Background: Prevalence and characteristics of fetal alcohol syndrome (FAS) and total fetal alcohol spectrum disorders (FASD) were studied in a second sample of three South African rural communities to assess change. Methods: Active case ascertainment focused on children with height, weight and/or head circumference ≤25th centile and randomly-selected children. Final diagnoses were based on dysmorphology, neurobehavioral scores, and maternal risk interviews. Results: Cardinal facial features, head circumference, and total dysmorphology scores differentiated specific FASD diagnostic categories in a somewhat linear fashion but all FASD traits were significantly worse than those of randomly-selected controls. Neurodevelopmental delays were significantly worse for children with FASD than controls. Binge alcohol use was clearly documented as the proximal maternal risk factor for FASD, and significant distal risk factors were: low body mass, education, and income; high gravidity, parity, and age at birth of the index child. FAS rates continue to extremely high in these communities at 89–129 per 1000 children. Total FASD affect 196–276 per 1000 or 20–28% of the children in these communities. Conclusions: Very high rates of FASD persist in these general populations where regular, heavy drinking, often in a binge fashion, co-occurs with low socioeconomic conditions. Fetal alcohol syndrome (FAS) was first defined by Jones and Smith in 1973 ,3,4. TheCommunities studied to date in ZA have the highest reported general-population rates of FASD in the world . From anThe sample presented here represents a second study of FASD (commenced in 2011) in a highly rural area with three small towns located one to two hours away from SC1. The study was initiated to assess whether there was change in the prevalence of FASD. The previous study in this region  includedn = 1448) enrolled in all 32 primary schools of the three (BAR) communities; consent was received for 1083 (74.8%). Per ZA ethics requirements, written assent was also obtained from children seven years and older.As before , this stA three-tier process of screening, data collection, and diagnosis was instituted for all consented children . All conIOM diagnostic criteria for FASD diagnoses are summarized in For example, in the previous study of these communities, 88% of the mothers of children with FAS directly reported drinking during the index pregnancy . ChildreAll randomly-selected control candidates and all children with significant traits common in a diagnosis within the continuum of FASD were advanced to cognitive testing in Tier III. Teachers completed Achenbach Teacher Report Forms (TRF) for each child  to charaAlso in Tier III, mothers of every child that could be located and consented were interviewed  regarding maternal risk for FASD in the index pregnancy. Time-line-follow-back methods ,41 have In multidisciplinary case conferences, findings from each domain  were formally reviewed in oral presentations by the examiners of each child and interviewers of each mother. All other clinical team members listened while viewing photographs of each child projected on a screen to refresh everyone’s memory of the child. After review and discussion, final IOM criteria were reviewed and assessed, and a final diagnosis was made by the dysmorphologists with consensus of the entire clinical team.Case control analyses compare results across diagnostic groups and controls. Data were processed with Excel   and analIn Average total dysmorphology scores  summarizCognitive testing and behavioral checklist results  characteMaternal alcohol consumption during pregnancy is detailed in Seventy percent of mothers of children with PFAS, reported drinking in the index pregnancy, drinking averages of: 6.6, 6.7 to 7.5 drinks per drinking day during the respective trimesters; but they drank fewer days per week than mothers of FAS and ARND children. Among mothers of children with FASD, the PFAS group binged least in every trimester, drank less frequently, and was less likely to report drinking at all in 2nd and 3rd trimesters.p < 0.012 was set to control for Type I familywise error. The analysis included 432 children for whom mothers’ education and family income were reported.Partial correlation analysis measured associations between maternal drinking and cognitive/behavioral measures, OFC, and total dysmorphology scores after adjusting for household income and mother’s education . Transfor ranging from −0.134 to −0.219. Thus, each drinking variable accounts for only about 3% of the variance in the child’s cognitive or behavioral scores. Binge drinking measures of drinks per drinking day and 3+ and 5+ drinks per occasion correlated most with head circumference and accounted for 5–6% of the variance in head size. More drinking is associated with smaller head size. Behavioral measures correlated in the expected direction, but not with cognitive measures. Again, the signs of the correlations were all in the expected direction, but were not particularly strong.Controlling for two measures of socioeconomic status (SES), all three measures of drinking during pregnancy  correlated significantly with all cognitive and behavioral scores using Bonferroni-adjusted values. Verbal and non-verbal IQ and Digit Span performance were significantly lower with prenatal drinking. Behavior problems and inattention were greater with prenatal drinking. However, none of these partial correlations were particularly strong once adjusted for SES, with r ranging from 0.206 to 0.256. Prenatal drinking was significantly associated with total dysmorphology score as well (r ranging from 0.168 to 0.215). No statistically significant associations were noted between paternal drinking problems and child traits after adjusting for socioeconomic measures and mothers’ weekly quantity of drinking during pregnancy.Maternal drinking measures correlated most highly with the child’s OFC after adjusting for income and education: with partial A significantly higher percentage of FASD mothers than non-FASD mothers reported smoking during the index pregnancy . The quaIn n = 1448) for the low estimate, and the total number with consent (n = 1088) for the upper estimate. Oversampling of small children provided the greatest probability of including virtually every child with FAS or PFAS. The rate of FAS with this technique is 89.1–118.6 per 1000, and the rate of total FASD is 196.1–261.0 per 1000.Final diagnoses of children and the first estimation of prevalence are presented in the left side of A second rate was calculated from the 87 cases of FASD found within the 276 children who entered the study via random selection as candidates for the control group  were performed blinded by professionals in their individual disciplines; therefore, linking each domain for the final diagnosis is important. Furthermore, it is vital to link the traits and diagnoses of the children to the quantity, frequency, and gestational timing of maternal alcohol use in the prenatal period. This was accomplished by direct maternal reports in 87% of the FASD cases and in the remaining cases by collateral reports from relatives, neighbors, and other knowledgeable individuals. Both case control and correlation analyses linked drinking to child traits: more drinking during the index pregnancy led to suppressed growth and physical development, more minor anomalies, and poorer performance on cognitive and behavioral measures. The latter association of drinking and cognitive performance, while significant, is not as strong as the link with microcephaly and dysmorphology overall, has also been demonstrated with complex multivariate structural models ,50. DysmThis multiple-method, multi-disciplinary, comprehensive study of FASD in the general population of three communities had limitations. First, since the populations of these communities are substantially unique in the world, extrapolating directly from the exact findings, measures, and values for most variables should only be done with caution. Second, maternal interviews for the index pregnancies were administered to the mothers seven years post-partum. Recall may have been negatively affected for some variables, although several studies show otherwise ,52. ThirThe rates of FASD in these towns and surrounding rural areas were stable between samples collected two years apart and remain higher than reported in a general population anywhere. Recreational binge drinking on the weekends is the primary, proximal risk factor for FASD, and a very high prevalence of FASD results. The traits of children with FAS, PFAS and ARND are clearly different from one another and from normal controls on most every measure. Furthermore, multiple distal maternal risk factors exist to varying degrees across this population, and the presence or absence of many of these factors differentiates the severity of child outcomes across groups. The rate of FAS remains extremely high, at 89–129 per 1000. The rates of PFAS and ARND are again high and similar to the previous sample. The total FASD rate of 20–28% in this sample represents the highest prevalence ever reported for a general population.


Salmonella enterica serovar Typhimurium strain LT2, which are important in cellular invasion during infection.Sialidases, which are widely distributed in nature, cleave the α-ketosidic bond of terminal sialic acid residue. These emerging virulence factors degrade the host glycan. We report here the release of seven sialidase and one sialic acid transporter deletion in  Sialidases are widely distributed among microbes and are one of the least characterized and ill-defined glycosyl hydrolases. Sialidases have been associated with several diseases. Sialidases play a critical role in microbiology by mediating metabolism, adherence, and infection, and they are important regulators of alternate complement pathway activation, red blood cell destruction, cell growth, cell adhesion, and tumor metastasis in mammalian systems –5. Recen–ΔnanH and ΔSTM1252) from Salmonella enterica serovar Typhimurium LT2 have the same domains and function as sialidases, but they are structurally very different, indicating domain shuffling and lack of structural conservation; therefore, this difference led to different invasion phenotypes during the in vitro infection of differentiated colonic epithelial cells (Caco-2)  .http://www.100kgenomes.org) is a large-scale sequencing consortium that offers the use of new next-generation sequencing methods to provide cutting-edge methods for pathogen detection and control in the food supply. This project is focused on producing genomes of pathogenic isolates from the environment, plants, animals, and humans worldwide, providing new insights into the genetic diversity of Salmonella spp. and other foodborne pathogens. These seven sialidase and one sialic acid transporter mutant strain were constructed in the Weimer Laboratory    as descrCA, USA) . CultureCA, USA) . GenomicCA, USA) , checkedCA, USA) . The 350CA, USA) , 13 wereCA, USA) –16 at thhttp://www.ncbi.nlm.nih.gov/sra); genome assemblies can be found in NCBI GenBank (All sequences are publicly available and can be found at the 100K Pathogen Genome Project (NCBI PRJNA186441) in the Sequence Read Archive ( GenBank .


