A multifunctional fluorescent probe for sequential detection of hydrogen sulfide and pH in foodstuffs, living cells and mice.

BACKGROUND: Cancer as a leading cause of premature death worldwide has become a major threat to human health due to the high incidence and mortality. Monitoring tumor markers are reliable and significantly important for early detection of cancers. In complex biological systems, it is of great urgency but still remains challenging to conceive a fluorescent probe with multiple tumor markers detection property. Hydrogen sulfide (H2S) and pH are two target biomarkers for diagnosis of early cancer. The preparation of a novel probe with H2S and pH dual detection functions is highly anticipated. RESULTS: Herein, a novel sequential detection probe HTPQ-HS for H2S and pH has been developed. In this system, HPQ (2-(2 -hydroxyphenyl)-4(3H)-quinazolinone) structure combined with triphenylamine is applied as the fluorophore, and 2, 4-dinitrophenylsulfonyl group is used as the recognition group. In the presence of H2S, HTPQ-HS is transformed into product HTPQ-OH which shows fluorescence enhancement (29-fold) at 525 nm in less than 4 min and further displays repeatable acid-base responsive ability. HTPQ-HS is able to sequentially response to H2S and pH in living cells and does not react directly with pH. Owing to the low cytotoxicity, HTPQ-HS is able to detect exogenous and endogenous H2S in colon cancer cells and mice, monitor H2S in inflammation model and in foodstuffs. As the environment changes from acidic to alkaline, the fluorescence intensity ratio (I470/I530) of product HTPQ-OH changes remarkably, illustrating the ratiometric fluorescent responsiveness to pH. SIGNIFICANCE AND NOVELTY: A multifunctional fluorescent probe HTPQ-HS for sequential detection of H2S and pH is synthesized. Probe HTPQ-OH realizes the monitoring of dynamic changes in intracellular pH and displays prospective application in security printing. We expect that our work could offer an important guidance on the development of multifunctional fluorescent probes for visualizing H2S and pH in biology and environment.

Research on Pathogenic Fungi and Mycotoxins in China (Volume II).

The presence of pathogenic fungi and contamination of mycotoxins in food and feed pose significant threats and challenging issues to food in the world [...].

Self-Assembled DNA Composite-Engineered Mesenchymal Stem Cells for Improved Skin-Wound Repair.

The direct use of mesenchymal stem cells (MSCs) as therapeutics for skin injuries is a promising approach, yet it still faces several obstacles, including limited adhesion, retention, and engraftment of stem cells in the wound area, as well as impaired regenerative and healing functions. Here, DNA-based self-assembled composites are reported that can aid the adhesion of MSCs in skin wounds, enhance MSC viability, and accelerate wound closure and re-epithelialization. Rolling-circle amplification (RCA)-derived DNA flowers, equipped with multiple copies of cyclic Arg-Gly-Asp (cRGD) peptides and anti-von Willebrand factor (vWF) aptamers, act as robust scavengers of reactive oxygen species (ROS) and enable synergistic recognition and adhesion to stem cells and damaged vascular endothelial cells. These DNA structure-aided stem cells are retained at localized wound sites, maintain repair function, and promote angiogenesis and growth factor secretion. In both normal and diabetes-prone db/db mice models with excisional skin injuries, facile topical administration of DNA flower-MSCs elicits rapid blood vessel formation and enhances the sealing of the wound edges in a single dose. DNA composite-engineered stem cells warrant further exploration as a new strategy for the treatment of skin and tissue damage.

Impact of Lysine Succinylation on the Biology of Fungi.

Post-translational modifications (PTMs) play a crucial role in protein functionality and the control of various cellular processes and secondary metabolites (SMs) in fungi. Lysine succinylation (Ksuc) is an emerging protein PTM characterized by the addition of a succinyl group to a lysine residue, which induces substantial alteration in the chemical and structural properties of the affected protein. This chemical alteration is reversible, dynamic in nature, and evolutionarily conserved. Recent investigations of numerous proteins that undergo significant succinylation have underscored the potential significance of Ksuc in various biological processes, encompassing normal physiological functions and the development of certain pathological processes and metabolites. This review aims to elucidate the molecular mechanisms underlying Ksuc and its diverse functions in fungi. Both conventional investigation techniques and predictive tools for identifying Ksuc sites were also considered. A more profound comprehension of Ksuc and its impact on the biology of fungi have the potential to unveil new insights into post-translational modification and may pave the way for innovative approaches that can be applied across various clinical contexts in the management of mycotoxins.

