The surfaces of nano-calcium silicate (n-CS)/polyetheretherketone (PK) composites were treated with abrasive paper and sand-blasting, and the surfaces performances of the as-treated composites were studied. The results showed that the surface roughness, hydrophilicity and mineralization of the simulated body fluid (SBF) of the composites surfaces were significantly improved, and the properties of the composites treated by with sand-blasting were better than those treated with abrasive paper. Moreover, the treated composites significantly promoted osteoblasts responses, such as cell attachment, spreading, proliferation and alkaline phosphatase (ALP) activity, compared to un-treated composites, and the cellular responses to the composites treated with sand-blasting were better than those treated with abrasive paper. The results suggested that surface treatment with sand-blasting was an effective method to greatly improve the surface bioperformances of the n-CS/PK composite, and this treated composite with improved bioactivity and cytocompatibility might be a promising implant material for orthopedic applications. Moreover, PK implants were found to have biomechanical properties  that resemble the natural bone of human beings, which could effectively reduce stress shielding that is often observed in conventional metallic implants, such as biomedical titanium alloys, cobalt-bases alloys and stainless steel4. However, PK implants are comprised of bioinert materials that cannot bond to bone tissue (osseointegration) after they are implanted in vivo, and an encapsulation of fibrous tissue will form that isolates implants from the surrounding bone, which would cause loosening/abscission of the implants from host bone and ultimate failure of the implants6.Since receiving US Food and Drug Administration (FDA) approval in the late 1990s, polyetheretherketone (PK) has been extensively used as an implantable material for spinal, trauma, and orthopedic applications due to its good mechanical properties, chemical resistance, high thermal stability and biocompatibility, etc7. During the past few years, to enhance the osseointegration performance of PK, bioactive coatings, such as plasma spraying and plasma ion deposition, have been applied to modify PK to create bioactive surfaces for implants with improved biological properties10. However, the fragility and low mechanical strength as well as the weak bonding to the PK substrates of the coating have caused unsatisfactory osseointegration between the implants and bone tissue. Moreover, various bioactive materials, such as bioglass, hydroxyapatite, titania and calcium silicate, have been incorporated into PK to develop bioactive composites for bone implants12.Osseointegration can ensure that the implants bond with natural bone through biochemical reactions at the interface between the materials and the bone, which favors the implants fixation in host bone14. It was found that although incorporation of bioactive materials into PK improved the mechanical performance and bioactivity of the composites, most of the bioactive materials were dispersed into the PK matrix, and the composite surface was significantly covered by PK . Thus, the surface bioactivities of these composites were lower than bioactive materials  alone. Surface treatment offers an effective way to enhance surface biological properties while preserving the advantages of the materials. Therefore, proper surface treatments should be presented as alternative solutions. In this study, a nano-calcium silicate (n-CS)/polyetheretherketone (PK) composite was fabricated and surface treated using abrasive paper and sand-blasting. The effects of surface treatment of bone implants comprised of n-C/PK composite on surface morphology/microstructure, hydrophilicity, mineralization and osteoblast behaviors were evaluated.In previous studies, we prepared several PK-based bioactive composites, including hydroxyapatite, titania and calcium silicate, and we investigated their chemical-physical-biological propertiesFigure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 15. However, PK is a bioinert material that does not promote cell adhesion and growth, and PK implants can barely bind to host bone directly, which impedes the use of PK in clinical treatments16.Polyetheretherketone (PK) has attracted increasing attention as an alternative material for hard tissue repair due to its good biocompatibility, mechanical properties and chemical stability, and it is currently used in orthopedic clinicsConsequently, in this study, calcium silicate was incorporated into PK to develop n-CS/PK biocomposites (CSPKC) to enhance the bioactivity of PK, and the surfaces of the composites were treated with abrasive paper or sand-blasting. The results indicated that only a few n-CS particles were exposed on the CSPKC surface, which was covered by PK. However, after treatment with abrasive paper, the composites showed smooth and flattened surfaces, and many n-CS particles were exposed on the surfaces (most PK on the surfaces was removed). Thus, the surface roughness of Ra values of the composites decreased  compared to CSPKC (1.58 μm). Clearly, the meshes of the abrasive paper had some effects on the surface roughness of the composites (the surface roughness of the composites increased with the decrease in meshes of the abrasive paper), but there were no obvious differences.Moreover, a large number of n-CS particles were exposed on sb-CSPK surfaces after the PK was removed by sand-blasting and many pores  were made on the surfaces. Thus, the Ra of the surface roughness for sb-CSPK (3.82 μm) significantly increased compared to CSPKC, 12-CSPKC, 8-CSPKC and 4-CSPKC. The results suggested that sand-blasting was good way to treat composite surfaces because it made coarse surfaces with many micropores and several n-CS particles were exposed on the surfaces.17. In this study, The water contact angles of the composites surfaces significantly decreased after treatment with abrasive paper and sand-blasting as compared to CSPKC (87.5°). The water contact angles of sb-CSPK were the lowest (58.8°) compared to 12-CSPKC (78.5°), 8-CSPKC (74.8°) and 4-CSPKC (73°).Surface hydrophilicity can greatly affect the bioperformances of biomaterial implants18. Therefore, the n-CS/PK composites treated with abrasive paper or sand-blasting likely had good hydrophilicity that had positive effects on the cells responses.The results showed that the surface treatment with abrasive paper or sand-blasting significantly improved the hydrophilicity of the composite surfaces, and sand-blasting proved to be a viable way to improve the hydrophilicity of the composite. It is well-known that PK is a hydrophobic polymeric material, while n-CS is a hydrophilic inorganic material. The improved hydrophilicity was likely due to the n-CS particles exposed on the composite surfaces after treatment. It has been reported that biomaterials surfaces are exposed to dilute serum and that more hydrophilic surfaces are better for cell attachment, spreading and proliferation than hydrophobic surfacesin vivo, and the bone-bonding ability of these biomaterials is often evaluated by examining apatite mineralization on the surfaces in SBF in vitro21. In this study, after the composites were immersed in SBF for 7 days, the apatite layers with many globular apatite particles covered the 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPKC surfaces. However, only small numbers of apatite particles were found on the CSPKC. In addition, the EDS demonstrated that Ca and P peaks appeared on the surfaces of CSPKC, 12-CSPKC, 8-CSPKC and 4-CSPKC and sb-CSPK, which indicated that the deposits were Ca-P substances.Bioactive materials, such as bioglass, hydroxyapatite (HA) and glass-ceramic A-W, have the ability to bond with bone tissues through the formation of an apatite-layer on surfaces The ion concentrations of Ca for 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPK increased on the first day, and then gradually decreased over 14 days after the composites were soaked in SBF, which indicated that the n-CS on the composite surfaces first dissolved and released Ca ions into SBF, and then the Ca-P substances were deposited. The formation of apatite will consume P ions. Thus, the P ions gradually decreased. In addition, the Si ions for 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPK gradually increased during the soaking time because of the dissolution of n-CS into the SBF. However, for CSPKC, small changes of Ca, P and Si ion concentrations in SBF were found during soaking time because only a small amount of these ions were released from n-CS dissolution into the SBF . Thus, a small amount of apatite was deposited on CSPKC.2+, which was exchanged with H+ in solution and formed silanol (Si-OH) groups22. The Si-OH groups were negatively charged, which attracted Ca2+ by electrostatic interaction23. With Ca2+ accumulated on the composites surfaces, the PO43− in solution was attracted by Ca2+ and deposited on the surfaces, which triggered the formation of an apatite nucleus on the surfaces. The apatite nucleus continued to grow by consuming the Ca and P ions in the surrounding fluid to form an apatite layer24. The results indicated that surface treatment of abrasive paper and sand-blasting had significantly effects on the mineralization of composites in vitro. Abundant CS particles were exposed on the composite surfaces after treatment, which was conducive to the formation of an apatite layer.The mechanism of apatite formation on the composite surfaces could be suggested as follows. After soaking into SBF, the composite initially released Ca25. The surface roughness and hydrophilicity of biomaterials had effects on cell responses, and rough surfaces and the hydrophilic surfaces of biomaterials were better for cell attachment and spreading than smooth surfaces and hydrophobic surfaces26. In this study, a greater amount of MC3T3-E1 cells grew on 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPK with more efficient spreading, and many fine filopodia of the cells were anchored in the composite surfaces, whereas the cells for CSPKC were spindle-shaped with a few filopodia. Furthermore, the sb-CSPK with the highest surface roughness and hydrophilicity had the highest cell attachment, while CSPKC with the lowest surface roughness and hydrophilicity had the lowest cell attachment. The results demonstrated that sb-CSPK with rough surfaces and hydrophilic surfaces treated with sand-blasting obviously promoted cell attachment and spreading.The initial cell attachment and spreading will influence the proliferation and differentiation of cells on implants surfacesThe proliferations of MC3T3-E1 cells