Bile acids (BAs) and glucocorticoids are steroid hormones derived from cholesterol that are important signaling molecules in humans and other vertebrates. Hydroxysteroid dehydrogenases (HSDHs) are encoded both by the host and by their resident gut microbiota, and they reversibly convert steroid hydroxyl groups to keto groups. Pairs of HSDHs can reversibly epimerize steroids from α-hydroxy conformations to β-hydroxy, or β-hydroxy to ω-hydroxy in the case of ω-muricholic acid. These reactions often result in products with drastically different physicochemical properties than their precursors, which can result in steroids being activators or inhibitors of host receptors, can affect solubility in fecal water, and can modulate toxicity. Microbial HSDHs modulate sterols associated with diseases such as colorectal cancer, liver cancer, prostate cancer, and polycystic ovary syndrome. Although the role of microbial HSDHs is not yet fully elucidated, they may have therapeutic potential as steroid pool modulators or druggable targets in the future. In this review, we explore metabolism of BAs and glucocorticoids with a focus on biotransformation by microbial HSDHs. Steroid hormones are signaling molecules derived from cholesterol that include glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, and bile acids (BAs) . SteroidHydroxysteroid dehydrogenases are nicotinamide adenine dinucleotide (phosphate) (NAD(P)(H))-dependent oxidoreductases that catalyze the reversible conversion of hydroxyl groups to keto groups on steroids . HSDHs aHydroxysteroid dehydrogenases are found in both host and microbial genomes, although more is known about the physiological function of host hydroxysteroid dehydrogenases, which are typically abbreviated HSDs in literature. In this review, host hydroxysteroid dehydrogenases are denoted “HSD” while bacterial enzymes are denoted “HSDH”. Host HSDs are key enzymes in the biosynthesis of steroids in steroidogenic tissues . They alHost HSDs are druggable targets important in the treatment of endocrine-dependent disorders, including cancers . Host-asHydroxysteroid dehydrogenases belong to one of the following three large and diverse protein superfamilies: short-chain dehydrogenase/reductase (SDR), medium-chain dehydrogenase/reductase (MDR), or aldo-keto reductase (AKR) ,14. ManyThe SDR superfamily is one of the largest, containing proteins spanning all three domains of life . SDR proThe MDR family is similar to the SDR family both in number of members and in function, although their structures have marked differences. MDR proteins contain Rossmann fold domains for NAD(P)(H) binding like SDRs, but they are ~350 residues long and many are metal-dependent . They arAKRs are NAD(P)(H)-dependent oxidoreductases acting on carbonyl groups or double bonds and are ~320 amino acids long. They are monomeric with diverse substrate recognition, including steroids, monosaccharides, and isoflavonoids. An ordered bi–bi kinetic mechanism has been shown for multiple AKR family members, where the cofactor is first to bind and last to leave . Most ha24 steroids that play an important role in host nutrition .,33.31,33(HSD3B7) ,37. Afte(HSD3B7) ,38,39. S-25 bond ,37. The nsferase ,40.24 BAs, C27 BAs, and C27 bile alcohols ClostridiB. desmolans and C cadaveris express 20β-HSDH [Bifidobacterium adolescentis [Clostridium scindens ATCC 35704 can convert cortisol to 20α-dihydrocortisol [C-20 reduced metabolites of cortisol have been observed in human urine, likely attributable to host enzymes that produce 20α- or 20β-dihydrocortisol and their derivatives ,187. How20β-HSDH , along wescentis . Additiocortisol . Thus, gEggerthella lenta (formerly Eubacterium lentum) [E. lenta 21-dehydroxylase has substrate specificity for 11-deoxycorticosterone, deoxycortisol, dehydrocorticosterone, and corticosterone [Human gut microbiota are also capable of 21-dehydroxylation of corticosteroids. 21-Dehydroxylase activity was first detected in  lentum) ,190. E. osterone ,192. Theosterone . Interesosterone  while thosterone .Host hydroxysteroid dehydrogenases have been established as important for biosynthesis and modulation of steroid hormones such as androgens, estrogens, and glucocorticoids for years . Since t20β-Dihydrocortisol is excreted in urine at rates comparable to that of free cortisol in healthy individuals ,187. UriStreptomyces hydrogenans [B. desmolans and C. cadaveris, organisms that were previously shown to have this activity in culture, have been identified [desE due to its involvement in the DesAB pathway and because it forms an operon with the desAB genes [B. desmolans and C. cadaveris are capable of cortisol side-chain cleavage, as well as 20β-oxidoreduction [B. desmolans ATCC 43058, which exhibits specificity for cortisol as substrate and is NAD(H)-dependent [Bifidobacterium scardovii ATCC BAA-773 and the urinary tract microbe Propionimicrobium lymphophilum ACS-093-V-SCH5 also express 20β-HSDH according to HPLC [P. lymphophilum has also been shown to encode desAB [desE in B. adolescentis strain L2-32 has been characterized. It is specific for cortisol and was crystallized in both the apo-form without any binding and the binary form with NADH bound at 2.2 and 2.0 Å, respectively [One of the first organisms studied expressing 20β-HSDH activity was the soil microbe rogenans . This enrogenans . More reentified ,184. TheAB genes ,184. Boteduction ,184. 20βde desAB . Additioectively .C. scindens along with steroid-17,20-desmolase activity [C. scindens ATCC 35704 was initially characterized from cell extracts and shown to be NAD(H)-dependent [desAB and desC, encoding steroid-17,20-desmolase and 20α-HSDH, respectively [C. scindens ATCC 35704 20α-HSDH was crystallized for further characterization of the enzymatic mechanism. Hybrid quantum mechanical molecular modeling simulations revealed a reaction mechanism involving a multistep proton relay, which was validated by site-directed mutagenesis experiments of active site and substrate binding residues [C. scindens ATCC 35704 20α-HSDH within the National Center for Biotechnology Information (NCBI) database uncovered two additional organisms, Denitratisoma oestradiolicum DSM 16959 and Intestinibacillus sp. Marseille-P4005, which may express 20α-HSDH, although activity has not yet been confirmed [Thus far, 20α-HSDH activity seems to be significantly less widespread than 20β-HSDH, with only one organism shown to exhibit the activity ,197. Redactivity . 20α-HSDependent . The genectively . Recentlresidues . An aminonfirmed .Clostridium scindens ATCC 35704 and the urinary microbe Propionimicrobium lymphophilum ACS-093-V-SCH5 can side-chain cleave both cortisol and glucocorticoid drugs [Microbial 20α- and 20β-HSDH may be important regulators of the steroid-17,20-desmolase/DesAB pathway. By competing for cortisol as substrate with DesAB, they would decrease the potential for 11β-OHAD formation. Microbial steroid-17,20-desmolase activity may be one of the important missing enzymes contributing to peripheral 11β-OHAD production in the body . Recent id drugs , suggestid drugs . This haid drugs . FurtherOverall, both host and microbial HSDHs play pivotal roles in BA and glucocorticoid metabolism. Research on the importance of HSDH-derived BAs on host physiology is in its infancy. However, the immense diversity of these BA metabolites, due to combinations of HSDH activity, means that the gut harbors a multitude of potential candidates for host receptor signaling. Gut microbial cortisol HSDHs are likely important regulators of steroid-17,20-desmolase activity, although additional research is needed to ascertain the physiological significance of 20α- and 20β-HSDH products. New microbial HSDHs are continually being discovered and characterized, which will allow mechanistic study of their impacts in disease models.Microbial HSDHs may have potential as therapeutic modulators in diseases such as colorectal cancer, liver cancer, castration-resistant prostate cancer, and polycystic ovary syndrome. However, to work toward therapeutics, we must first connect HSDH function to host phenotypes through mechanistic experiments, such as gnotobiotic animal studies ,201. Suc


Whittleia retiella  is a threatened salt marsh species of the bagworm moth family Psychidae. For its preservation it is necessary to develop efficient tools to survey its distribution and habitat requirements in order to use appropriate conservation methods. Such tools may be pheromone-based monitoring systems, which have documented efficacy in establishing the occurrence of cryptic insect species in nature. By using gas chromatography combined with electroantennographic detection (GC-EAD), we found two compounds in female W. retiella headspace samples and whole-body extracts that elicited electrophysiological activity in male antennae. Gas chromatograpy coupled with mass spectrometry (GC-MS) operating in electron impact (EI) mode and comparison of the analytical data with those of synthetic reference compounds showed the chemical structures of these putative pheromone components to be (1S)-1-methylpropyl (5Z)-dec-5-enoate and 1-methylethyl (5Z)-dec-5-enoate. Field assays using baits loaded with synthetic compounds revealed that conspecific males were attracted to (1S)-1-methylpropyl (5Z)-dec-5-enoate alone or in combination with 1-methylethyl (5Z)-dec-5-enoate, whereas 1-methylethyl (5Z)-dec-5-enoate neither attracted nor repelled males in the field assays when tested alone. This study shows the potential of using (1S)-1-methylpropyl (5Z)-dec-5-enoate for monitoring W. retiella to gather more detailed information about the geographic distribution and habitat needs of this rare moth.The online version of this article (10.1007/s10886-020-01145-x) contains supplementary material, which is available to authorized users. Insect sex pheromones have traditionally been studied for the purpose of pest control . Adult males of W. retiella have a wingspan of 7.5–8.5 mm, whereas females are wingless with rudimentary pairs of legs  to feed on and eventually pupate. This rare halobiotic moth flies at the end of April or the beginning of May -1-methylpropyl (5Z)-decenoate and 1-methylethyl (5Z)-decenoate, released by female W. retiella, which are sex pheromone component candidates based on the electrophysiological and behavioural activity they trigger in conspecific males. Syntheses and analytical data