Genome-wide identification of GA2ox genes family and analysis of PbrGA2ox1-mediated enhanced chlorophyll accumulation by promoting chloroplast development in pear.

BACKGROUND: Chlorophyll (Chl) is an agronomic trait associated with photosynthesis and yield. Gibberellin 2-oxidases (GA2oxs) have previously been shown to be involved in Chl accumulation. However, whether and how the PbrGA2ox proteins (PbrGA2oxs) mediate Chl accumulation in pear (Pyrus spp.) is scarce. RESULTS: Here, we aimed to elucidate the role of the pear GA2ox gene family in Chl accumulation and the related underlying mechanisms. We isolated 13 PbrGA2ox genes (PbrGA2oxs) from the pear database and identified PbrGA2ox1 as a potential regulator of Chl accumulation. We found that transiently overexpressing PbrGA2ox1 in chlorotic pear leaves led to Chl accumulation, and PbrGA2ox1 silencing in normal pear leaves led to Chl degradation, as evident by the regreening and chlorosis phenomenon, respectively. Meanwhile, PbrGA2ox1-overexpressing (OE) tobacco plants discernably exhibited Chl built-up, as evidenced by significantly higher Pn and Fv/Fm. In addition, RNA sequencing (RNA-seq), physiological and biochemical investigations revealed an increase in abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) concentrations and signaling pathways; a marked elevation in reducing and soluble sugar contents; and a marginal decline in the starch and sucrose levels in OE plants. Interestingly, PbrGA2ox1 overexpression did not prominently affect Chl synthesis. However, it indeed facilitated chloroplast development by increasing chloroplast number per cell and compacting the thylakoid granum stacks. These findings might jointly contribute to Chl accumulation in OE plants. CONCLUSION: Overall, our results suggested that GA2oxs accelerate Chl accumulation by stimulating chloroplast development and proved the potential of PbrGA2ox1 as a candidate gene for genetically breeding biofortified pear plants with a higher yield.

Determination of anthracnose (Colletotrichum fructicola) resistance mechanism using transcriptome analysis of resistant and susceptible pear (Pyrus pyrifolia).

BACKGROUND: Anthracnose, mainly caused by Colletotrichum fructicola, leads to severe losses in pear production. However, there is limited information available regarding the molecular response to anthracnose in pears. RESULTS: In this study, the anthracnose-resistant variety 'Seli' and susceptible pear cultivar 'Cuiguan' were subjected to transcriptome analysis following C. fructicola inoculation at 6 and 24 h using RNA sequencing. A total of 3186 differentially expressed genes were detected in 'Seli' and 'Cuiguan' using Illumina sequencing technology. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the transcriptional response of pears to C. fructicola infection included responses to reactive oxygen species, phytohormone signaling, phenylpropanoid biosynthesis, and secondary metabolite biosynthetic processes. Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway and phenylpropanoid biosynthesis were involved in the defense of 'Seli'. Furthermore, the gene coexpression network data showed that genes related to plant-pathogen interactions were associated with C. fructicola resistance in 'Seli' at the early stage. CONCLUSION: Our results showed that the activation of specific genes in MAPK, calcium signaling pathways and phenylpropanoid biosynthesis was highly related to C. fructicola resistance in 'Seli' and providing several potential candidate genes for breeding anthracnose-resistant pear varieties.

Actin-bundling protein fimbrin serves as a new auxin biosynthesis orchestrator in Arabidopsis root tips.

Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well-established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored. Using LC-MS/MS-based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots. AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton-26S proteasome-YUC8-auxin' axis that controls auxin levels. Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome.

Synthesis, preclinical evaluation and pilot clinical translation of [68Ga]Ga-PMD22, a novel nanobody PET probe targeting CLDN18.2 of gastrointestinal cancer.

PURPOSE: Claudin18.2 (CLDN18.2) is a novel target for diagnosis and therapy of gastrointestinal cancer. This study aimed to evaluate the safety and feasibility of a novel CLDN18.2-targeted nanobody, PMD22, labeled with gallium-68 ([68Ga]Ga), for detecting CLDN18.2 expression in patients with gastrointestinal cancer using PET/CT imaging. METHODS: [68Ga]Ga-PMD22 was synthesized based on the nanobody, and its cell binding properties were assayed. Preclinical pharmacokinetics were determined in CLDN18.2-positive xenografts using microPET/CT. Effective dosimetry of [68Ga]Ga-PMD22 was evaluated in 5 gastrointestinal cancer patients, and PET/CT imaging of [68Ga]Ga-PMD22 and [18F]FDG were performed head-to-head in 16 gastrointestinal cancer patients. Pathological tissues were obtained for CLDN18.2 immunohistochemical (IHC) staining and comparative analysis with PET/CT findings. RESULTS: Cell binding assay showed that [68Ga]Ga-PMD22 had a higher binding ability to AGSCLDN18.2 and BGC823CLDN18.2 cells than to AGS and BGC823 cells (p < 0.001). MicroPET/CT images showed that [68Ga]Ga-PMD22 rapidly accumulated in AGSCLDN18.2 and BGC823CLDN18.2 tumors, and high contrast tumor to background imaging was clearly observed. In the pilot study, the effective dose of [68Ga]Ga-PMD22 was 1.68E-02 ± 1.45E-02 mSv/MBq, and the CLDN18.2 IHC staining result was highly correlated with the SUVmax/BKGstomach of [68Ga]Ga-PMD22 (rs = 0.848, p < 0.01). CONCLUSION: A novel [68Ga]Ga-labeled nanobody probe targeting CLDN18.2, [68Ga]Ga-PMD22, was established and preliminarily proved to be safe and effective in revealing CLDN18.2-positive gastrointestinal cancer, providing a basis for the clinical translation of the agent. CLINICAL TRIAL REGISTRATION: This study was registered on the ClinicalTrials.gov (NCT05937919).

