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Abstract Background Acute kidney injury (AKI) is a common clinical disorder with complex etiology and poor prognosis, and currently lacks specific and effective treatment options. Mitochondrial dynamics dysfunction is a prominent feature in AKI, and modulation of mitochondrial morphology may serve as a potential therapeutic approach for AKI. Methods We induced ischemia–reperfusion injury (IRI) in mice (bilateral) and Bama pigs (unilateral) by occluding the renal arteries. ATP depletion and recovery (ATP-DR) was performed on proximal renal tubular cells to simulate in vitro IRI. Renal function was evaluated using creatinine and urea nitrogen levels, while renal structural damage was assessed through histopathological staining. The role of Drp1 was investigated using immunoblotting, immunohistochemistry, immunofluorescence, and immunoprecipitation techniques. Mitochondrial morphology was evaluated using confocal microscopy. Results Renal IRI induced significant mitochondrial fragmentation, accompanied by Dynamin-related protein 1 (Drp1) translocation to the mitochondria and Drp1 phosphorylation at Ser616 in the early stages (30 min after reperfusion), when there was no apparent structural damage to the kidney. The use of the Drp1 inhibitor P110 significantly improved kidney function and structural damage. P110 reduced Drp1 mitochondrial translocation, disrupted the interaction between Drp1 and Fis1, without affecting the binding of Drp1 to other mitochondrial receptors such as MFF and Mid51. High-dose administration had no apparent toxic side effects. Furthermore, ATP-DR induced mitochondrial fission in renal tubular cells, accompanied by a decrease in mitochondrial membrane potential and an increase in the translocation of the pro-apoptotic protein Bax. This process facilitated the release of dsDNA, triggering the activation of the cGAS-STING pathway and promoting inflammation. P110 attenuated mitochondrial fission, suppressed Bax mitochondrial translocation, prevented dsDNA release, and reduced the activation of the cGAS-STING pathway. Furthermore, these protective effects of P110 were also observed renal IRI model in the Bama pig and folic acid-induced nephropathy in mice. Conclusions Dysfunction of mitochondrial dynamics mediated by Drp1 contributes to renal IRI. The specific inhibitor of Drp1, P110, demonstrated protective effects in both in vivo and in vitro models of AKI.

Summary: Background: Earlier Omicron subvariants including BA.1, BA.2, and BA.5 emerged in waves, with a subvariant replacing the previous one every few months. More recently, the post-BA.2/5 subvariants have acquired convergent substitutions in spike that facilitated their escape from humoral immunity and gained ACE2 binding capacity. However, the intrinsic pathogenicity and replication fitness of the evaluated post-BA.2/5 subvariants are not fully understood. Methods: We systemically investigated the replication fitness and intrinsic pathogenicity of representative post-BA.2/5 subvariants (BL.1, BQ.1, BQ.1.1, XBB.1, CH.1.1, and XBB.1.5) in weanling (3–4 weeks), adult (8–10 weeks), and aged (10–12 months) mice. In addition, to better model Omicron replication in the human nasal epithelium, we further investigated the replication capacity of the post-BA.2/5 subvariants in human primary nasal epithelial cells. Findings: We found that the evaluated post-BA.2/5 subvariants are consistently attenuated in mouse lungs but not in nasal turbinates when compared with their ancestral subvariants BA.2/5. Further investigations in primary human nasal epithelial cells revealed a gained replication fitness of XBB.1 and XBB.1.5 when compared to BA.2 and BA.5.2. Interpretation: Our study revealed that the post-BA.2/5 subvariants are attenuated in lungs while increased in replication fitness in the nasal epithelium, indicating rapid adaptation of the circulating Omicron subvariants in the human populations. Funding: The full list of funding can be found at the Acknowledgements section.

