Isolation Techniques Research Articles

Streamlined Analysis of Maternal Plasma Indicates Small Extracellular Vesicles are Significantly Elevated in Early-Onset Preeclampsia

Preeclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity. While placental dysfunction is a core underlying issue, the pathogenesis of this disorder is thought to differ between early-onset (EOPE) and late-onset (LOPE) subtypes. As recent reports suggest that small extracellular vesicles (sEVs) contribute to the development of PE, we have compared systemic sEV concentrations between normotensive, EOPE, and LOPE pregnancies. To circumvent lengthy isolation techniques and intermediate filtration steps, a streamlined approach was developed to evaluate circulating plasma sEVs from maternal plasma. Polymer-based precipitation and purification were used to isolate total systemic circulating maternal sEVs, free from bias toward specific surface marker expression or extensive subpurification. Immediate Nanoparticle Tracking Analysis (NTA) of freshly isolated sEV samples afforded a comprehensive analysis that can be completed within hours, avoiding confounding freeze–thaw effects of particle aggregation and degradation.Rather than exosomal subpopulations, our findings indicate a significant elevation in the total number of circulating maternal sEVs in patients with EOPE. This streamlined approach also preserves sEV-bound protein and microRNA (miRNA) that can be used for potential biomarker analysis. This study is one of the first to demonstrate that maternal plasma sEVs harbor full-length hypoxia inducible factor 1 alpha (HIF-1α) protein, with EOPE sEVs carrying higher levels of HIF-1α compared to control sEVs. The detection of HIF-1α and its direct signaling partner microRNA-210 (miR-210) within systemic maternal sEVs lays the groundwork for identifying how sEV signaling contributes to the development of preeclampsia. When taken together, our quantitative and qualitative results provide compelling evidence to support the translational potential of streamlined sEV analysis for future use in the clinical management of patients with EOPE.

Exosomes in Osteoarthritis: A Review on Their Isolation Techniques and Therapeutic Potential.

Exosomes are the smallest extracellular vesicles (30-150nm) secreted by all cell types, including synovial fluid. However, because biological fluids are complex, heterogeneous, and contain contaminants, their isolation is difficult and time-consuming. Furthermore, the pathophysiology of osteoarthritis (OA)involves exosomescarryingcomplex components that cause macrophages to release chemokines and proinflammatory cytokines. This narrative review aims to provide in-depth insights into exosome biology, isolation techniques, role in OA pathophysiology, and potential role in future OA therapeutics. A literature search was conducted using PubMed, Scopus, and Web of Science databases for studies involving exosomes in the osteoarthritis using keywords "Exosomes" and "Osteoarthritis". Relevant articles in the last 15years involving both human and animal models were included. Studies involving exosomes in other inflammatory diseases were excluded. Despite some progress, conventional techniques for isolating exosomes remain laborious and difficult, requiring intricate and time-consuming procedures acrossvarious body fluids and sample origins. Moreover, exosomes are involved in various physiological processes associated with OA, like cartilage calcification, degradation of osteoarthritic joints, and inflammation. The process of achieving standardization, integration, and high throughput of exosome isolation equipment is challenging and time-consuming. The integration of various methodologies can be employed to effectively address specific issues by leveraging their complementary benefits. Exosomes have the potential to effectively repair damaged cartilage OA, reduce inflammation, and maintain abalance between the formation and breakdown of cartilage matrix, therefore showing promise as a therapeutic option for OA.

Research progress in isolation and identification of rumen probiotics.

With the increasing research on the exploitation of rumen microbial resources, rumen probiotics have attracted much attention for their positive contributions in promoting nutrient digestion, inhibiting pathogenic bacteria, and improving production performance. In the past two decades, macrogenomics has provided a rich source of new-generation probiotic candidates, but most of these "dark substances" have not been successfully cultured due to the restrictive growth conditions. However, fueled by high-throughput culture and sorting technologies, it is expected that the potential probiotics in the rumen can be exploited on a large scale, and their potential applications in medicine and agriculture can be explored. In this paper, we review and summarize the classical techniques for isolation and identification of rumen probiotics, introduce the development of droplet-based high-throughput cell culture and single-cell sequencing for microbial culture and identification, and finally introduce promising cultureomics techniques. The aim is to provide technical references for the development of related technologies and microbiological research to promote the further development of the field of rumen microbiology research.