The inclusion of animals into interventions for children with autism spectrum disorder (ASD) is a growing practice known as animal-assisted intervention (AAI). The choice of the animal to include in an intervention is often solely up to the interventionist and depends on their experience, subjective judgment, and ease of access to different animals. For individuals with ASD who are non-verbal and unable to indicate preferred stimuli or activities, incorporating preference into interventions has been linked to increases in positive behaviors and enhanced quality of life. We propose that animal choice based on a participant’s preference may enhance the experience of AAI and maximize its outcomes. A common technique used to reliably determine preferred interactions and activities in interventions for children with ASD is a stimulus preference assessment. The video-based multiple-stimulus without replacement (MSWO) procedure, in particular, allows for discrimination of complex stimuli that could not feasibly be presented all at once, which is the case when choosing an animal. Based on the well-documented reliability of this technique in the field of applied behavior analysis, we propose that a future direction in AAI is utilizing video-based MSWO to guide animal selection. Animals are increasingly included in interventions targeting children with neurodevelopmental disorders, including autism spectrum disorder (ASD) . ASD is The umbrella term AAI comprises any intervention using animals in support of human health or well-being, including animal-assisted therapy (AAT), animal-assisted activity (AAA), and animal-assisted education [AAE ]. DuringThe rationale supporting the inclusion of animals in services for with children with ASD is that an animal may provide a non-judgmental, soothing presence  that couA variety of animal species can be included in AAI, though domestic species are often recommended to both ensure the safety of the participants and maximize welfare for the animal. The most common species included are dogs, horses, small mammals , and domestic farm animals . BecausThe attitude of children toward animals varies from liking certain species to having a phobia of others and is significantly associated with their age, sex, ethnicity, background, and most importantly their personal experience . Even thResearch on the incorporation of individual preference in populations with neurodevelopmental disorders such as ASD has grown substantially in past decades and has been shown to be an important consideration for intervention success . While tBehavioral studies have found that incorporating choice into the daily lives of individuals with severe developmental disabilities can result in an increase in appropriate positive behaviors, decreases in problem or challenging behaviors, and enhanced task engagement and participation –30. FurtBased on the encouraging findings of behavioral research on choice and preference, we propose that incorporating choice into AAI has the potential to improve positive outcomes and engagement while reducing problem behavior by incorporating the participant’s desires and/or aversions into animal selection. By incorporating the widely used stimulus preference assessment (SPA) tool into the practice of AAI, low-functioning individuals can voice their own opinion prior to an animal interaction. Evaluating preference for an animal can increase the probability that a preferred animal is selected and individual autonomy respected. Furthermore, preference assessments may also help to rule out possible animal phobias that could increase the efficacy of AAI by avoiding discomfort and maximizing any positive outcomes to be gained. In this paper, we discuss the potential of SPAs for use in AAI, particularly to inform animal selection while offering suggestions and guidelines to both researchers and clinicians.Preference assessments first emerged in applied behavior analysis (ABA) research as a way to identify effective reinforcers in participants with low verbal communication. In one of the first attempts to assess preferences in non-verbal individuals, Pace et al.  designedThe structure and format of preference assessments have considerably varied over time . Early aWhether in a single, paired, or multiple stimuli format, preference assessments have previously been limited by size and modality; that is, in order to be presented on a surface the choice items must be both small and physically available. While this is practical with toys and other tangible items such as food, this method is logistically challenging when desired stimuli are either not present at the time of assessment, too large to be presented in this format , or represent a leisurely activity which cannot be represented by a single item . With the development of evaluation methods that are increasingly efficient and sensitive to practitioner needs, researchers have addressed this limitation by presenting non-tangible and non-accessible stimuli with alternative formats such as printed words, static pictures, and most recently with dynamic video formats .Video technology has emerged as a particularly useful tool for interacting with individuals with severe disabilities , 45. As Preliminary research suggests that animals may be incorporated into SPA. A recent study by Protopopova et al. recently used SPA with children with intellectual and developmental disabilities, rewarding the achievement of academic tasks with the access to leisure items or a dog . The doginteraction can be captured rather than the animal alone. When using pictures, an individual’s repeated choice of a picture of a rabbit over a picture of a cat might be attributable to unknown characteristics of the picture’s contents. However, repeated choice of a video of stroking a rabbit over stroking a cat might parse out the particular interest of the participant’s desire to touch the animal rather than to simply view the animal.A key characteristic of video-based preference assessments is their ability to feature more salient characteristics of the stimuli than pictorial, static formats. The incorporation of video assessments into the field of behavior analysis interventions has allowed researchers to broaden the utility of preference assessments into areas previously unavailable to incorporate choice in daily life, such as work preference and job selection , 49. ThiAnother primary advantage of using video-based preference assessments in AAI is that an individual can complete a session without needing to be directly exposed to animals. Preliminary research suggests that during preference assessments, obtaining contingent access to an activity after the choice is not necessary. For example, Clark et al. presented items via video to children with ASD in a paired-stimulus video format without providing access contingent on selection and found that even if the individual did not physically have access to the item, highly preferred stimuli did function as reinforcers . BrodheaThe above findings have important implications for feasibility because it seems unlikely that a clinician wishing to incorporate animal interactions into a therapy or intervention program would have access to a wide variety of animals at one time. Even in the case of larger therapy centers specialized in AAI, where the access to many sizes and species of animals is not a problem, presenting multiple animals at the same time may be logistically difficult and detrimental to animal welfare. Thus, it is more appropriate and plausible that an animal is introduced after the individual has completed a brief assessment and a preferred animal was identified. At that point, the activity or therapeutic intervention has been appropriately designed and maximizes the efficient use of both the animals’ and humans’ time.Another potential benefit for implementing a preference assessment prior to designing an AAI involves avoiding unknown or unpredictable phobias. Animals can be a common fear among children, especially among low-functioning or non-verbal individuals. Though teachers and parents can report a known phobia, studies have shown that caregiver reports can often be unreliable , 39. As Although video preference assessments might have advantages for use in AAI, their implementation raises several considerations. First, picture or video-based assessments are limited by the prerequisite of the individual’s ability to associate the animal seen on video with a live, tangible animal. This ability to associate an object seen on video and a tangible object is referred to as video-to-object and object-to-video correspondence. In previous research comparing two matched tangible and pictorial preference assessments, Clevenger and Graff found that only individuals who were capable of picture-to-object and object-to-picture matching skills had similar preference hierarchies . It is rSecond, a limitation of the implementation of SPAs into the field of AAI and a critical area for research is the untested assumption that a highly preferred animal interaction presented in a video will translate to an effective in-person activity for the individual receiving the intervention. Although a plethora of research has investigated the predictive validity and reliability of reinforcers identified from SPAs of all modalities , 54, furThe specifics of the protocol to incorporate SPA into AAI remain to be developed and assessed for feasibility and relevance. Practical elements to determine are the design of the SPA, the medium used to present the SPA, and the content and format of the videos. A brief multiple-stimulus paradigm with several video stimuli is the gold standard in the field of ABA and would be adapted to showing videos of human–animal interactions.The content of the video should reflect the animal with which the participant may interact, in a context representative of the actual interaction that is offered. Depending on the goal of the SPA and the choice presented, the videos could represent animals from different species , or different individual animals of the same species . The behaviors and the types of interactions with humans that are presented in the videos should be directed by the goals or nature of the intervention. For example, the videos could show the animals eating, being groomed, or playing with people. In addition, capturing the animals’ natural noises may increase the fidelity of the video to the animals and help participants make an informed choice.The videos should be presented in a format such that participants can easily select or point to a preferred stimulus. In the field of ABA, computers and more recently handheld tablets have been used to identify preferred stimuli. An example of an effective design could incorporate an adaptive program that simultaneously presents multiple previews of videos of animal interactions and allows the participant to select a video to play in full screen for a few seconds. When back to the main screen, the participant can choose any other video to watch next. Each time a video has been played, it disappears from the main screen. When all animals have been selected, all of the videos are available from the main screen again for a second round of viewing and eventual preference selection.Before any large-scale implementation, researchers should test any animal preference assessment method for predictive validity. A