middle- to old-aged Taiwanese men, subjects with MetS had significantly lower serum TT and SHBG levels compared to those without MetS. As the number of MetS components increased, both serum TT and SHBG levels decreased gradually with significant trend. However, no significant difference of serum FT levels was noted in subjects with and without MetS. Subjects in the lowest quartile of TT and SHBG levels are exposed to 1.58 and 3.22 times higher risk of having MetS respectively, when compared to those in the highest quartile of TT and SHBG levels after adjusting for age and lifestyle factors Table .15. According to their analysis, SHBG was the strongest and FT the weakest predictor for both prevalent and incident MetS15. In our study, both low TT and SHBG were significant predictors for the risk of prevalent MetS, but FT showed no significant association. Several cross-sectional studies also reported no significant difference in FT level between subjects with and without MetS21. In the longitudinal studies of Kupelian et al.22 and Li et al.23, only TT and SHBG, but not FT were significant predictors for the risk of incident MetS in men. These results suggest that the impact of FT on MetS may be weak or confounded by several factors like discrepancy in the measurement of FT, definition of MetS and baseline characteristics of the different study populations24. Further studies are needed to clarify the real relationship between FT and MetS.Recently, Brand and colleagues performed an updated meta-analysis from 20 observational studies and showed that men with low concentrations of TT, SHBG or FT were more likely to have prevalent MetS  and incident MetS  after adjusting for age and lifestyle factors17. In our study, we also found that serum SHBG levels were significantly correlated with adiponectin and leptin levels even after controlling for age, lifestyle factors, BMI and TT level. Both adiponectin and leptin levels were significantly associated with all individual MetS components.Although both TT and SHBG levels are significant predictors for the risk of MetS in our initial analyses, only SHBG retains its significance after adjustment of both TT and SHBG levels. Some studies have reported SHBG as a dominant predictor for prevalent and incident MetS, independent of TT, while TT lost its significance as a predictor for the risk of MetS after adjustment of SHBG25. Aside from our study, only limited epidemiologic studies have evaluated the relationships among SHBG, adiponectin and leptin in men28. Gannage´-Yared et al. first reported that serum SHBG level was positively correlated with adiponectin level and negatively correlated with leptin level in men26. The relationship between SHBG and adiponectin persisted after adjustment for waist or BMI, while SHBG lost its relationship with leptin after adjustment for BMI26. Vanbillemont et al. used dual-energy X-ray absorptiometry to determine body composition and found that SHBG remained significantly associated with adiponectin after adjustment for body composition, while SHBG lost its association with leptin after adjustment for body composition28. In our study, we found that SHBG was significantly correlated with both adiponectin and leptin levels after adjusting for BMI in a large sample of middle- to old-aged Taiwanese men. The discrepancies reported in above epidemiologic studies might be owing to ethnic variations. The impact of race/ethnicity on the association of SHBG with adiponectin and leptin should be clarified by further studies. In addition, Simó et al. recently reported that adiponectin can increase hepatic SHBG production by upregulating hepatocyte nuclear factor 4 levels via changes in the hepatic lipid content in their in vitro study by using HepG2 cells29. Further studies are also needed to elucidate other possible biological mechanisms.Adiponectin and leptin are two important cytokines secreted from adipose tissues that play important roles in the pathogenesis of MetS. Adiponectin has been found to have antidiabetic, anti-atherogenic, and anti-inflammatory properties, while leptin can regulate body weight by modulating appetite and energetic balance, and upregulate proinflammatory cytokines that are associated with insulin resistance and endothelial dysfunction30. In addition to early reports of the association between genetic polymorphisms of the SHBG gene and the risk of type 2 diabetes14, Pan et al. and Xita et al. also found that genetic polymorphisms of the SHBG gene were significantly associated with the risk of MetS31. Recently, Wang et al. used Mendelian randomization that utilizes genetic variants or polymorphisms as proxies for exposures, to evaluate the potential causality of SHBG on the metabolic measures and