Nanoscale insights into hematology: super-resolved imaging on blood cell structure, function, and pathology.

Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

The influence of biological rhythms on the initial onset of status epilepticus in critically ill inpatients and the study of its predictive Model.

This study aims to explore the relationship between the circadian rhythms of critically ill patients and the incidence of Status Epilepticus (SE), and to develop a predictive model based on circadian rhythm indicators and clinical factors. We conducted a diurnal rhythm analysis of vital sign data from 4413 patients, discovering significant differences in the circadian rhythms of body temperature, blood oxygen saturation, and heart rate between the SE and non-SE groups, which were correlated with the incidence of SE. We also employed various machine learning algorithms to identify the ten most significant variables and developed a predictive model with strong performance and clinical applicability. Our research provides a new perspective and methodology for the study of biological rhythms in critically ill patients, offering new evidence and tools for the prevention and treatment of SE. Our findings are consistent or similar to some in the literature, while differing from or supplementing others. We observed significant differences in the vital signs of epileptic patients at different times of the day across various diagnostic time groups, reflecting the regulatory effects of circadian rhythms. We suggest heightened monitoring and intervention of vital signs in critically ill patients, especially during late night to early morning hours, to reduce the risk of SE and provide more personalized treatment plans.

RNA m6A modification facilitates DNA methylation during maize kernel development.

N6-methyladenosine (m6A) in mRNA and 5-methylcytosine (5mC) in DNA have critical functions for regulating gene expression and modulating plant growth and development. However, the interplay between m6A and 5mC is an elusive territory and remains unclear mechanistically in plants. We reported an occurrence of crosstalk between m6A and 5mC in maize (Zea mays) via the interaction between mRNA adenosine methylase (ZmMTA), the core component of the m6A methyltransferase complex, and decrease in DNA methylation 1 (ZmDDM1), a key chromatin-remodeling factor that regulates DNA methylation. Genes with m6A modification were coordinated with a much higher level of DNA methylation than genes without m6A modification. Dysfunction of ZmMTA caused severe arrest during maize embryogenesis and endosperm development, leading to a significant decrease in CHH methylation in the 5' region of m6A-modified genes. Instead, loss of function of ZmDDM1 had no noteworthy effects on ZmMTA-related activity. This study establishes a direct link between m6A and 5mC during maize kernel development and provides insights into the interplay between RNA modification and DNA methylation.

Experience of Disease Acceptance in Chinese Patients with Newly Diagnosed Crohn's Disease: A Descriptive Qualitative Study.

Background: High levels of disease acceptance are important predictors of improved psychological well-being, treatment outcomes, and enhanced quality of life. Relatively few studies have focused on the process of disease acceptance in patients with Crohn's disease (CD), particularly those who are newly diagnosed. Purpose: To explore the disease acceptance process in newly diagnosed CD patients. Patients and Methods: A descriptive qualitative approach was employed. Sixteen CD patients from 2 tertiary hospitals in Hangzhou, Zhejiang were recruited through purposive sampling using a maximum variation strategy. Semi-structured interviews were conducted. The interviews were transcribed verbatim and analysed using conventional content analysis. Results: Five phases of the psychosocial process of the "acceptance journey" of newly diagnosed CD patients emerged from the data analysis: (1) praying for the illness to not be CD; (2) not being able to accept CD; (3) having to accept CD; (4) knowing that CD should be acceptable; and (5) starting to accept CD. Patients at the stage of "starting to accept CD" are more proactive and motivated to face the disease, and their overall acceptance of the disease is higher than that of the previous stages. However, by the end of the interview, 2 patients remained at the stage of "having to accept CD", and 3 patients remained at the stage of "knowing that CD should be acceptable". Two patients entered the stage of "starting to accept CD" and then reverted back to one of the previous stages. Conclusion: The "acceptance journey" of newly diagnosed CD patients is dynamic, individual and reversible. Traditional Chinese cultural values such as respect for authority, the philosophy of wu-wei and family responsibility contribute to the acceptance of CD in Chinese patients. Hence, there is a need to provide early and culturally tailored psychological support or interventions according to the stages of acceptance.

Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate.

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6 Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

Pyridine-containing substrate analogs are restricted from accessing the human cytochrome P450 8B1 active site by tryptophan 281.

The human oxysterol 12α-hydroxylase cytochrome P450 8B1 (CYP8B1) is a validated drug target for both type 2 diabetes and nonalcoholic fatty liver disease, but effective selective inhibitors are not yet available. Herein, steroidal substrate-mimicking compounds with a pyridine ring appended to the C12 site of metabolism were designed as inhibitors, synthesized, and evaluated in terms of their functional and structural interactions with CYP8B1. While the pyridine nitrogen was intended to coordinate the CYP8B1 active site heme iron, none of these compounds elicited shifts in the CYP8B1 Soret absorbance consistent with this type of interaction. However, when CYP8B1 was cocrystallized with the pyridine-containing compound with the 3-keto-Δ4 steroid backbone most similar to the endogenous substrate, it was apparent that this ligand was bound in a channel leading to the active site, instead of near the heme iron. Inspection of this structure suggested that tryptophan 281 directly above the heme might restrict active site binding of potential inhibitors with this design. This hypothesis was supported when a CYP8B1 W281F mutation did allow all three compounds to coordinate the heme iron as designed. These results indicated that the design of next-generation CYP8B1 inhibitors should be compatible with the low-ceiling tryptophan immediately above the heme iron.

Routine analysis of pesticides in foodstuffs: Emerging ambient ionization mass spectrometry as an alternative strategy to be on your radar.

Pesticides residues in foodstuffs are longstanding of great concern to consumers and governments, thus reliable evaluation techniques for these residues are necessary to ensure food safety. Emerging ambient ionization mass spectrometry (AIMS), a transformative technology in the field of analytical chemistry, is becoming a promising and solid evaluation technology due to its advantages of direct, real-time and in-situ ionization on samples without complex pretreatments. To provide useful guidance on the evaluation techniques in the field of food safety, we offered a comprehensive review on the AIMS technology and introduced their novel applications for the analysis of residual pesticides in foodstuffs under different testing scenarios (i.e., quantitative, screening, imaging, high-throughput detection and rapid on-site analysis). Meanwhile, the creative combination of AIMS with high-resolution mass analyzer (e.g., orbitrap and time-of-flight) was fundamentally mentioned based on recent studies about the detection and evaluation of multi-residual pesticides between 2015 and 2021. Finally, the technical challenges and prospects associated with AIMS operation in food industry were discussed.

Advances on removal of organophosphorus pesticides with electrochemical technology.

Widespread use of organophosphorus pesticides (OPs), especially superfluous and unreasonable use, had brought huge harm to the environment and food chain. It is because only a small part of the pesticides sprayed reached the target, and the rest slid across the soil, causing pollution of groundwater and surface water resources. These pesticides accumulate in the environment, causing environmental pollution. Therefore, in recent years, the control and degradation of OPs have become a public spotlight and research hotspot. Due to its unique advantages such as versatility, environmental compatibility, controllability, and cost-effectiveness compatibility, electrochemical technology has become one of the most promising methods for degradation of OPs. The fundamental knowledge about electrochemical degradation on OPs was introduced in this review. Then, a comprehensive overview of four main types of practical electrochemical technologies to degrade pesticides were presented and evaluated. The knowledge contained herein should conduce to better understand the degradation of pesticides by electrochemical technology, and better exploit the degradation of pesticides in the environment and food. Overall, the objective of this review is to provide comprehensive guidance for rational design and application of electrochemical technology in the degradation of OPs for the safety of the environment and food chain in the future.

Highly sensitive sensing and quantitative detection of sulfate ion with a SERS chip-based on boric acid's Lewis effect.