first step would be to compare the choice identified during the SPA before and after the participant has met the animals. If the same animal is preferred both before and after the participant has had a chance to interact with all the animals presented in the SPA, it is likely that the stimuli presented were representative. This would confirm the feasibility of identifying a preferred animal from a video.If the predictive validity of an SPA for AAI is established, the next step will be to examine the clinical relevance of identifying a preferred animal during a therapeutic intervention or AAA. We hypothesize that preferred animals will yield more positive outcomes from AAI, as participants may feel a stronger sense of bonding or affinity with animals with which they chose. This assumption could be tested by comparing the outcomes of interventions preceded by an SPA or not, and within interventions preceded by an SPA, led with an animal either identified as preferred or non-preferred. Better outcomes from the intervention with the animal identified as preferred would then be a sign that the use of SPA offers a significant enhancement to AAI.Stimulus preference assessments have been widely implemented in the field of ABA in order to incorporate the desires of those with communicative difficulties to interventions. With heavily validated, tested, and refined methodology, SPAs may be a useful tool for the field of AAI. However, although SPAs have been widely used and empirically researched in the field of ABA, it is important to note that this is a currently unexplored and untested strategy for AAI. While some clinician guidelines for implementing AAI do suggest to control for the potentially confounding variables of pet ownership, animal aversion, and any allergies , there iIn summary, we have proposed that integrating preference assessment methodology into AAI practices is a promising way to maximize positive outcomes for individuals who are severely disabled or experience communicative difficulty. Moreover, beyond simply assessing preference, it also may act as a way to rule out AAI as a possible treatment for an individual with ASD who is averse to animals or might not explicitly benefit from or exhibit positive emotions from an animal interaction. Further, behaviors that indicate aversion such as avoidance  or a lacBy incorporating individual preferences into AAI, clinicians will be able to better identify and avoid animals that a participant might be averse to and/or incorporate animals for which the individual might have a particular affinity. Ultimately, an ideal use of SPAs in the field of AAI could aid in increasing the efficiency and efficacy of the interventions that include animals, possibly by even determining its feasibility before starting. Additionally, we hope that the exploration of this practice will eventually allow for a broader application of incorporating animals into improving the mental health and well-being of those with ASD, who might have been restricted from AAI in the past because of their communicative limitations. Applications include the choice of a preferred animal prior to AAI, and the choice of activities during AAI. While the use of SPAs in identifying animals for therapeutic interventions is still exploratory, we believe that there are benefits to be gained from implementing this practice and encourage AAI researchers to consider the effects of incorporating preference and aversions into their research.NG and KR are co-first authors; both wrote the body of the text and articulated the main ideas presented in this paper. MB provided extensive guidance and expertise in the area of applied behavior analysis. MO provided expertise in the area of animal-assisted intervention.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The reproductive strategies of vertebrates are diverse. Seahorses (Pisces: Syngnathidae) possess the unique characteristic of male pregnancy; i.e., males, not females, incubate embryos in a specialized structure called a ‘brood pouch’. The brood pouch is formed along the ventral midline of the tail. The lumen of the brood pouch is surrounded by loose connective tissue, called pseudoplacenta, and dermis.Hippocampus abdominalis to gain generalizable insights into this process in seahorses. First, we employed several staining methods to characterize the pseudoplacenta and dermis of the brood pouch of mature male seahorses. The pseudoplacenta is composed mainly of reticular fibers, while the dermis is composed mainly of collagenous fibers. Further observations showed that pouch formation is initiated by linear projections of epithelia on both ventrolateral sides of the body. These projections elongated toward the ventral midline, eventually fused together, and then formed a baggy structure composed of a single dermis layer with neither smooth muscle nor pseudoplacenta. Finally, the pseudoplacenta was formed, together with two layers of dermis and smooth muscle. Thus, a fully developed brood pouch was established. The morphology of the luminal epithelium also changed during pouch formation. We analyzed the localization of C-type lectins as markers; haCTL II was localized in both the outer and luminal epithelia of the brood pouch throughout development in the male seahorse, whereas haCTL IV, which was not detected in the early stage of seahorse development, became localized only in the luminal epithelium as development proceeded.We visualized and evaluated the morphology of brood pouch formation in We categorized the processes of brood pouch formation during male seahorse development into three stages: (1) the early stage, characterized by formation of a baggy structure from the primordium; (2) the middle stage, characterized by the differentiation and establishment of brood pouch-specific tissues; and (3) the late stage, characterized by a fully formed pouch with developing blood vessels and a pouch fold ultimately capable of carrying and incubating embryos.The online version of this article (10.1186/s40851-017-0080-9) contains supplementary material, which is available to authorized users. Reproductive strategies vary greatly among vertebrate groups. Fishes in particular exhibit a wide variety of reproductive modes. For example, some species of gobies and sticklebacks protect their eggs until hatching by making nests, and some species of cichlids protect eggs and hatched-out fry within their mouths . SpeciesThe female seahorse deposits eggs into the brood pouch and the male ejaculates sperm at the entrance of the pouch. Fertilization occurs immediately during egg transfer . The malH. comes, C-type lectins are expressed in the brood pouch; however, their precise expression sites have not yet been determined. C-type lectins are in a type of carbohydrate-binding protein, the activity of which is mediated by conserved carbohydrate recognition domains (CRDs). Analyses of complimentary DNA (cDNA) libraries made from Hippocampus kuda and H. comes revealed that C-type lectins were commonly abundant in brood pouch of both species [The pseudoplacenta is rich in blood vessels, suggesting father-to-embryo interaction through epithelia on the lumen, similar to mother-to-embryo interaction in mammalian placenta. The epithelium facing the lumen of the pouch is considered to be derived from epithelium that covers the outser surface of the body. To better understand the timing of such changes of the outer epithelium into luminal epithelium, we sought to identify molecular markers that are expressed specifically in the luminal epithelium. It has recently been reported that, in  species , 16. TheHippocampus abdominalis. To understand the pathway of pouch formation, we first characterized brood pouch tissues in mature male seahorses by employing hematoxylin and eosin staining and Masson’s trichrome staining to identify fibrous collagen. The latter was used to stain H. kuda brood pouch dermis [In the present study, we observed brood pouch formation in the pot-bellied seahorse h dermis . We furtHippocampus abdominalis, aged 20–30 days , two months , three months , five months , six months , seven months , and eight months , as well as several mature adults, were obtained from Seahorse Ways Co. Ltd., Kagoshima, Japan, for use in the present study. Seahorses were anesthetized in ethyl 3-aminobenzoate methanesulfonate (MS-222) to obtain tissues. This study was performed in accordance with the guidelines of the Animal Experiment Committee of Sophia University, Tokyo.Pot-bellied seahorses, Body portions including the brood pouch were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C for 24 h. The samples were dehydrated in a stepwise manner using 25, 50, 70, 90, 95 and 100% ethanol, and then embedded in paraffin following the standard protocol . Thin .For Masson’s trichrome staining, sections were deparaffinized and rehydrated through 100%, 95%, and 70% alcohol. After washing in distilled water, sections were stained in Weigert’s iron hematoxylin solution for 5 min. After washing in distilled water, sections were stained in Biebrich scarlet-acid fuchsin solution for 5 min. After washing in distilled water, sections were immersed in 2.5% phosphomolybdic-2.5% phosphotungstic acid solution for 30 min. The sections were then transferred to aniline blue solution and stained for 5 min, rinsed briefly in distilled water, and immersed in 1% acetic acid solution for 5 min. After washing in distilled water, the sections were dehydrated through 100% isopropyl alcohol, and finally cleared in Clear-Rite 3 prior to mounting.Reticulin silver staining was performed with a Reticulum II Staining Kit (Roche) according to the manufacturer’s protocol. In brief, the deparaffinized and rehydrated sections were oxidized in acidified potassium permanganate solution for 3 min, rinsed in distilled water, decolorized with 1% oxalic acid for 2 min, and again rinsed in distilled water. Next, the sections were treated with 2% ammonium iron sulfate, rinsed in distilled water, and impregnated with silver carbonate solution. After rinsing again in distilled water, the sections were reduced with 0.4% formalin for 10 min, then treated with 1% gold chloride overnight. After fixing with 2% aqueous sodium thiosulfate for 5 min and washing in water for 5 min, the sections were counterstained with nuclear fast red for 1 min.Elastica van Gieson staining was performed with an Elastic Stain Kit  according to the manufacturer’s protocol. In brief, deparaffinized and rehydrated sections were treated with elastic stain solution for 10 min, differentiated in ferric chloride for 5 min, and treated with van Gieson’s counterstain.Hippocampus comes CTL genes. Nucleotide sequences of the primers are as follows:First-strand cDNA was synthesized from RNA extracted from the brood pouch of a mature male using a SMART™ RACE cDNA Amplification Kit . 