Methods and Protocols最新文献

From 2015 to 2023, we conducted a comprehensive study in the 11,893-hectare hunting area managed by the Marcal-Bitvaközi Hunting Company, characterised by its substantial wild boar population. The research was carried out across various settings, including a free-range wild boar garden during large-scale hunts and free-living areas during individual hunts. We examined 216 wild boars in total, with 173 individuals from free-living areas and 43 from free-range areas. Throughout the sample collection process, we encountered numerous technical challenges that are infrequently detailed in the professional literature, often mentioned only tangentially. This oversight in existing publications neglects the significance of addressing field sampling difficulties, which are crucial for ensuring the precision and accuracy of research. This paper details the equipment requirements, sampling methodologies, and practical solutions to streamline fieldwork. While our primary focus was on endoparasitic infections of the stomach and small intestine, the described methodologies and findings are broadly applicable to research involving all internal organs.

3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is a paucity of data regarding microbiome health and outcomes in the pediatric HSCT setting. We developed and thoroughly validated an affordable high-performance liquid chromatography/fluorescence detector (HPLC-FLD) method to quantify 3-IS in urine for use in the pediatric setting. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm × 5 μm) with a mobile phase consisting of pH 4.0 acetic acid-triethylamine buffer and acetonitrile (88:12, v/v), eluted isocratically at 1 mL/min. 3-IS fluorescence detection was set at excitation/emission of 280 and 375, respectively. The method was fully validated according to FDA-specified limits including selectivity, linearity (0.10 to 10.00 mg/L, r² > 0.997), intra- and inter-day accuracy, and precision. 3-IS stability was confirmed after three freeze-thaw cycles, for short- and medium-term on a benchtop and at 4 °C and for long-term up to 60 days at -20 °C. The validated method was used to quantify 3-IS in urine samples from HSCT pediatric patients.

Pressure ulcers are a frequent issue involving localized damage to the skin and underlying tissues, commonly arising from prolonged hospitalization and immobilization. This paper introduces a mathematical model designed to elucidate the mechanics behind pressure ulcer formation, aiming to predict its occurrence and assist in its prevention. Utilizing differential geometry and elasticity theory, the model represents human skin and simulates its deformation under pressure. Additionally, a system of ordinary differential equations is employed to predict the outcomes of these deformations, estimating the cellular death rate in skin tissues and underlying layers. The model also incorporates changes in blood flow resulting from alterations in skin geometry. This comprehensive approach provides new insights into the optimal bed surfaces required to prevent pressure ulcers and offers a general predictive method to aid healthcare personnel in making informed decisions for at-risk patients. Compared to existing models in the literature, our model delivers a more thorough prediction method that aligns well with current data. It can forecast the time required for an immobilized individual to develop an ulcer in various body parts, considering different initial health conditions and treatment strategies.

Hereditary breast/ovarian cancer (HBOC) syndrome is caused by the inheritance of monoallelic germline BRCA1/2 gene mutations. If BRCA1/2 mutation carriers are identified before the disease develops, effective actions against HBOC can be taken, including intensive screening, risk-reducing mastectomy and salpingo-oophorectomy, and risk-reducing medications. The Italian National Prevention Plan mandates the creation of regional BRCA genetic testing programmes. So far, however, only informal data have been reported on their implementation. We have designed a study aimed at evaluating the results of a population-based programme for risk assessment and genetic counselling and testing for BRCA1/2-related HBOC that is underway in the Emilia-Romagna region (northern Italy). The programme-which is entirely free-includes basic screening with an estimate of the likelihood of carrying a BRCA1/2 mutation using a familial risk assessment tool, a closer examination of women with suspected risk increase, an assessment of the need for further genetic counselling and, if needed, genetic testing and risk-reducing interventions. In this paper, the design of the programme and the protocol of the study are presented. The study has an observational, historical cohort design. Eligible are the women found to be at an increased risk of HBOC (profile 3 women). The main objectives are (i) to determine the precision of the programme in measuring the level of risk of HBOC for profile 3 women; (ii) to determine the characteristics of profile 3 women and their association with the risk management strategy chosen; (iii) to compare the age at onset, histologic type, tumour stage, molecular subtype, and prognosis of breast/ovarian cancers observed in the cohort of profile 3 women with the features of sporadic cancers observed in the general female population; (iv) to determine the level and the determinants of adherence to recommendations; and (v) to determine the appropriateness and timing of risk-reducing surgery and medications. Investigating the quality and results of the programme is necessary because the best practices in risk assessment and genetic counselling and testing for BRCA1/2-related cancer and the challenges they encounter should be identified and shared. The study has the potential to provide sound empirical evidence for the factors affecting the effectiveness of this type of service.

