Targeting MMP9 in CTNNB1 mutant hepatocellular carcinoma restores CD8+ T cell-mediated antitumour immunity and improves anti-PD-1 efficacy.

OBJECTIVE: The gain of function (GOF) CTNNB1 mutations (CTNNB1 GOF ) in hepatocellular carcinoma (HCC) cause significant immune escape and resistance to anti-PD-1. Here, we aimed to investigate the mechanism of CTNNB1 GOF HCC-mediated immune escape and raise a new therapeutic strategy to enhance anti-PD-1 efficacy in HCC. DESIGN: RNA sequencing was performed to identify the key downstream genes of CTNNB1 GOF associated with immune escape. An in vitro coculture system, murine subcutaneous or orthotopic models, spontaneously tumourigenic models in conditional gene-knock-out mice and flow cytometry were used to explore the biological function of matrix metallopeptidase 9 (MMP9) in tumour progression and immune escape. Single-cell RNA sequencing and proteomics were used to gain insight into the underlying mechanisms of MMP9. RESULTS: MMP9 was significantly upregulated in CTNNB1 GOF HCC. MMP9 suppressed infiltration and cytotoxicity of CD8+ T cells, which was critical for CTNNB1 GOF to drive the suppressive tumour immune microenvironment (TIME) and anti-PD-1 resistance. Mechanistically, CTNNB1 GOF downregulated sirtuin 2 (SIRT2), resulting in promotion of ß-catenin/lysine demethylase 4D (KDM4D) complex formation that fostered the transcriptional activation of MMP9. The secretion of MMP9 from HCC mediated slingshot protein phosphatase 1 (SSH1) shedding from CD8+ T cells, leading to the inhibition of C-X-C motif chemokine receptor 3 (CXCR3)-mediated intracellular of G protein-coupled receptors signalling. Additionally, MMP9 blockade remodelled the TIME and potentiated the sensitivity of anti-PD-1 therapy in HCC. CONCLUSIONS: CTNNB1 GOF induces a suppressive TIME by activating secretion of MMP9. Targeting MMP9 reshapes TIME and potentiates anti-PD-1 efficacy in CTNNB1 GOF HCC.

High ratio of resident to exhausted CD4 + T cells predicts favorable prognosis and potentially better immunotherapeutic efficacy in hepatocellular carcinoma.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) are significantly implicated in regulating the tumor immune microenvironment (TIME) and immunotherapeutic response. However, little is known about the impact of the resident and exhausted status of TILs in hepatocellular carcinoma (HCC). METHODS: Single-cell RNA sequencing data was applied to discover resident and exhausted signatures of TILs. Survival outcomes, biological function, immune infiltration, genomic variation, immunotherapeutic efficacy, and sorafenib response were further explored the clinical significance and molecular association of TILs in HCC. Moreover, a candidate gene with predictive capability for the dismal subtype was identified through univariate Cox regression analysis, survival analysis, and the BEST website. RESULTS: Single-cell analysis revealed that CD8 + T, CD4 + T, and NK cells were strongly associated with resident and exhausted patterns. Specific resident and exhausted signatures for each subpopulation were extracted in HCC. Further multivariate Cox analysis revealed that the ratio of resident to exhausted CD4 + T cells in TIME was an independent prognostic factor. After incorporating tumor purity with the ratio of resident to exhausted CD4 + T cells, we stratified HCC patients into three subtypes and found that (i) CD4 residencyhighexhaustionlow subtype was endowed with favorable prognosis, immune activation, and sensitivity to immunotherapy; (ii) CD4 exhaustionhighresidencylow subtype was characterized by genome instability and sensitivity to sorafenib; (iii) Immune-desert subtype was associated with malignant-related pathways and poor prognosis. Furthermore, spindle assembly abnormal protein 6 homolog (SASS6) was identified as a key gene, which accurately predicted the immune-desert subtype. Prognostic analysis as well as in vitro and in vivo experiments further demonstrated that SASS6 was closely associated with tumor prognosis, proliferation, and migration. CONCLUSIONS: The ratio of resident to exhausted CD4 + T cells shows promise as a potential biomarker for HCC prognosis and immunotherapy response and SASS6 may serve as a biomarker and therapeutic target for prognostic assessment of HCC.

