Neglected contributors to the transmission of bacterial antibiotic resistance in drinking water: Extracellular antibiotic resistance genes and the natural transformation.

Antibiotic resistance genes (ARGs) have increasingly gained recognition as an "emerging contaminant" that poses a threat to the biosafety of drinking water. However, previous researches have primarily focused on the intracellular state of ARGs and rarely investigated the ecological characteristics (e.g., distribution and origin), environmental behavior (spread), and risks of extracellular form (eARGs) within drinking water systems. Therefore, this review evaluated isolation strategies and extraction methods for recovering eARGs from drinking water, elucidated the distribution characteristics of eARGs, and examined their impact on the antibiotic resistome from source water to tap water. We emphasized that chlorination and biological treatments significantly contribute to the prevalence and persistence of eARGs in drinking water. Moreover, we highlighted the role of biological reactors (e.g., biofilter, biological activated carbon) and drinking water distribution systems in facilitating the natural transformation of eARGs while significantly contributing to bacterial antibiotic resistance (BAR) propagation. Finally, we summarized the current risk assessment systems for ARGs and critically address remaining challenging questions necessary for better forecasting health risks associated with eARGs in drinking water environments. Collectively, this review enhances the understanding of ecological characteristics and environmental behavior of eARGs in drinking water while providing important implications for controlling and reducing BAR contamination not only in drinking water but also in other aquatic environments.

Evaluating the Efficacy of the Diluted Schirmer Method for Tear Collection in Dry Eye Syndrome Patients.

PURPOSE: This study assesses the diluted Schirmer method's effectiveness in collecting tears from dry eye syndrome patients, aiming to identify the most suitable tear collection technique for them. METHODS: A prospective study. Tear samples were collected from patients with dry eye syndrome and healthy individuals using two methods: (1) Direct Schirmer Method: Schirmer strips were directly inserted into the eye to collect tears. (2) Diluted Schirmer Method: After instilling physiological saline into the eye and waiting for 30 s to ensure thorough mixing with tears, Schirmer strips were used for collection. Tear samples from both groups were analyzed and compared for total protein and cytokine levels (IL-1ß, IL-6, IL-8, TNF-α). RESULTS: (1) The study included 32 participants: 16 with dry eye syndrome (4 males, 12 females, average age 34.92 ± 10.13 years) and 16 healthy controls (5 males, 11 females, average age 32.25 ± 9.87 years). (2) The diluted Schirmer method produced a significantly larger tear volume compared to the direct method (p < 0.05), with lower Visual Analogue Scale (VAS) scores indicating less discomfort (p < 0.05). (3) The average total protein content of the two groups was 51.70 ± 3.166 ng measured by Direct Schirmer method, and the average total protein content of the Diluted Schirmer method was 50.05 ± 3.263 ng. There was no statistical difference between the two groups. (t = 1.051, p = 0.3098) (4) The concentrations of total tear protein and various cytokines measured by both methods were higher in the dry eye group compared to the normal group, with statistically significant differences (p < 0.05). Both methods reflected consistent changes in tear protein profiles. CONCLUSION: The diluted Schirmer method can comfortably collect an adequate volume of tear samples in a short time and consistently reflect changes in tear proteins, making it an effective method for tear collection in patients with dry eye syndrome.

Do Concurrent Peri-Implantitis and Periodontitis Share Their Microbiotas? A Pilot Study.

The microbial compositions from concurrent peri-implant and periodontal lesions were compared, since the results reported in the literature on the etiological relationship between these oral pathologies are contradictory. Microbial compositions from nine patients were evaluated using Illumina MiSeq sequencing of 16S rRNA gene amplicons and Principal Components Analysis. Comparisons between the use of curettes or paper points as collection methods and between bacterial composition in both pathologies were performed. Paper points allowed the recovery of a higher number of bacterial genera. A higher bacterial diversity was found in peri-implantitis compared to periodontal samples from the same patient, while a greater number of operational taxonomic units (OTUs) were present in the corresponding periodontal samples. A higher abundance of oral pathogens, such as Porphyromonas or Treponema, was found in peri-implantitis sites. The opposite trend was observed for Aggregatibacter abundance, which was higher in periodontal than in peri-implantitis lesions, suggesting that both oral pathologies could be considered different but related diseases. Although the analysis of a higher number of samples would be needed, the differences regarding the microbial composition provide a basis for further understating the pathogenesis of peri-implant infections.