Voriconazole is the first‐line treatment for invasive aspergillosis. Its pharmacokinetics exhibit considerable inter‐ and intra‐individual variability. The purpose of this study was to investigate the effects of CYP2C19, CYP2C9, CYP3A4, and FMO3 genetic polymorphisms and sex on the pharmacokinetics of voriconazole in healthy Chinese adults receiving single‐dose and multiple‐dose voriconazole, to provide a reference for its clinical individualized treatment. A total of 123 healthy adults were enrolled in the study, with 108 individuals and 15 individuals in the single‐dose and multiple‐dose doses, respectively. Plasma voriconazole concentrations were measured using a validated LC‐MS/MS method, and pharmacokinetics parameters were calculated using the non‐compartmental method with WinNonlin 8.2. CYP2C19, CYP2C9, CYP3A4, and FMO3 single‐nucleotide polymorphisms were sequenced using the Illumina Hiseq X‐Ten platform. The results suggested that CYP2C19 genetic polymorphisms significantly affected the pharmacokinetics of voriconazole at single doses of 4, 6, and 8 mg/kg and multiple doses of voriconazole. CYP3A4 rs2242480 had a significant effect on AUC0‐∞ (area under the plasma concentration‐time curve from time 0 to infinity) and MRT (mean residence time) of voriconazole at a single dose of 4 mg/kg in CYP2C19 extensive metabolizer. Regardless of the CYP2C19 genotype, CYP2C9 rs1057910 and FMO3 rs2266780 were not associated with the pharmacokinetics of voriconazole at three single‐dose levels or multiple doses. No significant differences in most voriconazole pharmacokinetics parameters were noted between male and female participants after single and multiple dosing. For patients receiving voriconazole treatment, CYP2C19 genetic polymorphisms should be genotyped for its precision administration. In contrast, based on our study of healthy Chinese adults, it seems unnecessary to consider the effects of CYP2C9, CYP3A4, and FMO3 genetic polymorphisms on voriconazole pharmacokinetics. [ABSTRACT FROM AUTHOR]

The rapid and accurate estimation of leaf area index (LAI) through remote sensing holds significant importance for precise crop management. However, the direct construction of a vegetation index model based on multi-spectral data lacks robustness and spatiotemporal expansibility, making its direct application in practical production challenging. This study aimed to establish a simple and effective method for LAI estimation to address the issue of poor accuracy and stability that is encountered by vegetation index models under varying conditions. Based on seven years of field plot trials with different varieties and nitrogen fertilizer treatments, the Kalman filter (KF) fusion method was employed to integrate the estimated outcomes of multiple vegetation index models, and the fusion process was investigated by comparing and analyzing the relationship between fixed and dynamic variances alongside the fusion accuracy of optimal combinations during different growth stages. A novel multi-model integration fusion method, KF-DGDV (Kalman Filtering with Different Growth Periods and Different Vegetation Index Models), which combines the growth characteristics and uncertainty of LAI, was designed for the precise monitoring of LAI across various growth phases of rice. The results indicated that the KF-DGDV technique exhibits a superior accuracy in estimating LAI compared with statistical data fusion and the conventional vegetation index model method. Specifically, during the tillering to booting stage, a high R2 value of 0.76 was achieved, while at the heading to maturity stage, it reached 0.66. In contrast, within the framework of the traditional vegetation index model, the red-edge difference vegetation index (DVIREP) model demonstrated a superior performance, with an R2 value of 0.65, during tillering to booting stage, and 0.50 during the heading to maturity stage, respectively. The multi-model integration method (MME) yielded an R2 value of 0.67 for LAI estimation during the tillering to booting stage, and 0.53 during the heading to maturity stage. Consequently, KF-DGDV presented an effective and stable real-time quantitative estimation method for LAI in rice. [ABSTRACT FROM AUTHOR]