Succinylated acetyl-CoA carboxylase contributes to aflatoxin biosynthesis, morphology development, and pathogenicity in Aspergillus flavus.

Acetyl-CoA carboxylase (ACC), which catalyzes acetyl-CoA to produce malonyl-CoA, is crucial for the synthesis of mycotoxins, ergosterol, and fatty acids in various genera. However, its biofunction in Aspergillus flavus has not been reported. In this study, the accA gene was deleted and site-mutated to explore the influence of ACC on sporulation, sclerotium formation, and aflatoxin B1 (AFB1) biosynthesis. The results revealed that ACC positively regulated conidiation and sclerotium formation, but negatively regulated AFB1 production. In addition, we found that ACC is a succinylated protein, and mutation of lysine at position 990 of ACC to glutamic acid or arginine (accAK990E or accAK990R) changed the succinylation level of ACC. The accAK990E and accAK990R mutations (to imitate the succinylation and desuccinylation at K990 of ACC, respectively) downregulated fungal conidiation and sclerotium formation while increasing AFB1 production, revealing that the K990 is an important site for ACC's biofunction. These results provide valuable perspectives for future mechanism studies of the emerging roles of succinylated ACC in the regulation of the A. flavus phenotype, which is advantageous for the prevention and control of A. flavus hazards.

Role of RNA Modifications, Especially m6A, in Aflatoxin Biosynthesis of Aspergillus flavus.

RNA modifications play key roles in eukaryotes, but the functions in Aspergillus flavus are still unknown. Temperature has been reported previously to be a critical environmental factor that regulates the aflatoxin production of A. flavus, but much remains to be learned about the molecular networks. Here, we demonstrated that 12 kinds of RNA modifications in A. flavus were significantly changed under 29 °C compared to 37 °C incubation; among them, m6A was further verified by a colorimetric method. Then, the transcriptome-wide m6A methylome and m6A-altered genes were comprehensively illuminated through methylated RNA immunoprecipitation sequencing and RNA sequencing, from which 22 differentially methylated and expressed transcripts under 29 °C were screened out. It is especially notable that AFCA_009549, an aflatoxin biosynthetic pathway gene (aflQ), and the m6A methylation of its 332nd adenine in the mRNA significantly affect aflatoxin biosynthesis in A. flavus both on media and crop kernels. The content of sterigmatocystin in both ΔaflQ and aflQA332C strains was significantly higher than that in the WT strain. Together, these findings reveal that RNA modifications are associated with secondary metabolite biosynthesis of A. flavus.

Engineered extracellular vesicles for ischemic stroke: a systematic review and meta-analysis of preclinical studies.

BACKGROUND: This systematic review and meta-analysis aimed to evaluate the efficacy of engineered extracellular vesicles (EEVs) in the treatment of ischemic stroke (IS) in preclinical studies and to compare them with natural extracellular vesicles (EVs). The systematic review provides an up-to-date overview of the current state of the literature on the use of EEVs for IS and informs future research in this area. METHODS: We searched PubMed, EMBASE, Web of Science, Cochrane Library, and Scopus databases for peer-reviewed preclinical studies on the therapeutic effect of EEVs on IS.Databases ranged from the inception to August 1, 2023. The outcome measures included infarct volumes, neurological scores, behavioral scores, apoptosis rates, numbers of neurons, and levels of IL-1ß, IL-6, and TNF-α. The CAMARADES checklist was used to assess the quality and bias risks of the studies. All statistical analyses were performed using RevMan 5.4 software. RESULTS: A total of 28 studies involving 1760 animals met the inclusion criteria. The results of the meta-analysis showed that compared to natural EVs, EEVs reduced infarct volume (percentage: SMD = -2.33, 95% CI: -2.92, -1.73; size: SMD = -2.36, 95% CI: -4.09, -0.63), improved neurological scores (mNSS: SMD = -1.78, 95% CI: -2.39, -1.17; Zea Longa: SMD = -2.75, 95% CI: -3.79, -1.71), promoted behavioral recovery (rotarod test: SMD = 2.50, 95% CI: 1.81, 3.18; grid-walking test: SMD = -3.45, 95% CI: -5.15, -1.75; adhesive removal test: SMD = -2.60, 95% CI: -4.27, -0.93; morris water maze test: SMD = -3.91, 95% CI: -7.03, -0.79), and reduced the release of proinflammatory factors (IL-1ß: SMD = -2.02, 95% CI: -2.77, -1.27; IL-6: SMD = -3.01, 95% CI: -4.47, -1.55; TNF-α: SMD = -2.72, 95% CI: -4.30, -1.13), increasing the number of neurons (apoptosis rate: SMD = -2.24, 95% CI: -3.32, -1.16; the number of neurons: SMD = 3.70, 95% CI: 2.44, 4.96). The funnel plots for the two main outcome measures were asymmetric, indicating publication bias. The median score on the CAMARADES checklist was 7 points (IQR: 6-9). CONCLUSIONS: This meta-analysis shows that EEVs are superior to natural EVs for the treatment of IS. However, research in this field is still at an early stage, and more research is needed to fully understand the potential therapeutic mechanism of EEVs and their potential use in the treatment of IS. PROSPERO REGISTRATION NUMBER: CRD42022368744.