Based on the Lewis acid's coordination principle, a surface-enhanced Raman scattering (SERS) chip strategy had been developed for the ultrasensitive quantitation of SO42-. Through the immobilization of silver nanoparticles (Ag NPs) and the construction of the boric acid-based sensing unit, the chip system displayed outstanding merits on the direct sensing of SO42-, e.g., simple operation, ultra-high sensitivity, reproducibility, excellent selectivity and specificity. Moreover, an accurate evaluation was obtained by ratiometric calculations on characteristic peaks (1382 and 1070 cm-1) for quantitative detection of SO42-. The detection limit was down to 10 nM. Tap water, beer, and mineral water samples were tested, and high recoveries were achieved (97.12-110.12%). Besides, such SERS chip also displayed strong applicability for the evaluation of SO32-. Therefore, this SERS chip provided a promising idea for the quantification of trace amounts of SO42- and SO32- in the fields of food safety and environmental monitoring.

Recent advances of three-dimensional micro-environmental constructions on cell-based biosensors and perspectives in food safety.

The development and application of cell-based biosensors (CBBs) provides a convenient strategy for rapid detection of target analytes. The CBBs had been widely applied in the fields of food safety, environment monitoring, and medicine diagnosis due to their advantages of short response time, easy operation, low toxicity, and portability. However, the CBBs based on two-dimensional (2D) cultured cells in-vitro suffer from a lower cell viability and isolated physiology, which had blocked the accurate evaluations of these biosensors. With the development of nanotechnology and three-dimensional (3D) printing technology, cells fixed in a 3D biosensor or a 3D microenvironment have shown great improvement in the sensitivity and detection authenticity than conventional CBBs. To promote the further development of CBBs, in this paper, we reviewed the related technologies used to construct 3D bionic cell chips including organic/inorganic agents and operating approaches suitable for constructing 3D cell cultural microenvironment. Then, the applications of 3D bionic cell chip based on microbial and mammalian cell biosensors in food safety field were discussed during recent ten years. Finally, the current challenges and further directions were summarized and prospected.

The structure and characterization of human cytochrome P450 8B1 supports future drug design for nonalcoholic fatty liver disease and diabetes.

Human cytochrome P450 8B1 (CYP8B1) is involved in conversion of cholesterol to bile acids. It hydroxylates the steroid ring at C12 to ultimately produce the bile acid cholic acid. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. Herein, the human CYP8B1 protein was generated and used to identify and characterize interactions with a series of azole inhibitors, which tend to be poorly selective P450 inhibitors. Structurally related miconazole, econazole, and tioconazole bound with submicromolar dissociation constants and were effective inhibitors of the native reaction. CYP8B was cocrystallized with S-tioconazole to yield the first X-ray structure. This inhibitor bound in the active site with its azole nitrogen coordinating the heme iron, consistent with inhibitor binding and inhibition assay data. Additionally, the CYP8B1 active site was compared with similar P450 enzymes to identify features that may facilitate the design of more selective inhibitors. Selective inhibitors should promote a better understanding of the role of CYP8B1 inhibition in normal physiology and disease states and provide a possible treatment for nonalcoholic fatty liver disease and type 2 diabetes.

Progress on aptamer-based SERS sensors for food safety and quality assessment: methodology, current applications and future trends.

It is well known that food safety has aroused extensive attentions from governments to researchers and to food industries. As a versatile technology based on molecular interactions, aptamer sensors which could specifically identify a wide range of food contaminants have been extensively studied in recent years. Surface-enhanced Raman spectroscopy integrated aptamer combines the advantages of both technologies, not only in the ability to specifically identify a wide range of food contaminants, but also in the ultra-high sensitivity, simplicity, portable and speed. To provide beneficial insights into the evaluation techniques in the field of food safety, we offer a comprehensive review on the design strategies for aptamer-SERS sensors in different scenarios, including non-nucleic acid amplification methods ("on/off" mode, sandwich mode, competition model and catalytic model) and nucleic acid amplification methods (hybridization chain reaction, rolling circle amplification, catalytic hairpin assembly). Meanwhile, a special attention is paid to the application of aptamer-SERS sensors in biological (foodborne pathogenic, bacteria and mycotoxins) and chemical contamination (drug residues, metal ions, and food additives) of food matrix. Finally, the challenges and prospects of developing reliable aptamer-SERS sensors for food safety were discussed, which are expected to offer a strong guidance for further development and extended applications.

