Pan-cancer analysis of CDKN2A alterations identifies a subset of gastric cancer with a cold tumor immune microenvironment.

BACKGROUND: Although CDKN2A alteration has been explored as a favorable factor for tumorigenesis in pan-cancers, the association between CDKN2A point mutation (MUT) and intragenic deletion (DEL) and response to immune checkpoint inhibitors (ICIs) is still disputed. This study aims to determine the associations of CDKN2A MUT and DEL with overall survival (OS) and response to immune checkpoint inhibitors treatment (ICIs) among pan-cancers and the clinical features of CDKN2A-altered gastric cancer. METHODS: This study included 45,000 tumor patients that underwent tumor sequencing across 33 cancer types from four cohorts, the MSK-MetTropism, MSK-IMPACT, OrigiMed2020 and TCGA cohorts. Clinical outcomes and genomic factors associated with response to ICIs, including tumor mutational burden, copy number alteration, neoantigen load, microsatellite instability, tumor immune microenvironment and immune-related gene signatures, were collected in pan-cancer. Clinicopathologic features and outcomes were assessed in gastric cancer. Patients were grouped based on the presence of CDKN2A wild type (WT), CDKN2A MUT, CDKN2A DEL and CDKN2A other alteration (ALT). RESULTS: Our research showed that CDKN2A-MUT patients had shorter survival times than CDKN2A-WT patients in the MSK MetTropism and TCGA cohorts, but longer OS in the MSK-IMPACT cohort with ICIs treatment, particularly in patients having metastatic disease. Similar results were observed among pan-cancer patients with CDKN2A DEL and other ALT. Notably, CDKN2A ALT frequency was positively related to tumor-specific objective response rates to ICIs in MSK MetTropism and OrigiMed 2020. Additionally, individuals with esophageal carcinoma or stomach adenocarcinoma who had CDKN2A MUT had poorer OS than patients from the MSK-IMPACT group, but not those with adenocarcinoma. We also found reduced levels of activated NK cells, T cells CD8 and M2 macrophages in tumor tissue from CDKN2A-MUT or DEL pan-cancer patients compared to CDKN2A-WT patients in TCGA cohort. Gastric cancer scRNA-seq data also showed that CDKN2A-ALT cancer contained less CD8 T cells but more exhausted T cells than CDKN2A-WT cancer. A crucial finding of the pathway analysis was the inhibition of three immune-related pathways in the CDKN2A ALT gastric cancer patients, including the interferon alpha response, inflammatory response, and interferon gamma response. CONCLUSIONS: This study illustrates the CDKN2A MUT and DEL were associated with a poor outcome across cancers. CDKN2A ALT, on the other hand, have the potential to be used as a biomarker for choosing patients for ICI treatment, notably in esophageal carcinoma and stomach adenocarcinoma.

Self-Competitive Growth of CsPbBr3 Planar Nanowire Array.

Semiconductor planar nanowire arrays (PNAs) are essential for achieving large-scale device integration. Direct heteroepitaxy of PNAs on a flat substrate is constrained by the mismatch in crystalline symmetry and lattice parameters between the substrate and epitaxial nanowires. This study presents a novel approach termed "self-competitive growth" for heteroepitaxy of CsPbBr3 PNAs on mica. The key to inducing the self-competitive growth of CsPbBr3 PNAs on mica involves restricting the nucleation of CsPbBr3 nanowires in a high-adsorption region, which is accomplished by overlaying graphite sheets on the mica surface. Theoretical calculations and experimental results demonstrate that CsPbBr3 nanowires oriented perpendicular to the boundary of the high-adsorption area exhibit greater competitiveness in intercepting the growth of nanowires in the other two directions, resulting in PNAs with a consistent orientation. Moreover, these PNAs exhibit low-threshold and stable amplified spontaneous emission under one-, two-, and three-photon excitation, indicating their potential for an integrated laser array.

Dual-Catalyzed Stereodivergent Electrooxidative Homocoupling of Benzoxazolyl Acetate.