Exosomes: the next-generation therapeutic platform for ischemic stroke.

Current therapeutic strategies for ischemic stroke fall short of the desired objective of neurological functional recovery. Therefore, there is an urgent need to develop new methods for the treatment of this condition. Exosomes are natural cell-derived vesicles that mediate signal transduction between cells under physiological and pathological conditions. They have low immunogenicity, good stability, high delivery efficiency, and the ability to cross the blood-brain barrier. These physiological properties of exosomes have the potential to lead to new breakthroughs in the treatment of ischemic stroke. The rapid development of nanotechnology has advanced the application of engineered exosomes, which can effectively improve targeting ability, enhance therapeutic efficacy, and minimize the dosages needed. Advances in technology have also driven clinical translational research on exosomes. In this review, we describe the therapeutic effects of exosomes and their positive roles in current treatment strategies for ischemic stroke, including their anti-inflammation, anti-apoptosis, autophagy-regulation, angiogenesis, neurogenesis, and glial scar formation reduction effects. However, it is worth noting that, despite their significant therapeutic potential, there remains a dearth of standardized characterization methods and efficient isolation techniques capable of producing highly purified exosomes. Future optimization strategies should prioritize the exploration of suitable isolation techniques and the establishment of unified workflows to effectively harness exosomes for diagnostic or therapeutic applications in ischemic stroke. Ultimately, our review aims to summarize our understanding of exosome-based treatment prospects in ischemic stroke and foster innovative ideas for the development of exosome-based therapies.

Aspects of conceptualization, development, and simulation of an extracellular vesicles' bulk isolation device for cosmetic and therapeutic applications

This paper discusses the concept and model to develop a prototype extracellular vesicle bulk-isolation device (EBID). It proposes an engineering design and potential ways to mitigate limitations in the currently available extracellular vesicle (EV) isolation techniques. A more convenient isolation technique is developed and presented to ensure production-scale isolation and purification of the specific size of EV (approximately 0.1 to 0.34 µm) from a large quantity of cell-culture media or biological fluid sources. A more appropriate design concept was established based on the chitosan-assisted precipitation of EV techniques and the membrane filtration technology-based prototype. After that, a novel theoretical model for bulk isolation of EVs was developed and presented using pressure-driven circulation and particulate flow in the EBID loop, where the EV production results from the biofluid flowing through the EBID membrane filter module (MFM). Governing equations are coupled with Darcy's law to investigate the biofluid flow behavior in the EBID MFM, therefore simulating the isolation process of EVs from their biofluids. The flow characteristics of the EV isolation process within the EBID MFM are theoretically simulated and discussed for improved isolation yield with a high degree of quality and purity.

Isolation of Myenteric and Submucosal Plexus from Mouse Gastrointestinal Tract and Subsequent Co-Culture with Small Intestinal Organoids.

Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus-both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field.

Genotypic and Phenotypic Characteristics of Lactic Acid Bacteria Associated with Forage Plants in the Native Grassland of Western Inner Mongolia and Their Application for Alfalfa Silage Fermentation.