Designing and Evaluating a Portable UV-LED Vane Trap to Expedite Arthropod Biodiversity Discovery.

A novel design of a portable funnel light trap (PFLT) was presented for collecting insects in ecological studies. The trap consists of a compact plastic box equipped with a light source and power source, along with two plastic polypropylene interception vanes. The PFLT costs 18.3 USD per unit and weighs approximately 300 g. A maximum of six PFLT units can be packed in one medium-sized backpack (32 cm × 45 cm × 15 cm, 20 L), making it easier to set up multiple units in remote areas wherein biodiversity research is needed. The low cost and weight of the trap also allows for large-scale deployment. The design is customizable and can be easily manufactured to fit various research needs. To validate the PFLT's efficacy in collecting insects across different habitat types, a series of field experiments were conducted in South Korea and Laos, where 37 trials were carried out. The PFLT successfully collected 7497 insects without experiencing battery issues or damage by rain or wind. Insect compositions and abundances differed across the three sampled habitat types: forests, grasslands, and watersides. This new FLT trap is an important tool for studying and protecting insect biodiversity, particularly in areas wherein conventional light traps cannot be deployed.

Bridging the Gap between Field Experiments and Machine Learning: The EC H2020 B-GOOD Project as a Case Study towards Automated Predictive Health Monitoring of Honey Bee Colonies.

Honey bee colonies have great societal and economic importance. The main challenge that beekeepers face is keeping bee colonies healthy under ever-changing environmental conditions. In the past two decades, beekeepers that manage colonies of Western honey bees (Apis mellifera) have become increasingly concerned by the presence of parasites and pathogens affecting the bees, the reduction in pollen and nectar availability, and the colonies' exposure to pesticides, among others. Hence, beekeepers need to know the health condition of their colonies and how to keep them alive and thriving, which creates a need for a new holistic data collection method to harmonize the flow of information from various sources that can be linked at the colony level for different health determinants, such as bee colony, environmental, socioeconomic, and genetic statuses. For this purpose, we have developed and implemented the B-GOOD (Giving Beekeeping Guidance by computational-assisted Decision Making) project as a case study to categorize the colony's health condition and find a Health Status Index (HSI). Using a 3-tier setup guided by work plans and standardized protocols, we have collected data from inside the colonies (amount of brood, disease load, honey harvest, etc.) and from their environment (floral resource availability). Most of the project's data was automatically collected by the BEEP Base Sensor System. This continuous stream of data served as the basis to determine and validate an algorithm to calculate the HSI using machine learning. In this article, we share our insights on this holistic methodology and also highlight the importance of using a standardized data language to increase the compatibility between different current and future studies. We argue that the combined management of big data will be an essential building block in the development of targeted guidance for beekeepers and for the future of sustainable beekeeping.

A comparative study of canine epididymal sperm collection techniques and cryopreservation.

Introduction: An optimized collection method and freezing protocol for preservation of epididymal spermatozoa remains a topic of interest to many scientists. The current study focused on the collection and preservation of canine epididymal spermatozoa. During the process of collection of canine epididymal spermatozoa, blood content can occur, which may affect sperm cryopreservation in a negative way. Here, we compared first two epididymal sperm collection techniques [epididymal mincing (EM) and single incision epididymal sperm aspiration (SESA)]; and next we tried to solve the issue of blood content using an erythrocyte lysis buffer (ELB). Methods: Hence spermatozoa were collected after weighing the epididymides, either by EM or SESA, and sperm quality assessed prior to and post freezing (concentration, total sperm output (TSO), motility, viability and morphology). Next, new sperm samples were collected from eight epididymides by EM and subjected either to a standard freezing protocol or to an ELB treatment freezing protocol. Post-thaw sperm parameters (concentration, TSO, motility, viability and morphology), including intracellular reactive oxygen species (ROS) and lipid peroxidation were assessed. The correlation between the weight of the epididymis and the TSO was evaluated based on the collection technique, and differences in sperm parameters were detected both within different collection techniques and between different pre-freezing treatment protocols. Results: There was a very strong correlation between the weight of the epididymis and the TSO for the EM technique (p = 0.002, R2 = 0.6), along with an increased sperm motility with EM compared to SESA (median 80%, inter-quartile range (IQR) 88-65 and median 67.5%, IQR 72.5-52.5, respectively; (p = 0.002). Post-thaw samples subjected to ELB treatment freezing protocol had lower motility and higher intracellular ROS compared to the standard freezing protocol (motility: median 56.25%, IQR 60-48.75 and median 70%, IQR 72.5-63, respectively; p = 0.01; ROS: median 78.5%, IQR 81.25-75.5 and median 70%, IQR 70.5-68.75, respectively; (p = 0.04). Discussion: The results indicated that EM is a better technique to harvest epididymal spermatozoa despite the presence of some blood content. Furthermore, the ELB treatment should not be implemented to remove those red blood cells prior to cryopreservation of epididymal spermatozoa in dogs.

