Gorgonia ventalina with copepods of the genus Sphaerippe from the Caribbean and (2) the scleractinian coral Stylophora pistillata with copepods of the genus Spaniomolgus from the Saudi Arabian part of the Red Sea. We show that bacterial communities in these two systems were substantially different with Actinobacteria, Alphaproteobacteria, and Betaproteobacteria more prevalent in samples from Gorgonia ventalina, and Gammaproteobacteria in Stylophora pistillata. In Stylophora pistillata, normal coral microbiomes were enriched with the common coral symbiont Endozoicomonas and some unclassified bacteria, while copepod and gall-tissue microbiomes were highly enriched with the family ME2  or Rhodobacteraceae. In Gorgonia ventalina, no bacterial group had significantly different prevalence in the normal coral tissues, copepods, and injured tissues. The total microbiome composition of polyps injured by copepods was different. Contrary to our expectations, the microbial community composition of the injured gall tissues was not directly affected by the microbiome of the gall-forming symbiont copepods.Corals harbor complex and diverse microbial communities that strongly impact host fitness and resistance to diseases, but these microbes themselves can be influenced by stresses, like those caused by the presence of macroscopic symbionts. In addition to directly influencing the host, symbionts may transmit pathogenic microbial communities. We analyzed two coral gall-forming copepod systems by using 16S rRNA gene metagenomic sequencing: (1) the sea fan  The structural basis of reefs is a complex system, termed coral holobiont, consisting of a core animal, coral polyp, symbiotic unicellular algae of the genus Symbiodinium, fungi, protists, viruses and prokaryotes – the coral microbiota4. With the development of culture-independent methods of high-throughput genome sequencing, the diversity and importance of the coral microbiome for the holobiont fitness has become evident8, although the direct mechanisms of interactions and functions of the microbial community are not well understood9. It has been shown that bacteria associated with corals play a role in nitrogen fixation10, synthesis of metabolites such as vitamins11, cycling of carbon, sulfur, and phosphorus9, and resistance to diseases, due to the antibiotic production and to the competition with pathogens for nutrients and space12. Moreover, environmental changes have been shown to cause shifts in the coral microbiome composition. Such shifts can be vital for fast adaptation to changing environmental stress conditions12 and play a role in the evolution of a coral holobiont7. On the other hand, changes induced by the stress can shift composition of the coral microbiome toward coral pathogens14. In particular, considerable changes in the microbiome composition and metabolism accompany bleaching18, and the microbiome composition is predictive of the corals stress tolerance19. Hereby, the establishing of a “healthy” coral microbiome and finding potential etiological agents or groups of agents associated with coral mortality are issues of importance, especially due to the recent degradation of coral reefs induced by human activities and climate shifts23. It is possible, however, that coral mortality is associated not with one group of agents but with the loss of a stable, healthy microbiome and subsequent, diverse stochastic changes in the microbial communities dominated by opportunistic bacteria or r-strategists24.Coral reefs are one of the most complex marine ecological systems, with biodiversity comparable to that of the rainforests30 and are usually dominated by Proteobacteria, mainly Gammaproteobacteria and Alphaproteobacteria33, with different dominating representative genera and species from these classes32. Bacteria of the genus Endozoicomonas  have been shown to reside in diverse marine hosts varying from sponges to fish all over the world34 while being one of the dominant associated taxa of the stony coral Stylophora pistillata in the Red Sea35 and in temperate gorgonians39. Other Oceanospirillales, such as those of the family ME2, and some Spirochaetales have been shown to be the dominant associated taxa in the precious deep-water octocoral Corallium rubrum 40.Based on the 16S rRNA gene amplicon massive sequencing, it has been shown that coral prokaryotic communities exhibit almost no overlap with dominating bacterial taxa in the surrounding reef waterRhodobacteraceae43 , Vibrio spp.48, Bacteriodetes13, Cyanobacteria , Fusobacteria13, Verrucomicrobiaceae47 and to changes in the species interactions and richness53. Opportunistic bacteria can come from a variety of sources — they can be minor groups present in the healthy coral, be transmitted with water54, or come from adjacent algae microbiomes, being advantageous for algae in the alga-coral competition55. One more source of such bacteria can be provided by invertebrates, like copepods. It has been shown that the microbiomes of free-living copepods, which, similar to the microbiomes of corals, are dominated by Gammaproteobacteria, Firmicutes, Actinobacteria, Cyanobacteria, and other Proteobacteria56, may contain bacteria that are potentially pathogenic for corals, such as Vibrio spp.58. White plague of corals is associated with small crabs of the family Cryptochiridae, which live in small pits or galls inside the host corals59, and both the crab and diseased coral microbiomes are dominated by Alphaproteobacteria, mainly Roseobacter, unlike the microbiomes of healthy corals60).Сoral injuries and diseases often lead to shifts in the coral microbial community towards a higher ratio of opportunistic or potentially pathogenic bacteria, like 62. Some of the symbiotic copepods have been reported in galls or cysts of corals66. However, the potential impact of copepods inducing galls to the state of coral hosts remains unknown. Some copepods could potentially act as vectors for the transmission of coral or fish pathogens68, which in theory might confer benefits for copepods, helping them to overcome host protection mechanisms. In search of such pathogens or microbial complexes specific to symbiotic copepods and coral galls induced by them, we applied 16S rRNA gene metagenomic sequencing to two recently discovered copepod-coral systems  with copepods of the genus Sphaerippe Grygier, 1980 (Copepoda: Poecilostomatoida: Lamippidae) from the Caribbean island Sint Eustatius66 and the scleractinian coral Stylophora pistillata Esper, 1797  with the copepods of the genus Spaniomolgus Humes & Stock, 1972 (Copepoda: Poecilostomatoida: Rhynchomolgidae) from the Saudi Arabian part of the Red Sea65. In both systems studied here, copepods were located within a gall or a modified polyp66. In our knowledge, this is the first detailed analysis of a microbial community of copepods as symbionts of corals. Our expectation is that if copepods are associated with the spread of disease to the coral, the microbiome of the symbiont copepod should share more bacterial species with the diseased coral gall tissue than with the healthy tissues of the same coral colony.Diverse and abundant symbiotic copepods are found associated with most of the inspected host corals, but the type and strength of the associations are not well-studied).Overall, about 150,000 reads were obtained per sample  after chimaera and error checking Table .Table 1SThe diversity of the prokaryotic communities expressed as three different metrics of alpha diversity, namely the number of OTUs per sample , the Shannon-Wiener index, and the Simpson index, was not statistically different between the healthy and diseased tissues, and symbiotic copepods for both coral species, nor between the species , with OTU-based Bray-Curtis distances between the species ranging from 0.64 to 0.96 and within the species from 0.48 to 0.93. No significant differences were observed between the types of substrate for the microbiome  , with distances between the substrates ranging from 0.48 to 0.93, and within from 0.51 to 0.84. The level of similarity between the microbiome types was the same for both coral host species .The community composition of the samples was significantly different between the coral host species , nor from the microbiome of the symbiotic copepod . Similarly, the microbial composition of the diseased tissues in Stylophora pistillata was neither significantly different from the healthy tissues in the same coral colony , nor from the microbiome of the symbiotic copepod . Yet, a plot of the differences between the samples at the genus level suggests some potential effect of the microbiome of the symbiotic copepod on the gall tissues, at least for one gall tissue of Stylophora pistillata that clusters with the microbiomes of the symbiotic copepods  and Gorgonia ventalina (Alcyonacea) were collected at the Saudi Arabian Red Sea  and the Dutch Caribbean island Sint Eustatius , respectively74.The corals of Each coral was photographed underwater, placed in a separate plastic bag and brought to the surface. The parts of corals with galls were dissected with sterilized needles in sterilized Petri dishes using dissecting microscope Olympus SZX 7, then rinsed several times and preserved in a 95% solution of ethanol. One copepod individual found in the gall was selected per gall of the coral colony. The copepods have been rinsed in ethanol, some of the copepods present in the samples have been inspected by scanning electron microscopy (SEM) in order to detect the presence of microbes.75.For scanning electron microscopy (SEM) analyses, copepods were dehydrated through graded ethanol concentrations, critical point dried, mounted on aluminum stubs, coated with gold, and examined in a JEOL scanning electron microscope at the Laboratory of Electron Microscopy DNA from the ethanol-preserved copepods, galls, or a normal coral tissue was extracted simultaneously using a standard silica-based DNA extraction kit . The DNA extraction was conducted according to the manufacturer’s protocol for the fresh blood samples.76. The 515F primer was labeled with sample-specific Multiplex Identification DNA-tags (MIDs)  and 806R (5′-GACTACHVGGGTWTCTAAT-3′)s) Table .25 µl PCR reactions contained 12,5 µl 2x Taqman Environmental Mastermix 2.0 (ThermoFisher), 10 pMol/µl V4-F-MID primer and 10 pMol/µl V4-R-trP1 primer (Sigma Aldrich), 1 µl template DNA and PCR grade water. PCR was performed as follows: heated lid 110 °C, 95 °C × 10 mins, followed by 40 cycles of 95 °C × 15 s, 50 °C × 20 s, 60 °C × 30 s, followed by 60 °C × 4 mins and held at 12 °C.Negative controls with PCR grade water occurred without amplification. PCR products were quantified in the QIAxcel (QIAGEN). PCR products were pooled in an equimolar concentration. The pool was cleaned using AMPure magnetic bead-based purification system (Beckman Coulter). The clean pool was quantified using the Bioanalyser (Agilent). The amplicon library was sequenced using an Ion 314 Chip by an Ion Torrent Personal Genome Machine (Life Technologies) at the Naturalis Biodiversity Center following manufacturer protocol.77. Long reads were trimmed to 300 bp, first 10 bp and low quality (Phred <20) ends of reads were trimmed, and then reads shorter than 140 bp were removed with Trimmomatic78. On average 8.5% of reads were removed. For OTU definition we used CD-HIT-EST79 with similarity level 0.987 that was more restrictive than the commonly used 0.97 threshold and was more likely to group together only reads from the same species81. All single-read OTUs were filtered out (approximately 97500 OTUs accounting for 4.3% of reads). We used two common approaches for accounting for different sequencing depth between samples: (1) normalizing OTU sizes by dividing them by the total number of reads in each sample, and (2) construction of rarefied samples that contained equal numbers of reads by random sub-sampling of the reads. Both methods produced similar results and following82 we used normalized OTUs for the beta-diversity analysis. Since some methods for estimating alpha-diversity require absolute numbers, we used rarefaction for all such analyses. Representative sequences from each OTU were scanned for possible chimeras with DECIPHER83 and 3056 minor OTUs  were marked as such. We assigned taxonomy to each read the Mothur software package84 with standard parameters and SILVA (version 128) as the reference database85. If at least 75% of reads from an OTU shared the same taxonomy, it was transferred to the whole OTU. All OTUs classified as chloroplast or eukaryote-related were removed. Rarefaction curves, alpha and beta diversity were calculated using the package vegan for R86. To estimate the alpha diversity, we used the Shannon–Wiener index, the Simpson index, and the observed number of species. For the beta diversity analysis, we used the Brain-Curtis dissimilarity and the Hellinger distance, the latter better accounting to low-abundant OTUs. The hierarchical clustering and principal component analyses were performed with built-in R functions based on the OTU or taxon distribution between the samples. Significance of differences between the microbiome compositions was tested with ADONIS implemented in the package vegan for R. The models included the coral holobiont species, the type of substrate for the microbiome , and the statistical interaction between the coral species and the substrate type.The quality of reads was analyzed with FastQChttp://www.ncbi.nlm.nih.gov/bioproject/433804). Other data are available in the Supplementary Data file.Sequence data determined in this study are available at NCBI under BioProject Accession PRJNA433804 (Supplementary information