5′ RACE PCR and its nested PCR, and 3′-RACE PCR were performed using gene-specific primers designed from nucleotide sequences of the three C-type lectin (CTL) fragments obtained from PCR using primers designed in conserved regions of haCTL I 5′-RACE (for 1st PCR): 5′-ATCGGTCCATATGTAGTCTTCAGCCAGCAG-3′.haCTL I 5′-RACE (for nested PCR): 5′-GTCAGGAGGAGGATCATTCTGGAAGGTGGT-3′.haCTL I 3′-RACE: 5′-GGAGCCTGGTCTTTCAAAAAGATTCAGTCC-3′.haCTL II 5′-RACE (for 1st PCR): 5′-GTGATGATGGAGCTTCATTGTGACTGAAGG-3′.haCTL II 5′-RACE (for nested PCR): 5′-GCTCTTGGACGTTACAAATTGGTTGTCAGG-3′.haCTLII 3′-RACE: 5′-AAATTCAACGACCTTCTCTGTCCTAAAGGG-3′.haCTL IV 5′-RACE (for 1st PCR): 5′-GGTGTATTTCACAACTGAGCCATCGGTCCA-3′.haCTL IV 5′-RACE (for nested PCR): 5′-CTGCCAAAACCCTTCGAGTCTCGAAAACTG-3′.haCTL IV 3′-RACE: 5′-GGACAACCGCTGTTACATCTACCAGAGTCC-3′.We searched for homologous genes for C-type lectin  with the BLAST program from GenBank using their amino acid sequences. In total, 89 genes were used in the phylogenetic analysis. The accession numbers are listed in Additional file One mature male seahorse was dissected to isolate the liver, intestine, heart, gill, muscle, brain, eye, gallbladder, kidney, and brood pouch, and one mature female seahorse was dissected to isolate the ovaries. The RNAs were extracted using RNAiso  according to the manufacturer’s instructions. The brood pouch sample isolated from a second mature seahorse was divided into two parts: the dermis along with outer epithelium, and the pseudoplacenta along with the luminal epithelium. Next, for cDNA construction, we used 0.5 μg of the RNAs to synthesize cDNA using PrimeScript Reverse Transcriptase (Takara Bio Inc.) and a poly-dT primer. The cDNA fragments were amplified with EmeraldAmp® PCR Master Mix (Takara Bio Inc.) using the primer sets shown below. The cDNA amounts added to the PCR cocktail were normalized by the intensity of the amplified β-actin band.haCTL I-F: 5′-GGAGCCTGGTCTTTCAAAAAGATTCAGTCC-3′.haCTL I-R: 5′-GTTGCAACAACAGTGTGGCAAGG-3′.haCTL II-F: 5′-TCGTTCAGCAACTGATTGTCGCC-3′.haCTL II-R: 5′-GACTGGAGAGATGCTCTTGGACG-3′.haCTL IV-F: 5′-TCTGGTCTCCATTCGTGATACCG-3′.haCTL IV-R: 5′-TTGCCAATAATTCAGCATCGTGG-3′.β-actin-F 5′-ACACCTTCTACAACGAGCTG-3′.β-actin-R 5′-GTACAGGTCCTTACGGATGT-3′.Escherichia coli strain BL21 with a C-terminal His tag. Nucleotide sequences of the primers are as follows:The haCTL II and haCTL IV were composed of a signal peptide and the CRD. The CRD was amplified from haCTL IV cDNA, using primers containing the restriction enzyme sites BamHI and NdeI, and cloned into the pET3c vector for expression in F: 5′-CATATGTGGAATCGGTGTCCACATGGCTGG-3′.R: 5′-GGATCCTTAATGATGATGATGATGGATGCCAGAGCCGCCAAGGAATTT-3′.2) and incubated at 4 °C for two days. This protein solution was dialyzed four times against 25 mM Tris–HCl buffer (pH 8.0) with 1 mM CaCl2. The folding mixture was loaded onto an Ni-NTA Superflow  and eluted once with 20 mM Tris–HCl buffer (pH 8) containing 0.3 M imidazole and 0.15 M NaCl.Inclusion bodies (insoluble protein aggregates), obtained according to a previously described method , were diInclusion bodies of haCTL II were not obtained according to the above method. Instead, the CRD of haCTL II was amplified by PCR using primers containing restriction enzyme sites , and then ligated into the pMAL-c2X vector  to obtain a fusion protein of haCTL II and maltose-binding protein (MBP). Nucleotide sequences of the primers are as follows:F: 5′-GAATTCCCTTCAAAAAAATTAAACGACCTT-3′.R: 5′-GTCGACTTAGTGGAACAAACGGTGCGCTTC-3′.E. coli strain BL21 containing expression plasmids were cultured, and then cell lysate was collected following a previously described method [d method . BecauseThe recombinant proteins were dialyzed against PBS, mixed with an equivalent volume of Complete Freund’s Adjuvant, and injected into a rabbit (for haCTL II) or a mouse (for haCTL IV) three times at 2-week intervals. One week after the last immunization, antisera were obtained. Specificity of the antisera against the haCTL II or haCTL IV was checked by western blotting using recombinant proteins.Sections of two late stage and one early stage animals were first placed sequentially in each of the following treatments: two times, 10 min each, Clear-Rite 3; two times, 5 min each, 100% ethanol; 5 min, 95% ethanol; 5 min, 70% ethanol. Sections were then washed in water for 5 min, and blocked in blocking buffer  for 1 h. Incubation with a primary antibody (1/100 dilution in blocking buffer) was performed for 1 h, then sections were washed three times with PBS for 5 min each. Thereafter, sections were incubated for 30 min in 500-fold dilution of alkaline phosphatase (AP)-conjugated secondary antibody (Thermo Fisher Scientific) in blocking buffer, and then washed three times in PBS for 5 min each. Finally, AP protein was visualized by adding NBT/BCIP solution to the sections for 10 min, then washing for 5 min in PBS. Once stained, the samples were observed with a light microscope  equipped with a digital camera .Hippocampus abdominalis males is located along the ventral midline of the tail posterior to the anus. We first observed the histology of the pouch after hematoxylin and eosin (HE) staining. As shown in the cross-section in Fig. The brood pouch of mature The lumen of the brood pouch opens to the outside through a small pore at the anterior of the pouch. We further observed the epithelial layer around the entrance of the pouch after HE staining Fig. . No notaH. kuda has been stained blue using Masson’s trichrome (MT) [H. abdominalis: the outer stratum spongiosum was stained light blue  . MT alsolue Fig. , while tlue Fig. . The blulue Fig.  was muchlue Fig. . Smooth lue Fig. . We obse H. kuda .Next, we further employed Elastica van Gieson staining on samples of four adult seahorses Fig. . In all Finally, reticulin silver staining clearly distinguished pseudoplacenta from dermis Fig. . The fibTo understand how the brood pouch is established during the development of the male seahorse, we observed 20 juveniles (body length 5.6 ± 0.7 cm) 20–30 days after birth. In 10 of 20 fish, structures regarded as part of the early stage of brood pouch formation were observed on the ventral side of the tail. The remaining 10 animals showed no visible signs of a brood pouch, thus making their sex identification impossible, and two of them began to form primordia of the pouch within one week of the initial observation. The brood pouch formation process was observed as follows: In the earliest stage, a white plate structure was observed just posterior to the anus  Early stage : the brood pouch as a baggy structure is formed from the primordium. The process of formation was observed in sections after HE staining Fig. . Dermal (2) Middle stage : various tissues began to differentiate and become visible in the mature brood pouch. At 3–5 months after birth, the dermis of the ventral side began to develop into two layers: the stratum compactum and stratum spongiosum Fig. . After M3) Late stage : the pouch fold, the structure that will hold the eggs during incubation, was formed in the pseudoplacenta Fig. . Many bl Late staHippocampus comes [H. abdominalis by RT-PCR. Cross-species comparisons enabled us to identify the H. abdominalis genes homologous to H. comes CTL reported previously  (76%), and was named hcCTL IV. The sequence analysis revealed that all the cloned haCTLs consisted of a signal sequence and 133–142 amino-acid residues of the CRD   were expressed in the brood pouch of us comes . Using pPhylogenetic analysis of fish group-VII CTLs revealed that haCTL I, II and IV and hcCTL I–IV belonged to the same clade  was colored black by Elastica van Gieson stain in two of four individuals. These results indicate that the two layers of dermis are composed mainly of collagenous fibers, and that the outer dermis contains a small amount of elastin fibers. These fiber structures lend aid to not only strength but also elasticity of the brood pouch.The brood pouch of male pot-bellied seahorse, During brood pouch formation, smooth muscle differentiates within the dermis, and the muscle especially abundant around pouch entrance is closely related to mating behavior. The unique courtship and mating rituals of seahorses have been described elsewhere . CourtshH. abdominalis and found to be localized in the outer epithelium and/or luminal epithelium of the male’s brood pouch. In several fishes, lectins are reportedly localized in the mucous cells of the dermis. Examples include C-type lectin AJL from eel Anguilla japonica, and pufflectin from pufferfish Takifugu rubripes [In this study, three types of C-type lectins  were cloned from rubripes –27. Amonrubripes . Howeverrubripes . In partrubripes .The luminal epithelium is transformed drastically in gene expression and in morphology during development. Large mucous granules were observed as equally abundant in the luminal and outer epithelia in the early stage of brood pouch formation. As development proceeded, the number of mucous granules in the luminal epithelium decreased. Along with this morphological change, haCTL IV was specifically localized in the luminal epithelium of the pouch. It is assumed that the brood pouch of seahorses exerts specific functions, particularly waste removal, gas exchange, osmoregulation and protection of embryos during incubation . In the Hippocampus abdominalis Additional file 2:Figure S2. Sagittal sections of the brood pouch at the early stage. Serial sagittal sections of the body at an early stage of brood pouch formation, after hematoxylin and eosin staining (a-g), reticulin silver staining (h-j), and Masson’s trichrome staining . The ventral side of the body is on the left and the dorsal side on the right. The lettered boxes in a, h, and k indicate sites of the high-magnification views. Scale bars: a–f, h, k = 100 μm; g, i, j, l = 20 μm. (TIFF 12606 kb)Additional file 3:Figure S3. Sagittal sections of the brood pouch at the late stage. Serial sagittal sections of the body at the late stage of brood pouch formation were observed after hematoxylin and eosin staining (a–f) together with their high magnification view of pseudoplacenta (i) and dermis (j). Sections were further observed using reticulin silver staining , and Masson’s trichrome staining . The ventral side of the body is on the left and the dorsal side on the right. The lettered boxes in d, e, g, and h indicate sites of high magnification. Scale bars: a–h = 1 mm; i–l = 40 μm. br, body ring; pp, pseudoplacenta; sc, stratum compactum; sm, smooth muscle; ss, stratum spongiosum. (TIFF 39781 kb)Additional file 4:Table S1. Amino acid sequence similarity between Hippocampus abdominalis  and H. comes (hcCTL I-IV) C-type lectins. (DOCX 16 kb)