The absence of periodic structures and uncertainty concerning active sites in traditional single-atom catalysts (SACs) consistently impede the understanding of the coordination environment and its impact on the peroxymonosulfate (PMS) activation mechanism. In this study, we develop well-defined FeN4+4 active-site configurations featuring robust and ordered N-coordinated Fe single-atomic centers within fully π-conjugated polyphthalocyanine frameworks (CPFs). CPF–FeN4+4 functions as an outstanding PMS activator, exhibiting remarkable efficiency in the degradation of bisphenol A (BPA) with a rate constant of 1.87 min−1, surpassing the majority of state-of-the-art SAC-based PMS systems. Mechanistic scrutiny unveils heightened chemisorption and electron transfer dynamics between PMS and CPF–FeN4+4, facilitating a 1O2-dominated selective oxidation pathway. The distinctive FeN4+4 active sites, integrated into π-conjugated frameworks, expedite S–O bond cleavage in PMS, thereby reducing the energy barrier for the formation of *HSO3 and *O2 (1O2 precursors). The charge redistribution in CPF–FeN4+4 engenders a dual-pump-driven, electron-fast shuttle path, involving electron-rich Fe centers and electron-poor C surrounding adjacent benzene rings, ensuring the continuous production of 1O2. This study not only delineates precise active sites at the atomic level for PMS activation but also advances the evolution of a highly promising catalytic oxidation system tailored for practical environmental purification. [ABSTRACT FROM AUTHOR]

The incidence of Hepatocellular carcinoma (HCC) and HCC‐related deaths have remarkably increased over the recent decades. It has been reported that β‐catenin activation can be frequently observed in HCC cases. This study identified the integrin‐linked kinase‐associated phosphatase (ILKAP) as a novel β‐catenin‐interacting protein. ILKAP is localized both in the nucleus and cytoplasm and regulates the WNT pathway in different ways. First, it is demonstrated that ILKAP activates the WNT pathway in HCC cells by increasing the protein level of β‐catenin and other proteins associated with the WNT signaling, such as c‐Myc and CyclinD1. Next, it is shown that ILKAP promotes the metastasis of HCC both in vitro and in vivo in a zebrafish xenograft model. It is also found that ILKAP dephosphorylates the GSK3β and CK1, contributing to the reduced ubiquitination of β‐catenin. Furthermore, it is identified that ILKAP functions by mediating binding between TCF4 and β‐catenin to enhance expression of WNT target genes. Taken together, the study demonstrates a critical function of ILKAP in metastasis of HCC, since ILKAP is crucial for the activation of the WNT pathway via stabilization of β‐catenin and increased binding between TCF4 and β‐catenin. [ABSTRACT FROM AUTHOR]

Aims We sought to explore the latent classifications of psychosocial adaptation in young and middle-aged patients with acute myocardial infarction (AMI) and analyse the characteristics of different profiles of AMI patients. Methods and results A cross-sectional study was performed in 438 Chinese young and middle-aged patients with AMI. The investigation time was 1 month after discharge. Three different self-report instruments were distributed to the participants, including the Psychosocial Adjustment to Illness Scale, the Perceived Stress Scale, and the Social Support Rating Scale. The seven dimensions of the Psychosocial Adjustment to Illness Scale were then used to perform a latent profile analysis. All participants signed informed consent forms in accordance with the ethical principles of the Declaration of Helsinki. Finally, a total of 411 young and middle-aged AMI patients were enrolled. Three distinct profiles were identified, including the 'well-adapted group' (44.8%), 'highlight in psychological burdens group' (25.5%), and 'poorly adapted group' (29.7%). The influencing factors included stress perception, social support, occupational type, and marital status (P < 0.05). Conclusion The psychosocial adaptation of young and middle-aged AMI patients can be divided into three profiles. Clinical nurses can carry out individualized psychological interventions according to the characteristics of patients in different potential profiles to improve the psychosocial adaptation of patients and the prognosis of their disease. [ABSTRACT FROM AUTHOR]