Genomic and co-expression network analyses reveal candidate genes for oil accumulation based on an introgression population in Upland cotton (Gossypium hirsutum).

KEY MESSAGE: Integrated QTL mapping and WGCNA condense the potential gene regulatory network involved in oil accumulation. A glycosyl hydrolases gene (GhHSD1) for oil biosynthesis was confirmed in Arabidopsis, which will provide useful knowledge to understand the functional mechanism of oil biosynthesis in cotton. Cotton is an economical source of edible oil for the food industry. The genetic mechanism that regulates oil biosynthesis in cottonseeds is essential for the genetic enhancement of oil content (OC). To explore the functional genomics of OC, this study utilized an interspecific backcross inbred line population to dissect the quantitative trait locus (QTL) interlinked with OC. In total, nine OC QTLs were identified, four of which were novel, and each QTL explained 3.62-34.73% of the phenotypic variation of OC. The comprehensive transcript profiling of developing cottonseeds revealed 3,646 core genes differentially expressed in both inbred parents. Functional enrichment analysis determined 43 genes were annotated with oil biosynthesis processes. Implementation of weighted gene co-expression network analysis showed that 803 differential genes had a significant correlation with the OC phenotype. Further integrated analysis identified seven important genes located in OC QTLs. Of which, the GhHSD1 gene located in stable QTL qOC-Dt3-1 exhibited the highest functional linkages with the other network genes. Phylogenetic analysis showed significant evolutionary differences in the HSD1 sequences between oilseed- and starch- crops. Furthermore, the overexpression of GhHSD1 in Arabidopsis yielded almost 6.78% higher seed oil. This study not only uncovers important genetic loci for oil accumulation in cottonseed, but also provides a set of new candidate genes that potentially influence the oil biosynthesis pathway in cottonseed.

Synthesis and Preliminary Study of 99mTc-Labeled HYNIC-FAPi for Imaging of Fibroblast Activation Proteins in Tumors.

Fibroblast activation protein (FAP) is an emerging target for cancer diagnosis. Different types of FAP inhibitor (FAPI)-based radiotracers have been developed and applied for tumor imaging. However, few FAPI tracers for single photon emission computed tomography (SPECT) imaging have been reported. SPECT imaging is less expensive and more widely distributed than positron emission tomography (PET), and thus, 99mTc-labeled FAPIs would be more available to patients in developing regions. Herein, we developed a FAPI-04-derived radiotracer, HYNIC-FAPi-04 (HFAPi), for SPECT imaging. 99mTc-HFAPi, with a radiochemical purity of >98%, was prepared using a kit formula within 30 min. The specificity of 99mTc-HFAPi for FAP was validated by a cell binding assay in vitro and SPECT/CT imaging in vivo. The binding affinity (Kd value) of 99mTc-HFAPi for human FAP and murine FAP was 4.49 and 2.07 nmol/L, respectively. SPECT/CT imaging in HT1080-hFAP tumor-bearing mice showed the specific FAP targeting ability of 99mTc-HFAPi in vivo. In U87MG tumor-bearing mice, 99mTc-HFAPi had a higher tumor uptake compared with that of HT1080-hFAP and 4T1-mFAP tumor models. Interestingly, 99mTc-HFAPi showed a relatively high uptake in some murine joints. 99mTc-HFAPi accumulated in tumor lesions with a high tumor-to-background ratio. A preliminary clinical study was also performed in breast cancer patients. Additionally, 99mTc-HFAPi exhibited an advantage over 18F-FDG in the detection of lymph node metastatic lesions in breast cancer patients, which is helpful in improving treatment strategies. In short, 99mTc-HFAPi showed excellent affinity and specificity for FAP and is a promising SPECT radiotracer for (re)staging and treatment planning of breast cancers.

Noninvasive Evaluation of Tumoral PD-L1 Using a Novel 99mTc-Labeled Nanobody Tracer with Rapid Renal Clearance.