Collection of environmental DNA from stemflow for monitoring arboreal biodiversity: Preliminary validation using lichens.

The forest canopy harbors a diverse array of organisms. However, monitoring their biodiversity poses challenges due to limited accessibility and the vast taxonomic diversity. To address these challenges, we present a novel method for capturing arboreal biodiversity by harnessing stemflow as a source of DNA from organisms inhabiting trees. Our method involves encircling the tree trunk with gauze, directing the stemflow along the gauze into a funnel, and collecting it in a plastic bag. We employed dual collection systems to retrieve environmental DNA (eDNA) from the stemflow: the gauze trap, designed to capture macroscopic biological fragments, and the plastic bag trap, which collected the stemflow itself. The trapped fragments and stemflow were separately filtered, and eDNA was subsequently extracted from the filter membranes. To validate our method, we focused on foliose lichens, which are easily observable on tree surfaces. We performed eDNA metabarcoding and successfully detected a majority of the observed foliose lichen species, including those not identified through visual observation alone.•We have developed a non-invasive and straightforward method for monitoring arboreal biodiversity by collecting eDNA from stemflow, which has been validated using lichens for its efficacy.•This cost-effective approach minimizes disruptions to tree ecosystems and is expected to provide an efficient means of sampling and monitoring arboreal organisms.

Comparison of cystocentesis versus home sampling to determine urinary protein: Creatinine ratio and urine specific gravity in cats.

BACKGROUND: Urinalysis is necessary for the diagnostic evaluation of chronic kidney disease in cats. Performing cystocentesis is not always feasible, but data comparing urine obtained by cystocentesis in the clinic with voided samples collected at home are lacking in cats. OBJECTIVES: To compare urinary protein:creatinine ratio (UPC) and urine specific gravity (USG) and to detect clinically relevant changes in proteinuria substage or urine concentration between urine collected at home and in-clinic by cystocentesis in cats. ANIMALS: Ninety-two healthy and diseased client-owned cats. METHODS: Prospective study. Owners collected voided urine at home and within 1 to 15 hours, cystocentesis was performed in the clinic. RESULTS: In a subset of motivated owners, 55% succeeded in collecting urine at home. Overall, UPC was higher (mean ±SD difference = 0.09 ±0.22; P < .001) and USG was lower (mean ±SD difference = -0.006 ±0.009; P < .001) in cystocentesis samples than in voided urine. Substantial agreement existed between sampling methods for UPC (weighted к = 0.68) and USG (к = 0.64) categories. A different proteinuria substage (UPC < 0.2, 0.2-0.4, >0.4) was present in paired urine samples from 28% of cats. In 18% of cats, urine concentrating ability (USG < or ≥1.035) differed between both samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Home sampling of urine is a valid alternative to cystocentesis in cats. However, because clinically relevant differences in UPC and USG were present in 28% and 18% of cats, respectively, by the same collection method for monitoring each cat is advised.

Integrated MCDM Approaches for Exploring the Ideal Therapeutic Plastic Disposal Technology: Probabilistic Hesitant Fuzzy Domain.

The probabilistic hesitant fuzzy set (PHFS) is a useful extended version of the hesitant fuzzy set (HFS), which allows decision-makers greater freedom in espousing their preferences through the use of hesitant evidence in the real DM method. As the implications for individuals and global concerns have grown, efficient clinical diagnosis of medical waste has been a major challenge, particularly in developing countries. Medical waste can be disposed of in a variety of ways. The essential thing is to decide which strategies work best. The optimal healthcare plastic waste disposal (HCPWD) option is a MCDM method involving a wide range of qualitative characteristics. The MCDM technique (ARAS) is then described, whereby the criterion weights are assessed using the recommended entropy weighted method (EWM) proportion and score function in order to increase the process utilisation. Moreover, the above-described approach is used to address a real-world problem by determining the optimal treatment option for healthcare waste (HCW) disposal. Finally, a feasibility analysis is given to support the stated viewpoint on HCPWD options being prioritised.

