A wide range of neuropsychiatric disorders, from schizophrenia to drug addiction, involve abnormalities in both the mesolimbic dopamine system and the cortical salience network. Both systems play a key role in the detection of behaviorally relevant environmental stimuli. Although anatomical overlap exists, the functional relationship between these systems remains unknown. Preclinical research has suggested that the firing of mesolimbic dopamine neurons may activate nodes of the salience network, but in vivo human research is required given the species-specific nature of this network.2/3 receptor ligand 11C-PHNO, n = 23) and dopamine synthesis capacity  within the ventral striatum. Resting-state functional magnetic resonance imaging was also undertaken in the same individuals to investigate salience network functional connectivity. A graph theoretical approach was used to characterize the relationship between dopamine measures and network connectivity.We employed positron emission tomography to measure both dopamine release capacity (using the DDopamine synthesis capacity was associated with greater salience network connectivity, and this relationship was particularly apparent for brain regions that act as information-processing hubs. In contrast, dopamine release capacity was associated with weaker salience network connectivity. There was no relationship between dopamine measures and visual and sensorimotor networks, indicating specificity of the findings.Our findings demonstrate a close relationship between the salience network and mesolimbic dopamine system, and they are relevant to neuropsychiatric illnesses in which aberrant functioning of both systems has been observed. SEE COMMENTARY ON PAGE 366Resting-state functional magnetic resonance imaging (rfMRI) has demonstrated that activity within networks of brain regions is temporally correlated even in the absence of explicit external demands The salience network plays a key role in identifying the most relevant internal and external stimuli to guide behavior appropriately Dopamine neurons also play a role in the identification of behaviorally relevant environmental stimuli. Mesolimbic dopamine neurons  have been proposed to signal reward prediction errors, which signal the discrepancy in the observed and predicted value of a stimulus The need to develop an integrative understanding regarding the roles of the salience network and the mesolimbic dopamine system has been previously stressed To address this, we employed positron emission tomography (PET) to measure both dopamine synthesis capacity and dopamine release capacity, and rfMRI to evaluate salience and default mode networks at rest in the same participants. Based on recent preclinical findings that stimulation of dopamine neurons projecting to the limbic striatum activates regions of the salience network In addition, we identified within these networks regions that played the most important role in information processing (“hub nodes”). Hubs support the rapid integration of information across a complex system and as such can be considered an optimal target via which a network input may efficiently maximize its influence in a coordinated fashion Given the preclinical emphasis on the mesolimbic dopamine projection, we focused on dopamine measures within the limbic striatum. However, we also explored the relationship between network connectivity and dopamine function in the associative and sensorimotor  striatum. In addition, to provide a control condition, we investigated the relationship between striatal dopamine function and the visual and sensorimotor networks , where we did not expect a relationship to be present.The experimental approach is summarized in We first investigated whether network connectivity was associated with measures of dopamine function and identified specific nodes that were associated with dopamine function. We then separately classified nodes as information-processing hubs solely based on their pattern of rfMRI connectivity, and we determined whether dopamine-associated nodes overlapped significantly with these hub nodes.In addition, the visual and sensorimotor networks were examined as control networks, as they are not directly involved in salience processing and show a lack of activation in preclinical studies of mesolimbic dopamine effects 18F]fluoro-L-phenylalanine (18F-DOPA). 18F-DOPA PET measures the rate constant Kicer for 18F-DOPA uptake, transport into synaptic vesicles, and its conversion into 18F-dopamine, thus providing a measure of dopamine synthesis capacity Participants underwent a PET scan with the ligand 3,4-dihydroxy-6-[Kicer [1/min]) et al.A region-of-interest analysis was performed to determine the limbic striatum influx constant .2/3 receptor ligand [11C]-(+)-4-propyl-9-hydroxy-naphthoxazine (11C-(+)-PHNO). A placebo scan gives a measure of baseline D2/3 receptor availability , while a scan following dexamphetamine administration allows quantification of the change in BPND due to competition from increased synaptic dopamine concentrations. The percentage reduction in D2/3 receptor availability between placebo and dexamphetamine scans thus provides an index of dopamine release capacity. We calculate the percent change in BPND as follows:Participants underwent two PET scans with the D11C-(+)-PHNO administration, so that scan acquisition coincided with the expected time of peak action ND was measured in the same regions as in experiment 1.Either placebo or 0.5 mg/kg dexamphetamine was administered orally 3 hours before Participants also underwent an rfMRI scan using a Siemens MAGNETOM Verio 3T scanner .Participants had no previous or current history of psychiatric illness .Time series were extracted from 333 predefined nodes of interests of the Gordon cortical atlas. The salience and default mode network nodes of the Gordon atlas are displayed in et al. as implemented in the cocor (1.1-3) package for R 3.3.2 For each participant and each network, average network strength was defined as the mean z-transformed Pearson’s correlation coefficient between all network nodes  t = 1.3–3.1, equivalent to p = .2–.005 for n = 23), where weaker thresholds will capture subnetworks showing a widespread diffuse relationship with dopamine function, and more stringent thresholds identify smaller clusters showing the strongest relationship.To identify whether specific nodes show a significant relationship with limbic dopamine synthesis capacity, we used the network-based statistic to investigate salience, default mode, sensorimotor, and visual networks separately see  (46). Wi, betweenness centrality (47), and participation coefficient (48). We termed a node that ranked highly on all three metrics a “combination hub” A is the set of dopamine-associated nodes and B is the set of combination-hub nodes) We next asked whether dopamine-associated nodes were statistically more likely to be combination hubs. We quantified the overlap of dopamine-associated nodes and combination hubs using the Dice similarity coefficient (where t = 1.3–3.1) and then averaged to give a single score -PHNO dopamine-associated nodes.We also investigated whether there was a significant overlap between 18F-DOPA study . Twenty-three participants took part in experiment 2, the 11C-(+)-PHNO study .Twenty-one participants took part in experiment 1, the rp = .51, p = .017, rp = .44–.62)  (The correlations between edge strength and limbic dopamine synthesis capacity are displayed in the lower triangle of .44–.62) . In contp = .16) .Figure 3z = −2.7, p = .008). Furthermore, salience–default mode balance  correlated with dopamine synthesis capacity   .rp = .46, p = .034) but not the sensorimotor striatum   or sensorimotor  networks.When the relationship between salience network average strength and dopamine synthesis capacity in other striatal regions was investigated, the findings were significant for the associative striatum ( = .053) . As hyporp = −.42, p = .049) (rp = −.24 to −.52) . The difference between these two correlations was not significant , and salience–default mode balance did not correlate significantly with dopamine release capacity .The correlations between edge strength and limbic dopamine release capacity are displayed in the upper triangle of  = .049) , a findito −.52) . There wrp = −.5, p = .015) but showed no relationship with the sensorimotor striatum   or sensorimotor  networks. Interestingly, however, in an exploratory analysis, dopamine release capacity within the sensorimotor striatum showed a significant relationship with sensorimotor network average strength .As in experiment 1, salience network strength was significantly associated with dopamine release capacity in the associative striatum (p = .44) . FurtherThere was no relationship between rfMRI motion and either network strength or dopamine measures , although this was not significant across a wide range of thresholds .p > .29 for all thresholds) or sensorimotor (p > .38) networks.When dopamine synthesis capacity in other striatal subdivisions was examined, the findings were again significant for the associative but not sensorimotor striatum . The speWe identified subnetworks within the salience network showing a significant negative relationship with limbic dopamine release capacity . No defap > .12 all thresholds) or sensorimotor subnetwork  was associated with limbic dopamine release capacity.Dopamine release in other regions was examined, and similarly to experiment 1, significant results were observed for the salience network with the dopamine measure in the associative striatum but not sensorimotor striatum . As befoIn both experiments, these findings were seen in various parcellations and methods of node assignment .18F-DOPA subnetworks and combination hubs across a range of thresholds is shown in We next investigated whether the dopamine-associated nodes identified in the previous step overlapped significantly with nodes that were classified as information-processing hubs. Within the salience network, we found that regardless of how many nodes were defined as hubs within our range of investigation , these nodes were likely to be dopamine-associated nodes, and this overlap was significantly more likely than expected by chance for all hub thresholds , and thiThe Dice coefficient between combination hubs and the dopamine-associated nodes within the default mode network was numerically greater than the Dice coefficient of the random network at all thresholds, but this difference was statistically significant only for certain hub thresholds and parcellations see .11C-(+)-PHNO subnetworks were numerically greater than the mean overlap expected of the random network, but this difference was statistically significant only for certain thresholds and parcellations -PHNO– and 18F-DOPA–associated nodes ranged from 0.36 at the most stringent network-based statistic threshold where equal node networks existed (number of nodes = 11) to 0.92 at the least stringent threshold (number of nodes = 36). None of these overlaps was greater than would be expected by chance . We then investigated which nodes were in dopamine-associated networks at the most stringent threshold and were also combination hubs (ranked in the top 11/40 nodes in both experiments). Only two nodes fulfilled these criteria; these were located bilaterally in the insula -PHNO preferentially bind to the high-affinity state of the D2 receptor 2 receptors in a high-affinity state as well as the level of dopamine release. Future studies combining antagonist and agonist radiotracers would help determine the potential influence of interindividual differences in the proportion of D2 receptors in the high-affinity state. A tentative hypothesis that unites our findings assumes that in a healthy individual, dopamine synthesis capacity reflects a summary measure of tonic dopamine neuron firing and appropriate adaptive phasic firing, while release capacity reflects that individual’s propensity for spontaneous phasic firing in the absence of behaviorally relevant stimuli We hypothesized that release and synthesis capacity would capture similar facets of a single construct—the activity of an individual’s mesolimbic dopamine system. Our finding of divergent relationships between these two measures and salience network connectivity does not support this interpretation. Both release and synthesis capacity are complex signals, and the relationship between the two is not clear Preclinical research has often focused on the dopamine neurons of the ventral tegmental area. The limbic striatum is a major projection target for these neurons and, as such, an appropriate region of focus. In rodents, however, the mesolimbic pathway is proportionally larger than in humans, and therefore, although the associative striatum receives dopaminergic innervation from the nigra, parts of the human midbrain-associative striatum pathway are homologous to the rodent mesolimbic pathway Structural and functional abnormalities within the salience network are a common biological substrate of mental illness and exist transdiagnostically across a broad range of disorders, including depression, schizophrenia, and Parkinson’s disease Given that the firing of mesolimbic neurons has been shown to provoke widespread neural activity in regions receiving no direct dopaminergic innervation 11C-(+)-PHNO and 18F-DOPA are unable reliably characterize dopamine function outside of the striatum, and it was therefore not possible to test whether a relationship between direct dopaminergic innervation of network nodes and network connectivity also existed.Likewise, the precise site of relevant dopaminergic activity is not clear. We used an eyes-closed resting-state scan, and some networks have shown greater reliability when participants have kept eyes open. The differences are relatively small, however, and therefore unlikely to have significantly influenced our findings The use of acute dopaminergic challenges during simultaneous PET and MRI would allow for the study of intraindividual effects of dopaminergic release on network average strength and organization and may help to disentangle some of these issues. Studies in clinical populations, such as individuals with schizophrenia, where measures of both dopaminergic and network function may show wider ranges Measures of dopamine function showed strong associations with salience network connectivity, and in the case of dopamine synthesis capacity, this was particularly the case for nodes that were identified as information-processing hubs within the salience network. These findings are relevant to developing integrated models of brain function in health and disease and for the development of treatments that attempt to restore network function via neurochemical modulation.


