Enrichment Protocol Research Articles

Effect of bacterial DNA enrichment on detection and quantification of bacteria in an infected tissue model by metagenomic next-generation sequencing

Before implementing metagenomic next-generation sequencing (mNGS) in the routine diagnostic laboratory, several challenges need to be resolved. To address strengths and limitations of mNGS in bacterial detection and quantification in samples with overwhelming host DNA abundance, we used the pig muscle tissue spiked with a home-made bacterial mock community, consisting of four species from different phyla. From the spiked tissue, we extracted DNA using: (i) a procedure based on mechanical/chemical lysis (no bacterial DNA enrichment); (ii) the Ultra-Deep Microbiome Prep (Molzym) kit for bacterial DNA enrichment; and (iii) the same enrichment kit but replacing the original proteinase K treatment for tissue solubilization by a collagenases/thermolysin digestion and cell filtration. Following mNGS, we determined bacterial: ‘host’ read ratios and taxonomic abundance profiles. We calculated the load of each mock-community member by combining its read counts with read counts and microscopically-determined cell counts of other co-spiked bacteria. In unenriched samples, bacterial quantification and taxonomic profiling were fairly accurate but at the expense of the sensitivity of detection. The removal of ‘host’ DNA by the modified enrichment protocol substantially improved bacterial detection in comparison to the other two extraction procedures and generated less distorted taxonomic profiles as compared to the original enrichment protocol.

Improved Detection of Herpesviruses from Diluted Vitreous Specimens Using Hydrogel Particles.

Infectious uveitis is a sight-threatening infection commonly caused by herpesviruses. Vitreous humor is often collected for molecular confirmation of the causative agent during vitrectomy and mixed in large volumes of buffered saline, diluting the pathogen load. Here, we explore affinity-capture hydrogel particles (Nanotrap®) to concentrate low abundant herpesviruses from diluted vitreous. Simulated samples were prepared using porcine vitreous spiked with HSV-1, HSV-2, VZV and CMV at 105 copies/mL. Pure undiluted samples were used to test capturing capability of three custom Nanotrap particles (red, white and blue) in a vitreous matrix. We found that all particles demonstrated affinity to the herpesviruses, with the Red Particles having both good capture capability and ease of handling for all herpesviruses. To mimic diluted vitrectomy specimens, simulated-infected vitreous were then serially diluted in 7 mL TE buffer. Diluted samples were subjected to an enrichment protocol using the Nanotrap Red particles. Sensitivity of pathogen detection by qPCR in diluted vitreous increased anywhere between 2.3 to 26.5 times compared to non-enriched specimens. This resulted in a 10-fold increase in the limit of detection for HSV-1, HSV-2 and VZV. These data demonstrated that Nanotrap particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix.

VarAmpliCNV: analyzing variance of amplicons to detect CNVs in targeted NGS data.

Computational identification of copy number variants (CNVs) in sequencing data is a challenging task. Existing CNV-detection methods account for various sources of variation and perform different normalization strategies. However, their applicability and predictions are restricted to specific enrichment protocols. Here, we introduce a novel tool named varAmpliCNV, specifically designed for CNV-detection in amplicon-based targeted resequencing data (Haloplex™ enrichment protocol) in the absence of matched controls. VarAmpliCNV utilizes principal component analysis (PCA) and/or metric dimensional scaling (MDS) to control variances of amplicon associated read counts enabling effective detection of CNV signals. Performance of VarAmpliCNV was compared against three existing methods (ConVaDING, ONCOCNV and DECoN) on data of 167 samples run with an aortic aneurysm gene panel (n = 30), including 9 positive control samples. Additionally, we validated the performance on a large deafness gene panel (n = 145) run on 138 samples, containing 4 positive controls. VarAmpliCNV achieved higher sensitivity (100%) and specificity (99.78%) in comparison to competing methods. In addition, unsupervised clustering of CNV segments and visualization plots of amplicons spanning these regions are included as a downstream strategy to filter out false positives. The tool is freely available through galaxy toolshed and at: https://hub.docker.com/r/cmgantwerpen/varamplicnv. Supplementary Data File S1: https://tinyurl.com/2yzswyhh; Supplementary Data File S2: https://tinyurl.com/ycyf2fb4. Supplementary data are available at Bioinformatics online.