Use of ion chromatographic pulsed amperometric method (IC-PAD) for measuring aqueous sulfide in synthetic and real domestic wastewater.

Sulfide detection in domestic wastewater is widely demanded, as sulfide induces odour nuisance and wastewater assets corrosion. However, traditional sulfide detection methods are usually plagued by the limited detection range or interference from impurities. To address these constraints, this study improved the ion chromatographic pulsed amperometric method (IC-PAD) and tested its validity for use in domestic wastewater. Prior to sulfide detection, sulfide-containing sample collection usually requires the use of sulfide antioxidant buffers (SAOB) to minimize sulfide loss. Different sample matrixes require different SAOB recipes, which increases complexity and uncertainty when measuring different environmental samples. Therefore, this study also developed a more convenient and generic sample collection method without the addition of SAOB. The results indicated that the proposed SAOB-free sample collection method could minimize the sulfide loss during sample collection. The IC-PAD method showed a wide linear detection range up to 10 mg-S/L. The detection limit was 3 µg-S/L. Matrix effect studies showed that 1 g/L glucose, formate, acetate, methanol, ethanol, propionate, butyrate, lactate, or sulfate had no evident interference on sulfide measurement. However, 5 mM phosphate buffer led to interference, but reducing the KOH eluent concentration from 62 to 30 mM avoid this interference. Wolfe's vitamin mixture and Wolfe's modified mineral mixture could cause diminutive interference equivalent to 2.53 ± 1.32 µg-S/L sulfide. Moreover, the interference caused by chloride indicated that the IC-PAD method is more applicable for measuring sulfide in low-chloride wastewater. To this end, the IC-PAD method showed high accuracy and precision in the real domestic wastewater samples with chloride concentration of 68 mg/L. The recovery was higher than 97% and the relative standard deviation (RSD) was lower than 1.2%. This study demonstrated the potential use of IC-PAD method for measuring sulfide in real domestic wastewater and possible interference from the solution matrix to be considered.

Towards an effective framework for integrating patient-reported outcomes in electronic health records.

Background: In the past decade, electronic modalities are increasingly deployed to integrate patient-reported outcomes into electronic health records. Most popularly, patient portals are used for remote questionnaires, and tablets are provided to patients in-office in case they need help. They are both useful. But some barriers are still in the way, which place burdens on patients and clinicians in the process of routine data collection. Objective: This study aims to describe a portable and scalable framework which can simplify the patient-reported outcome integration by mitigating the related burdens. Methods: A framework was proposed to use a modular approach to replace the tethered approach. The framework was open-sourced on GitHub. After development and testing, it was evaluated on an instrument with 24 questions in a real clinical setting. Patients were randomly selected in every modality-based group. For objective analysis, completion time and response rate were collected. No-show data was collected and analyzed. For subjective analysis, the NASA Task Load Index was used to measure workload, and the Net Promoter Score was used to assess user satisfaction. Results: The model could contain 46,656 questions. A quick response code could store 1120 encoded items. For remote visits, the response rate was improved compared to the portal group (76.6% vs. 61.1%). The completion time was reduced by 37.5% when compared to the tablet group and was reduced by 43.4% when compared to the portal group. The workload for clinicians and patients was both reduced significantly (p < 0.001). A higher Net Promoter Score was rated by both clinicians (89.3%) and patients (86.5%). Compared to the portal group, the no-show rate was reduced (11.7% vs. 8.6%). Conclusions: Collecting patient-reported outcomes over a quick response code appears to be an alternative modality to enable a simplified integration. This study provides new insights to collect patient-reported outcomes with interoperability and substitutability in mind.

Measurement of anti SARS-CoV-2 RBD IgG in saliva: validation of a highly sensitive assay and effects of the sampling collection method and correction by protein.