Deficiency of complement factor I is a rare immunodeficiency that typically presents with increased susceptibility to encapsulated bacterial infections. However, non-infectious presentations including rheumatological, dermatological and neurological disease are increasingly recognized and require a high-index of suspicion to reach a timely diagnosis. Herein, we present two contrasting cases of complement factor I deficiency: one presenting in childhood with invasive pneumococcal disease, diagnosed using conventional immunoassays and genetics and the second presenting in adolescence with recurrent sterile neuroinflammation, diagnosed via a genomic approach. Our report and review of the literature highlight the wide spectrum of clinical presentations associated with CFI deficiency and the power of genomic medicine to inform rare disease diagnoses. The human complement system is a tightly regulated cascade of soluble and membrane-bound proteins that maintains immune homeostasis by clearing apoptotic cells and immune complexes, and facilitates host defense via the opsonization and direct killing of microbial pathogens. Monogenic deficiencies of complement factors can result in susceptibility to infection by encapsulated bacteria and, in some cases, systemic lupus erythematosus-like disease, glomerulonephropathies and vasculitides.Complement factor I (CFI) is an 88 kDa glycoprotein that is principally produced by the liver. It circulates in a zymogen-like state prior to activation by furin , which cThe understanding of the spectrum of disease associated with deficiency of complement factor I has broadened considerably in recent years. Herein, we report two contrasting cases of complement factor I deficiency; one presenting with spontaneous pneumococcal peritonitis in childhood and the other presenting with recurrent, sterile neuroinflammation in adolescence.A 2-year-old girl (patient A) presented to the emergency department with a 24-h history of lethargy, fever and abdominal pain. One week previously, she had suffered from a mild gastroentritis-like illness but had recovered fully. She was a dichorionic-diamniotic twin born at 33 weeks, was previously well and fully vaccinated according to the UK schedule, including neonatal BCG. There was no family history of immunodeficiency .S. pneumoniae, a strain not contained within the 13-valent pneumococcal conjugate vaccine administered to children in the UK.On admission, she had a distended abdomen, tachycardia, pyrexia (39°C) and raised inflammatory markers; she was admitted and treated as suspected appendicitis. At laparotomy, frank pus was found in the abdomen but the appendix appeared grossly normal. A diagnosis of spontaneous bacterial peritonitis was made. Blood cultures from admission grew serotype 10A Routine investigation of pediatric invasive pneumococcal disease in our center is based on the protocol described by Gashinard et al. . The patCFI (NM_000204.4) in the proband, which revealed compound heterozygous variants  presented to the emergency department with a 3 day history of gradual onset frontal headache, blurred vision and slurred speech, followed by several tonic-clonic seizures in short succession, deteriorating into coma. Her family reported preceding upper respiratory tract infection symptoms. She was admitted and treated as presumed meningoencephalitis. MRI neuroimaging showed diffuse, confluent cerebral and cerebellar white matter high signal changes, oedema, and mass effect without DWI change . She hadCFI gene was the only gene to show the expected compound heterozygosity.CSF sampling showed an inflammatory picture , but no bacterial or viral pathogens were detected by routine culture or PCR. C3 was borderline low and acute phase proteins remained normal during her illness. There was no improvement following treatment with empirical antibiotics and antivirals but a slow recovery ensued following pulsed methylprednisolone, with no residual neurological though very mild cognitive deficit. Whole genome sequencing (WGS) was undertaken in the proband and the proband's unaffected mother to achieve a unifying diagnosis. Filtering of all the variants identified by WGS based on quality metrics, deleteriousness, inheritance pattern and biological function led to a short list of 5 genes that were investigated further , 2. Of tCFI  lying within the CFI FIMAC domain  analysis was performed , an apprin silico prediction, factor I levels were measured in the proband and found to be undetectable. Commensurate reductions in functional activity of the classical and alternative pathway were also identified  affecting 81.25% of individuals. The remainder present with a range of rheumatological, neurological and dermatological manifestations. Within the genetically confirmed cases, there is a slight female preponderance (2.2:1) and the deficiency typically presents in childhood, although significant diagnostic delay, as seen in Patient B, can occur.Complete CFI deficiency is a rare immunodeficiency with 32 genetically characterized cases arising in 27 separate kindreds in the literature . By far CFI and the resulting clinical phenotype of complete CFI deficiency. Factor I activity is dependent on its heavy chain which contains complement regulatory elements, its light chain which contains the serine protease domain, hydrophobic interactions between the heavy and light chain and the presence of a co-factor   , 25. DifPatient A, who presented with invasive pneumococcal disease in childhood, had biallelic mutations predicted to truncate CFI within the FIMAC and SRCR domains. Neither allele produces a protein with any enzymic function, reflected by the patient's low C3, AP50, and CH50. Susceptibility to encapsulated bacterial infection arises due to a secondary deficiency in C3 and inefficient bacterial opsonization.The management of infectious complications of complete CFI deficiency is well-established and relies on the optimization of vaccination against encapsulated bacteria and appropriate use of prophylactic antibiotics. As CFI synthesis occurs in the liver, bone marrow transplantation is not a variable strategy to correct CFI deficiency. There is no empirical evidence demonstrating the effectiveness of antibiotic prophylaxis and vaccination in CFI deficiency, however, reports suggest infection frequency reduces with age with some individuals eventually becoming asymptomatic , 26. PriRecurrent sterile neuroinflammation without a significant burden of infection, as seen in patient B, is an unusual presentation of CFI deficiency. The variants identified in patient B are predicted to affect the interface between CFI and C3b. The P64 residue of CFI directly contacts C3b V1658 and forms part of a larger hydrophobic patch on CFI involving residues I55, V60, L63, P64, Y65, F82, P83, and L91 that interacts with the hydrophobic residues V1657, V1658, F1659, and the methylene groups K1570 and K1576 of C3b  secondary to complete CFI deficiency: a 10 year old female initially responded to high-dose corticosteroids and intravenous immunoglobulin (IVIG) but this was ineffective during a relapse of the condition aged 17. Anakinra significantly improved neurological parameters and radiological appearances on that occasion and was subsequently tapered. The same physicians successfully used a combination of high-dose corticosteroids, IVIG and anakinra to treat a 10 year old with ALHE; weaning of anakinra was not possible due disease relapses . Direct eactions .In conclusion, we report two cases of CFI deficiency with quite different presentations and routes taken to diagnosis. Common to both cases was the observation of low C3 and normal C4, indicative of dysregulated complement consumption. Cognizance of this unusual profile of complement tests and the range of presentations of complete CFI deficiency may facilitate timely diagnosis of this rare immunodeficiency.All datasets analyzed for this study are included in the manuscript and/or the Full informed consent for publication was obtained from affected individuals or parents prior to the publication of this report. Patient A was enrolled in the study The genetic and functional characterization of patients with Primary Immune Deficiencies, Infections and Inflammatory conditions  and patient B was enrolled in the study Molecular Genetic and Analysis and Clinical Studies of Individuals and Families at Risk of Genetic Disease approved by the West Midlands Research Ethics Committee, reference 13/WM/0466.AS, AJP, HA, and SP provided clinical care for the individuals described in this manuscript. The Oxford Clinical Whole Genome Sequencing (OxClinWGS) consortium performed the whole genome sequencing. A full list of consortium members is available in the supplementary data. AS, ATP, JT, and SP analyzed the data and wrote the manuscript. All authors provided critical appraisal of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


About 1.1 billion people smoke tobacco globally and tobacco-related health care costs 1.8% of GDP in many countries. The majority of people are unable to quit smoking despite pharmacological intervention, highlighting the need to understand the pathophysiology associated with tobacco smoking to aid the development of new therapeutics. The reinforcing effects of tobacco smoking are thought to be mediated by the dopamine system. However, the nature of dopamine dysfunction seen in smokers is unclear.To determine the nature and robustness of the evidence for dopaminergic alterations in smokers.The entire MEDLINE, EMBASE, and PsycINFO databases were searched for studies from inception date to November 18, 2018. In vivo human molecular imaging studies of dopamine measures  in tobacco smokers were selected. Demographic, clinical, and imaging measures were extracted from each study and meta-analyses, and sensitivity analyses were conducted.p = 0.03). However, there was no difference in D2/3 receptor availability in smokers relative to controls . There were insufficient studies for meta-analysis of other measures. However, findings from the published studies indicated blunted dopamine release and lower D1 receptor availability, while findings for dopamine synthesis capacity were inconsistent.Fourteen studies met inclusion criteria comprising a total sample of 219 tobacco smokers and 297 controls. The meta-analysis showed a significant reduction in dopamine transporter availability in the smokers relative to controls with an effect size of − 0.72  contains supplementary material, which is available to authorized users. It is estimated that tobacco-related health care costs 1.8% of GDP in many countries  AND (dopamine OR dopamine release OR dopamine synthesis OR dopamine availability OR dopamine transporter OR dopamine reuptake OR dopamine receptor) AND (smoking OR nicotine OR nicotine dependence OR tobacco