Size-Based Method for Enrichment of Circulating Tumor Cells from Blood of Colorectal Cancer Patients.

Circulating tumor cells (CTCs) are precursors of the metastatic cascade, which is responsible for 90% of all cancer-related deaths. CTCs arise from solid tumors and travel through the bloodstream and lymphatic system. Developments in the isolation and analysis of CTCs promise potential biomarker assays for detection and monitoring of cancer through a minimally invasive procedure. Despite this, the precise role of CTCs in metastasis remains poorly characterized, mainly due to the low density of CTCs (1-10 CTCs per 10 mL of blood) present in patient blood and the lack of robust methods for their isolation in a standard laboratory setting. CellSearch is currently the only FDA-approved CTC enrichment protocol, but limitations of this EpCAM-based method include cost, availability, and the use of a single surface marker for capture. To address these limitations, we have optimized an existing method, MetaCell, which exploits the differences in size of CTCs compared to other blood cells for CTC enrichment from blood. MetaCell contains a membrane with 8 μm pores, and blood is filtered through this kit by capillary action and CTCs, which are typically larger than the pores and remain on top of the membrane, while most leukocytes pass through the pores. The membrane along with these CTCs can be detached and transferred to 6-well plates for culturing or directly used for characterization. Here, we provide a detailed protocol for enrichment of CTCs using the filtration device MetaCell and a procedure for characterization ofCTCs by immunohistochemical staining.

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.

Evaluation of short and long term enrichment of Artemia franciscana with mixed algae or DHA oil emulsion for improving Dicentrarchus labrax larvae aquaculture

Setting up nutritional requirements with high-quality, time-saving marine finfish larvae is a big issue for increasing aquaculture profit. In the current study, we evaluated the nutritional value of two enrichment diets [DHA oil emulsion-enriched Artemia (Art-DHA-Selco) and mixed algae-enriched Artemia (Art-MA)] and two-timing protocol (2 & 24 h) for serving European Sea bass. Larval growth performance and survival percentage, as well as biochemical compositions of the enriched Artemia and for the fed larvae after 25 rearing days, were determined. The results revealed that survival percentage (S%), feed conversion ratio (FCR), and protein efficiency ratio (PER) was insignificantly affected by the two factors (diets and time) or the interaction between them. Otherwise, growth in terms of final body weight (FBW), specific growth rate (SGR) as well as productive protein value (PPV) differed significantly at (P ≤ 0.01) among treatments in both factors. Concerning biochemical analysis, the highest protein value was recorded in Art-MA at 24 h. In contrast, lipid and carbohydrate levels were considerably higher in Art-MA 2 h than in the Art-DHA. That decrease in protein content, FBW, and SGR in the 2 h period in the MA treatment may attribute to the smaller size of the Artemia metanauplii in the 2 h than those after 24 h. That can be overcome by starting with Artemia metanauplii at the end with Instar II before enrichment process so that the size is big enough to give better results. The results revealed that PUFAs percentages were significantly highest especially EPA percentage in the enriched Art-MA 2 h followed by its correspondence Art-MA at 24 h compared with Art-DHA or un-enriched ones. Otherwise, the fatty acid composition (FA) of post-larvae surpassed greatly in EPA and n3/n6 ratios as diet reflection in the algal short-term duration. Based on the results and the documented drawbacks of DHA-Selco, the authors recommended using mixed algae in the enrichment protocol for feeding larval stages over the short term.

Boronate affinity paper spray mass spectrometry for determination of elevated levels of catecholamines in urine

The analysis of catecholamines, such as dopamine, epinephrine and norepinephrine in urine can be used in the diagnosis of certain pathologies, such as hormone-producing tumors. Here, a fast and simple quantitative boronate affinity paper spray tandem mass spectrometric (PS-MS/MS) method is established, which can improve selectivity and reduce ion suppression without needing any instrumental chromatography. We use here the property of boronic acids, which can selectively bind ortho-diol-containing compounds under alkaline conditions. Paper tip modification and catechol enrichment protocols were developed to selectively bind, clean up and subsequently desorb such catecholamines. Standard catecholamine solutions, as well as human urine samples were analyzed with the PS-MS(/MS) method, which is fast, cheap and easy-to-operate compared to HPLC-MS/MS. Despite its high simplicity, boronate affinity PS-MS/MS exhibits good performance compared to HPLC-MS/MS in human urine analysis in terms of precision (2.1%–7.2% vs. 1.1%–2.9%) and accuracy (−10.2%–9.3% vs. −4.8%–5.1%), and a physiologically relevant limit of detection (0.027–0.12 μg mL−1). The boronate affinity PS-MS/MS clearly achieved the detection limits that would allow the fast analysis of urine samples for clinical purposes, such as screening for pheochromocytoma (exceeding 0.5 μg mL−1).