Perinatal cohort studies with a prospective longitudinal design are critical for determining the effects of early-life exposures on offspring's health outcomes. The Sri Lanka Mother and Newborn Growth cohort study aims to investigate the impact of maternal nutritional and psychosocial factors on newborns' birth weight in the Sri Lankan context. This paper presents the methodology of participant recruitment, follow-ups, an overview of measurements, and planned data analyses. This study included a nationally representative sample of Sri Lankan pregnant women recruited in their first trimester of pregnancy. Follow-up assessments were conducted once during the second and third trimesters of pregnancy and after the baby's birth, prospectively tracking the women's dietary intake, mental health, hemoglobin concentrations, and gestational weight gain data. Once the participants delivered their babies, the data on gestational age, sex of the newborn, birth weight, length and occipitofrontal circumference at birth, and mode of delivery were collected. Between August 2022 and August 2023, we recruited 2000 first-trimester pregnant women to the cohort and continued to follow up with them until the baby's birth. The response rates were 90.4%, 81.4%, and 75.2% in the first, second, and third follow-ups. We plan to analyze the data in July 2024. We expect this study to provide valuable insights into various early-life exposures affecting neonatal birth weight. The study's findings will serve as a valuable information resource for a broader scientific community, enabling the development of effective policies to prevent low-birth-weight deliveries in low-resource settings.

Staphylococcus aureus and Acinetobacter baumannii are opportunistic pathogens, and both are involved in different oral infections. This work aimed to analyze the phytochemical composition of Myrciaria cauliflora hydroethanolic extract and to evaluate its antimicrobial and antibiofilm action against Staphylococcus aureus (ATCC 6538) and Acinetobacter baumannii (ATCC 19606; multi-resistant clinical strains 58004, 50098, 566006, and H557). Myrciaria cauliflora hydroethanolic extract was prepared, and the content of soluble solids, flavonoids, and phenols was quantified. High-performance liquid chromatography (HPLC) was performed later. The minimum inhibitory concentration was determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute, standard M7-A6, and subsequently, its minimum bactericidal concentration was determined. Then, the most effective concentrations were analyzed against biofilms. Statistical analysis was performed using the ANOVA method with Tukey's test. The soluble solids content in the prepared hydroethanolic extract of M. cauliflora was 2.22%. Additionally, the total flavonoid content, measured using the quercetin standard curve, was 0.040 mg/mL. Furthermore, the total phenol content, determined using the gallic acid standard curve, was 0.729 mg/mL. HPLC analysis presented peaks of gallic acid (11.80 m), p-coumaric acid (12.09 m), cinnamic acid derivative (19.02 m), and ellagic acid (29.83 m). The extract demonstrated antimicrobial and antibiofilm action against all tested strains. However, the most effective antibacterial concentration against all the tested bacteria was 5.55 mg/mL. Therefore, these chemical components justify that M. cauliflora hydroethanolic extract is effective in reducing biofilm formation in S. aureus (standard strain) and A. baumannii (standard and clinical strains).