insulin resistance in three Finnish population-based cohorts. They also found that SHBG is strongly associated with multiple circulating lipids and metabolites, and prospectively associated with the development of insulin resistance and DM even after adjustment for baseline T levels32. Those results suggest a causal role for SHBG in the risk of MetS. Further studies are needed to elucidate whether SHBG is merely a biomarker of MetS or could play an active role in the development and progression of MetS.The principal function of SHBG has traditionally been that as a transport protein for sex steroids it can regulate circulating concentrations of free (unbound) hormones and their transport to target tissues. However, recent studies have suggested that SHBG might influence the risk of MetS via (1) mediation of steroid hormone signal transduction at the plasma membrane (2) production of a direct effect in endothelial cells through the SHBG receptor and (3) direct effects on the cell by binding to its receptor, acting as a hormoneThere are some limitations to this study. First, our design is a cross-sectional study, so we cannot establish cause and effect relationships, but only investigate associations. Second, our results are based on single measurements of serum T levels. Although we have attempted to minimize natural diurnal variation in T levels by always drawing blood samples between 8:00 and 11:00 AM on the day of the screening, the results from single measurement may not accurately represent real serum T levels of study subjects.In conclusion, the results from a large sample of middle- to old-aged Taiwanese men reveal that subjects with lower serum TT or SHBG levels, but not FT levels are exposed to a higher risk for MetS. In addition, SHBG served as a major predictor for the risk of MetS and was correlated with serum adiponectin and leptin levels that are independent of T. Further studies are needed to elucidate the real role of SHBG in the pathogenesis of MetS and possible mechanisms associated with serum adiponectin and leptin levels.34. Ethics approval following the Declaration of Helsinki was authorized by the Institutional Research Ethics Committee of Kaohsiung Medical University Hospital and informed written consent was obtained from each participant before their participation. Exclusion criteria included: (1) subjects who had current malignancy, advanced liver and/or renal disease, major psychiatric disorder, or substance abuse disorders; (2) subjects who were using hormones, anti-androgen treatment, antifungal drugs, or steroidal agents; (3) subjects who had incomplete evaluation35.The cross-sectional data of Taiwanese men older than 40 years were collected from a free health screening held between August 2010 and February 2012 by a medical center in Kaohsiung City, Taiwan. Subject selection has been described in detail previously34. Subjects were classified as alcohol drinkers or cigarette smokers if they had regularly consumed any alcoholic beverage ≥1 times per week, or had smoked ≥10 cigarettes per week respectively, for at least 6 months. Current users were those who were still using any of these substances within one year before the interview34. A habit of regular exercise was defined as participants having exercised aerobically for a minimum of 20 min and perspired, performing this activity regularly more than 1 time per week within one year before the interview36.Each participant was interviewed by trained researchers using a structured questionnaire to collect detailed medical, surgical and psychosexual history, current medications and lifestyle data2)) was calculated as body weight divided by the square of body height.Physical measurements, including blood pressure, body weight (kg), height (cm) and waist circumference (WC) were recorded for each participant. Blood pressures were measured twice after resting for > 15 min by well‑trained nurses using a mercury sphygmomanometer, and the average values were included in analyses. WC was measured at the midpoint between the inferior costal margin and the superior border of the iliac crest on the mid-axillary line. Body mass index : 3.4%~8.3% and 1.78%~6.21% respectively; Inter-assay CV: 3.0%~6.2% and 6.90%~9.25% respectively)33. TT and SHBG levels were determined using a DPC Immulite analyzer . Free testosterone (FT) level was calculated from serum TT, SHBG, and albumin levels according to the Vermeulen formula37.After more than 8 hours fasting overnight, all participants had 20 cc blood samples drawn between 8:00 and 11:00 AM on the day of the screening for analyses of serum glucose, lipid panels, routine biochemical profiles, adipocytokines, and hormone levels. All analytical methods have been described in detail