dependence)”. In addition, the reference lists in the included studies and relevant review papers were screened to search for additional studies. Further details of study selection are provided in the supplementary Fig. The inclusion criteria were as follows: 1) original molecular imaging studies that indexed dopamine receptors, or dopamine transporters and/or dopamine release or synthesis; 2) included a group of regular (daily) tobacco smokers; and 3) reported data for the whole striatum or a striatal sub-region. We excluded studies which did not have a healthy control group or that included subjects with CNS co-morbidity. For studies with an overlap in participants, we included the study with the largest sample size without potentially missing any subject and excluded the smaller study from the meta-analysis to avoid duplication of subjects, consistent with previous molecular imaging meta-analyses , imaging characteristics , route of administration of drug challenge, and modeling method.I2 value . A significance level of p < 0.05 (two-tailed) was taken as significant.The main outcome measure was the effect size calculated as Hedges’ g for the dopaminergic index for the whole striatum in the smokers using a random effects model. Publication bias was assessed by visual inspection of funnel plots and tested with a regression test for funnel plot asymmetry (mixed-effects meta-regression model). Trim and fill analysis was conducted to impute potentially missing studies. Heterogeneity was estimated using the n = 2) and dopamine synthesis (n = 3)  , indicating high heterogeneity between studies. The regression test for funnel plot asymmetry was not significant . However, visual inspection of the funnel plot revealed asymmetry, indicating possible publication bias. The trim-and-fill analysis indicates two missing studies on the left side of the funnel plot .The p = 0.23) , indicating heterogeneity was low. The regression test for funnel plot asymmetry was not significant . However, a visual inspection of the funnel plot revealed asymmetry, indicating possible publication bias. The trim-and-fill analysis indicated that there were potentially one missing studies on the left side of the funnel plot .The Two studies reported D1 receptor availability, which used [11C] SCH23390 to compare smokers with controls . Our sensitivity analyses of the dopamine D2/3 receptor availability showed consistent results, and we noted low heterogeneity. However, there was significant heterogeneity in the dopamine transporter finding.There were insufficient studies to meta-analyze findings on dopamine synthesis in smokers, and the results of studies were inconsistent, indicating further, large studies are needed to determine if dopamine synthesis capacity is altered by smoking. Similarly, there were too few studies for meta-analyses of dopamine release or D1 receptor levels, although findings indicated blunted dopamine release and lower D1 levels in smokers. However, there were only two studies for each of these dopamine measures, and the studies had small sample sizes. Thus, while results indicate blunted dopamine release and D1 levels in smokers, further studies are needed before the consistency and robustness of these alterations can be determined.Our finding that D2/3 receptor levels are unaltered is consistent with post-mortem evidence, which also shows unaltered D2 receptor levels in smokers  polymorphism of DAT gene (SLC6A3) and DAT availability  nicotine exposure reduces basal dopamine level Table S1(DOCX 32.6 kb)


Our results showed that with the exception of nanofibers formed from L-PRP prepared at 800 + 400× g, all other microenvironments were favorable for h-AdMSC proliferation. Here, we present a reproducible protocol for the standardization of L-PRP and fibrin nanofibers useful in clinical practices with known platelet/leukocyte ratios and in vitro evaluations that may predict in vivo results.Leukocyte and platelet-rich plasma (L-PRP) is an autologous product that when activated forms fibrin nanofibers, which are useful in regenerative medicine. As an important part of the preparation of L-PRP, the centrifugation parameters may affect the release of soluble factors that modulate the behavior of the cells in the nanofibers. In this study, we evaluated the influences of four different centrifugation conditions on the concentration of platelets and leukocytes in L-PRP and on the anabolic/catabolic balance of the nanofiber microenvironment. Human adipose-derived mesenchymal stem cells (h-AdMSCs) were seeded in the nanofibers, and their viability and growth were evaluated. L-PRPs prepared at 100×  In the past few years, the benefits of autologous leukocyte- and platelet-rich plasma (L-PRP) have been evidenced in the treatment of many types of diseases ,3,4,5,6.g (10 min and 25 °C). The concentration patterns allowed for the identification of specific centrifugation ranges to obtain distinct platelet/leukocyte and the associate lymphocyte/granulocyte ratios. As blood centrifugation is a separation process based on the size and density of the components as well as the packing behavior of the erythrocytes, different recoveries of platelets and leukocytes could be obtained in L-PRP from a first spin -2,5-diphenyltetrazolium bromide (MTT)  for 4 h at 37 °C. Then, MTT was replaced with dimethyl sulfoxide (DMSO), the samples were left in a shaker for 30 min, and the absorbance was measured at 595 nm. In order to calculate the maximum specific rate of cell growth .The results are presented here as the mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey’s test was used with a 95% confidence level (


Ursus thibetanus); however, food availability in autumn strongly impacts bear nutrition and likely impacts cementum widths as well. This study aimed to test how cementum annuli widths and the formation of false annuli were influenced by hard mast production. We established two hypotheses: (1) cementum annuli widths become narrower in poor mast years owing to inadequate nutritional conditions and (2) false annuli occur more frequently in poor mast years. We used teeth samples from male bears to avoid reproductive influences and separated width data into “adult” and “subadult” groups. We calculated the proportional width index (PWI) and used linear mixed models to estimate the masting effects on PWI. Generalized linear mixed models estimated the masting effects on false annuli frequency. True annuli widths and false annuli formation showed no significant relationship with mast production in adults. In subadults, poor mast production weak negative influence on false annuli formation. These new data resolve previous questions, allowing us to deduce that cementum annuli widths are a reliable index of reproductive success in female bears.Cementum annuli widths in mammals are is influenced by the nutrition of mammals. Reproductive stress has been is suggested to reduce the width of lead to narrower cementum annuli widths in female Asian black bears ( For some organisms, banded growth lines on specific body parts document periodic growth . For exaNumerous studies have shown that cementum annuli are present in a number of mammal species, and it is possible to estimate the age of an individual by counting the cementum annuli  have been used for many years to provide an estimation of age [The cementum annuli of Asian black bears  [The first objective of our study was to test how the widths of cementum annuli are affected by fluctuating nutritional conditions owing to changes in hard mast production in autumn each year. The level of hard mast production has an impact on the nutritional status of the American black bear (ericana) . Additioericana) , 9, our To clarify these hypotheses, firstly, it was necessary to identify the seasons during which light bands were formed. Poor nutritional conditions owing to lactation in the spring and summer have an impact on cementum annuli widths , but harWe used data from the annuli widths of adult male and subadult male bears because these bears do not give birth and raise offspring, so they would not be influenced by nutritional deficiencies due to reproduction. However, in our analyses, we separated these data into adult and subadult periods because there are differences in nutritional conditions related to cementum deposition between adult and young bears . In JapaWe used the first premolar teeth from 85 male bears killed, a method that minimizes as much as possible the pain and distress to the animal shall be used in accordance with “Welfare and Management of Animals Act” (Ministry of the Environment), because of nuisance behavior such as trying intrude human settlement in Tochigi Prefecture, Central Japan, from 2007 to 2014. These captures were conducted under the permission obtained for the present study from the Tochigi Prefecture Government according to “Wildlife Protection and Proper Hunting Act” (Ministry of the Environment) and “Specified Wildlife Conservation and Management Plan” (Tochigi Prefecture Government), which covered the controlled killing of bears. Permission numbers of the Tochigi Prefecture Government permits are 19–8, 19–13, 19–36, 19–37, 20–4, 20–10, 20–31, 20–35, 21–1, 21–10, 21–16, 22–6, 22–11, 22–12, 22–20, 22–21, 22–25, 22–27, 22–28, 22–40, 22–42, 22–43, 22–55, 23–6, 23–7, 23–10, 23–11, 23–16, 23–18, 23–19, 23–20, 23–25, 23–30, 24–5, 24–9, 24–11, 24–12, 24–13, 24–16, 24–18, 24–19, 24–25, 24–30, 24–33, 24–37, 24–44, 24–45, 24–46, 24–48, 24–49, 24–50, 24–52, 24–53, 25–2, 25–4, 25–12, 25–14, 25–15, 25–16, 25–24, 26–2, 26–3, 26–24, 26–29, 26–78. In our study area, euthanasia is selected mainly to kill bears. Bears are euthanized as soon as possible after capture; a shotgun is used, so that the cerebral hemispheres and brainstem are sufficiently disrupted by the projectile to induce immediate loss of consciousness and subsequent death [We used a total of 85 bears to identify the periods of cementum annuli formation for estimating ages and 64 bears to analyze the relationship between cementum annuli widths and hard mast production. The dataset was divided into two groups, namely adult period  and subadult period , on the basis of age at the time of sexual maturation . We subtWe treated the teeth using the methods of Ohtaishi et al. . The colFalse annuli are often observed in teeth from both males and females, where dark bands appear broken or light-colored, making it impossible to distinguish a true annulus from a false annulus , 25. We We observed the edges of the cementum annuli from all individuals n = 85;  to deterQuercus crispula in early September from 2006 to 2013 in the Ashio-Nikko Mountains, Tochigi Prefecture [Q. crispula is the dominant hard mast species and staple food for bears; only the masting of Q. crispula influences bear behavior in this area, even though there are other