This study focuses on the development and optimization of a water removal device for biogenic volatile organic compounds (BVOCs) from plant emissions. BVOCs play a crucial role in various ecological processes and have potential therapeutic effects on human health. However, it is challenging to accurately detect and analyze BVOCs due to their very low concentrations and interference by water vapor. This study systematically evaluates different filler materials and ratios to alleviate water vapor interference while maintaining BVOCs' integrity. The experimental results demonstrate that the combination of MgSO₄ + Na₂SO₄ mixed filling and CuSO₄ layered filling in a 3:3:1 ratio can effectively improve the collection efficiency and detection accuracy of BVOCs. Meanwhile, the effectiveness of the device in improving the detection of volatile compounds in plant samples is also confirmed by the VOC verification experiments on Michelia maudiae and Cinnamomum camphora tree species after mechanical damage. The experimental results show that the device is effective in improving the detection of volatile compounds in plant samples. The findings provide a powerful technical means for exploring the role of BVOCs in environmental monitoring and scientific research.

Unresectable, metastatic, advanced urothelial carcinoma (aUC) is an aggressive disease and is treated with platinum-containing first-line chemotherapy, followed by immune checkpoint inhibitors and antibody-drug conjugates. Response to first-line chemotherapy is a vital priority in sequential treatment strategies because a better response to first-line chemotherapy is associated with a better response to subsequent therapies. Gemcitabine plus carboplatin chemotherapy is conventionally recommended for cisplatin-ineligible patients. This multicenter, single-arm prospective trial will investigate whether dose-dense methotrexate, vinblastine, doxorubicin, and carboplatin (DD-MVACarbo) chemotherapy is superior to gemcitabine plus carboplatin chemotherapy in terms of efficacy in platinum-naïve, cisplatin-ineligible patients with aUC. After screening and registration, a total of 46 patients will be treated with this novel chemotherapy regimen. The primary endpoint is the objective response rate. The secondary endpoints include disease control rate, patient-reported outcomes, and adverse events. No evidence of this novel intervention is available as of July 2024. The results are expected to change the standard of care and improve the management of patients with aUC.

With the advent of "next-generation" sequencing and the continuous reduction in sequencing costs, an increasing amount of genomic data has emerged, such as whole-genome, whole-exome, and targeted sequencing data. These applications are popular not only in mega sequencing projects, such as the 1000 Genomes Project and UK BioBank, but also among individual researchers. Evolutionary genetic analyses, such as the dN/dS ratio and Tajima's D, are demanded more and more for whole-genome-level population data. These analyses are often carried out under a uniform custom bin size across the genome. However, these analyses require subdivision of a genomic region into functional units, such as protein-coding regions, introns, and untranslated regions, and computing these genetic measures for large-scale data remains challenging. In a recent investigation, we successfully devised a method to address this issue. This method requires a multi-sample VCF file containing population data, a reference genome, target regions in the BED file, and a list of samples to be included in the analysis. Given that the targeted regions are extracted in a new VCF file, targeted population genetic analysis can be performed. We conducted Tajima's D analysis using this approach on intact and pseudogenes, as well as non-coding regions.

The first step in anthropological study is the positive identification of human remains, which can be a challenging undertaking when bones are broken. When bone pieces from different species are mixed together, it can be crucial to distinguish between them in forensic and archaeological contexts. For years, anthropology and archaeology have employed the histomorphological analysis of bones to evaluate species-specific variations. Based on variations in the dimensions and configuration of Haversian systems between the two groups, these techniques have been devised to distinguish between non-human and human bones. All of those techniques concentrate on a very particular kind of bone, zone, and segment. Histomorphometric techniques make the assumption that there are size, form, and quantity variations between non-humans and humans. The structural components of Haversian bones are significant enough to use discriminant function analysis to separate one from the other. This review proposes a comprehensive literature analysis of the various strategies or techniques available for distinguishing human from non-human bones to demonstrate that histomorphological analysis is the most effective method to be used in the case of inadequate or compromised samples.