Methamphetamine addiction is mimicked in rats that self-administer the drug. However, these self-administration (SA) models do not include adverse consequences that are necessary to reach a diagnosis of addiction in humans. Herein, we measured genome-wide transcriptional consequences of methamphetamine SA and footshocks in the rat brain. We trained rats to self-administer methamphetamine for 20 days. Thereafter, lever-presses for methamphetamine were punished by mild footshocks for 5 days. Response-contingent punishment significantly reduced methamphetamine taking in some rats  but not in others . Rats also underwent extinction test at one day and 30 days after the last shock session. Rats were euthanized one day after the second extinction test and the nucleus accumbens (NAc) and dorsal striatum were collected to measure gene expression with microarray analysis. In the NAc, there were changes in the expression of 13 genes in the SRvsControl and 9 genes in the SRvsSS comparison. In the striatum, there were 9  affected genes in the SRvsSS comparison. Among the upregulated genes was oxytocin in the NAc and CARTpt in the striatum of SR rats. These observations support a regional role of neuropeptides in the brain after a long withdrawal interval when animals show incubation of methamphetamine craving. Its abuse is associated with negative impact on the brain and peripheral organs3. These adverse consequences include abnormal blood flow and brain structural pathologies6, with consequent neuropsychological deficits7. Although a diversity of drugs can cause the neuropsychiatric syndromes designated as addictions, it is important to identify specific neurobiological processes involved in causing compulsive taking of individual substances. This is relevant to the development of pharmacological agents against addictions because individual substances may impact the brain differentially4. This suggests the importance of identifying and detailing the potential impact of these drugs on distributed systems both within and beyond the so-called reward circuitry8 because changes in these systems might influence or dictate treatment approaches11. These statements suggest that addiction may occur as the result of repeated exposure to drugs that lead to neuropathological changes in distributed networks of potentially dissociable reward and non-reward pathways in the mammalian brain8. This conclusion is supported by accumulated evidence from epigenetic, transcriptional, neuroimaging, and neuropsychological studies that have indeed provided support for a role of various brain regions in the development and maintenance of addicted states14. This suggestion also envisions the development of preclinical models that include additional criteria because, by definition, addiction diagnoses include more than just drug SA. Improved models may allow for the identification of diverse neuroanatomical structures, epigenetic and transcriptional events, and memory processes that might be involved in the promotion of addiction to specific substances such as methamphetamine.Methamphetamine (METH) addiction is a biopsychosocial disorder with a very high prevalence throughout the world4. This statement thus hints to the need to decipher molecular changes that may be predictive of resilience to and/or of risk to develop an addictive state9. Elucidation of these molecular signatures has the potential to help in the development of pharmacological interventions to modify the clinical manifestations of substance use disorders. To address these issues, we have begun to use a model that uses footshocks to differentiate rats that continue to self-administer methamphetamine compulsively from those that significantly reduce their intake in the presence of punishment17. In the present study, we have used this model to investigate transcriptional changes in the nucleus accumbens (NAc) and dorsal striatum after a month of forced abstinence.A better understanding of how individual drugs differentially impact specific brain regions within these distributed networks will require more in-depth knowledge of the specific biochemical and molecular consequences of each addictive drug on different brain regionsFigure During the training plus punishment phase, footshock intensity was increased from 0.18 to 0.30 A over a period of 5 days Fig. . The incWe also measured cue-induced reinstatement at withdrawal days 2 and 30 Fig.  and founWe used the Affymetrix array platform that contain a total of 68,842 probes to measure transcriptional changes in the NAc and dorsal striatum of animals euthanized one day after the second extinction test. These probes consist of 24,753 protein coding, 28,724 noncoding, and unassigned pseudogenes. The analysis of raw array data revealed that 9044 genes were expressed in the NAc and 9754 genes were expressed in the dorsal striatum. This was determined by comparing the average signal values of transcripts in the control animals to the average signal value of the bioB gene in the control animals. Gene with average signals above the average signal value of the bioB gene were considered as expressed in the specific brain region. The expressed genes were included in further analyses of differential gene expression between the experimental groups. There were 181 genes differentially expressed in the NAc and 304 genes were differentially expressed in the dorsal striatum.The results of the microarray analyses in the NAc are shown in Figs The results of the striatal microarray data are shown in Figs To validate the changes in oxytocin observed in the microarray data, we ran quantitative PCR using RNAs from the various groups of rats. Figure We also validated the expression of striatal CARTpt that showed increased expression between SR and SS in the microarray data Table . Figure We also validated the expression of some genes that showed changes in rats that were yoked to the METH SA to receive similar number of footshocks as the METH SA animals Fig. . Figure Figure 24. We had reasoned that drug-induced alterations of these neuroanatomical systems may negatively impact information processing and, over time, might have led to disturbed responses to environmental cues7, based on their different connections to various brain regions implicated in addiction25. Indeed, we found significant increases in oxytocin mRNA levels in the NAc, but not in the dorsal striatum, 30 days after stopping METH SA. These results are consistent with those of Baracz et al.26 who had reported significant increases in plasma oxytocin levels immediately after and at 15 days of withdrawal from METH self-administration. The observation that animals with reduced METH intake in the presence of footshocks did not show significant increases in oxytocin mRNA levels suggest that these observations may be related specifically to compulsive METH takers that continue to take the drug despite the footshocks (SR rats). The changes in oxytocin mRNA are also not secondary to footshocks alone since the yoked animals did not show any increases in oxytocin in comparison to control rats. We also found significant increases in CARTpt mRNA levels in the dorsal striatum but not in the NAc of compulsive METH takers, suggesting that different neurotransmitter systems may be activated in different brain regions during protracted forced abstinence from METH self-administration.The aim of the present study was to use an unbiased genome-wide approach to identify transcriptional changes in shock-sensitive and -resistant rats that had all undergone METH self-administration training and footshocks. We also compared gene expression in rats that were yoked to the METH SA animals to control for the effects of footshocks during the shock phase of the study. Our aim was to compare gene expression in the NAc and dorsal striatum because these two brain regions are known for their involvement in different aspects and phases of compulsive drug-taking behaviors and during withdrawal from METH SA28 and in some psychopathological states including stress, affective disorders, and schizophrenia30. A potential role for oxytocin in addiction has also been investigated by several groups of scientists34. For example, intracerebroventricular injections of oxytocin were shown to reduce METH-induced increased locomotor activity35 and conditioned place preference (CPP)36 in mice. Carson et al.32 have also reported that peripheral oxytocin administration can dose-dependently reduce the number of self-administered METH infusions by rats. Oxytocin also reduced locomotor activity and drug-induced reinstatement32. Follow-up studies have also documented that infusions of oxytocin in the NAc can reduce METH CPP37 and METH-primed reinstatement31. These studies are consistent with the recent report that oxytocin, given peripherally or intracerebrally, can reduce METH demand and seeking by rats33. When taken together, these observations implicate an important role of this neuropeptide in the effects of this psychostimulant. This conclusion is consistent with the report that METH can cause increased plasma oxytocin levels and decreased oxytocin receptors in the NAc of rodents31. Thus, our findings of increased oxytocin mRNA levels in the NAc may constitute compensatory responses to replace oxytocin that might have been released from the NAc during compulsive METH taking.Oxytocin is a centrally active neuropeptide that is involved in social bonding/attachment38. CARTpt is a brain/gut neuropeptide initially identified by differential display in the dorsal striatum of rats that were injected with either cocaine or amphetamine39. In the NAc, CARTpt was reported to be localized in D1-containing neurons not in D2 neurons40. In addition to the NAc, CARTpt is widely distributed in the brain42 and appears to play a role in the acute and chronic effects of some drugs of abuse44. For example, intra-NAc administration of CART was shown to attenuate hyperactivity induced by either cocaine or amphetamine46. The effects of CARTpt were reported to be dependent on the modulation of both dopamine (DA) D1 and D2 receptors47. Intra-accumbal CARTpt injections also attenuated cocaine self-administration48. Non-contingent injection of METH caused significant increases in CARTpt mRNA levels in the NAc of rats euthanized at either 2 hours or one month later49. When taken together with our present observations, these results support the notion that CARTpt may play a substantial role in regulating certain aspects of stimulant-induced behaviors44 both acutely and after prolonged abstinence.We also found significant increases in CARTpt mRNA levels in the striatum, but not in the NAc, after 30 days of withdrawal from METH see Fig. . These r54. These studies had investigated perturbances in dopaminergic systems during short periods of time after application of single or multiple footshocks. In the present study, gene expression was assessed after one month of withdrawal and, yet, we still observed significant changes in FMO2, PDK4 and PTPRO mRNA levels in two DA terminal fields. Although the reasons for these changes remain to be determined, these findings suggest that footshock punishment can have substantial transcriptional effects in these brain regions in a fashion that might affect brain metabolism since FMO2 is a member of a class of enzymes (FMO1–5) that are localized in the membranes of endoplasmic reticulum and are involved in metabolizing drugs, regulating fat and cholesterol metabolism, and breaking down of other dietary components56. Importantly, the presence of FMOs in the brain57 and the increased expression that we observed in the striatum suggests that FMO2 might play a role in the longterm effects of footshocks on the metabolic health of this brain structure. Parenthetically, FMO2 expression is increased by manipulations that increase animal longevity58.In view of the novelty of these observations, it is of interest to briefly discuss the findings that some genes showed significant changes in their expression in animals that had received many non-contingent footshocks  complex (PDC)62. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl-coA63 and is inactivated via PDK-dependent phosphorylation59. PDKs are expressed in the brain64 and astrocytes appear to contain higher expression of PDK2 and PDK4 whereas neurons contain higher levels of PDK165. PDKs may be involved in the generation and progression of several neurodegenerative processes that involve inflammation and abnormal mitochondrial functions66. Thus, our findings of increased PDK4 mRNA in the NAc and dorsal striatum suggest that footshocks might have activated mechanisms meant to further downregulate PDC function in glial cells to modify biochemical responses to repeated painful stimuli. This notion is consistent with the report that glial responses to inflammatory stimuli were attenuated in PDK4 knockout mice60.Another important metabolic enzyme affected by repeated footshocks in both the striatum and NAc is PDK4, a member of a class of 4 enzymes (PDK1-PDK4)68. Protein tyrosine phosphatases (PTPs) are subdivided into receptor and non-receptor phosphatases and are expressed in the central nervous system69. Thus, it was of interest that the mRNA expression of a protein phosphatase, PTPRO, was significantly increased in rats that received large number of non-contingent footshocks. PTPRO was first identified as a membrane protein that is expressed in the brain70 and is involved in the regulation of axonal guidance and neurodevelopment73. The observed increased in PTPRO in the yoked rats suggests that footshocks might have led to activation of mechanisms known to be involved in the regulation of PTPRO-dependent neurodevelopmental processes and, by extension, shock-induced synaptic plasticity in adult rats. In any case, the observations of footshock-induced changes in gene expression are consistent with the idea that adverse consequences can have longterm effects on the brain.Protein phosphorylation/dephosphorylation processes are key regulatory processes that serve to activate or inactivate proteins by the actions of protein kinases and protein phosphatasesOur present observations support the notion that accumbal oxytocin is a potentially important neuropeptide in the manifestation of METH addiction. Our data also identify a potential role of striatal CARTpt in regulating rat behaviors after protracted withdrawal from compulsive METH taking. When taken together, the present findings point to a potentially significant role of a diversity of neuropeptides in different stages of METH addiction. This conclusion supports the need for further clinical evaluation of these neuropeptides or their analogs in clinical situations.Guide for the Care and Use of Laboratory Animals (ISBN 0–309–05377–3) and were approved by the National Institute of Drug Abuse Animal Care and Use Committee.We used male Sprague-Dawley rats , weighing 350–400 g in the beginning of the study in all experiments. We housed animals in a humidity and temperature-controlled (22.2 ± 0.2 °C) room with free access to food and water. Our procedures followed the 74. We attached the catheters to a modified 22-gauge cannula that was mounted to their skulls with dental cement. We injected buprenorphine  one time after surgery to relieve pain and allowed the rats to recover for 5–7 days before methamphetamine self-administration training. During the recovery, training and punishment phases of the experiment, we flushed the catheters every 24–48 h with gentamicin  and sterile saline.We anesthetized rats with ketamine and xylazine  and inserted silastic catheters into the jugular veins, as described previously74. On the first day of training, we brought rats to the self-administration room and chronically housed them in self-administration chambers. Animals had free access to food and water that were available in water bottles and feeders hanging on the walls of all self-administration chambers. We trained rats to self-administer dl-methamphetamine HCl (NIDA) or saline during three 3-h sessions/day  for 20 days under a fixed-ratio-1 with 20-s timeout reinforcement schedule. Presses on the retractable active lever activated the infusion pump. Active lever presses were also accompanied by a 5-s compound tone-light cue. Presses on inactive (stationary) lever had no reinforced consequences. We connected the catheters of rats to a modified cannula  attached to a liquid swivel  using a polyethylene-50 tubing that was protected by a metal spring. Rats self-administered methamphetamine for 5 days a week with weekends off. During the 2 days off, rats remained housed in self-administration chambers but were disconnected from intravenous self-administration lines. Also, the levers were retracted and all other cues were removed. Rats self-administered methamphetamine at a dose of 0.1 mg/kg/infusion over 3.5 s (0.1 ml/infusion). To prevent overdose, we limited the number of infusions per 3-h session to 50. Control rats self-administered saline under the same conditions. We started the self-administration sessions at the onset of the dark cycle and sessions began with the insertion of the active lever and the illumination of a red house light that remained on for the duration of the session. At the end of each 3-h session, the house light was turned off, and the active lever was retracted.We performed the training procedure for methamphetamine self-administration essentially as describedDuring the training plus punishment phase, rats continued methamphetamine self-administration every day  under the fixed ratio-1 with 20-s timeout reinforcement schedule that was used during training. For methamphetamine-trained rats, 50% of the reinforced lever-presses also resulted in the concurrent delivery of a 0.5-s footshock through the grid floor. We set the initial footshock at 0.18 mA and increased the shock intensity by 0.06 mA to a final value of 0.3 mA . Additionally, some control rats were yoked to the shocked animals so that each time animals in the methamphetamine SA group received a contingent shock, these rats also received a non-contingent shock and a saline infusion. Thus, by the end of the behavioral experiments, there were rats yoked to the corresponding shock-sensitive and -resistant rats, namely yoked SS (YSS) and yoked SR (YSR), respectively.At the end of the training plus punishment phase, rats were returned to the animal vivarium and individually housed with no access to METH. Intravenous catheters were covered using dust caps and rats had access to home-cage food and water ad libitum. Cue-induced drug craving was then assessed at days 2 and 30 of withdrawal. To test cue-induced craving, rats were brought back to their corresponding SA chambers on the morning of each test. Each test consisted of a single 1-h session during which presses on the drug-associated lever resulted in contingent presentations of the tone and light cues previously paired with METH. However, no METH was available during these tests. Cue-induced drug seeking behavior was assessed using a within-subject design such that all rats tested on day 2 were also tested on day 30 of withdrawal. Animals were euthanized one day after the second cue-induced methamphetamine craving test.GeneChip™ Rat Transcriptome Array 1.0 (RTA 1.0.) . RNA samples showed no degradation with all RINs being greater than 8.4 . We assessed RNA integrity using an Agilent 2100 Bioanalyzer . We obtained RNA integrity numbers . The cRNA and single-stranded cDNA size distribution was determined using an Agilent Bioanalyzer to confirm that the cRNA was the correct size range (200–2000 nt) and the single-stranded cDNA was the correct size (approximately 400 nt). Quality control processes were also used for gene expression analysis including but not limited to hybridization controls, labeling controls, internal control genes (housekeeping controls), global array metrics, and algorithm parameters. Hybridization, labelling, scanning, and data extraction were also performed using standard Affymetrix protocols. Differentially expressed genes were chosen if there were ± 1.7-fold changes (p < 0.05) based on pairwise comparisons using Affymetrix software.Genes were selected for validation by real-time quantitative polymerase chain reaction (qPCR) essentially as previously described [15]. Briefly, individual total RNA from 6 rats per group was reverse-transcribed into cDNA using Advantage RT for PCR kit . Gene-specific PCR primers were generated by the LightCycler probe design software v. 2.0  and purchased from the Synthesis and Sequencing Facility of Johns Hopkins University . High Capacity cDNA Reverse Transcription Kit  was used with a ViiA 7 instrument . The relative amounts of messenger RNA were normalized to means of clathrin, ornithine decarboxylase antienzyme 1 (OAZ1), and tubulin.We analyzed the behavioral data with the statistical program SPSS and followed significant effects (p < 0.05) with SPSS post-hoc contrasts with the repeated measures ANOVA. For the training and shock phases, the dependent variables were the number of METH or saline infusions during 20 training days and 5 footshock days. The PCR data were analyzed by one-way ANOVA followed by Fischer’s protected least-significant difference test (PLSD) using StatView . The null hypothesis was rejected at p < 0.05.Gene expression data have been deposited at the NCBI under the accession #GSE95571.Suplementary Tables