The expression level of PD-L1 in tumor tissue is considered one of the effective biomarkers to guide PD-1/PD-L1 therapy. Quantifying whole-body PD-L1 expression by SPECT imaging may help in selecting patients that potentially respond to PD-1/PD-L1 therapy. Nanobody is the smallest antibody fragment with antigen-binding ability that is well suited for radionuclide imaging. Nevertheless, high retention of radioactivity in the kidney may limit its clinical translation. The present study aimed to screen, design, and prepare a nanobody-based SPECT probe with rapid renal clearance to evaluate the PD-L1 expression level in vivo noninvasively. A phage library was constructed by immunizing alpaca with recombinant human PD-L1 protein, and 17 anti-PD-L1 nanobodies were screened by the phage display technique. After sequence alignment and flow cytometry analysis, APN09 was selected as the candidate nanobody, and a GGGC chelator was attached to its C-terminus for 99mTc labeling to prepare a SPECT imaging probe. The affinity and specificity of 99mTc-APN09 were evaluated by protein and cell-binding experiments, and SPECT imaging and biodistribution were performed in a mouse model with bilateral transplantation of A549 and A549PD-L1 tumors. The ability of 99mTc-APN09 to quantify the PD-L1 expression level in vivo was validated in tumor models with different PD-L1 expression levels. 99mTc-APN09 had a radiochemical purity higher than 99% and a binding equilibrium dissociation constant of 21.44 ± 1.65 nM with hPD-L1, showing high affinity. SPECT imaging results showed that 99mTc-APN09 could efficiently detect PD-L1-positive tumors within 0.5 h, and the quantitative results of SPECT were well correlated with the expression level of PD-L1 in cell lines. SPECT imaging and biodistribution results also showed that 99mTc-APN09 was rapidly cleared from the kidney in 2 h postinjection. 99mTc-APN09 was a simple and stable tool for visualizing PD-L1 expression in the whole body. In addition, due to its significant reduction in renal retention, it has better prospects for clinical translation.

Co-localization and analysis of miR477b with fiber length quantitative trait loci in cotton.

Cotton is an important cash crop for the textile industry. However, the understanding of natural genetic variation of fiber elongation in relation to miRNA is lacking. A miRNA gene (miR477b) was found to co-localize with a previously mapped fiber length (FL) quantitative trait locus (QTL). The miR477b was differentially expressed during fiber elongation between two backcross inbred lines (BILs) differing in FL and its precursor sequences. Bioinformatics and qRT-PCR analysis were further used to analyse the miRNA genes, which could produce mature miR477b. Cotton plants with virus-induced gene silencing (VIGS) constructs to over-express the allele of miR477b from the BIL with longer fibers had significantly longer fibers as compared with negative control plants, while the VIGS plants with suppressed miRNA expression had significantly shorter fibers. The expression level of the target gene (DELLA) and related genes (RDL1 and EXPA1 for DELLA through HOX3 protein) in the two BILs and/or the VIGS plants were generally congruent, as expected. This report represents one of the first comprehensive studies to integrate QTL linkage mapping and physical mapping of small RNAs with both small and mRNA transcriptome analysis, followed by VIGS, to identify candidate small RNA genes affecting the natural variation of fiber elongation in cotton.

Transcriptome Analysis and Identification of Genes Associated with Cotton Seed Size.

Cotton seeds, as the main by-product of cotton, are not only an important raw material for edible oil and feed but also a source of biofuel. The quality of cotton seeds directly affects cotton planting and is closely related to the yield and fiber quality. However, the molecular mechanism governing cotton seed size remains largely unexplored. This study investigates the regulatory mechanisms of cotton seed size by focusing on two cotton genotypes, N10 and N12, which exhibit notable phenotypic variations across multiple environments. Developing seeds were sampled at various stages (5, 20, 30, and 35 DPA) and subjected to RNA-seq. Temporal pattern clustering and WGCNA on differentially expressed genes identified 413 candidate genes, including these related to sugar metabolism that were significantly enriched in transcriptional regulation. A genetic transformation experiment indicated that the overexpression of the GhUXS5 gene encoding UDP-glucuronate decarboxylase 5 significantly increased seed size, suggesting an important role of GhUXS5 in regulating cotton seed size. This discovery provides crucial insights into the molecular mechanisms controlling cotton seed size, helping to unravel the complex regulatory network and offering new strategies and targets for cotton breeding to enhance the economic value of cotton seeds and overall cotton yield.

Genome-Wide Analysis of Aquaporins Gene Family in Populus euphratica and Its Expression Patterns in Response to Drought, Salt Stress, and Phytohormones.