on 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPK was significantly higher compared to CSPKC, and the proliferation on sb-CSPK was significantly higher than 12-CSPKC, 8-CSPKC and 4-CSPKC. Moreover, more actin filaments linking adjacent cells grew on 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPK, which were polygonal and clustered, whereas small numbers of cells were found to grow on CSPKC. It is known that ALP activity is a key marker of osteogenic differentiation of osteoblasts in the early stages. Higher levels of ALP activity secretion illustrated more rapid and earlier differentiation of cells cultured on the materials, and osteogenic differentiation of the cells around the interface between bone and implants played a crucial role in the new bone formation process. In this study, the ALP activities of MC3T3-E1 cells on 12-CSPKC, 8-CSPKC, 4-CSPKC and sb-CSPK were significantly higher than CSPKC, and the ALP activity of cells on sb-CSPK was significantly higher than 12-CSPKC, 8-CSPKC and 4-CSPKC. The results demonstrated that the sb-CSPK treated with sand-blasting effectively promoted the proliferation and differentiation of MC3T3-E1 cells.28. Moreover, appropriate concentrations of Si and Ca ions released from Ca-Si based biomaterials played important roles in stimulating the proliferation and differentiation of osteoblasts29. In this study, the results revealed that the apatite layers appeared on the composite surface, and Si and Ca ions were released from the composite after treatment with abrasive paper and sand-blasting, which promoted the osteoblast proliferation and differentiation. Moreover, the pH of the solution containing 12-CSPKC, 8-CSPKC and 4-CSPKC slightly increased to 7.64, 7.70 and 7.75, while the pH of the solution for sb-CSPKC sharply increased to 7.8 due to the dissolution of n-CS and the formation of a weak alkaline environment. Previous studies have shown that weak alkaline conditions would provide a better micro-environment for cell proliferation and subsequently differentiation30.Studies have shown that the formation of the apatite layer on the biomaterial surface was beneficial to osteoblast proliferation and facilitated osteoblasts differentiation to form an extracellular matrix composed of biological apatite and collagenTherefore, it could be suggested that the mechanism of the positive response of MC3T3-E1 cells to the composites after treatment could be attributed to the improved surface roughness, hydrophilicity, apatite layer formation, Ca and Si ion release, and weak alkaline micro-environment. In short, an n-CS/PK composite treated with abrasive paper and sand-blasting could greatly improve bioperformance, which would facilitate the attachment, proliferation and differentiation of osteoblasts. The n-CS/PK biocomposite treated with sand-blasting might be a promising implanted material for orthopedic applications.In this study, bone implants made of n-CS/PK composites were surface treated using abrasive paper and sand-blasting. The results showed that surface treatments (n-CS was exposed on the composite surfaces) obviously decreased the water contact angles while greatly improving the mineralization ability of the composites in SBF compared to un-treated composites. In addition, the surface roughness of the composite treated by sand-blasting was obviously enhanced compared to abrasive paper. Moreover, the composites treated with abrasive paper and sand-blasting significantly promoted cell attachment, spreading and proliferation, and improved the ALP activity of MC3T3-E1 cells compared to un-treated composite; moreover, the sand-blasting treatment was better than the abrasive paper. With improved surface roughness, hydrophilicity, bioactivity and cytocompatibility, the n-CS/PK biocomposite treated with sand-blasting might be a promising implanted material for orthopedic applications.2O (30 mL) and HCl solution  in a water bath for 1 hour. Then, magnesium nitrate hexahydrate (9.6 g) was added to the solution, and tetraethyl orthosilicate  was added dropwise to the solution with magnetical stirring at 50 °C for 5 hours. The obtained white suspension was placed under a fume hood at room temperature for 24 hours for precipitation, which was then isolated by centrifugation, washed thoroughly with deionized water, and dried at 60 °C. The obtained powders were calcinated in air at 600 °C for 6 hours at a heating rate of 1 °C/min to obtain nano-magnesium silicate (n-MS). PK powders were obtained from Victrex Manufacturing Ltd. . The n-CS/PK composite with 40 w% n-CS was produced (the mixture of n-CS and PK powders) using an injection-molding machine  with an injection temperature of 380 °C.First, 4 g of P123  was dissolved by stirring in a HThe n-CS/PK composite was cut into discs (Φ10 × 2 mm), and the sample surfaces were treated using abrasive paper or sand-blasting. The n-CS/PK composite was CSPKC, the abrasive paper treatments with 1200#, 800# and 400# mesh were called 12-CSPKC, 8-CSPKC and 4-CSPKC, respectively, and treatment with sand-blasting was called sb-CSPKC. The surface-treated composite samples were cleaned with deionized water for 2 hours in an ultrasonic oscillator . After drying at 37 °C overnight, the samples were sterilized with ethylene oxide for 3 hours, and the sterilized samples were sealed in sterile containers for subsequent use.The hydrophilic properties of the surfaces of n-CS/PK composite samples before and after treatment with abrasive paper or sand-blasting were determined by measuring the water contact angles using the sessile drop method with a drop-shape analysis system . The surface morphology/microstructure and chemical composition of n-CS/PK composite samples before and after treatment were characterized using scanning electron microscopy  and energy dispersive spectrometry . The surface morphology/microstructure and surface roughness of Ra  were determined with InfiniteFocus Optical 3D surface metrology .in vitro mineralization of the composite samples before and after treatment with abrasive paper or sand-blasting in simulated body fluid (SBF) was evaluated by examining the apatite formation at different times31. The samples (Φ10 × 2 mm), including CSPKC (a), 12-CSPKC (b1), 8-CSPKC (b2), 4-CSPKC (b3) and sb-CSPKC (c), were immersed into SBF (pH = 7.4) at 37 °C for 1, 3, 5, 7 and 14 days. At the end of each time point, the samples were taken out and rinsed 3 times with deionized water and dried overnight. Then, the surfaces of the composites were analyzed with SEM and EDS. Moreover, the changes in ion concentrations of Ca, P and Si in the solution after the samples were soaked in SBF for different times were tested by ICP-AES . The pH variation of the solution after the samples were soaked in SBF during the entire period was monitored by using a pH meter .The 2 and 95% air. The culture medium was changed every 3 days.The MC3T3-E1 cells  were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum , 1% penicillin  and streptomycin sulfate  at 37 °C in a humidified atmosphere with 5% CO4/cm2 with three empty wells containing 1 mL of DMEM as blank controls. At each time point, the samples were gently rinsed three times with phosphate-buffered saline  to remove unattached cells and transferred to a new 24-well plate. A total of 50 μL of CCK-8 solution  was added to each well and incubated for 3 h. After that incubation, 100 μL of the supernatant was transferred into a 96-well plate and read at 450 nm using a microplate reader . The mean absorbance value/optical density (OD) obtained from the blank control was subtracted from the ODs of the test groups.The cell counting kit-8 (CCK-8) assay was used to analyze cell attachment to the samples at 6, 12 and 24 hours. The cultured MC3T3-E1 cells were digested with 0.25% trypsin , resuspended and seeded into a 24-well plate at a density of 3 × 10After culturing on the samples for 24 hours, the MC3T3-E1 cells were fixed with 2.5% glutaraldehyde for 15 min and washed 3 times with PBS. The samples were dehydrated with an ethanol gradient at volume fractions of 30%, 50%, 70%, 90% and 100%, which were then replaced by hexamethyldisilazane . Finally, the samples were air-dried and observed with SEM.4/cm2, and the detection time points were 1, 3 and 5 days. The other detailed procedures were nearly similar to the procedures of the cell attachment test. The modified OD values on days 3 and 5 were normalized to the OD values on day 1 because the numbers of attached cells on the different materials were different on day 1.Cell proliferation was investigated using the CCK-8 assay. The seeding density of the cells was 1 × 10The cell morphology on the samples was observed via confocal laser scanning microscopy (CLSM). After the cells were cultured on the samples for 5 days, the cells were gently washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min and stained with rhodamine phalloidin  for 30 min. The filamentous actin of the cell cytoskeleton was visualized using a CLSM .4/cm2 in a six-well plate containing samples (Φ10 × 2 mm). After the cells were cultured for 24 hours, the culture medium was changed to an osteogenic inductive culture medium supplemented with 100 nM dexamethasone (Sigma-Aldrich), 50 μg/mL ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate sodium (Sigma-Aldrich). After 7 and 14 days, ALP activity was determined by quantifying the amount of p-nitrophenol using a microplate test kit , and the ODs were measured using a microplate reader at 405 nm. Furthermore, the total protein content was determined using the BCA protein assay kit  according to the manufacturer’s provided. The ALP activity was finally normalized to the corresponding content of total protein.The MC3T3-E1 cells were seeded on the composite samples at a density of 3 × 10All data are presented as the means ± standard deviations, and all experiments were repeated three times. Statistical significance between different groups was analyzed using a two-way analysis of variance (ANOVA) test, and multiple comparisons were performed using the least significant difference (LSD) test. A p value < 0.05 was defined as significant, and a p value < 0.01 was defined as highly significant.Dataset 1


Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world from America, Europe, Asia to Africa and Australia. Some species of Bartonella are pathogenic for humans. Their main vectors are blood-sucking arthropods such as fleas, ticks and blood-feeding flies. One such dipteran able to transfer vector-borne pathogens is the deer ked (Lipoptena cervi) of the family Hippoboscidae. This species acts as a transmitter of Bartonella spp. in cervid hosts in Europe.The bacteria of the genus Lipoptena cervi) were collected from 26 red deer (Cervus elaphus) hunted in January 2014. A short fragment (333 bp) of the rpoB gene was used as a marker to identify Bartonella spp. in deer ked tissue by PCR test. A longer fragment (850 bp) of the rpoB gene was amplified from 21 of the positive samples, sequenced and used for phylogenetic analysis.In the present study, 217 specimens of deer ked (Lipoptena cervi infection with Bartonella spp. was 75.12% (163/217); 86.67% (104/120) of females and 60.82% (59/97) of males collected from red deer hunted in the Strzałowo Forest District in Poland  were infected. The nucleotide sequences from 14 isolates (Bartonella sp. 1) showed close similarity to Bartonella schoenbuchensis isolated from moose blood from Sweden (GenBank: KB915628) and human blood from France (GenBank: HG977196); Bartonella sp. 2 (5 isolates) and Bartonella sp. 3 (one isolate) were similar to Bartonella sp. from Japanese sika deer (GenBank: AB703149), and Bartonella sp. 4 (one isolate) was almost identical to Bartonella sp. isolated from Japanese sika deer from Japan (GenBank: AB703146).The overall prevalence of Bartonella spp. in deer keds (Lipoptena cervi) in Poland by molecular methods. Bartonella sp. 1 isolates were most closely related to B. schoenbuchensis isolated from moose from Sweden and human blood from France. The rest of our isolates (Bartonella spp. 2–4) were similar to Bartonella spp. isolated from Japanese sika deer from Japan.To the best of our knowledge, this is the first report to confirm the presence of The online version of this article (10.1186/s13071-017-2413-0) contains supplementary material, which is available to authorized users. Bartonella comprises small, Gram-negative bacteria which act as obligate intracellular parasites of vertebrates. About 30 species, as well as three subspecies, have been described [Bartonella species , is composed of wild mammals, which usually only possess asymptomatic bacteraemia. The prevalence of bacteremia in wild animals is often very high, ranging from 50 to 95% in selected rodent and ruminant populations [Bartonella infection has been recorded in cattle in three different countries  and in buffalo  from Thailand [Bartonella infection in these regions varies between 10 and 90%. Similar investigations in Japan found no infection in cattle, but recorded a prevalence of Bartonella sp., 67.5% in Honshu sika deer  and 50% in Yezo sika deer (Cervus nippon yesoensis) [The genus escribed . The zooulations , 3 and sulations . A good Thailand . The preBartonella species are considered as human pathogens and causative agents of zoonotic diseases: B. bacilliformis, the agent of Carrion’s disease and Verruga peruna is the chronic delayed stage of infection; B. quintana, the agent of trench fever and bacillary angiomatosis; B. henselae, the agent of cat scratch disease and of bacillary angiomatosis; B. clarridgeiae, B. elizabethae, B. vinsonii subsp. arupensis, B. vinsonii subsp. berkhoffii, B. alsatica, B. koehlerae and B. washoensis all cause endocarditis [Bartonella infection most often manifests as various cardiovascular, neurological and rheumatologic conditions [Many carditis –8. The snditions .Bartonella schoenbuchensis, which is found in cattle, wild animals (such as the cervids) and humans [Capreolus capreolus) in Germany [B. schoenbuchensis was first detected in France [Bartonellosis has been observed in humans throughout Europe, Asia and North America. One of the most important species is d humans –12. It h Germany , in catt Germany  and in hBartonella are transmitted by blood-sucking arthropods, such as lice, flies, fleas and ticks, many of which are emerging pathogens of humans and animals [B. schoenbuchensis in Lipoptena cervi collected from red deer and roe deer. Halos et al. [Bartonella spp. in hippoboscide flies, and suggested that flies can be a vector for pathogens. Other authors [Bartonella spp. in wingless adult deer keds. animals , 13–15.  animals  identifis et al.  reported authors , 17 haveLipoptena cervi are blood-sucking parasites which belong to a highly specialized family of louse flies (Diptera: Hippoboscidae) [Hippobosca equina), parasitizing cows and horses, deer keds (Lipoptena cervi and Lipoptena fortisetosa), parasitizing cervids, and sheep ked (Melophagus ovinus), which ectoparasites of sheep [Lipoptena fortisetosa in Europe could be introduced with Japanese sika deer (Cervus nippon). This cervids were introduced to England in 1860, and their number and range have since increased [Lipoptena have been identified: L. cervi and L. fortisetosa [oscidae) , 19. Thioscidae) , 21. In oscidae) . In Euroof sheep , 23. Lipncreased . From Entisetosa , 26.Lipoptena (L. cervi and L. fortisetosa) are cervids , the insects can attack a wide range of animals, including bovids , carnivores  and suids (Sus scrofa) [Lipoptena cervi in Europe are red deer (Cervus elaphus), roe deer (Capreolus capreolus), moose  and sika deer (Cervus nippon), while Japanese sika deer (Cervus nippon) are the predominate host in Japan, and white-tailed deer (Odocoileus virginianus), moose  and cattle (Bos taurus) in North America [Although the dominant hosts for two species of  scrofa) . The mos America , 28, 29.Lipoptena cervi is found swarming in large numbers on the host. Deer keds most frequently bite animal hosts; they can land on humans, but rarely bite in this case. Nevertheless, some authors have reported ked bites on humans [Generally, n humans , 31.Lipoptena cervi begins with free-ranging, winged adult deer keds that search for a suitable host (cervids). The winged deer keds usually suck blood from the host and mate; however, after landing on a host, they can crawl into the fur, shed their wings and become permanently associated with the host. The larvae develop up to stage 3 (L3) in the oviduct and are then deposited in the cervid fur as a white prepupa, which immediately starts to pupate. The fully developed pupa drops to the ground and remains there until August-September, when a new generation of winged adult deer keds can appear [Lipoptena cervi can overwinter on the host, and most deer keds can live to one year [The life-cycle of n appear , 32, 33.one year .Lipoptena cervi are attracted to large moving dark-colored objects when actively searching for a host [Lipoptena cervi only fly for short distances and frequently attack accidental hosts such as humans or dogs [The winged adult r a host . Lipopte or dogs .Lipoptena cervi is a potential vector for other zoonotic pathogens, such as Anaplasma, Borrelia and Rickettsia species [Bartonella spp. in all development stages of deer ked . These data show vector competence for transmission of Bartonella spp. by Lipoptena cervi. In America, Bartonella sp. has been reported in Lipoptena cervi from white-tailed deer in Massachusetts [Lipoptena mazamae from white-tailed deer (Odocoileus virginianus) in Georgia and South Carolina (USA) [L. mazamae, could be transmitted mechanically from female white-tailed deer to their offsprings [Bartonella sp. has also been found in ticks (Ixodes ricinus) parasitizing roe deer in Poland [ species . Korhone species  showed fchusetts , and in na (USA) . Additiofsprings . Bartonen Poland .Bartonella spp. DNA in Lipoptena cervi by PCR test and verification of prevalence of this infection in adult wingless males and females. Molecular characterization of specimens of Bartonella spp. was done by analysis of partial (850 bp) rpoB gene sequences.The main goal of the study was detection of Lipoptena cervi) were collected from 26 red deer (Cervus elaphus) hunted in January 2014 in the Strzałowo Forest District . After collection, all insects were preserved in 70% ethanol for further morphological and molecular processing. Identification of deer ked (L. cervi) species and sex were conducted using appropriate identification key [Living deer keds  according to the manufacturer’s protocol.rpoB gene of Bartonella spp. PCR reactions were conducted according to Paziewska et al. [rpoB gene with second set of primers (1400F and 2300R) [For PCR test the primers rpoR and rpoF were used. These primers amplify a 333 bp fragment of the a et al. . 21 of 1d 2300R)  , 1 μl of dNTPs (10 mM), 0.5 μl of each primer (20 mM), and 5 μl of 10× Taq DNA polymerase buffer . In the negative control, nuclease-free water was added to the PCR mix instead of the tested DNA.PCR reactions were conducted in a 50 μl reaction mixture containing 2 μl of DNA template, 0.5 U (0.1 μl) of RUN .DNA amplification (1400F/2300R) was performed using the DNA Engine PTC-200 Thermal Cycler  using the following program: initial denaturation was performed at 94 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and extension at 72 °C for 60 s. The final extension was performed at 72 °C for 7 min and then kept at 10 °C in a thermocyclerThe PCR products were visualized on a 1.0% agarose gel stained with ethidium bromide. Visualization was performed using ChemiDoc, MP Lab software . The resulting product was compared using the Nova 100 bp DNA Ladder Novazym . The PCR amplicons were purified using a QIAEX II Gel Extraction Kit , sequenced in both directions by Genomed (Poland) and contiguous sequences assembled using ContigExpress, Vector NTI Advance 11.0 . The derived sequences were submitted to the GenBank database under the accession numbers MF580655–MF580675.Lipoptena cervi, DNA from four specimens , L700F and L1213R primers were used to amplify and sequence 412 bp fragments of 16S rDNA , and 38.4 μl of deionized water.In order to perform PCR amplification, the following mixture reaction was used: 4 μl of DNA extract was added to 46 μl of reaction mixture consisting of 0.1 μl of Allegro Taq Polymerase DNA (5 U/μl) , 0.5 μl dNTPs (10 mM), 1 μl of each primer (20 mM), 5 μl of 10× .DNA amplification (L700F/L1213R) was performed using the DNA Engine PTC-200 Thermal Cycler  using the following program:an initial denaturation was performed at 92 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and extension at 72 °C for 30 s. The final extension was performed at 72 °C for 5 min and then kept at 12 °C in a thermocyclerrpoB gene sequence data was based on an alignment of 804 bp (268 amino acids) using the GTR + I + G model. The GTR models were chosen on the basis of jModelTest version 2.1.4 [We used Bayesian inference (BI) analysis with MrBayes version 3.2 . Analysion 2.1.4 , 44 usinCervus elaphus). All insects were identified as Lipoptena cervi using morphological features [Bartonella spp. infection was 75.12% (163/217) by PCR test. In the tested group of L. cervi, a greater proportion of females (86.67%) was found to be positive for Bartonella spp. than males (60.82%).In total, 217 deer keds were collected from 26 red deer (features . The preBartonella sp. 