This study was conducted to investigate the genotypic and phenotypic characteristics of lactic acid bacteria (LAB) associated with forage plants in the native grassland of western Inner Mongolia and to evaluate their effects on alfalfa silage fermentation. Forage plants and their spontaneous fermentation silages were analysed using culture-based techniques for LAB isolation; the phenotypic properties and 16S rDNA and pheS or rpoA gene sequences of the isolates were evaluated; alfalfa was ensiled with four additive combinations: Lactiplantibacillus plantarum subsp. plantarum (GI19), Lact. plantarum subsp. plantarum and Pediococcus pentosaceus (GI19+GI51), GI19 and 20 g/kg fresh matter of sucrose (GI19+S), and GI19+GI51+S, for 60 d. A total of 73 strains belonging to 16 species were isolated. All isolates grew at 5-45 °C and in 3.0% NaCl, and most of them grew in 6.5% NaCl. Enterococcus faecalis and Lact. plantarum were 26.03% and 17.81% of the total isolates, respectively. All additives improved the silage quality, while GI19+S was more effective for alfalfa ensiling with a higher lactic acid content and lower pH, undesirable microorganism counts, and acetic acid and NH3-N contents than remnant additives. In conclusion, the LAB species were diverse, and most of them possessed good cryotolerance and osmotolerance; GI19+S was the optimal inoculant for alfalfa fermentation improvement.

Exploring hematopoietic stem cell population in human milk and its benefits for infants: A scoping review

Objective: To comprehensively explore hematopoietic stem cells (HSCs) in human milk, understanding their molecular markers, isolation methods, benefits for infants, and potential medical applications. Methods: We conducted a scoping literature review following the PRISMA-ScR guidelines. This review included studies investigating HSCs in human milk, utilizing molecular markers such as CD34+, CD113+, and CD117+ for characterization. Both in vitro and in vivo studies exploring the morphology, function, and clinical implications of these cells were considered. The diverse range of papers reviewed were indexed in PubMed, Science Direct, Scopus, Sage Journals, and Google Scholar, published between 2010 and 2023. Results: This scoping review explored 577 articles and selected 13 studies based on our inclusion criteria, focusing on HSCs in human milk. Most studies dilute samples prior to HSC isolation, followed by detection using markers such as CD34+, CD113+, and CD117+, with flow cytometry serving as the primary analysis tool, focusing on their isolation and detection methods. While no definitive benefits have been conclusively established, there is a strong belief in the potential of HSCs to positively impact infant immunity, growth, and tissue repair. Conclusions: This review presents significant evidence supporting the presence of HSCs in human milk, identified by markers such as CD34+, CD113+, and CD117+. These cells show considerable potential in enhancing infant health, including immunity, tissue repair, cognitive development, and gastrointestinal health. Despite methodological variations in isolation and detection techniques, the collective findings underscore the potential clinical relevance of HSCs in human milk. Moreover, this review highlights the non-invasive accessibility of human milk as a source of HSCs and emphasizes the need for further research to unlock their therapeutic potential.

Advanced functional photochromic wearables with fast photo-responsivity, long color-retention, and multi-environmental stability

Smart photochromic wearables have demonstrated attractive prospects in many emerging fields. However, their lower functional performance limits their applications. Herein, we have fabricated a novel photochromic wearable fabric via covalent grafting and chemical isolation technique grafting WO3 nanoflowers onto cellulose. In the charming system, exceptional photochromic properties of WO3 serve as the color-changing material. Besides, incorporated potassium persulfate as a fast-fading reagent is capable of slow oxygen release upon heating. These combined components give the photochromic fabric a rapid response time of 2 s, a fading time of 5 min, and an unprecedented long color-retention (>7 days). Moreover, this photochromic fabric exhibits outstanding rewritability (100 cycles) and exceptional water resistance, maintaining superb photochromic performance even after undergoing standard washing (50 cycles). Moreover, the activation barriers to the photochromic mechanism for the minimum energy pathway were calculated, which provides a theoretical basis for hydrophobic design. The fabric not only imparts water and oil repellence and antifouling properties but also has chemical stability in multi-environments. Notably, the fabrication process is cost-effective, environmentally friendly, and amenable to large-scale production. Such fabrics hold tremendous promise in household products, public facilities and wearable healthcare technologies.