Aquaporins (AQPs) play an essential role in membrane water transport during plant responses to water stresses centered on conventional upstream signals. Phytohormones (PHs) regulate plant growth and yield, working with transcription factors to help plants withstand environmental challenges and regulate physiological and chemical processes. The AQP gene family is important, so researchers have studied its function and regulatory system in numerous species. Yet, there is a critical gap the understanding of many of their molecular features, thus our full knowledge of AQPs is far-off. In this study, we undertook a broad examination of the AQP family gene in Populus euphratica via bioinformatics tools and analyzed the expression patterns of certain members in response to drought, salt, and hormone stress. A total of 22 AQP genes were examined in P. euphratica, and were categorized into four main groups, including TIPs, PIPs, SIPs, and NIPs based on phylogenetic analysis. Comparable exon-intron gene structures were found by gene structure examination, and similarities in motif number and pattern within the same subgroup was determined by motif analysis. The PeuAQP gene family has numerous duplications, and there is a distinct disparity in how the members of the PeuAQP family react to post-translational modifications. Abiotic stress and hormone responses may be mediated by AQPs, as indicated by the abundance of stress response elements found in 22 AQP genes, as revealed by the promoter's cis-elements prediction. Expression pattern analysis reveals that selected six AQP genes from the PIP subgroup were all expressed in the leaves, stem, and roots with varying expression levels. Moreover, qRT-PCR analysis discovered that the majority of the selected AQP members were up- or down-regulated in response to hormone treatment and abiotic stress. Remarkably, PeuAQP14 and PeuAQP15 appeared to be highly responsive to drought stress and PeuAQP15 exhibited a high response to salt stress. The foliar application of the phytohormones (SA, IAA, GA3, MeJA, and ABA) were found to either activate or inhibit PeuAQP, suggesting that they may mitigate the effects of water shortage of poplar water stress. The present work enhances our knowledge of the practical roles of AQPs in stress reactions and offers fundamental information for the AQP genes in poplar species. It also highlights a direction for producing new varieties of poplar species with drought, salt, and hormone tolerance and holds substantial scientific and ecological importance, offering a potential contribution to the conservation of poplar species in arid regions.

The dominant influencing factors of desertification and ecological risk changes in Qinghai Area of Qilian Mountains National Park: Climate change or human activity?

Transitional features of desert environments partially determine the risks associated with ecosystems. Influenced by climate change and human activities, the variability and uncertainty of desertification levels and ecological risks in the Qinghai Area of Qilian Mountain National Park (QMNPQA) has become increasingly prominent. As a critical ecological barrier in northwest China, monitoring desertification dynamics and ecological risks is crucial for maintaining ecosystem stability. This study identifies the optimal monitoring model from four constructed desertification monitoring models and analyzes spatiotemporal changes in desertification. The spatial and temporal changes in ecological risks and their primary driving factors were analyzed using methods such as raster overlay calculation, geographic detector, cloud model, and trend analysis. The main conclusions are as follows: The desertification feature spatial model based on GNDVI-Albedo demonstrates better applicability in the study area, with an inversion accuracy of 81.24%. The levels of desertification and ecological risks in QMNPQA exhibit significant spatial heterogeneity, with a gradual decrease observed from northwest to southeast. From 2000 to 2020, there is an overall decreasing trend in desertification levels and ecological risks, with the decreasing trend area accounting for 89.82% and 85.71% respectively, mainly concentrated in the southeastern and northwestern parts of the study area. The proportion of areas with increasing trends is 4.49% and 7.05% respectively, scattered in patches in the central and southern edge areas. Surface temperature (ST), Digital Elevation Map (DEM), and Green normalized difference vegetation index (GNDVI) are the most influential factors determining the spatial distribution of ecological risks in QMNPQA. The effects of management and climatic factors on ecological risks demonstrate a significant antagonistic effect, highlighting the positive contributions of human activities in mitigating the driving effects of climate change on ecological risks. The research results can provide reference for desertification prevention and ecological quality improvement in QMNPQA.

Spartina alterniflora invasion altered phosphorus retention and microbial phosphate solubilization of the Minjiang estuary wetland in southeastern China.

Spartina alterniflora invasion is considered a critical event affecting sediment phosphorus (P) availability and stock. However, P retention and microbial phosphate solubilization in the sediments invaded with or without S. alterniflora have not been fully investigated. In this study, a sequential fractionation method and high-throughput sequencing were used to analyze P transformation and the underlying microbial mechanisms in the sediments of no plant (NP) zone, transition (T) zone, and plant (P) zone. Results showed that except for organic phosphate (OP), total phosphate (TP), inorganic phosphate (IP), and available phosphate (AP) all followed a significant decrease trend from the NP site to the T site, and to the P site. The vertical decrease of TP, IP, and AP was also observed with an increase in soil depth. Among the six IP fractions, Fe-P, Oc-P, and Ca10-P were the predominant forms, while the presence of S. alterniflora resulted in an obvious P depletion except for Ca8-P and Al-P. Although S. alterniflora invasion did not significantly alter the alpha diversity of phosphate-solubilizing bacteria (PSB) harboring phoD gene, several PSB belonging to p_Proteobacteria, p_Planctomycetes, and p_Cyanobacteriota showed close correlations with P speciation and IP fractions. Further correlation analysis revealed that the reduced soil pH, soil TN and soil EC, and the increased soil TOC mediated by the invasion of S. alterniflora also significantly correlated to these PSB. Overall, this study elucidates the linkage between PSB and P speciation and provides new insights into understanding P retention and microbial P transformation in the coastal sediment invaded by S. alterniflora.

