bluuebunny/crossref_metadata_2025 huggingface dataset:
Acanthus mollis Formulations for Transdermal Delivery: From Hydrogels to Emulsions
Authors: Matos Patrícia , Batista Maria Teresa , Veiga Francisco , Figueirinha Artur , Figueiras Ana
Published: December 2023

Topical formulations of Acanthus mollis L. leaf and the optimization of the release of their active compounds and their topical bioavailability were investigated for the first time. In vitro, the release of active compounds from three formulations—an oil-in-water cream and two hydrogels (Carbopol 940 and Pluronic F-127)—was determined using Franz diffusion cells. Detection and quantification of the compounds was performed via high-performance liquid chromatography with a photodiode array (HPLC-PDA). DIBOA, a bioactive compound of this medicinal plant, exhibited release kinetics of the Weibull model for the Carbopol and Pluronic F-127 formulation, identifying it as a potential active agent to optimize the topical distribution of the formulations. The implications extend to applications in inflammation treatment and tyrosinase inhibition, suggesting that it can make a significant contribution to addressing skin conditions, including melanoma and various inflammatory diseases.
First Contiguous Genome Assembly of Japanese Lady Bell (Adenophora triphylla) and Insights into Development of Different Leaf Types
Authors: Kang Ji-Nam , Lee Si-Myung , Choi Ji-Weon , Lee Seung-Sik , Kim Chang-Kug
Published: December 2023

Adenophora triphylla is an important medicinal and food plant found in East Asia. This plant is rich in secondary metabolites such as triterpenoid saponin, and its leaves can develop into different types, such as round and linear, depending on the origin of germination even within the same species. Despite this, few studies have comprehensively characterized the development processes of different leaf types and triterpenoid saponin pathways in this plant. Herein, we provide the first report of a high-quality genome assembly of A. triphylla based on a combination of Oxford Nanopore Technologies and Illumina sequencing methods. Its genome size was estimated to be 2.6 Gb, and the assembled genome finalized as 2.48 Gb, containing 57,729 protein-coding genes. Genome completeness was assessed as 95.6% using the Benchmarking Universal Single-Copy Orthologs score. The evolutionary divergence of A. triphylla was investigated using the genomes of five plant species, including two other species in the Campanulaceae family. The species A. triphylla diverged approximately 51-118 million years ago from the other four plants, and 579 expanded/contracted gene families were clustered in the Gene Ontology terms. The expansion of the β-amyrin synthase (bAS) gene, a key enzyme in the triterpenoid saponin pathway, was identified in the A. triphylla genome. Furthermore, transcriptome analysis of the two leaf types revealed differences in the activity of starch, sucrose, unsaturated fatty acid pathways, and oxidoreductase enzymes. The heat and endoplasmic reticulum pathways related to plant stress were active in the development of round type leaf, while an enhancement of pyrimidine metabolism related to cell development was confirmed in the development of the linear type leaf. This study provides insight into the evolution of bAS genes and the development of different leaf types in A. triphylla.
Proteomics Analysis to Explore the Resistance Genes of Silkworm to Bombyx mori Nuclear Polyhedrosis Virus
Authors: Ouyang Gui , Qian Heying , Sun Juan , Yang Runhuan , Gui Tao , Wang Wenbing , Liu Qiang , Chen Anli
Published: December 2023

The resistance of silkworms to Bombyx mori nuclear polyhedrosis virus (BmNPV) is controlled by a major dominant gene and multiple modifying genes. Given the presence of modified genes, it is difficult to determine the main gene by positional cloning. In this study, the main anti-BmNPV gene of BmNPV-resistant silkworm variety N was introduced into the susceptible variety Su to breed the near-isogenic line SuN with BmNPV resistance. The infection process of BmNPV in the hemolymph of Su and SuN was analyzed using the cell analysis system TissueFAXS PLUS. According to the law of infection and proliferation, hemolymph was extracted every 6 h for two-dimensional electrophoresis (2-DE) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Seven DEPs were found in comparisons between Su and SuN by 2-DE analysis. Among them, acid phosphatase, storage protein, and phenoloxidase can prevent pathogen invasion, which may play a role against BmNPV. Polyamine oxidase plays an important role in energy metabolism, which may be indirectly involved in the process of resisting BmNPV. Most of the transcriptional expression profiles of the seven DEPs were consistent with the 2-DE results. This study can provide a reference for the identification of anti-BmNPV genes and the breeding of BmNPV-resistant silkworm varieties.
Mechanisms of Rapid Karyotype Evolution in Mammals
Authors: Brannan Emry O. , Hartley Gabrielle A. , O’Neill Rachel J.
Published: December 2023