hard mast species present with annual changes in mast production [Q. crispula tree and averaged them for each year during the study period.The hard mast crop size was investigated by visual assessments of seed production for efecture , 26. Q. oduction ; therefoxi) for each age i from all individuals without separating the light and dark bands. In each of the eight sections, we used imaging software  to take 20 measurements at intervals of 50 μm; thus, the number of data points recorded per annulus for each individual was 160. After the measurements had been taken, we averaged the 160 data points, calculated proportional growth layer group width (PW) [i -th annulus was formed. PW considers the variation in cementum deposition that may occur between individuals (varying tooth sizes) or the variation due to aging, and is expressed asFirst, we measured the cementum annuli widths (dth (PW) , and suri (PWim) and then performing a log transformation of the data.Second, we calculated the proportional width index (PWI) using the above-determined PW to eliminate these effects . SpecifiWe used segment linear regression to make sure whether the decreasing patterns of cementum annuli widths showed a tendency similar to that seen in other mammals . We set We used linear mixed models (LMMs) because PWI is supposed to follow a normal distribution . To clarWe used generalized linear mixed models (GLMMs) for the previous and current year masting models for both the adult and subadult groups to determine whether the frequency of false annuli increased owing to poor masting. We replaced PWI values with false annuli data  as the response variable. The probability distribution followed a binominal distribution pattern, and individual IDs were defined as random effects. We used the lme4 package  to creatUntil September, light bands were formed in the teeth of all individuals, and no dark bands were formed. Formation of dark bands was observed in October (in 8 of 24 bears), November (0 of 3), and January (1 of 1) .We selected the 64 bears and estimated age to analyze the relationship between cementum annuli widths and hard mast production. The most common age of the bears in our study was 5 years followed by 6 year-olds .We confirmed the decreasing pattern in cementum formation using PW values that were calculated in the process of determining PWI. Decreases in cementum annuli widths were observed with increases in age . The resX2 = 4.94, df = 1, P = 0.03).We found that the false annuli formed accounted for 52.2% of the total number of annuli . In addition, we determined that the ratio of false annuli to total annuli was significantly different between adults and subadults using the Z-test for the equality of two proportions (-1) from hard mast production each year because the AIC values from both the previous year masting model and the current year masting model were higher than that from the null model for both adult and subadult periods ; there were therefore no significant effects of hard mast production on the occurrence of false annuli   .Larger random effects were seen in the subadult models than in the adult models Tables  and 6; tOur results showed that there were decreasing width patterns and periods of light- or dark-band formation in the cementum annuli of premolar teeth of Asian black bears, similar to other mammals. In addition, we found that hard mast production had no impact on cementum patterns for adult period, but a small impact on subadult period.Light bands were formed from May to November whereas dark bands were formed from October to next year of May. These seasons correspond to active and inactive periods for bears throughout the year. It is believed that there are three potential factors causing the formation of annuli in mammals . The firThe annual width (PW) decreases with age, as has been shown in the studies verifying similarly , 36, 37.There was no significant relationship between the calculated cementum annulus index (PWI) and hard mast production for adult male bears. The effects of hard mast production were not reflected in cementum annuli formation because the energy intake from hard mast during the autumn hyperphagia period was used to accumulate fat preferentially rather than to grow bone and muscle , 39. On False annuli were observed here in male teeth, as well as in studies of polar bear teeth , 25, andHow and when bears take in food are reflected in the formation of the cementum annuli; therefore, this food intake information is a prerequisite in surveys of the relationship between nutritional status influenced by food availability and cementum annuli formation. In the future, we need to conduct feeding experiments in captive bears to obtain accurate information of this nature. Our results showed that the abundance of food resources in autumn did not influence cementum annuli width in male brown bears; therefore, in females, parturition and lactation are the most likely causes of decreased annuli width.S1 FileThe dataset of proportional growth layer group width (PW), proportional width index (PWI), presence or absence of false annulus and mast energy of hard mast for each individual and each year.(ZIP)Click here for additional data file.


Group antimicrobial administration is used to control disease in livestock, but we have little insight into how this impacts antimicrobial resistance (AMR) gene dynamics. Here, a longitudinal study was carried out during a single production cycle on a commercial pig unit with high historic and current antimicrobial usage. Quantitative PCR, 16S rRNA gene metabarcoding and shotgun metagenomic sequencing were used to track faecal AMR gene abundance and diversity and microbiome alpha diversity. Shotgun metagenomic sequencing identified 144 AMR genes in total, with higher AMR gene diversity present in young pigs compared to dry sows. Irrespective of in-feed antibiotic treatment or changes in microbiome diversity, mean AMR gene copy number was consistently high, with some AMR genes present at copy numbers comparable to the bacterial 16S rRNA gene. In conclusion, AMR gene prevalence and abundance were not influenced by antibiotic use, either during the production cycle or following whole-herd medication. The high levels of certain genes indicate they are widely disseminated throughout the microbial population, potentially aiding stability. Despite the high and relatively stable levels of resistance genes against the main antimicrobials used, these compounds continue to control production limiting diseases on this unit. This has led to concerns about the anthropogenic selection of antimicrobial-resistant bacteria in livestock systems4, particularly due to the potential transfer of antimicrobial resistance (AMR) genes from livestock to humans7 and into the environment9. In the United Kingdom, 52% of all antimicrobials sold for livestock were used in the pig and poultry sectors, with tetracyclines being reported as the highest sold antibiotic class10. Given the substantial use of antimicrobials in pig production, the association between antimicrobial use and AMR has been an area of intensive study. Specifically, antimicrobial administration has been associated with increased AMR in sentinel bacteria such as Escherichia coli13, Staphylococcus aureus15, and Campylobacter16, Salmonella17 and Enterococcus18 species. Molecular studies have revealed a higher richness and diversity of AMR genes in pigs administered oxytetracycline in-feed19, with farm origin being associated with AMR gene abundances in faeces at slaughter using both quantitative PCR20 and metagenomic21 datasets. However, there are currently few longitudinal studies of AMR in livestock systems, specifically for pig production22, with information on antimicrobial use in these systems often derived from national figures, rather than from farm medicines records21.Antimicrobial agents are used regularly in many agricultural systems worldwide to improve the health, welfare and productivity of livestockHere, we examine microbial population dynamics and both AMR gene abundance and diversity on a single pig farm during a six-month production cycle. Faecal samples were collected weekly from a batch of young pigs that were followed from birth to slaughter. Group treatments during this time included acidified water, dietary zinc supplementation and a prolonged period of in-feed chlortetracycline, followed by tylosin administration. Faecal samples from dry sow accommodation containing unmedicated pigs were taken in parallel. Finally, faecal samples were taken from dry sows during and after a partial depopulation event which involved antibiotic administration to every pig on the farm to assess if this practice impacted on AMR gene abundance. Microbiome alpha diversity, AMR gene abundance and AMR gene diversity were measured by 16S rRNA gene metabarcoding, quantitative PCR (qPCR) and shotgun metagenomic sequencing, respectively.10. Detailed antimicrobial usage records and additional herd information are available in Supplementary Materials In order to study the impact of antimicrobial usage on AMR gene abundance and diversity, a 600 sow Landrace x Large White commercial farrowing to finishing unit (United Kingdom) with high historic and current antimicrobial usage was recruited. In the three months prior to the study period, a total of 389.1 mg/Population Correction Unit (PCU) of antibiotics were used on this farm. This compares to a UK average in pigs of 183 mg/PCU in 2016 and 131 mg/PCU in 2017, making this a high antibiotic usage farmFaecal samples were taken from this unit during two time periods (Phase 1 and Phase 2) as described below. All samples were collected in universal tubes and stored at −20 °C on-site, prior to batch transportation to the laboratory on dry ice and storage at −80 °C until processing.th October 2016 to 5th April 2017 (W1 to W25), when the studied batch were sent for slaughter. The sampling design and periods of in-feed medication are summarised in Fig. Pooled faecal samples from the pen floors containing medicated young pigs (referred to as “young pig accommodation”) were collected weekly from just prior to farrowing on 26In an effort to improve health status and reduce antimicrobial use, a partial depopulation was carried out on this farm in May 2017. This involved removing all the young pigs  from the unit, fumigating the young pig accommodation with formaldehyde and administering in-feed chlortetracycline and tiamulin to all the breeding sows for a period of six weeks. In order to establish the impact of this management practice on AMR gene abundance and diversity, faecal samples were obtained from the dry sow accommodation during the final week of antibiotic administration (W36), two months after treatment (W45) and five months after treatment (W57) (n = 6).DNA extractions were carried