At parallel fibre terminals in the cerebellar cortex, glutamate released outside of the active zone can activate AMPA receptors on juxtaposed Bergmann glial cell processes. This process is termed “ectopic” release, and allows for directed transmission to astroglial cells that is functionally independent of synaptic transmission to postsynaptic Purkinje neurons. The location of ectopic sites in presynaptic terminals is uncertain. Functional evidence suggests that stimulation of parallel fibres at 1 Hz exhausts ectopic transmission due to a failure to rapidly recycle vesicles to the ectopic pool, and so would predict a loss of vesicles in the near vicinity of extrasynaptic glial processes. In this study we used this stimulation protocol to investigate whether the distribution of vesicles within the presynaptic terminal is altered after suppression of ectopic release. Stimulation at 1 Hz had only a minor impact on the distribution of vesicles in presynaptic terminals when analysed with electron microscopy. Vesicle number and terminal size were unaffected by 1 Hz stimulation, but the relative abundance of vesicles in close proximity to the active zone was marginally reduced. In contrast, the fraction of vesicles facing glial membranes was unchanged after suppression of ectopic transmission. 1 Hz stimulation also resulted in a small but statistically-significant increase in the distance between glial membrane and presynaptic terminal, suggesting withdrawal of glial membranes from synapses is detectable in ultrastructural anatomy within minutes. These results raise doubts about the location of ectopic release sites, but indicate that neuron-glial association varies on a dynamic time scale. Closesmission –8. Genetsmission –5.A series of studies have determined that transmission from presynaptic parallel and climbing fibre terminals to the extrasynaptic Bergmann glia is functionally independent of transmission to the postsynaptic Purkinje neurons. Unitary events are desynchronized , the magDespite this wealth of functional evidence for independent routes for transmission to neurons and glia, the location of ectopic release sites remains uncertain. Matsui et al.  reportedCollectively, these reports suggest that structural rearrangements of the presynaptic terminal and glial sheath, and redistribution of presynaptic vesicle pools, may accompany changes in the strength of ectopic transmission to Bergmann glia.In this study, we stimulated parallel fibres at different frequencies, to investigate how the ultrastructure of the parallel fibres synapses are affected by activity-dependent depression of ectopic transmission. We recorded from individual Bergmann glia with the whole-cell patch clamp technique, using Lucifer yellow to guide identification of stimulation sites in cerebellar slices post-fixation with electron microscopy, and so determine the abundance and distribution of presynaptic vesicles and extrasynaptic glial processes at synapses that had been stimulated at 0.033 Hz (which does not depress ectopic transmission), and 1 Hz (which does depress ectopic transmission). By comparison of synapses stimulated in these ways, we tested the prediction that depression of ectopic transmission would be associated with a loss of vesicles positioned at sites opposite extrasynaptic glial processes. We also examined whether depression of ectopic transmission was associated with a change in the coverage of synapses by glial processes.The results showed small but detectable changes in the fraction of vesicles located within close proximity of the active zone and an increase in the distance between terminal and glial envelope, in synapses stimulated at 1 Hz compared to 0.033 Hz. In contrast, there was no detectable difference in the fraction of vesicles juxtaposed opposite glial membranes, casting doubt of the identity of such vesicles as the hypothesised ectopic pool.Rats (age 16–20 days) were humanely killed by cervical dislocation. All experiments were performed according to policies on the care and use of laboratory animals of British Home Office and European Community laws. The University of Nottingham Animal Welfare and Ethical Review Body approved the experiments. All efforts were made to minimize animal suffering and reduce the number of animals used.2 (1), MgCl2 (1), MgSO4 (2), NaHCO3 (26), glucose (10), ascorbic acid (0.4), and NaH2PO4 (1.25). Slices were allowed to recover for 1 hour at 32°C, with a further 30 min at room temperature, in aCSF containing (mM): NaCl (126), KCl (3), NaH2PO4 (1.2), NaHCO3 (25), glucose (15), MgSO4 (2), and CaCl2 (2). For recording, slices were transferred to an immersion chamber and perfused with aCSF containing a lower concentration of MgSO4 (1 mM). All solutions were continuously bubbled with carbogen  throughout.Transverse cerebellar slices (300 μm) were prepared from 16- to 20-day old Wistar rats of either sex. Animals were humanely killed by cervical dislocation, decapitated, and the cerebellum rapidly excised and sliced using a vibrating microtome (Leica VT1000S) in chilled, sucrose-based artificial cerebrospinal fluid (aCSF) containing (mM): sucrose (206), KCl (2.8), CaCl4 (4), ATP (4), GTP (0.2), phosphocreatine (9), and pH to 7.4 with 1 M KOH, plus 1 mg/mL Lucifer yellow. Whole-cell voltage clamp recordings were made from Bergmann glia  somata in the Purkinje cell layer. Currents were low pass filtered at 4–5 kHz and sampled at 25 kHz, using Spike2 software . Series resistances ranged from 5 to 15 MΩ and were uncompensated.Borosilicate recording electrodes were manufactured as previously described . InternaParallel fibres were stimulated with a patch electrode (~1–2 MΩ) filled with bath solution and positioned in the midpoint of the molecular layer, connected to an isolated constant current stimulator . Stimulus was delivered as a pair of pulses with a 100 ms interval at a frequency of either 0.033 Hz or 1 Hz; a stimulus protocol that has been shown to be the most effective mechanism for depression of ectopic release , due to Samples were processed according to a protocol modified from Marra et al. . Followi4Cl, and washed three times in PBS. Slices were then returned to the recording chamber (prefilled with PBS) and orientated to match the image acquired at the outset of the protocol.Slices were rapidly fixed in a calibrated microwave  in a fixative solution containing 6% paraformaldehyde and 2% glutaraldehyde prepared in PBS. Slices were then blocked for 1 hour in 100 mM glycine solution, rinsed for 1 minute in 100 mM NHPBS was replaced with 1 mg/mL 3,3'-diaminobenzidine (DAB) solution prepared in oxygenated PBS and incubated in the dark for 10 minutes. This was replaced with fresh oxygenated DAB solution and the photoconversion reaction initiated by illumination with high intensity blue light through a 40X objective lens . The DAB solution was bubbled with carbogen throughout illumination via a fine bore capillary tube, and the conversion reaction time ranged from 10 to 45 minutes. Conversion reactions were monitored every 5–10 minutes and terminated when a defined dark region was visible on the slice . Slices Peak identification in frequency histograms was carried out in Matlab by curve fitting using a kernel distribution function with bandwidth determined using Silverman’s rule. Area under the fitted curve was estimated using a trapezoidal numerical integration at the location determined using the ‘findpeaks’ function.Slices were incubated for 1 hour in 1.5% potassium ferrocyanide/1% osmium tetroxide (wt/vol) in CB, washed with CB (5 × 10 minutes), and incubated for 1 hour in 1% osmium tetroxide in CB.en bloc for 1 hour in 4% uranyl acetate in 70% ethanol before being dehydrated in rising concentrations of ethanol . Slices were then further dehydrated by overnight incubation in a 1:1 solution of propylene oxide and resin . Resin infiltration was performed by transferring the samples into freshly prepared resin for a period of 12 hours after which the resin was changed and incubated for a further 12 hours. Samples were then flat embedded between two sheets of ACLAR® film  and cured for 48 hours at 60°C. The embedded sample was then affixed to a resin blank and sectioned from the photoconverted face using an ultramicrotome (70 nm thickness) and embedded in a copper mesh grid.Slices were then washed again with CB (3 × 10 minutes) before a 10 minute incubation in 50% ethanol. Samples were then stained Ultrathin samples were imaged by transmission electron microscopy  using the previous slice images to assist with locating the loaded cell in each slice. Ten high-power images of synapses located within the arborisation of the loaded Bergmann glial cell were captured for each ultrathin section, and analysed by researchers blinded to the stimulation paradigm employed for each sample.http://rsb.info.nih.gov/ij; Wayne Rasband, National Institutes of Health, Bethesda, MD). Freehand tools were used to trace the perimeter of each presynaptic terminal, the postsynaptic density, and extrasynaptic glial membranes that abutted the terminal  to compare the number of vesicles within <50 nm of active zone and glial facing membrane in terminals stimulated at 0.033 Hz and 1 Hz. Aggregate data are the mean ± s.e.m. of multiple cells as indicated in figure legends.To examine synapse anatomy before and after depression of ectopic transmission, we adopted the following strategy : a stimuSynapses within this stimulated region were identified manually, and images taken at 43,000X magnification . ConsistTo quantify vesicle distribution in detail, the periphery of presynaptic terminal was traced, the active zone identified as the region of the terminal directly apposed to the postsynaptic density, and the centre of each identifiable vesicle within the terminal was marked. Extrasynaptic glial process that surrounded the presynaptic terminal were also traced. This procedure yielded a map of the presynaptic terminal, with the active zone and glial-facing membranes colour coded . The Eucp > 0.2, single sample t test), whereas 1 Hz stimulation depressed it to 25% of initial amplitude   generatemplitude . This reStimulation at the different frequencies had no obvious impact on the gross anatomy of the terminals . The aveThe distribution of vesicles throughout the terminal was analysed in detail . For theIn contrast, the distribution of vesicles relative to the glial-facing membrane appeared to be a single population with a skewed Gaussian distribution . VesicleTo explicitly test the central prediction of our hypothesis–that glial-facing vesicles are the ectopic pool and that stimulation at 1 Hz depletes that pool–we quantified the number of vesicles within 50 nm  of the terminal membrane at both active zone and glial facing sites . This anp < 0.02, Fisher’s exact tests, Unstimulated slices appeared to have a larger fraction of vesicles within 50 nm of both the active zone and glial facing membrane following stimulation at either frequency Click here for additional data file.