previously38.An individual was diagnosed with MetS if he was positive for at least three of the five following criteria: (1) WC ≥ 90 cm; (2) high density lipoprotein (HDL) cholesterol < 40 mg/dL or lipid medication use; (3) triglyceride (TG) ≥ 150 mg/dL or use of lipid‑lowering therapy; (4) blood pressure (BP) ≥ 130 /85 mm Hg or use of antihypertensive medication; (5) fasting blood glucose (FBG) ≥ 100 mg/dL or diagnosed as type 2 DM, in accordance with the modified criteria from the National Cholesterol Treatment Adult Treatment Panel III (NCEP ATP-III) definition proposed by the Bureau of Health Promotion in Taiwant-test. Correlations between biochemical variables and individual MetS components were analyzed by Spearman’s correlation. Any variables significantly associated with the risk of MetS in the initial analyses were further examined in multivariate regression analyses. Serum T and SHBG levels were examined as quartiles based on the entire population to determine the independent risk of MetS and their association with serum adipocytokine levels. Serum adipocytokine levels were log-transformed to be normally distributed before analyses. To examine the robustness of our analyses, sensitivity test were also performed to exclude subjects with DM in multivariate regression analyses. SPSS version 18.0  was used for all statistical analyses.Quantitative data have been represented as mean ± standard deviation (SD) and categorical data have been represented by number (n) and percentage. To quantify the differences between subjects with and without MetS, qualitative variables were compared using the chi-square test and Fisher’s exact test, while quantitative variables were compared using the Student’s


Rock outcrop is an important habitat supporting plant communities in karst landscape. However, information on the restoration of higher biotic populations on outcrops is limited. Here, we investigated the diversity, biomass changes of higher vascular plants (VP) and humus soil (HS) on karst outcrops during a restoration process. We surveyed VP on rock outcrops and measured HS reserved by various rock microhabitats in a rock desertification ecosystem (RDE), an anthropogenic forest ecosystem (AFE), and a secondary forest ecosystem (SFE) in Shilin County, southwest China. HS metrics (e.g. quantity and nutrients content) and VP metrics  were higher at AFE than at RDE, but lower than at SFE, suggesting that the restoration of soil subsystem vegetation increased HS properties and favored the succession of VP on rock outcrops. There was significantly positive correlation between VP metrics and HS amount, indicating that the succession of VP was strongly affected by availability and heterogeneity of HS in various rock microhabitats. Thus, floral succession of rock subsystem was slow owing to the limited resources on outcrops, although the vegetation was restored in soil subsystem. Depend on the local rainfall and the purity of bedrock, the land surface may consist of various morphologies of rock outcrops and rock-soil patterns4  comprising high heterogeneity8. These microhabitats can intercept and gather soil particles, animal residues, vegetation litter and other exotic substances transported by rainfall and air flow from great distances10. We refer to these mixed substances as the humus soil on rock outcrops (HS) in this study  on rock outcrops also fix atmospheric carbon (C) and nitrogen (N) to maintain HS fertility and rock outcrop subsystem productivity22. Yet there has been, and remains, some confusion concerning the relationship between such microhabitats’ conditions and the rock-dwellers. Particularly, little quantitative research addresses the HS and its ecological function on rock outcrops. In addition, the physical environment of rock subsystems is stressful for plant growth because of low water and nutrients availability, intense solar radiation, great temperature fluctuations, and the shallow soil layer23. Especially during the dry season in semihumid region (e.g. Shilin), plant growth suffers from severe drought stress24. Floral succession in rock subsystems can be considered as an important aspect when evaluating karst degradation and restoration.Plants on rock outcrops have been proven to substantially contribute to regional biodiversity26. At an early stage of vegetation succession, various types of algae29, fungi30, and cyanobacteria and heterotrophic bacteria colonize where conditions are most favorable20. Successively mosses, lichens32, and other higher plants become established in different successional periods33. Biological changes of soil subsystems also greatly affect the successional process of rock vegetation. Angelini and Silliman34 demonstrated that primary