The development of a reliable strategy for stereodivergent radical reactions that allows convenient access to all stereoisomers of homocoupling adducts with multiple stereogenic centers remains an unmet goal in organic synthesis. Herein, we describe a dual-catalyzed electrooxidative C(sp3)-H/C(sp3)-H homocoupling with complete absolute and relative stereocontrol for the synthesis of molecules with contiguous quaternary stereocenters in a general and predictable manner. The stereodivergent electrooxidative homocoupling reaction is achieved by synergistically utilizing two distinct chiral catalysts that convert identical racemic substrates into inherently distinctive reactive chiral intermediates, dictate enantioselective radical addition, and allow access to the full complement of stereoisomeric products via simple catalyst permutation. The successful execution of the dual-electrocatalytic strategy programmed via electrooxidative activation provides a significant conceptual advantage and will serve as a useful foundation for further research into cooperative stereocontrolled radical transformations and diversity-oriented synthesis.

An NADH/NAD+-favored aldo-keto reductase facilitates avilamycin A biosynthesis by primarily catalyzing oxidation of avilamycin C.

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes. Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD+ or NADPH/NADP+. These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD+-favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD+ in vivo. Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes, which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. More importantly, AviZ1 exhibits a unique NADH/NAD+ preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD+ in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.

DkDTX1/MATE1 mediates the accumulation of proanthocyanidin and affects astringency in persimmon.

Proanthocyanidins (PAs) is a kind of polyphenols widely distributed in plants, and their astringent properties can protect plants from herbivores and regulate fruit taste. There is a great difference in PA composition between astringent (A)-type and nonastringent (NA)-type persimmon. Here, we studied the potential of DkDTX1/MATE1 in regulating PAs composition through its preferred transport in persimmon fruit. The results of fluorescence microscope showed that the DkDTX1/MATE1 green fluorescence overlapped with the blue light emitted by PA. Overexpression of DkDTX1/MATE1 in persimmon leaves not only significantly increase the concentrations of PA, but also upregulated the expression of PA biosynthesis pathway genes. Further overexpression of DkDTX1/MATE1 in persimmon fruit discs and stable genetic transformation of DkDTX1/MATE1 also led to PA concentrations increased. Molecular docking and transporter assays showed that DkDTX1/MATE1 preferentially transported catechin, epicatechin gallate and epigallocatechin gallate. DkDTX1/MATE1 mainly bound to the PA precursors via serine at position 68. Our findings indicate that DkDTX1/MATE1 play a role in the accumulation of PAs in early stage of fruit development and affects the astringency of persimmon through preferential transport PA precursors, which provided a theoretical basis for the future use of metabolic engineering to regulate the composition of PAs in persimmon.

Development of small-molecular-based radiotracers for PET imaging of PD-L1 expression and guiding the PD-L1 therapeutics.

PURPOSE: Programmed cell death protein ligand 1 (PD-L1) is a crucial biomarker for immunotherapy. However, nearly 70% of patients do not respond to PD-L1 immune checkpoint therapy. Accurate monitoring of PD-L1 expression and quantification of target binding during treatment are essential. In this study, a series of small-molecule radiotracers were developed to assess PD-L1 expression and direct immunotherapy. METHODS: Radiotracers of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were designed based on a 2-methyl-3-biphenyl methanol scaffold and successfully synthesized. Cellular experiments and molecular docking assays were performed to determine their specificity for PD-L1. PD-L1 status was investigated via positron emission tomography (PET) imaging in MC38 tumor models. PET imaging of [68Ga]Ga-D-pep-PMED was performed to noninvasively quantify PD-L1 blocking using an anti-mouse PD-L1 antibody (PD-L1 mAb). RESULTS: The radiosyntheses of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were achieved with radiochemical yields of 87 ± 6%, 82 ± 4%, and 79 ± 9%, respectively. In vitro competition assays demonstrated their high affinities (the IC50 values of [68Ga]Ga-D-PMED, [68Ga]Ga-D-PEG-PMED, and [68Ga]Ga-D-pep-PMED were 90.66 ± 1.24, 160.8 ± 1.35, and 51.6 ± 1.32 nM, respectively). At 120 min postinjection (p.i.) of the radiotracers, MC38 tumors displayed optimized tumor-to-muscle ratios for all radioligands. Owing to its hydrophilic modification, [68Ga]Ga-D-pep-PMED had the highest target-to-nontarget (T/NT) ratio of approximately 6.2 ± 1.2. Interestingly, the tumor/liver ratio was hardly affected by different concentrations of the inhibitor BMS202. We then evaluated the impacts of dose and time on accessible PD-L1 levels in the tumor during anti-mouse PD-L1 antibody treatment. The tumor uptake of [68Ga]Ga-D-pep-PMED significantly decreased with increasing PD-L1 mAb dose. Moreover, after 8 days of treatment with a single antibody, the uptake of [68Ga]Ga-D-pep-PMED in the tumor significantly increased but remained lower than that in the saline group. CONCLUSION: PET imaging with [68Ga]Ga-D-pep-PMED, a small-molecule radiotracer, is a promising tool for evaluating PD-L1 expression and quantifying the target blockade of PD-L1 to assist in the development of effective therapeutic regimens.