SLC25A24) gene was suggested to be the common genetic basis of both syndromes, it has been proposed recently that they be integrated into a single disorder under the name of Fontaine progeroid syndrome (FPS).Gorlin–Chaudhry–Moss syndrome (GCMS) and Fontaine–Farriaux syndrome (FFS) are extremely rare genetic disorders that share similar clinical manifestations. Because a de novo missense mutation of the solute carrier family 25 member 24 (SLC25A24 analysis revealed a heterozygous mutation reported previously, NM_013386:c.650G > A, p.[Arg217His]. After screening her family for the identified mutation, she was confirmed as being a de novo case of FPS caused by an SLC25A24 mutation.A 9-year-old Korean girl presented with typical clinical features of FPS. She had generalized loose skin with decreased subcutaneous fat, skin wrinkling on the forehead and limbs, skull deformities and a peculiar facial appearance with microphthalmia and midface hypoplasia, anomalies of the digits and nails, a large umbilical hernia and a nearly normal developmental outcome. She exhibited prenatal and postnatal growth retardation together with short stature, and records showed that her height and weight were invariably under − 2.0 SD from birth to the age of 10 years. SLC25A24, as well as 10 years of clinical follow-up, including growth and developmental achievements.We describe a Korean girl with typical clinical findings of FPS and a de novo mutation in  Gorlin–Chaudhry–Moss syndrome (GCMS) was first described in 1960 in two girls who had clinical manifestations of craniosynostosis, midface hypoplasia, oligodontia, hypertrichosis, patent ductus arteriosus, anomalies of the eyes and hypoplasia of the labia majora –4 and itSLC25A24 were suggested as the molecular aetiology of both GCMS and FFS by two independent groups in 2017 , in The patient was born at 31 weeks and 6 days of gestational age to unrelated healthy parents. No abnormality was found on prenatal sonographic screening and the pregnancy was uneventful until oligohydramnios was observed at 26 weeks, which led to delivery at a birth weight of 1.08 kg (− 1.8 SD) by emergency caesarean section. She was transferred to the neonatal intensive care unit after birth and remained in care for 3 months.ATP6V0A2 were performed. However, the results of these analyses were normal.Physical examination showed that she had generalized loose skin, hypertrichosis and an umbilical hernia. She had a dysmorphic face with epicanthus inversus, depressed nasal bridge and low hairline. A congenital skull defect with anterior fontanelle widening and a softened occipital and parietal calvarium were observed. In addition, she had hypoplastic nails and syndactyly of the left 4th and 5th fingers. Based on her clinical phenotype, cutis laxa was initially suspected and conventional karyotyping and direct sequencing of At the age of 4 months, she visited our hospital for the evaluation of multiple congenital malformations, including congenital skull deformity and hypoplasia of fingers and nails. At that time, her head circumference was 34.3 cm (− 3.8 SD) and radiologic and sonographic findings showed interparietal widening and mild dilatation of the lateral ventricles. Simple radiographs of both hands revealed the absence of the distal phalanx in the 5th fingers and hypoplastic distal phalanges of both sets of 2nd to 4th fingers. Pure tone audiometry revealed mild conductive hearing loss. However, magnetic resonance imaging of the brain and orbit did not find any abnormalities, including the previously noted mild lateral ventricular dilatation. Abdominal sonography and echocardiography showed no structural anomalies, with the exception of a large umbilical hernia.At the age of 4 years, she underwent a series of surgical procedures, including division of syndactyly of the left 4th and 5th fingers, entropion repair of both upper eyelids and repair of the umbilical hernia. She needed regular ophthalmologic follow-up because of esotropia and severe hyperopia. Her early psychomotor development was delayed as she sat unsupported at 18 months, spoke several words at 24 months and walked independently when she was 36 months old. However, catch-up development allowed her to reach the level of her peers and she is now doing well in school.At the age of 8 years, she visited the paediatric genetics clinic of our institution for the identification of the underlying genetic aetiology of her dysmorphic features. Her height, weight and head circumference were 115.0 cm (− 3.0 SD), 15.0 kg (− 4.9 SD) and 48.5 cm (− 2.0 SD), respectively. She had progeroid features with loose skin and wrinkling on the forehead and limbs, as well as generalized hypertrichosis, especially on the forehead and back. A dysmorphic facial appearance with microphthalmia, epicanthus inversus, midface hypoplasia, narrow forehead and low hair line , also presents with the typical characteristics of FPS, including prenatal growth restriction and subsequent short stature, decreased subcutaneous fat, coronal synostosis, umbilical hernia at birth, short digital phalanges and normal development –17. Howeart Fig. . ConsideBecause the majority of patients with FPS are expected to have a nearly normal lifespan, the improvement of their quality of life by correcting deformities and managing medical issues is important. One report described the surgical correction of craniofacial malformations in a 7-year-old patient with GCMS using internal distraction devices . AnotherThis report presented a Korean case of FPS with genetic confirmation, as well as long-term growth follow up. We monitored serially the growth profile of our patient and found that her short stature was not caused by GH deficiency. We also organized chronologically the demographic information of related syndromes, including GCMS, FFS and PLWS. Even though FPS is an extremely rare disorder, efforts aimed at providing medical support based on its pathophysiology, as well as investigating unresolved questions, including the mechanism underlying the intersexual differences observed, should continue.Additional file 1. Detail and data of chromosomal microarray testing including non-pathogenic CNVs.


The best available technology for preventing the formation of acid drainage water from the sulfidic waste rock at mine closure aims to limit the oxygen access to the waste. There is, however, a concern that contaminants associated with secondary minerals become remobilized due to changing environmental conditions. Metal(loid) mobility from partially oxidized sulfidic waste rock under declining and limited oxygen conditions was studied in unsaturated column experiments. The concentrations of sulfate and metal(loid)s peaked coincidently with declining oxygen conditions from 100 to < 5 sat-% and to a lesser extent following a further decrease in the oxygen level during the experiment. However, the peak concentrations only lasted for a short time and were lower or in the similar concentration range as in the leachate from a reference column leached under atmospheric conditions. Despite the acid pH (~ 3), the overall quality of the leachate formed under limited oxygen conditions clearly improved compared with atmospheric conditions. In particular, the release of As was two orders of magnitude lower, while cationic metals such as Fe, Cu, Mn, and Zn also decreased, although to a lesser extent. Decreased sulfide oxidation is considered the primary reason for the improved water quality under limited oxygen conditions. Another reason may be the immobility of Fe with the incorporation of metal(loid)s in Fe(III) minerals, in contrast to the expected mobilization of Fe. The peaking metal(loid) concentrations are probably due to remobilization from solid Fe(III)-sulfate phases, while the relatively high concentrations of Al, Mn, and Zn under limited oxygen conditions were due to release from the adsorbed/exchangeable fraction. Despite the peaking metal(loid) concentrations during declining oxygen conditions, it is clear that the primary remediation goal is to prevent further sulfide oxidation. Rather than treating the formed AMD, the best available technology for the remediation of sulfidic mine wastes at mine closure is to prevent, or at least to minimize, the oxidation of sulfides and, thereby, the AMD formation  and a combination of four acid digestion , aqua regia digestion , and lithium borate fusion (for remaining elements) . The digests were analyzed for major and trace elements by inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) in both cases. Concentrations of F and Cl in the waste rock were determined by using ion selective electrodes . The sodium peroxide provides complete chemical analyses of resistant minerals without volatilization due to lower fusion temperature compared with lithium borate. However, except for Ni and W, the two analytical approaches showed an overall good agreement. Only the results from the sodium peroxide fusion (SGS), done on the same assays as the sequential extraction are reported here, with the exception of Na, F, Cl, S, and C that are based on ALS Global Table . The for(i)Optical microscopy of polished thin sections in transmitted and reflected light mode. Eight thin sections consisting of at least four waste rock particles each were prepared.(ii)θ/2θ Bragg–Brentano, radiation Kα1 of Cu (λ = 1.5406 Å), 45 kV–40 mA. Subsequent interpretation of the obtained diffractograms was done using XPert Highscore® software.X-ray diffraction (XRD) of pulverized samples at the Centres Científics i Tecnológics at the Universitat de Barcelona (CCiT-UB) using a diffractometer PANalytical X’Pert PRO MPD Alpha1 and the following settings: (iii)d = 1 μm) at 0.2 mW with 45-s integration time and three repetitions.Raman spectroscopy using a Bruker Senterra equipped with an Olympus BXFM optical microscope at the Luleå University of Technology. Raman spectra were obtained using a 532-nm laser beam ((iv)Scanning electron microscopy and energy-dispersive spectroscopy (SEM–EDS) using a ZEISS Merlin HR-SEM at Luleå University of Technology. The conditions for measurements were 20-keV accelerating voltage and 1.1-nA beam current.The mineralogy of primary and secondary mineral phases in the waste rock was studied via a multidiscipline approach:(i)Step 1—de-ionized water leach for the water-soluble fraction, including dried pore water, metal salts, and gypsum(ii)Step 2—ammonium acetate leach (pH 4.5) for the exchangeable fraction, but also dissolving carbonates and smectite(iii)Step 3—cold oxalic acid leach (pH 3) in the dark to dissolve easily reducible Fe–Mn(–Al) (hydr)oxides and hydroxysulfates(iv)Step 4—hot oxalate extraction (pH 3) at 80 °C to reduce remaining Fe(–Mn–Al) (hydr)oxidesIn order to gain quantitative information on the element distribution in primary and secondary mineral phases, and under which conditions the elements may become mobilized, the waste rock samples were also subjected to seven-step sequential extractions at the SGS laboratory . The sequential extraction scheme by Dold  is adaptThe sum of the element solubilized in the steps 1 to 4 is referred to as extractable fraction in the following sections, while a residual fraction is defined as the difference between the total element concentration and the sum of the steps 1–4. Such residual fraction includes sulfide and silicate minerals Dold .Leaching experiments were carried out in free-draining leach columns constructed from Perspex glass tubes diameter 19 cm), with caps made of polypropylene , and electrical conductivity (EC) had stabilized, one of the columns was sealed from the atmosphere. The amount of wetting water used per cycle was 430 mL, expect for 600 mL used in the first cycle. This matches the future precipitation rates in northern Sweden, predicted to increase according to future climate change scenarios from the Swedish Meteorological Office SMHI , assumin2 per cycle, thereby being fairly similar to a well-functioning dry cover system  attached to the columns at two different levels and calibrated against fully air-saturated (100 sat-%) and O2-free water (0 sat-%). The O2-sensor data are only considered semiquantitative because humidity variations in the columns were not measured but are known to have a large impact on the O2 saturation, and only a limited number of calibrations could be carried out during the experiment. During the first 12 months of leaching, no effort was