1: MF580662–MF580675) obtained in this study share over 99% similarity with B. schoenbuchensis isolated from moose blood from Sweden (GenBank: KB915628) and from human blood from France (GenBank: HG977196). Five sequences Bartonella sp. 2 (MF580657–MF580661) showed 94.6% similarity with Bartonella sp. 3, and 94.4% with Bartonella sp. from Japanese sika deer from Japan (AB703149). Bartonella sp. 3 (MF580656) showed 99.7% similarity with another samples isolated from Japanese sika deer from Wakayama Prefecture Japan (AB703149). Bartonella sp. 4 (MF580655) was 99,7% similar with Bartonella sp. isolated from Japanese sika deer  from Nara Prefecture Japan (AB703146). These results are summarized in Table Fourteen sequences (Lipoptena cervi collected in the Czech Republic (AF322437). The sequences derived during this study were submitted to the GenBank database under the accession numbers MF541726–MF541729.Four 16S rDNA sequences of deer ked were obtained, which were identical with Bartonella species in deer keds (Lipoptena cervi) obtained from red deer (Cervus elaphus). Dehio et al. [L. cervi appears to be a natural reservoir supporting the transmission of Bartonella schoenbuchensis. The largest group of isolates (Bartonella sp. 1) showed closed similarity with Bartonella schoenbuchensis. Our data indicate a high prevalence of Bartonella spp. A prevalence of 75.12% was recorded in the whole tested group, a result similar to global data; however, higher values were observed in Finland (90%) and France (94%) [Our findings indicate the presence of o et al.  noted thce (94%) , 37.Bartonella spp. infection in male and female deer keds. Females were more frequently infected than males; 86.67% of wingless females were infected by Bartonella, similar to a study from Hungary (76.0%), while only 60.82% of wingless males were infected, also similar to a result in Hungary (58%) [To the best of our knowledge, the present paper is the first report in Poland to identify differences in the prevalence of ry (58%) .Bartonella species are able to colonize or survive more efficiently as females than males, but the molecular mechanism for this remains unknown. The same authors [Bartonella spp. infection in female deer keds might lead to more female offspring than uninfected females, possibly resulting in the observed asymmetry in the female:male ratio of infected individuals.The reason for such a difference in infection prevalence is not clear; however, it might be associated with the multiple bites needed for larvae production (20–25 larvae per year) . De Brui authors  suggesteLipoptena cervi can be potentially dangerous for animals and humans. A study conducted in Finland found that L. cervi can transmit some pathogens by biting, and this is associated with hair-loss epizootics in moose [By transmitting pathogens, in moose . Since tin moose .Bartonella sp. 1, see Table Bartonella schoenbuchensis sample isolated from moose in Sweden (KB915628). Bartonella schoenbuchensis could be found in vectors, such as L. cervi or L. mazamae, or in definitive hosts like cattle, wild ruminants and humans [B. schoenbuchensis and deer ectoparasites may relate to similar cases in humans. Lipoptena cervi has fed on humans under experimental conditions and in the natural environment [B. schoenbuchensis during occasional biting by L. cervi, hunters, forestry workers and cross-country runners, among others, are at increased risk of infection [L. cervi has not yet been definitively demonstrated to transmit B. schoenbuchensis through bites, it is possible that hunters are at risk to infection of Bartonella schoenbuchensis or Anaplasma phagocytophilum following exposure to deer blood [Fourteen of the sequences  and human (HG977196). Bartonella schoenbuchensis  was previously reported from roe deer (C. capreolus) in Poland [In Massachusetts, a  mazamae , which sn Poland .Bartonella sp. 2 (5 isolates) and Bartonella sp. 3 (MF580656) demonstrated high similarity to sequences obtained from Japanese sika deer from Japan  is very similar (99.7%) to Bartonella sp. isolates collected from Japanese sika deer .Bartonella sp. samples, isolated from different prefectures, comprise a group distinct from other samples of Bartonella on GenBank; however, further testing is required utilizing other genes, including gltA and ftsZ, to confirm whether they could be regarded as a distinct species.Some Japanese Bartonella spp. can be transmitted by deer ked in central Europe, and the prevalence of this pathogen is very high. In this area, Lipoptena cervi are infected by various species of the genus Bartonella.Our data confirm that Additional file 1: Table S1.rpoB DNA and amino acid sequences variability among species/sequences of Bartonella used in phylogenetical analysis. Above diagonal: number of variable sites in the 268 amino acid gene rpoB sequences. Below diagonal: number of variable sites in the nucleotides gene rpoB sequences. The percentage of variable sites for each gene fragment between 2 species/sequences is given in parenthesis. Each species isolates/sequence information are provided in Table Pairwise comparison of partial (804 bp) mitochondrial gene


Streptococcus salivarius strains to mucus (HT29-MTX) and non-mucus secreting (Caco-2/TC7) human intestinal epithelial cells were investigated. Ability to adhere to these two eukaryotic cell lines greatly differs between strains. The presence of mucus played a major factor in adhesion, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface. Only one S. salivarius strain (F6-1), isolated from the feces of a healthy baby, was found to strongly adhere to HT-29 MTX cells at a level comparable to that of Lactobacillus rhamnosus GG, a probiotic strain considered to be highly adherent. By sequencing the genome of F6-1, we were able to identify 36 genes encoding putative surface proteins. Deletion mutants were constructed for six of them and their adhesion abilities on HT-29 MTX cells were checked. Our study confirmed that four of these genes encode adhesins involved in the adhesion of S. salivarius to host cells. Such adhesins were also identified in other S. salivarius strains.The adhesion properties of 14 The online version of this article (10.1007/s00253-018-8794-y) contains supplementary material, which is available to authorized users. Streptococcus salivarius is a Gram-positive bacterium that is closely related to Streptococcus thermophilus and Streptococcus vestibularis  of humans  (hereafter denoted as GM17) and conserved at − 20 °C in GM17 supplemented with 20% (w/v) of glycerol . Either erythromycin (5 μg mL−1) or spectinomycin (500 μg mL−1)  was added to GM17 medium for the selection of mutants.In previous works, we examined the susceptibility of commensal and clinical L. rhamnosus GG (LGG) (ATCC® 53103™) was included as a positive control (Laparra and Sanz Escherichia coli strains (Table −1) or spectinomycin (50 μg mL−1). The F6-1 strain characterized further in this study has been deposited at the CIRM  under the accession number CIRMBP-1193.ns Table  used forns Table  at 37 °CS. salivarius strains were centrifuged at 4500 rpm for 10 min, and the pellets were washed three times with 5 mL of a peptone water solution (casein peptone 0.1% (p/v); Merck Millipore, Molsheim, France). The pellets were then resuspended in peptone water solution to obtain bacterial suspensions adjusted to OD600nm = 0.8. A volume of 5 mL of these cultures was incubated at 37 °C for 5 h in a steady position. Aggregation was scored positive when clearly visible sand-like particles formed due to the precipitation of cells at the bottom of the tubes, leaving a clear supernatant. The aggregation ability was confirmed by turbidimetric methods at λ = 600 nm. The aggregation percentage was calculated by the following expression:The cultures (5 mL) of the 14 t0 of the bacterial suspension / ODt5 of the upper suspension after 5-h incubation) × 100.Auto-aggregation coefficient time at 5h (ACT) = 1 − (ODAuto-aggregation assays were performed in triplicate. Negative controls (without bacteria) confirmed the medium sterility.S. salivarius F6-1 cells by a standard protocol for Gram-positive bacteria after lysozyme (10 mg mL−1)/mutanolysin (700 u mL−1) treatment of bacterial cells  in appropriate buffer per 50 μL reaction volume. Classic PCR amplifications were performed using the following cycling parameters: 5 min at 98 °C, followed by 30 cycles of 30 s at 98 °C, 30 s at 58 °C and 1 min at 72 °C, and finally 5 min at 72 °C. Cycling conditions for the overlap PCR were 3 min at 98 °C, 15 cycles of three steps , 30 cycles of three steps , and a final extension for 10 min at 72 °C. The quantity and the size of amplified products were verified by electrophoresis on 1% agarose gels.Seven ts Table . Brieflyts Table  of aboutts Table . A seconts Table . The crots Table , and 0.0Two different intestinal epithelial cell lines were used in this study: (i) the non-mucus-secreting Caco-2/TC7 cells , Merck) and re-suspended in Dulbecco’s Modified Eagle Medium without antibiotics at a final concentration of 109 CFU mL1− (OD600nm of about 12) for S. salivarius strains and 1010 CFU mL1− (OD600nm of about 90) for LGG. The initial number of viable bacterial cells was determined by plating on GM17 agar for S. salivarius and MRS agar  for LGG.Adhesion experiments were performed as previously described by Kebouchi et al.  with minP value <0.05. For the bacterial adhesion of F6-1 mutants, the comparison was performed with the wild-type F6-1 strain.Experimental data were analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey’s test. R 3.1.2 software was used for the statistical analysis. Results were expressed as means ± SEM. Differences were considered statistically significant when S. salivarius strains used in this work were sequenced using an Illumina HiSeq2000 sequencer by Beckman Coulter Genomics . De novo assemblies were performed using CLC Genomics Workbench (CLC Bio) using default parameters. Scaffold of the genomes was built by using the Genome Finishing module of CLC Genomics Workbench with the S. salivarius JIM8777 genome as reference. Some assembly gaps were filled by doing PCR and sequencing. Multifasta files (one per genome) representing all translated CDSs of the studied genomes were generated. Among proteins, the ones being putatively secreted proteins were identified by their peptide signal sequence using the software Signal P 4.1 Server . Amino acid domains of each putative surface protein were retrieved with the CD-search tool from NCBI (www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) . One group includes the four S. salivarius strains B35, F6-1, L25, and L64, which show an auto-aggregation coefficient time (ACT) at 5 h below 