foundation species facilitate secondary foundation species by increasing habitat complexity and quality to enhance biodiversity. The restoration of degraded karst areas may take so long that human interventions, including reforestation, water and soil conservation are required to accelerate the restoration process26. Previous studies have concentrated on the ecological significance of vegetation restoration in soil subsystems but data concerning floral changes in rock subsystem are limited, and no study addresses the effects of soil subsystem vegetation on plants of rock outcrops.The succession of life in (and on) the rock outcrops plays an important role in natural rocky desertification restorationIn this study, we described the vascular plants (VP) and HS on rock outcrops across three karst landscapes, assuming that they were the three typical stages in restoration of rocky desertification. We aim to answer the following questions: (1) How much is the amount of HS on rock outcrops and how does it impact on VP? (2) Do the diversity, density, lifeforms, species composition, and distribution of VP on rock outcrops differ in rocky desertification restoration? (3) Did the rock outcrop subsystem restore following revegetation in soil subsystem? (4) What factors drive rock vegetation succession?2) than in the AFE (39.36 ± 16.27 g/m2), while for the RDE it was only 3.1 times higher than in the AFE , of which 88.5% were spermatophytes, and 11.5% were ferns. SFE had the highest species richness and RDE had the lowest . Most species (approximately 77.5~96.0%) had less than 50 individuals. Average species per subplot ranged from 0.92 to 3.53 and decreased in the order: SFE > RDE > AFE Table , and shoThe VP communities showed significant differences among different ecosystems in the NMDS ordination scatterplot Fig. . Each ofHerbs were the dominant species in each ecosystem, followed by shrubs, lianas and trees Fig. . More thCorrelation analysis showed that the numbers of individuals, species richness, and biomass of VP were positively related to the amount of HS, canopy coverage, rock outcrops ration, and relative humidity Table . HS on rA large proportion of vascular individuals were biased in favor of the RW, RS and RP microhabitats at SFE Fig. . Yet mos36. Much research has focused on soil-vegetation restoration processes23. However, there is no information concerning the rock-vegetation conversion and its relationship with the soil retained on rock outcrops. Furthermore, there is no research addressing the changing pattern of VP and HS on the highly fragile rock subsystem during the restoration of vegetation in soil subsystem. Our data indicated that a divers VP can be found on rock outcrops with declining density at the various microhabitats holding different amounts of HSs in the SFE. With the removal of forest from the soil sub-ecosystem, VP and HS will be highly diminished. On the other hand, the loss of VP and HS will be reversed with the restoration of tree plantations in the soil subsystem.Over the large karst area, such practice as reforestation in the soil subsystem was usually adopted to control rocky desertification and accelerate ecosystem restoration19. However, little research considers the changing pattern along the vegetative gradient on the nearby soil subsystem. Our results suggested that VP metrics obviously changed with the degradation and restoration of soil subsystem vegetation , desiccation-tolerant (Selaginella tamariscina), or deep rooted (Ficus tikoua)37. Similarly, the VP also changed with the restoration of soil subsystem vegetation. These changes in community structure and composition followed a trend towards SFE but remained different from it. Certain life forms  and space distribution of VP at AFE were in an intermediate stage of developmental sequence between RDE and SFE.Vascular flora on rock outcrops have a broad distribution in a range of habitats from savannas to humid tropical forestsion Fig. , Table 1ion Fig. , the speion Fig. . The VP HS also changed following the soil subsystem vegetation degradation, as did the restoration process, in both quantity and quality. Average amount and nutrient content of HS at RDE were significantly lower than at SFE Fig. . Almost 10, much water, redundant organic carbon and nutrients were transported to nearby soil subsystems in local karst landscape38.The availability and heterogeneity of HS are important factors for the slow succession of VP since a strong correlation was found between HS and VP metrics Table . Our res39. On average, it takes 300–73800 years to form 1 cm thick soil based on different estimation methods involving the dissolution test of carbonate rock39. The magnitude of quantity and quality in HS, and