made to remove O2 from the MQ water used to irrigate the column sealed from the atmosphere. In order to minimize contamination by O2, wetting and leachate sampling were done with the aid of an Ar gas and a filtration equipment was directly connected to the outlet. From the leaching cycle 51 onwards, the in-going MQ water was degassed by bubbling an N2 gas through the water for 20 min before wetting and leachate sampling were done under Ar gas flow, visually controlled by gas bubble generation from a discharge tube from the inlet water bypass.The amount of oxygen transported to the column dissolved in the ingoing water corresponds to ca. 0.1 mmol O4) were not further treated. Samples for cation and Fe redox species determinations were preserved with nitric (1 mL 65% Merck Suprapur HNO3 per 100 mL sample) and hydrochloric (0.5 mL 30% Merck Suprapur HCl per 100 mL sample), respectively. The samples were stored in dark and cold (4 °C) until analyses. Additionally, selected samples of unfiltered leachate were collected for the determination of total element concentrations.Leachate mass was determined by weight, and leachate pH, oxidation–reduction potential (ORP), and electric conductivity (EC) were measured immediately upon sampling in the unfiltered leachate. The electrodes used were a glass electrode (SenTix® 82) for pH, Pt electrode (SenTix® ORP) for ORP, and TetraCon® 925 for EC. The electrodes were calibrated and checked against standard pH buffers, and EC and ORP standard solutions, respectively. Redox potential (Eh) with respect to a standard hydrogen electrode (SHE) was calculated from the ORP readings. For determination of dissolved solute concentrations, leachate was filtered through 0.2-μm cellulose nitrate filters (Whatman). The filters had been cleaned with acetic acid (5%) and thoroughly rinsed and soaked in MQ water prior to sampling. Anion samples  at the accredited ALS Scandinavia in Luleå, Sweden. The anion samples were analyzed for F and Cl using ion chromatography (IC) and potentiometric titrations at the ALS Czech Republic. Sulfate was analyzed by several methods, including gravimetry (ALS Czech Republic), turbidimetry s released to leachate upon oxidation of sulfide minerals are either transported away or sequestered by precipitation or co-precipitation to secondary minerals or by sorption processes on either secondary or primary minerals. The fraction of As, Cu, Pb, and Zn solubilized in the extraction steps 1–4, thereby associated with the waste rock by sorption, ion exchange, or secondary minerals prior to leaching experiments, varies over a large range from ca. 5% (for As) to over 40% (for Cu) -containing minerals . No Fe or other metal(loid)s, with the exception of Mn, were detected in step 1. It is possible that other water-soluble phases, or alternatively dried pore water, were also present in the waste rock at low concentrations below the detection limited, as indicated by the elevated metal concentrations during the initial 1–3 leaching cycles “”.Step 2 not only solubilizes weakly adsorbed and exchangeable forms but also dissolves carbonates and clays Dold . In addi3O)Fe3(OH)6(SO4)2, alunite (KAl3(SO4)2(OH)6), or basaluminite (Al4(SO4)(OH)10 × 4H2O), also dissolved. These minerals commonly occur in a mine environment  and ferrihydrite  is clearly higher than the solubilized Fe/S ratio (2). Solubilization of Al and K in step 3 suggests that other minerals, such as jarosite  were the predominant hosts of extractable As, Cu, and Pb and also contained an important fraction of the extractable Zn and Mn. Arsenic was only detected in step 3, representing easily reducible phases such as poorly ordered ferrihydrite and schwertmannite. Copper, Mn, Pb, and Zn were, at least to some extent, also associated with the step 4 dissolving goethite, more ordered ferrihydrites, and jarosite. The association of metal(loid)s including As, Cu, and Pb, as well as Mn and Zn, with schwertmannite, ferrihydrites, and goethite is well established. Arsenic forms an oxyanion which tends to adsorb onto positively charged Fe(III) mineral surfaces at an acid pH and has a particularly high affinity to schwertmannite due to the formation of inner-sphere complexes s most prone to remobilization under declining oxygen conditions with the sequential are those associated with the extraction step 3. The more resistant and crystalline phases extracted in step 4 require strongly reducing conditions. Therefore, As, Cu, and Pb, together with Mn and Zn, are expected to mobilize under anoxic conditions, along with and major elements associated with these fractions, namely Fe, S, and Al. Note that also decrease in pH concentration leads to the dissolution of minerals solubilized in steps 3 and 4 as their solubility is strongly dependent on pH. Additionally, Mn, Zn, Al, and Fe associated with step 2 may become remobilized under acid conditions, but also in response to changing solution composition. An overall improvement of water quality is, however, expected in a long term due to the limited oxidation of sulfides under anoxic conditions and decreased dissolution of gangue minerals  and more favorable conditions for the adsorption of cationic metals under less acidic conditions.The extent and the rate at which the different reactions will proceed, and therefore the leaching behavior and water quality at mine closure, are difficult to predict based on solid phase characterization only. Column leaching experiments provide insight into the water quality expected upon cover application and to what extent the metal(loid)s become mobilized under changing chemical conditions. The partially oxidized waste rock originated from pilot-scale experimental leaching cells, collected after 5 years of leaching and having produced leachate with very high concentrations of S, Fe, As, Cu, S, Pb, and Zn, with the concentration levels depending on the water saturation and experimental conditions s, show an overall increase and reach their maximum between cycles 30 and 50, corresponding to ca. 9–12 months of leaching . After this, oxygen declined from 100 to 0 sat-% within 10 cycles of the sealing without further aid , no effort was made to remove oxygen from the ingoing MQ water, and therefore, the oxygen dissolved in MQ water entered the column during each leaching cycle “”; Fig. 6xpected “” and thexpected “”. Leacha2 ingress to the column. Still, traces of O2 (< 1 sat-%) were detected by the continuous measurements in association with wetting and sampling but disappeared within 1–2 days , degassing of the in-going water was initiated in order to minimize Oays Fig. . In respays Fig. .2 conditions. Sulfide oxidation does not re-accelerate similarly to the atmospheric conditions, in good agreement with the low Fetotal and Fe(III) concentrations. However, the quick disappearance (ca. 1 day) of the O2 introduced at each cycle shows ongoing oxidation s, which at least to some extent may then become incorporated into newly forming Fe(III) hydroxides, thereby resulting in peaking metal(loid) concentrations. Arsenic and Cu concentrations peak prominently, followed by decreasing concentrations to a low background level within a few cycles Fig. . Other mArsenic is the element of major concern associated with the waste rock, and its concentrations in the leachate reached a very low to a low level SEPA  under liOf the cationic metals, Cu showed somewhat similar behavior to As, with significantly lower overall concentrations under limited oxygen concentrations compared with atmospheric conditions, and temporally elevated concentrations associated with declining oxygen conditions. Unlike As, the maximum Cu concentrations reached under the two experimental conditions were similar. Similarly to As, Cu was associated predominantly with the reducible fraction (steps 3 and 4) in addition to residual/sulfide fraction, and its association with the solid phase did not change during the experiment Fig. . In addiThe maximum concentrations and release rate of Zn decreased less than those of Cu and As. Zinc concentrations also peaked during declining oxygen conditions, although not as clearly as those of As and Cu Fig. . DespiteConcentrations of Pb in the leachate produced in the present study (< 0.004 μmol/L) under both atmospheric and limited oxygen conditions are low compared with the concentrations reported for the leachate produced from the same waste rock previously s were released to the leachate under atmospheric conditions, even though the waste rock had previously been exposed to oxidation and consisted of relatively large particles used in the experiments. Despite the high concentrations of As, Fe, S, Cu, and Zn in the leachate produced upon leaching of the waste rock under atmospheric conditions, their release to the leachate corresponds to < 1% of the total element present in the waste rock for As, Fe, S and to 3–4% in case of Cu and Zn Table . AssuminOnly a fraction of the total amount of metal(loid)s associated with the reducible fractions became mobilized under limited oxygen conditions Figs.  and 10. Acid pH conditions were still maintained after 1 year of leaching under limited oxygen conditions relevant to those for a well-functioning cover system, and pH-raising measures may need to be considered. An explanation for such low pH may be buffering by Fe(III) minerals, where the Fe system buffers not only leachate pH but also leachate Eh and sulfate concentrations s with their release under atmospheric conditions “”, it is Extrapolation of these experimental results to field conditions is challenging due to the particle size effects and the different biogeochemical and hydrogeological conditions in the field. There are some important differences between the experimental approach and what is expected upon application of a dry cover. Limited oxygen conditions, relevant to those for a well-functioning cover system, prevailed in the sealed column after cycle 20 or so. In a waste rock dump, any oxygen transported through the cover is expected to be consumed by sulfide oxidation in the top part of a high column of waste rock, thereby hindering oxygen transport to the deeper parts of the dump. Therefore, the environmental conditions in the transport path of water in the dump vary and affect the leachate composition. In the experiments, however, such a process is limited by the small size and height of the column. Moreover, an actual dry cover will prevent not only the ingress of oxygen but also water percolation, thereby limiting contaminant transport. In the experiments, waste rock was leached under unsaturated conditions in order to allow sampling and analyses of leachate in order to study element release during declining oxygen conditions. Ultimately, water is required as the transport media for metal(loid)s, and in the absence of water, no transport of metals occurs, except with the water already present in the waste rock dump. Compared with the column experiment, the percolating water in the dump will be transported over longer distances, and therefore, the water/rock ratio and contact time will differ from those in the present study. Despite these differences, such column leaching approach provides useful insights into the leaching behavior of metal(loid)s under environmental conditions expected upon remediation. Sequential extractions, although providing quantitative information on element distribution in primary and secondary minerals, offer limited insights into leachate quality. Taking advantage of aggressive chemicals to quantitatively dissolve different minerals phases, sequential extractions tend to overestimate the amount of potentially mobilize elements compared with natural settings. Therefore, leaching tests as those presented here complement such data, giving more realistic insights into reaction rates and water quality upon mine closure.Partially oxidized highly sulfidic waste contains a limited amount of secondary minerals and produces highly acidic leachate with high concentrations metal(loid)s, highlighting the need for preventing sulfide oxidation.Total concentrations in the waste rock did not reveal which elements are of concern, and leaching tests are required to identify these.Although acid pH (~ 3) conditions were sustained, the quality of leachate produced under limited oxygen conditions relevant to those expected for a well-functional cover system was clearly improved compared to the leachate formed under atmospheric conditions.Concentrations of As decreased the most, followed by Fe and Cu, as well as Al, Zn, and Mn. The concentrations of Cu and Zn remain problematic.A short-term negative impact on water quality due to peaking metal(loid) concentrations, likely caused by dissolution Fe(III)-sulfate minerals, occurred coincidently with declining oxygen conditions but was only of short duration and concentration levels remained clearly lower, or in the similar range, as the concentrations observed under atmospheric conditions.The sustained low pH, most likely due to secondary mineral buffering, plays an important role for metal(loid) mobility, and release of cationic elements from the exchangeable fraction is one of the reasons behind the maintained elevated concentrations of at least Al, Mn, and ZnOnly a fraction of secondary minerals and associated metal(loid)s was released during the leaching testing. Leaching tests, such as those presented here, complement sequential extractions and mineralogical analysis and give useful insights into reaction rates and leachate quality upon mine closure.Although prevention of sulfide oxidation remains the main focus of remediation of partially oxidized waste rock, accumulation of secondary minerals and adsorbed metal(loid)s complicate the future remediation by, e.g., sustaining acidic pH and buffering metal(loid) concentrations and pose difficulties for predicting future water quality.Metal(loid) mobility from partially oxidized sulfidic waste rock was studied under declining and limited oxygen conditions in unsaturated column experiments with the aim to understand water quality at mine closure upon cover application. The study leads to following conclusions:


Obese women with polycystic ovary syndrome (PCOS) may face additional barriers in achieving weight loss. We aimed to compare the effects of the hypocaloric low glycemic index (LGI) diet on anthropometric variables and insulin resistance in women with and without PCOS and investigate the effect of this diet on the clinical and hormonal features of PCOS women.This interventional study was carried out at the Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Of 108 women invited for the purpose of the present study, 62 participants  followed a 24-week energy restricted LGI diet. Anthropometric, biochemical, hormonal and clinical measurements were documented at baseline, 12 weeks and 24 weeks with intervention.P = 0.83). In PCOS women, significant reduction in total testosterone , FAI  and increase in SHBG  were observed. Menstrual irregularity was improved in 80% of women with PCOS and a significant decrease (32.1%) in occurrence of acne was reported.The percentages of weight loss achieved by both the PCOS and non-PCOS groups did not differ significantly (PCOS: -8.04% vs. non-PCOS: -8.09%). No significant difference in decrease of homeostatic model assessment of insulin resistance (HOMA-IR) was observed between the two groups .This study is registered in the Iranian Randomized Clinical Trials Registry (IRCT, code:  Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age; its pooled prevalence varies from to 7 to 12% based on the various criteria used for its definition .In a general population, the energy restricted low glycemic index (LGI) diet has led to greater improvement in insulin resistance and glucose metabolism . EvidencThe international carbohydrate quality consortium (ICQC) propose the benefits of a low GI diet for participants with hyperinsulinism ; howeverThe aim of this study is to compare the effects of the hypocaloric LGI diet on anthropometric variables and insulin resistance in PCOS women with non-PCOS ones and to investigate the effect of this diet on the clinical and hormonal features of PCOS women.Written informed consent was obtained from all participants and the study was approved by the ethics committee of the Research Institute of Endocrine Sciences . This study is registered in the Iranian Randomized Clinical Trials Registry .This study was carried out at the Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran from 2016 to 2018. One hundred and eight overweight/obese women, aged 18-40 years, including 50 PCOS and 58 non-PCOS women, eumenorrheic non-hirsute controls, were invited to participate in the present study. The PCOS group were recruited from PCOS women, attending the Reproductive Endocrinology Research Center, Shahid Beheshti University of Medical Sciences (SBUMS). PCOS was defined using Rotterdam criteria by the presence of two or more of the following: 1) Oligo- and/or anovulation, 2) Hyperandrogenemia and/or hyperandrogenism 3) Polycystic ovaries (PCO) . Non-PCO2 BMI groups; hence PCOS cases were categorized into six age and BMI sub-groups. Finally, a total number of 73 women, including 33 PCOS and 40 BMI and age matched non-PCOS controls agreed to participate.Age and BMI levels of invited non-PCOS controls were matched with PCOS cases; to do this, participants were subdivided into < 25, 25–30 and over 30 year- old age groups, and further into BMI 25–30 and over 30 kg/m2 and a deficit of 500 kcal  that possibly caused an approximately 0.5 kg weight loss per week. Target macronutrient composition was 50% of energy from carbohydrate (CHO) with low and medium GI [The energy restricted LGI diet was calculated for an assumed BMI of 22 kg/medium GI , 20% of All participants were instructed to consume lean meat, whole grains, low fat dairies, non-starchy vegetables, vegetable oils and were prohibited from eating fast foods or food high in salt. In order to increase compliance with the diet, a food menu was prepared for each participant based on their energy requirement and eating habits. Fortnightly counselling visits were provided to participants to train them in recording daily dietary intakes and food compliance. To assess food compliance, a 3 day dietary food record (2 working days and one weekend day) was completed twice a month. Energy and nutrient intake of each food item was calculated using Nutritionist IV software. The US Department of Agriculture food composition table (USDA) was used as the nutrient database.The International Table of glycemic index (GI) and glycemic load (GL) was used to calculate GI values . GlycemiHeight was measured to the nearest 1 cm, without shoes in a standing position using a portable height meter measuring device. Weight was measured with a digital scale with an accuracy of 100 g. BMI was calculated by dividing weight by the square of height in meters. Waist circumference was measured using a tape at the narrowest part between the last rib and pelvic crest at the end of exhalation. Hip circumference was measured at the widest part and the waist to hip ratio was calculated. An overnight fasting venous blood sample was obtained from each participant, between the 2nd - 5th days of their spontaneous menstrual cycles and in amenorrheic women with PCOS at baseline and at weeks 12 and 24 of the intervention. It has been shown that the free androgen index (FAI) has a good correlation with free testosterone, measured by the physical separation method , and calAfter 15 min of sitting, blood pressure (BP) was measured on the left arm twice, and the average was recorded. Modified Ferriman Gallwey scoring method  was usedParticipants were asked to record their menses calendars 6 months before the commencement of study and during intervention. Menstrual cycles less than 21 days or over 35 days for at least 3 successive cycles were considered as menstrual irregularity. A change from irregular to regular cycles were considered as improvement in menstrual cyclicity.All participants were requested to sustain their baseline physical activity. The short form of the International Physical Activity Questionnaire (IPAQ)  was compMet level × days per week × minutes of activity .A sample of 60 participants (30 per group) was needed to detect a 5% weight loss in the PCOS group compared to 10% in the non-PCOS controls, with a two-sided 5% significance level, a power of 80% and loss to follow up rate of 15%.2 test. Baseline characteristics are presented as mean ± standard deviation for normal distributed variables. Differences in baseline descriptive characteristics of study groups were explored using t-test analysis. The Mann-Whitney U test was applied to compare the baseline values of variables with skewed distributions.Continuous variables were checked for normality using the one-sample Kolmogorov-Smirnoff test and normal plot; categorical variables are expressed as percentages, and are compared using Pearson’s χThe Generalized Estimation Equation (GEE) method was used to compare mean values of dietary intake and physical activity at three study time points  of intervention between groups. GEE analysis was also conducted to define the effects of the energy restricted LGI diet overtime on various factors, including anthropometric, hormonal and metabolic parameters in both groups and regularity of menstrual cycles, improvement in hirsutism and acne among PCOS participants. McNemar test was used to define the changes in percentage of irregular menstrual cycles between baseline and end of intervention and logistic regression analysis was conducted to identify the impact of influencing factors on the improvement of menstrual irregularity.n = 26, refused to participate, n = 9) and 73 participants meeting the study inclusion criteria were enrolled in the study ; of these 62 participants  completed 24-weeks of energy restricted LGI diet. The loss to follow-ups was 15% in both groups; there was no difference between the attrition in cases and controls , frequency of menstruation in the last 6 months (P < 0.001), acne status and hirsutism score (P < 0.001) , carbohydrate , fat percent , GI  and GL  were highly significant within each group, but were not significantly different between groups. Fiber  and percent of protein intake  were increased in both groups but were not significantly different  and non-PCOS ; there was also a significant change in BMI, compared to baseline, in the PCOS group  and in the non-PCOS group . Total weight reduction did not differ significantly between the two groups , with both groups achieving approximately the same percentage of weight loss (8.04% in PCOS vs. 8.09% in non-PCOS). Neither did physical activity levels in both groups differs significantly at baseline and during intervention  and non PCOS women , a reduction however not statistically significant between PCOS and non PCOS women . No significant difference in changes of HOMA-IR (p = 0.83), and fasting blood glucose (p = 0.73) were observed during follow up in the PCOS, compared to the non-PCOS controls.Compared to baseline, after 24-weeks of the energy restricted LGI diet, a significant reduction in insulin levels was detected in both groups of PCOS ; patients with PCOS had a higher reduction in their diastolic blood pressure compared to those of non-PCOS (P < 0.001)  Table .Table 3P < 0.001)  .For PCOS participants, an increase in SHBG , respectively. At baseline, of 28 PCOS women (85. 7%), 24 had menstrual irregularities; after 24 weeks of the energy restricted diet, 80% of women reported regular menstrual cycles (P < 0.001). Logistic regression analysis showed that odds of regularity of menstrual cycles were increased by reduction in BMI and HOMA-IR Z-scores . During the study, Ferriman–Gallwey score decreased from 7.89 ± 5.19 to 6.62± 4. 6 (P = 0.001) at week 24 of intervention. At baseline, 50% of participants with PCOS had acne and a 32.1% reduction (p = 0.004) in acne was reported at the end of follow up. At baseline, among PCOS women with acne, distributions of acne severity  were 50, 21. 4%, 25, and 3. 6%, respectively; values improved to 82.1, 10. 7%, 3. 6%, and 0% after 24 weeks of intervention, respectively (p < 0.001).In the PCOS group, at baseline, the mean intervals of menstrual cycles were 51.36 ± 15. 