We update a systematic review to map the prevalence of K540E and A581G mutations in 294 surveys of infected humans across Africa from 2004-present. Interpreting these data is complicated by multiclonal infections in humans, especially in high transmission areas. We extend statistical methods to estimate the frequency, i.e. the proportion of resistant clones in the parasite population at each location, and so standardise for varying transmission levels. Both K540E and A581G mutations increased in prevalence and frequency in 60% of areas after 2008, highlighting the need for ongoing surveillance. Resistance measures within countries were similar within 300 km, suggesting an appropriate spatial scale for surveillance. Spread of the mutations tended to accelerate once their prevalence exceeded 10% (prior to fixation). Frequencies of resistance in parasite populations are the same or lower than prevalence in humans, so more areas would be classified as likely to benefit from IPT if similar frequency thresholds were applied. We propose that the use of resistance frequencies as well as prevalence measures for policy decisions should be evaluated.Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine in vulnerable populations reduces malaria morbidity in Africa, but resistance mutations in the parasite  Pfdhfr gene) confer resistance to pyrimethamine while mutations in the dihydropteroate synthetase (Pfdhps gene) confer resistance to sulphadoxine. The association between these mutations and SP efficacy is complex1 and is affected by factors such as individuals’ immunity to malaria2, but the following associations have been found. In East Africa a triple mutant dhfr allele containing N51I, C59R, and S108N mutations combined with a double mutant dhps (A437G + K540E) (together known as the quintuple mutant) is associated with clinical and parasitological SP treatment failure6, and reduces the prophylactic period provided by SP6. However, protection by IPT with SP in pregnant women against low birth weight outcomes is sustained even in areas with high levels of the quintuple mutant9, and infants benefit from IPT in areas with intermediate , though not high  levels of the quintuple mutant11. In certain East African foci, resistance has intensified because parasites have acquired an additional dhps A581G mutation. One such area reported 86% clinical failure12 and loss of protective efficacy of IPTi13 and IPTp14, and a meta-analysis concluded that IPTp efficacy was reduced when the prevalence of the 581G mutation was over 10%15. Hence the surveillance and reporting of 540E and 581G has a central role in IPTp-SP policy decisions.Intermittent preventive treatment in infants (IPTi) and pregnant women (IPTp) with sulphadoxine–pyrimethamine (SP) is recommended by the World Health Organization (WHO) in areas of moderate-to-high malaria transmission in sub-Saharan Africa but wide regional variations in drug resistance critically influence the success of this intervention. In East Africa, parasites have a higher threshold of SP tolerance than those found in West Africa. Mutations in dihydrofolate reductase 38. This worked well on simulated data, and was also applied to field data in Uganda31. However, even mean MOI data are usually not available from sites with resistance surveillance data, and sometimes only the prevalence of resistance is reported, not the proportion of mixed resistant-wild type infections.The frequency of resistance in parasites in a survey is often estimated by simply excluding mixed wild type-resistant infections from analysis, and presenting the proportion of remaining individuals who have a pure resistant infection. However, this can mean a considerable proportion of samples are excluded in areas of moderate-high transmission and medium resistance prevalence40. We then update a systematic review of the prevalence of the 540E and 581G mutations in infected humans across Africa and apply our method to also estimate the frequency of these mutations in the parasite population in each area. We map mutation prevalence and frequency and quantify spatiotemporal trends in SP resistance.Here, we further develop these previously published methods in order to estimate the frequency of resistance mutations in the parasite population from the reported prevalence of resistance in infected humans in routine surveillance data, first validating our methods using a large, detailed dataset from southern Tanzania42 of the distribution of dhps 540E and 581G mutations in Africa, including all published studies which assessed the prevalence of the mutation in P. falciparum-infected individuals in endemic areas (full data available in Supplementary data www.drugresistancemaps.org). We excluded samples of patients recently treated with SP as part of the study. The previous review was completed in 2011 and the most recent samples in that review were collected in 2008 (the average time lag between sample collection and publication is 5 years41). The updated review includes studies published up to March 2016, although the most recent surveys reported in those publications were carried out in 2013. The review generated 182 new measures of the 540E mutation and 157 of the 581G mutation . Firstly, we estimated the frequency of 540E in each cluster and year using an existing method by Hastings et al. (Method 2A)32 which utilises the full information on resistance markers and MOI for each individual in the Tanzanian test dataset. We found that the estimated frequencies in the Tanzanian parasite populations were consistently lower than the prevalence of resistance mutations in humans, as expected . Frequencies estimated using this method 3A correlated well with the estimates made using the full individual MOI data . In the Tanzania test dataset this method gave relatively similar frequencies to those estimated from analysis of the full individual information  gave a good fit to the data  or estimated slide prevalence from the Malaria-Atlas Project44 in the same year as the survey (Method 5A). These mean MOI values were then used to generate a distribution of MOI in the population, assuming a Poisson distribution as before. Frequencies estimated using both these methods (Methods 4A & 5A) correlated very well with the estimates made using the full test dataset . Instead we used the fact that the average number of parasite clones per person increases with malaria transmission intensityA and 5A.set Fig. . AssuminWe next tried estimating frequencies using data from all individuals, but assumed that we only know the prevalence of infections carrying at least one resistance marker (Method 8A), not the prevalence of mixed wild type-resistant infections, as is often the case with published molecular marker surveys (mixed infections may be counted as resistant). However, frequencies estimated using only this information differed considerably from frequencies estimated from the full individual dataset (Method 2A) Fig. , indicat19. We incorporated imperfect detectability into the frequency estimation method which uses the full individual data (Method 2), as a baseline for comparison. We also chose to incorporate detectability into method 5 which estimated frequencies well using only information on the proportion of mixed infections and the Malaria-Atlas-Project-estimated slide prevalence, i.e. with the most parsimonious assumptions and data. We tested 3 different assumptions about detectability: (A) that 100% of clones are detected, as described above, (B) following a published method by Hastings et al.19, that resistant or wild type clones in an infection are not detected if they constitute less than a given proportion (the ‘genotyping sensitivity limit’) of the total clones or (C) that the probability of missing any clone is constant regardless of the MOI of an infection . We use these letters A, B and C to denote these different detectability assumptions. We first compared these different detectability assumptions when the frequencies were estimated from the full individual Tanzanian test dataset. Assumption C gave the best fit of the model to the test dataset (Method 2C), with the previously published method19 (Method 2B) fitting next best and the assumption of 100% detection (Method 2A) fitting worst . The probability of detecting each clone estimated by method 2C was 0.54 (95% CI 0.25–0.77), and the best fitting genotyping sensitivity limit in the published method (2B) was 0.33. Including imperfect detection improves the fit of the model to the proportion of mixed infections  of 46% (range 28–64%) at the time of the surveys. Interestingly, using frequency rather than prevalence for a 50% 540E threshold (the current IPTi policy recommendation) would only cause 6 out of 126 survey results to be additionally under the threshold. This is because resistance generally spreads rapidly at intermediate prevalence so is only at these levels for a short time with only 13% of surveys showing a prevalence between 30–70% . The median rate of change of 581 G prevalence was lower than 540E at 1.1% , but this may reflect low initial prevalence .We find evidence for continued spread of dhps mutation frequencies, giving a clearer picture of what is happening in the parasite population and enabling comparison across areas and time points with different transmission intensity and MOI distributions. Current IPTi policy is based on the prevalence of resistance markers in infected individuals, and the same is suggested for IPTp policy16. It will be critical to further characterise the relationships between clinical impact of IPTi and IPTp with SP and dhfr and dhps mutations and the biological mechanism of SP action54, and ongoing studies are addressing this issue. In the meantime, high prevalence of resistance markers may preclude implementation of IPTi  in some areas. The clinical relevance of mutation frequencies as opposed to prevalence in humans requires further research (see Introduction for more discussion of this issue). We suggest that frequency might be an important indicator of drug prophylactic effect given the prevalence of monoclonal infections within mosquitoes, as well as indicating the chance of clearing particular clones within mixed infections, but this needs to be better characterised. Given these uncertainties, we suggest it would be worth re-evaluating data to inform whether frequency thresholds as well as prevalence thresholds could be useful for policy guidelines. Mixed wild type-resistant infections, which create the difference between prevalence and frequency measures, are most common in high transmission settings, exactly where IPTp and IPTi have the highest public health impact. Frequency provides a lower threshold, as it is equal to or lower than prevalence, therefore the intervention would be recommended in more areas. For example, in our systematic review, we found that 33% more surveys had 581 G mutations under a 10% frequency threshold compared with a 10% 581 G prevalence threshold. However more research would be needed to identify whether frequencies are informative and if so, what a suitable frequency threshold would be.We show that frequencies of resistance in the parasite population could be estimated well from standard aggregate survey data on resistance markers in infected humans (see Supplementary Information for the full R code), without individual data or MOI data, as long as the proportion of mixed wild type-resistant