Earthquake Resisting Building by using Base Isolation

Abstract: Natural disasters have been always been a part of human life since the beginning of time, causing destruction to our lives and properties. Earthquake plays a major role in wreaking this havoc upon us. Various challenges have occurred to withstand structures on earth due to sudden moment of ground it also results in collapse of structure, due to inappropriate design consideration of structure without any seismic resistance. Various techniques to make the building earthquake resistant as well as various shapes and materials are being considered to achieve required strength to resist seismic forces. Various techniques like shear walls, bearings and base isolation are used to withstand the structure against seismic forces. In this project we have used base isolation technique, it is also known as seismic base isolation or base isolation system is a prominent technique to protect the structure against earthquake.

Single Visit Internal Perforation Repair Using a Novel HCSC and an Internal Isolation Technique: A Case Series

Single Visit Internal Perforation Repair Using a Novel HCSC and an Internal Isolation Technique: A Case Series

EFS-XGBoost: A robust framework for precision classification of COVID-19 cases

The emergence of the novel COVID-19 virus has had a profound impact on global healthcare systems and economies, underscoring the imperative need for the development of precise and expeditious diagnostic tools. Machine learning techniques have emerged as a promising avenue for augmenting the capabilities of medical professionals in disease diagnosis and classification. In this research, the EFS-XGBoost classifier model, a robust approach for the classification of patients afflicted with COVID-19 is proposed. The key innovation in the proposed model lies in the Ensemble-based Feature Selection (EFS) strategy, which enables the judicious selection of relevant features from the expansive COVID-19 dataset. Subsequently, the power of the eXtreme Gradient Boosting (XGBoost) classifier to make precise distinctions among COVID-19-infected patients is harnessed.The EFS methodology amalgamates five distinctive feature selection techniques, encompassing correlation-based, chi-squared, information gain, symmetric uncertainty-based, and gain ratio approaches. To evaluate the effectiveness of the model, comprehensive experiments were conducted using a COVID-19 dataset procured from Kaggle, and the implementation was executed using Python programming. The performance of the proposed EFS-XGBoost model was gauged by employing well-established metrics that measure classification accuracy, including accuracy, precision, recall, and the F1-Score. Furthermore, an in-depth comparative analysis was conducted by considering the performance of the XGBoost classifier under various scenarios: employing all features within the dataset without any feature selection technique, and utilizing each feature selection technique in isolation. The meticulous evaluation reveals that the proposed EFS-XGBoost model excels in performance, achieving an astounding accuracy rate of 99.8%, surpassing the efficacy of other prevailing feature selection techniques. This research not only advances the field of COVID-19 patient classification but also underscores the potency of ensemble-based feature selection in conjunction with the XGBoost classifier as a formidable tool in the realm of medical diagnosis and classification.

Optimization of protocols for blood-derived extracellular vesicles for studies in liver diseases

Aim: Extracellular vesicles (EVs) have gained significant attention for their diagnostic and therapeutic potential in various diseases, including liver disorders. This study focuses on optimizing the isolation and characterization of small EVs from plasma and serum samples in patients with liver diseases, aiming to advance our understanding and potential clinical applications of EVs. Methods: Blood samples were collected from patients with end-stage liver disease (ESLD) enlisted in the TransplantLines Cohort and Biobank Study, and healthy donors. We employed differential ultracentrifugation (DUC) to evaluate three distinct protocols: a 3-step DUC, a washing step omitted [samples without washing (WW)], and a contaminant-depleted plasma (CDP) protocol. RNA isolation methodologies were compared, involving the use of TRI-reagent or the commercial AllPrep DNA/RNA kit. Further insights into EV composition were obtained through proteomic analyses, comparing samples subjected to traditional cell lysis (L) with those processed without lysis (NL). Results: We successfully isolated EVs from both plasma and serum samples as confirmed by the presence of specific EV markers, including CD9, CD63, CD81, and tumor susceptibility gene 101 (TSG-101). While some contaminants remained, such as albumin and lipoproteins, the protocol selected to continue EVs analysis was the 3-step protocol. Transmission electron microscopy (TEM) and nanotracking analysis (NTA) further confirmed EVs presence. RNA extraction was achieved using TRI-reagent, but not with the commercial kit highlighting the importance of selecting an appropriate method for RNA isolation. Finally, proteomics analysis showed that lysed samples were significantly more enriched in proteins compared to non-lysed samples, although protein variability was still present in both groups. Conclusions: Optimizing EV isolation techniques is essential for harnessing their potential in liver disease diagnosis and therapy. Further refinement of purification methods, a deep characterization of our cohort and understanding the variability and cargo within EVs will be crucial for future biomarker discovery and therapeutic applications in liver-related diseases.