OBJECTIVES: To develop and evaluate a new highly sensitive assay to detect IgG anti-SARS-CoV-2 RBD in saliva samples. METHODS: A two-step sandwich type immunoassay based on the amplified luminescent proximity homogeneous technology was developed and an analytical validation was performed. As a part of this validation, the influence of factors, such as different sampling conditions (stimulated saliva and passive drool) and the correction of values by total protein content, in the ability of saliva to detect increases in antibodies after an immune stimulus and be an alternative to serum, was evaluated. For this purpose, paired samples of saliva and serum at different times after vaccination were used. RESULTS: Saliva concentrations were lower than serum, but both fluids showed similar kinetics, with higher correlations when saliva was obtained by passive flow and the results were not corrected by protein. CONCLUSIONS: The developed method showed a good analytical performance and can properly measure antibody concentrations in saliva of vaccinated individuals. However, saliva could have a lower sensitivity compared to serum at initial stages of the immune response and also when the antibody response decreased after a stimulus.

Investigation of Whole and Glandular Saliva as a Biomarker for Alzheimer's Disease Diagnosis.

Salivary Aß40, Aß42, t-tau, and p-tau 181 are commonly employed in Alzheimer's disease (AD) investigations. However, the collection method of these biomarkers can affect their levels. To assess the impact of saliva collection methods on biomarkers in this study, 15 healthy people were employed in the morning with six saliva collection methods. The chosen methods were then applied in 30 AD patients and 30 non-AD controls. The levels of salivary biomarkers were calculated by a specific enzyme-linked immunosorbent assay. The receiver operating characteristic was utilized to assess salivary biomarkers in AD patients. The results demonstrated that the highest levels of salivary Aß40, Aß42, t-tau, and p-tau were in different saliva collection methods. The correlations between different saliva biomarkers in the same collection method were different. Salivary Aß40, Aß42, t-tau, and p-tau had no significant association. Salivary Aß42 was higher in AD than in non-AD controls. However, p-tau/t-tau and Aß42/Aß40 had some relevance. The area under the curve for four biomarkers combined in AD diagnosis was 92.11%. An alternate saliva collection method (e.g., USS in Aß40, UPS in Aß42, t-tau, SSS in p-tau 181) was demonstrated in this study. Moreover, combining numerous biomarkers improves AD diagnosis.

Identifying small RNAs and Analyzing Their Association with Gene Expression Using Isolated Arabidopsis Male Meiocytes.

Meiosis is a specialized cell division that generates gametes and is essential for sexual reproduction. Studying meiosis in plants, like the model flowering plant Arabidopsis thaliana, contributes to our understanding of the fundamental biology of reproductive biology and has practical implications for improving economically important crop species. In this chapter, we provide a detailed protocol for capillary collection of Arabidopsis male meiocytes followed by total RNA extraction, RNA-Seq, and bioinformatics analysis of small-RNAs (sRNAs) including analysis of sRNA cluster that correlate with genomic features.

Correlations of Salivary and Blood Glucose Levels among Six Saliva Collection Methods.

BACKGROUND: Saliva has been studied as a better indicator of disorders and diseases than blood. Specifically, the salivary glucose level is considered to be an indicator of diabetes mellitus (DM). However, saliva collection methods can affect the salivary glucose level, thereby affecting the correlation between salivary glucose and blood glucose. Therefore, this study aims to identify an ideal saliva collection method and to use this method to determine the population and individual correlations between salivary glucose and blood glucose levels in DM patients and healthy controls. Finally, an analysis of the stability of the individual correlations is conducted. METHODS: This study included 40 age-matched DM patients and 40 healthy controls. In the fasting state, saliva was collected using six saliva collection methods, venous blood was collected simultaneously from each study participant, and both samples were analyzed at the same time using glucose oxidase peroxidase. A total of 20 DM patients and 20 healthy controls were arbitrarily selected from the above participants for one week of daily testing. The correlations between salivary glucose and blood glucose before and after breakfast were analyzed. Finally, 10 DM patients and 10 healthy controls were arbitrarily selected for one month of daily testing to analyze the stability of individual correlations. RESULTS: Salivary glucose levels were higher in DM patients than healthy controls for the six saliva collection methods. Compared with unstimulated saliva, stimulated saliva had decreased glucose level and increased salivary flow. In addition, unstimulated parotid salivary glucose was most correlated with blood glucose level (R2 = 0.9153), and the ROC curve area was 0.9316, which could accurately distinguish DM patients. Finally, it was found that the correlations between salivary glucose and blood glucose in different DM patients were quite different. The average correlation before breakfast was 0.83, and the average correlation after breakfast was 0.77. The coefficient of variation of the correlation coefficient before breakfast within 1 month was less than 5%. CONCLUSION: Unstimulated parotid salivary glucose level is the highest and is most correlated with blood glucose level, which can be accurately used to distinguish DM patients. Meanwhile, the correlation between salivary glucose and blood glucose was found to be relatively high and stable before breakfast. In general, the unstimulated parotid salivary glucose before breakfast presents an ideal saliva collecting method with which to replace blood-glucose use to detect DM, which provides a reference for the prediction of DM.