infections was reported. We mapped 39. Furthermore MOI varies by age55. Another potential confounder is that some surveys include largely asymptomatic malaria infections in the community, whilst others recruit patients with symptomatic malaria. Clinical samples may have higher levels of resistance, probably reflecting treatment in the informal sector before seeking care. Further work to quantify the impact of these other factors would be beneficial to standardise resistance measures. The test dataset from Tanzania that we used to validate our methods for estimating frequencies was a survey of asymptomatically infected individuals of all ages. We cannot be sure how accurate the method is when applied to different samples. However, similar methods worked well on clinical samples in other settings31.Whilst using frequency measures increases the comparability of surveys where transmission intensities were different, there are additional factors that we did not control for. The age of individuals in each survey varied considerably and is likely to influence the probability that someone carries resistant parasites, since intake of treatment is different across age groups47, but in West Africa, 581 G was detected in the absence of 540E. The public health implications of the latter genotype are unclear and further research is required to characterise its phenotype. We also did not map dhfr genes, since these are not the focus of current policy10 and few studies reported the full dhfr and dhps haplotypes. Several methods have been developed to estimate haplotypes of parasites, which employ efficient methods such as Markov-chain Monte Carlo or Expectation-Maximization algorithms to maximise the likelihood35, but these all require individual patient data on MOI and multiple resistance markers. Ideally future resistance surveillance studies would measure MOI and estimate frequencies as part of the routine analysis.We did not estimate haplotypes at the 540 and 581 loci because we did not have individual data and the degree of linkage disequilibrium is unknown. In East Africa, 581 G usually occurs together with 540E56 and resistance frequencies within infected individuals, enabling quantification of parasites with different genotypes and distinguishing haplotypes in mixed infections58. Whole genome data can be used to estimate MOI, for example using the estMOI software59. These techniques would provide extremely useful data but may not be available for routine surveillance of resistance markers for some time. The analyses presented in this paper show that robust estimation of frequencies of single SNPs is possible from routine surveillance data without more resource-intensive molecular methods. These methods can be applied in future to standardise data on other resistance markers, in particular artemisinin resistance-associated mutations at kelch 13 and mutations conferring resistance to partner drugs in artemisinin combinations therapies, such as the emerging piperaquine-resistant strains in Cambodia which represent a severe public health threat.Rapid advances in genetics and genetic analysis are improving techniques for measuring MOIP. falciparum-infected individuals in a specific geographical area and the date of sampling. We collected information on the location, date, sample size, reporting of mixed infections and age range.We searched PubMed in March 2016 using the terms “malaria” and at least one of “dhps”, “pfdhps”, “540” or “581”, screening all studies published from 2011 onwards, when the previous systematic review was completed. We included studies which reported the prevalence or frequency of the 540E and/or 581G mutation in et al.39. In brief, survey data were from cross-sectional surveys conducted in 2004 and 2007 during a cluster-randomized trial of IPT with SP in infants. The intervention was implemented in 2005. Blood samples were collected on filter paper, and mutations at codon 540 of the dhps gene were detected by polymerase chain reaction (PCR) sequence-specific oligonucleotide probing (SSOP). Infections were counted as mixed if peaks could be detected above the noise threshold. MOI was assessed by polymorphisms in microsatellite markers PolyA, PfPk2, and TA109, and was calculated as the maximum number of observed variants.Full details of the test dataset used to compare different methods for estimating frequency are given in Pearce 45. For each of the 24 clusters in the IPTi trial and for each survey in the systematic review, estimates were extracted using the year and longitude-latitude of the survey location. In order to estimate mean MOI in the area using the relationship in Fig. 60.Estimates of slide-prevalence in 2–10 year olds were taken from raster files published by the Malaria Atlas Projectp is defined as the proportion of parasite clones which have a resistance marker. Following the equations by Hastings et al.19, this is calculated as:The frequency of resistance ci is the number of resistant clones and ni is the total number of clones in individual i. The prevalence of resistance Prew(res) is defined as the proportion of infected humans who are carrying at least one resistant parasite clone. The relationship between the prevalence of resistance in humans Prev(res) and the frequency of resistance in the parasite population p, following Hastings et al.19 is:where n is the MOI of each individual infection, p is the frequency of resistance and dn is the proportion of samples which have an MOI of n. The prevalence of the wildtype Prev(wt) is calculated using the same equation, replacing the frequency of resistance p with the frequency of the wild type (1 − p). The proportion of human with mixed wildtype-resistant infections P(mix) is calculated from these as:where P(wt) or all resistant parasites P(res) is calculated by subtracting P(mix) from Prev(wt) or Prev(res), respectively.The proportion of infections with all wild type The frequency which maximised the likelihood of the observed prevalence of a molecular marker in any given survey was estimated, contrasting methods which make different assumptions and also those which use detailed individual datasets versus those which use various summary measures more often available from published surveys. The details of each method and the data used by each are as follows:Frequency is estimated simply by excluding mixed wild type-resistance infections from analysis and calculating the proportion of infections which contain only resistant clones . This assumes that the distribution of MOI is the same in all-resistant as in all-wild type infections. It results in a lower sample size for estimating frequency.32 which uses the full data where MOI and presence/absence of resistance markers is known for each individual (this was applied to the test dataset from Tanzania only). Here dn is simply the observed proportion of individuals with a given MOI in the data.The published Malhaplofreq algorithmdn is assumed to follow a zero-truncated Poisson distribution, which would be observed when individuals are each infected at the same average rate. The zero-truncation allows for only analysing data from infected individuals. We fit the equations for P(res), P(wt) and P(mix) described above to the observed proportion of all-resistant, all-wildtype and mixed infections in each survey, respectively, using multinomial likelihood. These assumptions are similar to those used by Taylor et al.31 but because we analyse only one SNP at a time, we could use standard methods to maximise the likelihood.This method can be applied when only the prevalence of resistant and mixed infections, and the mean MOI in the study population are known. Here, As method 3, except the sample mean MOI is unknown whilst the local slide-prevalence of malaria is known. We estimate mean MOI using the relationship in Fig. 44.As method 3, but the mean MOI and the local slide prevalence are unknown. We use estimated slide-prevalence from the Malaria Atlas Project based on the location and year of the surveydn is assumed to follow a zero-truncated negative binomial distribution where some individuals are at higher risk of infection than others, assuming a relatively high level of variation (dispersion parameter = 0.5).As method 5, but As method 5, but the mean MOI, as well as the frequency, is allowed to vary during the fitting procedure.As method 5, but the proportion of mixed infections is unknown, only the prevalence of resistance (counting mixed infections as resistant) is known. We fit equation For each of the methods 2–8, we further assessed three different assumptions about the sensitivity of detection of parasite clones:A) Perfect 100% sensitivity.et al.32. Minority clones present at a density under a ‘genotyping sensitivity limit’ in mixed infections are assumed to remain undetected. This method modifies equation B) That the probability of detecting a clone is dependent on the MOI of the infection, as described in the method by Hastings v is 0 if c/n is less than the genotyping sensitivity limit and 1 otherwise. We varied the assumed genotyping limit simultaneously with the frequency to maximise the likelihood of the observed data.where q of being detected, and calculated the proportion of resistant infections as follows. The probability of an all-resistant infection for a given value of c is the probability that all susceptible clones are missing multiplied by the probability that at least one resistant clone is non-missingC) That the probability of detecting a clone is independent of the MOI of the infection. We assumed that each clone, resistant or wild type, has a certain probability cSumming over all possible values of Equation and when the assumption is made of a Poisson-distributed MOI with mean µ:p and q simultaneously, allowing p to be different in each cluster and year but assuming q was constant. We obtained confidence intervals using profile likelihood. The sensitivity of methods used to detect MOI is much higher than the methods used to detect resistance markers39, and therefore we assumed no effect of imperfect detection on MOI measures.When fitting to the Tanzanian dataset, we estimated For each analysis we specify which of the methods 1–8 is used by its number, and which detectability assumption is used by the letters A, B and C.Supplementary InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