5%. The other group contains 10 strains  characterized by an ACT at 5 h higher than 58%.The auto-aggregation abilities of 14 S. salivarius strains were tested on two human intestinal epithelial cell lines, Caco-2/TC7 (non-mucus secreting) and HT29-MTX (secreting mucus). Results presented in Fig. L. rhamnosus GG (LGG) and show that the S. salivarius strains display statistically different adhesion capacities. The less adherent strains are T93 on Caco-2/TC7 (0.30 ± 0.04 CFU per Caco-2/TC7 cell) and B50 on HT29-MTX (4.9 ± 0.4 CFU per HT29-MTX cell). The most adherent strains are B50 on Caco-2/TC7 (29.4 ± 7.8 CFU per Caco-2/TC7 cell) and F6-1 on HT29-MTX (128.0 ± 8.6 CFU per HT29-MTX cell). All the S. salivarius strains show an adhesion capacity to Caco-2/TC7 and HT29-MTX cells lower than LGG , except F6-1, which shows an adhesion capacity to HT29-MTX cells which is 1.3 times more than LGG  present statistically different adhesion capacities with the two cell lines (P < 0.05) . Most of these 11 strains, except B50 and N20, showed better adhesion ability to HT29-MTX cells than to Caco-2/TC7 cells. The three other strains  display no significant difference of adhesion capacity to both cell lines.Figure S. salivarius strain with the best adhesion capacity to the HT29-MTX cells, presumably due to the surface adhesins. Thus, a focus was made on this commensal strain, which was isolated from the feces of a healthy baby of 15 months. To be present on the outside of the bacterial cell, adhesins have to translocate across the cytoplasmic membrane before being exposed at the cell surface. Genes encoding putative surface proteins with a signal peptide were first identified in the genome sequence of F6-1. Thirty-six CDS encoding such proteins have been detected in the genome of F6-1 (Table F6-1 is the -1 Table . The 26 ΔsrtA mutant. Its adhesion capacity to HT29-MTX cells (13.4 ± 0.9 CFU per HT29-MTX cell) is 7.5 times lower than that of F6-1 wild type , suggesting that it is a zinc metalloprotease. Streptococcus zinc metalloproteases are less described but Novak et al.  and to act as a virulence factor in this bacterium , F6-1 SALIVA_1472 (MucPB domain), and F6-1 SALIVA_1473 (G5 domains) proteins were separately replaced by a spectinomycin resistance gene to generate the F6-1 Δ1457, F6-1 Δ1472, and F6-1 Δ1473 mutants. As shown in Fig. S. salivarius F6-1 and is found to bind to fibronectin but not to collagen or submaxillary mucin in Streptococci , F6-1 SALIVA_0576 (FctA and collagen_bind domains), and F6-1 SALIVA_1475 (Antigen_C and FctA domains) proteins were separately replaced by a spectinomycin resistance gene to generate the F6-1 Δ0442, F6-1 Δ0576, and F6-1 Δ1475 mutants. As shown in Fig. S. salivarius sequenced genomes. These include 22 genomes already available in the NCBI database and 11 newly sequenced ones corresponding to S. salivarius strains tested in our aggregation/adhesion study.Thus, our results show the involvement of four putative adhesins in the attachment of F6-1 to HT29-MTX cells, the three LPxTG proteins F6-1 SALIVA_1457, F6-1 SALIVA_1472, and F6-1 SALIVA_1473 and a protein devoid of LPxTG motif, F6-1 SALIVA_1475. Therefore, homologs of these F6-1 proteins were searched in all the translated CDSs of 33 SALIVA_1457 gene has two internal assembly gaps, which cannot be filled because of sequence repetitions. Such problem was also observed for 14 other sequences of F6-1 SALIVA_1457 homologs which are partial (data not shown). Thus, complete (7) or partial (14) sequences of F6-1 SALIVA_1457 homologs were detected in only 21 of the 33 strains (data not shown). The seven complete proteins have sizes between 1134 aa (HSISS4 strain) and 2300 aa (JIM8777 strain). They exhibit more than 86% of identity with F6-1 SALIVA_1457. They all include an LPxTG motif and a ser_adhes_Nterm domain (TIGR04224) as in F6-1 SALIVA_1457.The sequence of the F6-1 S. salivarius strains examined  or partial (5) sequences of F6-1 SALIVA_1473 homologs were detected in the 33 other strains examined (data not shown). The complete proteins have sizes between 1953 aa (strains 1270 and B63) and 2105 aa  and display more than 90% of identity with F6-1 SALIVA_1473. The proteins showing the closest relationship with F6-1 SALIVA_1473 are those of JIM8777, B50, and L64 strains . In total, complete (17) or partial (16) sequences of F6-1 SALIVA_1475 homologs were detected in all the 33 S. salivarius strains (data not shown). The 17 complete proteins have sizes between 2220 aa (F1–4 strain) and 5423 aa (L25 strain). They show more than 70% of identity with F6-1 SALIVA_1475. All of these 17 proteins show the same domains as F6-1 SALIVA_1475 (Antigen_C and FctA), but their copy number is variable, from 2 (F1–4 strain) to 14 (B50 strain) for the Antigen_C domain and from 8 (F1–4 strain) to 20 (B50 strain) for the FctA domain.The sequence of the F6-1 S. salivarius strains varies considerably between strains. Such variability was previously observed with S. thermophilus on HT-29  may reflect their excellent mucus-adhesive capacity which can be ascribable to strain-specific receptors at their surface.The second finding is that most of the Bifidobacterium longum or Lactobacillus reuteri . Altogether, these results suggest that S. salivarius strains might perform their adhesion and aggregation by different mechanisms involving various surface molecules. This was observed for Lactococcus lactis BGKP1, in which the aggregation and the adhesion to gastric-type mucin proteins involve the aggregation factor AggL and the mucin-binding protein MbpL, respectively  and pig gastric mucins , a family of glycosylated adhesins highly conserved in Gram-positive bacteria and mediating bacterial-host interactions . Furthermore, biofilm formation is dependent on the copy number of G5 domains; five or more G5 domains are required in SasG to support biofilm formation of S. aureus  I/II family that are cell wall-anchored proteins produced by most indigenous species of oral streptococci


A previous paper from our lab Shalom,  presente A previous paper from out lab Shalom,  argued tThe current review attempts to perform a similar analysis for the lateral prefrontal cortex (LPFC), shifting from the relevant medial prefrontal areas to their lateral counterparts: lateral BA 11/BA 47 (perception), lateral BA 10/BA 46 (memory), lateral BA 9 (emotion), and lateral BA 8 (motor) Figure . It brinThere is evidence that perceptual selection and inhibition are supported by neural networks involving the lateral part of BA 11/47 in the LPFC. In a model proposed by Zsuga et al. , the orbLateral BA 11/47 has been also shown to be involved in the maintenance of visual stimuli in working memory and guidance of visual selection. In one study, Soto et al. , compareFinally, according to Price , lateralThere is evidence that children and adults with ADHD have problems with inhibiting irrelevant visual and auditory stimuli. For example, ADHD adults have shown more interference effects  than controls when asked to complete a recall task while ignoring a background noise  and lateral BA 9. For example, in a study by Hallam et al. , particiIn addition, two different meta-analyses were used to examine fMRI studies assessing emotion regulation. Buhle et al.  found thSeveral studies found emotion regulation deficits in children with ADHD. For example, several studies show that children with ADHD have a harder time suppressing their emotions than their typically developing (TD) peers. For example, in one study children underwent a peer competition task, in which half were requested to hide their emotions and half were not. According to assessments made by naive judges, results show that ADHD children were less able to mask their emotions than their peers  in which participants are required to press a key when a stimulus is presented, but refrain from executing that key press if the stimulus is followed shortly by a signal. Studies show that inhibiting the key press involves activation in lateral BA 8 (Matthews et al., There is robust evidence of motor selection and inhibition deficits in children with ADHD. Many studies to date have shown that children, adolescents and adults with ADHD show poorer performance on different versions of the stop signal task (e.g., Rubia et al., There is also considerable evidence showing specific atypicalities in lateral BA 8 activation in ADHD participants performing tasks which require motor selection and inhibition. For example, two separate meta-analyses have explored fMRI studies assessing motor inhibition in children and adults with ADHD (Hart et al., The current paper presents evidence that the lateral part of the prefrontal cortex is involved in the inhibition, selection and manipulation of motor, emotional, memory, and perceptual-sensory information, a function that is helpful in everyday life through the ability to ignore distraction, select and retrieve specific information from our memories, identify and regulate our emotional state or plan a situation appropriate motor response. It uses this model to classify challenges encountered by people with ADHD, linking different types of difficulties in selection/inhibition to different subareas of the lateral prefrontal cortex.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous research investigating attention and impulse control in individuals with Attention-Deficit/Hyperactivity Disorder (ADHD) has largely ignored the symptomatic differences among the three subtypes of ADHD: ADHD-Inattentive Type, ADHD-Hyperactive/Impulsive Type, and ADHD-Combined Type. The present study examined attention and impulse control by focusing on these subtypes. Based on their self-reported symptoms of ADHD, participants belonged to one of four groups: ADHD-Inattentive, ADHD-Hyperactive/Impulsive, ADHD-Combined, and control. Cortical activity was recorded from participants during performance of a Go/NoGo task. The event-related potentials (ERP) measured at frontal and posterior sites discriminated between the control group and participants with symptoms of ADHD. The control group consistently exhibited a higher P3 amplitude than all the ADHD groups. The main difference occurred at the frontal site, indicating that individuals with ADHD symptoms have deficits in the anterior attentional system, which mediates signal detection. Behavioral measures of signal sensitivity revealed that the ADHD-Inattentive and the ADHD-Hyperactive/Impulsive groups had more difficulty with the attention-demanding Go/NoGo respond-to-target task, while behavioral measures of response bias indicated that the ADHD-Hyperactive/Impulsive and the ADHD-Combined groups responded more liberally in the inhibition-demanding Go/NoGo suppress-to-target task.