PP2C phosphatases Ptc1 and Ptc2 dephosphorylate PGK1 to regulate autophagy and aflatoxin synthesis in the pathogenic fungus Aspergillus flavus.

IMPORTANCE: Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Mesenchymal Stem Cell-Derived Exosomes Promote Recovery of The Facial Nerve Injury through Regulating Macrophage M1 and M2 Polarization by Targeting the P38 MAPK/NF-Kb Pathway.

Facial nerve (FN) injury seriously affects human social viability and causes a heavy economic and social burden. Although mesenchymal stem cell-derived exosomes (MSC-Exos) promise therapeutic benefits for injury repair, there has been no evaluation of the impact of MSC-Exos administration on FN repair. Herein, we explore the function of MSC-Exos in the immunomodulation of macrophages and their effects in repairing FN injury. An ultracentrifugation technique was used to separate exosomes from the MSC supernatant. Administrating MSC-Exos to SD rats via local injection after FN injury promoted axon regeneration and myelination and alleviated local and systemic inflammation. MSC-Exos facilitated M2 polarization and reduced the M1-M2 polarization ratio. miRNA sequencing of MSC-Exos and previous literature showed that the MAPK/NF-κb pathway was a downstream target of macrophage polarization. We confirmed this hypothesis both in vivo and in vitro. Our findings show that MSC-Exos are a potential candidate for treating FN injury because they may have superior benefits for FN injury recovery and can decrease inflammation by controlling the heterogeneity of macrophages, which is regulated by the p38 MAPK/NF-κb pathway.

Identifying subgroups of patients with type 2 diabetes based on real-world traditional chinese medicine electronic medical records.

Introduction: Type 2 diabetes (T2D) is a multifactorial complex chronic disease with a high prevalence worldwide, and Type 2 diabetes patients with different comorbidities often present multiple phenotypes in the clinic. Thus, there is a pressing need to improve understanding of the complexity of the clinical Type 2 diabetes population to help identify more accurate disease subtypes for personalized treatment. Methods: Here, utilizing the traditional Chinese medicine (TCM) clinical electronic medical records (EMRs) of 2137 Type 2 diabetes inpatients, we followed a heterogeneous medical record network (HEMnet) framework to construct heterogeneous medical record networks by integrating the clinical features from the electronic medical records, molecular interaction networks and domain knowledge. Results: Of the 2137 Type 2 diabetes patients, 1347 were male (63.03%), and 790 were female (36.97%). Using the HEMnet method, we obtained eight non-overlapping patient subgroups. For example, in H3, Poria, Astragali Radix, Glycyrrhizae Radix et Rhizoma, Cinnamomi Ramulus, and Liriopes Radix were identified as significant botanical drugs. Cardiovascular diseases (CVDs) were found to be significant comorbidities. Furthermore, enrichment analysis showed that there were six overlapping pathways and eight overlapping Gene Ontology terms among the herbs, comorbidities, and Type 2 diabetes in H3. Discussion: Our results demonstrate that identification of the Type 2 diabetes subgroup based on the HEMnet method can provide important guidance for the clinical use of herbal prescriptions and that this method can be used for other complex diseases.

Sensitive and rapid detection of tetrodotoxin based on gold nanoflower-and latex microsphere-labeled monoclonal antibodies.