Chromosome reshuffling events are often a foundational mechanism by which speciation can occur, giving rise to highly derivative karyotypes even amongst closely related species. Yet, the features that distinguish lineages prone to such rapid chromosome evolution from those that maintain stable karyotypes across evolutionary time are still to be defined. In this review, we summarize lineages prone to rapid karyotypic evolution in the context of Simpson’s rates of evolution—tachytelic, horotelic, and bradytelic—and outline the mechanisms proposed to contribute to chromosome rearrangements, their fixation, and their potential impact on speciation events. Furthermore, we discuss relevant genomic features that underpin chromosome variation, including patterns of fusions/fissions, centromere positioning, and epigenetic marks such as DNA methylation. Finally, in the era of telomere-to-telomere genomics, we discuss the value of gapless genome resources to the future of research focused on the plasticity of highly rearranged karyotypes.
Attitudes and Opinions of Teachers on Student Descriptive Assessment: A Case Study on the Teachers of Geology-Geography at Secondary School (Greece)
Authors: Louvaris Konstantinos , Psychogiou Maria , Triantaphyllou Maria V. , Antonarakou Assimina
Published: December 2023

This paper presents an initial study of teachers’ perspectives and practices on students’ assessment methods regarding the subject of Geology-Geography taught in junior secondary education in Greece. While the application of descriptive assessment is recorded in other European countries, the main focus of the research is the current situation in Greece, as well as the willingness of science teachers to adopt an alternative form of evaluation and the definition of the respective circumstances. The methodology that was followed in this research included questionnaires that were sent to science teachers. Their answers were processed with the IBM SPSS 23 statistical software. The results reveal that most teaching staff surveyed positively acknowledge the need for an effective appraisal system. Their views and opinions on how student assessment contributes to the overall educational procedure were also recorded during the survey. Statistical analysis of the raw data shows that the views and opinions of teaching staff were not differentiated due to their level of experience or their level of education but only due to their field of specialization. The analysis also points to a range of additional factors that impact teaching staff’s preferences and perceptions of the effectiveness of different student appraisal and evaluation approaches. Such factors create skepticism among teachers towards alternative assessment methods, such as descriptive assessment. While this is preliminary research, it raises important issues regarding effective evaluation methods that would promote students’ development.
Teaching Strategies for Developing Clinical Reasoning Skills in Nursing Students: A Systematic Review of Randomised Controlled Trials
Authors: Pérez-Perdomo Ana , Zabalegui Adelaida
Published: December 2023

Background: Clinical reasoning (CR) is a holistic and recursive cognitive process. It allows nursing students to accurately perceive patients’ situations and choose the best course of action among the available alternatives. This study aimed to identify the randomised controlled trials studies in the literature that concern clinical reasoning in the context of nursing students. Methods: A comprehensive search of PubMed, Scopus, Embase, and the Cochrane Controlled Register of Trials (CENTRAL) was performed to identify relevant studies published up to October 2023. The following inclusion criteria were examined: (a) clinical reasoning, clinical judgment, and critical thinking in nursing students as a primary study aim; (b) articles published for the last eleven years; (c) research conducted between January 2012 and September 2023; (d) articles published only in English and Spanish; and (e) Randomised Clinical Trials. The Critical Appraisal Skills Programme tool was utilised to appraise all included studies. Results: Fifteen papers were analysed. Based on the teaching strategies used in the articles, two groups have been identified: simulation methods and learning programs. The studies focus on comparing different teaching methodologies. Conclusions: This systematic review has detected different approaches to help nursing students improve their reasoning and decision-making skills. The use of mobile apps, digital simulations, and learning games has a positive impact on the clinical reasoning abilities of nursing students and their motivation. Incorporating new technologies into problem-solving-based learning and decision-making can also enhance nursing students’ reasoning skills. Nursing schools should evaluate their current methods and consider integrating or modifying new technologies and methodologies that can help enhance students’ learning and improve their clinical reasoning and cognitive skills.
Telehealth Competencies: Training Physicians for a New Reality?
Authors: Cruz-Panesso Ilian , Tanoubi Issam , Drolet Pierre
Published: December 2023

In North America, telehealth increased by 40% between 2019 and 2020 and stabilized at 40% in 2021. As telehealth becomes more common, it is essential to ensure that healthcare providers have the required skills to overcome the challenges and barriers of this new modality of care. While the COVID-19 pandemic has accelerated the design and implementation of telehealth curricula in healthcare education programs, its general adoption is still a major gap and an important barrier to ensuring scaling up and sustainability of the telesshealth practice. Lack of experienced faculty and limited curricular time are two of the most common barriers to expanding telehealth education. Overcoming the barriers of telehealth curricula implementation may require moving away from the classic expert model of learning in which novices learn from experts. As the adoption of telehealth curricula is still in its early stages, institutions may need to plan for faculty development and trainee education at the same time. Questions regarding the timing and content of telehealth education, the interprofessional development of curricula, and the identification of optimal pedagogical methods remain open and crucial. This article reflects on these questions and presents telesimulation as an ideal instructional method for the training of telehealth competencies. Telesimulation can provide opportunities for practical training across a range of telehealth competencies, fostering not only technical proficiency but also communication skills and interprofessional collaboration.
Clarifying Key Concerns about the Dating of Holy Relics: The Holy Chalice of the Last Supper at the Cathedral of Valencia
Authors: Zarzo Manuel
Published: December 2023