of these compounds and compounds used for reference are also presented in this work.In the present study, we identify two compounds, .Specimens were collected in 2010 and 2012 from the salt marshes and grasslands by the North Sea coast at Westerhever, Nordfriesland, Germany. Larval and pupal cases made of small fragments of dry grass spun together were collected, and larvae were sorted by weight and separated by sex. Generally, females were heavier than males. Larvae were kept in Petri dishes together with living plants of -1 for several hours (range: 17–24 hr) during 4 consecutive days, using a KNF NMP830KNDC air pump . Long and sequential collection times were justified since the calling time of the females during the day was unknown. The PQ filters were changed every day. After collection, the filters were extracted with 2 × 150 μl redistilled n-hexane, which was collected in 1.5 mL glass vials and stored at −20 °C until further use. In addition, 15 females were removed from their old pupal cases and extracted for 30 min in 1 mL n-hexane, which was later concentrated to 300 μl under N2 and stored in 1.5 mL glass vials at −20 °C until further use.Between 6 and 42 adult females (less than one week old) were pooled into 20 mL glass scintillation vials with two holes made in the lids. One hole was fitted with an activated charcoal filter through which air entered the vial, while the other was fitted with an odour collection filter made from Teflon tubing (60 mm with I.D. 3.0 mm O.D. 4.0 mm) and containing 25 mg Porapak Q (PQ) mesh size 80/100  as adsorbent material. Air was pumped through the vial and the collection filter at 0.2 L/min−1, and the injector temperature was 250 °C. The GC effluent was split at a 1:1 ratio between the flame ionisation detector (FID) and the antennal preparation. The oven temperature was maintained at 50 °C for 1 min after injection and then increased to 210 °C at a rate of 10 °C·min−1 and a final hold of 10 min. Additional analyses were performed on an HP 6890 N gas chromatograph equipped with a non-polar HP-5 column . For these analyses, an antenna was mounted between two glass capillaries filled with Beadle-Ephrussi ringer solution and connected to a pre-amplifier (10 × gain) (Syntech). The GC effluent passed through a Gerstel ODP-3 transfer line, which was temperature adjusted in tandem with the GC oven temperature. It was mixed with charcoal-filtered and humidified air before reaching the antenna, placed 0.5 cm apart from the glass tube outlet. Hydrogen was used as carrier gas at an average linear flow of 45 cm·s−1 and an injector temperature of 225 °C. The GC effluent was split 1:1 in a Gerstel 3D/2 low dead volume fourway-cross  with 4 psi of nitrogen added through the fourth connection. The oven temperature was maintained at 50 °C for 3 min after injection and increased by 10 °C·min−1 to 230 °C, with a final hold of 10 min. All recordings were performed using the GC-EAD Pro ver. 4.1 software (Syntech).In order to screen for putative pheromone components, headspace samples and whole-body extracts of female moths (less than one week old) were analysed using combined gas chromatography and electroantennographic detection (GC-EAD) (Larsson and Svensson −1 (inlet pressure 172 kPa) at an injector temperature of 225 °C. Analyses started at 30 °C with a 3 min hold and programmed to 250 °C at a rate of 8 °C·min−1. The transfer line temperature started at 150 °C and was then set to remain 10 °C above the oven temperature. Sample injections of 2 μL of extracts were made manually in splitless mode (split vent opened after 0.5 min). Headspace samples were also analysed using an HP 5890II gas chromatograph linked to an HP 5972 mass spectrometer equipped with an HP-INNOWax column . Helium was used as the carrier gas at a velocity of 40 cm·s−1 and an injector temperature of 220 °C. After injection in splitless mode, the oven temperature was maintained at 50 °C for 2 min and then increased by 10 °C·min−1 to 250 °C, with a final hold for 10 min.Tentative identification of the female-produced, antennally active compounds was performed by GC-MS with column types matching the columns used for GC-EAD recordings. An HP-5 ms capillary column  was mounted in a GC 6890 N model GC interfaced to a 5975 mass selective detector . The carrier gas was helium with a constant flow rate of 1.8 mL·min−1), and the split/splitless injector was operated in splitless mode for 2.5 min at 250 °C. The transfer line was maintained at 250 °C, and the MS source was set to 230 °C. Two different capillary columns were used: i) a 30 m × 0.25 mm I.D. × 0.25 μm film thickness fused silica capillary column VF-23 ms (Agilent Technologies); temperature program from 50 °C to 230 °C at a rate of 10 °C·min−1, then held at 230 °C for 10 min and, ii) a 30 m × 0.25 I.D. × 0.25 μm film thickness fused silica capillary column CP-WAX 58 (Agilent Technologies); initial temperature 50 °C, held for 2 min, then increased to 100 °C at a rate of 10 °C·min−1, and then held at 100 °C for 35 min.More detailed investigations of insect extracts were performed on a Hewlett-Packard 6890 N GC using a HP 5973 mass spectrometer operating in electron impact mode . Helium was used as the carrier gas (flow rate = 1.0 mL·min−1). The split/splitless injector was operated in splitless mode and set at 250 °C. The transfer line was maintained at 250 °C, and the MS source was set to 230 °C. The GC was equipped with a fused silica capillary column (30 m × 0.25 mm ID × 0.25 μm film thickness) coated with Cyclosil-B (Agilent Technologies). Separations were performed using a temperature program starting at 50 °C (2 min hold), then increased to 250 °C at a rate of 5 °C·min−1.For the separation of enantiomers, a Hewlett-Packard 6890 N GC linked to a HP 5973 mass spectrometer was used with helium as the carrier gas . Compounds were analysed by comparing the mass spectra of natural products with data reported in the NIST MS 2.0 mass spectral library, using reverse and forward match values. The postulated structures were also confirmed by comparing retention times and mass spectra of the natural compounds with those of synthetic samples.E)-Dec-4-enoic acid was obtained from Penta International Corporation company, (4Z)-dec-4-en-1-ol and (5E)-dec-5-en-1-ol were purchased from TCI Europe, and (5Z)-dec-5-en-1-ol was obtained from Bedoukian Research Inc., Danbury, CT, USA. Candida rugosa lipase (CRL), (5-carboxypentyl)triphenylphosphonium bromide, and racemic butan-2-ol and propan-2-ol were purchased from Sigma-Aldrich, whereas (2R)-butan-2-ol and (2S)-butan-2-ol were obtained from Alfa-Aesar . All chemicals and solvents were of the highest available purity i.e. >95%.(4Z)-Dec-4-enoic acid, (5E)-dec-5-enoic acid, and (5Z)-dec-5-enoic acid were prepared via oxidation of (4Z)-dec-4-en-1-ol, (5E)-dec-5-en-1-ol, and (5Z)-dec-5-en-1-ol, respectively, according to Bowden , and a catalytic amount of sulfuric acid (2 M) were mixed in a round-bottom flask. The contents were heated on a boiling water-bath and stirred for different lengths of time. For details of the syntheses and analytical data see S)-1-methylpropyl (5Z)-dec-5-enoate and (2R)-1-methylpropyl (5Z)-dec-5-enoate. (5Z)-Dec-5-enoic acid and (2R)-butan-2-ol as well as (2S)-butan-2-ol were used according to Sabbani et al.  and the hexane allowed to evaporate in a fume hood after being partially adsorbed into the septum. Two sets of baits were prepared (Table Z)-dec-5-enoate. The second set, prepared in 2016, included a more limited number of blends comprising only the (S)-enantiomer. Each bait type was individually marked with a felt-tip pen. Baits were stored individually in the freezer when not in use and were brought out in the field for short periods (a few hours per day) during approximately one week during each season over several years.Lures with synthetic pheromone candidate compounds were prepared by dissolving known amounts of compounds by weight in redistilled W. retiella to synthetic baits were performed in 2015–2018 at several localities in Denmark in biotopes corresponding to preferred habitats of the species . Observations of flying W. retiella at these localities are rare, and populations were expected to be very small. Therefore, no attempts were made to trap any insects with baits, but were based on observations of free-flying males approaching the different lures. Attempts to observe flights and attraction of W. retiella were limited to days of suitable weather with sun and limited wind during the expected short flight season in May–June. Behavioural observations of male attraction were performed by placing a series of rubber septa on the ground with individual septa at a distance of 50–100 cm apart. Each septum was marked with an individual number or letter with a felt-tip pen, but the content of each septum was unknown to the observer. At each visit, the septa were displayed from one to several hours. Flying males approaching the septa were counted on each occasion, and their flight patterns with repeated approaches to individual septa were noted.Attempts to observe the attraction of male W. retiella when stimulated with female-produced compounds collected by headspace sampling or whole-body extraction. The GC-EAD analysis on a polar column (HP-INNOWax) showed that two compounds (X and Y), sampled with both methods, elicited electrophysiological activity in male antennae, indicating that they are candidates as sex pheromone components of W. retiella  of the two compounds. The observed losses of 42 amu from M+ = 212 and 56 amu from M+ = 226 produced the same fragment of m/z 170 for both X and Y . As seen in Fig. 3 ms. As Z)-tetradecenoate -dodecenoate -C12-precursor to (5Z)-decenoic acid. Consequently, we synthesised 1-methylpropyl (5Z)-dec-5-enoate, and found that its retention time matched that of Y. Nevertheless, to be on the safe side, we synthesized the (5E)-isomer as well as E/Z-isomers of esters carrying the double bond in positions 4 or 6 and compared the retention times of these compounds with that of Y  and m/z 152 (ketene fragment) -dec-5-enoate (2d’) coincided with that of compound Y in the female extract, whereas the other candidate compounds, including 1-methylpropyl (5E)-dec-5-enoate (2d), did not and were excluded from the investigation. As the mass spectra of 1-methylpropyl (5Z)-dec-5-enoate and that of the natural product Y were very similar, it was concluded that 1-methylpropyl (5Z)-dec-5-enoate represented compound Y, eluting at 8.27 min -dec-5-enoate has a chiral center and thus exists as two enantiomers -1-methylpropyl (5Z)-dec-5-enoate is noted -1-methylpropyl (5Z)-dec-5-enoate.The retention time of Y was compared with retention times of a reference ester mixture of known enantiomeric composition as proven by enantioselective GC Fig.  