Aims and method There is currently no trial or other scientific evidence informing the efficacy of any crisis intervention for people with borderline personality disorder (BPD). We aimed to assess the patterns of service use by patients with BPD taken on for crisis resolution and home treatment between 2010 and 2013. Patients with a diagnosis of BPD were identified and demographic and clinical data were collected.Results All patients were female, and a high proportion had recurrent presentations to crisis and home treatment services in Edinburgh. Many appeared to benefit from intensive home treatment, as measured by the Clinical Global Impression scale. A small number of patients (n = 5) were responsible for more than half of all referrals. Polypharmacy, or regular use of multiple medications, was common, with 62% of all patients receiving three or more regular medications.Clinical implications Crisis and home treatment services can be beneficial to most people with BPD in crisis. The high rate of polypharmacy seen in this study is of concern. It is present in 10% of out-patient mental health clinics, 15–20% of psychiatric in-patients, and 30–60% of clinical populations with a personality disorder. It occurs in an estimated 2% of the general population and has an estimated gender ratio of more than 3:1 for women/men.2 Because of the nature and potential consequences of these crises, the identification and utilisation of effective crisis management interventions with this population is of considerable importance.2The extent of the emotional and behavioural problems experienced by people with BPD varies considerably. Some people with BPD are able to sustain some relationships and occupational activities. Others, with more severe BPD, experience very high levels of emotional distress. They have repeated crises, which can involve self-harm and impulsive aggression, and can have high rates of comorbidity, including addictions. Despite this, people with BPD can be difficult to engage in treatment and frequently present to health services in crisis.3 and in 2007 described CRHT as a key step in implementing the National Service Framework, partly to ensure in-patient care was used only where necessary.3 National Health Service (NHS) services in Scotland were not constrained by the National Service Framework and did not incorporate functionalised teams such as assertive outreach, early intervention and CRHT teams until 2008.The Department of Health in England and Wales has recommended crisis resolution and home treatment (CRHT) in its best practice and policy implementation guides since 2002,4In this observational study, we evaluated the current patterns of service use in patients with BPD taken on by Edinburgh Intensive Home Treatment Team (IHTT), which is a CRHT that facilitates early discharge from hospital as well as providing intensive home-based care. It was established in 2008 and has been linked to a reduction in psychiatric admissions and positive feedback.a priori criteria. IHTT records the severity of the presenting mental disorder using the Clinical Global Impression Scale (CGI)2 at admission and clinicians note the improvement or lack thereof in the presenting condition via the CGI-I score at the point of discharge from IHTT. Prospective approval for this service evaluation project was obtained from the local clinical governance department.Data were collected during a retrospective examination of medical records of all patients who had primary ICD-10 diagnosis of BPD (code F60.3) taken on by Edinburgh Intensive Home Treatment Team (IHTT) between 2010 and 2013 (4 years). Using unique patient identifiers, each included e-case record was reviewed using 3The CGI has two components: the CGI-Severity (CGI-S), which rates illness severity, and CGI-Improvement (CGI-I), which rates change from the initiation of treatment (baseline). CGI-S is rated on the following 7-point scale: 1 normal/not at all ill, 2 borderline mentally ill, 3 mildly ill, 4 moderately ill, 5 markedly ill, 6 severely ill, 7 among the most extremely ill patients. CGI-I is similarly rated on a 7-point scale: ‘compared with the patient's condition at admission, this patient's condition is' 1 very much improved, 2 much improved, 3 minimally improved, 4 no change, 5 minimally worse, 6 much worse, 7 very much worse.patients' age and genderany medical comorbiditiescurrently prescribed medicationssource and reason for referral to IHTTthe nature of the crisis intervention by IHTTduration of treatment by IHTTCGI scores on entrance to and at exit from IHTTsubsequent service provider at discharge from IHTTnumber of repeat referrals/contact with the same patient .Data collected included:Each patient's regular medications were reviewed and classified into six groups: antidepressant drugs (selective serotonin reuptake inhibitors (SSRIs), serotonin and noradrenalin re-uptake inhibitors (SNRIs) and tricyclics (TCAs)), antiepileptic drugs used as mood stabilisers, antipsychotic drugs (first- and second-generation antipsychotics (FGAs and SGAs)), anxiolytics (benzodiazepines) and substitute treatment for opioid dependency (methadone).2 with Yates correction was used to test the association between CGI score and hospital admission. An unpaired t-test of the means of two groups was also used  to test whether there was a statistically significant reduction in CGI for those who were successfully discharged from IHTT compared with those who needed hospital admission.Pearson χThe total number of referrals for the 4-year period was 64, and 100% were female. The median age of patients was 39 years .n = 5) presented to emergency services frequently owing to repeated crises and suicidal thoughts – they were responsible for 53% (n = 34) of all referrals. These five patients were all women who had a traumatic background and multiple prior hospital admissions.Of the 64 referrals, the number of individual patients was only 27, as some patients required multiple contacts with the IHTT. Thirteen patients were responsible for 50 referrals . A small number of patients  were from the community owing to deterioration in mental health and increased suicidal behaviour; 66%  were discharged back into community following IHTT interventions. However, for the remaining 34% of referrals IHTT intervention was not enough to manage suicide risk and hospital admission was required , whereasP<0.0001; χ = 16, d.f. = 1 with Yates correction), as illustrated in The mean value of CGI of all patients at admission to IHTT was 3.3 (and similarly the mean CGI of the five patients who needed frequent IHTT intervention was 3.2). Using the CGI, an improvement in mental state and behaviour was documented in the majority of patients. The mean CGI improved from a baseline of 3.3 to 2.4 at the time of IHTT discharge in 39 patients. Twenty-two patients were admitted to hospital despite IHTT, with a mean baseline CGI of 4.2. There are significant associations between a lack of improvement as measured by CGI, and hospital admissions (t-test) showed a significant difference in CGI between those admitted to hospital and those who were not admitted (two-tailed P<0.0001 and 95% CI 1.4 to 2.2).Further analysis comparing those who needed hospital admission (mean CGI pre- and post-IHTT involvement: 3.6 and 4.2 respectively) with those who were successfully discharged from IHTT  to alleviate distress. There is no identified specific crisis intervention for this population. In the majority of cases IHTT interventions were successful as the mean CGI improved from baseline 3.3 to 2.4 in those patients who were discharged back into community. The remaining 34% of referrals required hospital admission despite IHTT interventions, with almost 41% needing re-admission owing to failed early discharge despite IHTT intervention.n = 5) were responsible for the majority of referrals to IHTT (51%), and were all women in their mid-40s (apart from one) who were regularly prescribed at least four different psychotropic medications. They all had a traumatic background and multiple prior hospital admissions. Interpersonal difficulties (mostly among family members) were usually noted as the main precipitating factor for crisis and increased suicidal thoughts. This small group of individuals were responsible for 50% of all the BPD hospital admissions during this period, illustrating the challenges of working with this population, and the disproportionate demand on the NHS. IHTT interventions and even hospital admissions were not always effective as patients were frequently referred back to IHTT, or indeed later re-admitted to hospital. The reasons for this are unclear, but may include only partial adherence to or rejection of therapeutic interventions.Relatively few patients  guidelines, and Abraham & Calabrese, recommend the use of SSRIs as first-choice treatment for affective dysregulation and impulsive-behavioural symptoms, and low-dose antipsychotics for cognitive-perceptual symptoms in BPD.6 However, Leib et al8 argue that the previous guidelines were based on research published only up to 1998 and recommend using anticonvulsants for affective dysregulation symptoms and impulsive-behavioural symptoms, and antipsychotics for cognitive-perceptual symptoms.7 Nevertheless, despite considering similar evidence to Leib et al, the National Institute for Health and Care Excellence (NICE) guidelines draw different conclusions and make clear recommendations against the use of any regular pharmacotherapy in this group:9‘Drug treatment should not be used specifically for borderline personality disorder or for the individual symptoms or behaviour associated with the disorder . Antipsychotic drugs should not be used for the medium- and long-term treatment of borderline personality disorder. (…) Short-term use of sedative medication may be considered cautiously as part of the overall treatment plan for people with borderline personality disorder in a crisis.’Despite this, in routine clinical practice clearly a broad spectrum of medications is used for the symptomatic treatment of BPD, as demonstrated in There has been much debate on the effectiveness of pharmacotherapy in treating different facets of BPD.4Compared with the conflicting advice on medication in BPD, the international guidelines are more in agreement regarding the role of psychotherapy. APA recommends psychotherapy as the core treatment for BPD (and more than one type of psychotherapy is effective), along with adjunctive medication.11 examining the effectiveness of partial hospitalisation on BPD showed that those who received individual or group psychoanalytic psychotherapy for 18 months had a significant fall in their symptoms  by 6 months, with continued improvement until the end of treatment at 18 months. In contrast, their control group (standard psychiatric care) showed limited change or even deterioration over the same period.11There does not appear to have been a previous study of crisis or home treatment for BPD. However, a randomised trial by Bateman & FonagyOur observational study showed that patients with BPD are frequent users of psychiatric services, and that some but not all respond well to home treatment as an alternative to hospitalisation. Indeed, the high re-admission rate for BPD suggests that hospital-based care is not always helpful. Despite the frequent crises experienced by this population and associated risks, there is a dearth of evidence on the best psychological and pharmacological treatments for BPD. More high-quality large studies in this area are needed.