Solvent-dependent chemoselective synthesis of different isoquinolinones mediated by the hypervalent iodine(III) reagent PISA.

Isoquinolinone is an important heterocyclic framework in natural products and biologically active molecules, and the efficient synthesis of this structural motif has received much attention in recent years. Herein, we report a (phenyliodonio)sulfamate (PISA)-mediated, solvent-dependent synthesis of different isoquinolinone derivatives. The method provides highly chemoselective access to 3- or 4-substituted isoquinolinone derivatives by reacting o-alkenylbenzamide derivatives with PISA in either acetonitrile or wet hexafluoro-2-isopropanol.

High in-vivo stability in preclinical and first-in-human experiments with [18F]AlF-RESCA-MIRC213: a 18F-labeled nanobody as PET radiotracer for diagnosis of HER2-positive cancers.

PURPOSE: [18F]AlF-RESCA was introduced as a core particularly useful for 18F-labeling of heat-sensitive biomolecules. However, no translational studies have been reported up to now. Herein, we reported the first-in-human evaluation of an 18F-labeled anti-HER2 nanobody MIRC213 as a PET radiotracer for imaging HER2-positive cancers. METHODS: MIRC213 was produced by E. coli and conjugated with ( ±)-H3RESCA-Mal. [18F]AlF-RESCA-MIRC213 was prepared at room temperature. Its radiochemical purity and stability of were determined by radio-HPLC with the size-exclusion chromatographic column. Cell uptake was performed in NCI-N87 (HER2 +) and MCF-7 (HER2-) cells and the cell-binding affinity was verified in SK-OV-3 (HER2 +) cells. Small-animal PET/CT was performed using SK-OV-3, NCI-N87, and MCF-7 tumor-bearing mice at 30 min, 1 h, and 2 h post-injection. For blocking experiment, excess MIRC213 was co-injected with radiotracer. Biodistribution were performed on SKOV-3 and MCF-7 tumor-bearing mice at 2 h post-injection. For clinical study, PET/CT images were acquired at 2 h and 4 h after injection of [18F]AlF-RESCA-MIRC213 (1.85-3.7 MBq/kg) in six breast cancer patients (3 HER2-positive and 3 HER2-negative). All patients underwent [18F]-FDG PET/CT within a week for tissue selection purpose. Distribution and dosimetry were calculated. Standardized uptake values (SUV) were measured in tumors and normal organs. RESULTS: MIRC213 was produced with > 95% purity and modified with RESCA to obtain RESCA-MIRC213. [18F]AlF-RESCA-MIRC213 was prepared within 20 min at room temperature with the radiochemical yield of 50.48 ± 7.6% and radiochemical purity of > 98% (n > 10), and remained stable in both PBS (88%) and 5% HSA (92%) after 6 h. The 2 h cellular uptake of [18F]AlF-RESCA-MIRC213 in NCI-N87 cells was 11.22 ± 0.60 AD%/105 cells. Its binding affinity Kd value was determined to be 1.23 ± 0.58 nM. Small-animal PET/CT with [18F]AlF-RESCA-MIRC213 can clearly differentiate SK-OV-3 and NCI-N87 tumors from MCF-7 tumors and background with a high uptake of 4.73 ± 1.18 ID%/g and substantially reduced to 1.70 ± 0.13 ID%/g for the blocking group (p < 0.05) in SK-OV-3 tumors at 2 h post-injection. No significant bone radioactivity was seen in the tumor-bearing animals. In all six breast cancer patients, there was no adverse reaction during study. The uptake of [18F]AlF-RESCA-MIRC213 was mainly in lacrimal gland, parotid gland, submandibular gland, thyroid gland, gallbladder, kidneys, liver, and intestines. There was no significant bone radioactivity accumulation in cancer patients. [18F]AlF-RESCA-MIRC213 had significantly higher tumor uptake in lesions from HER2-positive patients than that lesions from HER2-negative patients (SUVmax of 3.62 ± 1.56 vs. 1.41 ± 0.41, p = 0.0012) at 2 h post-injection. The kidneys received the highest radiation dose of 2.42 × 10-1 mGy/MBq, and the effective dose was 1.56 × 10-2 mSv/MBq. CONCLUSIONS: [18F]AlF-RESCA-MIRC213 could be prepared with high radiolabeling yield under mild conditions. [18F]AlF-RESCA-MIRC213 has relatively high stability both in vitro and in vivo. The results from clinical transformation suggest that [18F]AlF-RESCA-MIRC213 PET/CT is a safe procedure with favorable pharmacokinetics and dosimetry profile, and it is a promising new PET radiotracer for noninvasive diagnosis of HER2-positive cancers.

Immuno-PET of colorectal cancer with a CEA-targeted [68 Ga]Ga-nanobody: from bench to bedside.