Tetrodotoxin (TTX) could result in serious diseases due to its extremely high neurotoxicity. Thus, it is of great importance to measure TTX for food safety. In this study, an anti-TTX monoclonal antibody with good specificity and high affinity was used to develop the immunochromatographic test strips (ICTS). Gold nanoflower (AuNF) with multiple branches and latex microsphere (LM) with large particle size as signal reporters were employed for improving the sensitivity of test strips. Both AuNF and LM probes are stable, and the developed ICTS were specific to TTX, demonstrating no cross-reactivity with other marine toxins. The linear range of AuNF- and LM-based strips for TTX was 9.49-330.98 ng/mL and 5.40-443.19 ng/mL, respectively. The limit of detection (LOD) of AuNF- and LM-based strips was determined to be 9.49 ng/mL and 5.40 ng/mL, respectively. In summary, the developed ICTS based on AuNF and LM signal probes displayed enhancement of sensitivity and provided rapid and specific detection of TTX.

Small Extracellular Vesicles Maintain Homeostasis of Senescent Mesenchymal Stem Cells at Least Through Excreting Harmful Lipids.

Mesenchymal stem cells (MSCs) play an essential role in multiple physiological processes in vivo and a promising cell-based therapy for various diseases. Nonetheless, MSCs suffer from senescence with expansion culture, leading to a limitation for their clinical application. Recently, it was reported that small extracellular vesicles (sEVs) are involved in regulation of senescence in tumor cells and fibroblasts. However, the biological roles of sEVs in senescent MSCs (Sen MSCs) are poorly understood. In this study, we established a replicative senescence model of MSCs by successive passages and compared the phenotypic changes between presenescent MSCs (Pre-Sen MSCs) and Sen MSCs and found that Sen MSCs exhibited a diminished adipogenic and osteogenic differentiation potential and elevated senescence-associated secretory phenotype levels. In addition, we found that sEV secretion was increased in Sen MSCs, and inhibition of sEV secretion led to apoptosis, DNA damage, and decreased cell viability, suggesting that increased sEV secretion plays an important role in maintaining Sen MSC homeostasis. To further investigate the molecular mechanisms, metabolomic profiling of Pre-Sen MSC-derived sEVs (Pre-Sen-sEVs) and Sen MSC-derived sEVs (Sen-sEVs) was performed. The results showed that lipid metabolites were significantly increased in Sen-sEVs and these significantly upregulated lipid metabolites were shown to be toxic for inducing cellular senescence and apoptosis in previous studies. Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment of differential metabolites between Pre-Sen-sEVs and Sen-sEVs mainly in 25 signaling pathways, of which 21 metabolic pathways have been shown to be closely associated with senescence. Taken together, our findings suggested that increased sEV secretion maintains Sen MSC homeostasis, at least in part, by excreting harmful lipids, thus providing new insights into the regulation of senescence by sEVs.

Chemotherapy for the initial treatment of metastatic prostate adenocarcinoma and neuroendocrine carcinoma at diagnosis: real world application and impact in the SEER database (2004 -2018).

Background: Randomized controlled phase III trials have reported significant improvements in disease response and survival with the addition of chemotherapy to androgen deprivation therapy for men presenting with metastatic prostate cancer. We examined the implementation of such knowledge and its impact within the Surveillance, Epidemiology, and End Results (SEER) database. Method: The administration of chemotherapy for men with an initial presentation of metastatic prostate cancer from 2004 to 2018 in the SEER database and its association with survival outcomes was examined. Kaplan-Meier estimates were applied to compare survival curves. Cox proportion hazard survival models were used to analyze the association of chemotherapy and other variables with both cancer- specific and overall survival. Result: A total of 727,804 patients were identified with 99.9% presenting with adenocarcinoma and 0.1% with neuroendocrine histopathology. Chemotherapy as initial treatment for men with de novo distant metastatic adenocarcinoma increased from 5.8% during 2004-2013 to 21.4% during 2014-2018. Chemotherapy was associated with a poorer prognosis during 2004-2013 but was associated with improved cancer-specific (hazard ratio (HR) = 0.85, 95% confidence interval (CI): 0.78-0.93, p=0.0004) and overall survival (HR= 0.78, 95% CI: 0.71-0.85, p < 0.0001) during 2014-2018. The improved prognosis during 2014-2018 was observed in patients with visceral or bone metastasis and most impactful for patients aged 71-80 years. These findings were confirmed by subsequent propensity score matching analyses. Furthermore, chemotherapy was consistently provided to 54% of patients with neuroendocrine carcinoma at diagnosis from 2004 to 2018. Treatment was associated with improved cancer-specific survival (HR= 0.62, 95% CI: 0.45-0.87, p=0.0055) and overall survival (HR= 0.69, 95% CI: 0.51-0. 94, p=0.0176) during 2014-2018 but not significant in earlier years. Conclusion: Chemotherapy at initial diagnosis was increasingly employed in men with metastatic adenocarcinoma after 2014 and consistent with the evolution of National Comprehensive Cancer Network (NCCN) guidelines. Benefits for chemotherapy are suggested after 2014 in the treatment of men with metastatic adenocarcinoma. The use of chemotherapy for neuroendocrine carcinoma at diagnosis has remained stable, and outcomes have improved in more recent years. Further development and optimization of chemotherapy continues to evolve for men with de novo diagnosis of metastatic prostate cancer.