Arsenic (As) is a metal with potentially toxic effects on different organisms. Hydrogen sulfide (H2S) plays a vital role in mitigating heavy metal toxicity by reducing oxidative stress in plants and animals. However, the role of H2S in alleviating arsenic toxicity in yeast cells remains unclear. In this study, the role of NaHS (exogenous physiological H2S) in alleviating As-induced yeast cell death was investigated. Yeast cells in the logarithmic phase were pretreated with 0.05 mmol/L NaHS for 6 h, and then incubated in the YPD medium with or without 1 mmol/L As. After 12 h of treatment, relative survival rate, H2S content, oxidative stress biomarkers, and antioxidant machinery were measured. Our results showed that sodium arsenite-induced yeast cell death and pretreatment with 0.05 mmol/L NaHS significantly alleviated sodium arsenite-induced cell death. Under sodium arsenite conditions, the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) increased, accompanied by the inhibition of the catalase (CAT) activity and the downregulation of CTT1 expression. However, the activities of the superoxide dismutase (SOD) and glutathion peroxidase (GPX) increased, and the expression of SOD1 and GPX2 was markedly upregulated in the group treated with sodium arsenite. When yeast cells were pretreated with NaHS, the intracellular ROS and MDA levels decreased significantly, and the activities of SOD, CAT, and GPX increased significantly. This was associated with a significant increase in relative survival rate and H2S content compared to the arsenic treatment alone. Our findings indicate that NaHS alleviates sodium arsenite-induced yeast cell death, mainly by enhancing the antioxidant defense system. [ABSTRACT FROM AUTHOR]

The number of seedlings is an important indicator that reflects the size of the wheat population during the seedling stage. Researchers increasingly use deep learning to detect and count wheat seedlings from unmanned aerial vehicle (UAV) images. However, due to the small size and diverse postures of wheat seedlings, it can be challenging to estimate their numbers accurately during the seedling stage. In most related works in wheat seedling detection, they label the whole plant, often resulting in a higher proportion of soil background within the annotated bounding boxes. This imbalance between wheat seedlings and soil background in the annotated bounding boxes decreases the detection performance. This study proposes a wheat seedling detection method based on a local annotation instead of a global annotation. Moreover, the detection model is also improved by replacing convolutional and pooling layers with the Space-to-depth Conv module and adding a micro-scale detection layer in the YOLOv5 head network to better extract small-scale features in these small annotation boxes. The optimization of the detection model can reduce the number of error detections caused by leaf occlusion between wheat seedlings and the small size of wheat seedlings. The results show that the proposed method achieves a detection accuracy of 90.1%, outperforming other state-of-the-art detection methods. The proposed method provides a reference for future wheat seedling detection and yield prediction. [ABSTRACT FROM AUTHOR]

Abstract Cardiovascular diseases have been the leading cause of global mortality and disability. In addition to traditional drug and surgical treatment, more and more studies investigate tissue engineering therapeutic strategies in cardiovascular medicine. Collagen interweaves in the form of trimeric chains to form the physiological network framework of the extracellular matrix of cardiac and vascular cells, possessing excellent biological properties (such as low immunogenicity and good biocompatibility) and adjustable mechanical properties, which renders it a vital tissue engineering biomaterial for the treatment of cardiovascular diseases. In recent years, promising advances have been made in the application of collagen materials in blood vessel prostheses, injectable cardiac hydrogels, cardiac patches, and hemostatic materials, although their clinical translation still faces some obstacles. Thus, we reviewed these findings and systematically summarizes the application progress as well as problems of clinical translation of collagen biomaterials in the cardiovascular field. The present review contributes to a comprehensive understanding of the application of collagen biomaterials in cardiovascular medicine. Graphical abstract

Abstract The rhizosphere bacterium Pseudomonas protegens FD6, which is associated with the production of antibiotics such as pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), has strong antagonistic effects on phytopathogens. Hfq is a conserved RNA chaperone involved in the regulation of stress tolerance, antibiotic production, and bacterial virulence. Here, we determined the regulatory effects of Hfq on biocontrol traits in P. protegens FD6. Mutation of hfq in FD6 reduced the growth rate, swimming and swarming ability, and production of proteases, siderophores, and hydrogen cyanide (HCN). Transmission electron microscopy revealed that disruption of hfq led to the loss of flagella. Furthermore, Hfq exerted a suppressive effect on biofilm formation and PLT production while not affecting antagonistic activity and control effect against tomato bacterial wilt. However, Hfq positively regulated the production of 2,4-DAPG and activated the phlD expression. Additionally, mutation of hfq abolished the function of the type VI secretion system (T6SS) due to a decrease in the expression of T6SS-related genes. Overall, these results suggest that Hfq plays a pleiotropic role in modulating the expression of biocontrol trait-related genes in P. protegens FD6.