12 days and the mean menstrual frequency per year was 3.46 ± 0.99; after 24 weeks of the energy restricted diet these parameters were improved to 42.59 ± 16.34 days, The present study demonstrates that an energy restricted LGI diet has similar beneficial effects on the anthropometric and metabolic characteristics of overweight women with and without PCOS. Moreover, in PCOS women, both the regularities of menstrual cycles and the clinical and biochemical features of hyperandrogenism were improved after 6 months of implementing this diet.Although earlier studies suggest that PCOS is often associated with an increased risk of metabolic disorders, in particular obesity, it is unclear whether the energy restricted LGI diet can improve these disorders as well as non-PCOS women , 4. Our There is some evidence that the LGI diet delays absorption of carbohydrates and improves metabolic pathways and insulin resistance . StudiesIn the present study, women with PCOS, compared to their non-PCOS counterparts, have similar systolic and diastolic blood pressures at baseline. After the 24-week energy restricted LGI diet, a subtle reduction in systolic blood pressure was observed in both PCOS women and non-PCOS controls, although this change was not clinically important. This finding is consistent with those of another study, demonstrating that a low GI diet did not cause a reduction in blood pressure .We found that PCOS women had a significant reduction in serum levels of testosterone, FAI and an increase in SHBG, findings in agreement with another study that also showed the beneficial effects of weight loss on reproductive hormones . This stPrevious studies show the benefits of restriction of calories and the resulting weight loss in improving ovarian function and menstrual regularity , 32. In In this study, significant improvements in mean FG-scores were determined after 24 weeks of intervention, a result in line with those of some life style modification studies , 32, 33;In the present study, in agreement with others, we found a decrease in the occurrence and severity of acne . Recent Our study strengths include having a control group and simultaneous medication. Another strength of this study is high levels of adherence to the dietary plan and dropout rate of 15%; as this study implemented a diet based on participants’ dietary habits, increased number of follow ups, and emphasized modification of eating behavior, like omitting non-hunger-eating, rapid eating, food with high energy density, and consumption of food while watching TV, all of which improved the compliance of our participants. Our study had also assessments of physical activity before and during intervention, which may prevent the potential influencing role of different physical activity status on the beneficial effect of LGI diet in terms of clinical and endocrine variables. Age and BMI matching of cases and controls can prevent all the biases that could have arisen from differences in age and BMI.Our study has its limitations as well; we used self-reporting questionnaires, relying on participants’ reports on compliance with their prescribed diet. Adiposity and inflammatory markers were not assessed. We did not also assess the lipid profiles of PCOS patients; however, since previous studies showed that lipid lowering therapies can improve PCOS clinical and ovarian dysfunction abnormalities –39, we eStudy results demonstrate that the energy restricted LGI diet induces equally beneficial decrease in weight and insulin resistance in women with or without PCOS, by confirming the effect of energy restricted LGI diet in enhancement of endocrine and clinical variables in PCOS women. Improvement of menstrual irregularities in women with PCOS was associated with greater weight loss and improved HOMA. The efficacy of LGI diet in improving IR, hyperandrogenism, hirsutism, acne, menstrual irregularities in addition to its high dietary compliance make the LGI diet an optimal dietary choice for women with PCOS. For better comparison of the effects of this diet in PCOS women with their non PCOS counterparts, larger clinical trials with sufficient number of participants in each PCOS phenotype and measurements of other adiposity and body composition markers is highly recommended.


To assess x5x2 mm) with bonded brackets. The volunteers used the devices in two different moments of 14 days each. During the first 14 days, a product containing fluoride + nano-hydroxyapatite was applied twice , and for the other 14 days no prevention product was applied . In both groups, along the experiment, the blocks were dripped with 20% sucrose eight times daily. After the experiment, all the specimens were sectioned and examined for lesion depth analysis (µm) under polarized light microscopy, and for enamel longitudinal microhardness , at seven different depths . Eight volunteers wore palatal devices prepared with 6 bovine enamel blocks (5Under polarized light, group GNH presented significantly less demineralization depth (Fluoride + nano-hydroxyapatite can be an alternative preventive procedure for demineralization of the enamel adjacent to orthodontic brackets.  Many studies have been performed with the objective of preventing the formation of white spot lesions and also to diagnose them as soon as possible.in situ, the effect of a product containing fluoride associated to nano-hydroxyapatite, for the prevention of demineralization of the enamel adjacent to orthodontic brackets.Therefore, the objective of this study was to assess, in situ study involved a randomized design performed in a period of 28 days (two stages of 14 days each), during which eight volunteers wore a palatal intraoral device containing six specimens of bovine enamel with orthodontic brackets bonded to them . Acording to Gameiro et al,in situ study. The inclusion criteria for the volunteers were: dental students, aged 20-25 years, without use of antibiotics or any other medication that would reduce saliva flow, and free from active caries lesions.The study was approved by the Institutional Review Board of the State University of Maringá, Brazil. This x5x2 mm, from the flattest area of the buccal area. They were sterilized in a 2% formaldehyde solution for a month. They were then polished  using #320 sandpaper (1 min), followed by #600 and @1200 grit for 2 min each, at low speed. Polishing was completed with felt added with a diamond suspension of 1 µm, at high speed. The blocks were left immersed into deionized water for 12 h, to remove impurities. Bovine lower incisors from Nellore cattle, aged around 5 years, were extracted and immediately stored in 0.1% aqueous thymol solution. Teeth were cleaned and those with enamel stains, cracks and hypoplasia were discarded. They were sectioned  to obtain one block per tooth measuring 5The enamel blocks fixed to acrylic disks were taken to a microdurometer  to measure surface microhardness. Three indentations were conducted at the center of each block at a distance of 100 µm between them with a Knoop indenter (50g for 5s). The average of the three indentations was calculated to obtain the average microhardness value. Enamel blocks presenting microhardness below 200 KNH were discarded. Ninety-six blocks were selected, presenting an average microhardness of 303.80 KNH (SD = 47.33). Each one received a number and was randomly allocated to the two experimental groups.The studied groups were as follows: Control group CG (n = 48 enamel blocks), cariogenic challenge for 14 days - and did not receive any product application; Group GNH (n = 48 enamel blocks), experimental group - cariogenic challenge for 14 days with fluoride + nano-hydroxyapatite application on day 1 and 7.x6 mmx4 mm) at each side of the device. The blocks were fixed with sculpture wax and assembled in such a way that the enamel surface stayed 1 mm below the surface of the device  was applied according to the manufacturers instructions, and incisive brackets  were bonded to the center of the enamel blocks with composite resin . Then, a 0.016-in NiTi wire  was fitted to the slots of the three brackets on each side, and fixed with individuals elastic modules The removable palatal devices were fabricated with acrylic resin containing two cavities  contains the following active ingredients: sodium fluoride (9000 ppm), nano-calcium phosphate (in the form of hydroxyapatite), and potassium nitrate; and as inactive ingredients: distilled water, thickener, surfactant, humectant, flavor, sweetener, and preservative.-The volunteers were instructed to wear the palatal devices for 14 consecutive days, make an interval of 7 days, and use them for another consecutive 14 days. The palatal device should be used continuously (24 h/day), except during meals and when performing normal oral hygiene after meals . All volunteers received toothpaste without fluoride , soft toothbrushes , deionized water, gauze and 20% sucrose solution. They were instructed to brush their teeth three times daily but not to brush the enamel blocks. The volunteers were also instructed to remove the palatal device and drip 20% sucrose solution on each enamel block eight times daily.in situ period, the enamel blocks were repositioned onto their respective numbered acrylic disks and stored in plastic recipients moistened with absorbent paper and deionized water.During the first 14-day period, blocks received the treatment with fluoride + nano-hydroxyapatite, applied twice with the assistance of a microbrush on day 1 and 7 (group GNH). During the second period of 14 days, the enamel blocks did not receive any additional treatment (group CG). The same general instructions were repeated concerning the use, hygiene and the application of sucrose. After Enamel blocks were then longitudinally sectioned with a diamond disk assembled onto an electric sectioning machine. Half of the sample was submitted to the microhardness test, while the other half had the lesion depths analyzed by polarized light microscopy.2 of pressure). With the longitudinal sectioning plane facing the resin surface, the samples were submitted to a surface polishing. Then longitudinal microhardness was evaluated with a Knoop indenter with a load of 25 g per 5s, making indentations lines at the depths of 10 µm, 20 µm, 30 µm, 50 µm, 70 µm, 90 µm, and 110 µm under the bracket base, and at 30 µm and 130 µm from the edge of the bracket . The photographs were taken with maximum illumination, and the depth of each lesion was measured within an distance of 300 µm from the bracket edge. Three readings were taken  and the average of three measurements was calculated .ANOVA and Tukey post-test were used for intragroup comparisons, and Inter and intragroup comparisons at the different microhardness depth measurements are presented in For the intragroup comparisons, it was found that the microhardness presented main significant differences until 30 µm of depth , 4 and 6One specimen from CG and four from GNH broke during enamel cut preparation for the microscopy evaluation. Then 47 enamel slices from CG and 44 from GNH were examined under polarized microscopy. -,,in situ a product containing fluoride and nano-hydroxyapatite that reduced demineralization around brackets (Enamel demineralization around orthodontic appliances continues to be a common clinical problem, even in face of the application of fluoridated materials, which minimize but do not solve the problem completely.in situ model with intraoral devices has been frequently used, as it simulates the caries process that occurs within the oral cavity. Ideally, all the dental caries etiological factors such as dental substrate, diet, formation of biofilm and time should be involved.-The ,-,-,,The group that received fluoride + nano-hydroxyapatite demonstrated lesions significantly reduced under polarized optical microscopy , as wellMany studies reported from 40 to 50% reduction in demineralization around brackets under fluoride or hidroxiapatite-containing products applications. This seems to be less preventive effect than we found from the combination of both. Unfortunately, our study did not present a nano-hydroxiapatite nor a fluoride exclusive groups to isolate its effects from their association.in vitro and in vivo studies have already demonstrated a demineralization preventive or reducing potential of fluoride-containing products.,,,,,,,Previous in situ study, fluoride associated with nano-hydroxyapatite in situ effects demonstrated relevant enamel protection, and inspires a future in vivo study to verify its behavior in orthodontic patients.Considering the limitations of the Taking into consideration the methodology used and the data derived from the present study, it may be concluded that the tested product containing fluoride associated with nano-hydroxyapatite presented a preventive effect on the demineralization of enamel adjacent to orthodontic brackets, and it may be an alternative for the treatment of patients with high risk of caries.