Objective: Thrombosis of a site of anastomosis in microsurgical free tissue transfer can result in tissue necrosis. To salvage potentially failing free flap, various methods of monitoring the viability of tissue have been described. We report a novel method of monitoring free flaps using near-infrared spectroscopy.Methods: After microsurgical operation, we monitored the regional oxygen saturation of the flap with using the In-Vivo Optical Spectroscopy. A total of 57 patients participated in this study.Results: Of 57 cases, arterial insufficiency was detected in 1 case and venous insufficiency was detected in 3 cases. Regional oxygen saturation decreased before the flap color changed to a pale or congestive color. We could salvage these 4 patients by re-exploration.Conclusions: The postoperative monitoring with the In-Vivo Optical Spectroscopy is noninvasive, continuous, reliable, and reproducible. This technique could be one of the best methods for monitoring microsurgical free tissue transfers. These methods are roughly divided into noninvasive techniques and invasive techniques.12) of tissue that is placed directly beneath the sensor. This unique system allows clinicians to measure site-specific oxygen levels rather than requiring them to infer the data from systemic, whole body measures, such as blood pressure and pulse oximetry. Furthermore, this system uses 2 data channels, which allows clinicians to conveniently monitor multiple regions throughout the body. This system has been used widely by cardiovascular surgeons and anesthesiologists especially for monitoring regional cerebral oxygen saturation during cardiac surgery.13,The Oximetry Monitoring System  provides real-time monitoring of changes in regional oxygen saturation . Unlike parameters that measure only venous or arterial blood, INVOS technology includes contributions from a 3:1 ratio, thereby yielding a venous-weighted percent saturation. The INVOS system uses near-infrared light at wave lengths that are absorbed by hemoglobin (730 and 810 nm). Light travels from the sensor's light-emitting diode to either a proximal or a distal detector, thereby enabling separate data processing of shallow and deep optical signals. Data from the surface tissue are subtracted and suppressed, reflecting rSO2 in deeper tissues . This feature makes it possible to measure rSO2 of brain tissue under the skull bone. We applied this technology to free flap monitoring.The In-Vivo Optical Spectroscopy (INVOS) system noninvasively monitors site-specific adequacy of perfusion in the tissue directly beneath its sensors.Between 2009 and 2017, a total of 57 patients underwent free tissue transfer with monitoring of changes in regional oxygen saturation by INVOS. There were 21 male patients and 36 female patients. The ages of patients ranged from 20 to 82 years, with a mean age of 59 years. After the operation, we threaded the sensors of the INVOS to the flap and a control region. We selected the pectoralis major muscle as the control region and threaded the other sensor to the anterior part of the chest  to c. Th2  was 73.8 ± 10.6, while the average control area rSO2 (hereinafter CSO2) was 75.9 ± 10.0 . The average flap area rSO9 ± 10.0 . When cothe legs . When coer flaps . During 2 value of 88.6 and an average CSO2 value of 86.3. Case 2 was a 66-year-old male patient with cranial reconstruction using a latissimus dorsi flap. Monitoring was conducted for 6 days postoperatively, with an average FSO2 value of 67.3 and an average CSO2 value of 84. Case 3 was a 67-year-old male patient with foot reconstruction using a latissimus dorsi flap. Monitoring was conducted for 6 days postoperatively, with an average FSO2 value of 53.8 and an average CSO2 value of 48.9. All 3 cases progressed smoothly postoperatively. The postoperative FSO2 and CSO2 values are shown in Case 1 was a 54-year-old female patient with breast reconstruction using a deep inferior epigastric perforator flap. Monitoring was conducted for 7 days postoperatively, with an average FSO2 value dropped rapidly from the 70s to the 40s over approximately 15 minutes. At that time, no clear change in flap color was observed. The decline continued slowly after this as well and, when the FSO2 value dropped below 40 three hours after the patient was returned to his hospital room, arterial occlusion was diagnosed because of the pale color of the flap, after which arterial reanastomosis was performed. 2 value.A 63-year-old male patient with arterial occlusion underwent reconstruction surgery with a free latissimus dorsi flap to treat exposure of an artificial joint in the right knee. The descending genicular artery and the great saphenous vein were anastomosed with the thoracodorsal artery and the accompanying vein. Artificial dermis was affixed over the muscle flap and the INVOS was placed over the skin paddle. Thirty minutes after the patient was returned to his hospital room, the FSO2 began dropping from its previous level of about 60. At that time, no change in flap color was observed. Approximately 1 hour later, the FSO2 dropped to 15, the minimum value. Because the central skin island also presented with a congested color at that point, venous occlusion was diagnosed and venous reanastomosis was performed. 2 value when the venous thrombosis was present.A 63-year-old female patient with venous occlusion underwent reconstruction surgery with a free latissimus dorsi flap to treat a radiation ulcer in the left knee. The descending branch of the lateral circumflex femoral artery and the accompanying vein were anastomosed with the thoracodorsal artery and the accompanying vein. The skin paddle was divided and sutured with the peripheral side, which was positioned ventrally, and the center, which was positioned dorsally. The INVOS was placed on the central skin paddle. Immediately following the operation, the peripheral skin paddle showed a slight pattern of congestion, which became pronounced 1 day postoperatively. Since the flap color in the central skin island was good, this was deemed to be an intraflap blood flow issue, and the decision was made to monitor the patient's progress. Four days postoperatively, the FSO2 and CSO2, and the amplitude of that variation generally stayed within 20 points. Based on the observation that baseline variation in FSO2 and CSO2 tends to be linked, it is possible that the INVOS can detect slight changes in the patient's general condition, such as slight fluctuations in interstitial oxygen saturation due to blood pressure and oxygen administration. Although there was a wide range of absolute values for rSO2 depending on the patient, when broken down by flap type, rSO2 was high in abdominal flaps and low in latissimus dorsi flaps, while a pattern of low rSO2 in the legs was observed when broken down by reconstruction site. This led us to consider factors related to blood flow in the flap itself and factors related to blood flow in graft bed blood vessels. However, it is difficult to use the absolute value of rSO2 to monitor flap blood flow. Accordingly, it is important to monitor trends that indicate changes in flap blood flow.Baseline variation was observed in both FSO2 during arterial occlusions with an ischemia model of the brachioradialis muscle that used the INVOS 3100 and tourniquets in healthy adults. This study reported that the rSO2 value rapidly dropped from 75 to 25 within 10 minutes of the start of ischemia and, 1 minute after ischemia was corrected, surpassed the preischemia value by rising to 80, after which the value returned to 75. This is believed to be due to the fact that the rSO2 value in the INVOS reflects arterial inflow. Specifically, when the rSO2 value that dropped because of ischemia rose rapidly after the ischemia was corrected, large amounts of arterial blood continued to flow into the tissue over a short period of time immediately following the release. This resulted in a higher value than the value achieved when the preischemia value was temporarily reached. Ultimately, once the inflow returned to preischemic levels, the rSO2 value returned to equilibrium.Muellner et al2 increased for a few days following day 1 before achieving equilibrium. This is another indicator of arterial inflow in free flaps following anastomosis. In other words, we inferred that, although flap resistance limits the amount of arterial inflow immediately after anastomosis, an alternative venous return beyond that of the anastomosed veins subsequently formed within a few days of surgery, thereby contributing to a decrease in flap resistance. This ultimately increased arterial blood flow. According to Sakurai et al,2 indicates the amount of arterial blood flow that enters the flap.In many patients, we observed a pattern in which FSO2 value rapidly dropped over approximately 15 minutes, which may demonstrate the fact that arterial inflow decreases rapidly with arterial thrombosis. In addition, although the FSO2 value did not bottom out because of thrombus formation, we deduced that the patient's vasculature was not completely occluded and that a small amount of arterial blood continued to flow into the transplanted flap. In contrast, in case 5, where a venous thrombosis occurred, the speed of the decline was slower than the speed of decline in cases of arterial thrombosis, which required approximately 1 hour. This is likely due to the fact that since the expansion of the vascular network inside the flap allows a certain amount of arterial inflow to be accepted even after outflow tracts are blocked by venous thrombosis, the reduction of arterial inflow was milder than in arterial thrombosis. In addition, we inferred that FSO2 reached the minimum value because arterial inflow was completely blocked when the flap resistance reached its maximum value, at which time flap congestion was detected clinically. Although findings of flap ischemia are difficult to identify in cases of arterial thrombosis, in both arterial thrombosis and venous thrombosis, clinical changes in flap color seem to first occur when arterial inflow is almost completely blocked. The INVOS system is thought to be a sensitive system for detecting the process of thrombus formation and the progression of decreasing arterial inflow before arterial inflow is completely blocked. This suggests that the INVOS system is a valuable tool that enables the clinical diagnosis of thrombosis at a site of anastomosis and allows for earlier monitoring.In case 4, where arterial thrombosis occurred, the FSO2. In 1 case by suture removal, and in the other case by posture change, FSO2 returned to baseline. We considered that the cause of these vascular insufficiencies was tension caused by suture and local pressure, and we believed that we could salvage them before re-exploration by this method.We experienced 2 cases that did not demonstrate vascular thrombosis in spite of a rapid decrease in FSOThe characteristics of this system is requirement of threading sensors and measurement of regional oxygen saturation almost 2 or 3 cm beneath its sensors. Consequently, this is especially suitable for buried flap with no skin paddle and can be acceptable in case of skin graft over muscle flap, while it is unfitted for monitoring of shallow region in thin flap or intraoral reconstruction.2 value immediately following INVOS placement as a baseline value in actual clinical settings and directed nurses to contact us if there was a decrease of more than 20 points. Although 1 patient was labeled positive because of a gradual decrease over several days, this drop was likely due to changes in flap hemodynamics . Otherwise, there were no cases in which flap ischemia or congestion due to anastomosis-site thrombosis went undetected. If a rapid decrease of more than 20 points is positive and the 2 cases that did not demonstrate vascular thrombosis were false positive, the positive predictive value was 66.7% (4/6), the negative predictive value was 100% (51/51), and the number needed to treat is 28.5. In a systemic review by Chen et al,Based on these defining characteristics of the INVOS system, we used the FSOAn implantable Doppler probe is another effective monitoring method, and many reports on its usefulness have been published recently. This device provides a direct and real-time measurement of the flow of vessels and is more efficacious than clinical assessment in terms of flap failure rate, salvage rate, and sensitivity.Flap blood flow was monitored using a noninvasive mixed blood oxygen saturation monitor. This makes it possible to sensitively detect changes in intraflap blood flow before clinical signs appear, and it is considered useful as a continuous and noninvasive monitor for flap blood flow.


