Flow and On-Water Synthesis and Cancer Cell Cytotoxicity of Caffeic Acid Phenethyl Amide (CAPA) Derivatives.

Caffeic acid phenethyl ester (CAPE) is a phenolic natural product with a wide range of biological activities, including anticancer activity; however, the ester group of CAPE is metabolically labile. The corresponding amide, CAPA, has improved metabolic stability but limited anticancer activity relative to CAPE. We report the synthesis using flow and on-water Wittig reaction approaches of five previously reported and five novel CAPA analogues. All of these analogues lack the reactive catechol functionality of CAPA and CAPE. Cytotoxicity studies of CAPE, CAPA, and these CAPA analogues in HeLa and BE(2)-C cells were carried out. Surprisingly, we found that CAPA is cytotoxic against the neuroblastoma BE(2)-C cell line (IC50 = 12 µM), in contrast to the weak activity of CAPA against HeLa cells (IC50 = 112 µM), and the literature reports of the absence of activity for CAPA against a variety of other cancer cell lines. One novel CAPA analogue, 3f, was identified as having cytotoxic activity similar to CAPE in HeLa cells (IC50 = 63 µM for 3f vs. 32 µM for CAPE), albeit with lower activity against BE(2)-C cells (IC50 = 91 µM) than CAPA. A different CAPA analogue, 3g, was found to have similar effects against BE(2)-C cells (IC50 = 92 µM). These results show that CAPA is uniquely active against neuroblastoma cells and that specific CAPA analogues that are predicted to be more metabolically stable than CAPE can reproduce CAPA's activity against neuroblastoma cells and CAPE's activity against HeLa cells.

2-(3-Indolyl)acetamides and their oxazoline analogues: Anticancer SAR study.

We previously studied 2-aryl-2-(3-indolyl)acetohydroxamates as potential agents against melanoma. These compounds were ineffective in a mouse melanoma xenograft model, most likely due to unfavorable metabolic properties, specifically due to glucuronidation of the N-hydroxyl of the hydoxamic moiety. In the present work, we prepared a series of analogues, 2-aryl-2-(3-indolyl)acetamides and their oxazoline derivatives, which do not contain the N-hydroxyl group. We investigated the structure-activity relationship in both series of compounds and found that the 2-naphthyl is a preferred group at C-2 of the indole in the amide series, whereas the tetralin moiety is favorable in the same location in the oxazoline series. Overall, three compounds in the amide series have GI50 values as low as 0.2-0.3 µM and the results clearly indicate that the N-hydroxyl group is not necessary for high potency in vitro.

Identification of CDKN3 as a Key Gene that Regulates Neuroblastoma Cell Differentiation.

We conducted a high-content screening (HCS) in neuroblastoma BE(2)-C cells to identify cell cycle regulators that control cell differentiation using a library of siRNAs against cell cycle-regulatory genes. We discovered that knocking down expression of cyclin dependent kinase inhibitor 3 (CDKN3) showed the most potent effect in inducing neurite outgrowth, the morphological cell differentiation marker of neuroblastoma cells. We then demonstrated that CDKN3 knockdown increased expression of neuroblastoma molecular differentiation markers, neuron specific enolase (NSE), ßIII-tubulin and growth associated protein 43 (GAP43). We further showed that CDKN3 knockdown reduced expression of cell proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), and reduced colony formation of neuroblastoma cells. More importantly, we observed a correlation of high tumor CDKN3 mRNA levels with poor patient survival in the investigation of public neuroblastoma patient datasets. In exploring the mechanisms that regulate CDKN3 expression, we found that multiple strong differentiation-inducing molecules, including miR-506-3p and retinoic acid, down-regulated CDKN3 expression. In addition, we found that N-Myc promoted CDKN3 expression at the transcriptional level by directly binding to the CDKN3 promoter. Furthermore, we found that CDKN3 and two additional differentiation-regulating cell cycle proteins identified in our HCS, CDC6 and CDK4, form an interactive network to promote expression of each other. In summary, we for the first time discovered the function of CDKN3 in regulating neuroblastoma cell differentiation and characterized the transcriptional regulation of CDKN3 expression by N-Myc in neuroblastoma cells. Our findings support that CDKN3 plays a role in modulating neuroblastoma cell differentiation and that overexpression of CDKN3 may contribute to neuroblastoma progression.