B. By mited Fig. , which sZ)-dec-4-enoate (3c’) and 1-methylethyl (5Z)-dec-5-enoate (3d’) were almost identical, and the retention time of both compounds nearly coincided with that of X, but the the latter eluted slightly closer -dec-5-enoate was regarded to be the electrophysiologically active component X, eluting at 7.38 min.For structure elucidation of X, similar GC-MS investigations as for Y were performed using corresponding 1-methylethyl decenoates as reference compounds Fig. . It turnser Fig. . Howevern-hexane. Visits occurred to all baits loaded with the main component (1S)-1-methylpropyl (5Z)-dec-5-enoate at 100 μg, either as a single component or with additions of 1-methylethyl (5Z)-dec-5-enoate at doses from 10 to 50 μg. At two of the observation periods, visiting males also approached a bait containing 100 μg racemic 1-methylpropyl (5Z)-dec-5-enoate and 30 μg 1-methylethyl (5Z)-dec-5-enoate. No attraction was observed to baits containing (1R)-1-methylpropyl (5Z)-dec-5-enoate alone or in combination with 1-methylethyl (5Z)-dec-5-enoate. No flying males were ever observed apart from those attracted to the baits on display, and no other moth species were observed approaching the baits. Although bioassays often lasted for the better part of a day, all observations of males attracted to baits occurred between 12.00 and 14.00.In 2017, three tests at Mulbjerge/Dokkedal revealed attraction of several males to the baits . In 2018, one test at Mulbjerge/Dokkedal (4: May 14) brought two males approaching the baits of series 1, although both series were used, and one test at Kongenshus Hede (5: May 15) brought ten males using only the bait series 2. Males were clearly attracted to the baits and preferentially visited a subset of the baits on display (Table W. retiella consists of (1S)-1-methylpropyl (5Z)-dec-5-enoate, and possibly also 1-methylethyl (5Z)-dec-5-enoate. The field bioassays revealed a preference of males towards baits containing (1S)-1-methylpropyl (5Z)-dec-5-enoate (Table R)-1-methylpropyl (5Z)-dec-5-enoate, as the main constituents (Table S)-1-methylpropyl (5Z)-dec-5-enoate were among the most preferred baits at two occasions with the highest numbers of visiting males, suggesting that 1-methylethyl (5Z)-dec-5-enoate is not behaviourally active, but there is a possibility that the presence of the latter compound among adjacent baits may have contributed to the attraction. There is a recorded case where male moths, once activated by the complete blend of a female pheromone, display lower selectivity during the subsequent search for the pheromone source -1-methylbutyl decanoate as sex pheromone of Thyridopteryx ephemeraeformis (Haworth), and the same compound and four additional esters were found to constitute the sex pheromone of Oiketicus kirbyi (Guilding) . More recently, Gries et al. (Clania variegata (Snellen), and the most abundant compound was identified as (1S)-1-ethyl-2-methylpropyl 3,13-dimethylpentadecanoate. The use of long-chain fatty acid esters as sex pheromone components outside the bagworm family seems to be restricted, and so far only revealed for species belonging to the subfamily Procridinae within the Zyganidae, e.g. (1S)-1-methylpropyl (7Z)-tetradecenoate in Theresimima ampellophaga Bayle-Barelle -1-methylpropyl (7Z)-dodecenoate and (1R)-1-methylpropyl (9Z)-tetradecenoate in Illiberis rotundata Jordan -1-methylpropyl (5Z)-dec-5-enoate. The species has been recorded in Great Britain, France, Germany, Belgium, Holland, Denmark, and Sweden -1-methylpropyl (5Z)-dec-5-enoate applied on septa and the role of 1-methylethyl (5Z)-dec-5-enoate as potential pheromone component to improve the attraction of males to baits in the field.Monitoring with an efficient sex pheromone is a valuable conservation tool when gathering information about the geographic distribution and habitat requirements of rare and threatnend insect species. Detection of ESM 1(DOCX 24 kb)


For people with idiopathic generalized epilepsy, functional networks derived from their resting-state scalp electrophysiological recordings have shown an inherent higher propensity to generate seizures than those from healthy controls when assessed using the concept of brain network ictogenicity (BNI). Herein we tested whether the BNI framework is applicable to resting-state magnetoencephalography (MEG) from people with juvenile myoclonic epilepsy (JME).The BNI framework consists in deriving a functional network from apparently normal brain activity, placing a mathematical model of ictogenicity into the network and then computing how often such network generates seizures in silico. We considered data from 26 people with JME and 26 healthy controls.We found that resting-state MEG functional networks from people with JME are characterized by a higher propensity to generate seizures  than those from healthy controls. We found a classification accuracy of 73%.The BNI framework is applicable to MEG and was capable of differentiating people with epilepsy from healthy controls.The BNI framework may be applied to resting-state MEG to aid in epilepsy diagnosis. The diaThe low sensitivity of IEDs results from IEDs being typically rare events. This may be a consequence of their sources being deep in the brain and/or the extent of cortex involved in epileptic activity being undetectable at the scalp surface . Consequin silico  at a sampling rate of 600 Hz. We obtained approximately 5 minutes of MEG recordings per individual. The participants were instructed to sit steadily in the MEG chair with their eyes focused on a red dot on a grey background. Each individual also underwent a whole-brain T1-weighted magnetic resonance imaging (MRI) acquired using a General Electric HDx 3 T MRI scanner and an 8-channel receiver head coil  with an axial 3D fast spoiled gradient recalled sequence .To assess the presence of artefacts and interictal spike wave discharges, the MEG data was divided into 2 s segments and each segment was visually inspected. Artefact-free segments were identified and re-concatenated for each individual. We thus obtained concatenated recordings with a variable length ranging from 204 s to 300 s, and to avoid the potential impact of different recording lengths on our analysis, we only considered the first 200 s of each recording for every individual. The pre-processed data were then filtered in the alpha band (8–13 Hz) and down-sampled to 250 Hz. We focused on the alpha band because it has been shown to be the most informative for differentiating people with epilepsy from healthy controls .2.3http://www.ru.nl/neuroimaging/fieldtrip). We mapped the source signals into the 90 brain regions of the Automated Anatomical Label (AAL) atlas , and we obtained a volume conduction model from the MRI scan using a semi-realistic model . To recoL) atlas . For morL) atlas .2.4We divided the 200-s-long source reconstructed MEG recordings into 10, non-overlapping, 20 s segments. The choice of segment length was motivated by previous studies that aimed to distinguish people with epilepsy from controls using resting-state scalp EEG . For eac2.5To study the inherent propensity of a MEG functional network to generate seizures, we placed a canonical mathematical model of ictogenicity at each network node, i.e. at each of the 90 brain regions represented in the functional network .2.6The mathematical model allowed us to generate synthetic brain activity which fluctuated between the resting and the seizure states. To quantify this activity, we used the BNI . This finding confirms our hypothesis that resting-state functional networks from people with epilepsy have a higher propensity to generate seizures than those from healthy controls. Note also that for each individual, we observed that We considered resting-state MEG recordings from 26 people with JME and 26 healthy controls. To test whether The results in 4To date, the BNI framework has proved to be valuable for epilepsy diagnosis using scalp EEG in IGE , assessmResting-state MEG functional networks have been previously shown to be altered in people with epilepsy relative to healthy controls . For exaWe acknowledge that our study has some limitations. First, in order to truly test how MEG-based predictions compare to scalp EEG-based predictions, we would need both MEG and EEG data collected from the same participants. Future work should assess whether predictions based on both data modalities would deliver equivalent individual classification. Second, people with JME were taking anti-seizure medication, which may have potentially reduced the BNI in some JME individuals, making them indistinguishable from healthy individuals. Future studies should consider newly diagnosed drug-naïve individuals. This may be particularly important to also control for the effect of epilepsy duration and seizure frequency on BNI. Our results suggest that individuals with longer epilepsy duration and higher seizure frequency were more likely to be characterized by high BNI. On one hand this is an expected observation, i.e. BNI should be higher for individuals more prone to seizures and also those for which a longer disease may have had an impact on resting-state functional connectivity. On the other hand, these were individuals for which diagnosis could be less challenging. Third, we focused our analysis on differentiation of people with JME from healthy controls. We therefore cannot exclude the possibility that our findings are specific to JME. More comprehensive datasets will be needed to explore whether our findings generalize to other types of epilepsy.5By extending the application of the BNI framework to MEG, our results demonstrate that the BNI can be useful to interrogate different data modalities beyond EEG. We showed that resting-state MEG from people with JME is characterized by higher BNI than that from healthy controls. Our results suggest that BNI applied to resting-state MEG may aid in the diagnosis of JME.JT is co-founder and Director of Neuronostics.