Attention-deficit/hyperactivity disorder (ADHD) in the adult population is frequently associated with comorbid psychiatric diseases that complicate its recognition, diagnosis and management.The prevalence of ADHD in the general adult population is 2.5% and it is associated with substantial personal and individual burden. The most frequent comorbid psychopathologies include mood and anxiety disorders, substance use disorders, and personality disorders. There are strong familial links and neurobiological similarities between ADHD and the various associated psychiatric comorbidities. The overlapping symptoms between ADHD and comorbid psychopathologies represent challenges for diagnosis and treatment. Guidelines recommend that when ADHD coexists with other psychopathologies in adults, the most impairing condition should generally be treated first.Early recognition and treatment of ADHD and its comorbidities has the potential to change the trajectory of psychiatric morbidity later in life. The use of validated assessment scales and high-yield clinical questions can help identify adults with ADHD who could potentially benefit from evidence-based management strategies. Attention-deficit/hyperactivity disorder (ADHD) is a psychiatric disorder associated with considerable personal and societal burden. While ADHD is well recognized in the pediatric population, where it was first described as a clinical diagnosis in the 1930s . Often, As our diagnostic systems are evolving towards a more dimensional approach to the classification of mental disorders, so too is our understanding of adult ADHD . Today, Thus, the goals of this article are to review the available evidence on the prevalence, burden, and neurobiology of adult ADHD, to describe how a practical, dimensional approach can help clinicians identify ADHD in patients with complex presentations, and to inform appropriate management decisions in order to improve patient outcomes in this under-treated population. With the recognition that the evidence base continues to evolve and that there is a lack of quality evidence to guide the management of complex patient presentations, this article answers some common clinical questions based on available evidence as well as our collective experience in the management of adult ADHD and comorbidities.PubMed was searched for articles in English published between 1996 and 2016, using the following search terms: “adult ADHD” in combination with anxiety; addiction; affective dysregulation; alcohol; bipolar; burden; catecholamine deficit; cocaine; cognition; depression; dimension; dimensional; disability; executive functioning; functioning; guideline; heritability; life expectancy; mania; marijuana; mortality; neurobiology; nicotine; personality disorders; prevalence; prevention; recommendation; risk factors; and substance use. Clinical trials, guidelines, meta-analyses, and systematic reviews were selected by the authors for inclusion in this review.In total, 113 unique articles were identified; 8 were excluded at the title filtering stage, 7 at the abstract filtering stage, and another 7 after a full-text review. Overall, 22 articles were excluded and 91 were included. Exclusion criteria included: articles about disorders other than ADHD ; studies of pediatric or adolescent populations; and studies involving alternative therapies rather than psychological therapy or pharmacotherapy. Manual searches of the reference lists of identified articles and other interesting published works including authoritative texts were also selected, bringing the total number of articles included in this review to 150.ADHD has an estimated childhood prevalence of 4% to 7%  with incThe general population prevalence of ADHD in adults has been estimated to be 2.5%  , with adP ≤ 0.001) and were significantly less satisfied with their personal, social and professional lives [Adult ADHD is associated with profound functional and psychosocial disability, leading to serious personal and societal costs. Its most prominent feature is attentional dysfunction, associated especially with impairment in focused and sustained attention . Individal lives . ADHD isal lives , as wellal lives . Collegeal lives . Later ial lives , 33. Oneal lives .The detrimental effects of ADHD on overall health and safety provide additional imperative to appropriately recognize and manage this debilitating disorder. Adult ADHD has been associated with poorer driving and a higher incidence of traffic citations and motor vehicle accidents . A recenP < 0.001) [In addition to its substantial burden at the individual level, adult ADHD is often associated with considerable societal costs. Notably, there have been consistent associations between adult ADHD and unemployment , 37. In < 0.001) . The ass< 0.001) . A more < 0.001) . ADHD ha< 0.001) , with on< 0.001) . Another< 0.001) .Along with these functional and psychosocial impairments, ADHD is associated with a higher risk of developing mood and anxiety disorders. In many studies, ADHD has been associated with comorbid depression, anxiety disorders, bipolar disorder, and substance use disorder , 40–42. There are strong familial links between ADHD and psychiatric comorbidities such as bipolar disorder, suggesting a genetic contribution . One posInterestingly, the main neural pathway that modulates emotional affect comprises the limbic-cortical-striatal-pallidal-thalamic (LCSPT) circuits, consisting of connections between the orbital and medial prefrontal cortex (OMPFC), ventromedial striatum, ventral pallidum, hippocampal subiculum, mediodorsal and midline thalamic nuclei, and amygdala . These cAs such, one can imagine this low hedonic tone as a key feature shared by MDD and resulting from a shared dysfunction in monoamine signaling, particularly in the ventral striatum . In suppn = 3199), only 11% of adults with ADHD were receiving treatment [Overlapping symptomology between ADHD and mood, anxiety, or SUDs present several barriers to diagnosis and treatment. Studies have concluded that emotional dysregulation is a distinctive attribute of adult ADHD psychopathology, however these symptoms may be misdiagnosed as a mood disorder –68. Simireatment . In partreatment , which fBipolar DisorderThe most common psychiatric comorbidities that co-occur with ADHD in adults are depression, anxiety disorders, bipolar disorder, SUDs and personality disorders. The overlapping and distinctive features of these disorders are summarized in Fig. b)Dysthymia/DepressionADHD has a high prevalence of comorbidity with bipolar disorder. Rates of ADHD comorbidity in bipolar disorder have been estimated between 9.5% and 21.2%, and rates of comorbid bipolar disorder in ADHD at 5.1% and 47.1% . BipolarP < 0.03). This is not surprising, since serotonergic agents alone would not be expected to improve ADHD symptoms, which typically respond to catecholaminergic agents such as noradrenalin-dopamine reuptake inhibitors or psychostimulants. Purely serotonergic activity lowers dopamine and norepinephrine levels via effects on 5-HT2C and 5-HT2A interneurons, respectively [c)Anxiety DisordersADHD and dysthymia/depression co-occur frequently, with studies reporting prevalence rates of depression in individuals with ADHD ranging from 18.6%  to 53.3%ectively . As wellectively , 84.c)Ad)Substance Use DisorderThe risk for anxiety disorders is higher in individuals with ADHD than in the general population , 86 withPossibly the most common comorbid condition with ADHD is SUD, particularly alcohol and/or nicotine, cannabis and cocaine use . SubstanThe burden of comorbid ADHD with SUD is substantial. Studies have found that ADHD in individuals with SUD is associated with earlier onset of substance use, increased likelihood of suicide attempts, more hospitalizations, higher rates of poly-substance abuse, less likelihood of achieving abstinence, and lower rates of treatment adherence , 98, 99.e)Personality DisordersAlthough a link between treatment of ADHD with psychostimulants and later development of SUD has been proposed, this is not supported by the literature . NotablyThe literature on comorbid ADHD and personality disorders is sparse compared to other psychiatric comorbidities. Reports suggest that personality disorders are present in more than 50% of adults with ADHD, most commonly cluster B and C personality disorders, and 25% of individuals have two or more personality disorders . ImportaADHD and personality disorders often co-occur with other axis I disorders. For instance, 18% of adults with ADHD and depression and 23% of adults with ADHD and bipolar disorder are estimated to also have a personality disorder . The subThere is a paucity of clinical trials evaluating management strategies for personality disorders in general , let aloIt is important to determine if a patient presenting with one of the above-listed psychiatric disorders also has comorbid ADHD. This may be difficult, in part because of the difficulties associated with establishing a correct diagnosis. According to the DSM-IV-TR, ADHD is defined on the basis of the diagnosis being made before age 7 , but theHave you had long-standing and consistent problems with attention and distractibility?Have your current complaints been present over the last 10 or 20 years?If I could see you in the classroom when you were a child, what would you be like?Thus, we propose three key questions that clinicians can ask in order to help identify red flags suggestive of an ADHD diagnosis in complicated patients:If these questions suggest a possible positive ADHD diagnosis, an in-depth clinical interview should be undertaken using a screening instrument such as the Adult ADHD Self-Report Scale (ASRS), the Wender-Reimherr Adult Attention-Deficit Disorder Scale (WRAADDS), or the Conners Adult ADHD Rating Scales (CAARS). Another tool for assisting diagnosis is FAST MINDS  . Once a Several published articles have presented reviews and recommendations concerning treatment options and algorithms , 107. TrPharmacologic treatments for ADHD are usually divided into stimulants and non-stimulants. Stimulant medications include methylphenidate, mixed amphetamine salts, and lisdexamfetamine dimesylate. Non-stimulants used in ADHD treatment include atomoxetine and alpha-2-adrenergic agonists. Antidepressants such as venlafaxine and bupropion have also been evaluated as treatment options for ADHD, with some evidence of benefit in addressing ADHD symptoms , 109. OnAnother important treatment consideration is the potential for the effective treatment of ADHD to improve functional outcomes of patients with comorbid conditions. Many studies have reported improvements in comorbid psychiatric symptoms when ADHD is effectively treated. For instance, atomoxetine has been associated with improvements in both ADHD and comorbid anxiety  and deprPerhaps more exciting is the concept that early and optimal treatment of ADHD could potentially prevent the later development of psychiatric comorbidities. In a 10-year longitudinal follow-up study of male youths with ADHD, Biederman et al. (2009)  found thA final important treatment consideration is safety and tolerability. Both stimulant and non-stimulant medications have possible side effects, which must be taken into account. Common side effects of stimulants include headache, appetite suppression, nausea, dry mouth, mood fluctuations, difficulty sleeping, jitteriness, increased heart rate, and increased blood pressure . GeneralNon-pharmacological interventions play a central role in the management of ADHD. Evidence supports the superiority of multimodal approaches utilizing pharmacotherapy and psychosocial and/or behavioural interventions to target the core symptoms of ADHD and for the improvement of functional outcomes , 117. SiPsychotherapeutic modalities are also central to the management of the most common psychiatric comorbidities in adults with ADHD, namely SUD, depression, anxiety, and bipolar disorder. However, prospective studies of psychosocial strategies in comorbid ADHD populations are scarce. One study in 419 outpatients with ADHD, of which 38% had at least one comorbid axis I psychiatric disorder, showed that multimodal strategies are more effective than psychosocial or pharmacotherapeutic strategies alone for improving symptoms of ADHD, but the effects on depressive symptoms were not statistically significant . A smallWhat are the risks of prescribing psychostimulants to a patient with ADHD plus bipolar disorder or anxiety? Will it precipitate a switch to mania or an exacerbation of anxiety?b)What should I treat first: the ADHD or the comorbid psychiatric disorder?A review of previous studies supports stimulants as a first-line therapy for the treatment of ADHD symptoms in individuals with concurrent ADHD and bipolar disorder, given a lack of strong evidence that stimulants are linked to mania . Howeverc)What should I do if my patient is smoking marijuana, or has a history of cocaine use?The consensus in the literature is that the most severe, functionally impairing or destabilizing illness should be treated first, and comorbidities should be addressed in a stepwise fashion once the patient has responded to treatment , 69, 107Patients with ADHD and SUD require multimodal treatment, and ADHD treatment should be delayed until the SUD has been addressed . MarijuaAlthough there have been some concerns that stimulant use in the treatment of ADHD may cause increased cocaine cravings or use, this was not supported in a study of methylphenidate . If dived)Will treating ADHD improve outcomes in depression, bipolar disorder, or SUD?Marijuana abuse has been associated with deficits in cognition , 125. HoEffective treatment of ADHD has been shown to improve comorbid disorders including SUD , 95, 132e)Why have psychostimulants failed in trials of treatment-resistant depression?Finally, it should be reiterated that the best practice for comorbid ADHD and psychiatric disorders is the principle of treating the most serious or debilitating condition first and proceeding with treatment of subsequent residual symptoms in a stepwise manner . If deprf)Can early treatment of ADHD prevent the onset and/or mitigate the severity of depression, bipolar disorder, or SUD?Psychostimulants have been evaluated in clinical trials for the treatment of resistant forms of depression. Although rapid antidepressant effects have been observed, such effects tend to be transient . One ranThere is some evidence to suggest that early treatment of ADHD can have a protective effect on future development of depression, bipolar disorder, and SUDs. Halmøy et al. (2009) reported that individuals with ADHD who received stimulant treatment in childhood had lower rates of depression and bipolar disorder later in life compared with individuals who were not treated . A similg)Are psychostimulants addictive?Early ADHD treatment can also have beneficial effects that extend beyond comorbidities. For instance, Halmøy et al. (2009) reported that children who received ADHD treatment had rates of unemployment three times lower than individuals who did not receive treatment for their ADHD as children .g)Are ph)Are psychostimulants overprescribed and overused in college/university students?Psychostimulants such as methylphenidate and mixed amphetamine salts have been demonstrated to possess likeability and euphorogenic effects, and are therefore classified as Schedule II controlled substances with abuse liability potential . DespiteThere is some concern that college or university students may exaggerate ADHD symptoms in order to obtain a stimulant prescription, which they perceive as helpful in terms of academic performance . There iADHD is a prevalent psychiatric disorder in the adult population that is frequently unrecognized, under-diagnosed, and under-treated. Given that it is often comorbid with other psychopathologies including mood or anxiety disorders, substance use disorders, and personality disorders, adults presenting with symptoms of ADHD should be screened for these frequently comorbid conditions, and vice versa, in order to identify patients who could potentially benefit from optimal management of ADHD and its comorbidities. Although the clinical presentation of ADHD in adults can be variable and complex, it can often be identified using a few high-yield clinical questions, and the use of validated assessment scales in patients screening positive. Early and optimal treatment of ADHD has the potential to change the trajectory of psychiatric morbidity later in life and to substantially improve functional outcomes across the spectrum of psychiatric comorbidities. In general, when ADHD coexists with other psychiatric pathologies, the more severe disorder should be treated first according to evidence-based guidelines. In the coming years, research on the genetic and neurobiological basis of ADHD should continue to uncover fruitful avenues of treatment for this debilitating disorder and ultimately improve outcomes for patients and their families.