Development of Shortened miR-506-3p Mimics Exhibiting Strong Differentiation-Inducing Activity in Neuroblastoma Cells.

microRNA mimics are synthetic RNA molecules that imitate the mature miRNA duplexes and their functions. These mimics have shown promise in treating cancers. Nucleotide chemical modifications of microRNA mimics have been investigated and have improved the stability of miRNA mimics. However, the potential therapeutic benefit of mimic analogs based on sequence modifications has not been explored. miR-506-3p was identified as a differentiation-inducing microRNA in neuroblastoma cells, suggesting the potential of applying the miR-506-3p mimic in neuroblastoma differentiation therapy. In this study, we explored the possibility of developing shortened miR-506-3p analogs that can maintain differentiation-inducing activities comparable to the wild-type miR-506-3p mimic. We found that deleting up to two nucleotides at either the 3' end or within the middle region of the miR-506-3p sequence fully maintained the differentiation-inducing activity when compared to the wild-type mimic. Deleting up to four nucleotides from the 3' end or deleting three nucleotides in the middle positions diminished the differentiation-inducing activity, but the analogs still maintained differentiation-inducing activities that were significantly higher than the negative control oligo. The shortened analog designs potentially benefit patients from two perspectives: (1) the reduced cost of manufacturing shortened analogs, and (2) the reduced non-specific toxicity due to their smaller molecular sizes.

Discovery of 5-sulfonyltetrazoles as neuroblastoma differentiation agents.

Previously, we developed an innovative high-content screening (HCS) approach to quantify neuroblastoma cell differentiation based on neurite outgrowth, a morphological differentiation marker of neuroblastoma cells. Here, we report the utilization of this platform to identify 1-methyl-5-(ethylsulfonyl)-1H-tetrazole (3a) as a new neuroblastoma differentiation agent using the ChemBridge DiversetTM commercial synthetic small molecule compound library. We show that this activity can be extended to a group of analogues, which can be accessed via a short two-step synthetic sequence. A new analogue, 5-(allylsulfonyl)-1-methyl-1H-tetrazole (3c) was identified in this synthetic effort as a compound that has even more pronounced differentiation and cytotoxic activities than the original hit compound 3a.

Highly efficient bioconversion of icariin to icaritin by whole-cell catalysis.

BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.

Conversion of Natural Narciclasine to Its C-1 and C-6 Derivatives and Their Antitumor Activity Evaluation: Some Unusual Chemistry of Narciclasine.

During the search for a general, efficient route toward the synthesis of C-1 analogues of narciclasine, natural narciclasine was protected and converted to its C-1 enol derivative using a novel semi-synthetic route. Attempted conversion of this material to its triflate in order to conduct cross-coupling at C-1 resulted in a triflate at C-6 that was successfully coupled with several functionalities. Four novel compounds were fully deprotected after seven steps and subjected to evaluation for cytotoxic activity against three cancer cell lines. Only one derivative showed moderate activity compared to that of narciclasine. Spectral and physical data are provided for all new compounds.

The Function and Regulation of SAPCD2 in Physiological and Oncogenic Processes.