Since its discovery almost 70 years ago, the hologram has been considered to reproduce the most realistic three dimensional images without visual side effects. Holographic video has been extensively researched for commercialization, since Benton et al. at MIT Media Lab developed the first holographic video systems in 1990. However, commercially available holographic video displays have not been introduced yet for several reasons: narrow viewing angle, bulky optics and heavy computing power. Here we present an interactive slim-panel holographic video display using a steering-backlight unit and a holographic video processor to solve the above issues. The steering-backlight unit enables to expand the viewing angle by 30 times and its diffractive waveguide architecture makes a slim display form-factor. The holographic video processor computes high quality holograms in real-time on a single-chip. We suggest that the slim-panel holographic display can provide realistic three-dimensional video in office and household environments. Holographic displays that are both compact and produce realistic holograms without eyestrain are still difficult to realize. Here the authors implement a steering-backlight unit and a holographic video processor to produce a realistic holographic display in a slim panel. When real objects and holographic images are located in the same space, they can be perceived without inhomogeneity. Figure 3. One cannot clearly see the fairy image and the real hand at the same time, and will experience visual fatigue caused by an accommodation–vergence conflict2. Therefore, holographic displays are essential parts of future video systems including interactive 3D user interfaces.A holographic display uses light diffraction to create three-dimensional (3D) images in space4. Recently, nanophotonics5 and metasurfaces6 are also used to reconstruct static holograms. However, those holographic media are non-updatable or have a limited updating frequency7, causing a fundamental limitation for generating dynamic holograms. By using a spatial light modulator (SLM), which directly modulates the wavefront of light, it is possible to update holograms at video rate. Many researches have demonstrated holographic video systems so far, including anisotropic leaky mode8 and eye tracking9. The recent study on a holographic display was reported, including a full-colour 3D computer-generated hologram (CGH) calculated in real-time, and a focus-adjustable reconstructed image10. However, it has been demonstrated that vertical parallax only holograms using a bulky optical system. It is expected to require quite an amount of time to reach the mobile holographic video for practical applications.Because of its advantage in 3D image reproduction, the technology for static holograms is quickly developed to a high standard by using hologram recording materials such as silver halide and photopolymer16.To build a mobile holographic video display, the following barriers need to be overcome. First of all, there is the limitation of the space-bandwidth product (SBP), which determines both the size of holographic image and the viewing angle. The static holographic media can produce large holographic images with a large viewing angle, because the information of hologram is recorded in the sub-wavelength density and it can be recorded on the large size film. However, SBP is limited by the pixel size and the number of pixels when an SLM is used as a dynamic holographic medium. The SBP of the currently available SLM is generally a few hundred times less than the SBP of the static holographic media. It means that only a small size or a narrow viewing angle dynamic hologram can be realised. Second, to generate a large coherent backlight, complicated optical components and a considerable space is required for the manipulation of light. It is difficult to realise a holographic video display as slim as flat-panel displays commercialised nowadays. Last, the calculation of hologram in real-time typically requires huge computational cost, and the amount of computation increases as the SBP increases. Several studies have been carried out to optimise algorithms and to increase computation speed, but they still require clustered processors or high-performance parallel processing systems to calculate high-quality hologram at video frame rateIn this research, a real-time interactive slim-panel holographic video display is demonstrated for the first time. It resolves all the above issues of low SBP, bulky optical system, and enormous computational cost. To increase effective SBP  resolution can provide the viewing angle of 0.25° in 10-in. display or 30° in 0.1-in. display. For satisfying both large size and large viewing angle, the SBP should be expanded by increasing the number of pixels N. There are studies to increase the number of pixels N by tiling many SLMs17 or by increasing the resolution of a single SLM18. To achieve the 30° viewing angle in 10-in., 221-K horizontal resolution is required, which is ~100 times higher resolution than the FHD resolution. Apart from the difficulty in fabricating such an SLM, the computation of the CGH in real-time may not be feasible, because the total number of pixels increases ~13,000 times.In holographic display, SBP can be represented by the equation, In this research, we expand the effective SBP without increasing the number of pixels of the SLM. To overcome the small viewing angle of a large size SLM, we introduce S-BLU, which is able to tilt the angle of the backlight for reconstructing holograms as shown in Fig. W × θ = λ × N(1 + pSLM/pBD). The enhancement factor proportionally increases with the ratio of pixel pitches between SLM and BD. In our holographic video system, the pixel pitch of the BD, pBD, is designed as 2 µm while the pixel pitch of the SLM, pSLM is 58 µm. Then, the effective SBP is increased ~30 times without increasing the number of pixels in the SLM. Since the pBD can be reduced further independent to the SLM, using a BD is more scalable mean to expand SBP than directly increasing the number of pixels of the SLM.The BD, one of the key elements of S-BLU, plays the role of steering a beam. The BD consists of transmission phase arrays, which can provide the linear phase profile for tilting the transmission angle. By using the BD, an extra enhancement factor is added to the original SBP equation 19 and the C-BLU20 forms the uniform wavefront incident from the BD as shown in Fig. The S-BLU is the key component for expanding the viewing angle of holographic display. In Fig. In a typical beam steering, the maximum steering angle is reduced as the area of the light source increases due to the etendue. In our holographic video system, the etendue issue has been solved by using the waveguide structure of the C-BLU. Once the beam size of 14 mm × 140 mm is steered as wanted by the BDs, it is enlarged to 140 mm × 230 mm by duplicating inside of the waveguide without reduction of the maximum steered angle.D = 0.5 × L/tan(θ/2), where L is the horizontal length of the display and θ is the viewing angle. The minimum viewing distance is ~25 m when L is 22 cm  and θ is 0.25°. By using a lens of 1 m focal length, the viewing distance is reduced by 25 times. However, a conventional bulk lens is not appropriate for the slim-panel holographic video display in terms of its thickness and weight. The geometric phase lens (GPLs), which exploits the spatial anisotropy21, is implemented as a field lens. The diffraction efficiency of a GPL can reach 98% over the entire visible wavelength. The overall thickness of optical components including the GPL is 1 cm.One disadvantage of the small viewing angle is a long viewing distance where the diffracted lights from all pixels start to overlap. Thus, a lens is necessary to reduce the viewing distance by gathering those diffracted lights together. The minimum viewing distance is, 23. Layer-based method can generate high-quality holograms for real scenes. However, layer-based method using FFTs has been considered unsuitable for CGH hardware accelerators since FFT processors were difficult to build in large scale15. We built an IFFT-based holographic video processor , filter and scale unit (FSU), and 32 IFFT processors. The IFFT processors perform 2D IFFT computations required for the calculations of the optical field distribution on the eye lens and the hologram patterns for the SLM. The 32 IFFT processors can compute 32 1-D IFFTs at the same time. The 2D IFFTs are computed with the row–column method. The DPU prepares the data needed for the IFFT computations from various sources including the input RGB-D data. The FSU performs several filtering operations and miscellaneous functions. The FSU also performs pixel encoding, which converts a complex number into an integer, which is provided to the SLM.The holographic video processor uses fixed-point number representations for all data processing tasks including multiplications in the IFFT. The number of bits used to represent a number is kept at the lowest level. For example, when the Fourier coefficients are stored in the memory, only 32 bits are used to represent a complex number. The holographic video processor is built with a single-chip FPGA. The single-chip FPGA holographic video processor generates binocular holographic colour images with 3840 × 2160 pixels at the speed of 30 frames per second (fps).As the system bus, we used Advanced Microcontroller Bus Architecture Advanced Extensible Interface 4 (AMBA AXI4), which is widely used in smartphone application processors. This makes it easy to embed the holographic video processor into a smartphone application processor.The actual system implementation of the slim-panel holographic video display is shown in Fig. Two experiments are conducted to validate the dynamic colour holographic display. First, the accommodation effect is checked by placing voxels in the air with different depths. Second, a full-screen colour holographic video is played. Each scene consists of 8 million voxels and it is refreshed at 30 fps. The CGH is calculated by the single-chip holographic video processor from a 3D scenery source rendered in the Unity platform.Z-axis with different X–Y positions, so that overall shape forms the letter S, when they are seen at Z = 1 m position. By changing the focal plane of the camera at the distance of 0 and 0.3 m, different images are captured as shown in Fig. As shown in Fig. 25, the contrast ratio does not change regardless of the number of voxels in our case.As the depth of voxel increases, a minimum diameter of voxel slightly increases due to the finite spatial coherence of the backlight. The contrast of the letter S is ~200. Unlike the other SBP improving methodFigure In this paper, a novel slim-panel holographic video display is proposed mainly on a new S-BLU architecture and a single-chip real-time holographic video processor. The S-BLU expands the effective SBP by 30 times, while the holographic video processor calculates high-quality CGH at 30 fps. The overall system thickness is <10 cm. The thickness of the display parts is 1 cm. Based on the combination of these, we demonstrated the world’s first slim full-colour holographic video display using 10.1-in. conventional UHD LCD. Research is in progress to scale down the volume of the system suitable for mobile phones. The holographic video processor is also designed to be embedded in a mobile application processor. The proposed system will accelerate the adoption of the holographic video for mobile devices.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Video 1Supplementary Video 2