ERCC3 is likely involved in the genotoxic effects pathway of benzene. Epigenetic effects of benzene need more complex explanation in causative pathway leading to hematoxicity.We would like to thank Moshammer and Poteser for their comments. We agree with Moshammer and Poteser’s suggestion that epigenetic changes do lead to genetic instability and the methylation of promoter region of MAGE-1) cancer associated antigen gene and hypermethylation in p15 in workers exposed to benzene. We reported downregulation of p15 and p16 expressions were associated with DNA promoter hypermethylation in benzene poisoning (BP) patients [ERCC3 promoter region should be explored in a well controlled in vitro system to better understand the effects of benzene-induced hypermethylation on gene dysregulation.Regarding whether the methylation pattern is specific for benzene, Bollati et al.  found thpatients . Inductipatients . These fBLM, TP53, RAD51, WDR79, and WRN) that play a critical role in DNA repair and genomic maintenance were associated with highly significant reductions in the white blood cell count among benzene-exposed workers but not controls [In this study, we only observed an association between lower neutrophil and higher methylation in exposed workers. In a study of 250 benzene-exposed workers and 140 unexposed controls, single-nucleotide polymorphisms in genes . It is necessary to verify the correlation between methylation of ERCC3 gene and hematotoxicity in a larger cohort of benzene-exposed population.We agree that it is necessary to study hypermethylation patterns in a longitudinal manner starting after an episode of toxic neutropenia. We analyzed the methylation levels in the BP group compared with the non-BP group and found that there was no statistical significance between these two sub-groups. The toxicity of benzene discussed in this paper did not specifically apply to the BP group but rather to all benzene-exposed workers including BP. Although the BP workers had their poisoning episode approximately 20 years ago and afterwards were no longer exposed, they still display lower nuetrophil counts compared to the other exposed workers (2.55 ± 0.84 × 10We are thankful to Moshammer and Poteser for their kind suggestion. We thank the editor for giving us the opportunity to provide a reply to the letter.


The development of high-throughput sequencing technologies have allowed the possibility to investigate and characterise the entire microbiome of individuals, providing better insight to the complex interaction between different microorganisms. This will help to understand how the microbiome influence the susceptibility of secondary agents and development of disease. We have applied viral metagenomics to investigate the virome of lymph nodes from Swedish pigs suffering from the multifactorial disease postweaning multisystemic wasting syndrome (PMWS) as well as from healthy pigs. The aim is to increase knowledge of potential viruses, apart from porcine circovirus type 2 (PCV2), involved in PMWS development as well as to increase knowledge on the virome of healthy individuals. In healthy individuals, a diverse viral flora was seen with several different viruses present simultaneously. The majority of the identified viruses were small linear and circular DNA viruses, such as different circoviruses, anelloviruses and bocaviruses. In the pigs suffering from PMWS, PCV2 sequences were, as expected, detected to a high extent but other viruses were also identified in the background of PCV2. Apart from DNA viruses also RNA viruses were identified, among them were a porcine pestivirus showing high similarity to a recently (in 2015) discovered atypical porcine pestivirus in the US. Majority of the viruses identified in the background of PCV2 in PMWS pigs could also be identified in the healthy pigs. PCV2 sequences were also identified in the healthy pigs but to a much lower extent than in PMWS affected pigs. Although the method used here is not quantitative the very clear difference in amount of PCV2 sequences in PMWS affected pigs and healthy pigs most likely reflect the very strong replication of PCV2 known to be a hallmark of PMWS. Taken together, these findings illustrate that pigs appear to have a considerable viral flora consisting to a large extent of small single-stranded and circular DNA viruses. Future research on these types of viruses will help to better understand the role that these ubiquitous viruses may have on health and disease of pigs. We also demonstrate for the first time, in Europe, the presence of a novel porcine pestivirus. Viral metagenomics and high-throughput sequencing technology have allowed the detection and characterisation of novel viruses. The technology provides the possibility to investigate the aetiology of diseases of unknown cause as well as to characterise the virome of healthy individuals or of, for example, the environment , 2. It hPostweaning multisystemic wasting syndrome (PMWS) is a multifactorial disease associated with high replication levels of porcine circovirus type 2 (PCV2), primarily in the lymph nodes, leading to increased size of the lymph nodes and acute wasting with a rapid loss of weight. Occasionally also jaundice, paleness and respiratory problems, are recorded . HoweverWe have in a previous study used metagenomic to investigate lymph node samples from pigs suffering from PMWS in order to identify additional viral agents in these samples. A porcine bocavirus was found in the background of PCV2, as were Torque Teno sus virus 1 and 2 (TTSuV1 and 2) , but if Previously collected lymph nodes, stored at -80°C, were used in this study; samples 1 and 2 were subiliac lymph nodes collected from two healthy pigs bought from an SPF herd  in FebruThe tissues were mechanically homogenized in 1.5 ml of 1x DNase buffer (Roche) and centrifuged at 4000 rpm for 10 minutes. The supernatant was aliquoted to 200 μl volumes and 100U DNase (Roche) and 2 μg of RNase A (Invitrogen) was added to each aliquot. After a short vortex the samples were incubated for 2 hours at 37°C prior to DNA and RNA extraction.20.One half of the sample was used for DNA extraction and the other half for RNA extraction. DNA was extracted using QIAamp DNA mini kit (Qiagen) according to the manufacturers instruction and the DNA was eluted in 50 μl AE. RNA was extracted using a combination of Qiazol (Qiagen) and RNeasy mini kit and eluted in 30 μl RNase-free H-) reaction. The cDNA and DNA was amplified using AmpliTaq Gold polymerase as described elsewhere [The RNA was labelled with the PCR primer tag-sequence through the cDNA synthesis while the DNA was labelled through a Klenow Fragment (3′ → 5′exolsewhere . Prior tde novo assembly and both the created contigs as well as the remaining singletons were then subjected to blastn (database nt). The sequences that did not render a blastn result were subjected to blastx (database nr) searches. The Camera portal was used for all the blast searches [−4 was used as a cut-off value to enable detection of divergent viruses. For the taxonomic classification the top blast hit was used.The quality of the sequences from the 454-sequening run was checked using CLC-genomic workbench. The program was also used for searches . An e-vaPrimers  were desOn average, around 100 000–230 000 sequences were obtained for each sample with an average of 260 bp in length. However, only 4559 sequences were obtained from pig 5.The sequences were annotated through blastn and blastx searches using the Camera portal . In all In samples 1 and 2, collected 10 years prior to the first outbreak of PMWS in Sweden, a majority of the identified viral sequences belonged to different single-stranded DNA viruses. In pig 1, both PCV1 and PCV2 were identified as well as other unclassified circoviruses. The majority of the unclassified circovirus hits, showed closest similarity to circovirus NW2 identified in pork product in the USA , 25. TheIn lymph nodes 3 and 4, collected from healthy SPF pigs in 2004 a vast diversity of viruses was identified . In bothA different picture was seen in the lymph nodes from the pigs affected by PMWS, i.e. in pig 5 and 6 from 2004 and in pig 7 and 8 from 2007 . In thesWe selected three of the identified viruses for further analysis. Main focus was on those that had not previously been detected in Sweden and/or that showed a divergence to previously described viruses.Rhinolophus affinis—Rhinolophus affinis pestivirus 1 (Ra-PestV-1) [Pestiviral sequences was present in two of the samples, in particular in the sample from pig 8. Using PCR a region of around 1500 bp was amplified from this virus. This sequence showed closest similarity to an atypical porcine pestivirus (APPV)  that was detected in pigs in the USA in 2015 . On nuclPestV-1)  and to tet al. (2010) identified