out using the DNeasy PowerSoil Kit  following the manufacturer’s instructions. In parallel, approximately 1 g of homogenised faeces were weighed out and placed into a drying oven overnight at 60 °C. After re-weighing, the percentage of dry matter (% DM) was determined.tetB and tetQ (tetracycline), ermA and ermB (tylosin) and dfrA1 (trimethoprim). Additionally, quantification of the 16S rRNA gene was included as a proxy of overall bacterial load. Plasmids containing gene fragments of tetB, tetQ, ermA, ermB, dfrA1 and the 16S rRNA gene were generated in-house and used as standards for absolute quantification of gene copy number per gram of dry faeces. Further details on methodology and primer/probe sequences are shown in Supplementary Materials Five AMR genes associated with the main antibiotics used on the unit were targeted for longitudinal analysis based on initial end point screening of 36 AMR genes. These were 23 and in Supplementary Materials All DNA extracts were prepared for 16S rRNA gene metabarcoding targeting the V3 hypervariable region, as described in previous work24 and mothur25 (URL: https://www.mothur.org/wiki/MiSeq_SOP. Accessed January 2018) as described previously23. Here, unique sequences were binned into operational taxonomic units (OTUs) using a database-independent approach. A mean of 125,312 sequences per sample were retained after quality control and were subsampled to 10,000 sequences per sample for analysis. Both the Inverse Simpson index (ISI) and Shannon index (SI) were calculated for each sample to assess alpha diversity.The generated sequences were processed using cutadapt34 available in Supplementary Materials Three time points were selected that corresponded to differing antimicrobial treatment phases in the young pigs Fig. . TriplicTemporal changes in both gene copy number and alpha diversity indices were assessed statistically using repeated measures analysis of variance  with the data obtained from the dry sow and young pig accommodation faecal samples. To assess the impact of the antimicrobial treatments on gene copy number and alpha diversity indices in the young pig accommodation, least significant differences were used for multiple comparisons of means.For the dry sow analyses, the values calculated from samples collected between W1-W25 were included in the statistical models. For the young pig analyses, the values calculated from samples collected between W5-W25 were included in the statistical models, since W1 samples were obtained from pregnant sows only and samples obtained between W2-W4 were obtained when the piglets were still grouped by litter and were not yet assigned to their rearing pens. For the young pig analyses, the pen was included as a factor to assess any differences between the triplicate pen samples.Temporal fluctuations in AMR gene copy number and bacterial load (16S rRNA gene) can be observed in both the young pig Fig.  and dry dfrA1 counts occurred in the young pig accommodation between W5  0.80) and W7 , and a decrease in ermA between W5  and W7 . There were no marked changes in the levels of tetB, ermB and tetQ during acidified water and zinc administration.Prior to weaning and during co-housing (W1-W4), the levels of all five AMR genes were similar when comparing the nursing sow and young pig samples, in the presence of similar faecal bacterial load Fig. . During tetB gene copy number in the young pig accommodation between W9  and W10  with no significant changes in tetQ, ermA, ermB or dfrA1 levels being observed during chlortetracycline administration. Tylosin administration (W11-W25) did not have an effect on AMR gene counts in the young pig accommodation following withdrawal from chlortetracycline. Increases in the majority of AMR genes being observed were seen from W21 to W24 in both the young pig and dry sow accommodation, which coincided with an increase in bacterial load, suggesting that this increase was independent of antimicrobial administration  was associated with a decrease in 16S rRNA gene metabarcoding was carried out to provide context on faecal microbiota development in the young pigs and whether changes in alpha diversity were linked to AMR gene abundances. Notable stepwise increases in measures of alpha diversity (both SIs and ISIs) occurred during the first 4 weeks of life Fig. , highligA total of 144 AMR genes were detected on the farm Fig. , 21 of wWhen examining the proportion of reads associated with each antibiotic class, AMR genes associated with tetracycline, macrolide (MLS) and aminoglycoside resistance predominated across all samples Fig. . With a The biggest change in the proportion of reads associated with each antimicrobial class occurred on W9 in both sampling locations. In the young pig accommodation, there was a proportional increase in reads associated with cross-class resistance (CCR). In the dry sow accommodation, the increase was seen in reads associated with aminoglycoside and sulphonamide resistance. These proportional changes were accompanied by an increase in AMR genes, from 94 genes in the young pig accommodation on W7 to 120 genes on W9 and 32 genes in the dry sow accommodation on W7 to 60 genes on W9. The proportion of reads associated with tetracycline resistance declined between W7 and W9, despite the young pigs starting chlortetracycline treatment during this time.In the young pig accommodation, there were both high levels and numbers (i.e. >30) of genes conferring cross-class resistance (CCR) during zinc and acidified water (W7) and chlortetracycline administration (W9). This number dropped by the end of the production cycle, despite the young pigs having spent several months on in-feed antibiotics Fig. . Genes aTo improve the health status of the farm, all the young pigs were removed  and the sows treated with in-feed chlortetracycline and tiamulin. During this time period, all the pigs on the farm were treated with antibiotics simultaneously. During the last week of treatment (W36), and two (W45) and five (W57) months after cessation of treatment, bacterial load and AMR gene copy number in these samples did not change in response to the partial depopulation and antibiotic treatment Fig. .35, they have not provided the granularity of data and medicines usage history required to test these hypotheses. For this reason, we chose to undertake a longitudinal dissection of a single commercial pig unit, which included three group in-feed antibiotic treatment regimens and a partial depopulation event.When designing this study, our first hypothesis was that there would be a reservoir of AMR genes within the faecal microbiome, which would increase in both abundance and diversity in response to in-feed antimicrobial administration. Furthermore, we anticipated that AMR gene abundance would be inversely related to faecal microbiome diversity. Whilst large cross-sectional studies have been instrumental in furthering our understanding of AMR gene abundance and diversity across different livestock systems36 and phenotypic resistance in Escherichia coli isolates38 in unmedicated pigs, our initial expectation was that AMR gene abundances would markedly increase in response to antimicrobial administration as a result of selective pressure being exerted on specific genes39. In agreement with previous work, we showed high levels of all studied AMR genes in the non-medicated dry sows, presumably as a consequence of the strong selection pressure exerted by this farm’s history of high antimicrobial usage. The levels of the five AMR genes studied in the young pigs reflected those of the sow population, whilst prolonged exposure to two different in-feed antibiotic regimens in the young pigs and a combined in-feed antibiotic regimen in the dry sows had no marked effect on AMR gene counts, potentially suggesting that they have reached saturation within the faecal bacterial populations39. Curiously, the antibiotics used (chlortetracycline and tylosin) were still clinically effective on this farm, suggesting that despite a high abundance of resistance genes within the faecal microbiome, that they were not present, or at least not active, within the organisms of clinical interest, i.e. Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Actinobacillus pleuopneumoniae and Lawsonia intracellularis.Although previous work has demonstrated high baseline levels of specific AMR genestetQ and ermB) were present in higher copy numbers than the 16S rRNA gene at the majority of the studied time points. This is consistent with the work of other groups, which has demonstrated overall AMR gene abundances of 0.77–5.1440 and 0.54–3.1 gene copies41 per 16S rRNA gene in agricultural faecal and wastewater samples. It is worth noting that these AMR gene copy numbers are orders of magnitude higher than in samples obtained from other environments, such as sediments, soil and river water42. Collectively, these findings suggest that some bacteria are carrying multiple copies of particular AMR genes.Interestingly, some of the targeted AMR genes  which coincided with an increase in 16S rRNA gene copies, hence suggesting an as yet undefined environmental influence on bacterial load and consequently AMR gene abundance. A previous cross-sectional study highlighted that only 10–42% of the variation in AMR gene levels could be explained by factors included in statistical models , suggesting that AMR gene levels are strongly influenced by a variety of other elements48. However, changes in the microbiome associated with antimicrobial treatment were not observed. Previous work has demonstrated that sub-therapeutic administration of chlortetracycline and tylosin had no impact on alpha diversity indices49. Whilst changes in relative abundances of specific taxa have been observed in response to tylosin administration, it was previously reported that these shifts were temporary, suggesting that the gut microbiota post-weaning seems to be resilient to perturbation by antimicrobial agents47. Despite using markedly higher levels of chlortetracycline and tylosin , our results also showed that antimicrobial treatment did not impact on microbiome diversity in the young pigs.Similar to the studied AMR genes, fluctuations in microbiome alpha diversity were apparent in both dry sow and young pig accommodation samples. These changes were clearly more pronounced in the young pigs, as the alpha diversity of the faecal microbiome increased from nursing to finishing, which has been shown previously53, this was not associated with changes in AMR gene prevalence. We expected, as others have proposed, that changes in the microbiota would influence AMR gene levels54, but in fact the high levels of studied AMR genes in the young pigs reflected that of the farm’s sow population. The most obvious explanation for this is a combination of vertical and horizontal transmission of