Abstract Polyorchidism is an uncommon congenital condition often discovered incidentally, which may significantly impact male fertility. We present a case of a 34-year-old man with unilateral polyorchidism and associated severe asthenozoospermia and teratozoospermia. Despite normal hormonal levels and no detected genetic anomalies, the patient’s sperm showed mitochondrial damage, and his fertility remained compromised after conservative management and assisted reproductive attempts. This case underscores the intricate relationship between polyorchidism and male infertility, highlighting the need for personalized management strategies and further research into its etiology and impact.

Objective To explore the inhibitory effect of berberine （BBR） on contrast-induced nephropathy （CIN） and clarify the underlying mechanism. Methods Sprague-Dawley （SD） rats were utilized for constructing a rat CIN model. BBR treatment group （M+B） received a gavage of 200 mg/kg BBR while control group （Con） and model group （Mod） had an oral intake of the same volume of normal saline. Human kidney cortex proximal tubule epithelial cells （HK-2） were utilized for constructing a CIN cell model in vitro. No special treatment was offered to HK-2 cells in Con group. HK-2 cells in Mod group were treated with 50 g/L ioversol for 24 h and HK-2 cells in M+B group 50 g/L ioversol and 30 μmol/L BBR for 24 h. Akt agonist （SC79） and Akt inhibitor （MK2206） were utilized for confirming the effect of BBR on protein kinase B-forkhead box O3-nuclear factor erythroid 2-related factor 2 （Akt-Foxo3a-Nrf2） regulatory axis. Serum creatinine （Scr） detection and hematoxylin-eosin （HE） stain were employed for evaluating renal injury in rats. Annexin V-propidium iodide （annexin V-PI） stain was used for detecting cell apoptosis. And Western blot was utilized for detecting the protein expression of Akt-Foxo3a-Nrf2 axis.Results The level of Scr was higher in Mod group than that in Con group [（58.83 ± 7.96）μmol/L vs （23.52 ± 0.78）μmol/L，P＜0.05]. And it was lower in M+B group than that in Mod group [（39.64 ± 2.52）μmol/L vs （58.83 ± 7.96）μmol/L，P＜0.05]. HE stain indicated that kidney morphology was normal and renal tubular epithelial cells remained intact in Con group. In Mod group， structure of renal tubules was disarrayed with diffuse death of epithelial cells and obvious vacuolar degeneration of renal tubular epithelial cells. In M+B group， morphology of renal tubules was partially restored and vacuolar degeneration of renal tubule epithelial cells and cell death lessened. Western blot results indicated that， as compared with Con group， the expression of phospho-protein kinase B （p-Akt） was up-regulated in CIN rats and ioversol treated HK-2 cells. And the difference was statistically significant （P＜0.05）. In M+B group， the expression of p-Akt dropped as compared with Mod group （P＜0.05）. Annexin V-PI apoptosis assay indicated that， as compared with Con group， cell apoptosis spiked in Mod group [（12.65 ± 1.25）% vs （27.44 ± 0.73）%，P＜0.05]. As compared with Mod group， cell apoptosis declined in M+B group [（27.44 ± 0.73）% vs （24.81 ± 0.49）%，P＜0.05]. As compared with M+B group， cell apoptosis rose in BBR plus SC79 treatment group [（24.81 ± 0.49）% vs （27.50 ± 0.35）%，P＜0.05]. As compared with Mod group， cell apoptosis declined in MK2206 treatment group [（27.65 ± 0.56）% vs （25.20 ± 0.64）%，P＜0.05]. As compared with M+B group， MK2206 plus BBR further reduced ioversol-induced HK-2 cell apoptosis [（24.51 ± 0.52）% vs （21.24 ± 0.99）%，P＜0.05]. Conclusion BBR may suppress CIN in vivo and in vitro through regulating Akt-Foxo3a-Nrf2 axis in a p-Akt dependent mode.