The Suppressor APC Domain Containing 2 (SAPCD2) gene, also known by its aliases p42.3 and c9orf140, encodes a protein with an approximate molecular weight of 42.3 kDa. It was initially recognized as a cell cycle-associated protein involved in mitotic progression. Since the initial discovery of this gene, emerging evidence has suggested that its functions extend beyond that of regulating cell cycle progression to include modulation of planar polarization of cell progenitors and determination of cell fate throughout embryonic development. The underlying mechanisms driving such functions have been partially elucidated. However, the detailed mechanisms of action remain to be further characterized. The expression level of SAPCD2 is high throughout embryogenesis but is generally absent in healthy postnatal tissues, with restored expression in adult tissues being associated with various disease states. The pathological consequences of its aberrant expression have been investigated, most notably in the development of several types of cancers. The role of SAPCD2 in tumorigenesis has been supported by in vitro, in vivo, and retrospective clinical investigations and the mechanisms underlying its oncogenic function have been partially revealed. The potential of SAPCD2 as a diagnostic marker and therapeutic target of cancers have also been explored and have shown great promise. However, many questions pertaining to its oncogenic mechanisms as well as its value as a diagnostic marker and therapeutic target remain to be answered. In addition to its function as an oncogene, an involvement of SAPCD2 in other pathological processes such as inflammation has also been implicated and provides additional directions that warrant future investigation. This article reviews the current understanding of the normal cellular functions of SAPCD2 and the relevance of SAPCD2 in disease development with a primary focus on tumorigenesis. The mechanisms that regulate p43.2 expression, including the potential role of microRNAs in regulating its expression, are also reviewed. To the best of our knowledge, we are the first to comprehensively review the published findings regarding the physiological and pathological functions of this gene.

Scalable Synthesis and Cancer Cell Cytotoxicity of Rooperol and Analogues.

Plant polyphenols, such as the African potato (Hypoxis hemerocallidea)-derived bis-catechol rooperol, can display promising anticancer activity yet suffer from rapid metabolism. Embarking upon a program to systematically examine potentially more metabolically stable replacements for the catechol rings in rooperol, we report here a general, scalable synthesis of rooperol and analogues that builds on our previous synthetic approach incorporating a key Pd-catalyzed decarboxylative coupling strategy. Using this approach, we have prepared and evaluated the cancer cell cytotoxicity of rooperol and a series of analogues. While none of the analogues examined here were superior to rooperol in preventing the growth of cancer cells, analogues containing phenol or methylenedioxyphenyl replacements for one or both catechol rings were nearly as effective as rooperol.

[3 + 2]-Annulation of pyridinium ylides with 1-chloro-2-nitrostyrenes unveils a tubulin polymerization inhibitor.

Indolizines and pyrazolo[1,5-a]pyridines were prepared via [3 + 2]-cycloaddition of pyridinium ylides to 1-chloro-2-nitrostyrenes. The synthesized molecules were evaluated for antiproliferative activities against a BE(2)-C neuroblastoma cell line with several compounds decreasing the viability of cancer cells. Indolizine 9db showed higher potency than that of all-trans-retinoic acid, an approved cancer drug. Mechanistically, it was found to inhibit tubulin polymerization and it is thus proposed that the discovered chemistry can be exploited for the development of novel microtubule-targeting anticancer agents.

LIM domain only 1: an oncogenic transcription cofactor contributing to the tumorigenesis of multiple cancer types.

ABSTRACT: The LIM domain only 1 (LMO1) gene belongs to the LMO family of genes that encodes a group of transcriptional cofactors. This group of transcriptional cofactors regulates gene transcription by acting as a key "connector" or "scaffold" in transcription complexes. All LMOs, including LMO1, are important players in the process of tumorigenesis. Unique biological features of LMO1 distinct from other LMO members, such as its tissue-specific expression patterns, interacting proteins, and transcriptional targets, have been increasingly recognized. Studies indicated that LMO1 plays a critical oncogenic role in various types of cancers, including T-cell acute lymphoblastic leukemia, neuroblastoma, gastric cancer, lung cancer, and prostate cancer. The molecular mechanisms underlying such functions of LMO1 have also been investigated, but they are currently far from being fully elucidated. Here, we focus on reviewing the current findings on the role of LMO1 in tumorigenesis, the mechanisms of its oncogenic action, and the mechanisms that drive its aberrant activation in cancers. We also briefly review its roles in the development process and non-cancer diseases. Finally, we discuss the remaining questions and future investigations required for promoting the translation of laboratory findings to clinical applications, including cancer diagnosis and treatment.

Synergistic digestion of banana pseudo-stems with chicken manure to improve methane production: Semi-continuous manipulation and microbial community analysis.