bacteria at or shortly after birth56. The presence of comparable levels of AMR genes in both sows and piglets and the associated large differences in microbiome diversity suggest that the AMR genes studied appear to either be widespread across multiple taxa or highly concentrated within dominant taxa present throughout all stages of microbiota development.With respect to our second hypothesis, even when the young pig faecal microbiome was at its least diverse during the suckling period, the AMR gene levels were comparable to that of the nursing sows. Although microbiome diversity increased dramatically in the piglets during the first few weeks of life, as reflected in previous studies19. Specifically, genes associated with tetracycline and macrolide resistance predominated. This is not surprising given the history of high levels of tetracycline and macrolide use on this farm. Reassuringly, there was no evidence of co-selection between antimicrobial classes following chlortetracycline or tylosin administration. This is a significant finding and relevant to the principles of antimicrobial stewardship, where veterinary surgeons are actively discouraged from using fluoroquinolones and 3rd/4th generation cephalosporins, so as to minimise the risk that resistance to these critically important antibiotics in human medicine is selected for in livestock.Metagenomic sequencing revealed a diverse set of AMR genes in the presence and absence of antimicrobial treatment, which reflects findings in other recent work58.What was evident, were the large numbers of genes associated with cross-class resistance (CCR) in the young pig accommodation samples taken on W7 and W9 and how these reduced by W25 and were almost absent from the unmedicated sows. The presence of these genes was not associated with the administration of chlortetracycline and tylosin in this study. Although the current study design does not allow us to disentangle temporal effects versus the effect of different treatments, it is interesting to note that the CCR genes were already highly abundant at W7, following a period of in-feed zinc administration and the use of acidified water. Copper and zinc salts are commonly administered in-feed at supranutritional levels due to their antimicrobial properties, with increasing doses of zinc oxide being previously shown to increase the abundance of both tetracycline and sulphonamide resistance genes in weaned pigsThe metagenomic sequencing results for the dry sow accommodation on W9 were unexpected. All three biological replicates demonstrated an increase in the proportion of reads associated with sulphonamide and aminoglycoside resistance. The richness of AMR genes also doubled at this time point compared to W7, despite no antimicrobial administration to this group of pigs. An increase in AMR gene diversity was also seen in the young pig accommodation at the same time point, however the increase was seen in beta-lactam resistance genes, CCR genes and genes associated with other antimicrobial classes. This is unlikely to be an artefact, given that this observation was seen across two different locations at the same time and five of the six biological replicates. This shift appears to be a consequence of an undefined environmental factor affecting the whole farm, where the two groups of pigs respond differently with regards to faecal AMR gene diversity.The partial depopulation at the end of the study involved considerable antibiotic use, with every sow on the farm receiving in-feed tiamulin and chlortetracycline. The treated sows were followed for five months after this treatment, during a period where antibiotic use on the farm declined nearly twenty-fold. Despite such dramatic changes in antibiotic usage, there were no marked changes in AMR gene abundances. This therefore begs the question as to how long  it would take to see reductions in AMR gene levels following the reduction or cessation of antimicrobial use on farms with a previous history of high-level use. We propose that the long-term use of these treatments has led to wide and stable dissemination of relevant resistance genes throughout the gastrointestinal microbial population of the pigs.AMR gene abundance and diversity on this unit were high in both dry sows and young pigs, likely as a consequence of historic antimicrobial usage. In this context, in-feed antibiotic administration (chlortetracycline and tylosin) did not affect faecal AMR gene abundance or diversity, suggesting that genes under positive selection in the presence of these antibiotics have already become stably integrated into the faecal microbiome. This would also potentially explain the failure of AMR gene levels to decline following dramatic reductions in antimicrobial use on the farm. Acidified water and zinc supplementation were linked to an increase in AMR gene diversity, which importantly included CCR genes, which decayed quickly after their withdrawal. This study did not identify which bacteria carried these AMR genes or indeed which genes were being expressed. Future work therefore needs to determine in what organisms these genes are not only present, but also active.Additional methodology.


Kaposi sarcoma (KS) is a multicentric angioproliferative disorder of endothelial origin. It predominantly affects mucocutaneous sites but may also affect visceral organs. We are reporting a case of KS with unusual presentation. A 45-year-old male presented violaceous macules on the glans which appeared 3 weeks ago. Otherwise he reported fever and loss weight. Histological examination detected the presence of spindle cells and the positivity for Kaposi-associated Herpes virus-8 confirmed the lesions as Kaposi's sarcoma. Visceral sarcoma lesions were also present in the oesophagus and gastric fundus. HIV viral serology was positive. Treatment with antiretroviral therapy and bleomycine was initiated. Sarcomas of the penis are very uncommon, representing less than 5% of malignant tumors in this area. KS is the most common sarcoma of the penis and the second most common is leiomyosarcoma. Primary presentation of KS on the penis is not common but more often observed in patients with AIDs, whose lesions are the aggressive form, and only approximately 2-3% cases have shown penile KS lesions as first manifestation of disease as our patient. This case is important as it illustrates that disseminated KS was not to be predicted by the number or the external lesions.


The tetragonal zirconia nanofibers with high specific surface area prepared at 500 °C can be considered, for instance, as promising supports for heterogeneous catalysts, enhancing their activity.For the first time, zirconia nanofibers with an average diameter of about 75 nm have been fabricated by calcination of electrospun zirconium acetylacetonate/polyacrylonitrile fibers in the range of 500–1100 °C. Composite and ceramic filaments have been characterized by scanning electron microscopy, thermogravimetric analysis, nitrogen adsorption analysis, energy-dispersive X-ray spectroscopy, and X-ray diffractometry. The stages of the transition of zirconium acetylacetonate to zirconia have been revealed. It has been found out that a rise in calcination temperature from 500 to 1100 °C induces transformation of mesoporous tetragonal zirconia nanofibers with a high specific surface area (102.3 m Nanofibers are fibers with diameters in the nanometer range. They attract a great deal of attention due to their remarkable properties compared to their microfiber counterparts, namely high surface area, high surface-to-mass ratio, interconnected porous structure, and flexibility in surface functionalization .Among a number of nanofiber fabrication techniques, electrospinning is the most commonly used method due to its simplicity, adaptability, cost-efficiency, and versatility, as well as the capability to control the diameter, composition, and morphology of filaments. Besides, electrospinning is the only method for the large-scale fabrication of nanofibers .Electrospinning can be used for the production of ceramic nanofibers. This process includes the following stages: Preparation of a spinnable solution containing the ceramic precursor and a binding polymer; spinning of the prepared solution; calcination of the electrospun composite fibers to remove unwanted constituents and to obtain the desired ceramic phase; and finally, sintering at an elevated temperature to fabricate the final ceramic nanofibers with the required structure .2 [Among ceramic nanofibers, zirconia nanofibers have significant potential to be utilized in a variety of applications because of their high chemical and thermal stability, high ionic conductivity, biocompatibility, and polymorphism of ZrO2 . For exa2 ,5, impro2 ,7, incre2 ,9, and a2  and as s2 ,12.Due to its availability and reasonable price, zirconium oxychloride is frequently used as a ceramic precursor in the zirconia nanofiber fabrication process ,14,15,16Poly(vinyl pyrolidone) (PVP) is the most popular polymer employed as a matrix due to its high solubility in ethanol, water, and dimethylformamide (DMF), and its good compatibility with many metal alkoxides and salts . Using D5H7O2)4) (hereinafter referred to as ZrAA) is easily dissolvable in DMF and can be considered as an alternative to the above-mentioned zirconia precursors (zirconium oxychloride and zirconium acetate). In [Zirconium acetylacetonate (Zr(Cate). In ,23, simiThis paper for the first time reports zirconia nanofibers fabricated from electrospun composite ZrAA/PAN fibers, and the results of the comprehensive investigation of the filaments’ morphological evolution during heat treatment that is important for their practical use.A 10 wt. % polymer solution was prepared by adding 1 g PAN  into 9 g DMF, with periodic stirring by hand for 2 h at 50 °C. Then, 0.3 g ZrAA  was added into the solution under stirring in the ultrasonic bath for 6 min at 42 kHz at room temperature to prepare the transparent electrospinning solution.The prepared composite solution was transferred into a 10 ml plastic syringe and electrospun using the electrospinning machine NANON-01A  at a feeding rate of 1.2 mL/h through a 21 G blunt-tip needle. The following operative parameters were chosen to fabricate smooth and bead-free composite fibers: The accelerating voltage was 23 kV, and the distance between the needle tip and the flat collector covered by aluminum foil was 21 cm. The fibers were collected in the form of non-woven mats.As-spun mats were calcined at different temperatures in the range of 500–1300 °C for 1 h using two-stage heating: Heating to 500 °C with a heating rate of 1 °C/min, and then further heating to the target temperature with a heating rate of 5 °C/min. To prevent fiber destruction, a low heating rate at the first annealing stage was chosen to ensure delicate removal of