Plasmodium malariae infection are rare. This is the first systematic review and meta-analysis demonstrating the global prevalence and mortality of severe P. malariae infection in humans.Severe complications among patients with P. malariae infection cases in humans were retrieved from three public databases: PubMed, Scopus, and ISI Web of Science. The pooled prevalence estimate and 95% confidence interval (CI) of complications in patients with P. malariae malaria was analysed using the random-effects model provided in Stata software. The pooled odds ratio (OR) and 95% CI of severe malaria for P. malariae infection and Plasmodium falciparum infection were analysed using Review Manager software.The systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. All research articles published on the severity and mortality of P. malariae malaria. Out of 10,520 patients infected with P. malariae, the pooled prevalence estimate of severe P. malariae infection was 3% (95% CI 2–5%), with high heterogeneity (I2: 90.7%). Severe anaemia (3.32%), pulmonary complications (0.46%), and renal impairments (0.24%) were the most common severe complications found in patients with P. malariae infection. The pooled proportion of severe anaemia for P. malariae infection and P. falciparum infection was comparable among the four included studies . The pooled proportion of pulmonary complications was comparable between patients with P. malariae infection and those with P. falciparum infection among the four included studies . For renal complications, the funnel plot showed that the pooled proportion of renal complications for P. malariae infection and P. falciparum infection was comparable among the four included studies . The mortality rate of patients with P. malariae infection was 0.17% .Six studies were used to estimate the pooled prevalence of severe P. malariae infection developed severe complications, with a low mortality rate. Severe anaemia, pulmonary involvement, and renal impairment were the most common complications found in patients with P. malariae infection. Although a low prevalence and low mortality of P. malariae infection have been reported, patients with P. malariae infection need to be investigated for severe anaemia and, if present, treated aggressively to prevent anaemia-related death.This systematic review demonstrated that approximately two percent of patients with  Plasmodium species which can infect human beings, including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi [Anopheles mosquito [Malaria is a public health problem, with an estimated 219 million cases in 87 countries, and 435,000 malaria-related deaths were estimated in 2017 [P. falciparum and P. vivax [P. falciparum than P. vivax in Africa, while P. vivax is more prevalent than P. falciparum in South America [P. ovale infection is widespread principally in tropical Africa, whereas P. knowlesi infection occurs only in certain forested areas of Southeast Asian countries [P. malariae is frequently co-endemic with P. falciparum in sub-Saharan Africa, South America, Indonesia, Southeast Asia, and the western Pacific [P. malariae infections were mixed infections with P. falciparum [Most malaria diseases in humans are caused by P. vivax . Both sp America . P. ovalountries . P. mala Pacific . A previlciparum .Plasmodium species infection that develops rapidly and leads to death within hours or days [P. falciparum, while P. vivax or P. ovale can also induce severe complications resulting in death but less frequently than P. falciparum [P. malariae infection [P. malariae infection among patients using articles published in three research databases.Severe malaria is any complication of  or days , and the or days . Most selciparum . Severe nfection , includinfection –15, cerenfection , and pronfection . This isP. malariae cases in humans were retrieved from three public databases: PubMed, Scopus, and ISI Web of Science. The published articles were searched up to 31 December 2019. The search terms were “(severe OR complicated OR Complication) AND ” using Boolean operators “OR” or “AND”. EndNote software version X7  was used to process all references in the present study.The systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . All resP. malariae infection and any primary study published in the English language. Only articles reporting severe P. malariae mono-infection were included. Those included studies were used to analyse the pooled prevalence of severe P. malariae malaria. Malaria diagnosis was based on one of the following methods: microscopy, rapid diagnostic test (RDT), and polymerase chain reaction (PCR).The inclusion criteria were any study that reported severe P. malariae infection with no related topics/no complications, animal studies, drug studies, case reports and case series, editorials, letters, proceedings, short communications, experimental studies, malaria in pregnancy, co-infection with P. malariae and other Plasmodium species or other diseases, and review articles. Studies were also excluded if they reported incomplete data on severe P. malariae infection.Exclusion criteria were any study reporting Plasmodium species, were extracted into Microsoft Excel spreadsheets  for further analysis.Two reviewers (MK and KUK) selected the article independently. Any discrepancy between two reviewers about article selection was resolved by discussion or by requesting the opinion of the third reviewer (GDM). The data set of this systematic review and meta-analysis, including the name of the author(s), publication year, study period, age distribution, haemoglobin concentration, parasitaemia level, nephrotic syndrome, albuminuria, the number of all malaria cases for each species, mortality rate, and the number of severe cases of all P. malariae malaria were based on the WHO definition among the included studies [The criteria of severe  studies .A quality assessment was performed following the quality assessment tool developed by the Newcastle–Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses . The quaPlasmodium spp., were analysed using the frequency and percentage. Data in Microsoft Excel spreadsheets were transferred into Stata version 14  for calculating the pooled prevalence and 95% confidence interval (CI) of complications in patients with severe P. malariae malaria. The pooled prevalence estimate of severe P. malariae infection was calculated using the number of patients with severe malaria and the number of patients with P. malariae malaria. The pooled proportion of severe complications for P. malariae infection was calculated using the number of severe complications among patients with P. malariae, and the total number of severe complications among patients infected with all Plasmodium spp., which have been reported in the included studies. The estimation of the pooled prevalence was following a previous research guideline [P. malariae infection compared with non-malariae infection and were also determined using Review Manager (RevMan) . The degree of heterogeneity between the included studies was assessed using Cochran’s Q test and Moran’s I2 (inconsistency), with I2 values of more than 50% indicating substantial heterogeneity.Qualitative variables, including the number of patients with severity signs, the number of deaths, and the number of patients infected with uideline . The comP. malariae infection and to evaluate whether publication bias was the underlying cause of data heterogeneity in the included studies.Funnel plot analysis was used to demonstrate any symmetry in the funnel plots among patients with P. malariae, 89 book and book chapters, 24 conference papers, 39 editorials/letters/correspondence articles, 39 note articles, and 444 review articles. The full texts of a total of 326 articles were screened for eligibility, and 320 were excluded, including 81 non-severe P. malariae, 72 experimental studies, 62 non-P. malariae, 23 no full-text, 14 entomological studies, 13 mixed infection of P. malariae with other Plasmodium species, 11 Pregnant participants, 10 drug research and clinical trials, 7 animal studies, 7 non-English language, 7 case reports of severe P. malariae, 6 case reports of non-severe P. malariae, 5 case reports of non-P. malariae, and 2 knowledge assessment. Six articles were included in the final qualitative and quantitative analysis  developed severe P. malariae infection , P. vivax , P. ovale (1355 cases), and mixed infection . The crude severity rates of infections with other Plasmodium spp. were 4.57% P. falciparum (four studies), 1.26% P. vivax (five studies), 2.59% P. ovale (four studies), and 4.83% mixed infection. The number of severity signs of patients with P. malariae and other Plasmodium spp. infection are demonstrated in Table P. malariae infection . Severe anaemia was also the most common complication found in P. falciparum (3.86%), P. vivax (1.52%), P. ovale (0.66%), and mixed infection (4.73%). Pulmonary complications (0.46%) and renal impairment (0.24%) were the second and third most common complications found in patients with P. malariae infection, respectively.All six included studies reported a total of 10,520 patients with u et al. , while td et al. . Among tP. malariae infection and the total number of patients with P. malariae infection were used to estimate the pooled prevalence of severe P. malariae infection . The pooled prevalence estimate of severe P. malariae infection was 3% (95% CI 2–5%), with high heterogeneity , using random-effects analysis  [The number of patients with severe I 7–43%) .Fig. 2ThP. malariae, and the total number of severe complications among patients infected with all Plasmodium spp. were used to estimate the pooled proportion of severe complications for P. malariae infection in comparison to those with Plasmodium spp. . The pooled proportion of severe complications for P. malariae infection in comparison to those with Plasmodium spp. was demonstrated in Fig. P. malariae infection was 2% (95% CI 1–3%). The highest proportion of severe complications caused by P. malariae infection in comparison to other Plasmodium spp. was severe anaemia .The number of severe complications among patients with P. malariae infection and P. falciparum infection was comparable among the four included studies   [P. malariae infection was higher than that in P. falciparum infection in a study by Langford et al.  [The pooled OR of severe anaemia for 33–0.50) , and HwaP. malariae infection and those with P. falciparum infection among the four included studies   [P. malariae infection than in P. falciparum infection in the study by Chaparro et al.  [P. malariae infection and P. falciparum infection was comparable among the four included studies   [P. malariae infection than in those with P. falciparum infection in the study by Chaparro et al.  [The pooled proportion of pulmonary complications was comparable between patients with 03–0.55) , while p9–202.1) . For ren08–0.35) , while r94–69.1) .Fig. 5ThP. malariae infection was 0.17% (18 cases), as reported in the included studies by Hwang et al. [Plasmodium spp. reported by the included studies were P. falciparum , P. vivax , P. ovale , and 1 case of mixed infection (0.002%).The crude mortality rate of g et al. , and by g et al. . The cruAll six studies included in the present study were rated according to the NOS guidelines. Overall, five studies were rated good, with a maximum of nine stars, and one study was rated medium with eight stars because of their “Representativeness of the cases” . The rating details are provided in Table The funnel plot analysis to demonstrate the publication bias among the six included studies could not be performed, as the analysis required a minimum of 10 studies .P. malariae infection. The results demonstrated that the pooled prevalence estimate of severe P. malariae malaria was low (3%). These results were in accordance with a previous study indicating that severe P. malariae infection was a rare complication with less frequent occurrence in malarial patients [P. malariae infections is unknown. P. malariae is a slow-growing parasite infecting mature red blood cells [P. malariae is not the cause of malarial relapse from persistent liver-stage parasites, but the recrudescence of blood-stage parasites can persist for long periods without signs or symptoms. Even with appropriate treatment, chronic subclinical P. malariae infection can occur because of its extended prepatent period when the inadequate drug in the blood cannot eliminate newly emerging merozoites [P. malariae infections have no sign or symptoms of malaria, they have no need for health services during their asymptomatic infection [P. malariae infections visit the health service, P. malariae parasites are missed by light microscopy due to their low parasite density compared to that of P. falciparum parasites [P. malariae from other Plasmodium species using a microscopic method, and its prevalence may be underestimated, resulting in late disease presenting severe complications [P. malariae infection can easily be misdiagnosed when microscopy is used for detection and is often treated as a bacterial infection [P. malariae by microscopy in patients with mixed infection with P. falciparum in endemic areas where P. falciparum predominates is also difficult, as, in the area of malaria endemicity in Africa, infections of P. malariae can frequently be mixed with P. falciparum infections [P. malariae infection is rosette formation, i.e., the binding of two or more infected red cells to an uninfected red cell, which is associated