PURPOSE: An accurate diagnosis of colorectal carcinoma (CRC) can assist physicians in developing reasonable therapeutic regimens, thereby significantly improving the patient's prognosis. Carcinoembryonic antigen (CEA)-targeted PET imaging shows great potential for this purpose. Despite showing remarkable abilities to detect primary and metastatic CRC, previously reported CEA-specific antibody radiotracers or pretargeted imaging are not suitable for clinical use due to poor pharmacokinetics and complicated imaging procedures. In contrast, radiolabeled nanobodies exhibit ideal characteristics for PET imaging, for instance, rapid clearance rates and excellent distribution profiles, allowing same-day imaging with sufficient contrast. In this study, we developed a novel CEA-targeted nanobody radiotracer, [68 Ga]Ga-HNI01, and assessed its tumor imaging ability and biodistribution profile in preclinical xenografts and patients with primary and metastatic CRC. METHODS: The novel nanobody HNI01 was acquired by immunizing the llama with CEA proteins. [68 Ga]Ga-HNI01 was synthesized by site-specifically conjugating [68 Ga]Ga with tris(hydroxypyridinone) (THP). Small-animal PET imaging and biodistribution studies were performed in CEA-overexpressed LS174T and CEA-low-expressed HT-29 tumor models. Following successful preclinical assessment, a phase I study was conducted on 9 patients with primary and metastatic CRC. Study participants received 151.21 ± 25.25 MBq of intravenous [68 Ga]Ga-HNI01 and underwent PET/CT scans at 1 h and 2 h post injection. Patients 01-03 also underwent whole-body dynamic PET imaging within 0-40 min p.i. All patients underwent [18F]F-FDG PET/CT imaging within 1 week after [68 Ga]Ga-HNI01 imaging. Tracer distribution, pharmacokinetics, and radiation dosimetry were calculated. RESULTS: [68 Ga]Ga-HNI01 was successfully synthesized within 10 min under mild conditions, and the radiochemical purity was more than 98% without purification. Micro-PET imaging with [68 Ga]Ga-HNI01 revealed clear visualization of LS174T tumors, while signals from HT-29 tumors were significantly lower. Biodistribution studies indicated that uptake of [68 Ga]Ga-HNI01 in LS174T and HT-29 was 8.83 ± 3.02%ID/g and 1.81 ± 0.87%ID/g, respectively, at 2 h p.i. No adverse events occurred in all clinical participants after the injection of [68 Ga]Ga-HNI01. A fast blood clearance and low background uptake were observed, and CRC lesions could be visualized with high contrast as early as 30 min after injection. [68 Ga]Ga-HNI01 PET could clearly detect metastatic lesions in the liver, lung, and pancreas and showed superior ability in detecting small metastases. A significant accumulation of radioactivity was observed in the kidney, and normal tissues physiologically expressing CEA receptors showed slight uptakes of [68 Ga]Ga-HNI01. An interesting finding was that strong uptake of [68 Ga]Ga-HNI01 was found in non-malignant colorectal tissues adjacent to the primary tumor in some patients, suggesting abnormal CEA expression in these healthy tissues. CONCLUSION: [68 Ga]Ga-HNI01 is a novel CEA-targeted PET imaging radiotracer with excellent pharmacokinetics and favorable dosimetry profiles. [68 Ga]Ga-HNI01 PET is an effective and convenient imaging tool for detecting CRC lesions, particularly for identifying small metastases. Furthermore, its high specificity for CEA in vivo makes it an ideal tool for selecting patients for anti-CEA therapy.

The role of [99mTc]Tc-HFAPi SPECT/CT in patients with malignancies of digestive system: first clinical experience.

BACKGROUND: Recently, PET/CT imaging with radiolabelled FAP inhibitors (FAPIs) has been widely evaluated in diverse diseases. However, rare report has been published using SPECT/CT, a more available imaging method, with [99mTc]Tc-labelled FAPI. In this study, we evaluated the potential effect of [99mTc]Tc-HFAPi in clinical analysis for digestive system tumours. METHODS: This is a single-centre prospective diagnostic efficiency study (Ethic approved No.: XJTU1AF2021LSK-021 of the First Affiliated Hospital of Xi'an Jiaotong University and ChiCTR2100048093 of the Chinese Clinical Trial Register). Forty patients with suspected or confirmed digestive system tumours underwent [99mTc]Tc-HFAPi SPECT/CT between January and June 2021. For dynamic biodistribution and dosimetry estimation, whole-body planar scintigraphy was performed at 10, 30, 90, 150, and 240 min post-injection in four representative patients. Optimal acquisition time was considered in all the patients at 60-90 min post-injection, then quantified or semi-quantified using SUVmax and T/B ratio was done. The diagnostic performance of [99mTc]Tc-HFAPi was calculated and compared with those of contrast-enhanced CT (ceCT) using McNemar test, and the changes of tumour stage and oncologic management were recorded. RESULTS: Physiological distribution of [99mTc]Tc-HFAPi was observed in the liver, pancreas, gallbladder, and to a lesser extent in the kidneys, spleen and thyroid. Totally, 40 patients with 115 lesions were analysed. The diagnostic sensitivity of [99mTc]Tc-HFAPi for non-operative primary lesions was similar to that of ceCT (94.29% [33/35] vs 100% [35/35], respectively; P = 0.5); in local relapse detection, [99mTc]Tc-HFAPi was successfully detected in 100% (n = 3) of patients. In the diagnosis of suspected metastatic lesions, [99mTc]Tc-HFAPi exhibited higher sensitivity (89.66% [26/29] vs 68.97% [20/29], respectively, P = 0.03) and specificity (97.9% [47/48] vs 85.4% [41/48], respectively, P = 0.03) than ceCT, especially with 100% (24/24) specificity in the diagnosis of liver metastases, resulting in 20.0% (8/40) changes in TNM stage and 15.0% (6/40) changes in oncologic management. CONCLUSION: [99mTc]Tc-HFAPi demonstrates a greater diagnostic efficiency than ceCT in the detection of distant metastasis, especially in identifying liver metastases.