The Cathedral of Valencia, Spain, has preserved the so-called Holy Chalice of the Last Supper since 1437. It consists of a foot, a gold stem, and an upper cup carved in agate stone. Based on a pious tradition, this cup is supposed to be the one used by Jesus of Nazareth to institute the Eucharist. According to an archeological study published in 1960, this agate bowl was crafted around the 2nd–1st centuries BC, but people visiting this famous relic often wonder about its authenticity and about the evidence supporting it as a Hellenistic–Roman cup. Attempting to clarify this concern, a photographical characterization of the agate cup is presented and discussed. The main conclusions are the following: (i) The typology of the bowl is common to classical tableware, but the wall thickness, shape of the rim, and foot are typical of gemstone cups from the Hellenistic–Roman period. (ii) This cup would have been extremely valuable, which agrees with the location of the Cenacle in the aristocratic neighborhood of Jerusalem. This case study highlights the importance of further investigating the dating of historical objects as a key issue to support their authenticity.
Enhancing Growth, Yield, and Antioxidant Activity of Bitter Gourd (Momordica charantia L.) through Amino Acid Foliar Spray Application
Authors: El-Khayat Lamiaa , Elbagory Mohssen , Elsadek Mohamed , Ahmed Nevin , Mohamed Ibrahim , Omara Alaa El-Dein , Salim Nesrein
Published: December 2023

Bitter gourd has picked up noteworthy consideration for its pharmacological benefits. However, the impact of amino acids (AAs) dosage on growth, yield, and antioxidants is uncertain. In this study, we investigated the effects of foliar spraying bitter gourd with 100, 200, and 300 mg/L dosages of tryptophan (Trp), glutamine (Gln), and phenylalanine (Phe). The results revealed that Trp, at a dosage of 300 mg/L, produced the most substantial increase in plant length, followed by 300 mg/L Phe. Additionally, the highest values of the fresh dry weight of the plants, fresh weight of the first fruit, fruit number per plant, fresh weight of the fruits per plant, and total fruit output per hectare were seen at 300 mg/L of Trp, followed by 300 mg/L of Gln. Phe at 300 mg/L yielded the highest levels of total phenolics and total flavonoids, coupled with strong scavenging activity against 2,2-diphenyl-1-picrylhydrazyl. These outcomes show the potential of Trp and Gln foliar sprays to enhance bitter gourd growth, yield, and certain antioxidant compounds. These findings carry substantial implications for the enhancement of bitter gourd cultivation and quality. By revealing AA’s potential for improving bitter gourd, our research contributes to bolstering the agricultural sustainability of this remarkable crop.
Health Risk Assessment of Nitrate in Drinking Water with Potential Source Identification: A Case Study in Almaty, Kazakhstan
Authors: Sailaukhanuly Yerbolat , Azat Seitkhan , Kunarbekova Makhabbat , Tovassarov Adylkhan , Toshtay Kainaubek , Tauanov Zhandos , Carlsen Lars , Berndtsson Ronny
Published: December 2023

Infant mortality in Kazakhstan is six times higher compared with the EU. There are several reasons for this, but a partial reason might be that less than 30% of Kazakhstan’s population has access to safe water and sanitation and more than 57% uses polluted groundwater from wells that do not comply with international standards. For example, nitrate pollution in surface and groundwater continues to increase due to intensified agriculture and the discharge of untreated wastewater, causing concerns regarding environmental and human health. For this reason, drinking water samples were collected from the water supply distribution network in eight districts of Almaty, Kazakhstan, and water quality constituents, including nitrate, were analyzed. In several districts, the nitrate concentration was above the WHO and Kazakhstan’s maximum permissible limits for drinking water. The spatial distribution of high nitrate concentration in drinking water was shown to be strongly correlated with areas that are supplied with groundwater, whereas areas with lower nitrate levels are supplied with surface water sources. Based on source identification, it was shown that groundwater is likely polluted by mainly domestic wastewater. The health risk for infants, children, teenagers, and adults was assessed based on chronic daily intake, and the hazard quotient (HQ) of nitrate intake from drinking water was determined. The non-carcinogenic risks increased in the following manner: adult < teenager < child < infant. For infants and children, the HQ was greater than the acceptable level and higher than that of other age groups, thus pointing to infants and children as the most vulnerable age group due to drinking water intake in the study area. Different water management options are suggested to improve the health situation of the population now drinking nitrate-polluted groundwater.
Cold Exposures in Relation to Dysmenorrhea among Asian and White Women
Authors: Wu Tianying , Doyle Cassie , Ito Joy , Ramesh Neeraja , Ernest Deepali Karina , Crespo Noe C. , Hsu Fang-Chi , Oren Eyal
Published: December 2023