Development and Evaluation of [68Ga]Ga-Labeled Riboflavin Derivative for RFVT3-Targeted PET Imaging of Melanoma in Mice.

The limited progress in treatment options and the alarming survival rates in advanced melanoma emphasize the significant research importance of early melanoma diagnosis. RFVT3, a crucial protein at the core of energy metabolism reprogramming in melanoma, might play a pivotal role in early detection. In this study, [68Ga]Ga-NOTA-RF, based on riboflavin (RF), was rationally developed and validated, serving as an innovative tool for positron emission tomography (PET) imaging of RFVT3 expression in melanoma. The in vitro assays of RFVT3 specificity of [68Ga]Ga-NOTA-RF were performed on B16F10 melanoma cells. Then, PET imaging of melanoma was investigated in B16F10 allograft mouse models with varying volumes. Biodistribution studies are used to clarify the behavior of [68Ga]Ga-NOTA-RF in vivo. [68Ga]Ga-NOTA-RF was obtained with high radiochemical purity (>95%). A significant uptake (37.79 ± 6.86%, n = 4) of [68Ga]Ga-NOTA-RF was observed over time in B16F10 melanoma cells, which was significantly inhibited by RFVT3 inhibitors RF or methylene blue (MB), demonstrating the specific binding of [68Ga]Ga-NOTA-RF. At 60 min postinjection, the tumor-to-muscle (T/M) ratio of [68Ga]Ga-NOTA-RF was 4.03 ± 0.34, higher than that of the RF-blocked group (2.63 ± 0.19) and MB-blocked group (2.14 ± 0.20). The T/M ratios for three distinct tumor volumes-small (5 mm), medium (10 mm), and large (15 mm) were observed to be 5.25 ± 0.28, 4.03 ± 0.34, and 3.19 ± 0.55, respectively. The expression of RFVT3 was validated by immunohistochemical staining in various tumor models, with small B16F10 tumors exhibiting the highest expression. [68Ga]Ga-NOTA-RF demonstrates promising properties for the early diagnosis of melanoma and the examination of minute metastatic lesions, indicating its potential to assist in guiding clinical treatment decisions.

A proof-of-concept study on bioorthogonal-based pretargeting and signal amplify radiotheranostic strategy.