Preclinical evaluation and pilot clinical study of [68Ga]Ga-THP-APN09, a novel PD-L1 targeted nanobody radiotracer for rapid one-step radiolabeling and PET imaging.

PURPOSE: Programmed cell death protein-1/ligand-1 (PD-1/L1) blockade has been a breakthrough in the treatment of patients with non-small cell lung cancer (NSCLC), but there is still a lack of effective methods to screen patients. Here we report a novel 68 Ga-labeled nanobody [68 Ga]Ga-THP-APN09 for PET imaging of PD-L1 status in mouse models and a first-in-human study in NSCLC patients. METHODS: [68 Ga]Ga-THP-APN09 was prepared by site-specific radiolabeling, with no further purification. Cell uptake assays were completed in the human lung adenocarcinoma cell line A549, NSCLC cell line H1975 and human PD-L1 gene-transfected A549 cells (A549PD-L1). The imaging to image PD-L1 status and biodistribution were investigated in tumor-bearing mice of these three tumor cell types. The first-in-human clinical translational trial was registered as NCT05156515. The safety, radiation dosimetry, biodistribution, and correlations of tracer uptake with immunohistochemical staining and major pathologic response (MPR) were evaluated in NSCLC patients who underwent adjuvant immunotherapy combined with chemotherapy. RESULTS: Radiosynthesis of [68 Ga]Ga-THP-APN09 was achieved at room temperature and a pH of 6.0-6.5 in 10 min with a high radiochemical yield (> 99%) and 13.9-27.8 GBq/µmol molar activity. The results of the cell uptake study reflected variable levels of surface PD-L1 expression observed by flow cytometry in the order A549PD-L1 > H1975 > A549. In small-animal PET/CT imaging, H1975 and A549PD-L1 tumors were clearly visualized in an 8.3:1 and 2.2:1 ratios over PD-L1-negative A549 tumors. Ex vivo biodistribution studies showed that tumor uptake was consistent with the PET results, with the highest A549PD-L1 being taken up the most (8.20 ± 0.87%ID/g), followed by H1975 (3.69 ± 0.50%ID/g) and A549 (0.90 ± 0.16%ID/g). Nine resectable NSCLC patients were enrolled in the clinical study. Uptake of [68 Ga]Ga-THP-APN09 was mainly observed in the kidneys and spleen, followed by low uptake in bone marrow. The radiation dose is within a reliable range. Tumor uptake was positively correlated with PD-L1 expression TPS (rs = 0.8763, P = 0.019). Tumor uptake of [68 Ga]Ga-THP-APN09 (SUVmax) in MPR patients was higher than that in non-MPR patients (median SUVmax 2.73 vs. 2.10, P = 0.036, determined with Mann-Whitney U-test). CONCLUSION: [68 Ga]Ga-THP-APN09 has the potential to be transformed into a kit-based radiotracer for rapid, simple, one-step, room temperature radiolabeling. The tracer can detect PD-L1 expression levels in tumors, and it may make it possibility to predict the response of PD-1 immunotherapy combined with chemotherapy. Confirmation in a large number of cases is needed. TRIAL REGISTRATION: Clinical Trial (NCT05156515). Registered 12 December 2021.

Periconception Red Blood Cell Folate and Offspring Congenital Heart Disease : Nested Case-Control and Mendelian Randomization Studies.