Designer Exosomes for Targeted Delivery of a Novel Therapeutic Cargo to Enhance Sorafenib-Mediated Ferroptosis in Hepatocellular Carcinoma.

Sorafenib is one of the few effective first-line drugs approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common among individuals with HCC. Recent evidence indicated that the anticancer activity of sorafenib mainly relies on the induction of ferroptosis. Furthermore, in our study, genes that suppress ferroptosis, especially GPX4 and DHODH, were enriched in sorafenib-resistant cells and primary tissues and were associated with poor prognosis of HCC patients who received sorafenib treatment. Therefore, a new ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) capable of simultaneously silencing GPX4 and DHODH was created. Then, exosomes with high multi-siRNA loading and HCC-specific targeting were established by fusing the SP94 peptide and the N-terminal RNA recognition motif (RRM) of U1-A with the exosomal membrane protein Lamp2b. The results from the in vitro and in vivo experiments indicate that this tumor-targeting nano-delivery system (ExoSP94-lamp2b-RRM-multi-siRNA) could enhance sorafenib-induced ferroptosis and overcome sorafenib resistance. Taken together, HCC-targeted exosomes (ExoSP94-Lamp2b-RRM) could specifically deliver multi-siRNA to HCC tissues, enhance sorafenib-induced ferroptosis by silencing GPX4 and DHODH expression and consequently increase HCC sensitivity to sorafenib, which opens a new avenue for clinically overcoming sorafenib resistance from the perspective of ferroptosis.

Ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen based on a hairpin DNA probe and exonuclease I-assisted target recycling.

A novel ratiometric electrochemical aptasensor was constructed for the detection of carcinoembryonic antigen (CEA) based on a hairpin DNA (hpDNA) probe and exonuclease Ⅰ (Exo Ⅰ)-assisted target recycling amplification strategy. A thiolated methylene blue (MB)-labeled hpDNA as the internal control element was fixed on the surface of the gold nanoparticles (AuNPs)-modified gold electrode (AuE) through Au-S bonds. A ferrocene (Fc)-modified aptamer DNA (Fc-Apt) was partially hybridized with hpDNA to form a Fc-Apt/hpDNA duplex. Due to the specific recognition of Fc-Apt to CEA, the presence of CEA caused dissociation of Fc-Apt from the duplex, and further triggered the degradation process of Exo Ⅰ and recycling of CEA. Hence, the Fc tags were released from the electrode surface and the oxidation peak current of Fc (IFc) decreased while that of MB (IMB) remained stable owing to the distance between MB tags and the electrode unchanged. A linear relationship was observed between IFc/IMB and the logarithm of CEA concentration from 10 pg mL-1 to 100 ng mL-1 with a detection limit of 1.9 pg mL-1. Moreover, the developed aptasensor had been applied to detect CEA in diluted human serum with satisfactory results, indicating its great potential in practical applications.

A novel analytical strategy for the determination of perfluoroalkyl acids in various food matrices using a home-made functionalized fluorine interaction SPME in combination with LC-MS/MS.

In this study, a fluorine-fluorine interaction approach through fluoridating boron nitride nanosheets (BNNs) for sensing perfluoroalkyl acids (PFAAs) in multiple food matrices was developed. Through a facile hydrothermal fluorination modification, the BNNs were transferred into homogeneous fluorinated boron nitride nanoparticles (F-BNNs) with robust networks and specific surface area. After morphological modification, the particles displayed strong adsorption and sensing capabilities on PFAAs in both solid and liquid food matrix. Under the evaluation of mass spectrometry, F-BNNs based microextraction approach exhibited low method detection limits (MDLs) in the ranges of 0.9-3.9 pg mL-1 and 3.6-15.8 pg g-1 for milk and meat matrices, respectively, with satisfactory repeatability (RSD% <13.5%) and recoveries (77.7-110.5%). This work not only depicted a facile approach for preparing F-BNNs based SPME fiber, but also provided a routine analysis protocol for monitoring PFAAs in food systems.