Design, synthesis, and biological evaluation of novel donepezil-tacrine hybrids as multi-functional agents with low neurotoxicity against Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and deficits in cognitive domains. Low choline levels, oxidative stress, and neuroinflammation are the primary mechanisms implicated in AD progression. Simultaneous inhibition of acetylcholinesterase (AChE) and reactive oxygen species (ROS) production by a single molecule may provide a new breath of hope for AD treatment. Here, we describe donepezil-tacrine hybrids as inhibitors of AChE and ROS. Four series of derivatives with a ß-amino alcohol linker were designed and synthesized. In this study, the target compounds were evaluated for their ability to inhibit AChE and butyrylcholinesterase (BuChE) in vitro, using tacrine (hAChE, IC50 = 305.78 nM; hBuChE, IC50 = 56.72 nM) and donepezil (hAChE, IC50 = 89.32 nM; hBuChE, IC50 = 9137.16 nM) as positive controls. Compound B19 exhibited an excellent and balanced inhibitory potency against AChE (IC50 = 30.68 nM) and BuChE (IC50 = 124.57 nM). The cytotoxicity assays demonstrated that the PC12 cell viability rates of compound B19 (84.37 %) were close to that of tacrine (87.73 %) and donepezil (79.71 %). Potential therapeutic effects in AD were evaluated using the neuroprotective effect of compounds against H2O2-induced toxicity, and compound B19 (68.77 %) exhibited substantially neuroprotective activity at the concentration of 25 µM, compared with the model group (30.34 %). Furthermore, compound B19 protected PC12 cells from H2O2-induced apoptosis and ROS production. These properties of compound B19 suggested that it was a multi-functional agent with AChE inhibition, anti-oxidative, anti-inflammatory activities, and low toxicity and that it deserves further investigation as a promising agent for AD treatment.

Resolving complex structures at oncovirus integration loci with conjugate graph.

Oncovirus integrations cause copy number variations and complex structural variations (SVs) on host genomes. However, the understanding of how inserted viral DNA impacts the local genome remains limited. The linear structure of the oncovirus integrated local genomic map (LGM) will lay the foundations to understand how oncovirus integrations emerge and compromise the host genome's functioning. We propose a conjugate graph model to reconstruct the rearranged LGM at integrated loci. Simulation tests prove the reliability and credibility of the algorithm. Applications of the algorithm to whole-genome sequencing data of human papillomavirus (HPV) and hepatitis B virus (HBV)-infected cancer samples gained biological insights on oncovirus integrations. We observed four affection patterns of oncovirus integrations from the HPV and HBV-integrated cancer samples, including the coding-frame truncation, hyper-amplification of tumor gene, the viral cis-regulation inserted at the single intron and at the intergenic region. We found that the focal duplicates and host SVs are frequent in the HPV-integrated LGMs, while the focal deletions are prevalent in HBV-integrated LGMs. Furthermore, with the results yields from our method, we found the enhanced microhomology-mediated end joining might lead to both HPV and HBV integrations and conjectured that the HPV integrations might mainly occur during the DNA replication process. The conjugate graph algorithm code and LGM construction pipeline, available at https://github.com/deepomicslab/FuseSV.

Oviz-Bio: a web-based platform for interactive cancer genomics data visualization.

Genetics data visualization plays an important role in the sharing of knowledge from cancer genome research. Many types of visualization are widely used, most of which are static and require sufficient coding experience to create. Here, we present Oviz-Bio, a web-based platform that provides interactive and real-time visualizations of cancer genomics data. Researchers can interactively explore visual outputs and export high-quality diagrams. Oviz-Bio supports a diverse range of visualizations on common cancer mutation types, including annotation and signatures of small scale mutations, haplotype view and focal clusters of copy number variations, split-reads alignment and heatmap view of structural variations, transcript junction of fusion genes and genomic hotspot of oncovirus integrations. Furthermore, Oviz-Bio allows landscape view to investigate multi-layered data in samples cohort. All Oviz-Bio visual applications are freely available at https://bio.oviz.org/.

Analysis of high-risk factors for preoperative DVT in elderly patients with simple hip fractures and construction of a nomogram prediction model.