Микрочип для выделения экзосом с возможностью одновременной импрегнации его иматинибом: анализ in vitro

Exosomes, a small bilayer membrane derived from eukaryotic cells, have been identified as a useful natural delivery platform due to their suitable size, biocompatibility, structural stability, high loading capacity, and editable surface capability. Due to the difficulty of maintaining the highly pure exosome, several attempts have conducted the techniques for exosome isolation. In recent years, microstructures have found many applications in chemistry, biology, and medicine due to their high accuracy and low cost of materials. Soft lithography is a low-cost, fast, accurate, and yet widely used method of construction of Micron channels. In the present study, a soft lithography process has been performed to construct channels for exosome separation with immunoaffinity function. Both biochemical and biophysical categories tests were performed to examine the quality of extracted exosomes from different sources (serum, cell supernatant, and urine) and compared with the commercially available kit. Results showed that the current technique was capable to isolate exosomes with a high yield rate, purity, and low time consumption. All forms of the imatinib loaded exosomes exhibited the antitumor activity against KYO-1 cell line.

Recent Advances in Intelligent Algorithms for Fault Detection and Diagnosis.

Fault-finding diagnostics is a model-driven approach that identifies a system's malfunctioning portion. It uses residual generators to identify faults, and various methods like isolation techniques and structural analysis are used. However, diagnostic equipment doesn't measure the remaining signal-to-noise ratio. Residual selection identifies fault-detecting generators. Fault detective diagnostic (FDD) approaches have been investigated and implemented for various industrial processes. However, industrial operations make it difficult to implement FDD techniques. To bridge the gap between theoretical methodologies and implementations, hybrid approaches and intelligent procedures are needed. Future research should focus on improving fault prognosis, allowing for accurate prediction of process failures and avoiding safety hazards. Real-time and comprehensive FDD strategies should be implemented in the age of big data.

An overview of current research on cancer stem cells: a bibliometric analysis.

Cancer stem cells (CSCs) represent a potential mechanism contributing to tumorigenesis, metastasis, recurrence, and drug resistance. The objective of this study is to investigate the status quo and advancements in CSC research utilizing bibliometric analysis. Publications related to CSCs from 2010 to 2022 were collected from the Web of Science Core Collection database. Various analytical tools including CiteSpace, VOSviewer, Scimago Graphica, and GraphPad Prism were used to visualize aspects such as co-authorship, co-occurrence, and co-citation within CSC research to provide an objective depiction of the contemporary status and developmental trajectory of the CSC field. A total of 22,116 publications were included from 1942 journals written by 95,992 authors. Notably, China emerged as the country with the highest number of publications, whereas the United States exerted the most significant influence within the field. MD Anderson Cancer Center emerged as the institution making the most comprehensive contributions. Wicha M.S. emerged as the most prolific and influential researcher. Among journals, Cancers emerged as a focal point for CSC research, consistently publishing a wealth of high-quality papers. Furthermore, it was observed that most journals tended to approach CSC research from molecular, biological, and immunological perspectives. The research into CSCs encompassed a broad array of topics, including isolation and enrichment techniques, biomarkers, biological characteristics, cancer therapy strategies, and underlying biological regulatory mechanisms. Notably, exploration of the tumor microenvironment and extracellular vesicles emerged as burgeoning research frontiers for CSCs. The research on CSCs has garnered growing interest. A trend toward multidisciplinary homogeneity is emerging within the realm of CSCs. Further investigation could potentially center on the patients of extracellular vesicles and the tumor microenvironment in relation to CSCs.