Holstein calves' preference for potential physical enrichment items on different presentation schedules

Impoverished housing environments are thought to prevent motivated behaviors and may result in frustration. We first aimed to investigate an effective physical enrichment protocol to improve dairy calves' welfare and initially determine their use of various items. Thereafter, we aimed to determine dairy calves' preference for and ways of interacting with various items, and whether this was influenced by social housing. In experiment 1, at 21 ± 3 d of age, 27 individually housed calves were assigned alternately into 1 of 3 treatments: control (CON, no additional items), rotating enrichment (RE, one item each week on a rotating presentation schedule), and fixed enrichment (FE, 4 types of item at the same time). The items were stationary brushes, ropes, springs, nets filled with strawberry-scented hay, and dry teats. Calves' behaviors were observed from 4 to 7 wk of age using focal observations after feeding, followed by instantaneous scan sampling. Their behavioral responses to a novel object were assessed at 43 ± 1 d of age. In the instantaneous scans, calves in FE tended to interact with items more often than calves in RE. Calves in RE and FE expressed less non-nutritive oral behavior than those in CON. Latency to touch novel objects did not differ significantly between treatments. Calves in RE and FE interacted with nets filled with strawberry-scented hay more often than with other items in instantaneous scans. In experiment 2, 24 calves were assigned alternately into 8 individual pens and 8 pair pens at 2 d of age. All pens were provided with a stationary brush, plastic chain, net filled with strawberry-scented hay, and dry teat. Calves' behaviors were collected from 2 to 5 wk of age using instantaneous scan sampling. Calves interacted with nets filled with strawberry-scented hay more often than with other items. Pair housing reduced calves' interactions with items compared with individual housing. Individually and pair-housed calves' frequencies of overall interaction with items varied with time of day, with frequencies increasing to peaks at 0700, 1500, and 1900 h. Calves showed scratching, sniffing, sucking, butting, and hay intake toward nets filled with strawberry-scented hay and showed the first 3 behaviors toward stationary brushes, plastic chains, and dry teats. In conclusion, dairy calves are likely to prefer a fixed multi-item enrichment presentation schedule over a rotating schedule with a single enrichment item presented at one time. For the fixed multi-item enrichment presentation schedule, items were used more in individual pens than in pair pens, and a diurnal pattern was observed for use of the items. Nets filled with scented hay might be the most multifunctional and attractive item of the items tested.

Risk factors for stereotypic behaviour in captive ungulates.

Behavioural needs are highly motivated actions critical to a species survival and reproduction. Prolonged restriction of these behaviours can lead to stereotypic behaviours (SB) in captive animals, and this is particularly common in ungulate species. While risk factors for SB have been suggested for some ungulates, no study has integrated these findings to identify which aspects of ungulates' wild behavioural biology and captive husbandry are potential drivers for SB across this clade. We collated SB data from 15 236 individuals across 38 ungulate species from 95 sources, and determined species wild/free-ranging behaviour from 559 additional studies. Bayesian-phylogenetic statistical methods showed that ungulate behavioural needs relating to foraging and mating are particularly affected by captive environments, with promiscuous and browsing species showing the greatest prevalence of SB. Concentrate-only diets and lack of ad libitum feed substrates were also associated with high SB prevalence. This study identifies which ungulates are better suited to captive environments and which species require targeted husbandry, enrichment and breeding protocols in order to meet their behavioural needs. Our approach of applying Bayesian-phylogenetic inference to factors influencing SB within a clade can be used to identify other intrinsic and extrinsic risk factors of reduced animal health and welfare.

Mass Purification Protocol for Drosophila melanogaster Wing Imaginal Discs: An Alternative to Dissection to Obtain Large Numbers of Disc Cells.