In clinical practice, diagnostic hysteroscopy (DH) videos are recorded in full which are stored in long-term video libraries for later inspection of previous diagnosis, research and training, and as an evidence for patients’ complaints. However, a limited number of frames are required for actual diagnosis, which can be extracted using video summarization (VS). Unfortunately, the general-purpose VS methods are not much effective for DH videos due to their significant level of similarity in terms of color and texture, unedited contents, and lack of shot boundaries. Therefore, in this paper, we investigate visual saliency models for effective abstraction of DH videos by extracting the diagnostically important frames. The objective of this study is to analyze the performance of various visual saliency models with consideration of domain knowledge and nominate the best saliency model for DH video summarization in healthcare systems. Our experimental results indicate that a hybrid saliency model, comprising of motion, contrast, texture, and curvature saliency, is the more suitable saliency model for summarization of DH videos in terms of extracted keyframes and accuracy. The recent advancement in modern technology has shown promising results in human reproductive healthcare. One of the popular method of ensuring reproductive health is diagnostic hysteroscopy (DH), where the sensitive regions of female reproductive system (FRS) are assessed and visualized to diagnose uterine abnormalities  for the frame “DHF”. “l” refers to the scale of contrast and “N(p)” shows the neighborhood of the pixel “P”, which is 5 × 5. The value of ɳ is set to 3, indicating the levels of Gaussian pyramid (Liu et al. Contrast map is an effective measurement for finding the uniqueness of a region in a video frame, which has been widely used in computer vision algorithms (Ejaz et al. During DH examination, the gynecologists move the hysteroscope with a certain orientation to effectively visualize the areas of interests. The previously mentioned saliency detection models are less effective in handling DH frames with such abnormalities. In this context, curvature map is comparatively more effective due to its rotational-invariant property in finding the keyframes with abnormalities, which are captured from different orientations. Furthermore, the neuroscience and psychophysical research also dictates that curvature is an important factor in determining the saliency and improving the decision of gynecologists in selection of keyframes. The curvature map “CM” for a DH frame “DHF” can be calculated using Eqs. After computing the numerous saliencies for each frame, it is important to combine them to generate a fused saliency map for keyframes extraction. There are several ways to fuse the different saliencies such as linear fusion, linear weighted fusion, max fusion, and non-linear weighted fusion (Ejaz et al. After calculating the attention curve, the user/gynecologist is asked to specify the number of keyframes “NKF” for a given DH video. Accordingly, the video is divided into “NKF” number of shots. Within each shot, the frame with highest saliency score is determined as keyframe. By only changing the value of “NKF”, a different set of keyframes can be extracted, enabling gynecologists to analyze the DH video at different summarization levels.This sub-section illustrates the performance evaluation of various saliency detection models and general-purpose VS methods for DH videos abstraction. Experiments were performed on a set of DH videos according to Ejaz et al. , each ofTable Table Figure The general-purpose video summarization schemes are less suitable for hysteroscopic videos due to their significant similarity in color and texture, and absence of shot boundaries.Among the evaluated visual saliency models, a hybrid saliency detection model comprising of motion, texture, multi-scale contrast, and curvature is found as the best combination of visual saliencies for hysteroscopic video abstraction, considering its accuracy and extracted keyframes.During the process of diagnostic hysteroscopy, several hysteroscopic sessions are conducted for a single patient per day. Due to the large number of patients and their multiple hysteroscopic sessions, an enormous amount of hysteroscopic videos are collected. However, a limited number of frames are required for actual diagnosis process, whose manual extraction by gynecologists is comparatively difficult and time consuming due to large-sized hysteroscopic videos. To facilitate the gynecologists in browsing for desired diagnostically important contents, video summarization schemes are used. In this work, we have conducted a comprehensive study of numerous generic and domain-specific video summarization schemes for hysteroscopic videos. Further, we have investigated the performance of various visual attention models combined with domain knowledge for summarization of DH videos. Our findings based on numerous experiments are reported as follows:In future, we have intension to focus on minimizing the computational complexity of the system by extracting light-weight features from DH videos. Another possible future direction is to combine data hiding [watermarking (Liu et al.


Intestinal parasites are still a serious public health problem in the world, especially in developing countries. This study aimed to assess the prevalence of intestinal protozoa infections and associated risk factors among schoolchildren in Sanandaj City, Iran.This cross-sectional study involving 400 schoolchildren was carried out in 2015. Each student was selected using systematic random sampling method. Questionnaire and observation were used to identify possible risk factors. Fresh stool samples were observed using formal-ether concentration method.Blastocys hominis (21.3%) and Entamoeba coli (4.5%). Overall, 143 (35.9%) had single infections and 26 (6.4%) were infected with more than one intestinal protozoa, in which 23 (5.9%) had double intestinal protozoa infections and 3 (0.5%) had triple infections. A significant relationship was observed between intestinal protozoa infection with economic status, water resources for drinking uses, and the methods of washing vegetables (P<0.05).Five species of intestinal protozoa were identified with an overall prevalence of 42.3%. No cases of helminthes infection were detected. The predominant protozoa were Education programs on students and their families should be implemented for the prevention and control of protozoa infections in the study area. The prevalence rate of the intestinal parasites is high around the world, especially in developing countries. According to WHO, three billion people are infected; out of which, about 450 million are infected as result of these infections , 2. ThesThere are several environmental and socioeconomic factors contributed to the presence of IPIs in children. Among them are lack of education, poverty, poor sanitary conditions, the absence of potable water, inadequate disposal of excreta, and poor housing facilities , 9. In sCompared with other age groups, school-children have the highest morality rate because of IPIs . TreatinAlthough several studies were carried out on the distribution and prevalence of IPIs in Iran, still there are several localities lacking epidemiological information on IPIs. Sanandaj City, located in northwestern Iran, is one of such localities without any report in this regard. Thus, this study aimed to assess the prevalence of intestinal protozoa infections and associated risk factors among schoolchildren in Sanandaj, Iran.This cross-sectional study was carried out between Jan 5 to Feb 30, 2015, in Sanandaj City, northwestern Iran. The city height from sea level is 1450 to 1538 m. The average annual temperature ranges from −13.5 to 44 °C and the average annual rainfall is 3497 mm  test was used to assess relationship between risk factors and prevalence of IPIs. Crude Odds Ratio (COR) and 95% confidence interval (CI) were performed to determine the strength of the relationship between infection and risk factors. A P-value less than 0.05 were considered statistically significant.The date collected using questionnaires and parasitological examinations were analyzed using SPSS Ver. 20  statistical software. Chi-square  were males. The mean age of the study subject was 12.3 yr old (range 7–15 yr). According to their residence, 190 (47.5%) were from urban and 210 (52.5%) schoolchildren were from rural areas. The study population consisted of students from primary and secondary schools with the same percent of 200 (50%) .P>0.05) , and the least encountered protozoa were E. hystolitica (0%) (P>0.05) (P>0.05) . Five maica (0%) . Among 1ica (0%) . Among a(P>0.05) .P<0.05). Intestinal protozoa infections were independent of hand washing habit after toilet, parent’s educational status, occupation of parents, and grade of students (P>0.05).The results of the personal questionnaire were used to determine possible risk factors for IPIs prevalence. In univariate logistic regression analysis of risk factors potentially associated with protozoa infection prevalence , signifiThis study aimed to assess the prevalence of protozoa infection and determinant factors among the schoolchildren of Sanandaj City. There is no previous study on the prevalence of protozoa infection in the study area.This research found that the overall prevalence of intestinal protozoa infection was 42.3%; no case of helminthes infection was detected. It was a bit lower compared with other studies conducted among schoolchildren in other places. For example, the prevalence of intestinal protozoa infection was reported 95.95% in Ouagadougou region, Burkina Faso; 63.2% in Amalapuram, India; and 69.2% in North Gondar region of Ethiopia –18. PrevThe result of the present study is also comparable with previous studies conducted in different parts of Iran. A high prevalence (50.57% and 47.7%) was recorded among schoolchildren in Urmia and at Khorasan province, Iran, respectively , 22. TheB. hominis followed by E. coli and E. nana infections with the prevalence of 21.3, 4.5, and 3.8%, respectively. The least encountered protozoa were E. hystolitica. This finding is similar with the results of Balcioglu and Ben-shimol, who reported that the most common protozoa were B. hominis with prevalence of 35 and 11.8% respectively   . In geneme (IBS) , 30.P>0.05); although male (42.5%) had slightly higher prevalence rate compared with female (41.3%), which is the same as reported in another study (P>0.05). Intestinal protozoa infections are more prevalent in the age groups 13–15 yr than the younger counterparts in the study area are, but there was no significant difference (P>0.05). This might be related to the high activities and behavior of children in this age group; they usually play and move around covering wider territory whereby the possibility of acquiring infections is increased. Nonetheless, the result was adverse with some of the reports, which showed a marginally higher positive rate among younger children . The difference in prevalence rate might be due to culture, living standard, and category of the study population. Difference due to gender was not observed in this study . This may arise from the contamination of water with animals and human waste discharging into the wells (P>0.05). This might be due to high knowledge of children about the fecal-oral transmission of protozoa infection through their unwashed hands and relatively high rate of positive responses by the students and their parents (P>0.05) (P>0.05). Moreover, no significant association was observed between grade level of students and rate of protozoa infections. This was in contrast to another study  , 37, 38.er study . The limB. hominis among schoolchildren in Sanandaj City, while helminthes were no found. Thus, it is crucial to have prompt implementation of educational programs for students and their families. Physicians, school teachers and mass media should create awareness among school-children and their parents for effective prevention and control of protozoan infections in the study area.A relatively high prevalence of intestinal protozoa infections was revealed especially