in pork and beef and the protein sequence identity was high to those isolates (97–99%).Apart from PCV2/1 and TTSuV1/2 unclassified single-stranded circoviruses were identified through the annotation. The most prevalent of these were viruses with similarity to a circovirus originally identified in pork products in the US in 2009 (PorkNW2) . PCR andThe 454-sequence analysis indicated that, apart from the porcine bocavirus (PBoV group 1) previously demonstrated in Swedish pig , 18, a sIn this study, we show that a number of different single-stranded DNA viruses are circulating in pig populations in Sweden apart from the previously well-known PCV2, TTSuV1 and 2. In many cases a high level of viral co-infection appeared to be present in different individuals, this was especially apparent in the healthy individuals. Apart from the previously mentioned PCV2, TTSuV1 and 2, we discovered a virus showing high sequence identity  to a circovirus identified in pork products in the USA . This virus was identified in two lymph nodes from healthy pigs and from one PMWS pig. As the samples were collected in 1993, 2004 and 2007 this virus has apparently been present in Swedish pig herds for at least two decades.Linear single-stranded DNA viruses, including PBoV1 that have previously been identified in Swedish pig herds , 18, werPicornaviridae family. PTV-1 infections are often asymptomatic but can occasionally lead to clinical disease in the form of encephalomyelitis in pigs [Flaviviridae family identified in a bat metagenomic study from China [Not only DNA viruses were identified but also different RNA viruses. Pig 6 had sequences that showed 91% amino acid and 87% nucleotide sequence identity to a part of the VP-gene of porcine teschovirus 1 (PTV-1). This is a member of the  in pigs  However, in pigs . These tom China .There was an apparent difference in the amount of PCV2 sequences detected in the PMWS pigs compared to the healthy individuals. While high numbers of PCV2 sequences were found in PMWS samples, only a few were found in the lymph nodes from healthy individuals (with the exception of pig 2), although this is not a quantitative method this difference most likely reflect the massive PCV2 replication always seen in PMWS pigs. In fact, the percentage of PCV2 sequences was not higher than other viral sequences in most of the samples collected from healthy pigs. Many of the viruses found in healthy individuals were also present in the PMWS samples.Taken together, this study show for the first time the presence of the atypical porcine pestivirus APPV outside of the US and PBoV 3 in Sweden. Our study also shows that there is a high co-infection situation in healthy pigs, as well as in pigs with PMWS. This is in concurrence with other studies that have shown simultaneous identification of different RNA and DNA viruses in porcine faeces samples , 5. Futu


Arabidopsis thaliana, was made in understanding the structure and metabolism of starch and its conjunction. The process underlying the initiation of starch granules remains obscure, although this is a fundamental process and seems to be strongly regulated, as in Arabidopsis leaves the starch granule number per chloroplast is fixed with 5-7. Several single, double, and triple mutants were reported in the last years that showed massively alterations in the starch granule number per chloroplast and allowed further insights in this important process. This mini review provides an overview of the current knowledge of processes involved in the initiation and formation of starch granules. We discuss the central role of starch synthase 4 and further proteins for starch genesis and affecting metabolic factors.Starch is the primary storage carbohydrate in most photosynthetic organisms and allows the accumulation of carbon and energy in form of an insoluble and semi-crystalline particle. In the last decades large progress, especially in the model plant  Arabidopsis thaliana leaves.Starch is one of the most abundant biopolymers on earth. The entire process of starch biosynthesis encompasses: the generation of a suitable primer (glucosyl acceptor); the biosynthesis of a glucosyl donor (in most cases ADPglucose); the elongation of the primer by repetitive glucosyl transfer reactions via starch synthases (SS) and most probably via plastidial phosphorylase (see below); the introduction of branching points via branching enzymes and the removal of excess branching points via debranching enzymes; and the ordering of the glucans to the final physical organization. Starch granule synthesis in green algae, mosses, ferns, and higher plants occurs inside the plastids. In many other eukaryotic algae starch is formed in the cytosol, which is assumed to represent an earlier evolutionary state . This restarch initiation in a strict sense as a process in which a primer-like structure/initiation complex is generated, that can in principle be further used for polymerization reactions. This also includes the possibility that no starch granules are detectable since the primer-like structure/initiation complex is not further synthesized. The term starch formation indicate when these potential primer-like structures/initiation complexes are used to form starch granules, and so starch granules are detectable in chloroplasts.To some extent, the initial steps of the starch biosynthesis, the generation of a suitable primer , and the further elongation of the glucosyl acceptor by the action of glucosyl transferases is not precise to separate. The first elongation processes as well as the first branching/debranching events does not immediately result in a native starch granule rather generate a polyglucan. Furthermore, SS specifically interact with branching enzymes during the process of starch synthesis . But onlStarch synthases (SSs) are the enzymes responsible for elongation of amylose and amylopectin chains. These enzymes transfer glucosyl residue from ADPglucose to the non-reducing end of a preexisting glucan. It was discussed whether SSs are able to synthesize glucan chains without a glucan primer . Howeverss4 mutant reveals mostly one starch granule per chloroplast . Thus, immature leaves of ss4 have no detectable starch granules in contrast to mature leaves .dpe2/phs1 revealed mostly one starch granule per chloroplast when grown under 12 h light and 12 h dark conditions, similar to ss4. However, expression of SS4 was unaltered in this mutant . ptst2/ptst3 revealed also a high accumulation of ADPglucose (with ∼30-fold compared to wild type) that was twice of that of ptst2 (ss4/ss3 showed a similar high accumulation of ADPglucose as ss4 (with ∼100-fold increase compared to wild type). dpe2/phs1 and especially dpe2/phs1/ss4 also revealed mostly one starch granule per chloroplast but showed only a very moderate increase in ADPglucose content . Furthermore, dpe2/ss4 and phs1/ss4, each having one starch granule per chloroplast, revealed totally different ADPglucose values . Under continuous light, an increase in the granule number per chloroplast was observed for ss4, dpe2/phs1, and dpe2/phs1/ss4. But these observations were not accompanied by a reduction in the ADPglucose amounts  was observed in mex1/phs1 compared to wild type  the granule was large and spherical, whereas when 5-7 starch granules were monitored (under continuous light) the starch granule morphology was similar to wild type . The initiation of starch seems to depend on a chloroplastidial maltodextrin metabolism. At least PTST2 and PHS1 seem to be involved in this process. As this maltodextrin metabolism can be influenced by the ongoing starch turn-over and distinct enzymes of the starch metabolism  the involvement of further proteins/enzymes is highly likely. In yeast cells, starch-like synthesis was recreated by introduction of Arabidopsis SSs without plant enzymes related to synthesis of a primer, thus the intrinsic primers of yeast seems to be sufficient for the formation of starch like polyglucans. Furthermore, the experiments again showed that SS4 can be substituted by other SSs, as starch-like polyglucans were observed not exclusively when SS4 was introduced (In the last years we got more insights into starch granule biosynthesis. The appearance of starch granules is a rather complex mechanism influenced by various factors. According to this, it is very important to distinguish between the possibility to initiate starch and the formation of starch granules. It is quite clear that SSs, or more precisely SS4, is important in the formation of the typical number of starch granules per chloroplast in Arabidopsis 5-7, . Howevertroduced .All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.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.The reviewer NC and handling Editor declared their shared affiliation.