LncRNA LBX2-AS1 promotes colorectal cancer progression and 5-fluorouracil resistance.

BACKGROUND: Recent reports suggest that the long non-coding RNA LBX2 antisense RNA 1 (LBX2-AS1) acts as an important regulator in cancer progression, but its significance in colorectal cancer (CRC) remains undetermined. METHODS: LBX2-AS1 expression levels in CRC were determined from the GEPIA database and CRC tissues to investigate clinical relevance. meRIP-PCR assays investigated the molecular mechanisms underlying the function of m6A in LBX2-AS1. Loss of function experiments was used to define the role of LBX2-AS1 in the progression of CRC. The ceRNA function of LBX2-AS1 was evaluated by RNA immunoprecipitation. In vitro and PDX models were used to determine if LBX2-AS1 promotes 5-fluorouracil resistance. RESULTS: Data from the TCGA and our institutional patient cohorts established that LBX2-AS1 levels were significantly upregulated in most CRC tissues relative to normal adjacent colon tissues. Moreover, LBX2-AS1 levels were positively correlated with aggressive disease characteristics, constituting an independent prognostic indicator of overall patient survival. Mechanistic investigations suggested that the increased LBX2-AS1 in CRC was mediated by METTL3-dependent m6A methylation. In vitro experiments indicated that knockdown of LBX2-AS1 inhibited CRC proliferation, migration and invasion with this phenotype linked to LBX2-AS1-mediated regulation of AKT1, acting as a ceRNA to sponge miR-422a. Ex vivo analysis of patient-derived CRC xenografts showed that low LBX2-AS1 expression cases exhibited 5-FU responsiveness and clinical investigations confirmed that low LBX2-AS1 expression was associated with improved clinical benefits from 5-FU therapy. CONCLUSIONS: Together these results suggest that LBX2-AS1 may serve as a therapeutic target and predictor of 5-FU benefit in CRC patients.

Tropomodulin 3 modulates EGFR-PI3K-AKT signaling to drive hepatocellular carcinoma metastasis.

The mechanism of hepatocellular carcinoma (HCC) metastasis remains poorly understood. Tropomodulin 3 (TMOD3) is a member of the pointed end capping protein family that contributes to invasion and metastasis in several types of malignancies. It has been found to be crucial for the membranous skeleton and embryonic development, although, its role in HCC progression remains largely unclear. We observed increased levels of Tmod3 in HCCs, especially in extrahepatic metastasis. High Tmod3 expression correlated with aggressive carcinoma and poor patient with HCC survival. Loss-of-function studies conducted by us determined Tmod3 as an oncogene that promoted HCC growth and metastasis. Mechanistically, Tmod3 increases transcription of matrix metalloproteinase-2, -7, and -9 which required PI3K-AKT. Interaction between Tmod3 and epidermal growth factor receptor (EGFR) that supports the activation of EGFR phosphorylation, is essential for signaling activation of PI3K-AKT viral oncogene homolog. These findings reveal that Tmod3 enhances aggressive behavior of HCC both in vitro and in vivo by interacting with EFGR and by activating the PI3K-AKT signaling pathway.

Erratum: miR-365a-3p regulates ADAM10-JAK-STAT signaling to suppress the growth and metastasis of colorectal cancer cells: Erratum.

[This corrects the article DOI: 10.7150/jca.42731.].

Suppression of lung cancer metastasis-related protein 1 (LCMR1) inhibits the growth of colorectal cancer cells.

Lung cancer metastasis-related protein 1 (LCMR1) is a critical subunit of the mediator complex, and plays an important role in the elaborate regulation of gene transcription. However, the functional role of LCMR1 in colorectal carcinoma (CRC) has not been clarified. In this study, LCMR1 expression in CRC specimens was quantified by using the quantitative reverse transcription polymerase chain reaction method. The effect of downregulation of LCMR1 by lentivirus-mediated small hairpin RNA (shRNA) on CRC cell proliferation and tumorigenesis was explored. There was a higher expression of LCMR1 in CRC tissue in comparison with adjacent normal colon tissue (P < 0.05). LCMR1 gene was effectively knocked down in human CRC RKO and DLD-1 cells that infected with lentivirus delivering shRNA against LCMR1, which resulted in inhibition of cell proliferation and augmentation of G0/G1 phase proportion. Moreover, the tumorigenicity of RKO cells was also dramatically inhibited after LCMR1 was knocked down. In conclusion, our results suggest that LCMR1 promotes CRC cell growth, and lentivirus-mediated silencing of LCMR1 may contribute to the gene therapy for CRC.