Dysmenorrhea is highly prevalent, ranging from 16% to 91% among women, and it can lead to multiple reproductive disorders. However, risk factors associated with dysmenorrhea remain unexamined. Cold exposures can significantly disturb blood circulation and prostaglandin production in the uterus, leading to dysmenorrhea. This study investigated the relationship between cold exposures and dysmenorrhea, as well as potential disparities between Asians and Whites and the potential cultural influences on these associations. This was a cross-sectional survey among 197 Asian and 222 non-Asian women recruited from the U.S., with more than 40% from California. We assessed cold exposures, such as the frequency of consumption of cold water/drinks and ice cream, as well as room temperatures at home and public places, for both summer and winter over the past 12 months. The type of cold exposure associated with dysmenorrhea differs between Asian and White women. We found that among Asian women, a higher frequency of ice cream consumption in winter (beta = 1.19, p = 0.0002 when comparing high to low categories) was associated with dysmenorrhea; however, among White women, increased consumption of cold water/drinks in winter (beta = 0.49, p = 0.04 when comparing high to low categories) was also associated with dysmenorrhea. Higher home room temperatures in winter were associated with reduced severity of dysmenorrhea among White women but not among Asian women. All these associations supported our hypothesis and were stronger among women who lived in states with colder winters. However, there are a few exceptions. For instance, women who drank cold water/drinks less frequently during their menstrual period were more likely to experience more severe dysmenorrhea. In conclusion, this study provides crucial evidence to support the link between cold exposures and dysmenorrhea among Asians and Whites. The associations contradictory to our hypothesis are likely due to reserved causation influenced by Asian cultural practice. This paper sheds light on an understudied area that profoundly affects women’s quality of life.
Parallel Channel Identification and Elimination Method Based on the Spatial Position Relationship of Different Channels
Authors: Zhao Mingwei , Ju Xiaoxiao , Wang Ni , Wang Chun , Zeng Weibo , Xu Yan
Published: December 2023

Extracting a channel network based on the Digital Elevation Model (DEM) is one of the key research topics in digital terrain analysis. However, when the channel area is wide and flat, it is easy to form parallel channels, which seriously affect the accuracy of channel network extraction. To solve this problem, this study proposes a method to identify and eliminate parallel channels extracted by classical methods. First, the channel level in the study area is marked based on the flow accumulation data, and the parallel channels are then identified using the positional relationship between the different channel levels. Finally, the modification point of the identified parallel channels is determined to eliminate the parallel channels, with the help of the change relationship between the parallel channel and its upper-level channel. In this study, two watersheds in southeast China are selected as examples for method verification and analysis. Experimental results show that the parallel channel identification method proposed in this paper can accurately identify all parallel channels and eliminate the identified parallel channels one by one. The location relationship of the modified channels is consistent with the actual situation, indicating that the proposed method has good application potential in DEM-based channel extraction networks.
Targeting Key Inflammatory Mechanisms Underlying Heart Failure: A Comprehensive Review
Authors: Papamichail Adamantia , Kourek Christos , Briasoulis Alexandros , Xanthopoulos Andrew , Tsougos Elias , Farmakis Dimitrios , Paraskevaidis Ioannis
Published: December 2023

Inflammation is a major component of heart failure (HF), causing peripheral vasculopathy and cardiac remodeling. High levels of circulating inflammatory cytokines in HF patients have been well recognized. The hallmark of the inflammatory imbalance is the insufficient production of anti-inflammatory mediators, a condition that leads to dysregulated cytokine activity. The condition progresses because of the pathogenic consequences of the cytokine imbalance, including the impact of endothelial dysfunction and adrenergic responsiveness deterioration, and unfavorable inotropic effects on the myocardium. Hence, to develop possible anti-inflammatory treatment options that will enhance the outcomes of HF patients, it is essential to identify the potential pathophysiological mechanisms of inflammation in HF. Inflammatory mediators, such as cytokines, adhesion molecules, and acute-phase proteins, are elevated during this process, highlighting the complex association between inflammation and HF. Therefore, these inflammatory markers can be used in predicting prognosis of the syndrome. Various immune cells impact on myocardial remodeling and recovery. They lead to stimulation, release of alarmins and risk-related molecule patterns. Targeting key inflammatory mechanisms seems a quite promising therapy strategy in HF. Cytokine modulation is only one of several possible targets in the fight against inflammation, as the potential molecular targets for therapy in HF include immune activation, inflammation, oxidative stress, alterations in mitochondrial bioenergetics, and autophagy.
Transcriptomic Analysis of the Aged Nulliparous Mouse Ovary Suggests a Stress State That Promotes Pro-Inflammatory Lipid Signaling and Epithelial Cell Enrichment
Authors: Chacón Carlos , Mounieres Constanza , Ampuero Sandra , Urzúa Ulises
Published: December 2023