This study aimed to investigate the interactions between banana pseudo-stems (BPS) and chicken manure (CM) during anaerobic co-digestion (AcoD) in batch and semi-continuous experiments. The batch experiments results showed that the methane yield was the highest (193.7 mL/g VS) in AcoD with BPS: CM ratio of 4:1, which was increased by 57.2% and 66.1%, respectively. Semi-continuous experiments revealed that AcoD resulted in higher methane production. Monitoring of the system parameters indicated that AcoD could better adapt to the increasing organic loading rate, with better system stability and methane production efficiency. The microbial analysis illustrated that AcoD increased the relative abundance of hydrolytic bacteria such as Firmicutes, Patescibacteria, and Bacteroidetes. With regard to archaea, AcoD improved the abundance of Methanosaeta, the major acetoclastic methanogens. These changes in the microbial flora allowed AcoD to remain stable while efficiently producing methane and improved the BPS and CM processing efficiency.

Nitroalkanes as electrophiles: synthesis of triazole-fused heterocycles with neuroblastoma differentiation activity.

We discovered a reaction of nitroalkanes with 2-hydrazinylquinolines, 2-hydrazinylpyridines and bis-2,4-dihydrazinylpyrimidines in polyphosphoric acid (PPA) affording 1,2,4-triazolo[4,3-a]quinolines, 1,2,4-triazolo[4,3-a]pyridines and bis[1,2,4]triazolo[4,3-a:4',3'-c]pyrimidines, respectively. The reaction expands the scope of heterocyclic annulations involving phosphorylated nitronates, believed to be the electrophilic intermediates formed from nitroalkanes in PPA. Several of the synthesized triazoles showed promising anticancer activity by inducing differentiation in neuroblastoma cancer cells. Due to the urgent need for novel differentiation agents for neuroblastoma therapy, this finding warrants further evaluation of this class of compounds against neuroblastoma.

The PLAGL2/MYCN/miR-506-3p interplay regulates neuroblastoma cell fate and associates with neuroblastoma progression.

BACKGROUND: The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. METHODS: By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. RESULTS: We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. CONCLUSIONS: Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

ELF4 Is a Target of miR-124 and Promotes Neuroblastoma Proliferation and Undifferentiated State.

13-Cis-retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF-miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. IMPLICATIONS: miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4, which drives neuroblastoma proliferation and its undifferentiated phenotype.

Evaluation of squeezing pretreatment for improving methane production from fresh banana pseudo-stems.

Banana pseudo-stems (BPS) are an abundant and low-lignin-content lignocellulosic biomass for methane production. However, the high-water content in BPS increases the transport costs, and the resistant structure of BPS hinders methane production. In this study, squeezing of BPS as a pretreatment was evaluated for improving anaerobic digestion (AD). After 20-d digestion, methane production from squeezed BPS was 204.2 ± 6.2 mL/(g volatile solids (VS) of feedstock), which was 41.2% more than that from untreated BPS. This increase was mainly attributed to the improvement of physical properties (e.g. water absorbing capacity) and the change in the resistant structure of BPS after squeezing, which promoted good contact between microbes and substrate during AD. The measured methane production was described using a modified Gompertz model and the results showed that anaerobic process would take less time and occur faster when pretreated BPS was used as the substrate. The energy produced during AD of squeezed BPS, after deducting the energy used by the squeezer, resulted in an energy surplus of 26.2%.

Synthesis of Spiro[indole-3,5'-isoxazoles] with Anticancer Activity via a Formal [4 + 1]-Spirocyclization of Nitroalkenes to Indoles.

An acid-assisted [4 + 1]-cycloaddition of indoles with nitrostyrenes affords 4' H-spiro[indole-3,5'-isoxazoles] in a diastereomerically pure form. Several of these spirocyclic molecules exhibit promising anticancer activity by reducing viability and inducing differentiation of neuroblastoma cells.

microRNA-2110 functions as an onco-suppressor in neuroblastoma by directly targeting Tsukushi.