BACKGROUND: Radiotheranostics differs from the vast majority of other cancer therapies in its capacity for simultaneous imaging and therapy, and it is becoming more widely implemented. A balance between diagnostic and treatment requirements is essential for achieving effective radiotheranostics. Herein, we propose a proof-of-concept strategy aiming to address the profound differences in the specific requirements of the diagnosis and treatment of radiotheranostics. RESULTS: To validate the concept, we designed an s-tetrazine (Tz) conjugated prostate-specific membrane antigen (PSMA) ligand (DOTA-PSMA-Tz) for 68Ga or 177Lu radiolabeling and tumor radiotheranostics, a trans-cyclooctene (TCO) modified Pd@Au nanoplates (Pd@Au-PEG-TCO) for signal amplification, respectively. We then demonstrated this radiotheranostic strategy in the tumor-bearing mice with the following three-step procedures: (1) i.v. injection of the [68Ga]Ga-PSMA-Tz for diagnosis; (2) i.v. injection of the signal amplification module Pd@Au-PEG-TCO; (3) i.v. injection of the [177Lu]Lu-PSMA-Tz for therapy. Firstly, this strategy was demonstrated in 22Rv1 tumor-bearing mice via positron emission tomography (PET) imaging with [68Ga]Ga-PSMA-Tz. We observed significantly higher tumor uptake (11.5 ± 0.8%ID/g) with the injection of Pd@Au-PEG-TCO than with the injection [68Ga]Ga-PSMA-Tz alone (5.5 ± 0.9%ID/g). Furthermore, we validated this strategy through biodistribution studies of [177Lu]Lu-PSMA-Tz, with the injection of the signal amplification module, approximately five-fold higher tumor uptake of [177Lu]Lu-PSMA-Tz (24.33 ± 2.53% ID/g) was obtained when compared to [177Lu]Lu-PSMA-Tz alone (5.19 ± 0.26%ID/g) at 48 h post-injection. CONCLUSION: In summary, the proposed strategy has the potential to expand the toolbox of pretargeted radiotherapy in the field of theranostics.

3D printed guide-assisted percutaneous screw fixation for minimally displaced scaphoid waist fractures with delayed diagnosis or presentation.

OBJECTIVES: To Investigate the value of 3D printed guide-assisted percutaneous management of minimally displaced scaphoid waist fractures(Herbert's B2) with delayed diagnosis or presentation. METHODS: From October 2018 to February 2022, 10 patients with established delayed diagnoses and presentation of minimally displaced scaphoid waist fractures were treated with 3D printed guides assisted with percutaneous internal fixation without bone grafting. This technique was based on the patient's preoperative CT and imported into the software. Based on Boolean subtraction, the most centralized screw placement position was identified and a customized guide was produced. Intraoperative percutaneous insertion of the guide wire was assisted by the custom guide. RESULTS: All 10 patients were successful in one attempt. The fractures healed at a mean of 7.7 weeks postoperatively (range 6-10 weeks). At a mean follow-up of 7.7 months (6-13 months), patients had excellent recovery of wrist function with minimal pain reduction. There were no major postoperative complications and the patients all returned to their previous activities before the injury. CONCLUSIONS: Percutaneous internal fixation based on 3D printed guides is a safe and effective technique for delayed diagnosis or presentation of patients with minimally displaced fractures of the scaphoid waist. This method allows for easy insertion of screws and avoids multiple attempts.

Minimally Invasive Percutaneous Screw Guided by 3-Dimensional-Printed Guide for the Treatment of Scaphoid Fractures.

PURPOSE: This study aimed to explore the feasibility and efficacy of percutaneous fixation of minimally displaced scaphoid waist fractures using a 3-dimensional-printed guide in 10 cases. METHODS: Fractures were examined using preoperative computed tomography. The skin interface and bone models were reconstructed using computed tomography data. Guidewire insertion was assisted by a guide. Computed tomography was performed 4-6 weeks after surgery until healing of the fracture was confirmed. The mean follow-up period was 7 months (range, 6-9 months). The fracture healing time, grip strength, flexion-extension arc, patient-rated wrist evaluation, and Mayo wrist score were recorded. RESULTS: A total of 6 hands were in the dominant limb. The mean operation time was 41 minutes (range, 32-70 minutes). Three (30%) scaphoids healed at 6 weeks after surgery, 8 (80%) scaphoids healed at 8 weeks after surgery, and 100% scaphoids healed at 12 weeks after surgery. After correcting for hand dominance, the mean grip strength was 84% (range, 71% to 95%) of that of the contralateral side. The flexion-extension arc was 97% (range, 82% to 100%) of that of the contralateral side. The mean Mayo wrist score was 95 (range, 85-100), and pain decreased to minimal levels. All patients returned to their preinjury activities. CONCLUSIONS: Three-dimensional printing is an effective and feasible technology that can help guide intraoperative processes. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Serum amyloid A and risks of all-cause and cardiovascular mortality in chronic kidney disease: a systematic review and dose-response meta-analysis.