For additional comparison, adult mice (EtOHadults) with comparable drinking history (PND56-69) were subdivided into groups tested for anxiety/drinking either on PND70  or PND98 . Tissue from the nucleus accumbens shell (AcbSh) and central nucleus of the amygdala (CeA) was examined by immunoblotting for changes in the expression of glutamate-related proteins. EtOHadults exhibited some signs of hyperanxiety during early withdrawal (PND70), but not during protracted withdrawal (PND98). In contrast, EtOHadolescents exhibited robust signs of anxiety-l and depressive-like behaviors when tested as adults on PND70. While all alcohol-experienced animals subsequently consumed more alcohol than mice drinking for the first time, alcohol intake was greatest in EtOHadolescents. Independent of drinking age, the manifestation of withdrawal-induced hyperanxiety was accompanied by reduced Homer2b expression within the CeA and increased Group1 mGlu receptor expression within the AcbSh. The present data provide novel evidence that binge-drinking during adolescence produces a state characterized by profound negative affect and excessive alcohol consumption that incubates with the passage of time in withdrawal. These data extend our prior studies on the effects of subchronic binge-drinking during adulthood by demonstrating that the increase in alcoholism-related behaviors and glutamate-related proteins observed in early withdrawal dissipate with the passage of time. Our results to date highlight a critical interaction between the age of binge-drinking onset and the duration of alcohol withdrawal in glutamate-related neuroplasticity within the extended amygdala of relevance to the etiology of psychopathology, including pathological drinking, in later life.Binge-drinking is common in underage alcohol users, yet we know little regarding the biopsychological impact of binge-drinking during early periods of development. Prior work indicated that adolescent male C57BL6/J mice with a 2-week history of binge-drinking (PND28-41) are resilient to the anxiogenic effects of early alcohol withdrawal. Herein, we employed a comparable Drinking-in-the-Dark model to determine how a prior history of binge-drinking during adolescence (EtOH Adolescence is a critical period of accelerated neurodevelopment, which occurs between the ages of approximately 11–21 years in humans and conservative estimates of adolescence in rodents range from postnatal days (PNDs) 28–42 . During In both humans and animal models, adolescents typically consume larger quantities of alcohol than adults per drinking episode and adolescents also respond differently to alcohol than their adult counterparts . Adult dBinge-drinking is the most toxic pattern of excessive alcohol consumption and has been shown to produce a ‘kindling’ effect , wherebyStudies have revealed persistent alcohol-induced neurobiological changes within the extended amygdala – the subcortical macrostructure integrally involved in governing diverse emotional states . The extConsistent with existing human and animal research, previous work from our lab has demonstrated that adolescent mice exhibit minimal signs of negative affect during early (24 h) withdrawal, and are also resistant to changes in protein expression within the Acb  using thExperimental procedures were similar to those in our previous studies , 2016 anad libitum, except during the 2 h alcohol-drinking period. Adolescent drinkers (EtOHadolescents) began drinking at PND28, spanning the approximate period of early-mid adolescence in mice (adults) were PND56 at drinking onset and consisted of two subgroups: one group was behaviorally tested at PND70, after 1 day withdrawal (wd1EtOHadults), to match the age of the aforementioned EtOHadolescents mice and control for known age-related differences in basal behavior and protein expression (adults), to control for the effects of a 28-days withdrawal period upon behavior/protein expression. All control animals (PND70 and PND98) received only water prior to behavioral testing. Sample sizes were n = 9 for all groups. The experimental timeline for behaviorally tested animals is summarized in Figure 1.The animals used in this study were male C57BL/6J mice . Animals were housed in groups of 4 in standard Plexiglas cages, in a temperature-controlled vivarium (23°C), under a 12 h reverse light/dark cycle (lights off at 10 am). Food and water were available  in mice , and undpression . In a fon = 12/group) was used to generate brain tissue for immunoblotting, as a previous study from our laboratory showed that behavioral testing procedures induced cellular activation within Acb subregions .Beginning approximately 24 h following behavioral testing, all animals, including previously alcohol-naïve water drinkers, were subjected to 5 additional days of DID procedures in order to relate prior alcohol experience, age of first exposure, and affective state to alcohol consumption in adulthood.Behavioral testing consisted of the marble burying test, which was followed by the Porsolt forced swim test (FST). Both of these procedures were demonstrated to be particularly sensitive to the effects of alcohol withdrawal in our previous studies of mice , 2016. T2×1.25 cm tall) were placed in the animals’ home cage, six at each end. Animals were then left undisturbed for 15 min and video recorded for later analysis. At the end of the trial, a blind observer recorded the number of marbles at least 75% buried. Later, a blind observer reviewed the video footage and the latency to begin burying and the total time spent burying was recorded using a stopwatch.The marble-burying test was used as a measure of anxiety-induced defensive burying, as an increase in burying-related behavior serves as an index of anxiety  In our p++s), total time spent immobile, and the numbers of immobile episodes were monitored during a 6-min period using AnyMazeTM tracking software .The FST is a common measure of depression-like behaviors in laboratory animals, based on changes in active swimming ,b, 2001.The sucrose preference test is a common assay of anhedonia e.g., , used toFigures 7).Animals in the immunoblotting study were rapidly decapitated approximately 24 h following the final alcohol presentation to mirror the time-frame of the behavioral testing. Brains were removed and cooled on ice, then sectioned in 1 mm-thick coronal slice at the level of the striatum and amygdala. The AcbSh and CeA were bilaterally sampled from the slice located approximately 1.18 mm and -1.22 mm relative to Bregma, respectively, as shown in the mouse brain atlas of Western blotting was performed on whole tissue homogenates from the AcbSh and AcbC (AP +1.18 mm), and CeA and BLA (AP -1.34 mm)  as the between-subjects factor and day (14 days) as the within-subjects repeated measure to screen for potential group differences in alcohol consumption, which could confound alcohol-induced behavioral and neurobiological changes. The 5-day intake data was similarly analyzed with a drinking age  X day (5) repeated-measures ANOVA. A repeated measures ANOVA was also used determine if there was an effect of age/prior alcohol experience on the preference for a particular alcohol concentration, with drinking age  as the between-subjects factor and both day (14 or 5 levels) and concentration  as the within-subjects factors.Alcohol intake data from the 14-day drinking period were analyzed with a repeated measures analysis of variance (ANOVA), with drinking age (EtOHadolescents or wd1EtOHadults) as the between-subjects factor, and Tukey’s post hoc comparisons when appropriate; α = 0.05. All comparisons between wd28EtOHadults and age-matched control animals were conducted using independent samples t-tests with Bonferroni corrections for multiple comparisons, as these animals were run as a separate follow-up to the animals tested at PND70. Paired-samples t-tests were used to compare the average consumption during the first and second rounds of drinking in alcohol-experienced animals.Behavioral data for animals tested at PND70 were analyzed using between-subjects ANOVAs, with drinking  univariate ANOVA, while that for the animals subjected to our single-bottle procedure were analyzed using unpaired-samples t-tests. For all analyses, statistical outliers were identified using the ±1.5∗IQR rule and omitted from analyses. There were no statistical outliers excluded from the behavioral data. Outlier exclusion resulted in n’s of 10–12 per group for the immunoblotting data . All statistics and calculations were performed using SPSS v.21 statistical software.The immunoblotting data for the animals subjected to our 4-bottle-choice drinking procedures were analyzed using a drinking age  = 0.14, p = 0.87], there was a significant age × day within-subjects interaction . Further analysis revealed that over days 1–7, wd1EtOHadults drank more alcohol than EtOHadolescents (p = 0.001) but the converse occurred over days 8–14 (p < 0.001). This shift was reflected by a similar drinking-age × concentration × day interaction  for concentration preference, with EtOHadolescents exhibiting greater preference for lower concentration during the first week and a shift to a preference for higher concentration during the second week compared to wd1EtOHadults (Table 1). The repeated measures ANOVA also showed a significant drinking-age × concentration interaction  and post hoc analysis revealed that wd1EtOHadults had a lower preference for the 5% concentration and a higher preference for the 20% concentration compared to EtOHadolescents . There was a trend toward higher preference for the 40% in adolescents compared to adults (p = 0.078). During the subsequent 5-day drinking period, there were no significant main effects or interactions between age/prior alcohol experience or concentration (p’s > 0.10).Although the repeated measures ANOVA showed no between-subjects differences in the total amount of alcohol consumed by EtOHF = 0.39, p = 0.68] and an overall analysis of all alcohol-drinking animals revealed no differences between cohorts used for behavioral testing or tissue collection .There were no differences in alcohol intake amongst the animals used for tissue collection  and post hoc analysis revealed that while wd1EtOHadults showed increased sucrose preference , EtOHadolescents showed decreased preference (p = 0.04).The ANOVA showed significant group differences in sucrose preference , the latency to start burying , and total number of marbles buried . Both wd1EtOHadults and EtOHadolescents spent more time burying compared to water controls . EtOHadolescents also had a shorter latency to start burying (p = 0.022) and buried more marbles overall (p = 0.001). However, wd1EtOHadults did not differ significantly from controls on these factors (p’s > 0.1). There were no differences between wd28EtOHadults and age-matched control animals on any behavioral factor tested .In the marble burying test, there were significant group differences in total time spent burying , total time spent immobile , and the latency to first immobility . Both wd1EtOHadults and EtOHadolescents had significantly fewer immobile episodes  compared to control animals, but EtOHadolescents spent significantly more time immobile (p = 0.008), while adults spent less (p = 0.04). EtOHadolescents also had a shorter latency to first immobility (p < 0.001) but wd1EtOHadults did not (p > 0.20). Despite having fewer immobile episodes, EtOHadolescents spent more time immobile, compared to control animals, thus reflecting an overall increase in immobility with longer time spent immobile per episode.In the FST, there were group differences found for the number of immobile episodes . Post hoc tests showed that both wd1EtOHadults and EtOHadolescents consumed more alcohol than first-time drinkers . Additionally, EtOHadolescents drank more than