Abstract Deep-sea trenches representing an intriguing ecosystem for exploring the survival and evolutionary strategies of microbial communities in the highly specialized deep-sea environments. Here, 29 metagenomes were obtained from sediment samples collected from Kermadec and Diamantina trenches. Notably, those samples covered a varying sampling depths (from 5321 m to 9415 m) and distinct layers within the sediment itself (from 0~40 cm in Kermadec trench and 0~24 cm in Diamantina trench). Through metagenomic binning process, we reconstructed 982 metagenome assembled genomes (MAGs) with completeness >60% and contamination 90%, while an additional 331 were >80%. Phylogenomic analysis for the MAGs revealed nearly all of them were distantly related to known cultivated isolates. The abundant bacterial MAGs affiliated to phyla of Proteobacteria, Planctomycetota, Nitrospirota, Acidobacteriota, Actinobacteriota, and Chlorofexota, while the abundant archaeal phyla affiliated with Nanoarchaeota and Thermoproteota. These results provide a dataset available for further interrogation of diversity, distribution and ecological function of deep-sea microbes existed in the trenches.

Abstract Lymph node metastasis (LNM) is a typical marker in oral squamous cell carcinoma (OSCC) indicating poor prognosis. Pathological examination by artificial image acquisition and analysis, as the main diagnostic method for LNM, often takes a week or longer which may cause great anxiety of the patient and also retard timely treatment. However, there are few efficient fast LNM diagnosis methods in clinical applications currently. Our previous study profiled the proteomics of extracellular vesicles (EVs) derived from postoperative drainage fluid (PDF) and showed the potential of detecting specific EVs that expressed aspartate β-hydroxylase (ASPH) for LNM diagnosis in OSCC patients. Considering that the analysis of ASPH+ PDF-EVs is challenging due to their low abundance (counting less than 10% of total EVs in PDF) and the complex EV isolation process of ultra-centrifugation, we developed a facile platform containing two microfluidic chips filled with antibody-modified microbeads to isolate ASPH+ PDF-EVs, with both the capture and retrieval rate reaching around 90%. Clinical sample analysis based on our method revealed that a mean of 6 × 106 /mL ASPH+ PDF-EVs could be isolated from LNM+ OSCC patients compared to 2.5 × 106 /mL in LNM- OSCC ones. When combined with enzyme-linked immunosorbent assay (ELISA) technique that was commonly used in clinical laboratories in hospitals, this microfluidic platform could precisely distinguish postoperative OSCC patients with LNM or not in several hours, which were validated by a double-blind test containing 6 OSCC patients. We believe this strategy has promise for early diagnosis of LNM in postoperative OSCC patients and finally helps guiding timely and reasonable treatment in clinic.