Expression of TMED3 is independently associated with colorectal cancer prognosis.

Colorectal cancer (CRC) is the third most common cancer type and one of the deadliest cancers worldwide. Transmembrane p24 trafficking protein 3 (TMED3) has previously been indicated to suppress CRC metastasis, but its clinical significance has remained undetermined. In the present study, the expression of TMED3 was indicated to be elevated at the mRNA and protein levels in CRC tumor samples relative to that in para-cancerous healthy tissue samples (P<0.05). Furthermore, Kaplan-Meier survival analysis revealed a significant negative association between elevated TMED3 protein levels and overall survival of patients with CRC (P<0.001, log-rank test). Multivariate Cox regression analysis additionally determined that elevated TMED3 expression in primary CRC tumors was an independent predictor of poor prognosis (P<0.05). These results revealed that elevated TMED3 expression in CRC was associated with patient survival outcomes, suggesting that TMED3 may be a potential prognostic biomarker for this cancer type.

[Lymph node metastasis and its risk factors in T1-2 staging invasive rectal carcinoma].

OBJECTIVE: To investigate the lymph node metastasis and its risk factors in T1-2 staging invasive rectal carcinoma. METHODS: The data of 1116 patients with rectal cancer treated with total mesorectal excision (TME) technique from January 2000 to April 2009 was analyzed retrospectively. The clinicopathological factors analyzed included gender, age, primary symptom type, number of symptoms, duration of symptom, synchronous polyps, preoperative serum carcino-embryonic antigen level, preoperative serum CA19-9 level, the distance of tumor from the anal verge, tumor size, tumor morphological type, tumor circumferential extent, tumor differentiation and tumor T staging. Statistical analysis was performed by using Logistic regression analysis and Chi-square test. RESULTS: A total of 1116 patients were enrolled, and 358 cases (32.1%) were classified as with T1-2 staging tumor. Two cases (5.6%, 2/36) in patients with a T1 staging tumor were found with lymph node metastasis, and 75 cases (23.3%, 75/322) in patients with a T2 staging tumor, respectively. Compared with patients with T3-4 staging tumor, lymph node metastasis rate of the patients with T1-2 staging tumor was significantly lower [21.5% (77/358) vs. 51.6% (391/758), P < 0.05]. Only the tumor T staging was found as the independent risk factor for the lymph node metastasis in patients with T1-2 staging tumor on multivariate Logistic regression analysis (odds ratio: 5.162; 95%CI: 1.212 to 21.991; P = 0.026). CONCLUSIONS: A substantial proportion of T1-2 staging rectal cancers harbor metastatic lymph nodes and the clinicopathological features except for T staging fail to predict the lymph node metastasis. Further research is warranted to identify the risk factors and guide the clinical practice in patient with T1-2 staging tumor.

Long noncoding RNA LINC00520 accelerates the progression of colorectal cancer by serving as a competing endogenous RNA of microRNA-577 to increase HSP27 expression.

The long noncoding RNA (lncRNA) LINC00520 is an important modulator of the oncogenicity of multiple human cancers. However, whether LINC00520 is involved in the malignant characteristics of colorectal cancer (CRC) has not been extensively studied until recently. Therefore, the present study aimed to detect LINC00520 expression in CRC and evaluate its clinical significance in patients with CRC. Functional experiments were conducted to test the biological roles and underlying mechanisms of LINC00520 in CRC progression. In this study, high-LINC00520 expression was verified in CRC tissues and cell lines, and this high expression was associated with patients' unfavorable clinicopathological parameters and shorter overall survival and disease-free survival. Functionally, interference of LINC00520 resulted in a significant decrease of CRC cell proliferation, migration, colony forming ability, and invasion. Mechanistically, LINC00520 functioned as a competing endogenous RNA by sponging microRNA-577 (miR-577) and thereby increasing heat shock protein 27 (HSP27) expression. Rescue experiments revealed that inhibiting miR-577 or restoring HSP27 could abrogate the effects of LINC00520 silencing on malignant phenotypes of CRC. LINC00520 functioned as an oncogenic lncRNA in CRC, and it facilitated CRC progression by regulating the miR-577/HSP27 axis, suggesting that the LINC00520/miR-577/HSP27 axis is an effective target in anticancer management.

The synthesis of N,N'-disulfanediyl-bis(N'-((E)-benzylidene)acetohydrazide) from (E)-N'-benzylideneacetohydrazide and S8.