BACKGROUND: Hip fractures are anatomically classified in relation to femoral neck, intertrochanteric or subtrochanteric fractures. Simple hip fractures discussed in this study are femoral neck fractures or intertrochanteric fractures, which are the most common types of hip fractures. Controversy remains regarding the value of biochemical indices of thrombosis in elderly patients with fractures. A retrospective study was conducted to investigate the index admission data in blood draws of elderly patients with hip fractures and their high-risk factors for deep venous thrombosis (DVT). A nomogram prediction model for DVT was established to facilitate a rapid, accurate, and effective prediction based on the results. METHODS: The data were based on 562 elderly patients undergoing hip fracture surgery, from whom 274 patients were selected for enrollment. The 274 patients were divided into two groups using preoperative vascular color Doppler ultrasonography. Chi-square tests, t-tests, and U tests were conducted, and logistic regression analysis was conducted showing different factors between the two groups. Independent risk factors with statistical significance (P < 0.05) were obtained, and the logistic regression equation and the new variable prediction probability_1 (PRE_1) were constructed. The receiver operating characteristic (ROC) curve of risk factors and PRE_1 was drawn to obtain the area under the curve (AUC) and truncation value of each risk factor. Finally, a nomogram prediction model was constructed using the R programming language to calculate the concordance index (C-index). RESULTS: Time from injury to hospitalization, platelet (PLT) count, D-dimer level, fibrinogen (FIB) level, and systemic immune-inflammatory index (SII) score were independent risk factors for preoperative DVT in elderly patients with hip fractures. The logistic regression equation and PRE_1 were constructed by combining the above factors. ROC analysis showed that the area under the curve for PRE_1 (AUC = 0.808) was greater than that of the other factors. The sensitivity of PRE_1 (sensitivity = 0.756) was also higher than that of the other factors, and the specificity of PRE_1 (specificity = 0.756) was higher than that of two other factors. Moreover, a predictive nomogram was established, and the results showed a high consistency between the actual probability and the predicted probability (C-index = 0.808), indicating a high predictive value in fractures accompanied by DVT. CONCLUSIONS: This study confirmed that SII score could be used as a risk factor in the prediction of DVT occurrence. A nomogram prediction model was constructed by combining 5 independent risk factors: time from injury to admission, PLT count, D-dimer level, FIB level, and SII score, which had high predictive values for fractures accompanied by DVT. This model use is limited to simple hip fracture.

Design, synthesis and biological evaluation of selective histone deacetylase 6 (HDAC6) inhibitors bearing benzoindazole or pyrazoloindazole scaffold as surface recognition motif.

A series of compounds were designed and synthesized based on the compound 11i bearing phenylpyrazole scaffold with histone deacetylase 6 (HDAC6) inhibitory activity. Most of the compounds showed considerable inhibitory activity against HDAC6 and compound A16 with good inhibitory activity was found therein. We further found that A16 had an inhibitory effect on inflammatory mediators (NO, TNF-α, IL-6) involved in inflammatory response and neuroendocrine regulation. In addition, A16 has a certain neuroprotective effect on PC12 cells injured by hydrogen peroxide. Acute toxicity assay showed that the LD50 of A16 was 274.47 mg/kg in mouse model. Furthermore, A16 displayed good stability properties in microsomes and plasma.

Construction of pH sensitive smart glutathione peroxidase (GPx) mimics based on pH responsive pseudorotaxanes.

Two organoselenium compounds, both of which were modified with two primary amine groups, were designed and synthesized to mimic the catalytic properties of glutathione peroxidase (GPx). It was demonstrated that the catalytic mechanism of the diselenide organoselenium compound (compound 1) was a ping-pong mechanism while that of the selenide organoselenium compound (compound 2) was a sequential mechanism. The pH-controlled switching of the catalytic activities was achieved by controlling the formation and dissociation of the pseudorotaxanes based on the organoselenium compounds and cucurbit[6]uril (CB[6]). Moreover, the switching was reversible at pH between 7 and 9 for compound 1 or between 7 and 10 for compound 2.

Whole-genome sequencing uncovers the genetic basis of chronic mountain sickness in Andean highlanders.