Abstract Background Family with sequence similarity 109, member B (FAM109B) is involved in endocytic transport and affects genetic variation in brain methylation. It is one of the important genes related to immune cell-associated diseases. In the tumor immune system, methylation can regulate tumor immunity and influence the maturation and functional response of immune cells. Whether FAM109B is involved in tumor progression and its correlation with the tumor immune microenvironment has not yet been disclosed. Methods A comprehensive pan-cancer analysis of FAM109B expression, prognosis, immunity, and TMB was conducted. The expression, clinical features, and prognostic value of FAM109B in low-grade gliomas (LGG) were evaluated using TCGA, CGGA, and Gravendeel databases. The expression of FAM109B was validated by qRT-PCR, immunohistochemistry (IHC), and Western blotting (WB). The relationship between FAM109B and methylation, Copy Number Variation (CNV), prognosis, immune checkpoints (ICs), and common chemotherapy drug sensitivity in LGG was explored through Cox regression, Kaplan–Meier curves, and Spearman correlation analysis. FAM109B levels and their distribution were studied using the TIMER database and single-cell analysis. The potential role of FAM109B in gliomas was further investigated through in vitro and in vivo experiments. Results FAM109B was significantly elevated in various tumor types and was associated with poor prognosis. Its expression was related to aggressive progression and poor prognosis in low-grade glioma patients, serving as an independent prognostic marker for LGG. Glioma grade was negatively correlated with FAM109B DNA promoter methylation. Immune infiltration and single-cell analysis showed significant expression of FAM109B in tumor-associated macrophages (TAMs). The expression of FAM109B was closely related to gene mutations, immune checkpoints (ICs), and chemotherapy drugs in LGG. In vitro studies showed increased FAM109B expression in LGG, closely related to cell proliferation. In vivo studies showed that mice in the sh-FAM109B group had slower tumor growth, slower weight loss, and longer survival times. Conclusions FAM109B, as a novel prognostic biomarker for low-grade gliomas, exhibits specific overexpression in TAMs and may be a potential therapeutic target for LGG patients.

Abstract Background Cystathionine accumulates selectively in 1p/19q-codeleted gliomas, and can serve as a possible noninvasive biomarker. This study aims to optimize the echo time (TE) of point-resolved spectroscopy (PRESS) for cystathionine detection in gliomas, and evaluate the diagnostic accuracy of PRESS for 1p/19q-codeletion identification. Methods The TE of PRESS was optimized with numerical and phantom analysis to better resolve cystathionine from the overlapping aspartate multiplets. The optimized and 97 ms TE PRESS were then applied to 84 prospectively enrolled patients suspected of glioma or glioma recurrence to examine the influence of aspartate on cystathionine quantification by fitting the spectra with and without aspartate. The diagnostic performance of PRESS for 1p/19q-codeleted gliomas were assessed. Results The TE of PRESS was optimized as (TE1, TE2) = (17 ms, 28 ms). The spectral pattern of cystathionine and aspartate were consistent between calculation and phantom. The mean concentrations of cystathionine in vivo fitting without aspartate were significantly higher than those fitting with full basis-set for 97 ms TE PRESS (1.97 ± 2.01 mM vs. 1.55 ± 1.95 mM, p

Abstract Background Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity. Results At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully. Conclusions We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.

Abstract The epigenetic regulation of N6-methyladenosine (m6A) has attracted considerable interest in tumor research, but the potential roles of m6A regulator-related genes, remain largely unknown within the context of gastric cancer (GC) and tumor microenvironment (TME). Here, a comprehensive strategy of data mining and computational biology utilizing multiple datasets based on 28 m6A regulators (including novel anti-readers) was employed to identify m6A regulator-related genes and patterns and elucidate their underlying mechanisms in GC. Subsequently, a scoring system was constructed to evaluate individual prognosis and immunotherapy response. Three distinct m6A regulator-related patterns were identified through the unsupervised clustering of 56 m6A regulator-related genes (all significantly associated with GC prognosis). TME characterization revealed that these patterns highly corresponded to immune-inflamed, immune-excluded, and immune-desert phenotypes, and their TME characteristics were highly consistent with different clinical outcomes and biological processes. Additionally, an m6A-related scoring system was developed to quantify the m6A modification pattern of individual samples. Low scores indicated high survival rates and high levels of immune activation, whereas high scores indicated stromal activation and tumor malignancy. Furthermore, the m6A-related scores were correlated with tumor mutation loads and various clinical traits, including molecular or histological subtypes and clinical stage or grade, and the score had predictive values across all digestive system tumors and even in all tumor types. Notably, a low score was linked to improved responses to anti-PD-1/L1 and anti-CTLA4 immunotherapy in three independent cohorts. This study has expanded the important role of m6A regulator-related genes in shaping TME diversity and clinical/biological traits of GC. The developed scoring system could help develop more effective immunotherapy strategies and personalized treatment guidance.