STS gene and SULT2A1 gene, dehydroepiandrosterone (DHEA) and its sulfated form (DHEA-S), and characteristics of attention-deficit/hyperactivity disorder (ADHD). We used cheek swabs to obtain the genomic DNA of 200 ADHD male probands (mean age: 8.7 years), 192 patients’ mothers and 157 patients’ fathers. Three SNPs in the STS gene  and one SNP in the SULT2A1 gene (rs182420) were genotyped. Saliva samples were collected from the ADHD patients to analyze DHEA and DHEA-S levels. The behavioral symptoms were evaluated with the Swanson, Nolan, and Pelham, and Version IV Scale for ADHD (SNAP-IV), and the neuropsychological function was assessed using the Conners’ Continuous Performance Tests (CPT). We found the C allele of rs2270112 within the STS gene to be over-transmitted in males with ADHD. Polymorphisms of rs182420 within the SULT2A1 gene were not associated with ADHD. In addition, the C allele carriers of rs2270112 demonstrated significantly higher DHEA-S levels than the G allele carriers. Levels of DHEA were positively correlated with attention as measured by the CPT. These findings support a potential role in the underlying biological pathogenesis of ADHD with regard to STS polymorphisms and neurosteroid levels.This study examined the relationships among polymorphisms of the  A) receptors and facilitates N-methyl-D-aspartate (NMDA) activity111214Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that affects approximately 3–10% school-age children around the world16STS) gene, located at Xp22.32, encodes a multi-pass membrane protein of the endoplasmic reticulum19STS gene exhibit abnormal behaviors and cognitive functions that are relevant to the inattention and hyperactivity that many ADHD patients experience2123The sex discrepancy in many ADHD characteristics is significant. The prevalence of ADHD is significantly higher in males (the ratios of boys to girls range from 2:1 to 9:1)1STS gene is correlated with androgen synthesis and metabolismSTS deficiency has been proposed as a risk factor for ADHD19et al.STS deficiency) and ADHD diagnosis/symptoms. Brookes et al.STS gene , as well as an allele specific haplotype of 12 SNPs, were found to be significantly associated with ADHDet al.STS were related to cognitive performance among ADHD males. However, said association between STS polymorphisms and ADHD has not yet been examined in a non-Caucasian population.In humans, the SULT2A1 gene, located at 19q13.3, encodes a member of the sulfotransferase familySULT2A1 gene may participate in ADHD pathophysiology19et al.SULT2A1 gene (rs2637125 and rs182420) may affect DHEA-S concentration in post-pubertal healthy boys. Although a genome-wide association study (GWAS) revealed that genetic variants of SULT2A1 do not affect individuals’ DHEA(S) concentrations or the DHEA/DHEAS ratioSULT2A1 influence neurosteroid and further correlate to ADHD susceptibility remains unclear.The STS and SULT2A1 genes are vital for catalyzing neurosteroids (DHEA or DHEA-S)19203319STS gene and the SULT2A1 gene, DHEA(S) levels, and ADHD characteristics. Genetic associations for complex diseases may be probed either with case-control studies of unrelated people or with family-based designs3636STS gene and SULT2A1 gene, salivary DHEA(S) levels, and ADHD characteristics.Evidence has already established that the The study protocol was approved by the Institutional Review Board of Chang Gung Hospital in Taiwan (IRB No: 101-4835A3). All procedures performed in this study involving participants were in accordance with the ethical standards of the institutional and/or national research committee and with the Helsinki Declaration. We obtained written informed consent from the parents or guardians of all the participating children.We recruited eligible outpatients with ADHD receiving treatment at the Department of Child Psychiatry at Kaohsiung Chang Gung Memorial Hospital in Taiwan. The admissibility criteria for entry into this study were as follows: (1) patients aged between 6 and 16 years who met the criteria for ADHD outlined in the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV); (2) without a history of neuropsychiatric diseases ; (3) without any known major physical illnesses  that require surgical intervention or long-term medication; (4) either newly diagnosed with ADHD or had an existing diagnosis but had not taken medication for ADHD for at least the prior 6 months; (5) of ethnic Han Chinese origin; and (6) with at least one biological parent that can provide the check swab sample and complete this study’s assessment. A total of 208 boys (mean age of 8.7 ± 2.1 years) met the inclusion criteria and agreed to participate in this study.Patients were interviewed by a child psychiatrist using the Chinese version of the Schedule for Affective Disorder and Schizophrenia for School-Age Children, Epidemiologic Version (K-SADS-E). The social and behavioral competence and ADHD symptoms of patients were evaluated with the Swanson, Nolan, and Pelham Questionnaire, Version IV for ADHD (SNAP-IV), which the patients’ parents and teachers completed. The ADHD patients then performed the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV), and the Conners’ Continuous Performance Tests (CPT) were performed by an experienced psychologist in a dedicated room for testing so that test condition variability was minimized. Patients’ parents were asked to fill out the 18-question Adult ADHD Self-Report Scale (ASRS).High molecular weight genomic DNA of ADHD patients and their biological parents were extracted from cheek swabs using routine procedures. The ADHD patients’ saliva samples were collected between 7:00 and 8:00 am using the passive drool method in order to analyze the neuroendocrine substrate levels. Patients were instructed to avoid excessive physical activity during the preceding 24 h and to fast overnight prior to saliva collection.STS gene (rs6639786 and rs2270112 in intron 1 and rs17268988 in intron 9) has been carried out in previous literature2930www.hapmap.org), the Han Chinese population has no reported polymorphisms for rs5934770, rs12861247, or rs17335568. Therefore, these SNPs were not considered for further analyses in this study.The genotyping of three SNPs in the SULT2A1 gene has been genotyped in previous literature34The polymorphism rs182420 in the in-vitro diagnostic quantitative determination of DHEA-S . The detection range of these methods for DHEA and DHEA-S was 2.2–1440 pg/ml and ≥0.05 ng/mL, respectively. The intra- and inter-assay coefficients of variation were 3.4–4.7% and 3.1–5.6% for DHEA and ≦7.8% and ≦14.9% for DHEA-S.Saliva was put in collecting tubes, immediately placed on ice, and stored at −80°C until assayed. We used an enzyme immunoassay (ELISA) to determine and quantify the DHEA in saliva  and also used an ELISA for the The K-SADS-E is a semi-structured diagnostic interview designed to assess current and past episodes of psychopathology in children and adolescents according to DSM-III-R and DSM-IV criteriaThe SNAP-IV is a 26-item questionnaire used to evaluate ADHD symptoms and severityThe ASRS is a self-administered questionnaire that evaluates ADHD symptoms in adults. The two-part questionnaire consists of inattention and hyperactivity/impulsivity subscales, each of which has nine indications that have persisted for at least 6 monthsThe WISC-IV is an individually administered and norm-referenced tool that aims to measure intelligence in children aged from 6 to 16The CPT is a 14-minute computerized test that mainly judges attention and impulse controlData were analyzed using the Statistical Package for the Social Sciences for Windows . Variables are presented as either mean ± standard deviation (SD) or frequency.p-value were calculated for each allele of interest.The transmission disequilibrium test (TDT) was originally developed to test for the transmission of associated marker locus by studying case-parent trios from a heterozygous parent to an affected childSTS gene were classified based on the allele identified on X-chromosome, and genotypes of rs182420 in the SULT2A1 gene were classified into C/T or T/T. Metric variables between genotypes were tested using the t-test or the Mann-Whitney U test. The Mann-Whitney U test was used to determine the potential difference in neurosteroid levels between patients with different genotypes. Spearman’s correlation coefficient was used to investigate the relationship between neurosteroid levels and ADHD characteristics. Two-tailed p-values < 0.05 were considered statistically significant. Bonferroni correction was further performed to adjust for multiple testing in the correlation matrix.Because data on the DHEA and DHEA-S levels exhibited significant positive skews and violated normal distribution, and the case numbers of the minor allele carriers were small, non-parametric statistics were used to analyze the data. Genotypes of the SNPs  within SULT2A1 gene had higher variability scores (p = 0.006) than those with T/T genotype.Of the 208 ADHD boys recruited in this study, 200 patients provided their genomic DNA with a cheek swab; the saliva samples of 160 patients were successfully collected; and 173 patients completed the clinical and neuropsychological assessments. STS gene (p = 0.020) was over-transmitted in males with ADHD. The other two SNP markers located in the STS gene (rs6639786 and rs17268988) and rs182420 in the SULT2A1 gene were not associated with ADHD , DHEA levels were only significantly correlated to Confidence Index and Hit RT SE. We observed no significant correlations between salivary DHEA(S) levels and behavioural symptoms.The relationships of salivary neurosteroid levels and ADHD characteristics are provided in STS gene was over-transmitted in Han Chinese boys with ADHD. Polymorphisms of rs182420 within the SULT2A1 gene were not associated with ADHD. Since the C allele of rs2270112 within the STS gene was over-transmitted in males with ADHD, we explored whether rs2270112 polymorphisms influence