BACKGROUND: Periconception folic acid supplementation has been suggested to protect against congenital heart disease (CHD), but the association between maternal red blood cell (RBC) folate, the gold-standard biomarker of folate exposure, and subsequent offspring CHD risk is lacking. OBJECTIVE: To quantify the association between periconception maternal RBC folate and offspring CHD risk. DESIGN: Prospective, nested, case-control study and 1-sample Mendelian randomization. (ClinicalTrials.gov: NCT02737644). SETTING: 29 maternity institutions in 12 districts of Greater Shanghai, China. PARTICIPANTS: All 197 mothers of offspring with CHD and 788 individually matched mothers of unaffected offspring from the SPCC (Shanghai Preconception Cohort). MEASUREMENTS: Maternal RBC folate was measured before or at early pregnancy. Odds ratios [ORs] were estimated using conditional logistic regression after adjustment for covariates. Mendelian randomization was done using the methylenetetrahydrofolate reductase (MTHFR) C677T as the genetic instrument. RESULTS: Case patients had lower median maternal RBC folate concentrations than control participants (714 nmol/L [interquartile range, 482 to 1008 nmol/L] vs. 788 nmol/L [557 to 1094 nmol/L]). Maternal RBC folate concentrations were inversely associated with offspring CHD (adjusted OR per 100 nmol/L, 0.93 [95% CI, 0.89 to 0.99]). The adjusted OR for mothers with periconception RBC folate of 906 nmol/L or more (vs. <906 nmol/L) was 0.61 (CI, 0.40 to 0.93). Mendelian randomization showed that each 100-nmol increase in maternal RBC folate concentrations was significantly associated with reduced offspring CHD risk (OR, 0.75 [CI, 0.61 to 0.92]). LIMITATION: Potential confounding due to unmeasured covariates in the nested case-control study. CONCLUSION: Higher maternal RBC folate is associated with reduced offspring CHD risk. For primary CHD prevention, higher target RBC folate levels than currently recommended for neural tube defect prevention may be needed and warrant further study. PRIMARY FUNDING SOURCE: National Key Research and Development Program of China, National Natural Science Foundation of China, China Postdoctoral Science Foundation, and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.

High-Quality Genome Resource of Gilbertella persicaria Causing Peach Soft Rot.

Peach soft rot caused by Gilbertella persicaria is an economically important disease. Here, we report a high-quality complete and annotated genome sequence of G. persicaria strain TFLB-J, isolated from peach fruit in Yuanyang county of Henan Province, China. The assembly consists of 91 scaffolds with an estimated genome size of 33.59 Mb and N50 length of 0.92 Mb, encoding 13,296 predicted protein-coding genes. The whole-genome sequence could provide gene resources for further study of pathogenic effectors and comparative genomics of peach soft rot pathogens.

First Report of Nothophoma quercina Causing Brown Spot Disease on 'Huanghua' Pear (Pyrus pyrifolia Nakai) in China.

Pear (Pyrus L.) is an important fruit tree in China, which has the largest cultivation area and yield in the world (Jia et al. 2021). In June 2022, brown spot symptoms were observed on 'Huanghua' pear (Pyrus pyrifolia Nakai, cv. Huanghua) leaves in the germplasm garden of Anhui Agricultural University (High Tech Agricultural Garden), Anhui, Hefei, China. The disease incidence was approximately 40% according to the percentage of diseased leaves among 300 leaves (50 leaves each were obtained from 6 plants). Initially, small, brown, round to oval lesions appeared on the leaves, the spots were gray in the central, and surrounded by brown to black margins. These spots rapidly enlarged, eventually causing abnormal leaf defoliation. To isolate the brown spot pathogen, symptomatic leaves were harvested, washed with sterile water, surface-sterilized with 75% ethanol for 20 s, and washed 3-4 times with sterile water. Leaf fragments were placed onto PDA medium and incubated at 25°C for 7 days to obtain isolates. The colonies exhibited white to pale gray aerial mycelium and reached a diameter of 62 mm after 7 days of incubation. Conidiogenous cells were characterized as phialides, and exhibited a doliform to ampulliform shape. Conidia displayed various shapes and sizes, ranging from subglobose to oval or obtuse, with thin walls, aseptate hyphae, and a smooth surface. They measured 4.2-7.9 × 3.1-5.5 µm in diameter. These morphologies were similar to Nothophoma quercina as reported previously (Bai et al. 2016; Kazerooni et al. 2021). For molecular analysis, the internal transcribed spacers (ITS), beta-tubulin (TUB2), and actin (ACT) regions were amplified using the primers ITS1/ITS4, Bt2a/Bt2b, and ACT-512F/ACT-783R respectively. The sequences of ITS, TUB2, and ACT were deposited in GenBank (accession numbers: OP554217, OP595395, and OP595396, respectively). A nucleotide blast search revealed high homology with N. quercina sequences: MH635156 (ITS: 541/541, 100%), MW672036.1 (TUB2: 343/346, 99%), FJ426914.1 (ACT: 242/262, 92%). A phylogenetic tree was constructed with ITS, TUB2 and ACT sequences based on neighbor-joining method using MEGA-X software, which showed the highest similarity with N. quercina. To confirm the pathogenicity, the leaves of three healthy plants were sprayed with spore suspension (106 conidia/mL), whereas control leaves were prayed with sterile water. The inoculated plants were covered with plastic bags and cultured in a growth chamber (90% relative humidity) at 25°C. Typical disease symptoms appeared on the inoculated leaves after 7-10 days, whereas no symptoms were observed on the control leaves. The same pathogen was re-isolated from the diseased leaves, according with Koch's postulates. Therefore, based on morphological and phylogenetic tree analyses, we confirmed that the causal organism for brown spot disease was N. quercina fungus (Chen et al. 2015; Jiao et al. 2017). To our knowledge, this is the first report of brown spot disease caused by N. quercina on 'Huanghua' pear leaves in China.