BACKGROUNDS: The available literature on the correlation between serum amyloid A (SAA) and prognosis of chronic kidney disease (CKD) are limited, and the findings from existing studies are inconclusive. This meta-analysis aimed to evaluate the available evidence regarding the link between SAA and risks of all-cause and cardiovascular mortality in CKD patients. Additionally, we aimed to investigate the potential dose-response relationships, provided that adequate data is accessible. METHODS: Pubmed and Embase were searched for related literature (last update: 12 July 2023). The pooled effect estimates were calculated using random- or fixed-effects models depending on heterogeneity among studies. RESULTS: This meta-analysis incorporated 8 studies encompassing 2331 CKD patients. The findings revealed an 85% increase in all-cause mortality risk [hazard risk (HR) 1.85, 95% confidence interval (CI) 1.29-2.65] and a 39% increase in cardiovascular mortality risk (HR 1.07, 95% CI 1.07-1.80) when comparing the highest tertile of baseline SAA levels to the lowest tertile. Furthermore, a positive linear relationship between SAA and all-cause mortality risk was observed (Pnon-linearity = 0.959), with a 17.7% increase in risk for each 10 mg/L SAA increase (HR 1.177, 95% CI 1.055-1.313). Similarly, a linear relationship between SAA and cardiovascular mortality risk was identified (Pnon-linearity = 0.477) with a 19.3% increase in risk for each 10 mg/L SAA increase (HR 1.193, 95% CI 1.025-1.388). CONCLUSIONS: This meta-analysis provided evidence that SAA levels are positively and linearly associated with risks of all-cause and cardiovascular mortality among CKD patients.

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon.

Proanthocyanidins (PAs) are specialized metabolites that influence persimmon fruit quality. Normal astringent (A)-type and non-astringent (NA)-type mutants show significant variation in PA accumulation, but the influencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by DkDTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pattern of DkDTX5/MATE5 was correlated with PA accumulation in NA-type fruit. Upregulation and downregulation of DkDTX5/MATE5 promoted and inhibited PA accumulation, respectively, in the NA-type fruit. Interestingly, transporter assays of Xenopus laevis oocytes indicated that DkDTX5/MATE5 preferentially transported the PA precursors catechin, epicatechin, and epicatechin gallate, resulting in their increased ratios relative to the total PAs, which was the main source of variation in PA accumulation between the A-type and NA-type. The allele lacking Ser-84 in DkDTX5/MATE5 was identified as a dominantly expressed gene in the A-type and lost its transport function. Site-directed mutagenesis revealed that DkDTX5/MATE5 binds to PA precursors via Ser-84. These findings clarify the association between the transporter function of DkDTX5/MATE5 and PA variation, and can contribute to the breeding of new cultivars with improved fruit quality.

DkMYB14 is a bifunctional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit.

Proanthocyanidins (PAs) are phenolic secondary metabolites that contribute to the protection of plant and human health. Persimmon (Diospyros kaki Thunb.) can accumulate abundant PAs in fruit, which cause a strong sensation of astringency. Proanthocyanidins can be classified into soluble and insoluble PAs; the former cause astringency but the latter do not. Soluble PAs can be converted into insoluble PAs upon interacting with acetaldehydes. We demonstrate here that DkMYB14, which regulates the accumulation of PA in persimmon fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting its insolubilization. We show that the amino acid Gly39 in the R2 domain and the ethylene response factor-associated amphiphilic repression-like motif in the C-terminal of DkMYB14 are essential for the regulation of both PA and acetaldehyde synthesis. The repressive function of DkMYB14 was lost after the mutation of either motif, and all activities of DkMYB14 were eliminated following the mutation of both motifs. Our results demonstrate that DkMYB14 functions as both a transcriptional activator and a repressor, directly repressing biosynthesis of PA and promoting its insolubilization, resulting in non-astringency in persimmon.

CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m6A-modified CREB1 mRNA.