Ovarian cancer (OC) incidence and mortality peaks at post-menopause while OC risk is either reduced by parity or increased by nulliparity during fertile life. The long-term effect of nulliparity on ovarian gene expression is largely unknown. In this study, we describe a bioinformatic/data-mining analysis of 112 coding genes upregulated in the aged nulliparous (NP) mouse ovary compared to the aged multiparous one as reference. Canonical gene ontology and pathway analyses indicated a pro-oxidant, xenobiotic-like state accompanied by increased metabolism of inflammatory lipid mediators. Up-regulation of typical epithelial cell markers in the aged NP ovary was consistent with synchronized overexpression of Cldn3, Ezr, Krt7, Krt8 and Krt18 during the pre-neoplastic phase of mOSE cell cultures in a former transcriptome study. In addition, 61/112 genes were upregulated in knockout mice for Fshr and for three other tumor suppressor genes (Pten, Cdh1 and Smad3) known to regulate follicular homeostasis in the mammalian ovary. We conclude that the aged NP ovary displays a multifaceted stress state resulting from oxidative imbalance and pro-inflammatory lipid signaling. The enriched epithelial cell content might be linked to follicle depletion and is consistent with abundant clefts and cysts observed in aged human and mouse ovaries. It also suggests a mesenchymal-to-epithelial transition in the mOSE of the aged NP ovary. Our analysis suggests that in the long term, nulliparity worsens a variety of deleterious effects of aging and senescence thereby increasing susceptibility to cancer initiation in the ovary.
Phylogenetic and Structure-Function Analyses of ENA ATPases: A Case Study of the ENA1 Protein from the Fungus Neurospora crassa
Authors: Aguilella Marcos , Garciadeblás Blanca , Fernández Pacios Luis , Benito Begoña
Published: December 2023

ENA transporters are a group of P-type ATPases that are characterized by actively moving Na+ or K+ out of the cell against their concentration gradient. The existence of these transporters was initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus Neurospora crassa was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Na+ or K+ ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.
Biochemical Analysis to Understand the Flooding Tolerance of Mutant Soybean Irradiated with Gamma Rays
Authors: Komatsu Setsuko , Zhou Tiantian , Kono Yuhi
Published: December 2023

Flooding stress, which reduces plant growth and seed yield, is a serious problem for soybean. To improve the productivity of flooded soybean, flooding-tolerant soybean was produced by gamma-ray irradiation. Three-day-old wild-type and mutant-line plants were flooded for 2 days. Protein, RNA, and genomic DNA were then analyzed based on oppositely changed proteins between the wild type and the mutant line under flooding stress. They were associated with cell organization, RNA metabolism, and protein degradation according to proteomic analysis. Immunoblot analysis confirmed that the accumulation of beta-tubulin/beta-actin increased in the wild type under flooding stress and recovered to the control level in the mutant line; however, alpha-tubulin increased in both the wild type and the mutant line under stress. Ubiquitin was accumulated and genomic DNA was degraded by flooding stress in the wild type; however, they were almost the same as control levels in the mutant line. On the other hand, the gene expression level of RNase H and 60S ribosomal protein did not change in either the wild type or the mutant line under flooding stress. Furthermore, chlorophyll a/b decreased and increased in the wild type and the mutant line, respectively, under flooding stress. These results suggest that the regulation of cell organization and protein degradation might be an important factor in the acquisition of flooding tolerance in soybean.
Optimized Recovery of Immature Germ Cells after Prepubertal Testicular Tissue Digestion and Multi-Step Differential Plating: A Step towards Fertility Restoration with Cancer-Cell-Contaminated Tissue
Authors: De Windt Sven , Kourta Dhoha , Kanbar Marc , Wyns Christine
Published: December 2023

Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal porcine ITT. Strategies for maximizing germ cell recovery, including a comparison of two enzymatic digestion protocols (P1 and P2) of ITT fragment sizes (4 mm3 and 8 mm3) and multi-step differential plating were explored. Cellular viability and yield, as well as numbers and proportions of DDX4+ germ cells, were assessed before incubating the cell suspensions overnight on uncoated plastics. Adherent cells were processed for immunocytochemistry (ICC) and floating cells were further incubated for three days on Poly-D-Lysine-coated plastics. Germ cell yield and cell types using ICC for SOX9, DDX4, ACTA2 and CYP19A1 were assessed at each step of the multi-step differential plating. Directly after digestion, cell suspensions contained >92% viable cells and 4.51% DDX4+ germ cells. Pooled results for fragment sizes revealed that the majority of DDX4+ cells adhere to uncoated plastics (P1; 82.36% vs. P2; 58.24%). Further incubation on Poly-D-Lysine-coated plastics increased germ cell recovery (4.80 ± 11.32 vs. 1.90 ± 2.07 DDX4+ germ cells/mm2, respectively for P1 and P2). The total proportion of DDX4+ germ cells after the complete multi-step differential plating was 3.12%. These results highlight a reduced proportion and number of germ cells lost when compared to data reported with other methods, suggesting that multi-step differential plating should be considered for optimization of immature germ cell recovery. While Poly-D-Lysine-coating increased the proportions of recovered germ cells by 16.18% (P1) and 28.98% (P2), future studies should now focus on less cell stress-inducing enzymatic digestion protocols to maximize the chances of fertility restoration with low amounts of cryo-banked human ITT.
Heterogeneity in Neutrophil Extracellular Traps from Healthy Human Subjects
Authors: Collins Margaret S. , Imbrogno Michelle A. , Kopras Elizabeth J. , Howard James A. , Zhang Nanhua , Kramer Elizabeth L. , Hudock Kristin M.
Published: December 2023

Neutrophil extracellular traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provide a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three-and-one-half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean NET total DNA, dsDNA, protein, LDH, neutrophil elastase (NE), and proteinase 3 (PR3) in isolated NETs were consistent across subjects. NET serine protease activity varied considerably within the same donor from day to day. The mean NET cathepsin G and MPO were significantly different across donors. IL-8 > IL-1RA > G-CSF were the most abundant cytokines in NETs. There was no significant difference in the mean concentration or variability of IL-8, IL-1RA, G-CSF, IL-1α, IL-1β, or TNF-α in different subjects’ NETs. NET DNA concentration was correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. The mean reduction in protease activity by protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in the barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.
Integrated Transcriptomic and Metabolomic Analyses Identify Critical Genes and Metabolites Associated with Seed Vigor of Common Wheat
Authors: Yang Zhenrong , Chen Weiguo , Jia Tianxiang , Shi Huawei , Sun Daizhen
Published: December 2023

Seed aging is a common physiological phenomenon during storage which has a great impact on seed quality. An in-depth analysis of the physiological and molecular mechanisms of wheat seed aging is of great significance for cultivating high-vigor wheat varieties. This study reveals the physiological mechanisms of wheat seed aging in two cultivars differing in seed vigor, combining metabolome and transcriptome analyses. Differences between cultivars were examined based on metabolomic differential analysis. Artificial aging had a significant impact on the metabolism of wheat seeds. A total of 7470 (3641 upregulated and 3829 downregulated) DEGs were detected between non-aging HT and LT seeds; however, 10,648 (4506 up and 6142 down) were detected between the two cultivars after aging treatment. Eleven, eight, and four key metabolic-related gene families were identified in the glycolysis/gluconeogenesis and TCA cycle pathways, starch and sucrose metabolism pathways, and galactose metabolism pathways, respectively. In addition, 111 up-regulated transcription factor genes and 85 down-regulated transcription factor genes were identified in the LT 48h group. A total of 548 metabolites were detected across all samples. Cultivar comparisons between the non-aged groups and aged groups revealed 46 (30 upregulated and 16 downregulated) and 62 (38 upregulated and 24 downregulated) DIMs, respectively. Network analysis of the metabolites indicated that glucarate O-phosphoric acid, L-methionine sulfoxide, isocitric acid, and Gln-Gly might be the most crucial DIMs between HT and LT. The main related metabolites were enriched in pathways such as glyoxylate and dicarboxylate metabolism, biosynthesis of secondary metabolites, fatty acid degradation, etc. However, metabolites that exhibited differences between cultivars were mainly enriched in carbon metabolism, the TCA cycle, etc. Through combined metabolome and transcriptome analyses, it was found that artificial aging significantly affected glycolysis/gluconeogenesis, pyruvate metabolism, and glyoxylate and dicarboxylate metabolism, which involved key genes such as ACS, F16P2, and PPDK1. We thus speculate that these genes may be crucial in regulating physiological changes in seeds during artificial aging. In addition, an analysis of cultivar differences identified pathways related to amino acid and polypeptide metabolism, such as cysteine and methionine metabolism, glutathione metabolism, and amino sugar and nucleotide sugar metabolism, involving key genes such as BCAT3, CHI1, GAUT1, and GAUT4, which may play pivotal roles in vigor differences between cultivars.
Probing the Cellular Fate of the Protein Corona around Nanoparticles with Nanofocused X-ray Fluorescence Imaging
Authors: Skiba Marvin , Guedes Gabriela , Karpov Dmitry , Feliu Neus , L. Cortajarena Aitziber , Parak Wolfgang J. , Sanchez-Cano Carlos
Published: December 2023