with microcirculatory blood flow obstruction in P. falciparum infection and has been reported in patients with P. malariae infection [This is the first systematic review and meta-analysis demonstrating the pooled prevalence estimate of severe patients . The mecod cells . It can od cells . P. malarozoites , 29. As nfection . Even tharasites , 32. It ications , 33. In nfection . The detfections . Anothernfection .P. malariae infection, as reported by the included studies. The included study by Douglas et al. [P. malariae infection was lower than that in non-P. malariae infection. They indicated that P. malariae infection was associated a lower mean haemoglobin level than infection with other Plasmodium spp., [P. malariae infections and demonstrated that the mean haemoglobin concentration was lower (9.0 g/dl) in patients with P. malariae infection than in those with infection with all other Plasmodium species [P. falciparum infection [P. falciparum and potentially cause a rapid drop in haemoglobin [P. malariae infection might be due to prolonged erythrocyte destruction and bone marrow suppression with a minimal reduction of erythrocyte lifespan by a low parasitaemia level [P. malariae infection in the included studies were Highland Papuans, who are the ethnic group with the highest risk of severe anaemia caused by nutritional and haematological factors [P. malariae infections was similar to that for P. falciparum and P. vivax infections after adjusting for ethnicity in the multivariable model [The present systematic review and meta-analysis demonstrated that severe anaemia was the most common complication in patients with s et al. , demonstum spp., . The inc species . Neverthnfection . This camoglobin . Previouia level , 22, 34.P. malariae infection, the included study by Langford et al., clarified that most patients with P. malariae infections were aged more than 15 years, which was older than those with P. falciparum, P. vivax, and mixed infections, reflecting the low transmission intensity in Indonesia [P. malariae infections were aged less than 5 years (10.9%), the most vulnerable group affected by malaria [P. malariae infection. The mechanism of pulmonary complications in patients with severe P. malariae infection is unknown. Lung injury in P. malariae malaria might be caused by microvascular sequestration, similar to P. falciparum infection, which can increase vascular permeability, resulting in fluid loading and progression to pulmonary oedema [P. malariae cytoadhere to endothelial cells of humans.Regarding the age distribution of patients with ndonesia . A low n malaria . This syy oedema . NeverthP. malariae infection, with a mixed IgM and IgG immune complex located at the renal basement membrane [P. malariae infection has long been recognized as the causative agent of nephrotic syndrome among untreated P. malariae infections [P. malariae for neurosyphilis in the 1930s [P. malariae infections [P. malariae infections, the proportion of nephrotic syndrome was higher in P. malariae infection than in infections with the other Plasmodium species [P. malariae-induced nephrotic syndrome can lead to progressive renal failure, particularly in young adults [P. malariae-induced acute renal failure after treatment with quinine was reported previously. In this case, treatment with intravenous quinine and four rounds of renal dialysis improved the renal function of the patient [P. malariae infection in an individual with thalassemia major remained undiagnosed for several months, and the individual eventually developed acute renal failure [Renal impairment and nephrotic syndrome were commonly present in patients with membrane . P. malafections , 38–40. he 1930s . Neverthfections . Althoug species . Previoug adults , 43. P.  patient . An unus failure .P. malariae infection is infrequent and is caused by multiple susceptibility genes: genes related to inflammation, including tumour necrosis factor (TNF), interleukin-6 (IL-6) and IL-10, macrophage migration inhibitory factor (MIF), angiotensin-converting enzyme (ACE), and catalase; genes for coagulation factors, including plasminogen activator inhibitor (PAI)-1; fibrinogen; coagulation factors II, V, VII, and XIII; and genes related to innate immunity, including toll-like receptor (TLR)-2, TLR-4, TLR-5, mannose-binding lectin (MBL), interleukin 1 receptor associated kinase 1 (IRAK-1), cluster of differentiation-14 (CD-14), toll interleukin-1 receptor-associated protein (TIRAP), and Nf-κB inhibitor (IκB) [P. malariae infection [Severe malaria caused by or (IκB) . In addinfection .P. vivax malaria prevalence globally. Second, although severe P. malariae infection was associated with a high burden of severe anaemia, the quantitative analysis of haemoglobin concentrations in P. malariae infection could not be performed, as it was reported quantitatively only in the included study by Douglas et al. [P. malariae infections with nephrotic syndrome. Fourth, the age distribution of patients with P. malariae infections could not be analysed, as only the included study by Langford et al. reported the age distribution of patients with P. malariae infections. Fifth, the parasitaemia level related to severe P. malariae infection could not be analysed, as only the included study by Bottieau et al. [P. malariae infection, those with P. malariae infection need to be investigated for anaemia and, if present, treated aggressively to prevent anaemia-related death.The present systematic review and meta-analysis had several limitations. First, the number of included studies was limited, which might affect the pooled estimate of severe s et al. . Third, s et al.  reportedu et al.  reportedP. malariae infection. Severe anaemia, pulmonary complications, and renal impairment were the common complications found in patients with P. malariae infection. Despite the low prevalence and mortality of P. malariae infection, infected patients need to be investigated for severe anaemia and, if present, treated aggressively to prevent anaemia-related death.This systematic review demonstrated the low prevalence and low mortality of severe malaria among patients with Additional file 1. Demographic and laboratory data.Additional file 2. Data for calculate the pooled prevalence estimate of severe P. malariae infection.Additional file 3. Data for calculate the pooled prevalence estimate of complications for P. malariae malaria.


Rodents perceive the emotional states of conspecifics using vision. In the present study, we demonstrated that exposure to the video-recorded distress of conspecifics induces stress responses in male C57BL/6J mice. A single exposure to a video-recorded scene of the social defeat stress (SDS) increased plasma corticosterone levels in these mice. This physiological change was suppressed by blocking the visual information, suggesting that vision plays a crucial role in inducing stress responses. Furthermore, after exposure to the video, there were increased numbers of c-Fos-positive neurons in the anterior cingulate cortex and other brain areas that are associated with the negative valence and empathy systems, but not in the regions related to the pain signaling. In addition, repeated exposure to SDS videos induced an apparent reduction in reward sensitivity in the sucrose preference test, but did not affect avoidance behaviour in the social interaction test or immobility behaviour in the forced swim test. Reduced reward sensitivity in mice reflects anhedonia, which is a core symptom of depression in humans. Our video SDS model therefore provides a unique opportunity to not only understand the mechanisms underlying stress-induced anhedonia, but also to screen effective candidate molecules for stress-related disorders with greater reproducibility. Visually broadcast news reports of disasters  or unpleasant situations  may also hurt many people simultaneously. A recent study of the Boston Marathon bombings demonstrated that indirect exposure through the media resulted in higher acute stress symptom scores than direct exposure3, suggesting that indirectly experiencing stressful stimuli has more of an impact on mental and physical health than was originally thought.Exposure to traumatic events, regardless if it is direct or indirect, causes severe mental damage to individuals and may be a risk factor for developing post-traumatic stress disorder5. In these studies, animals witnessing the stressed conspecifics display stress-related behavioural and physiological changes that are similar to those of actually stressed animals10. We have also recently reported that the emotional stress caused by witnessing SDS induces an apparent reduction in reward sensitivity, while experiencing SDS has no such effect10.Recent studies have successfully established animal models of vicarious stress, such as a witness foot shock model and a witness social defeat stress (SDS) model, by adding the concept of witnessing11 and mice14 display fear response such as freezing behaviours when they observe their conspecifics exposed to foot shock. In addition, rats that observe their cage-mates in pain display altered pain sensitivity15. It has also been reported that rats are able to discriminate between images of other rats’ painful and neutral facial expressions, and tend to avoid the former16. These findings suggest that the presentation of recorded negative visual images might stimulate negative emotions in rodents. However, it remains unclear whether rodents feel stressed when visually presented with recordings of conspecifics in fear and distress.Rodents perceive the emotional reactions of conspecifics using vision, and their behaviours are influenced by the emotional state of the conspecific. For example, ratsIn the present study, we therefore investigated whether exposure to the recorded distress of conspecifics is a stressful stimulus that induces physiological and behavioural changes in male C57BL/6J mice. First, we recorded SDS scenes and examined the effects of a single SDS video exposure on peripheral corticosterone levels to detect stress responses in mice. Then, we examined c-Fos expression in the brain as a marker for neuronal activity. Finally, we investigated the effects of 10 days of SDS video exposure on emotional behaviour using the social interaction test (SIT), elevated plus-maze test (EPM), forced swim test (FST), and sucrose preference test (SPT).t-test revealed a significant difference between the control and stress groups (t(8) = 2.343, p = 0.047). Mice exposed to the SDS video had significantly increased plasma corticosterone levels compared with controls    Fig. b. In addU test and unpaired t-test revealed significant differences between the stress and control groups in the anterior cingulate cortex  = 3.223, p = 0.007), prelimbic cortex  = 2.471, p = 0.028), nucleus accumbens  = 2.410, p = 0.026), bed nucleus of the stria terminalis  = 2.660, p = 0.019), hypothalamic paraventricular nucleus , supraoptic nucleus  = 2.209, p = 0.039), insular cortex  = 2.864, p = 0.013), basolateral amygdala  = 3.355, p = 0.005), central amygdala  = 3.349, p = 0.004), and ventral tegmental area  = 3.293, p = 0.005). There were no significant differences in the infralimbic cortex  = 1.557, p = 0.135), lateral septum  = 1.451, p = 0.162), periaqueductal gray  = 1.148, p = 0.264), and primary somatosensory cortex . Thus, SDS video exposure induced significantly increased neuronal activation in the ACC, PL, NAc, BNST, PVN, SON, IC, BLA, CeA, and VTA compared with the control group.We next investigated which brain areas were activated by a single SDS video exposure. Figure t(28) = 0.600, p = 0.553). Two-way ANOVA revealed a significant main effect of stress  = 4.926, p = 0.035) and day  = 13.273, p < 0.001), but there was no significant interaction effect between stress × day  = 1.929, p = 0.102). Repeated exposure to SDS videos led to significantly reduced weight gain in mice compared with the control group. In addition, there were no significant differences in the body weight on the day before the training session of the SPT between the groups  = 1.475, p = 0.151).To further investigate the effects of SDS video exposure, we repeatedly exposed the mice to SDS videos and assessed their daily body weight and emotional behaviours. Figure U = 72.000, p = 0.093). In addition, there were no significant differences in total distance between the groups, regardless of the presence or absence of the target mice (with target: t(28) = 1.736, p = 0.094; without target: t(28) = 1.584, p = 0.124)  = 0.106, p = 0.916). In addition, there were no significant