Herein we report an oxidative coupling reaction for N-S/S-S bond formation from (E)-N'-benzylideneacetohydrazide and S8 to furnish substituted N,N'-disulfanediyl-bis(N'-((E)-benzylidene) acetohydrazide). It provides a direct approach for the synthesis of disulfides with good yields.

Comparison of Fresh Frozen Tissue With Formalin-Fixed Paraffin-Embedded Tissue for Mutation Analysis Using a Multi-Gene Panel in Patients With Colorectal Cancer.

Background: Next generation sequencing (NGS)-based multi-gene panel tests have been performed to predict the treatment response and prognosis in patients with colorectal cancer (CRC). Whether the multi-gene mutation results of formalin-fixed paraffin-embedded (FFPE) tissues are identical to those of fresh frozen tissues remains unknown. Methods: A 22-gene panel with 103 hotspots was used to detect mutations in paired fresh frozen tissue and FFPE tissue from 118 patients with CRC. Results: In our study, 117 patients (99.2%) had one or more variants, with 226 variants in FFPE tissue and 221 in fresh frozen tissue. Of the 129 variants identified in this study, 96 variants were present in both FFPE and fresh frozen tissues; 27 variants were found in FFPE tissues only; 6 variants were found only in fresh frozen tissues. The mutation results demonstrated >94.0% concordance in all variants, with Kappa coefficient >0.500 in 64.3% (83/129) of variants. At the gene level, concordance ranged from 73.8 to 100.0%, with Kappa coefficient >0.500 in 81.3% (13/16) of genes. Conclusions: The results of mutation analysis performed with a multi-gene panel and FFPE and fresh frozen tissue were highly concordant in patients with CRC, at both the variant and gene levels. There were, however, some important differences in mutation results between the two tissue types. Therefore, fresh frozen tissue should not routinely be replaced with FFPE tissue for mutation analysis with a multi-gene panel. Rather, FFPE tissue is a reasonable alternative for fresh frozen tissue when the latter is unavailable.

miR-365a-3p regulates ADAM10-JAK-STAT signaling to suppress the growth and metastasis of colorectal cancer cells.

Purpose: MicroRNAs (miRNAs) are key regulators of the growth and development of a wide range of cancer types such as colorectal cancer (CRC). A number of previously studies have observed that the levels of miR-365a-3p expression are dysregulated in many cancers, but the specific role of this miRNA in CRC and its association with patient prognosis remains unclear. Methods: The expression of miR-365a-3p in CRC tissues and cell lines was detected by Real-time Quantitative polymerase chain reaction (RT-qPCR), while the relationship between miR-365a-3p expression and clinicopathological characteristics was further analyzed. After increasing the expression of miR-365a-3p by plasmid transfection in CRC cells, we further investigated the cell proliferation, invasion and migration by cell counting kit-8 (CCK-8), and Transwell assays. Epithelial-mesenchymal transition (EMT) pathway was also measured by western blotting. In addition, the relationship among miR-365a-3p, ADAM10 and JAK in CRC, was explored by luciferase reporter assay. Results: In the present study, we determined that CRC cells and clinical samples exhibited decreased miR-365a-3p expression, and this was associated with larger tumor size, lymph node metastasis, and local invasion. Patients with lower expression of miR-365a-3p had significantly decreased recurrence-free survival (RFS) and overall survival (OS) relative to those with higher levels of this miRNA. In a multivariate analysis, we confirmed that reduced miR-365a-3p levels were independently predictive of poorer CRC patient outcomes. In a functional study, we determined that elevated miR-365a-3p expression inhibited the ability of CRC cells to proliferate and metastasize in vitro and in vivo. We further identified ADAM10 as a direct miR-365a-3p target, resulting in the suppression of ADAM10 expression in cells expressing this miRNA and ADAM10 levels were in turn closely linked to JAK/STAT signaling. Conclusion: Our study suggested the ability of miR-365a-3p to inhibit the progression of CRC at least in part via suppressing ADAM10 expression and associated JAK/STAT signaling, thus identifying this signaling axis as a possible prognostic and therapeutic target in CRC.

MicroRNA-95-3p inhibits cell proliferation and metastasis in colorectal carcinoma by HDGF.