The hypoxic conditions at high altitudes present a challenge for survival, causing pressure for adaptation. Interestingly, many high-altitude denizens (particularly in the Andes) are maladapted, with a condition known as chronic mountain sickness (CMS) or Monge disease. To decode the genetic basis of this disease, we sequenced and compared the whole genomes of 20 Andean subjects (10 with CMS and 10 without). We discovered 11 regions genome-wide with significant differences in haplotype frequencies consistent with selective sweeps. In these regions, two genes (an erythropoiesis regulator, SENP1, and an oncogene, ANP32D) had a higher transcriptional response to hypoxia in individuals with CMS relative to those without. We further found that downregulating the orthologs of these genes in flies dramatically enhanced survival rates under hypoxia, demonstrating that suppression of SENP1 and ANP32D plays an essential role in hypoxia tolerance. Our study provides an unbiased framework to identify and validate the genetic basis of adaptation to high altitudes and identifies potentially targetable mechanisms for CMS treatment.

CBX4 suppresses CD8+ T cell antitumor immunity by reprogramming glycolytic metabolism.

Rationale: CD8+ T cells undergo a series of metabolic reprogramming processes during their activation and proliferation, including increased glycolysis, decreased aerobic oxidation of sugars, increased amino acid metabolism and increased protein synthesis. However, it is still unclear what factors regulate these metabolic reprogramming processes in CD8+ T cells in the tumor immune microenvironment. Methods: T cell chromobox protein 4 (CBX4) knock-out mice models were used to determine the role of CBX4 in CD8+ T cells on the tumor immune microenvironment and tumor progression. Flow cytometry, Cut-Tag qPCR, Chip-seq, immunoprecipitation, metabolite detection, lentivirus infection and adoptive T cells transfer were performed to explore the underlying mechanisms of CBX4 knock-out in promoting CD8+ T cell activation and inhibiting tumor growth. Results: We found that CBX4 expression was induced in tumor-infiltrating CD8+ T cells and inhibited CD8+ T cell function by regulating glucose metabolism in tumor tissue. Mechanistically, CBX4 increases the expression of the metabolism-associated molecule aldolase B (Aldob) through sumoylation of trans-acting transcription factor 1 (SP1) and Krüppel-like factor 3 (KLF3). In addition, Aldob inhibits glycolysis and ATP synthesis in T cells by reducing the phosphorylation of the serine/threonine protein kinase (Akt) and ultimately suppresses CD8+ T cell function. Significantly, knocking out CBX4 may improve the efficacy of anti-PD-1 therapy by enhancing the function of CD8+ T cells in the tumor microenvironment. Conclusion: CBX4 is involved in CD8+ T cell metabolic reprogramming and functional persistence in tumor tissues, and serves as an inhibitor in CD8+ T cells' glycolysis and effector function.

YTHDF2 Is a Therapeutic Target for HCC by Suppressing Immune Evasion and Angiogenesis Through ETV5/PD-L1/VEGFA Axis.

N6-methyladenosine (m6A) modification orchestrates cancer formation and progression by affecting the tumor microenvironment (TME). For hepatocellular carcinoma (HCC), immune evasion and angiogenesis are characteristic features of its TME. The role of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), as an m6A reader, in regulating HCC TME are not fully understood. Herein, it is discovered that trimethylated histone H3 lysine 4 and H3 lysine 27 acetylation modification in the promoter region of YTHDF2 enhanced its expression in HCC, and upregulated YTHDF2 in HCC predicted a worse prognosis. Animal experiments demonstrated that Ythdf2 depletion inhibited spontaneous HCC formation, while its overexpression promoted xenografted HCC progression. Mechanistically, YTHDF2 recognized the m6A modification in the 5'-untranslational region of ETS variant transcription factor 5 (ETV5) mRNA and recruited eukaryotic translation initiation factor 3 subunit B to facilitate its translation. Elevated ETV5 expression induced the transcription of programmed death ligand-1 and vascular endothelial growth factor A, thereby promoting HCC immune evasion and angiogenesis. Targeting YTHDF2 via small interference RNA-containing aptamer/liposomes successfully both inhibited HCC immune evasion and angiogenesis. Together, this findings reveal the potential application of YTHDF2 in HCC prognosis and targeted treatment.