X-ray fluorescence imaging (XRF-imaging) with subcellular resolution is used to study the intracellular integrity of a protein corona that was pre-formed around gold nanoparticles (AuNP). Artificial proteins engineered to obtain Gd coordination for detection by XRF-imaging were used to form the corona. Indications about the degradation of this protein corona at a cellular and subcellular level can be observed by following the Au and Gd quantities in a time and spatial-dependent manner. The extended acquisition times necessary for capturing individual XRF-imaging cell images result in relatively small sample populations, stressing the need for faster image acquisition strategies in future XRF-imaging-based studies to deal with the inherent variability between cells. Still, results obtained reveal degradation of the protein corona during cellular trafficking, followed by differential cellular processing for AuNP and Gd-labelled proteins. Overall, this demonstrates that the dynamic degradation of the protein corona can be tracked by XRF-imaging to a certain degree.
Depletion of Endothelial-Derived 2-AG Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-Proteome
Authors: Levine Aidan A. , Liktor-Busa Erika , Balasubramanian Shreya , Palomino Seph M. , Burtman Anya M. , Couture Sarah A. , Lipinski Austin A. , Langlais Paul R. , Largent-Milnes Tally M.
Published: December 2023

Mounting evidence supports the role of the endocannabinoid system in neurophysiology, including blood–brain barrier (BBB) function. Recent work has demonstrated that activation of endocannabinoid receptors can mitigate insults to the BBB during neurological disorders like traumatic brain injury, cortical spreading depression, and stroke. As alterations to the BBB are associated with worsening clinical outcomes in these conditions, studies herein sought to examine the impact of endocannabinoid depletion on BBB integrity. Barrier integrity was investigated in vitro via bEnd.3 cell monolayers to assess endocannabinoid synthesis, barrier function, calcium influx, junctional protein expression, and proteome-wide changes. Inhibition of 2-AG synthesis using DAGLα inhibition and siRNA inhibition of DAGLα led to loss of barrier integrity via altered expression of VE-cadherin, which could be partially rescued by exogenous application of 2-AG. Moreover, the deleterious effects of DAGLα inhibition on BBB integrity showed both calcium and PKC (protein kinase C)-dependency. These data indicate that disruption of 2-AG homeostasis in brain endothelial cells, in the absence of insult, is sufficient to disrupt BBB integrity thus supporting the role of the endocannabinoid system in neurovascular disorders.
Comprehensive Pan-Cancer Mutation Density Patterns in Enhancer RNA
Authors: Zhang Troy , Yu Hui , Jiang Limin , Bai Yongsheng , Liu Xiaoyi , Guo Yan
Published: December 2023

Significant advances have been achieved in understanding the critical role of enhancer RNAs (eRNAs) in the complex field of gene regulation. However, notable uncertainty remains concerning the biology of eRNAs, highlighting the need for continued research to uncover their exact functions in cellular processes and diseases. We present a comprehensive study to scrutinize mutation density patterns, mutation strand bias, and mutation burden in eRNAs across multiple cancer types. Our findings reveal that eRNAs exhibit mutation strand bias akin to that observed in protein-coding RNAs. We also identified a novel pattern, in which mutation density is notably diminished around the central region of the eRNA, but conspicuously elevated towards both the beginning and end. This pattern can be potentially explained by a mechanism involving heightened transcriptional activity and the activation of transcription-coupled repair. The central regions of the eRNAs appear to be more conserved, hinting at a potential mechanism preserving their structural and functional integrity, while the extremities may be more susceptible to mutations due to increased exposure. The evolutionary trajectory of this mutational pattern suggests a nuanced adaptation in eRNAs, where stability at their core coexists with flexibility at their extremities, potentially facilitating their diverse interactions with other genetic entities.
Over-Production of the Human SLC7A10 in E. coli and Functional Assay in Proteoliposomes
Authors: Galluccio Michele , Mazza Tiziano , Scalise Mariafrancesca , Tripicchio Martina , Scarpelli Martina , Tolomeo Maria , Pochini Lorena , Indiveri Cesare
Published: December 2023

The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C12E8 and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na+-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.
Animal Microbiomes as a Source of Novel Antibiotic-Producing Strains
Authors: Baranova Margarita N. , Pilipenko Ekaterina A. , Gabibov Alexander G. , Terekhov Stanislav S. , Smirnov Ivan V.
Published: December 2023

Natural compounds continue to serve as the most fruitful source of new antimicrobials. Analysis of bacterial genomes have revealed that the biosynthetic potential of antibiotic producers by far exceeds the number of already discovered structures. However, due to the repeated discovery of known substances, it has become necessary to change both approaches to the search for antibiotics and the sources of producer strains. The pressure of natural selection and the diversity of interactions in symbiotic communities make animal microbiomes promising sources of novel substances. Here, microorganisms associated with various animals were examined in terms of their antimicrobial agents. The application of alternative cultivation techniques, ultrahigh-throughput screening, and genomic analysis facilitated the investigation of compounds produced by unique representatives of the animal microbiota. We believe that new strategies of antipathogen defense will be discovered by precisely studying cell–cell and host–microbe interactions in microbiomes in the wild.
Visualizing the 4D Impact of Gold Nanoparticles on DNA
Authors: Abdelhady Hosam , Aleanizy Fadilah , Alqahtani Fulwah , Bukhari Abdullah , Soliman Sahar , Sau Samaresh , Iyer Arun
Published: December 2023