BACKGROUND: MicroRNAs (miRNAs) play an important regulatory role in carcinogenesis and cancer progression. MiR-95-3p has been reported to be an oncogene in hepatocellular carcinoma. However, the role of miR-95-3p in colorectal carcinoma (CRC) remains unclear. METHODS: miR-95-3p was validated in an independent validation sample cohort of 215 CRC tissues. Functional assays, Cell proliferation (MTT) assay colony formation, wound healing, transwell and animal xenograft assays were used to determine the oppressor role of miR-95-3p in human CRC progression. Furthermore, Bioinformatics analysis, western blotting and dual-luciferase reporter assay were used to determine the mechanism by which miR-95-3p suppresses progression of CRC cells. RESULTS: In this study, we found that miR-95-3p was downregulated in CRC tissues. The low level of miR-95-3p in CRC tumors was correlated with aggressive clinicopathological characteristics, and it predicted poor prognosis in CRC patients. The overexpression of miR-95-3p significantly inhibited CRC cell proliferation, colony formation and metastasis in vitro and in vivo. Bioinformatic analysis further identified hepatoma-derived growth factor (HDGF) as a novel target of miR-95-3p in CRC cells. These findings suggest that miR-95-3p regulates CRC cell survival, partially through the downregulation of HDGF. CONCLUSIONS: Therefore, the miR-95-3p/HDGF axis might serve as a novel therapeutic target in patients with CRC.

Rectal cancer patients with downstaging after neoadjuvant chemoradiotherapy and radical resection do not benefit from adjuvant chemotherapy.

BACKGROUND: Whether adjuvant chemotherapy is beneficial for rectal cancer patients who respond well to neoadjuvant chemoradiotherapy (NCRT) and undergo radical resection is controversial. This study aimed to assess the effect of adjuvant chemotherapy on the oncological outcomes of ypT0-2N0 rectal cancer patients after NCRT and radical resection, and identify the prognostic factors. METHODS: The clinical and pathological data of rectal cancer patients with ypT0-2N0 who underwent NCRT and radical resection between January, 2010 and June, 2018 were collected and retrospectively analyzed. The oncological outcomes of the chemotherapy (chemo) group and the non-chemotherapy (non-chemo) group were compared. Multivariate analysis, using a Cox proportional hazard model, was performed to identify independent predictors of oncological outcome. RESULTS: Of the 121 rectal cancer patients enrolled, 90 patients received postoperative adjuvant chemotherapy with no fewer than 3 cycles (the chemo group), and the other 31 patients with fewer than 3 cycles (the non-chemo group). There was no significant difference in the 5-year disease-free survival (DFS) or overall survival (OS) rates between the two groups (DFS: 79.1% vs. 82.9%, P=0.442; OS: 87.5% vs. 78.2%, P=0.667). cT4 is an independent risk factor for OS (HR =4.227, 95% CI: 1.128-15.838, P=0.02) and DFS (HR =4.878, 95% CI: 1.752-13.578). Preoperative consolidation chemotherapy with Capeox or FOLFOX after NCRT significantly improved the DFS rate (HR =0.212, 95% CI: 0.058-0.776, P=0.019). CONCLUSIONS: Rectal cancer patients with ypT0-2N0 who underwent NCRT and radical resection did not benefit significantly from postoperative adjuvant chemotherapy. For these patients, cT4 was an independent risk factor for OS and DFS. Preoperative consolidation chemotherapy with Capeox or FOLFOX after NCRT can significantly improve DFS.

Expression of annexin a5 is associated with higher tumor stage and poor prognosis in colorectal adenocarcinomas.

GOALS: To gain an insight into the putative role of annexin A5 (ANXA5) in the tumor stage and its clinical outcome. BACKGROUND: ANXA5 is a calcium-binding protein, which has been implicated in the carcinogenesis of several carcinomas. However, the role of ANXA5 in colorectal cancer (CRC) is unclear. STUDY: We investigated the expression of ANXA5 in colorectal adenocarcinoma. This study included 207 consecutive patients with sporadic CRC. Paired colorectal tissue samples and corresponding nonmalignant tissues were obtained by surgical resection. ANXA5 mRNA and protein expression in each tissue were assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical staining. Data were statistically correlated with pathologic parameters and clinical outcome. RESULTS: Real-time reverse transcriptase polymerase chain reaction showed that there is an up-regulation in the mRNA level of ANXA5 in tumors (P<0.001). Immunohistochemical study revealed that high ANXA5 expression was present in 40.58% (84 of 207) of tumors. Univariate analysis showed increased ANXA5 expression correlated with pT stage (P=0.008), liver metastasis (P=0.024), pathologic tumor-node-metastasis stage (P=0.015), Dukes' stage (P=0.017), recurrence (P=0.024), cancer-related death (P=0.028), recurrence-free probability (P=0.003), and overall survival (P=0.005). Multivariate analysis showed that ANXA5 expression and liver metastasis significantly correlated with recurrence-free probability (P=0.039 and P=0.048, respectively) and overall survival (P=0.012 and P=0.021, respectively) independent of pT stage and pN stage. CONCLUSIONS: From these findings ANXA5 expression seems to be related to the tumor stage and clinical outcome of CRC. Thus ANXA5 could serve as a prognostic marker for tumor progression.