differences in total distance and time in the closed arms between the groups  = 0.856, p = 0.399; time in the closed arms: t(28) = 0.816, p = 0.422)  = 1.430, p = 0.165). In addition, there were no significant differences in swimming and climbing counts between the groups  = 0.117, p = 0.907) . In addition, using a two-way ANOVA for water and sucrose consumption over 1 h in the SIT, we revealed that there were significant main effects of stress  = 7.653, p = 0.010) and taste  = 10.786, p = 0.003), and an interaction effect between stress × taste  = 7.520, p = 0.011). Post-hoc testing revealed no significant difference in water intake between the control and stress groups . However, the stress group had significantly lower sucrose intake compared with the control group . These findings indicate that repeated exposure to SDS videos induces significant decreases in sucrose preference in the SPT.Figure 18. These brain areas are reported to be involved in the negative valence system, which regulates fear, anxiety, aversion, depressive mood, and reward sensitivity22. Our findings suggest that indirect exposure to SDS via video is a stressor that drives the negative valence system in mice. On the other hand, an increase in c-Fos expression was not observed in the PAG and S1, which are related to the pain signalling. The absence of changes in such brain regions may represent the characteristics of the painless SDS video exposure.In Experiment 1, we demonstrated that male C57BL/6J mice have increased levels of corticosterone after a single exposure to SDS video. This result suggests that visual exposure to the SDS scene is a stressor that may activate the hypothalamic–pituitary–adrenal (HPA) axis in mice. In addition, single exposure to the SDS video led to activation (as measured by c-Fos expression) of a range of brain areas, including the ACC, PL, NAc, BNST, PVN, SON, IC, BLA, CEA, and VTA. This c-Fos expression pattern was similar to that reported after actual SDS10. It has been suggested that they recognise the emotional state of conspecifics in distress as their own stress, using visual, auditory, and chemosensory stimuli as cues. Interestingly, it has been reported that mice do not display stress-induced avoidance behaviour when visual information is blocked while they witness SDS9. Similarly, in the present study, stress-induced increases in corticosterone levels were suppressed when we blocked the visual information. Our result suggests that vision plays a crucial role in inducing stress-related various changes when exposed to stressful stimuli, either directly or indirectly.Rodents are reported to display various stress-related changes when directly witnessing SDS12. Therefore, it is possible that the dissociation between the direction of the image and the sound could activate the HPA. However, we have confirmed that the level of corticosterone was not increased by the exposure to pixelated SDS video, even though the direction of the image and the sound did not match. This finding suggests that the corticosterone response in the SDS video observer was not solely due to this dissociation. In addition, our result also suggests that the content, but not the intensity, of the video is important to activate the HPA axis.On the other hand, it is previously reported that an integration of auditory and visual information is important for vicarious fear23. It is therefore possible that the mice in the present study observed the facial expressions and defeat postures of their conspecifics in the SDS videos, and recognised them as stressful. We hypothesise that empathy may be a mechanism by which the mice were affected by the SDS videos and displayed stress-related changes. Empathy is one of the emotional processes by which individuals are affected by the state of another’s emotions, at least in humans24. Rodents are also known to display empathy-like behaviours14. For example, when mice observe conspecifics receiving electrical shocks, they exhibit freezing behaviours as if they themselves are receiving the shocks. In addition, witnessing another individual in pain activates the ACC neurons, which are the same neurons that experience self-pain at the single-cell level25. Furthermore, inactivation of the ACC results in impaired fear transmission13. Therefore, ACC activity is likely involved in the empathy-like behaviour. In the present study, c-Fos-positive neurons in the ACC were increased after exposure to a single SDS video, suggesting that the empathy-related circuit was also driven in this model. We therefore speculate that various brain areas associated with negative emotions and the HPA axis were also activated after exposure to the SDS video.It has been reported that rodents express emotion in their facial expressions, and that they change their behaviours by perceiving the emotional responses of conspecificsThere is another possibility: that the aggressive ICR which attacked the conspecific was the stressful stimulus that affected the subject mice. However, given the SIT results, where the subject C57BL/6J mice did not show avoidance behaviour toward the ICR mice, it is unlikely that the subject C57BL/6J mice were recognising the ICR mice themselves as a fear target.10. In the present study, however, repeated exposure to the SDS video did not affect these parameters. This discrepancy may be because the effects of the SDS video exposure are weaker than the effects of directly witnessing the actual SDS. In this study, we did not conduct a daily investigation of the sensory contact that the subject mice had with the aggressive ICR mice across the partition until the stress exposure the next day, although this is usually performed in a standard SDS paradigm26. The lack of sensory contact may therefore also contribute to the discrepancy. In addition, we must note that our SDS video did not contain ultrasonic vocalisation28 or alarm pheromones31 which are released by rodents under stressful conditions and could induce physiological and behavioural changes in other conspecifics. Since the integration of multiple sensory inputs is important for vicarious fear, the addition of pheromones and ultrasonic vocalizations to the SDS video could induce changes in the SIT and the FST. It is also interesting to examine c-Fos expression in the brain regions that mediate the pheromones or ultrasonic vocalizations in future studies.We have previously reported that witnessing actual SDS induces avoidance behaviour in the SIT and increases immobility in the FSTIn Experiment 2, mice in the stress group had lower gains in body weight during the SDS video exposure period. Interestingly, the SDS video group did not show any large changes in body weight gain until about day 6. One possible reason is that the effects of daily stress gradually accumulated, and changes in body weight gain appeared a few days after the start of video exposure. On the other hand, recovery of body weight of SDS video group seems to be faster in the last 2 days during the video exposure period. The adaptation to the SDS video may explain this tendency.33. Furthermore, mice witnessing actual SDS that showed increase in weight gain were also reported to have reduced sucrose preference10. Together, these results strongly suggest that exposure to SDS videos has significant effects on reward sensitivity. The mesolimbic dopamine neurons in the VTA are important for regulating the reward system and stress responses21. The reduced sucrose preference in the SPT caused by 10 days of video exposure might reflect a dysfunction of VTA dopamine neurons, which were activated after just a single acute video exposure . All mice were housed in standard polyolefin plastic cages at 23 ± 1 °C with a 12 h light–dark cycle with lights on at 8:00, and were allowed free access to food and water. The experiments were conducted in compliance with the Guidelines for the Care and use of Laboratory Animals, and received National Center of Neurology and Psychiatry Animal Care and Use Committee  approval. All behavioural experiments were performed between 13:00 and 17:00.26. Prior to the SDS experiment, ICR mice were screened for aggressive behaviours toward C57BL/6J mice according to the screening criteria, which we have previously published10. For the SDS experiment, an aggressive ICR mouse was placed into a transparent acrylic cage (10 cm × 23 cm × 12 cm) with their bedding, and a C57BL/6J mouse was then placed into the cage. The SDS session was recorded for 5 min from the side of the cage by a video camera, and different ICR and C57BL/6J mice were used for each session. Two independent evaluators assessed the severity of aggressive behaviours in these videos, based on the numbers of attacks and the facial expressions (narrowed eyes) of the ICR mice, and the defeat posture, freezing behaviour, and squealing of C57BL/6J mice. Of the 38 videos, 20 showing highly aggressive behaviours were selected by agreement between the evaluators. These 20 SDS videos were edited to create the ten 10-min videos used in this study.To prepare the SDS video, we performed SDS experiments according to the standard method for mice SDS modelsAs controls, we prepared ten 10-min videos in which a pair of C57BL/6J mice interacted in the same transparent acrylic cage. For the habituation video, a single C57BL/6J mouse was recorded in the cage for 10 min. Mice were presented with this habituation video to become accustomed to the equipment and environment before the effects of video exposure were examined.In addition, we also generated three pixelated videos  to mask the contents, while the intensities of those videos were preserved, by pixelating original videos to 8-pixel based images.After the habituation session, a C57BL/6J mouse was exposed to a SDS (stress group) or control (control group) video. We examined the effects of this single video exposure on peripheral corticosterone levels, measured 20 min after video exposure, and c-Fos expression in the brain, measured 90 min after video exposure.Then, we examined the effect of SDS video exposure on peripheral corticosterone levels under the condition when visual information is blocked by the opaque divider and only sound cues were available. In this experiment, after the habituation session with the divider, a mouse was exposed to SDS or control video with the divider.Finally, we examined the effect of the pixelated SDS video exposure on peripheral corticosterone levels. In this experiment, after the habituation session with the pixelated habituation video, a mouse was exposed to the pixelated SDS video or pixelated control video.After the habituation session, C57BL/6J mice were exposed to a SDS video  or control (control group) video, then returned to the home cage. This procedure was repeated for 10 consecutive days Fig. a. After C57BL/6J mice were individually placed into the acrylic cage, which was sandwiched between a laptop computer LCD monitor and a liquid crystal display Fig. . An audi10. Trunk blood was collected in individual heparin tubes  20 min after a single video exposure. Blood samples were centrifuged at 3500 × g for 90 s at 4 °C to obtain plasma and stored at − 80 °C until use. Plasma samples were diluted 100 times in assay buffer and analysed using a Corticosterone ELISA Kit  following the manufacturer’s instructions. All samples were measured in duplicate.The corticosterone assays were performed as previously described37. Ninety minutes after a single video exposure, mice were anaesthetised with sodium pentobarbital (50 mg/kg) and transcardially perfused with 0.02 M phosphate buffer  followed by fixative   for 1 h. Sections were incubated with anti-c-Fos antibody  in PBSTS for 24 h at 4 °C. After incubation with biotinylated anti-rabbit IgG antibody  for 1 h at room temperature, sections were incubated with Elite avidin–biotin peroxidase complex (Vector Laboratories) according to the manufacturer’s instructions. The immunostaining reaction was developed using the ImmPACT DAB peroxidase substrate kit (Vector Laboratories). Sections were mounted onto glass slides and air-dried overnight, counterstained with 0.5% methyl green, dehydrated in graded ethanol solutions, cleared with xylene, then coverslipped with VectaMount Mounting Medium (Vector Laboratories). Digital images of brain sections were taken using a microscope  and digitised by a camera . The Allen Brain Atlas38 and its mouse brain stereotaxic coordinates were used to define the brain area boundaries. The c-Fos immunopositive nuclei were counted using ImageJ v 1.52a software, developed at the National