A Competitive "On-Off-Enhanced On" AIE Fluorescence Switch for Detecting Biothiols Based on Hg2+ Ions and Gold Nanoclusters.

In this study, a novel "on-off-enhanced on" approach to highly sensitive rapid sensing of biothiols was developed, based on competitive modulation of gold nanoclusters (AuNCs) and Hg2+ ions. In our approach, the AuNCs were encapsulated into a zeolite imidazole framework (ZIF) for predesigned competitive aggregation-induced luminescence (AIE) emission. To readily operate this approach, the Hg2+ ions were selected as mediators to quench the fluorescence of AuNCs. Then, due to the stronger affinities between the interactions of Hg2+ ions with -SH groups in comparison to the AuNCs with -SH groups, the quenched probe of AuNCs@ZIF-8/Hg2+ displayed enhanced fluorescence after the Hg2+ ions were competitively interacted with -SH groups. Based on enhanced fluorescence, the probe for AuNCs@ZIF-8/Hg2+ had a sensitive and specific response to trace amounts of biothiols. The developed fluorescence strategy had limit of quantification (LOQ) values of 1.0 µM and 1.5 µM for Cys and GSH molecules in serum, respectively. This competitive AIE strategy provided a new direction for developing biological probes and a promising method for quantifying trace amounts of biothiols in serum. It could promote progress in disease diagnosis.

Maize decrease in DNA methylation 1 targets RNA-directed DNA methylation on active chromatin.

DNA methylation plays vital roles in repressing transposable element activity and regulating gene expression. The chromatin-remodeling factor Decrease in DNA methylation 1 (DDM1) is crucial for maintaining DNA methylation across diverse plant species, and is required for RNA-directed DNA methylation (RdDM) to maintain mCHH islands in maize (Zea mays). However, the mechanisms by which DDM1 is involved in RdDM are not well understood. In this work, we used chromatin immunoprecipitation coupled with high-throughput sequencing to ascertain the genome-wide occupancy of ZmDDM1 in the maize genome. The results revealed that ZmDDM1 recognized an 8-bp-long GC-rich degenerate DNA sequence motif, which is enriched in transcription start sites and other euchromatic regions. Meanwhile, 24-nucleotide siRNAs and CHH methylation were delineated at the edge of ZmDDM1-occupied sites. ZmDDM1 co-purified with Argonaute 4 (ZmAGO4) proteins, providing further evidence that ZmDDM1 is a component of RdDM complexes in planta. Consistent with this, the vast majority of ZmDDM1-targeted regions co-localized with ZmAGO4-bound genomic sites. Overall, our results suggest a model that ZmDDM1 may be recruited to euchromatic regions via recognition of a GC-rich motif, thereby remodeling chromatin to provide access for RdDM activities in maize.

Current Progress on Antibiotic Sensing Based on Ratiometric Fluorescent Sensors.

Antibiotics, which can be used as veterinary drugs, are widely used in the prevention and treatment of infectious diseases for animals. However, overuse of antibiotics had caused serious problems on food contamination and human harm. For control such public issues, several of techniques have been in recent years. Ratiometric fluorescent (RF) technique, as one of the most promising strategies for quantitatively evaluated analytes, had been extensively developed for the readily measurements on the two different fluorescent emission intensities. In this review, the construction strategies for recent RF sensors will be mainly focused on. Meanwhile, the recent advances and new tendencies for detection of antibiotics based on RF technique shall be introduced. Finally, outlooks on the opportunities and challenges for quantitative fluorescence sensing on antibiotics will be summarized.