Comparison of oral human papilloma virus detection methods among Hispanic adults.

OBJECTIVES: Oral human papilloma virus (HPV) infection is associated with nearly three-quarters of all oropharyngeal cancers in the United States. Research also suggests its association with periodontal disease. There are limited studies evaluating differences in HPV detection methods; however, oral rinse is considered the most sensitive detection method. We compared HPV detection by self-collected oral rinse versus self-collected cytobrush and assessed whether the strength of association between periodontitis and HPV is modified by the collection method. MATERIALS AND METHODS: Data from a cross-sectional study of Hispanic adults in Puerto Rico (n = 346) who provided oral rinse and cytobrush samples for oral HPV detection and were clinically evaluated for periodontitis. The agreement between the oral mouthwash and cytobrush methods was assessed using the Kappa (κ) statistic. Logistic regression models were used to determine if the association between HPV infection and other risk factors varied by oral sample collection method. RESULTS: HPV prevalence was slightly higher using cytobrush than oral rinse (5.8% vs. 4.3%). The sensitivity of cytobrush to detect oral HPV was 64.7%, and the specificity was 97.4%. We observed a κ of 0.61 (95% confidence interval [CI]: 0.45-0.78), indicative of fair to good agreement between the two collection methods. The association between oral HPV infection and periodontitis severity was stronger when using the oral rinse collection method (odds ratio [OR] = 3.23, 95% CI: 1.06-9.84); the association was not statistically significant for cytobrush (OR = 1.96, 95% CI: 0.68-5.65). CONCLUSIONS: These findings support the significance of choosing the most suitable collection method in oral HPV-related studies. Selecting the most appropriate collection method is an essential criterion in oral HPV-related studies.

Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics.

Integrative analysis of high-quality metagenomics and metabolomics data from fecal samples provides novel clues for the mechanism underpinning gut microbe-human interactions. However, data regarding the influence of fecal collection methods on both metagenomics and metabolomics are sparse. Six fecal collection methods (the gold standard [GS] [i.e., immediate freezing at -80°C with no solution], 95% ethanol, RNAlater, OMNIgene Gut, fecal occult blood test [FOBT] cards, and Microlution) were used to collect 88 fecal samples from eight healthy volunteers for whole-genome shotgun sequencing (WGSS) and untargeted metabolomic profiling. Metrics assessed included the abundances of predominant phyla and α- and ß-diversity at the species, gene, and pathway levels. Intraclass correlation coefficients (ICCs) were calculated for microbes and metabolites to estimate (i) stability (day 4 versus day 0 within each method), (ii) concordance (day 0 for each method versus the GS), and (iii) reliability (day 4 for each method versus the GS). For the top 4 phyla and microbial diversity metrics at the species, gene, and pathway levels, generally high stability and reliability were observed for most methods except for 95% ethanol; similar concordances were seen for different methods. For metabolomics data, 95% ethanol showed the highest stability, concordance, and reliability (median ICCs = 0.71, 0.71, and 0.65, respectively). Taken together, OMNIgene Gut, FOBT cards, RNAlater, and Microlution, but not 95% ethanol, were reliable collection methods for gut metagenomic studies. However, 95% ethanol was the best for preserving fecal metabolite profiles. We recommend using separate collecting methods for gut metagenomic sequencing and fecal metabolomic profiling in large population studies. IMPORTANCE The choice of fecal collection method is essential for studying gut microbe-human interactions in large-scale population-based research. In this study, we examined the effects of fecal collection methods and storage time at ambient temperature on variations in the gut microbiome community composition; microbial diversity metrics at the species, gene, and pathway levels; antibiotic resistance genes; and metabolome profiling. Our findings suggest using different fecal sample collection methods for different data generation purposes. OMNIgene Gut, FOBT cards, RNAlater, and Microlution, but not 95% ethanol, were reliable collection methods for gut metagenomic studies. However, 95% ethanol was the best for preserving fecal metabolite profiles.