Simple SummaryDrosophila melanogaster, also known as the fruit fly, is a widely used organism model, especially for genetic studies or as a model for pathologies. The Drosophila genome is well known and conserved within humans, thus allowing biologists to obtain numerous mutants and transgenic flies from the fruit flies. Gene function studies at the cellular and molecular levels are often performed using extracts of larval tissues. Due to their small size, it is difficult to dissect substantial amounts of these tissues for performing genomic or proteomic experiments. This paper develops a simple method to purify larval tissues en masse. This protocol preserves tissue integrity in the same way as manual dissection; the protocol is achievable by individual researchers and allows the purification of different samples simultaneously.Drosophila melanogaster imaginal discs are larval internal structures that become the external organs of the adult. They have been used to study numerous developmental processes for more than fifty years. Dissecting these imaginal discs for collection is challenging, as the size of third-instar larvae organs is typically less than 1 mm. Certain experimental applications of the organs require many cells, which requires researchers to spend several hours dissecting them. This paper proposes an alternative to dissection in the form of a mass enrichment protocol. The protocol enables the recovery of many wing imaginal discs by grinding large quantities of third-instar larvae and separating the organs using filtration and a density gradient. The wing imaginal discs collected with this protocol in less than three hours are as well preserved as those collected by dissection. The dissociation and filtration of the extract allow the isolation of a large amount of wing imaginal disc cells.

Rapid quantitation of feed rotifer ( Brachionus plicatilis ) fatty acids by Raman spectroscopy

Improving the lipid composition of feed-rotifers enhances the survival and growth rates of juvenile fish in aquaculture, but monitoring enrichment profiles using gas chromatography (GC) is slow, labour-intensive and requires expensive instrumentation. Here, we describe the potential of Raman spectroscopy as an alternative, more rapid tool for quantitation of rotifer fatty acid composition in an aquaculture setting. A compositionally diverse sample set was prepared by periodically subsampling rotifers undergoing enrichment using three different feeding regimens. Sampled rotifers were freeze-dried and their fatty acid content and composition was measured by GC–MS (mass spectrometry). Raman spectra (1064 nm excitation) were acquired with two instruments: a state-of-the-art Fourier transform (FT)-Raman spectrometer equipped with a liquid nitrogen-cooled Germanium detector; and a smaller, portable spectrometer equipped with an InGaAs detector. Fatty acid reference data acquired by GC–MS were related to structured variance in each Raman spectral data set using partial least squares regression. The resultant models could accurately quantitate total fatty acids (TFA) and total polyunsaturated fatty acids (PUFA) using both FT-Raman spectra (Root Mean Square Error [RMSEV] = 5.0 mg g−1 TFA and 6.1 mg g−1 PUFA) and spectra generated using the portable instrument (RMSEV = 4.2 mg g−1 TFA and 5.3 mg g−1 PUFA). Analysis times with the portable instrument were approximately 3 min, compared to 16 min with the FT-Raman system. Portable Raman spectroscopy may be a useful tool for high throughput screening of rotifer lipid composition when testing and optimizing enrichment protocols and developing microdiets for aquaculture species.

Comparison of microfluidic platforms for the enrichment of circulating tumor cells in breast cancer patients

PurposeCirculating tumor cells (CTCs) hold promise to be a non-invasive measurable biomarker in all cancer stages. Because the analysis of CTCs is still a technical challenge, we compared different types of microfluidic enrichment protocols to isolate these rare cells from the blood.MethodsBlood samples from patients with early and metastatic breast cancer (BC) were processed using the microfluidic Parsortix® technology employing (i) a single-step cell separation using the standard GEN3D6.5 microfluidic cassette, (ii) a two-step separation with an upfront pre-enrichment, and (iii) a two-step separation with a different type of cassette. In the enriched cells, the gene expression levels of CTC-related transcripts were assessed using quantitative real-time PCR (qPCR) by Taqman® and Lightcycler (LC) technology.Results23/60 (38.3%) BC samples were assigned as positive due to the presence of at least one gene marker beyond the threshold level. The prevalence of epithelial markers was significantly higher in metastatic compared to early BC (EpCAM: 31.3% vs. 7.3%; CK19: 21.1% vs. 2.4%). A high level of concordance was observed between CK19 assessed by Taqman® and LC technology, and for detection of the BC-specific gene SCGB2A2. An upfront pre-enrichment resulted in lower leukocyte contamination, at the cost of fewer tumor cells captured.ConclusionThe Parsortix® system offers both reasonable recovery of tumor cells and depletion of contaminating leukocytes when the single-step separation using the GEN3D6.5 cassette is employed. Careful selection of suitable markers and cut-off thresholds is an essential point for the subsequent molecular analysis of the enriched cells.