Institutes of Health39. We defined immunopositive nuclei as nuclei that were darker than the threshold determined by adjacent background stains in each brain region, and we counted the immunopositive nuclei within a 200 × 200 μm area.Immunohistochemical staining was performed as previously described, with minor modifications26. First, a C57BL/6J mouse was allowed to explore an open field arena (40 cm × 40 cm) for 2.5 min. Along one side of the arena was a circular wire cage (8 cm diameter) that remained empty during the first trial (target absent). The mouse was then removed from the testing arena and a novel ICR mouse was placed into the wire cage (target present). Last, the C57BL/6J mouse was returned to the arena and allowed to explore. The time spent in the interaction zone (a 14 cm × 25 cm area surrounding the cage) for each session was recorded and analysed using video tracking software . The social interaction ratio (SI ratio) was obtained by dividing the time spent in the interaction zone with the target present by the time spent in the interaction zone with the target absent.The SIT was performed as previously described10. The maze comprised two perpendicular intersecting runways. One runway had tall walls (closed arms), and the other runway had no walls (open arms). The runways were 5 cm wide and 25 cm long, and the closed walls were 15 cm tall. The maze was 50 cm from the floor. Mice were placed in the central area, facing one of the closed arms, and were allowed to explore for 5 min. The time spent in the open arms was recorded and analysed using video tracking software (Smart 3.0).The EPM was performed as previously described10. Mice were placed individually into 5 L beakers (27 cm × 19 cm) containing 3.5 L of water (23 ± 1 °C) for 6 min. An immobile posture was defined as stopping all active behaviours and floating in the water with minimal movement40. Movements during the 6 min were recorded using a video camera, and blinded experimenters recorded immobility as when mice spent more time in an immobile posture than in the performance of active behaviours over each 5 s period.The FST was performed as previously described10. This test was conducted over 3 days, which included a 2-day training period. Mice were trained to drink from two bottles (water and 1% sucrose) for 2 days. The position of the two bottles was balanced across the experimental mice to exclude potential side preference bias. On day 3, mice were deprived of food, water, and sucrose for 4 h. Afterwards, mice were again provided water and sucrose bottles for 1 h, and their liquid consumption was recorded. Sucrose preference was obtained by dividing sucrose consumption by total consumption (sucrose + water).The SPT was performed as previously describedt-test was used for normally distributed data, and Mann–Whitney U test was used for not normally distributed data. For comparing more than two groups, a two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test was used. Differences with p < 0.05 were considered statistically significant.All data are presented as the mean ± standard error of the mean (SEM). Data were analysed using IBM SPSS Statistics 21 . For comparing two groups, an unpaired


Background: We estimated the change in the prevalence of harms attributed by students to their drinking and to others’ drinking, over a decade of concerted effort by university authorities to reduce antisocial behaviour and improve student safety. Interventions included a security and liaison service, a stricter code of conduct, challenges to liquor license applications near campus, and a ban on alcohol advertising. Methods: We used a pre-post design adjusting for population changes. We invited all students residing in colleges of a New Zealand University to complete web surveys in 2004 and 2014, using identical methods. We estimated change in the 4-week prevalence of 15 problems and harms among drinkers, and nine harms from others’ drinking among all respondents. We adjusted for differences in sample sociodemographic characteristics between surveys. Results: Among drinkers there were reductions in several harms, the largest being in acts of vandalism (7.1% to 2.7%), theft (11% to 4.5%), and physical aggression (10% to 5.3%). Among all respondents (including non-drinkers), there were reductions in unwanted sexual advances (14% to 8.9%) and being the victim of sexual assault (1.0% to 0.4%). Conclusion: Alcohol-related harm, including the most serious outcomes, decreased substantially among college residents in this period of alcohol policy reform. In conjunction with evidence of reduced drinking to intoxication in this population, the findings suggest that strategies to reduce the availability and promotion of alcohol on and near campus can substantially reduce the incidence of health and social harms.  University students are renowned for ‘heavy episodic’, ‘binge’, or ‘risky single episode’ drinking, i.e., consuming enough alcohol, rapidly enough, to become intoxicated. This is often characterised as at least four or five drinks (40–60 g ethanol) within two hours. In a man of average body weight, without significant tolerance, this level of consumption produces a blood alcohol concentration between 0.05 and 0.10 g/dL [In studies making the comparison, which were mainly performed in the USA in the early 2000s, college students in their late teens and early 20s were found to consume a greater volume of alcohol and had a higher prevalence of consuming ≥5 drinks on a single occasion than thein non-student peers . A New ZIn a previous paper we estimated changes in drinking at a New Zealand university with a long history of risky drinking and related social disorder, finding no change in the prevalence of drinking per se, but large reductions in risky consumption over a 10 year period . For exaThe New Zealand experience contrasts with findings from surveys of nationally representative samples of college students between 1993 and 2001 in the USA. Wechsler and colleagues ,11,12.While drinking behaviour is an important outcome by which to judge the effects of changes in policy, it is arguably a means to the end of reducing the incidence and severity of various harms. Vandalism, physical aggression, and sexual assault are what motivated efforts to make the campus and surrounding environment safer for students and other members of the community. Accordingly, in this study we estimated the change in the prevalence of a range of health, social, and legal consequences of risky drinking, in the same population, between 2004 and 2014.The University of Otago Human Ethics Committee  approved the study protocols.http://ipru3.otago.ac.nz/demo/halls2004/). We employed the same methods and many of the same questions in a survey of the same colleges, as well as two that opened after 2004, in April-May 2014 .We invited University of Otago students living in the 12 Dunedin residential colleges in April-May 2004 to participate in a survey using a web-survey method described previously . We askeWe sent personalised letters inviting all residents  to participate by clicking on the hyperlink sent to their university e-mail account. In the following weeks, we sent up to one reminder letter and three reminder e-mails to non-respondents.We asked respondents who had consumed alcohol in the previous four weeks (who we defined as drinkers) if they had experienced a range of problems as a result of their drinking during that time . The folcluster command, we inflated standard errors to reflect clustering of respondents within colleges, and adjusted prevalence estimates using the margins command [We compared proportions of respondents experiencing the outcomes of interest in each survey, using Stata/SE 13.1 . To acco command . Of the 2497 students living in the Dunedin residential colleges in 2004, 1594 (64%) completed the questionnaire and a further 68 (3%) submitted responses that met a minimum data requirement. The corresponding figures for 2014 (N = 3262) were 1835 (56%) and 106 (3%).To investigate the effect of non-response we adopted an approach previously employed to quantify non-response bias in survey data . We estiWe then estimated differences in the proportion of respondents with these outcomes in 2004 versus 2014, based on the assumption that the true prevalence among non-respondents was 20%, 33%, 50%, or 100% higher than among respondents. The result was larger differences in estimates from 2004 to 2014 across the outcomes, ranging from <0.01%  to 1.3% . The proportion of residents reporting alcohol-related problems that they attributed to their own or others’ drinking declined markedly from 2004 to 2014, while “baby-sitting” drunk students increased. The results are consistent with the anecdotal accounts of college administrators that alcohol-related disorder declined across the decade, and that residents had been encouraged to assume greater responsibility for their peers, particularly when out drinking. In 2004 and 2014 the Dunedin colleges housed a similar proportion of students who had consumed alcohol in the preceding calendar year ; however, in 2014, a smaller proportion had consumed alcohol hazardously in the preceding year. This may reflect the greater proportion of 16–17 year-olds in the colleges in 2014 . While NIt is noteworthy that two thirds of the respondents in the 2014 survey were women. This reflects the fact that there are more women at the university and residing in the halls, and that women were more likely to respond. The study aimed to recruit a sample to represent the student population. Our previous analysis of selective non-response in this population suggests that it is likely to have biased estimates of the prevalence of alcohol-related harm downward to a small extent . In relaStrengths of the study include the equivalent timing of the surveys during the academic year, and the use of identical questions and survey methods, facilitating comparisons over time. Sensitivity analyses suggest that the difference in response fractions is unlikely to have substantially biased the estimates of change. The lack of a control setting limits the strength of inference one can make about the impact of changes at the university on alcohol consumption and harms in the colleges. However, the reductions in this study are consistent with those in the wider University of Otago population, and contrast with what appears to have occurred at other New Zealand universities .No significant change in alcohol policy occurred at the local government level in Dunedin during the study period. Localised strategies to improve public safety and reduce alcohol-related crime were focused on the Central Business District rather than North Dunedin, where most of the student population resides . The blood alcohol limit for drivers under 20 years of age was reduced from 0.03 g/dL to zero in 2011. However, the residential colleges in Dunedin are within walking distance of campus and alcohol outlets , so resiCampus Watch and other policy changes [Our previous research showed that drinking in the wider student population decreased at Otago compared with other universities from 2005 to 2013 [ changes . We arguThe marked decrease in drinking at on-licenced premises makes the pub closures in North Dunedin a likely explanation for at least part of the observed changes, given that drinking episodes in pubs are more likely to result in intoxication, particularly among men, than is drinking in other locations . QuantifOverall, there has been a shift among Otago students to drinking in fewer types of locations, with a marked shift away from drinking in pubs . This coReducing the tendency for pub drinking may have changed a prevalent pattern of multi-location drinking that could account for the reductions in vandalism, theft, physical aggression, and sexual assault we found in this study. Students may be exposed to fewer opportunities to commit, or be victims of, such acts , e.g., wThe findings are broadly concordant with previous research showing that policies restricting the availability and promotion of alcohol are the most effective at reducing alcohol consumption and harm among the general population . There iIn this period of alcohol policy reform, alcohol-related harm, including the most serious outcomes, decreased substantially among college residents. In conjunction with evidence of reduced drinking to intoxication in this population, the findings suggest that strategies to reduce the availability and promotion of alcohol on and around campus can substantially reduce the incidence of health and social harms.