wd1EtOHadults (p = 0.003). Both wd1EtOHadults and EtOHadolescents also exhibited higher average alcohol consumption overall compared to their previous 14-day average . There was no difference in intake between wd28EtOHadults and PND98 water control animals  and no increase in intake between the 14- and 5-day drinking period in wd28EtOHadults .During the subsequent 5-day drinking period following behavioral testing, the repeated measures ANOVA showed a significant effect of prior alcohol experience  and mGlu5 . Post hoc analysis showed that EtOHadolescents had increased mGlu1 expression relative to water controls (p = 0.04), with a similar trend seen in wd1EtOHadults (p = 0.09). wd1EtOHadults, but not EtOHadolescents, showed a significant increase in mGlu5 expression . There were no group differences in Homer2b expression within the AcbSh (non-significant immunoblotting results are from the AcbSh and CeA are summarized in Table 4).In the AcbSh, there were significant group differences in mGlu1 expression  and Homer 2b . Post hoc testing showed that both EtOHadolescents and wd1EtOHadults had decreased Homer2b expression relative to water controls . EtOHadolescents, but not wd1EtOHadults, showed a significant decrease in mGlu1 relative to water controls . There were no group differences found in mGlu5 expression (Table 4). There were no significant differences in mGlu1, mGlu5, or Homer2 within the AcbC or BLA .There were significant group differences in mGlu1 expression within the CeA  in alcohol-experienced animals versus water-drinking controls  and also observed a trend toward reduced CeA Homer2 expression  .Finally, when the immunoblotting data for the adult mice drinking under our single-bottle paradigm were compared, we replicated the reduction in CeA expression of mGlu1  withdrawal that are not apparent in adolescent drinkers . In the stressor , 2017. wh humans  and animh humans  with a hmeasures . As the measures , but alsadolescents exhibited signs of both hyperanxiety and depression during protracted withdrawal. In fact, EtOHadolescents demonstrated increased burying behavior across all measures in the marble-burying test and exhibited greater immobility in the FST, relative to wd1EtOHadults. Although general locomotion was not assessed in this study, it is unlikely that the FST results are attributable to suppressed locomotor activity, given the vigorous burying behavior exhibited in the marble-burying test. Based on conventional interpretations of the FST, this increased immobility is indicative of depressive-like behavior. Consistent with this interpretation, EtOHadolescents also showed significantly lower sucrose preference relative to both wd1EtOHadults and water control animals, supporting the presence of an anhedonic state. Interestingly, the difference in sucrose preference between EtOHadolescents and wd1EtOHadults suggests that an alcohol-induced preference for sweet liquids is either absent in EtOHadolescents or is masked by the manifestation of anhedonia.In contrast to adults with a 2-week binge-drinking history, EtOHadolescents consumed significantly more than wd1EtOHadults, despite the fact that wd1EtOHadults were earlier in withdrawal, when the presence of an alcohol deprivation effect is typically more pronounced .Within the CeA, EtOH the CeA , they co the CeA  and othe the CeA  indicati the CeA  and furt the CeA  reflecteThe functional relevance of the observed reduction in CeA mGlu1/Homer2 expression remains to be determined, particularly considering that negative affect is classically associated with amygdalar hyperactivation . HoweverAs the present study assayed protein expression in whole-cell homogenates, the site-specificity of these changes  remains to be determined. Nevertheless, the work of This study provides further basic science evidence to support a causal relationship between adolescent binge-drinking and negative outcomes manifested during protracted withdrawal in adulthood. Despite apparent insensitivity to the negative affective consequences of drinking during acute withdrawal, this study indicates that adolescent binge-drinkers are uniquely vulnerable to the latent maladaptive effects of alcohol upon emotionality that manifest in later withdrawal and shows that even a 2-week history of binge-drinking during the adolescent phase of neurodevelopment can have profound and enduring effects upon negative affect and subsequent drinking behavior, which are temporally related to molecular anomalies within brain regions regulating emotionality and negative reinforcement. This combination of negative affect and increased drinking likely contributes to the predisposition toward alcohol abuse and alcoholism later in life. Alcohol-induced dysregulation within extended amygdala structures regions offers a potential neurobiological correlate for the high comorbidity between substance abuse and mood disturbances. Additional research is necessary to characterize the progression and duration of these changes throughout the course of withdrawal in order to further our understanding of the ontogenetic differences in the etiology of alcoholism and its high rate of comorbidity with affective disorders.All of the research described in this report was approved by the Institutional Animal Care and Use Committee of the University of California, Santa Barbara.KL, MC, NS, and KS conducted the experiments. KL and KS analyzed the data. KL composed the manuscript. MC, NS, and KS edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arteriovenous malformations (AVMs) are characterised by tangles of dysplastic blood vessels which shunt blood from arteries to veins with no intervening capillary bed. It is not known at what stage of development and differentiation, AVM vessels became aberrant. To address this, we have analysed the expression of vascular differentiation, vascular maturation and brain capillary specific genes in AVM nidus.HEY2, DLL4, EFNB2, and COUP-TFII), vascular maturation (ENG and KLF2) and brain capillary specific genes (GGTP and GLUT1) on ten surgically excised human brain AVMs and ten normal human brain tissues.We performed immunohistochemistry and western blot analysis of vascular differentiation  increase in expression of these proteins in AVM vessels compared to control vessels. These findings were further confirmed by western blot analysis and found to be statistically significant (P < 0.0001 and P < 0.001) for all proteins except Hey2. Both immunostaining and western blot analysis revealed that AVM vessels express GGTP and GLUT1, markers specific to brain capillaries. Immunofluorescent staining demonstrated that expression of KLF2, a vascular maturation marker is significantly (P <0.001) decreased in AVM vessels and was further confirmed by western blot analysis (P < 0.001). Immunohistochemical and western blot analysis demonstrated that another vascular maturation protein Endoglin had high expression in AVM vessels compared to control vessels. The results were found to be statistically significant (P < 0.0001).Our findings suggest that vascular structures of AVMs co-express markers specific for arteries, veins and capillaries. We conclude that AVM nidus constitutes of aberrant vessels which are not terminally differentiated and inadequately matured. Arteriovenous malformation (AVM) of the brain is a devastating vascular malformation characterized by direct connections of dilated arteries and veins and a dearth of the normal capillary system. Feeding artery, draining vein and nidus are the three parts of the AVM lesion. These lesions can occur in any of the four lobes of the brain and patients with AVM present symptoms such as head ache, seizures, hemorrhage etc. Based on its location, size of lesion and type of venous drainage, surgical or nonsurgical treatment strategies are selected for AVM cure .de-novo formation of AVM have challenged the view on its congenital origin [Despite much understanding on normal vascular development, mechanisms in the formation of discrete AVM lesions are unclear. Reports on the l origin . Previoul origin . VasculaVarious hierarchical signaling molecules are involved in the early and late stages of vascular development. The initial stage of vascular development consists of the formation of a primitive vascular tube made of a single layer of endothelial cells. Predetermined genetic factors expressed in the endothelial cells decide the fate of the tube to develop as an artery or a vein , 5. The TGF-β and PDGF- β are two major genes involved in this process. TGF-β has two types of receptors, type I activin-like kinase (ALK) and type II serine/threonine kinase receptors. Both type1 and type II receptors of TGF-β interacts with type III co-receptor, Endoglin. Type I receptor, ALK and the co-receptor Endoglin are essential for promoting smooth muscle cell recruitment. Arterio-venous identity is lost in both ALK and Endoglin null embryos. Another paracrine signaling established between smooth muscle cells and endothelial cells through platelet- derived growth factor B (PDGF- β) also advances smooth muscle cell recruitment [The later stage of vascular development includes blood vessel maturation. A reciprocal interaction established between endothelial cells and smooth muscle cells is essential for vessel maturation. This interaction, recruits smooth muscle cells around the vascular tube and thus stabilize the blood vessel. ruitment , 9. Receruitment .In the normal brain, nervous and vascular systems maintain an inseparable relationship at cellular, molecular and anatomic levels –13. EndoHEY2, DLL4, EFNB2, COUP-TFII) and late phase of vascular development (ENG and KLF2), in arteriovenous malformations in the brain. We also investigated whether AVM nidus expresses brain capillary specific proteins such as GGTP and GLUT1.In this report, we present the result of a comprehensive analysis of expression of genes involved in both early . Ethical approval was obtained from Institutional Human Ethics Committees of SCTIMST (SCT/IEC/629/JUNE-2014) and Rajiv Gandhi Centre for Biotechnology (IHEC/01/2015/05), Thiruvananthapuram. Written informed consents were obtained from the study patients prior to surgery for the collection of tissue samples.Patients were in the age group of 22–35 years. Clinical profile of patients is given in AVM and control brain tissues were divided into three parts and stored in 10% neutral buffered formalin, 4% Paraformaldehyde (PFA) and RNA later solution. Tissues stored in 10% neutral buffered formalin were used for histological, immunohistochemical analysis and those stored in 4% PFA were used for immunofluorescence analysis. Proteins were isolated from tissues stored in RNA later solution.After two days of formalin fixation, tissues were processed and embedded in paraffin. Tissue sections of 5 µm thickness were cut from paraffin blocks and used for H & E staining , EVG staWe have done IHC scoring by manual method, as it was reported to be as significant as automated methods of IHC analysis . H-scoriAfter overnight fixation of tissues in 4% PFA, tissues were suspended in increasing concentrations of sucrose in phosphate buffer saline . Tissues were embedded in Optimal Cutting Temperature Compound (OCT) and were stored at -80°C. Later, sections of 5 µm thickness were taken using a cryotome. On the day of the staining, microscopic slides with tissue sections were fixed in acetone for one