microRNA-2110 (miR-2110) was previously identified as inducing neurite outgrowth in a neuroblastoma cell lines BE(2)-C, suggesting its differentiation-inducing and oncosuppressive function in neuroblastoma. In this study, we demonstrated that synthetic miR-2110 mimic had a generic effect on reducing cell survival in neuroblastoma cell lines with distinct genetic backgrounds, although the induction of cell differentiation traits varied between cell lines. In investigating the mechanisms underlying such functions of miR-2110, we identified that among its predicted target genes down-regulated by miR-2110, knockdown of Tsukushi (TSKU) expression showed the most potent effect in inducing cell differentiation and reducing cell survival, suggesting that TSKU protein plays a key role in mediating the functions of miR-2110. In investigating the clinical relevance of miR-2110 and TSKU expression in neuroblastoma patients, we found that low tumor miR-2110 levels were significantly correlated with high tumor TSKU mRNA levels, and that both low miR-2110 and high TSKU mRNA levels were significantly correlated with poor patient survival. These findings altogether support the oncosuppressive function of miR-2110 and suggest an important role for miR-2110 and its target TSKU in neuroblastoma tumorigenesis and in determining patient prognosis.

LMO1 functions as an oncogene by regulating TTK expression and correlates with neuroendocrine differentiation of lung cancer.

LMO1 encodes a protein containing a cysteine-rich LIM domain involved in protein-protein interactions. Recent studies have shown that LMO1 functions as an oncogene in several cancer types, including non-small cell lung cancer (NSCLC). However, the function of LMO1 in other histological subtypes of lung cancer, such as small cell lung cancer (SCLC), was not investigated. In analyzing the expression of LMO1 across a panel of lung cell lines, we found that LMO1 expression levels were significantly and dramatically higher in SCLC cells, an aggressive neuroendocrine subtype of lung cancer, relative to NSCLC and normal lung cells. In NSCLC cells, LMO1 mRNA levels were significantly correlated with expression of neuroendocrine differentiation markers. Our in vitro investigations indicated that LMO1 had the general property of promoting cell proliferation in lung cancer cells representing different histological subtypes, suggesting a general oncogenic function of LMO1 in lung cancer. In investigating the clinical relevance of LMO1 as an oncogene, we found that a high tumor level of the LMO1 mRNA was an independent predictor of poor patient survival. These results suggest that LMO1 acts as an oncogene, with expression correlated with neuroendocrine differentiation of lung cancer, and that it is a determinant of lung cancer aggressiveness and prognosis. By combining gene expression correlations with patient survival and functional in vitro investigations, we further identified TTK as mediating the oncogenic function of LMO1 in lung cancer cells.

miR-195 targets cyclin D3 and survivin to modulate the tumorigenesis of non-small cell lung cancer.

miR-195 has recently been reported to function as a tumor suppressor in various cancers, including non-small cell lung cancer (NSCLC). However, the mechanisms by which miR-195 represses the tumorigenesis of NSCLC cells are not fully understood. We performed a high-throughput screen using an miRNA mimic library and confirmed the identification of miR-195 as a tumor suppressor in NSCLC. We demonstrated that overexpression or induced expression of miR-195 in lung tumors slows tumor growth and that repression of miR-195 accelerates tumor growth. In addition, we found that knockout of miR-195 promotes cancer cell growth. We demonstrated that miR-195 targets cyclin D3 to cause cell cycle arrest at the G1 phase and that miR-195 targets survivin to induce apoptosis and senescence in NSCLC cells. Overexpression of cyclin D3 or survivin reverses the effects of miR-195 in NSCLC cells. Through the analysis of data from The Cancer Genome Atlas, we confirmed that the expression of miR-195 is lower in tumors than in adjacent normal tissues and that low expression of miR-195 is associated with poor survival in both lung adenocarcinoma and squamous cell carcinoma patients. Specifically, we found that BIRC5, which codes for survivin, is upregulated in both adenocarcinoma and squamous cell carcinoma tissues and that high expression of BIRC5 is associated with poor survival in adenocarcinoma, but not squamous cell carcinoma. In addition, the ratio of miR-195 level to BIRC5 level is associated with both recurrence-free and overall survival in lung adenocarcinoma. Our results suggest that the miR-195/BIRC5 axis is a potential target for treatment of lung adenocarcinoma specifically, and NSCLC in general.