The genotoxicity of AuNPs has sparked a scientific debate, with one perspective attributing it to direct DNA damage and another to oxidative damage through reactive oxygen species (ROS) activation. This controversy poses challenges for the widespread use of AuNPs in biomedical applications. To address this debate, we employed four-dimensional atomic force microscopy (4DAFM) to examine the ability of AuNPs to damage DNA in vitro in the absence of ROS. To further examine whether the size and chemical coupling of these AuNPs are properties that control their toxicity, we exposed individual DNA molecules to three different types of AuNPs: small (average diameter = 10 nm), large (average diameter = 22 nm), and large conjugated (average diameter = 39 nm) AuNPs. We found that all types of AuNPs caused rapid (within minutes) and direct damage to the DNA molecules without the involvement of ROS. This research holds significant promise for advancing nanomedicines in diverse areas like viral therapy (including COVID-19), cancer treatment, and biosensor development for detecting DNA damage or mutations by resolving the ongoing debate regarding the genotoxicity mechanism. Moreover, it actively contributes to the continuous endeavors aimed at fully harnessing the capabilities of AuNPs across diverse biomedical fields, promising transformative healthcare solutions.
Comprehensive Analysis of Highbush Blueberry Plants Propagated In Vitro and Conventionally
Authors: Mazurek Marzena , Siekierzyńska Aleksandra , Piechowiak Tomasz , Spinardi Anna , Litwińczuk Wojciech
Published: December 2023

In vitro culture allows the production of numerous plants with both desirable and undesirable traits. To investigate the impact of the propagation method on highbush blueberry plants, an analysis was performed on four groups of differentially propagated plants: in vitro with axillary (TC-Ax) or adventitious shoots (TC-Ad), conventionally (SC) and using a mixed method (TC/SC). The analysis included plant features (shoot length and branching, chlorophyll and fluorescence and DNA methylation) and fruit properties (antioxidant compounds). The data obtained indicated significant differences between plants propagated conventionally and in vitro, as well as variations among plants derived from in vitro cultures with different types of explants. SC plants generally exhibited the lowest values of morphological and physiological parameters but produced fruits richest in antioxidant compounds. TC/SC plants were dominant in length, branching and fluorescence. Conversely, TC-Ax plants produced fruits with the lowest levels of antioxidant compounds. The methylation-sensitive amplified polymorphism (MSAP) technique was employed to detect molecular differences. TC-Ad plants showed the highest methylation level, whereas SC plants had the lowest. The overall methylation level varied among differentially propagated plants. It can be speculated that the differences among the analysed plants may be attributed to variations in DNA methylation.
Genome-Wide Identification and Expression Analysis of Calmodulin and Calmodulin-like Genes, Revealing CaM3 and CML13 Participating in Drought Stress in Phoebe bournei
Authors: Fu Ningning , Wang Li , Han Xiao , Yang Qi , Zhang Yuting , Tong Zaikang , Zhang Junhong
Published: December 2023

Calmodulin (CaM) and calmodulin-like (CML) proteins are major Ca2+ sensors involved in the regulation of plant development and stress responses by converting Ca2+ signals into appropriate cellular responses. However, characterization and expression analyses of CaM/CML genes in the precious species, Phoebe bournei, remain limited. In this study, five PbCaM and sixty PbCML genes were identified that only had EF-hand motifs with no other functional domains. The phylogenetic tree was clustered into 11 subgroups, including a unique clade of PbCaMs. The PbCaMs were intron-rich with four EF-hand motifs, whereas PbCMLs had two to four EF-hands and were mostly intronless. PbCaMs/CMLs were unevenly distributed across the 12 chromosomes of P. bournei and underwent purifying selection. Fragment duplication was the main driving force for the evolution of the PbCaM/CML gene family. Cis-acting element analysis indicated that PbCaMs/CMLs might be related to hormones, growth and development, and stress response. Expression analysis showed that PbCaMs were generally highly expressed in five different tissues and under drought stress, whereas PbCMLs showed specific expression patterns. The expression levels of 11 candidate PbCaMs/CMLs were responsive to ABA and MeJA, suggesting that these genes might act through multiple signaling pathways. The overexpression of PbCaM3/CML13 genes significantly increased the tolerance of yeast cells to drought stress. The identification and characterization of the CaM/CML gene family in P. bournei laid the foundation for future functional studies of these genes.