CREB3 suppresses hepatocellular carcinoma progression by depressing AKT signaling through competitively binding with insulin receptor and transcriptionally activating RNA-binding motif protein 38.

cAMP responsive element binding protein 3 (CREB3), belonging to bZIP family, was reported to play multiple roles in various cancers, but its role in hepatocellular carcinoma (HCC) is still unclear. cAMP responsive element binding protein 3 like 3 (CREB3L3), another member of bZIP family, was thought to be transcription factor (TF) to regulate hepatic metabolism. Nevertheless, except for being TFs, other function of bZIP family were poorly understood. In this study, we found CREB3 inhibited growth and metastasis of HCC in vitro and in vivo. RNA sequencing indicated CREB3 regulated AKT signaling to influence HCC progression. Mass spectrometry analysis revealed CREB3 interacted with insulin receptor (INSR). Mechanistically, CREB3 suppressed AKT phosphorylation by inhibiting the interaction of INSR with insulin receptor substrate 1 (IRS1). In our study, CREB3 was firstly proved to affect activation of substrates by interacting with tyrosine kinase receptor. Besides, CREB3 could act as a TF to transactivate RNA-binding motif protein 38 (RBM38) expression, leading to suppressed AKT phosphorylation. Rescue experiments further confirmed the independence between the two functional manners. In conclusion, CREB3 acted as a tumor suppressor in HCC, which inhibited AKT phosphorylation through independently interfering interaction of INSR with IRS1, and transcriptionally activating RBM38.

Bone Lesion-Derived Extracellular Vesicles Fuel Prometastatic Cascades in Hepatocellular Carcinoma by Transferring ALKBH5-Targeting miR-3190-5p.

Bone is the second leading metastatic site for hepatocellular carcinoma (HCC). Patients with HCC and bone metastasis suffer poor quality of life and reduced survival time. Extracellular vesicles (EVs) are widely involved in HCC formation and metastasis. However, the communication between primary HCC and bone lesions mediated by EVs remains unclear and the possible effect of bone metastasis on the progression of HCC remains largely unknown. Here, bone-metastasized HCC-derived EVs (BM-EVs) are found to localize to orthotropic HCC cells and promote HCC progression. Mechanistically, miR-3190-5p (miR-3190) is upregulated in intracellular HCC cells isolated from bone lesions as well as in their derived EVs. miR-3190 in BM-EVs is transferred into orthotopic tumor cells and enhances their metastatic capacity by downregulating AlkB homolog 5 (ALKBH5) expression. Decreased level of ALKBH5 exacerbates the prometastatic characteristics of HCC by modulating gene expression in N6-methyladenosine-dependent and -independent ways. Finally, antagomir-miR-3190-loaded liposomes with HCC affinity successfully suppress HCC progression in mice treated with BM-EVs. These findings reveal that BM-EVs initiate prometastatic cascades in orthotopic HCC by transferring ALKBH5-targeting miR-3190 and miR-3190 is serving as a promising therapeutic target for inhibiting the progression of HCC in patients with bone metastasis.

FOXP2 confers oncogenic effects in prostate cancer.

Identification oncogenes is fundamental to revealing the molecular basis of cancer. Here, we found that FOXP2 is overexpressed in human prostate cancer cells and prostate tumors, but its expression is absent in normal prostate epithelial cells and low in benign prostatic hyperplasia. FOXP2 is a FOX transcription factor family member and tightly associated with vocal development. To date, little is known regarding the link of FOXP2 to prostate cancer. We observed that high FOXP2 expression and frequent amplification are significantly associated with high Gleason score. Ectopic expression of FOXP2 induces malignant transformation of mouse NIH3T3 fibroblasts and human prostate epithelial cell RWPE-1. Conversely, FOXP2 knockdown suppresses the proliferation of prostate cancer cells. Transgenic overexpression of FOXP2 in the mouse prostate causes prostatic intraepithelial neoplasia. Overexpression of FOXP2 aberrantly activates oncogenic MET signaling and inhibition of MET signaling effectively reverts the FOXP2-induced oncogenic phenotype. CUT&Tag assay identified FOXP2-binding sites located in MET and its associated gene HGF. Additionally, the novel recurrent FOXP2-CPED1 fusion identified in prostate tumors results in high expression of truncated FOXP2, which exhibit a similar capacity for malignant transformation. Together, our data indicate that FOXP2 is involved in tumorigenicity of prostate.

Both simulation and sequencing data reveal coinfections with multiple SARS-CoV-2 variants in the COVID-19 pandemic.