Patients with medically unexplained physical symptoms (MUPS) are common in primary care, with a spectrum from mild to moderate and chronic MUPS. The burden of chronic MUPS is high, and early identification of moderate MUPS patients is important to prevent chronicity. The PRESUME screening method to identify moderate MUPS patients in primary care was developed, but insight in prognostic accuracy is needed. Therefore, our objective is to determine the prognostic accuracy for identification of moderate MUPS patients using the screening method with 5 year follow-up.n = 114.185), patients were identified with mild, moderate or chronic MUPS in 2008 (index year), using routine care data. In 5 years follow-up we calculated predictive values and odds ratio’s for sustained MUPS related symptoms.The PRESUME screening method consists of three subsequent steps based on consultation frequency, exclusion of medical/psychiatric diagnosis and identification of MUPS. In a random 10% sample of patients from the Julius General Practitioners Network  were identified as having mild, moderate or chronic MUPS. On average 55.5% of the moderate MUPS patients in 2008, still had MUPS related symptoms or developed chronic MUPS in 5 year follow-up. Positive predictive values for maintaining MUPS related symptoms or worsening was 67% after 1 year, and 48.7% after 5 years for moderate MUPS patients.The prognostic accuracy of the PRESUME screening method using electronic medical record data for identification of moderate MUPS patients is moderate. However, it might be a useful method to identify patients at increased risk of moderate MUPS, if combined with a validity check by the GP.The online version of this article (10.1186/s12875-019-0950-7) contains supplementary material, which is available to authorized users. Medically unexplained physical symptoms (MUPS) are a serious problem in primary care . Common MUPS can be regarded as a continuum with a spectrum from mild, to moderate, and persisting or chronic MUPS , 5, 6. SDespite the low prevalence of chronic MUPS, the burden is substantial . The impAlthough previous research has identified several modifiable risk factors for the development of chronic MUPS , 14, GPsRecently, a new screening method  was developed to identify patients with an increased risk of mild, moderate or chronic MUPS using electronic medical record (EMR) data Fig. . In a vaTherefore, the objective of this study is to determine the prognostic accuracy of the PRESUME screening method in identifying patients with an increased risk of moderate MUPS. Secondly, transitions between MUPS subgroups for patients with an increased risk of moderate MUPS as well as transitions of MUPS subgroups to an anxiety and/or a depressive disorder or medical diagnosis over a 5 year follow-up period will be assessed.In this prognostic cohort study we identified patients with an increased risk of MUPS  using historical data from electronic medical records of general practitioners, and followed them up over a period of 5 years to gain a prospective value of patients with moderate MUPS using PRESUME.This study was conducted with routine health care data as collected within the Julius General Practitioners Network (JGPN) database, which was approved by the medical ethical committee of University Medical Center Utrecht (file#99–240). JGPN comprises data from 72 primary care practices with 215 GPs in the central part of the Netherlands. This represent the average Dutch primary care practice and GP, where 49% of the GPs is male with an average age of 48 years , 20. DatGPs did not receive specific training on coding, but before EMR data was extracted, all included primary care practices signed a collaboration agreement that care registration is based on the standards and guidelines that apply within the profession of the GP . GPs areIn 2008 the database consisted of 114.185 patients between 18 and 65 years. The patient population is a representative sample of the Dutch population . Four tiThe PRESUME screening method was used to identify patients with MUPS symptoms in three subgroups according to severity and disease impact. The method is based upon three subsequent steps Fig. . In the In order to assess the prognostic value for identifying an increased risk of sustained moderate MUPS, the index cohort 2008) were followed up for 5 years and reclassified according to the PRESUME in each follow-up year (2009–2013). Furthermore, the percentage of patients that developed a depressive and/or an anxiety disorder or a medical explained diagnosis . Descriptive statistics were used to describe the patient population. Differences in baseline characteristics  between subgroups were investigated using Pearson’s Chi-square and Kruskal-Wallis statistics.For determination of the stability of the patients with an increased risk of moderate MUPS identified in the index year using PRESUME, transitions between MUPS subgroups over 5 years follow-up were determined, per year separately. It was hypothesized that at least 25% of the patients will still have an increased risk of mild or moderate MUPS (MUPS related symptoms) or developed chronic MUPS after 5 years follow-up.To determine the prognostic value of PRESUME in predicting an increased risk of sustained MUPS diagnosis, positive and negative predictive values and odds ratios were calculated after one and 5 years follow-up. Accuracy was considered high when predictive values were > 75%, moderate accuracy with predictive values between 50 and 75% and low accuracy with predictive values < 50%.Based on previous research , 34, ourn = 729) had a medical explained diagnosis  or an established psychiatric diagnosis . Of the remaining 1344 patients, 789 (58.7%) were identified with an increased risk of MUPS and classified in one of the MUPS subgroups , 2.073 patients (18.2%) had more than five encounters in 2008. Of these, 35.1% (n = 26) had developed chronic MUPS  (see Table n = 273), 13.5% (n = 37) developed a depressive and/or an anxiety disorder in 5 years follow-up, compared to 1.4% (n = 156) of the patients without MUPS, 12.3% (n = 56) of the patients identified at increased risk of mild MUPS and 19.6% (n = 12) of the patients with chronic MUPS  developed a depressive and/or an anxiety disorder, of which a depressive disorder was most frequently diagnosed n = 15; 55.5% n = 273), 15.8% (n = 43) developed a medical explained diagnosis in the same ICPC chapter as the MUPS related symptoms in the index year, as compared to 2.1% (n = 231) of the patients without MUPS, 11.6% (n = 53) of the patients identified at increased risk of mild MUPS and 16.4% (n = 10) of the patients with chronic MUPS during 5 years follow-up. Of all patients who developed a medical diagnosis during follow-up, most diagnosis regarded in the ICPC chapter L . The risk for development of a medical diagnosis in patients within one of the MUPS subgroups is significantly higher compared with the non MUPS group.Of the 11.419 patients, 337 patients (2.9%) were diagnosed with a confirmed medically diagnosis during follow-up  of the patients identified at increased risk of mild MUPS in 2008, 15.8% (n = 43) of those identified at increased risk of moderate MUPS and 16.4% (n = 10) of the patients with chronic MUPS was labelled with a medical diagnosis in the same ICPC chapter as in which they had MUPS in 2008, during the 5 years follow-up. In short, almost 50% of the patients identified at increased risk of MUPS will develop a medical diagnosis. However, this percentage is probably an overestimation since 54 of the 106 patients (50.9%) who were diagnosed with a medical diagnosis during follow-up, also still had MUPS related symptoms. Consequently, the MUPS related symptoms cannot be explained by the medical diagnosis, and the medical diagnosis seems not always anatomically be related to the MUPS related symptoms. Nevertheless, patients identified at increased risk of moderate MUPS according to the PRESUME screening method might have an established medical diagnosis, since we only exclude patients with chronic obstructive pulmonary disease, hypertension or diabetes mellitus in the second step of the PRESUME screening method. Therefore, to ensure that we have identified patients in the right stage of MUPS, also due to the moderate prognostic accuracy, GPs should perform a validity check and filter out patients with an established medical diagnosis, to prevent that patients are incorrectly offered treatment for MUPS.Of all patients within one of the MUPS subgroups in 2008, we hypothesized that less than 5% would develop a medical diagnosis during 5 years. Our results proved otherwise: 11.6% Additional file 2:Medically explained diagnoses. A list of International Classification of Primary Care codes of medically explained diagnoses used to determine the percentage of patients that developed a medically explained diagnosis during the 5 year follow-up period. (DOCX 20 kb)


Praying mantids are predators that consume a wide variety of insects. While the gut microbiome of carnivorous mammals is distinct from that of omnivores and herbivores, the role of the gut microbiome among predatory insects is relatively understudied. Praying mantids are the closest known relatives to termites and cockroaches, which are known for their diverse gut microbiota. However, little is known about the mantid gut microbiota or their importance to host health. In this work, we report the results of a 16S rRNA gene-based study of gut microbiome composition in adults and late-instar larvae of three mantid species. We found that the praying mantis gut microbiome exhibits substantial variation in bacterial diversity and community composition. The hindgut of praying mantids were often dominated by microbes that are present in low abundance or not found in the guts of their insect prey. Future studies will explore the role of these microbes in the digestion of the dietary substrates and/or the degradation of toxins produced by their insect prey. The gut microbiota of insects and other animals is known to play key roles in shaping host health. Gut microbes have been shown to assist in digestion and nutrient absorption, provide a layer of protection against opportunistic pathogens, and can metabolize certain toxins and/or environmental pollutants –3. The gTenodera sinensis), have been used to study and model predator behavior [Praying mantids are sympatric general predators that typically hunt based on prey size. Praying mantids primarily subsist on a diet of small insects including fruit flies, crickets, and caterpillars, although adult praying mantids are known to consume small birds or small reptiles , 31–34. behavior –40. Recebehavior . Howeverbehavior , 33, 42.behavior , 15. Sevbehavior , 16–18. behavior . Chinesebehavior .Published work on the gut microbiome has demonstrated that most insects host simple gut microbial communities that are highly variable among individuals of the same species . InsectsT. sinensis and 