Trajectory analysis of circulating tumor cells through contorted deterministic lateral displacement array for unruptured trapping: a simulation study

During the multistage process of cancer spread, fragments of cells branch out from the primitive tumor tissue and scatter throughout the body by blood flow stream or lymph, which are termed as circulating tumor cells (CTCs), regarded as an important biomarker for early cancer diagnosis. Deterministic lateral displacement (DLD) is one of the most efficient passive type particle isolation techniques which functions on asymmetrical laminar flow diversion around the placed microposts forming an array, based on the critical dimeter of the device. In this simulation study, complete trajectory analysis of CTCs and WBCs has been performed using COMSOL Multiphysics software with respect to the angular orientation of sample inlet channel. A simulated model of an asymmetric DLD array-based microfluidic device with latest empirical expression for critical diameter has been presented with optimized sample inlet orientation so as to maintain perfect separation efficiency of CTCs along with arrangement for unruptured trapping of CTCs and WBCs. The approximate CTC isolation efficiency was obtained above 95% for sample inlet inclinations up to 0.5°, and further tilt showed reduction in efficiency. Migration angle of CTC trajectory for the modeled device was obtained around 15° for both the sample inlet orientations leading to least intermingling of CTCs and WBCs. A smaller number of CTC–WBC interaction points was observed for clockwise sample inlet tilt compared to the anticlockwise displacement reducing possibilities of cell rupture. The shear rate around central microposts was found to be higher as well in clockwise sample inlet orientation. This simulation study establishes that sample inlet clockwise tilting of maximum around 0.5°–0.7° leads to better CTC isolation and provides better chances of unruptured trapping of cells at the designated outlets. The results of this study provide an approach toward further optimization of DLD devices' functioning and, thus, could help fabricate better DLD-based microfluidic devices for efficient trapping of CTCs.

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages.

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

Analysis of maternal Fc gamma receptor IIIb isoantibodies using immunomagnetic negative selected neutrophils.

The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/μL vs. 4.5 × 103/μL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.

85 years counteracting an invasion: chestnut ecosystems and landscapes survival against ink disease

A retrospective examination of early pest and disease outbreaks, such as ink disease, offers new insights into their impact on ecosystems and landscapes. Ink disease, caused by Phytophthora spp., is one of the most destructive diseases affecting Castanea sativa Mill. It first appeared in Europe in the early 18th century, with the initial recorded case in Italy (Piedmont) dating back to 1845. However, its spread instilled significant concerns in several chestnut-growing regions primarily devoted to fruit production. In 1938, a comprehensive assessment of Phytophthora xcambivora outbreaks was conducted in the Bolognese Apennines (Italy), providing a detailed overview of chestnut cultivation status. Thirty-six disease foci were documented, and laboratory tests confirmed the presence of ink disease. To understand the disease’s impact on chestnut ecosystem and landscape over the past 85 years, the 1938 sites were revisited to assess chestnut persistence and phytosanitary status, with Phytophthora species detected using isolation and molecular techniques. Monitoring data revealed that C. sativa still thrives in all but one site, with its wood seemingly able to coexist in dynamic equilibrium with the disease. While P. xcambivora was still detected in several foci, the extent of damage appeared limited. The potential for natural control, likely influenced by the complexity of soil biota, along with the natural spread of hypovirulence in chestnut blight and biological control of the Asian chestnut gall wasp, could explain the continued presence of chestnut in the investigated area and potentially throughout the Italian chestnut range, despite nearly two centuries of ink disease prevalence. Nevertheless, ongoing monitoring is essential to dynamically comprehend the factors at play and their efficacy, particularly in the context of climate change and the possible spread of other Phytophthora species. The survival of chestnut ecosystems amidst ink disease underscores the preservation of the economic, ecological, and landscape values associated with these woodlands.