BACKGROUND: Aberrant expression of circular RNAs (circRNAs) contributes to the initiation and progression of human malignancies, but the underlying mechanisms remain largely elusive. METHODS: High-throughput sequencing was performed to screen aberrantly expressed circRNAs or miRNAs in colorectal cancer (CRC) and adjacent normal tissues. A series of gain- and loss-of-function studies were conducted to evaluate the biological behaviors of CRC cells. RNA pulldown, mass spectrometry, RIP, qRT-PCR, Western blot, luciferase reporter assays and MeRIP-seq analysis were further applied to dissect the detailed mechanisms. RESULTS: Here, a novel circRNA named circEZH2 (hsa_circ_0006357) was screened out by RNA-seq in CRC tissues, whose expression is closely related to the clinicpathological characteristics and prognosis of CRC patients. Biologically, circEZH2 facilitates the proliferation and migration of CRC cells in vitro and in vivo. Mechanistically, circEZH2 interacts with m6A reader IGF2BP2 and blocks its ubiquitination-dependent degradation. Meanwhile, circEZH2 could serve as a sponge of miR-133b, resulting in the upregulation of IGF2BP2. Particularly, circEZH2/IGF2BP2 enhances the stability of CREB1 mRNA, thus aggravating CRC progression. CONCLUSIONS: Our findings not only reveal the pivotal roles of circEZH2 in modulating CRC progression, but also advocate for attenuating circEZH2/miR-133b/IGF2BP2/ CREB1 regulatory axis to combat CRC.

Genome editing of SlMYB3R3, a cell cycle transcription factor gene of tomato, induces elongated fruit shape.

Fruit shape is an important trait that attracts consumers, and the regulation of genes related to cell division is crucial for shaping multicellular organs. In Arabidopsis, MYB3R transcription factors, which harbor three imperfect repeats in the N-terminus, control organ growth by regulating cell division. However, the function of MYB3Rs in tomato remains unknown. Here, we characterized tomato SlMYB3R3, which was preferentially expressed in flowers and placed in a subclade with two Arabidopsis cell cycle suppressors (MYB3R3/5). slmyb3r3 knockout mutants were generated using the CRISPR/Cas9 system. Morphological observation of the slmyb3r3 mutants showed that fruits that were elongated and occasionally peanut-like in shape were formed, which was caused by significantly increased cell numbers in the longitudinal direction. Transcriptome and yeast one-hybrid assay results suggested that SlMYB3R3 acted as a suppressor of cell-cycle-related genes by binding to the mitosis-specific activator (MSA) motifs in their promoters. Taken together, knock out of the suppressor SlMYB3R3 leads to elongated fruit, which results from the altered cell division pattern at the ovary stage, by regulating cell-cycle-related genes in an MSA-dependent manner. Our results suggest that SlMYB3R3 and its orthologs have the potential to change fruit shape as part of the molecular breeding of fruit crops.

Bi(OTf)3-Catalyzed Alkyl-Intercepted Meyer-Schuster Rearrangement of Propargylic Alcohols for the Synthesis of 1,2,3,5-Tetrasubstituted Pentane-1,5-diones.

An alkyl intercepted Meyer-Schuster rearrangement reaction with α,ß-unsaturated ketones as the electrophiles was first investigated, which provided a facile method to construct 2-methylene-pentane-1,5-diones. Then the in situ generated 2-methylene-pentane-1,5-diones underwent a Michael addition to give diverse 2-malononitrile methyl substituted pentane-1,5-diones in a one-pot fashion. This transformation was reliable on a gram scale. The high yield, convenient experimental operation, and 100% atom economy made it a valuable method for the construction of 1,2,3,5-tetrasubstituted pentane-1,5-dione derivatives.

DkmiR397 Regulates Proanthocyanidin Biosynthesis via Negative Modulating DkLAC2 in Chinese PCNA Persimmon.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, DkLAC2, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene DkLAC2 in 'Eshi 1' persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves. Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of DkLAC2. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2, whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type (WT) and overexpression of DkLAC2, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.

Enantioselective Nickel-Catalyzed Electrochemical Radical Allylation.