minute. Antigen was retrieved in citrate buffer, non specific binding was prevented by blocking with 3% bovine serum albumin (BSA). Immunofluorescence staining was attempted for KLF2  antibody. Primary antibody incubation was followed by addition of Alexafluor conjugated rabbit secondary antibody . Nuclear staining was done using Hoechst 33342 . Later, sections were mounted in fluorochrome and excited for fluorescence using a fluorescent microscope .Proteins were isolated from whole tissue using radio immunoprecipitation assay (RIPA) buffer as described earlier . ProteinAll results are expressed as mean ± SEM. Graph Pad PRISM version 6.07 was used for statistical analysis. Student’s t- test was done to analyze difference in protein expression levels. P value less than 0.05 was considered as statistically significant.In control tissues, blood vessels and the surrounding neural tissue had normal histological features . Histolo also known as CD-31 is an endothelial cell specific marker. In our study, we observed that it is specifically expressed in the endothelial cell lining of AVM and control vessels Figs . SecondaImmunohistochemical staining with α-SMA and PECAM-1 antibodies in AVM vessels revealed that endothelial cells of certain AVM vessels express α-SMA . We found differential expression of genes involved in the early and late stage of vascular development in these malformed vessels.ENG and KLF2 [Vascular development consists of an early stage of vasculogenesis and a later stage of vascular maturation. The fate of an endothelial cell, either to be an artery or to be a vein is determined in the early stage of vascular development , 5. Exprand KLF2 –10.Histological analysis showed that AVM nidus is a collection of aberrant vascular structures depicted as hypertrophic veins: with increased thickness of media layer; arterial like structures with reduced tunica media layer and small arteriole and venules like vessels. In AVM nidus, EVG staining revealed the presence of large arteries and dysplastic veins with discontinuous internal elastic lamina layer.α-SMA staining demonstrated aberrancy in the smooth muscle layer of AVM associated blood vessels. We observed that endothelial cells of certain AVM vessels expressed α-SMA. We confirmed our above finding by staining those blood vessels with PECAM-1, a membrane glycoprotein specifically expressed in endothelial cells. Moreover, there are previous reports on α-SMA expression in endothelial cells . In certEphrinB2 is the key determinant of arterial endothelial cell fate . The EphVascular fate of an endothelial cell for venous differentiation is determined by COUP-TFII , 36, 37.COUP-TFII gene leads to over expression of Notch signaling pathway genes [It is well known that Notch signaling and subsequent arterialisation pathways are suppressed by COUP-TFII , 37. In HEY2, DLL4, EFNB2 and vein specific gene COUP-TFII in endothelial cells of AVM vessels as observed in our study is a novel finding. Simultaneous expression of these genes in the endothelial cells of the nidus could be the basis for deregulation of artery-vein fate determination in AVM. We speculate that in the endothelial cells of AVM, default mechanisms of artery and vein fate determination are disrupted and result in the genesis of blood vessels, which are neither arteries nor veins. A recent report on defective characteristics of AVM derived endothelial cells supports our hypothesis [Previous studies had identified that endothelial cell of AVM vessels expresses either artery or vein marker , 33, 34.pothesis , 40.et al where they reported that AVM nidus structures especially AV shunts can be formed from preexisting normal capillaries. They identified that Notch4 over expression in neonatal mice leads to the formation of AV shunts from normal capillaries [A critical feature of AVM is that it is characterized by a complex nidus structure, instead of a normal capillary system . To undeillaries . On anotillaries –48. Exprillaries , 18, 43.ENG gene, there is a 50% reduction of Endoglin in AVM blood vessels compared to normal vessels [Endoglin maintains vascular integrity by promoting recruitment of vascular smooth muscle cells towards the endothelial cell network , 9. In a vessels . Interes vessels . HoweverKLF2 gene is reduced in conditions of disturbed flow of blood when compared to conditions of normal laminar flow [KLF2 induces recruitment of smooth muscle cells by PDGF- βduring vascular maturation. KLF2 deficient embryos have normal endothelial cell development pattern. Smooth muscle cell recruitment is defective in these embryos and thus vascular integrity is affected . A novelnar flow , 52. As Our study demonstrates that AVM structures have overlapping features of vascular identity. AVM nidus structures express markers specific to arteries and veins as well as capillaries. Genes involved in vessel maturation are also differentially expressed in AVM structures compared to control vessels. Thus AVM vessels can be considered as aberrant vessels, which are not terminally differentiated and inadequately matured possibly because of a developmental arrest. This study, to our knowledge, is the first comprehensive gene expression analysis of vascular structures of AVM.S1 Fig(A) α-SMA staining is seen in the endothelial cell lining of AVM nidus (arrow) and there is much less expression of α-SMA (arrowhead) in tunica media layer. 20X magnification.  40X magnification images of α-SMA staining showing circular (arrow) and longitudinal smooth muscle cells (arrowhead) in AVM vessels. (E) PECAM-1 is expressed in the endothelial cell lining of AVM vessels. (B) Secondary isotype controls for PECAM-1 staining. 20X magnification.(TIF)Click here for additional data file.S2 Fig(A) Hey2 is expressed in the endothelial cell lining (arrowhead) of AVM vessels. (B) Hey2 is expressed in micro vessels in control brain (arrowhead). Neuronal cells in control brain express Hey2 (arrow). (C) COUP-TFII is seen in the endothelial cell lining (arrowhead) of AVM vessels. (D) In control brain tissue GLUT1 expression is much less in the artery (arrowhead). (E) There is no expression of GGTP in control artery (arrowhead). (F) Endoglin is highly expressed in the microvessels in control brain (arrowhead). Magnification (A) and (C) 40X; (B and F) 10X and (D and E) 20X.(TIF)Click here for additional data file.S3 FigThere is less expression of KLF2 in AVM vessels . Magnification 60X.(TIF)Click here for additional data file.


Plasmodium malariae is the only human malaria parasite species with a 72-hour intraerythrocytic cycle and the ability to persist in the host for life. We present a case of a P. malariae infection with clinical recrudescence after directly observed administration of artemether/lumefantrine. By using whole-genome sequencing, we show that the initial infection was polyclonal and the recrudescent isolate was a single clone present at low density in the initial infection. Haplotypic analysis of the clones in the initial infection revealed that they were all closely related and were presumably recombinant progeny originating from the same infective mosquito bite. We review possible explanations for the P. malariae treatment failure and conclude that a 3-day artemether/lumefantrine regimen is suboptimal for this species because of its long asexual life cycle. Plasmodium falciparum. This trend has often been associated with increased recognition of the burden of malarial disease caused by the other Plasmodium species  has been central to this approach, with proven efficacy against multidrug-resistant P. malariae is well-recognized . By using whole-genome sequencing of isolates from the initial and the recrudescent infections, we show that the 2 major P. malariae haplotypes, constituting ≈90% of the parasite load in the initial infection, were cleared successfully by AL, whereas a third haplotype, constituting a minority subpopulation in the initial infection, survived and recrudesced.We report a case of a P. malariae and negative for all other Plasmodium species  with all stages of asexual development visible on the blood film , who had been a resident in Australia for 5 years sought care at Royal Darwin Hospital  on March 1, 2015, with a 4-day history of fevers and headaches. He had returned to Australia 56 days previously after a 2-week trip to Uganda visiting friends and relatives , but by day 11, his repeat blood film examination and his aldolase rapid diagnostic test results were negative.P. malariae with a parasite count of 3,332/µL  and recrudescent (PmUG02) infection  Table 1,eviously  Table 2.eviously  Figure 2Searching solely for SNPs that distinguish PmUG01 and PmUG02, we identified 2,631 variants after filtering  Table 2.Comparison of the distribution of the alternate allele frequencies throughout the genome of the initial and recrudescent strains  revealedP. malariae samples  also increased in frequency in the recrudescent strain. Testing by using additional  samples  Figure 5To clarify the relationships of the different haplotypes with each other, we classified every genome region by whether any of the 3 haplotypes were identical to each other . Approximdr2) gene, 1 of which was in the ABC transporter domain, and 2 in the ABC transporter domain of ABC transporter C family member 2, present in the recrudescent strain (H2) but not the other strains , all 4 were microscopy negative at day 28, with no follow-up beyond this time , the evidence that latent exoerythrocytic stages do not occur in P. malariae is limited  occurs in 3 human malaria parasite species (Although inadequate drug absorption , panel BP. falciparum, resistance to artemether is acquired through mutations in the propeller domain of K13 (P. falciparum resistance to lumefantrine is associated with mutations and copy number variation in the Pfmdr1 gene (mdr2, potentially involved in artemisinin resistance (P. falciparum (P. malariae, and rapid elimination of lumefantrine all might have contributed to the observed treatment failure in our patient.Finally, the minority clone with H2 haplotype might have recrudesced because of a fitness advantage over the other clones/haplotypes , panel GP. malariae recrudescence occurring in a non–malaria-endemic country after adequately administered AL. Whole-genome sequencing data revealed that the monoclonal recrudescence consisted of a minor haplotype that accounted for ≈10% of the initial infection and that all the haplotypes in the initial infection were related to each other and therefore probably originated from the same infective mosquito bite. Although the haplotypes were closely related, the genomic data suggest that >4 parental haplotypes were ingested by the mosquito, indicating considerable diversity and transmission of P. malariae. This case raises concerns about the adequacy of ACTs with a short half-life partner drug, such as AL, in treating P. malariae infections and suggests that optimal ACTs to treat P. malariae should include a slowly eliminated partner drug. Our findings reinforce the importance of a longer duration of follow-up monitoring of patients infected with P. malariae for late recrudescence.In conclusion, we have described a case of Plasmodium malariae infection diagnosed and treated at Royal Darwin Hospital, Darwin, Northern Territory, Australia, March–April 2015.Extended methods and additional results for case of a patient with recrudescence