A rodent obstacle course procedure controls delivery of enrichment and enhances complex cognitive functions

Enrichment in rodents affects brain structure, improves behavioral performance, and is neuroprotective. Similarly, in humans, according to the cognitive reserve concept, enriched experience is functionally protective against neuropathology. Despite this parallel, the ability to translate rodent studies to human clinical situations is limited. This limitation is likely due to the simple cognitive processes probed in rodent studies and the inability to control, with existing methods, the degree of rodent engagement with enrichment material. We overcome these two difficulties with behavioral tasks that probe, in a fine-grained manner, aspects of higher-order cognition associated with deterioration with aging and dementia, and a new enrichment protocol, the ‘Obstacle Course’ (OC), which enables controlled enrichment delivery, respectively. Together, these two advancements will enable better specification (and comparisons) of the nature of impairments in animal models of complex mental disorders and the potential for remediation from various types of intervention (e.g., enrichment, drugs). We found that two months of OC enrichment produced substantial and sustained enhancements in categorization memory, perceptual object invariance, and cross-modal sensory integration in mice. We also tested mice on behavioral tasks previously shown to benefit from traditional enrichment: spontaneous object recognition, object location memory, and pairwise visual discrimination. OC enrichment improved performance relative to standard housing on all six tasks and was in most cases superior to conventional home-cage enrichment and exercise track groups.

Efficacy and Comparison of Different Strategies for Selenium Biofortification of Tomatoes

At appropriate concentrations, selenium (Se) is beneficial for humans. Tomato appears to be one of the best commodities for producing Se-biofortified fruit for dietary supplementation. To assess the efficacy of different enrichment protocols, a total of four on-plant and off-plant trials were conducted. Hydroponically grown tomato plants were sprayed with: (i) chemically synthesized Se nanoparticles (SeNPs) at 0, 1, and 1.5 mg Se L−1 at blooming; (ii) sodium selenate (Na2SeO4) or SeNPs solution at 0, 5, and 10 mg Se L−1 when the fruit entered the immature green stage. With regard to the off-plant trials, harvested mature green fruit were immersed in Na2SeO4 solution: (iii) at 0, 5, 10, and 20 mg Se L−1 for 15 s under a vacuum; (iv) at 0, 40, and 80 mg Se L−1 for 1 h. Spraying Na2SeO4 induced higher Se accumulation in plant tissue than SeNPs: both protocols were effective in enriching tomatoes. Postharvest Se enrichment via vacuum infiltration caused textural damage, whereas passive immersion in solution induced fruit Se accumulation without causing any damage. SeNPs appear to be quantitatively less effective than Na2SeO4, but might be environmentally safer. Elemental Se carried by NPs may be more easily incorporated into organic forms, which are more bioavailable for humans. Passive immersion may represent an alternative Se-enrichment strategy, allowing for the biofortification of harvested tomato fruit directly, with lower risks of environmental pollution.

A gene-based capture assay for surveying patterns of genetic diversity and insecticide resistance in a worldwide group of invasive mosquitoes.

Understanding patterns of diversification, genetic exchange, and pesticide resistance in arthropod disease vectors is necessary for effective population management. With the availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for a large number of genetic markers. To this end, the targeting of gene sequences of known function can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens such as avian malaria parasites that are detrimental to birds. Here we describe the development of a targeted, gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we sequenced samples from several members of the complex, as well as from distinct populations of the relatively under-studied Culex quinquefasciatus. The data generated was then used to examine taxonomic divergence and population clustering between and within these mosquitoes. We also used this data to investigate genetic variants present in our samples that had previously been shown to correlate with insecticide-resistance. Broadly, our gene capture approach successfully enriched the genomic regions of interest, and proved effective for facilitating examinations of taxonomic divergence and geographic clustering within the Cx. pipiens complex. It also allowed us to successfully survey genetic variation associated with insecticide resistance in Culex mosquitoes. This enrichment protocol will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of these ubiquitous and increasingly damaging disease vectors.