A simplified and appealing strategy via a chiral catalyst to facilitate the electrochemical process and provide asymmetric induction of radical reactions is particularly desirable and would have potential applications in electrosynthesis, organic chemistry, and drug discovery. Here, we introduced a novel electrolytic system that diverts the standard ionic reactivity to new catalytic functions, enabling mechanistically distinct single-electron transfer-based enantioselective routes to exhibit a brand-new pattern of reactivity-electricity-driven asymmetric catalysis as a privileged chiral platform for enantioselective radical allylation. The nickel-catalyzed activation of nucleophiles triggered a single-electron transfer process to provide a chiral catalyst-bound radical cation intermediate, which could be applied as an alternative strategy for the development of stereocontrolled radical reactions.

Comparative transcriptome analysis reveals regulatory network and regulators associated with proanthocyanidin accumulation in persimmon.

BACKGROUND: Proanthocyanidins (PAs) are important plant secondary metabolites that confer flavor, nutritional value, and resistance to pathogens. Persimmon is one of the PA richest crops. Mature fruits can be inedible because of the astringency caused by high PA levels and need to go through a de-astringency treatment before consumption. The molecular basis for PA accumulation is poorly known, particularly transcriptional regulators. We characterised three genotypes ('Luotiantianshi' (LT), 'Mopanshi' (MP), and 'Youhou' (YH)) with different PA accumulation patterns using an approach that combined PacBio full-length sequencing and Illumina-based RNA sequencing to build high-quality full-length transcriptomes. Additionally, we analysed transcriptome dynamics of the three genotypes (LT, MP, and YH) at four key fruit developmental stages. RESULTS: A total of 96,463 transcripts were obtained. We identified 80,075 protein-coding sequences (CDSs), 71,137 simple sequence repeats (SSRs), and 27,845 long noncoding RNAs (lncRNAs). Pearson correlation coefficient (PCC), principal component analysis (PCA), and differentially expressed transcripts (DETs) analyses indicated that the four different developmental stages within a genotype exhibited similar transcriptome activities. A total of 2,164 transcripts specific to each fruit developmental stage were detected. The transcripts specific to early stages were attributed to phenylpropanoid and flavonoid biosynthesis. Co-expression network analyses revealed MEbrown and MEblue modules were strongly associated to PA accumulation. From these two modules, 20 hub TFs are potential regulators for PA accumulation. Among them, Cluster_78388 (SBP protein), Cluster_63454 (bZIP protein), and Cluster_66595 (MYB protein) appear to involve in the PA biosynthesis in Chinese genotypes. CONCLUSIONS: This is the first high-quality reference transcriptome for commercial persimmon. Our work provides insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout fruit development.

Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A.

Low-molecular-mass thiols in organisms are well known for their redox-relevant role in protection against various endogenous and exogenous stresses. In eukaryotes and Gram-negative bacteria, the primary thiol is glutathione (GSH), a cysteinyl-containing tripeptide. In contrast, mycothiol (MSH), a cysteinyl pseudo-disaccharide, is dominant in Gram-positive actinobacteria, including antibiotic-producing actinomycetes and pathogenic mycobacteria. MSH is equivalent to GSH, either as a cofactor or as a substrate, in numerous biochemical processes, most of which have not been characterized, largely due to the dearth of information concerning MSH-dependent proteins. Actinomycetes are able to produce another thiol, ergothioneine (EGT), a histidine betaine derivative that is widely assimilated by plants and animals for variable physiological activities. The involvement of EGT in enzymatic reactions, however, lacks any precedent. Here we report that the unprecedented coupling of two bacterial thiols, MSH and EGT, has a constructive role in the biosynthesis of lincomycin A, a sulfur-containing lincosamide (C8 sugar) antibiotic that has been widely used for half a century to treat Gram-positive bacterial infections. EGT acts as a carrier to template the molecular assembly, and MSH is the sulfur donor for lincomycin maturation after thiol exchange. These thiols function through two unusual S-glycosylations that program lincosamide transfer, activation and modification, providing the first paradigm for EGT-associated biochemical processes and for the poorly understood MSH-dependent biotransformations, a newly described model that is potentially common in the incorporation of sulfur, an element essential for life and ubiquitous in living systems.