3 Tenodera angustipennis), with an average depth of 552 sequences per sample [A 2014 study used 454 pyrosequencing of 16S rRNA genes to complete a survey of the gut microbiome from 305 insects across multiple orders . This sur sample . Due to r sample . No otheBugs in Cyberspace  where they were reared on a diet of Drosophilia. Three species, with ten nymphs per species, were represented: T. sinensis, Hierodula venosa, and Deroplatys lobata. Nymphs were kept isolated in plastic cups, with a pipe cleaner for perching and a cotton ball damp with water. Nymphs were initially fed daily and raised on a mixed diet of Drosophilia melanogaster, Drosophilia hydei, and Periplaneta americana nymphs. After several molts, surviving praying mantids were exclusively fed P. americana every 1–2 days until their sacrifice. In nearly all cases, mantids were observed to consume prey insects in their entirety, including the gut and gut contents. All insects were offered prey within 24 hours of sacrifice.30 fourth-instar praying mantis nymphs were purchased from The initial aim of the work was to sacrifice healthy adult mantids at one week past their final molt. Given high rates of insect loss, particularly following the final adult molt, some adults and nymphs were sacrificed early when their overall condition and activity levels suggested that they were unlikely to survive to the target age Tables  and S1. P. americana were provided by the University of Georgia’s entomology department. They are maintained in mixed-age, mixed-sex colonies in aquarium tanks on a diet of dog food  ad libitum. Healthy adult cockroaches were selected at random from the tank for dissection, and all were in excellent condition at time of sacrifice  was used to extract microbial DNA from stored hindgut samples. After thawing on ice, samples were pulverized with a sterile microcentrifuge pestle. Pulverized samples were centrifuged for 10 min at 5,000 g, the supernatant was discarded, and the pellet was resuspended in 100 μL of 1XTE Buffer. 10 μL lysozyme (as supplied by kit) was added to the resuspended sample before incubation at 37°C for 30 min. After incubation, approximately 25 mg of glass beads (as supplied by the kit) was added to each sample. Samples were bead beaten for 5 min at 3,000 rpm using a vortex mixer with a horizontal adapter. Next, 100 μL BTL buffer and 20 μL proteinase K solution (as supplied by the kit) were added to each sample before incubation at 55°C while shaking at 600 rpm for 1 h. After incubation, the manufacturer’s protocol (June 2014 version) was followed beginning at step 11. The final DNA concentrations (ranging from 40 to 800 ng/μL) and A260/A280 were measured using a NanoDrop Lite spectrophotometer .GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) primers. The initial reaction contained 1X Q5 buffer , 200 μM dNTPS, 0.5 μM 515F, 0.5 μM 806R, 2 ng template DNA, and 0.02 U/μL Q5 Hot Start High-Fidelity DNA polymerase (NEB) for a total reaction volume of 10 μL. PCR conditions were 98°C for 30 s for denaturation followed by 15 cycles at 98°C for 10 s, 52°C for 30 s, and 72°C for 30 s. The final extension step was 72°C for 2 min.A previously described two-step PCR method was used to prepare the amplicon library for sequencing . During Immediately after the final extension, 9 μL of the initial reaction product was reamplified using barcoded 515F and 806R primers  S2 Tabl. The iniAmplicons were visualized on a 2% w/v agarose gel. PCR for failed samples was redone with an additional 5 cycles during the initial reaction . AmplicoBlattabacterium (cockroach endosymbiont found in fat body cells) were removed. The remaining sequences were clustered using the OptiClust [The Mothur software package was used to analyze 16S rRNA gene sequences generated during this experiment . To alloptiClust  method iData generated by Mothur was imported into R for further analysis . We utilThe sequences generated from this experiment were submitted to the NCBI Sequence Read Archive and are available under BioProject accession numbers SRP132948 and SRP132487.16S rRNA gene libraries were prepared and sequenced from hindguts of praying mantids fed a diet of laboratory-raised cockroaches and from hindguts of laboratory-raised prey cockroaches. This 16S rRNA library resulted in a total of 1,426,892 raw sequences, with 939,355 remaining after quality control filtering .Praying mantids were found to harbor diverse gut microbiota. As a whole, these insects exhibited lower alpha diversity and higher beta diversity in gut microbiome composition than their cockroach prey. Although the mean alpha diversity observed in mantids (Shannon Index: 4.4) was lower than in cockroaches (Shannon Index: 6.7), there was no significant difference in the means . However, variance in alpha diversity was significantly higher . In contrast, we did not find significant relationships between gut microbiome composition and praying mantis age or condition .Inter-individual variation among praying mantids was substantially higher than observed in cockroach prey by both weighted and unweighted beta diversity measures  and S1. Firmicutes, Proteobacteria, and Bacteriodetes were the predominant phyla present in the hindgut microbiota of all insects. All hindguts also contained a high proportion of microbes from other, less abundant phyla including Actinobacteria, Verrucomicrobia, and Tenericutes. Variability among individual insects was higher in praying mantids, with non-predominant phyla composing 3–40% of the gut microbiota in praying mantids and only 5–13% in cockroaches .The Streptococcaceae, Lactobacillaceae, and Enterococcaceae families were prevalent among praying mantids, representing 75% of all Firmicutes found in praying mantis hindgut samples . In contWhile Enterobacteriaceae were found only within praying mantids, other bacterial lineages were common to both praying mantids and their cockroach prey. For example, the majority of Bacteriodetes in both insect orders are members of the Flavobacteriaceae 1, Bacteroidaceae, and Porphyromonadaceae gut group families . These fAt the 97% OTU level, few lineages were shared between praying mantids and cockroaches. Only 7 OTUs were found in all insect microbiomes in the study, with 52 shared among all praying mantids (but not cockroaches) and 19 shared among all cockroaches  Tables. D. melanogaster and D. hydei until large enough to safely consume cockroach nymphs. It is also possible that unique microbes were acquired from nymphal cockroach prey, as only adult cockroaches were included in the comparison dataset. However, work by Carrasco et al. demonstrates that cockroach nymphs older than two weeks possess a gut microbiota that is highly similar to that of adult cockroaches [Our work suggests that praying mantids have a unique, host-associated gut microbiota that exhibits a distinct phylogenetic profile from the gut microbiota of prey and is dominated by lineages that are either not found or not abundant in cockroaches. This result is in contrast to emerging work on the caterpillar gut microbiome, which demonstrates some insects have no resident microbiota and instead harbor transient, diet-associated microbiota . Potentikroaches .Drosophila sp.  are known to harbor extensive individual-to-individual variability in their gut microbiota [Many of the predominant bacterial lineages found in mantid guts showed substantial variance in overall abundance across individuals. Due to the destructive sampling techniques used, it is impossible to say whether this variability is the result of inter-individual variability that is paired with intra-individual temporal stability or whether the observed variability is the result of frequent microbial blooms and temporal variability within the guts of individual mantids. While diet was held constant for these mantids, potential sources of variability may include time since last meal (which varied from immediately before sacrifice up to 24 hours before sacrifice) which may have implications for the loss rate of ‘transient’ prey-associated microbes. Variability in gut microbiota composition may also have resulted from variability in microbes introduced early in life, as crobiota . In addiDrosophila are not known to produce or carry toxins, in the wild mantids routinely prey on insects, such as caterpillars, that utilize alleochemical defense mechanisms [Lactobacillus and Enterococcus sp., including Lactobacillus lactis and Enterococcus faecalis [Lactobacillus and Enterococcus sp. were present in the gut microbiota of multiple mantids and three of the 52 core mantid OTUs were Lactobacillus sp.Although there was high variability in the gut microbiota among the sampled mantids, a core community of 52 OTUs was found in every mantid hindgut . The OTUchanisms , 16–18. chanisms , 19–22. faecalis , 19. OTUOne goal of this work was to determine the extent to which the gut microbiome of praying mantids resemble those of other members of superorder Dictyoptera. Our results suggest that, unlike cockroaches and termites, the praying mantis possess a gut microbial community that is highly variable between individuals, with lower average alpha diversity and higher beta diversity than observed in cockroaches. This difference may be due to the predatory diet and/or solitary lifestyle of mantids. Previous work has suggested that omnivorous insects have more diverse gut microbiota than insects that were solely carnivorous or herbivorous, which might explain in part the higher alpha diversity observed in cockroaches . AlternaIn conclusion, this work suggests that mantids possess a diverse and highly variable gut community microbiota that is unique in composition from that of their prey. Overall, this gut microbiome is more typical of that observed in other insects than the highly diverse and stable microbiota found in other members of the superorder Dictyoptera. This work represents the first in-depth examination of the praying mantis gut microbiome and provides a foundation for future work on the origins of the predator gut microbiome and its role in host health and nutrition.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigBoxplots show Shannon diversity indicies (left), and weighted (middle) and unweighted (right) dissimilarities among praying mantid and cockroach gut microbial communities at 97% sequence identity. Libraries were resampled to a depth of the sample with the fewest sequences (3901). For each group, the bars delineate the means, the hinges represent the lower and upper quartiles, the whiskers extend to the most extreme values (which are no more than 1.5 times the interquartile range from the box), and outliers are plotted, if present.(TIF)Click here for additional data file.S2 FigEach bar represents an individual insect gut. All phyla that represent ≥1% of sequences from any one sample are listed in the barplot, all other phyla are grouped together under 'Other'.(TIF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.