The Effects of Rearing in a Shelved Environment on Behavioral and Physiological Markers of Welfare in Rats (Rattus norvegicus)

Early-life experiences are critical modifiers of development. An important component of early-life experience is the nature of maternal interactions, which can be modified by stress. During rearing, mothers are typically allocated to single-level cages where they are readily accessible to pups, a potentially stressful scenario not reflective of nature. Accordingly, mothers regularly removed from the rearing environment interact differently with their offspring, leading to long-term changes in offspring physiology and behavior. Such changes commonly include modifications within the hypothalamic-pituitary-adrenal axis, of which corticosterone is a major component. Modifications in the hypothalamic-pituitary-adrenal axis may also be manifested through changes in affective behavior and assessed via tests such as the open field and elevated plus maze as well as via ultrasonic vocalization (USV) analysis. As a means of assessing the impact of rearing in a shelved environment, we allocated mothers to standard single-level cages or cages with an integrated shelf, which allowed the mother to temporarily escape pups. While there were no differences in fecal cortico­sterone, behavior in the elevated plus maze, or USVs, male rats reared in standard cages weighed more, and all standard single-level housed rats spent more time in the center of the open field. The observed differences indicate that allocating nursing mothers to shelved environments throughout the postnatal period has long-lasting effects on offspring behavior that must be considered when establishing dam enrichment protocols.

High-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing.

Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.

Enriched environment causes epigenetic changes in hippocampus and improves long-term cognitive function in sepsis

Sepsis is defined as a life-threatening organ dysfunction caused by an inappropriate host response to infection. The presence of oxidative stress and inflammatory mediators in sepsis leads to dysregulated gene expression, leading to a hyperinflammatory response. Environmental conditions play an important role in various pathologies depending on the stimulus it presents. A standard environment condition (SE) may offer reduced sensory and cognitive stimulation, but an enriched environment improves spatial learning, prevents cognitive deficits induced by disease stress, and is an important modulator of epigenetic enzymes. The study evaluated the epigenetic alterations and the effects of the environmental enrichment (EE) protocol in the brain of animals submitted to sepsis by cecal ligation and perforation (CLP). Male Wistar rats were divided into sham and CLP at 24 h, 72 h, 10 days and 30 days after sepsis. Other male Wistar rats were distributed in a SE or in EE for forty-five days. Behavioral tests, analysis of epigenetic enzymes:histone acetylase (HAT), histone deacetylase (HDAC) and DNA methyltransferase (DNMT), biochemical and synaptic plasticity analyzes were performed. An increase in HDAC and DNMT activities was observed at 72 h, 10 days and 30 days. There was a positive correlation between epigenetic enzymes DNMT and HDAC 24 h, 10 days and 30 days. After EE, HDAC and DNMT enzyme activity decreased, cognitive impairment was reversed, IL1-β levels decreased and there was an increase in PSD-95 levels in the hippocampus. Interventions in environmental conditions can modulate the outcomes of long-term cognitive consequences associated with sepsis, supporting the idea of the potential benefits of EE.

P-332 Management of Thin Endometrium by hysteroscopic instillation of G-CSF mobilized progenitor cells combined with platelet-derived growth factor concentrate into the sub endometrial region-A case series