SWD1 epigenetically chords fungal morphogenesis, aflatoxin biosynthesis, metabolism, and virulence of Aspergillus flavus.

As the main producer of aflatoxins, Aspergillus flavus is also one of the most important causes of invasive and non-invasive aspergillosis. Therefore, it is crucial to unravel the regulatory mechanisms of growth, metabolism, and pathogenicity of A. flavus. SWD1 is highly conserved across species for maintaining COMPASS methyltransferase activity, but the bio-function of SWD1 in A. flavus has not been explored. Through genetic analysis, this study revealed that SWD1 is involved in fungal morphogenesis and AFB1 biosynthesis by regulating the orthodox pathways through H3K4me1-3. Stresses sensitivity and crop models analysis revealed that SWD1 is a key regulator for the resistance of A. flavus to adapt to extreme adverse environments and to colonize crop kernels. It also revealed that the WD40 domain and 25 aa highly conserved sequence are indispensable for SWD1 in the regulation of mycotoxin bio-synthesis and fungal virulence. Metabolomic analysis inferred that SWD1 is crucial for the biosynthesis of numerous primary and secondary metabolites, regulates biological functions by reshaping the whole metabolic process, and may inhibit fungal virulence by inducing the apoptosis of mycelia through the inducer sphingosine. This study elucidates the epigenetic mechanism of SWD1 in regulating fungal pathogenicity and mycotoxin biosynthesis, and provides a potential novel target for controlling the virulence of A. flavus.

A functional intact SUMOylation machinery in Aspergillus flavus contributes to fungal and aflatoxin contamination of food.

SUMO adducts occur in Aspergillus flavus, and are implicated in fungal biology, while the underlying mechanism and the SUMOylation apparatus components in this saprophytic food spoilage mould, remain undefined. Herein, genes encoding SUMOylation cascade enzymes in A. flavus, including two heterodimeric SUMO E1 activating enzymes, a unique SUMO E2 conjugating enzyme, and one of SUMO E3 ligases, were identified and functionally analyzed. Global SUMO adducts immunoassay, multiple morphological comparison, aflatoxin attributes test, fungal infection and transcriptomic analyses collectively revealed that: E1 and E2 were essential for intracellular SUMOylation, and contributed to both stress response and fungal virulence-related events, including sporulation, colonization, aflatoxins biosynthesis; the primary E3 in this fungus, AfSizA, might serve as the molecular linkage of SUMOylation pathway to fungal virulence rather than SUMOylation-mediated stress adaptation. These findings demonstrated that SUMOylation machinery in A. flavus was functionally intact and contributed to multiple pathobiological processes, hence offering ideas and targets to control food contamination by this mycotoxigenic fungus.

Oncologic safety of the pedicled submental island flap for reconstruction in oral tongue squamous cell carcinoma: An analysis of 101 cases.

OBJECTIVE: To evaluate whether the pedicle submental island flap (SIF) can be safely used in the oral tongue squamous cell carcinoma (OTSCC) patients with pathologically node-positive (pN+) neck, especially pN+ at level I. METHODS: Retrospectively, 101 OTSCC patients with SIF reconstruction were enrolled. Oncological outcomes included the total locoregional recurrence, the SIF related locoregional recurrence (SRLR) which referred to the local recurrence at flap and ipsilateral neck recurrence at level I, recurrence free survival (RFS), overall survival (OS), and disease specific survival (DSS). RESULTS: Sixty-one patients were pathologically node-negative (pN0) and 40 were pN+. Thirteen patients experienced locoregional recurrence, of which 5 had a SRLR. The pN+ group had a significantly higher locoregional recurrence rate, lower 5-year RFS, OS and DSS than pN0 group (P < 0.05). Patients with pN0 had a significantly higher neck RFS when compared to those with pN+ either at level I (P = 0.005) or at other levels (P < 0.001). However, the neck RFS was similar between the two subgroups of pN+ (P = 0.550). Especially, patients with pN+ at level I had a significantly higher SRLR rate (P = 0.006) compared to those with pN0 at level I. Multivariate analysis showed that pN+ was an unfavorable factor for tumor recurrence and OS. CONCLUSION: Our data did not support the use of SIF in OTSCC patients with pN+ neck at level I due to an significantly increased SRLR rate compared to those with pN0 neck at level I.