associated ecological function at AFE cannot reach the levels within the regional natural forests (e.g. SFE). Therefore, the floral succession on rock outcrops remained slow in plantation forest. Additionally, HS tended to amass at the “funnel” shaped microhabitats (RC and RP), whereas few residua were detained at the rain-washed places  have a strong influence on plant community structure during the restoration of rock desertification.The low HS quantity can be attributed to the extremely slow rate of soil formation because little residue results from carbonate rock weatheringces Fig. . The fin40. Porembski43 reported many species on inselbergs distributed in ephemeral flush plot and a shallow depression with more nutrients and soil. Our study showed that distinctive species were usually found in diverse microhabitats with a shallow soil layer, forming a patchy size distribution ranging from a few centimeters across to hundreds of square centimeters. This agrees with other studies where numerous individual plants were biased in favor of rocky habitats where considerable amounts of HS was easily accumulated44. Outcrops which appear bare at a distance, however, can bear many vascular plant species, some of which have the striking ability to grow on a surface with almost no soil40. SFE reflected this; there was very little HS in RW but many VP. More than 160 g/m² of HS occurred at RP with only relatively few individuals  which may facilitate the establishment of subsequent species by improving the habitat. Clements et al.45 claimed the presence of karst microhabitats supported high floral diversity. Both primary communities and diverse topographies result in a difference of microclimate, and further affect the structure and distribution of VP. Additionally, seed and establishment limitations restrict plant recruitment at early stages during ecological restoration46. Compared with soil subsystem vegetation, more stressful establishment conditions  resulted in slow flora succession on outcrops.Many factors are likely to influence the succession of plants in rocky habitats. Angelini and Silliman47. Rock outcrop is a critical component of karst landscapes and typically supports the vast majority of its entire floristic diversity45, even in the severe environment of RDE. Certain attempts have been made to alleviate the rate of environmental degradation in the vulnerable karst area19, and have made good progress5. Most of the eco-efficiency indexes that evaluate rocky desertification control, mainly come from soil subsystem vegetation, such as coverage, biomass, and diversity indexes48. Due to the different VP variation pattern during rocky desertification restoration, its succession should be taken into consideration when constructing an evaluation index system of rocky desertification restoration, especially in areas with a high rock-to-soil area ratio.Ecological restoration is not just a matter of planting trees, it involves assisting the restoration of a damaged and destroyed ecosystem49. The karst landscapes consist of rock gaps, rock ditches, small rock caves and rock slots. Altitudes range from 1600 to 2200 m above sea level. A subtropical plateau monsoon climate prevails with a mean annual precipitation of 967.9 mm, 80–88% of which falls between May and October. Mean annual temperature is 16.2 °C, mean maximum temperature of the warmest month (July) is 20.7 °C, and mean minimum temperature of the coldest month (January) is 8.2 °C 36. Red earth and calcareous soil are the primary soil types.This study was performed in Shilin County , Yunnan Province, southwest China, where karst landforms are composed of Permian, Carboniferous and Devonian carbonate rocksSophora viciifolia, Spiraea salicifolia, Diospyros duetorum, Bidens pilosa, Heteropogon contortus, Themeda triandra. Rock outcrops are exposed. The rock surface is covered by cyanobacteria film, and a few VP occur in microhabitats formed on rock outcrops.A rocky desertification ecosystem (RDE), where most of the trees of the primary forest were removed or cut because of human activities , followed by the establishment of shrubs and herbs, such as Pinus yunnensis, Koelreuteria paniculata, Photinia × fraseri, and Pyracantha fortuneana which were planted after a land preparation on a RDE site in 2005. Currently, these trees are higher than most of rock outcrops. The site is 4 km away from the RDE. Tian et al.29 investigated the species and communities of epilithic cyanobacteria films in this area. There are more VP on rock outcrops compared with RDE. No restoration measures were applied to the vegetation on rock subsystem and they are the result of succession.An anthropogenic forest ecosystem (AFE), with trees including Neolitsea