Abstract Study question Can hysteroscopic instillation of G-CSF mobilized progenitor cells combined with platelet derived growth factor concentrate into subendometrial region help patient with thin endometrium conceive? Summary answer Hysteroscopic subendometrial administration of stem cells represents a safe effective alternative method for patients recommended surrogacy, fail to improve despite using all possible treatment options. What is known already Thin endometrium is still a challenge in ART, and is considered sub-optimal for transfer and associated with lower implantation and pregnancy rates. For such patients and their treating physicians, chronically thin endometrium results in repeated expensive IVF cycles, cycle cancellations, unplanned cryopreservation of embryos and, in most extreme cases in the utilization of gestational carriers. Strategies like hysteroscopic adhesiolysis, estrogen, vasoactive chemical measures, and infusion of growth factors into the uterus, have little or no effect. Recently, many studies have successfully used stem cells or PRP alone or in combination for rejuvenation of reproductive tissues and organ failure. Study design, size, duration Case series study involving 20 patients with persistent non-responsive thin endometrium were included and underwent frozen embryo transfer over a period of 18 months from July 2020-December 2021 at A4 fertility center, Chennai Participants/materials, setting, methods 20 patients with thin endometrium and more than two implantation failures were included. Two doses of subcutaneous G-CSF were given on Menstrual Cycle Day 3&4, followed by 60ml venous blood aspirated on MCD 5. Seragen’s selective enrichment protocol was used to prepare circulating endothelial progenitor cells and growth factors concentrate. With 2.9mm hysteroscope and single lumen ovum pickup needle harvested cells and growth factors were implanted in sub-endometrial zone on all four walls of cavity. Main results and the role of chance 20 patients with thin endometrium of less than 7mm and previously more than two implantation failures were included. In spite of routine HRT with Estradiol valerate and intrauterine PRP instillation, endometrium was non-responsive. After another failed attempt using letrozole for ovulation induction to improve endometrial thickness, we ventured into the option of hysteroscopic stem cell instillation. Average endometrial thickness increased from 6.35±0.6mm to 7.32±0.7mm. Out of 20 patients who underwent FET, 12(60%) became pregnant of whom 2(10%) delivered successfully. 6(30%) uneventful pregnancies are ongoing and 4(20%) had miscarriage and 8 patients didn’t conceive are recruited for ET in the next cycle. Many studies have successfully used stem cells or PRP for rejuvenation of reproductive tissues. When these growth-promoting cells and growth factor from platelets are delivered into basal layer which is origin of endometrial cells quality of the endometrium can be improved. While bone marrow is excellent source for obtaining stem cells to treat endometrial pathologies, main challenge is invasiveness of collection. To overcome this, we have adopted G-CSF mobilization to mobilize endothelial progenitor cells from bone marrow into circulation for easy collection and concentration. Limitations, reasons for caution Further research is needed to provide opportunity for women with refractory thin endometrium to conceive without surrogacy. Our study provides promising information for future randomized, controlled trials with large sample size in this field.This study has limitation of being small size with the women serving as their own historical controls. Wider implications of the findings We demonstrated that the proposed combination of two less invasive yet proven therapeutics combined with targeted delivery could be new ray of hope in females recommended for surrogacy or who fail to improve despite all possible treatment options and reduces emotional and financial burden associated with surrogacy and repeated IVFcycles. Trial registration number Not Applicable

Preliminary investigation of microorganisms potentially involved in microplastics degradation using an integrated metagenomic and biochemical approach

Plastic pollution is becoming an emerging environmental issue due to inappropriate disposal at the end of the materials life cycle. When plastics are released, they undergo physical and chemical corrosion, leading to the formation of small particles, commonly referred to as microplastics. In this study, a microbial community derived from the leachate of a bioreactor containing a mixture of soil and plastic collected during a landfill mining process underwent an enrichment protocol in order to select the microbial species specifically involved in plastic degradation. The procedure was set up and tested on polyethylene, polyvinyl chloride, and polyethylene terephthalate, both in anaerobic and aerobic conditions. The evolution of the microbiome has been monitored using a combined approach based on microscopy, marker-gene amplicon sequencing, genome-centric metagenomics, degradation assays, and GC–MS analyses. This procedure permitted us to deeply investigate the metabolic pathways potentially involved in plastic degradation and to depict the route for microplastics metabolization from the enriched microbial community. Six enzymes, among the ones already identified, were found in our samples (alkane 1-monooxygenase, cutinase, feruloyl esterase, triacylglycerol lipase, medium-chain acyl-CoA dehydrogenase, and protocatechuate 4,5-dioxygenase) and new enzymes, addressed as MHETases most probably for the presence of the catalytic triad (His-Asp-Ser), were detected. Among the enzymes involved in plastics degradation, alkane 1-monooxygenase was found in high copy number (between ten and 62 copies) in the metagenomes that resulted most abundant in the microbiome enriched with polyethylene, while protocatechuate 4,5-dioxygenase was found between one and eight copies in the most abundant metagenomes of the microbial culture enriched with polyethylene terephthalate. Degradation assays, performed using both bacterial lysates and supernatants, revealed interesting results on polyethylene terephthalate degradation. Moreover, this study demonstrates to what extent different types of microplastics can affect the microbial community composition. The results obtained significantly increase the knowledge of the plastic degradation process.