ß2-microglobulin functions as an endogenous NMDAR antagonist to impair synaptic function.

Down syndrome (DS) is a neurological disorder with multiple immune-related symptoms; however, crosstalk between the CNS and peripheral immune system remains unexplored. Using parabiosis and plasma infusion, we found that blood-borne factors drive synaptic deficits in DS. Proteomic analysis revealed elevation of ß2-microglobulin (B2M), a major histocompatibility complex class I (MHC-I) component, in human DS plasma. Systemic administration of B2M in wild-type mice led to synaptic and memory defects similar to those observed in DS mice. Moreover, genetic ablation of B2m or systemic administration of an anti-B2M antibody counteracts synaptic impairments in DS mice. Mechanistically, we demonstrate that B2M antagonizes NMDA receptor (NMDAR) function through interactions with the GluN1-S2 loop; blocking B2M-NMDAR interactions using competitive peptides restores NMDAR-dependent synaptic function. Our findings identify B2M as an endogenous NMDAR antagonist and reveal a pathophysiological role for circulating B2M in NMDAR dysfunction in DS and related cognitive disorders.

N-Myristoyltransferase, a Potential Antifungal Candidate Drug-Target for Aspergillus flavus.

The filamentous fungus Aspergillus flavus causes devastating diseases not only to cash crops but also to humans by secreting a series of secondary metabolites called aflatoxins. In the cotranslational or posttranslational process, N-myristoyltransferase (Nmt) is a crucial enzyme that catalyzes the myristate group from myristoyl-coenzyme A (myristoyl-CoA) to the N terminus or internal glycine residue of a protein by forming a covalent bond. Members of the Nmt family execute a diverse range of biological functions across a broad range of fungi. However, the underlying mechanism of AflNmt action in the A. flavus life cycle is unclear, particularly during the growth, development, and secondary metabolic synthesis stages. In the present study, AlfNmt was found to be essential for the development of spore and sclerotia, based on the regulation of the xylose-inducible promoter. AflNmt, located in the cytoplasm of A. flavus, is also involved in modulating aflatoxin (AFB1) in A. flavus, which has not previously been reported in Aspergillus spp. In addition, we purified, characterized, and crystallized the recombinant AflNmt protein (rAflNmt) from the Escherichia coli expression system. Interestingly, the crystal structure of rAlfNmt is moderately different from the models predicted by AlphaFold2 in the N-terminal region, indicating the limitations of machine-learning prediction. In conclusion, these results provide a molecular basis for the functional role of AflNmt in A. flavus and structural insights concerning protein prediction. IMPORTANCE As an opportunistic pathogen, A. flavus causes crop loss due to fungal growth and mycotoxin contamination. Investigating the role of virulence factors during infection and searching for novel drug targets have been popular scientific topics in the field of fungal control. Nmt has become a potential target in some organisms. However, whether Nmt is involved in the developmental stages of A. flavus and aflatoxin synthesis, and whether AlfNmt is an ideal target for structure-based drug design, remains unclear. This study systematically explored and identified the role of AlfNmt in the development of spore and sclerotia, especially in aflatoxin biosynthesis. Moreover, although there is not much difference between the AflNmt model predicted using the AlphaFold2 technique and the structure determined using the X-ray method, current AI prediction models may not be suitable for structure-based drug development. There is still room for further improvements in protein prediction.

Acupuncture Case Registry Study: Rationale, Implementations, and Achievements.

The acupuncture case registry study is focusing on acupuncture therapy data from patient cases. The main objective is to collect real-world data and integrate clinically meaningful outcome evaluation indicators to uncover and evaluate real-world acupuncture efficacy and safety, explore factors affecting acupuncture efficacy, and provide real-world evidence to complement RCTs. Since the International Acupuncture Case Registry data collection system's establishment in 2017, 16 projects have been underway, including two acupuncture specialty therapies and 15 diseases. Data from 3404 patients included extensive information on the diagnosis and treatment of acupuncture and the evaluation of its efficacy. In order to serve as a guide for future studies, this article discusses the value of and rationale for establishing acupuncture case registry studies, how to distinguish them from patient registries, and crucial techniques for implementing registry studies in terms of applications, patient recruitment, costakeholder collaboration, data collection and management, study quality control, and ethics.