homilantha, Olea yunnanensis, Cyclobalanopsis glaucoides and Pistacia weinmannifolia, with mixed deciduous species such as Albizia mollis, Carpinus mobeigiana and Pistacia chinensis. It was a god forest of a local village, the forest is dense, has a clear vertical stratification, and outcrops are covered by the tree canopy. Cyanobacteria, lichen, bryophytes, and many VP live on the rocks50. The site is 26.5 km away from RDE, within the natural reserve of Shilin Geopark.A secondary forest ecosystem (SFE), is an evergreen broadleaved forest dominated by Three typical local ecosystems were selected for this study:51. For AFE, the soil subsystem vegetation has almost been restored after a period of more than 20 years. The time was confirmed by the record of Stone Forest Scenic Area Administration. There were no trees in soil subsystem before restoration according to the description of local people, although there was no detailed record about it. The previous situation of AFE could be regarded as same as RDE. Vegetation is well preserved in the SFE with slight human disturbance. All three ecosystems are located on the same karst rock base, and have similar geographical features. We conceptualized the RDE as the degradation stages of SFE, and the AFE and SFE as the ecological restoration stages of RDE by employing a space-for-time substitution method52. The coordinates and environmental conditions of the study sites are shown in Table The specific degradation process of RDE is difficult to ascertain for the complicated reasonsComprehensive fieldwork was undertaken between October and November 2014. Six transects of 10 m wide were established along the hill slope within each of the three ecosystems and then each transect was divided into 10 plots with plastic line Fig. . At the Within each 2 m × 2 m subplot, all vascular species (VP) growing on rock outcrops were identified. Some plants were too small to identify. We only recorded the recognizable individuals more than one centimeter high. Next, all the recorded VP were collected, sealed within bags and brought back to laboratory, dried for 72 h at 70 °C to attain constant weight to calculate biomass. Such biomass obtained for the rock outcrops must be regarded as conservative. This is because a few shrubs and lianas had roots extending into soil, but only the part that was growing on rock outcrops was collected and counted as biomass.53, and further grouped into deciduous and evergreen, and into heliophyte and sciophyte54 for comparison among the three ecosystems.Based on our investigation and analysis, the VP were grouped into life forms of trees, shrubs, herbs and lianas8. Their specific features are shown in Table The microhabitats on rock outcrops were divided into six categories: rocky crevice (RC), rocky gully (RG), rocky pit (RP), rocky surface (RS), rocky terrace (RT), rocky wall (RW)3-HClO4.The HS samples from each transect were combined into one sample (six samples for each ecosystem), passed through a 0.25 mm sieve, and subjected to chemical analysis. Total N was determined using a Vario MAX CN elemental analyzer. The P and K content were determined with an ICP-AES  after mixing with HNO55.VP diversity was estimated using (1) α-diversity, evaluated as species richness per subplot, (2) γ-diversity, the total species numbers in each karst outcrops, (3) Shannon-Wiener index, (4) Simpson index55. Moreover, to estimate the differences in VP community across ecosystem types, a similarity matrix of 85 plots (eliminated without plants) × 61 species was subjected to non-metric multidimensional scaling (NMDS) with function metaMDS56 in the package vegan57 in R 3.3.2 58. NMDS is a good ordination method because it can use rank information and map ranks non-linearly onto ordination space to measure community dissimilarities based on species data.We calculated VP similarities using Sorensen’s similarity index58.One-way ANOVA, followed by a Tukey test for multiple pair-wise comparison, were used to assess the differences in diversity of VP among the three study sites. All data were checked for homogeneity of variances using Bartlett’s test and normality using the Shapiro-Wilk test and when the assumptions could not be satisfied after transformation, comparisons of non-parametric data were made using the Kruskal-Wallis test and Wilcoxon rank sum test. We performed all calculations and statistical analyses in R 3.3.2 Kruskal-Wallis test and Wilcoxon rank sum test were used to assess the difference in the quantity and nutrients concentration of HS among ecosystems. Spearman’s correlation coefficients were used to express the relationship between physical factors and number of individuals, species richness and biomass.Supplementary information