STS gene function, reflected by patients’ nerurosteroids levels. Among boys with ADHD, the C allele carriers of rs2270112 had significantly higher DHEA-S levels than the G allele carriers. In addition, we found that the salivary levels of DHEA were correlated with several neuropsychological indices measured by the CPT.Our results reveal that rs2270112 within the STS gene and the SULT2A1 gene in ADHD among a non-Caucasian population. A previous study using family-based designSTS gene were significantly associated with ADHD. Furthermore, another study using case-control designSTS gene were associated with cognitive function in ADHD males. In contrast, we found that only one SNP (rs2270112) of the STS gene was over-transmitted in ADHD boys. The inconsistent findings may be attributed to discrepancies of sample size, study designs, or possible ethnic differences in genetic variants. For example, several SNPs of the STS gene that have been associated with Caucasian ADHD patients  do not exhibit polymorphisms in the Han Chinese population. Additionally, it has been reported that the G allele at rs17268988 is the minor alleleSTS gene in males with ADHD.To the best of our knowledge, this study is the first to investigate the genetic variants of the STS gene was significantly associated with higher DHEA-S levels in boys with ADHD. The STS protein is a microsomal enzyme that converts DHEA-S into its unsulfated form of DHEA, which participates in a neurosteroid biosynthesis pathway of the brainSTS genetic variants affect DHEA(S) concentrations is still scarce2652STS gene with the synthesis or transformation of DHEA(S) levels in patients with ADHD, our result suggest the rs2270112 may be linked to the neurobiology pathogenesis of ADHD8We found that the C allele of rs2270112 at SULT2A1 gene were not associated with ADHD, and did not influence the salivary DHEA or DHEA-S levels in ADHD boys. However, Garcia-Anguita et al.SULT2A1 gene may differ between ethnicities. The SULT2A1 gene has been reported as a catalyst for the sulfate conjugation of DHEASULT2A1 appear to have effects on individuals’ DHEA(S) have been inconsistent in previous studies34SULT2A1 gene plays a role in the susceptibility of ADHD.Our study’s results reveal that the polymorphisms of rs182420 in the A and NMDA activity657The results of this study show that several indexes measured by CPT were positively correlated with salivary DHEA levels, particularly Confidence Index and Hit RT SE. These findings are in line with our previous investigations in which an independent cohort was recruited10489212223STS and SULT2A1 genes also play a role in female ADHD patients.The subjects recruited in our study were all males. Compelling evidence have revealed that male and female ADHD patients have distinct clinical manifestations64666769STS gene and SULT2A1 gene in this study were chosen based on the findings of previous literature. However, several SNPs that have been found to be associated with ADHD in Caucasian populations did not exhibit polymorphisms in our Han-Chinese study sample. Therefore, advanced techniques, such as next-generation sequencing, may be more beneficial for discovering the genetic variants associated with ADHDThis study has several limitations. First, the case number was small, and that reduced statistic power lowers the likelihood of finding a potential association between genetic variants and ADHD. Second, the target SNPs of the STS gene polymorphisms and DHEA(S) levels may related to ADHD susceptibility. Among Han Chinese boys with ADHD, polymorphisms of rs2270112 within the STS gene were over-transmitted, and are associated with DHEA-S concentration. Salivary levels of DHEA were positively correlated with attention measured by a neuropsychological test. Future research with a considerably larger sample size is needed to determine how neurosteroids and their associated genes are involved in the underlying biological mechanisms of ADHD.In conclusion, our findings suggest that How to cite this article: Wang, L.-J. et al. Polymorphisms of STS gene and SULT2A1 gene and neurosteroid levels in Han Chinese boys with attention-deficit/hyperactivity disorder: an exploratory investigation. Sci. Rep.7, 45595; doi: 10.1038/srep45595 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


While psychiatric comorbidities of attention-deficit/hyperactivity disorder (ADHD) have been extensively explored, less attention has been paid to somatic conditions possibly associated with this disorder. However, mounting evidence in the last decade pointed to a possible significant association between ADHD and certain somatic conditions, including obesity. This papers provides an update of a previous systematic review on the relationship between obesity and ADHD (Cortese and Vincenzi, Curr Top Behav Neurosci 9:199–218,  Attention-deficit/hyperactivity disorder (ADHD) is a major public health issue. It is one of the most frequent childhood-onset psychiatric conditions, with an estimated prevalence exceeding 5% in school-age children . It has Whereas the comorbidity between ADHD and psychiatric disorders has been extensively explored , the assCortese et al. •• first Although the present paper is not intended to be a systematic review with a formal and quantitative appraisal of the quality of the studies, we performed a systematic search for original peer-reviewed papers in a set of electronic databases, including PubMed, Ovid databases , and ISI Web of Knowledge . The search terms and syntax for the search in PubMed were (ADHD OR Attention-Deficit/Hyperactivity Disorder OR Attention Deficit Hyperactivity Disorder OR Hyperkinetic Syndrome) AND (obes* OR overweight). The search terms and syntax were adapted for each of the other electronic databases. References from each paper were examined to find additional studies possibly missed in the electronic search.We searched for studies reporting information on (1) the prevalence of obesity in individuals with ADHD, (2) the prevalence of ADHD in individuals with obesity, (3) possible mechanisms underlying the putative association between ADHD and obesity, and (4) the implications of the possible association between ADHD and obesity for the clinical management of individuals with both conditions. Regarding criteria no. 1 and no. 2, we included only studies that used either a formal diagnosis of ADHD or in which the diagnosis of ADHD was self-reported. In order to avoid possible bias in the estimation of the prevalence of ADHD in individuals with obesity or of obesity in individuals with ADHD, we did not include studies in which participants presented only with ADHD symptoms above a cutoff on any scale for ADHD. However, we did not apply this exclusionary criterion when considering studies on the possible mechanisms linking ADHD and obesity, since a dimensional approach can still be informative in this respect. We did not apply any language restriction. We searched for reports published from January 1st, 2012, to July 16th, 2016.The search retrieved 3412 potentially pertinent hits. After excluding references not meeting our criteria, we retained a total of 41 –50 studiDetails of the studies retained in our review are presented in Tables Cortese and Vincenzi  reviewedCortese and Vincenzi  presenteTaking together the two types of studies , it is fair to state that evidence supports a bidirectional relationship between ADHD and obesity, irrespective of possible confounding factors.When Cortese and Vincenzi  wrote thOver the past 4 years, there have been remarkable progresses in the understanding of the possible mechanisms linking ADHD and obesity. In fact, we located 28 studies –50, 58 . SeveralImportantly, in the last 4 years, longitudinal studies have explored the direction of the link between ADHD and obesity. Three studies retrieved in our search showed that ADHD chronologically precedes, and likely contributes to, weigh gain , 35•, 49Cortese and Vincenzi  cited thUnfortunately, since then, no other studies have been published directly testing, by means of a randomized design, the effects of ADHD screening and treatment of obesity outcomes. However, evidence from recent studies, including those retrieved in our search , supOver the past 4 years, there has been an increasing interest for the relationship between ADHD and obesity. Studies that addressed the questions: “Is obesity (or overweight) more frequent in individuals with, compared to those without, ADHD?” or “Is there a significant relationship between ADHD and obesity/overweight?” provide overall mixed findings, likely due to heterogeneity in diagnostic methods for ADHD and obesity, population characteristics , and medication status. However, meta-analytical evidence controlling for these confounding factors support a significant association between nontreated ADHD and obesity.Remarkably, in the past 4 years, a large number of studies have contributed to our insight on the factors underlying the links between ADHD and obesity. Such body of research has pointed to the role of abnormal (dysregulated) eating patterns, decreased physical activity, sleep disruption, and psychiatric comorbidities, including conduct disorder. Preliminary evidence has also revealed possible common genetic underpinnings. Importantly, longitudinal studies have been published that show how ADHD may be a risk for the future development of obesity, although the reverse causal link cannot be ruled out.Given the epidemic of obesity, if ADHD does contribute to it, understanding how and to which extent the treatment of comorbid ADHD in individuals with obesity may lead to long-term weight loss in individuals with obesity, improving adherence to diet programs is fundamental. There is a paucity of studies on this issue and we believe that this should receive further attention in future research. This line of research has ultimately the potential to improve the clinical management and, as a consequence, the quality of individuals with both ADHD and obesity.