SARS-CoV-2 is a single-stranded RNA betacoronavirus with a high mutation rate. The rapidly emerging SARS-CoV-2 variants could increase transmissibility and diminish vaccine protection. However, whether coinfection with multiple SARS-CoV-2 variants exists remains controversial. This study collected 12,986 and 4,113 SARS-CoV-2 genomes from the GISAID database on May 11, 2020 (GISAID20May11), and Apr 1, 2021 (GISAID21Apr1), respectively. With single-nucleotide variant (SNV) and network clique analyses, we constructed single-nucleotide polymorphism (SNP) coexistence networks and discovered maximal SNP cliques of sizes 16 and 34 in the GISAID20May11 and GISAID21Apr1 datasets, respectively. Simulating the transmission routes and SNV accumulations, we discovered a linear relationship between the size of the maximal clique and the number of coinfected variants. We deduced that the COVID-19 cases in GISAID20May11 and GISAID21Apr1 were coinfections with 3.20 and 3.42 variants on average, respectively. Additionally, we performed Nanopore sequencing on 42 COVID-19 patients and discovered recurrent heterozygous SNPs in twenty of the patients, including loci 8,782 and 28,144, which were crucial for SARS-CoV-2 lineage divergence. In conclusion, our findings reported SARS-CoV-2 variants coinfection in COVID-19 patients and demonstrated the increasing number of coinfected variants.

Human papillomavirus integration perspective in small cell cervical carcinoma.

Small cell cervical carcinoma (SCCC) is a rare but aggressive malignancy. Here, we report human papillomavirus features and genomic landscape in SCCC via high-throughput HPV captured sequencing, whole-genome sequencing, whole-transcriptome sequencing, and OncoScan microarrays. HPV18 infections and integrations are commonly detected. Besides MYC family genes (37.9%), we identify SOX (8.4%), NR4A (6.3%), ANKRD (7.4%), and CEA (3.2%) family genes as HPV-integrated hotspots. We construct the genomic local haplotype around HPV-integrated sites, and find tandem duplications and amplified HPV long control regions (LCR). We propose three prominent HPV integration patterns: duplicating oncogenes (MYCN, MYC, and NR4A2), forming fusions (FGFR3-TACC3 and ANKRD12-NDUFV2), and activating genes (MYC) via the cis-regulations of viral LCRs. Moreover, focal CNA amplification peaks harbor canonical cancer genes including the HPV-integrated hotspots within MYC family, SOX2, and others. Our findings may provide potential molecular criteria for the accurate diagnosis and efficacious therapies for this lethal disease.

Functional organization of the maternal and paternal human 4D Nucleome.

Every human somatic cell inherits a maternal and a paternal genome, which work together to give rise to cellular phenotypes. However, the allele-specific relationship between gene expression and genome structure through the cell cycle is largely unknown. By integrating haplotype-resolved genome-wide chromosome conformation capture, mature and nascent mRNA, and protein binding data from a B lymphoblastoid cell line, we investigate this relationship both globally and locally. We introduce the maternal and paternal 4D Nucleome, enabling detailed analysis of the mechanisms and dynamics of genome structure and gene function for diploid organisms. Our analyses find significant coordination between allelic expression biases and local genome conformation, and notably absent expression bias in universally essential cell cycle and glycolysis genes. We propose a model in which coordinated biallelic expression reflects prioritized preservation of essential gene sets.

One-Pot Synthesis of Renewable Phthalic Anhydride from 5-Hydroxymethfurfural by using MoO3 /Cu(NO3 )2 as Catalyst.

Herein, a synthetic pathway to renewable phthalic anhydride (PA) from 5-hydroxymethfurfural (HMF) in one pot is reported. The commonly available catalysts MoO3 and Cu(NO3 )2 play a crucial role in integrating the multiple steps of the reaction, namely decarbonylation of HMF to active furyl intermediate (AFI), oxidation of HMF to maleic anhydride (MA), Diels-Alder cycloaddition of AFI and MA, and subsequent dehydration, in one pot. Under mild reaction conditions, a 63.2 % yield of PA is obtained from HMF. Compared with the currently reported route to renewable PA based on the Diels-Alder cycloaddition of biomass-derived MA and furan, this convenient one-pot synthesis represents a great improvement in efficiency.