the decomposition products of the ceramic precursor and binding polymer.2 at the relative pressure of 0.995. The thermogravimetric (TG) analysis was performed on the thermal analyzer EXSTAR TG/DTA7200  in air atmosphere with a heating rate of 10 °C/min.The surface texture, diameter, and elemental composition of the fibers were analyzed by the scanning electron microscope (SEM) Merlin  coupled with an energy-dispersive X-ray spectrometer (EDS) INCA Energy 350X-Max 80 . XRD patterns were recorded in the 20–80° 2θ range by the X-ray diffractometer (XRD) D2 Phaser  using CuKα1 monochromatic radiation. XRD patterns were assigned using the PDF-2 Diffraction Database File compiled by the International Centre for Diffraction. The phase content was determined from XRD patterns by the Rietveld method in the TOPAS software . SEM, EDS, and XRD measurements were carried out at room temperature. Nitrogen adsorption–desorption isotherms at 77 K were registered with a gas sorption analyzer Autosorb iQ-C . Specific surface areas of the nanofibers were determined with the Brunauer–Emmett–Teller (BET) method. Total pore volume was calculated through the quantity of adsorbed NThe electrospinning process of the prepared solution resulted in randomly distributed cylindrical bead-free ZrAA/PAN fibers with smooth surfaces a. Fiber 2 nanofibers to 71 ± 6 nm, due to their sintering. Within the measurement error, no further filament shrinkage is observed in the range of 800–1100 °C. At 1100 °C, the average diameter of ceramic nanofibers is 70 ± 3 nm, and zirconia grains attain a size equal to the fiber diameter  and zirconia precursor  3 formation, which is confirmed by the observed weight loss of about 42% at 290 °C in the TG curve of ZrAA. The next weight loss before 325 °C is mainly attributed to oxidative stabilization of PAN. During the stabilization process, the cyclization of the nitrile groups and cross-linking of the chain molecules are followed by dehydrogenation [3COO)3 decomposition leading to ZrO(CH3COO)2 occurs in the range of 290–355 °C (with a weight loss of about 54% at 355 °C in the TG curve of ZrAA). This explains the further weight drop in the TG curve of filaments as the temperature rises above 325 °C. The final weight loss in the TG curve of fibers, observed in the range of 355–500 °C, is associated with polymer combustion and ZrO2 formation (with a 75% weight loss at 500 °C in the TG curve of ZrAA). So, we can conclude that the transformation process of ZrAA to ZrO2 goes through the following stages: ZrAA → Zr(OH)(CH3COO)3 → ZrO(CH3COO)2 → ZrO2.The TG curve of as-spun ZrAA/PAN fibers is characterized by several weight losses. The first weight loss before 165 °C results from the removal of the residual solvent from the composite fibers, since it is not observed in the TG curves of ZrAA and PAN. The second one occurs in the range of 165–290 °C and is related to Zr(OH) zirconia. The average t-ZrO2 grain size can be estimated as 8 nm from the characteristic peak at 30.2°, using the Scherrer equation.The formation and evolution of the filament crystalline structure with increases in calcination temperature is shown in 2 become sharper and narrower with a rise in calcination temperature to 800 °C, and reflections at 34.6°, 50.7°, and 59.3° are visualized. This indicates that the crystallinity is higher and the grain size is larger for zirconia nanofibers fabricated at higher calcination temperatures. The average t-ZrO2 grain size becomes 21 nm at 800 °C. At 800 °C, monoclinic (m-ZrO2) zirconia with characteristic peaks at 28.2° and 31.5° appears, and the t-ZrO2 content decreases from 100% to 67%. From the reflection at 28.2°, the average m-ZrO2 grain size is estimated to be about 25 nm via the Scherrer equation. A rise in calcination temperature from 800 to 1100 °C further stimulates m-ZrO2 content growth from 33% to 91%. The intensities of m-ZrO2 main peaks at 28.2° and 31.5° increase, and a large number of m-ZrO2 low-intensity peaks appear in the XRD pattern. The average m-ZrO2 grain size attains 70 nm, which is confirmed by SEM measurements  to non-porous m-ZrO2 nanofibers of almost the same diameter with a low value of specific surface area (8.3 m2/g). It is determined that the transition of electrospun zirconium acetylacetonate/polyacrylonitrile fibers to zirconia nanofibers is accompanied with the following stages of the ceramic precursor transformation: Zr(C5H7O2)4 → Zr(OH)(CH3COO)3 → ZrO(CH3COO)2 → t-ZrO2 → t-ZrO2+m-ZrO2 → m-ZrO2.A novel combination of the ceramic precursor (zirconium acetylacetonate) and the binding polymer (polyacrylonitrile) for zirconia nanofiber fabrication via electrospinning was successfully tested. The produced ceramic filaments are real nanofibers, with a diameter of about 75 nm. It is revealed that a rise in calcination temperature from 500 to 1100 °C induces transformation of mesoporous t-ZrO2 nanofibers with high specific surface area prepared at 500 °C may be considered as perspective supports for heterogeneous catalysts, simultaneously enhancing the activity of the latter. For example, it is known that Ag and Rh supported on t-ZrO2 are far more active in the hydrogenation of carbon dioxide to methanol [2.The t-ZrOmethanol  and in tmethanol  than Ag


Hyperprolactinemia (HPRL) is a classical side effect of antipsychotic drugs primarily attributed to blockade of dopamine D2 receptors (DRD2s) on the membranes of lactotroph cells within the pituitary gland. Certain antipsychotic drugs, e.g. risperidone, are more likely to induce HPRL because of relative accumulation within the adenohypophysis. Nevertheless, due to competition for pituitary DRD2s by high dopamine levels may limit antipsychotic-induced HPRL. Moreover, the activity of prolactin-producing lactotrophs also depends on other hormones which are regulated by the extra-pituitary activity of dopamine receptors, dopamine transporters, enzymes of neurotransmitter metabolism and other factors. Polymorphic variants in the genes coding for these receptors and proteins can have functional significance and influence on the development of hyperprolactinemia.DRD1, DRD2, DRD3, DRD4, the dopamine transporter SLC6A3 and dopamine catabolizing enzymes MAOA and MAOB was investigated in a population of 446 Caucasians  with a clinical diagnosis of schizophrenia (according to ICD-10: F20) with and without HPRL who were treated with classical and/or atypical antipsychotic drugs. Additive genetic model was tested and the analysis was carried out in the total group and in subgroup stratified by the use of risperidone/paliperidone.A set of 41 SNPs of genes for dopamine receptors MAOB gene and HPRL in men was found in the total group. Furthermore, the rs40184 and rs3863145 variants in SLC6A3 gene appeared to be associated with HPRL in the subgroup of patients using the risperidone/paliperidone, but not with HPRL induced by other antipsychotic drugs.One statistically significant association between polymorphic variant rs1799836 of MAOB and SLC6A3 may have consequences on the modulation of prolactin secretion. A further search for genetic markers associated with the development of antipsychotic-related hyperprolactinemia in schizophrenic patients is needed.Our results indicate that genetic variants of The online version of this article (10.1186/s12881-019-0773-3) contains supplementary material, which is available to authorized users. Schizophrenia is a serious polymorphic mental disorder, characterized by positive , negative  and cognitive symptoms . Treatment of schizophrenic illness usually involves the long-term usage of antipsychotic drugs , which hProlactin (PRL) is a polypeptide hormone that contains 199 amino acids and is categorized as part of the growth hormone family . ProlactFactors involved in the regulation of prolactin secretion can be divided into two groups: 1. prolactin-inhibiting – dopamine (DA), gamma-aminobutyric acid, gastrin, somatostatin, gonadotropin-binding protein and 2. prolactin-stimulating – serotonin, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (TRG), vaso-intestinal peptide, opiates, neurotensin and substance P, oxytocin, angiotensin 2 , 7.Dopamine holds a predominant role in the regulation of prolactin secretion. Dopamine, which is released by tuberoinfundibular neurons, normally acts at specific receptors on lactotrophes to tonically inhibit prolactin secretion and maintain physiologically normal prolactin levels in the blood . These dThe hypothalamic dopaminergic neurons, which provide DA to the anterior pituitary gland, are themselves regulated by feedback from PRL through a ‘short-loop feedback mechanism’. Elevation of serum levels of prolactin increases hypothalamic dopamine synthesis  and the All typical antipsychotic medications are associated with sustained hyperprolactinemia due to their high affinity for the D2 receptor and their slow dissociation from the receptor once bound, but the atypicals clozapine and quetiapine differ quite dramatically in their propensity to cause prolonged high prolactin levels, because they are loosely bound to D2 receptors . The dopAnother way to explain hyperprolactinemia is the ability of antipsychotic-drugs to cross the blood-brain barrier and to affect the activity of prolactin-inhibiting and prolactin-stimulating factors. This capacity partly depends upon affinity to the drug efflux transporter P-glycoprotein, which prevents drugs to pass the blood brain barrier , 15. ThiPRL elevations with antipsychotic medication generally are dose dependent. However, antipsychotics having a high potential for PRL elevation  can have a profound impact on PRL levels even at relatively low doses, while PRL levels with antipsychotics having a minimal effect on PRL, in most cases, can remain unchanged (quetiapine) or reduce (aripiprazole) over all dosages. Although tolerance and decreases in PRL values after long-term administration of PRL-elevating antipsychotics can occur, the elevations, in most cases, remain above the upper limit of normal .In conclusion, antipsychotic drugs may affect prolactin release directly by blocking dopamine D2 receptors on lactotrophs, but modulate this activity by affecting dopamine levels within the hypothalamic-pituitary portal blood. Moreover, antipsychotics can modulate intracerebral mechanisms involved in the regulation of prolactin release. As the main targets for the therapeutic actions of neuroleptics in schizophrenia are components of cerebral dopamine and – to a lesser extent – serotonin neurotransmission, the authors aimed at identifying the association of neuroleptic hyperprolactinemia with polymorphism of genes that determine the activity of various components of dopaminergic transmission – enzymes that catalyze the synthesis and metabolism of dopamine, its storage, release into the synaptic cleft and reverse capture, as well as the structure