The Possibility of Analyzing Endometrial Receptivity Using Cells from Embryo Transfer Catheters.

It is very important to investigate the expression of endometrial receptive markers in the endometrium during implantation. Therefore, we examined whether it would be possible to analyze endometrial receptivity using cells from embryo transfer catheters. A total of 81 cycles from 81 consenting patients were enrolled in this study. The tip of the embryo transfer (ET) catheter was cut and immersed in a dedicated reagent. Confirmation of cell distribution was carried out using a Papanicolaou stain and immunocytochemistry. Protein expression was carried out by immunocytochemistry. The expressions of estrogen receptor α, progesterone receptor, and homeobox A10 mRNA were analyzed using quantitative reverse transcription-polymerase chain reaction. We analyzed the relationship between the gene expression profiles associated with pregnancy from endometrial cells. Samples collected from the ET catheter showed clear staining for endometrial cells. Most of the cells were endometrial epithelial cells. Cervical cells were not observed. The protein expression was also confirmed. Three genes were analyzed that are associated with endometrial receptivity. Progesterone receptor expression was 1.4-fold (p<0.05) and homeobox A10 was 2.8-fold (p<0.01) higher in patients who became non-pregnant group, compared to the pregnant group. Estrogen receptor α expression tended to be higher in the non-pregnant group (p=0.18). Our results suggest that endometrial receptivity can be evaluated using cells obtained from the ET catheter. This method may be useful for elucidating the cause of implantation failure by comparing a receptive and non-receptive endometrium at the time of ET.

Optimal Collection Methods for Asian Longhorned Ticks (Ixodida: Ixodidae) in the Northeast United States.

The Asian longhorned tick, Haemaphysalis longicornis Neumann, is an invasive species in the United States. Since its earliest recorded presence in West Virginia in 2010, H. longicornis has been reported from 15 states. While its public health significance in the United States is unclear, globally it transmits pathogens that infect livestock and humans, causing economic losses and substantial morbidity. Management and control of H. longicornis requires knowledge of its biology, ecology, and distribution. Here, we address the need for effective collection methods for host-seeking H. longicornis as an important step for accurately assessing tick abundance and potential disease risk. The number of H. longicornis collected were compared across three collection methods (dragging, sweeping, CO2 traps) and three tick check distances (5 m, 10 m, and 20 m) were compared for dragging and sweeping. Field collections were conducted from June through August 2019 in Westchester County, New York, and ticks were grouped by life stage to assess collection method efficiency. Results indicated that implementing shorter (5 m) tick check distance was ideal for adult and nymphal collections. The dragging method proved better than sweeping for adult collections; however, there was no significant difference between the methods for nymphal collections, at any tick check distance evaluated. CO2 traps attracted H. longicornis, but additional research is necessary to devise an effective tick retaining method before the traps can be implemented in the field. The results are presented to inform and support H. longicornis surveillance and control programs across the nation.

Comparison of murine retroorbital plexus and facial vein blood collection to mitigate animal ethics issues.

BACKGROUND: Blood collection is an important procedure used in animal experiments. Blood collection methods that reduce pain, injury, and stress in experimental animals are important with regard to animal ethics. Various comparative studies of blood collection methods have been reported; however, there are no comparative studies on serial blood collection considering animal ethics. To suggest simple methods that minimize pain during serial blood collection, we compared the retroorbital plexus (RP) and facial vein (FV) blood collection methods performed by both experienced and novice groups. The experienced and novice groups collected up to 0.4 mL of blood via the RP and FV methods every second day for 2 weeks. After blood collection, all mice were evaluated by corticosterone concentrations for stress, hematological, immunological, and histological analyses. RESULTS: We found that the FV methods reduced the collection time, pain, distress, tissue damage and lasting harms without anesthesia. Corticosterone concentrations in the peripheral blood were decreased in mice subjected FV methods compare with those subjected to RP methods. The proportion of granulocytes and monocytes, such as macrophages in the peripheral blood and spleen, was decreased in mice subjected to FV methods compared with that in mice subjected to RP methods in both experienced and novice groups. White blood cells were infiltrated in RP areas with severe tissue damage and inflammation. CONCLUSIONS: With respect to animal ethics, we suggest that the FV method, a simple and fast technique that can easily be performed by both experienced and novice researchers, is suitable for serial blood collection.