of its receptors.According to the literature data genetic variants of dopaminergic receptors, transporters and enzyme may have functional consequences on the respond of antipsychotic treatment and development of side effects of therapy.G198A and G1263A [DRD1 is considered as a potential pharmacogenomic marker for treatment response to antipsychotic drugs [In the structure of the dopamine receptor D1 , two single nucleotide polymorphisms associated with the risk of developing schizophrenia have been identified: d G1263A . These sd G1263A . In seveic drugs .DRD2 were evaluated to determine whether any of them controlled DRD2 expression [Taq1A, represented by two alleles – A1 and A2 in the region of the promoter that regulates gene expression, has been studied most. It was found that reduced dopamine D2 receptor binding in the human striatum associated with the A1 allele [Taq1A polymorphism with schizophrenia was reported [A1 allele is associated with a predisposition to the development of dyskinesias on the background treatment with neuroleptics [ANNK1, which encodes protein kinase RKK2. DRD2/ANKK1 genotypes are associated with susceptibility to second generation antipsychotic-induced akathisia [At least 23 single nucleotide polymorphisms within pression . The pol1 allele . The assoleptics . The stukathisia .Polymorphism of other subtypes of dopamine receptors of the second type, D3 and D4, also, according to a number of researchers, is associated with a risk of developing schizophrenia spectrum disorders –28. TherSLC6A3) appears to be one of the most important candidate genes for affecting mental disorders, as it is the target of many psychostimulants that cause symptoms similar to the positive symptoms of schizophrenia. Controversial information was obtained by studying the effect of polymorphism in SLC6A3 on pharmacogenetics of adverse events in schizophrenia treatment [The dopamine transporter gene  and catechol-O-methyltransferase (COMT) play a leading role. There are two isoforms of monoamine oxidase – MAOA and MAOB, whose genes are located on the X chromosome. MAOA catalyzes monoamine neurotransmitters including 5-hydroxytryptamine , norepinephrine and dopamine, while MAOB deaminates 2-phenylethylamine and also dopamine . The asspression .DRD2 and dopamine transporter SLC6A3 [DRD1, DRD2, DRD3 and COMT [DRD1, COMT, DRD2, and DRD3 [There is a series of studies with the search for associations of polymorphisms of the genes of dopamine receptors and metabolizing enzymes in various combinations and response or side effects to antipsychotic therapy: r SLC6A3 ; DRD1, Dand COMT ; DRD1, Cand DRD3 .DRD1, DRD2, DRD3, DRD4), 12 polymorphic variants of dopamine transporter SLC6A3, as well as 3 polymorphisms of monoamine oxidase A and B and here we present new data on the association between them and HPRL in antipsychotic drug-treated patients with schizophrenia from West Siberia, Russian Federation.Based upon reviewing the literature we selected a set of 26 polymorphisms in dopamine receptors genes ) than the men (mean ± SD 45.2 ± 13.9 vs. 37.8 ± 11.9 years). Among the women, 86 were > 50 years of age. Women suffered from the disease for a significantly longer  period of time (17.6 ± 12.5 vs. 13.1 ± 10.0 years). The median antipsychotic daily dose was 500 mg CPZeq  in men and 320 mg CPZeq  in women .The total sample consisted of 446 patients . The women were significantly older  according to the predefined criteria , 39. DemOne hundred ninety-one patients were treated with conventional antipsychotics in oral and/or long-acting formulations. The most common conventional antipsychotic was haloperidol, which was used in 110 patients, but other treatments included oral chlorpromazine, chlorprothixene, trifluoperazin, and zuclopenthixol, and/or long-acting formulations of haloperidol-, zuclopenthixol-, and flupenthixol-decanoate. A total of 176 patients were treated with atypical antipsychotics: risperidone, clozapine, quetiapine, olanzapine, amisulpride, paliperidone, and sertindole. Different combinations of classical and atypical drugs were used by 79 patients. 76 patients were treated with risperidone/paliperidone.To compare antipsychotic medications, all dosages were converted into chlorpromazine equivalents (CPZeq) .Blood samples were taken 8 h after overnight fasting in tubes containing EDTA for DNA extraction and in tubes with CAT (clot activator) to obtain serum (BD Vacutainer). Blood with EDTA was stored in several aliquots at -20 °C until DNA isolation. Blood samples with CAT were centrifuged for 30 min at 1500 rpm at 4 °C to obtain serum.The PRL concentration was measured in serum using the AccuBind ELISA Microwells kit . In this microplate immunoenzymatic assay, the ELISA has a sensitivity of 0.004 ng/well. This is equivalent to a sample containing 0.150 ng/ml PRL. The upper limits for normal PRL concentration were set at ≤20 ng/ml for men and ≤ 25 ng/ml for non-pregnant, non-nursing women , 40. ForDNA was isolated from the leukocytes in whole peripheral blood from patients with mental disorders using the standard phenol-chloroform method.DRD1, DRD2, DRD3, DRD4, and SLC6A3 genes in the Genome Analysis Facility, Dept. Genetics (Head: Prof. Dr. C. Wijmenga), University Medical Center Groningen on the MassARRAY® Analyzer 4 (Agena Bioscience™) using the set SEQUENOM Consumables iPLEX Gold 384. DNA sample preparation for SEQUENOM MassARRAY® Analyzer 4 includes several steps: a standard PCR reaction to obtain the amplification products, a shrimp alkaline phosphatase (SAP) reaction to neutralize the unincorporated dNTPs in the amplification products, the PCR iPLEX Gold extension reaction, and then placing the samples on a special chip (SpectroCHIP Array) using NanoDispenser RS1000 prior to loading them into the analyzer. SNPs in MAOA, MAOB genes were genotyped in the Mental Health Research Institute, Tomsk National Research Medical Center of the Russian Academy of Sciences, using the fluorogenic 5′-exonuclease TaqMan technology performed on the real-time polymerase chain reaction system “StepOnePlus” .Genotyping was carried out for DRD1, DRD2, DRD3, DRD4 . The Mann-Whitney test was used to compare qualitative traits and χ2 test for categorical traits.We analyzed associations between the polymorphisms and HPRL using logistic regression models including HPRL as a dependent categorical variable and polymorphisms as the predictors. Age, sex, and CPZeq were used as covariates. Additive genetic model was tested and Odds ratios (OR) along with 95% confidence intervals (CI) were calculated regarding the risk of HPRL for the rare allele vs common allele. Prior to the analysis, the polymorphisms were filtered out in case of minor allele frequency below 5% or Hardy-Weinberg equilibrium (HWE) All calculations were performed in the R statistical environment using basic R functions and the SNPassoc package .SLC6A3 genes we excluded 4 SNPs  with Hardy-Weinberg equilibrium p-value < 0.001, thus leaving 34 SNPs for the analysis of associations with HPRL. In the total group of patients none of the studied genetic markers localized in autosomal chromosomes are associated with HPRL  studied whether obesity is associated with genetic variants that increase the availability of dopamine. Low activity genotypes at both the  of 5.01 . AntipsyOur preliminary hypothesis was that genetic variants of dopamine receptors may have functional consequences on the modulation of PRL secretion and may play an important role in the development of hyperprolactinemia, but the results of the study indicate that there is no association in the group of patients.SLC6A3 gene are associated with risperidone/paliperidone induced HPRL, but not with HPRL induced by other antipsychotic drugs. The solute carrier family 6 (neurotransmitter transporter), member 3 (SLC6A3) gene encodes the dopamine transporter DAT. The difference between risperidone/paliperidone and other antipsychotics might indicate that, although dopamine transporters are present within the adenohypophysis [However we found that rs40184 and rs3863145 of pophysis  and may pophysis , the phapophysis  and thusSLC6A3 plays a critical role in controlling dopamine transmission  through the accumulation of dopamine in extracellular space which is major site of action of psychostimulant drugs [SLC6A3 functioning may lead to inter-individual variability of response to antipsychotic drug treatment. Sjoholm H. et al. (2004) [SLC6A3 binding sites in the schizophrenia patients who were being treated with dopamine D2-receptor blocking antipsychotics. Single locus analysis showed significant association of nine variants from SLC6A3, PIP4K2A and BDNF genes with incomplete antipsychotic response in schizophrenia patients with high severity [BDNF_rs7103411-BDNF_rs1491851-SLC6A3_rs40184 in severely ill incomplete responders [SCL6A3 functioning can be expected and these may also influence PRL secretion.The DAT protein is expressed in the membrane of neurons, where it transports dopamine from the synaptic cleft back into neurons for reuse. nt drugs –60. The . (2004)  reportedseverity . The mulsponders . Hence, Our new data could be taken into account when therapy with risperidone/paliperidone or different antipsychotic therapy is considered regarding the risk of developing HPRL.MAOB gene in (hemizygous) men and rs40184 and rs3863145 variants in the SLC6A3 gene which codes for the dopamine transporter in patients using risperidone/paliperidone. Strength of our study is the relatively large patient population and careful assessment of hyperprolactinemia. A relative weakness is the sex difference between the HPRL versus non-HPRL groups and the relatively high age of women as this may increase the number of post-menopausal women in the HPRL-group. However, this also makes the associations which we found more likely to exist. A further search for genetic markers associated with the development of antipsychotic-related hyperprolactinemia in schizophrenic patients is needed.In conclusion, we found an association of antipsychotic-induced HPRL with rs1799836 of the Additional file 1:Table S1. Logistic regression analysis for HPRL as a dependent categorical variable and polymorphisms as the predictors and age, sex, CPZeq as covariates for all patients. (DOC 65 kb)Additional file 2:Table S2. Genotype and allele frequencies for MAO gene polymorphisms. (DOC 45 kb)Additional file 3:Table S3. Genotype and allele frequencies for all polymorphisms of studied genes in the subgroup of patients using the risperidone/paliperidone. (DOC 275 kb)Additional file 4:Table S4. Lewontin’s D’ statistics to measure pairwise linkage disequilibrium between SLC6A3 SNPs. (XLS 40 kb)Additional file 5:Table S5. Analysis of association between HPRL and polymorphisms in MAO genes for female/male patients in risperidone/paliperidone group. (DOC 34 kb)