[Factors associated with anastomotic leakage after anterior resection in rectal cancer].

OBJECTIVE: To analyze the factors associated with anastomotic leakage after anterior resection in rectal cancer with the technique of total mesorectal excision (TME). METHODS: From January 2005 and December 2007, 738 consecutive patients with rectal cancer underwent anterior resection. The data of those patients was collected and reviewed retrospectively. The associations between anastomotic leakage and 9 patient-related variables as well as 7 surgical-related variables were examined. RESULTS: Low rectal cancer (located 7 cm or less above the anal edge), non-specialized surgeon and transanal tube use were the risk factors associated with anastomotic leakage on univariate analysis. The anastomotic leakage rate of low-rectal cancer was significantly higher than that of high-rectal cancer (5.9% vs. 0.9%, P = 0.003). The anastomotic leakage rate of the cases operated by colorectal surgeon was significantly lower than that of the cases operated by non-specialized surgeon (3.9% vs. 11.3%, P = 0.031). There was a tendency for colorectal surgeons to operate on a greater proportion of low rectal cancer than non-specialized surgeons (72.1% vs. 52.8%, P = 0.003). The leakage rate of transanal tube group was unexpectedly higher than that in patients without transanal tube (14.5% vs. 3.6%, P < 0.001). On multivariate logistic regression analysis, diabetes mellitus (P = 0.027), distance less than 1 cm from tumor to distal resection margin (P = 0.009) and defunctioning stoma (P = 0.031) were also associated with anastomotic leakage rate besides low rectal cancer, non-specialized surgeon and transanal tube use. In a further analysis of 522 patients with low rectal cancer, the leakage rate of defunctioning stoma group was significantly lower than that of non-stoma group (2.9% vs. 8.5%, P = 0.007). By contract, the leakage rate of transanal tube group was still higher than that in patients without transanal tube (15.1% vs. 4.9%, P = 0.008) because of its poor protective effect as well as the selection bias. CONCLUSIONS: Low-rectal cancer, non-specialized surgeons and diabetes mellitus are risk factors of anastomotic leakage after rectal surgery. A defunctioning stoma was effective in preventing leakage after low-rectal cancer surgery.

Downregulation of miR-196-5p Induced by Hypoxia Drives Tumorigenesis and Metastasis in Hepatocellular Carcinoma.

In hepatocellular carcinoma (HCC), the hypoxic tumor microenvironment can drive enhance tumor malignancy and recurrence. The microRNA (miRNA) miR-196-5p has been shown to modulate the progression of several cancer types, but its roles in HCC remain uncertain. In the present report we observed significant miR-196-5p downregulation in HCC tissues and cells, and we found that the expression of this miRNA significantly impaired the proliferation and metastatic potential of HCC in vitro and in vivo. We identified high-mobility group AT-hook 2 (HMGA2) as a miR-196-5p target gene that was associated with the ability of miR-196-5p to modulate the progression of HCC. Expression of miR-196-5p and HMGA2 were correlated with the clinical characteristics and poor outcomes in patients with HCC. Finally, we found that hypoxic conditions were linked with reduced miR-196-5p expression in the context of HCC. Together these results highlight the role for miR-196-5p as an inhibitor of the proliferation and metastasis of HCC via the targeting of HMGA2, with this novel hypoxia/miR-196-5p/HMGA2 pathway serving as a potential target for future therapeutic intervention.

MicroRNA-212-3p inhibits the Proliferation and Invasion of Human Hepatocellular Carcinoma Cells by Suppressing CTGF expression.

MicroRNA-212-3p inhibits several human cancers but its effects on hepatocellular carcinoma (HCC) remain unclear. In this study, we show that miR-212-3p is down-regulated in HCC cell lines and tissues, and correlates with vascular invasion (p = 0.001), and the absence of capsule formation (p = 0.009). We found that miR-212-3p influenced the epithelial to mesenchymal transition (EMT) of HCCLM3 and Huh7 cells. Mechanistically, miR-212-3p repressed cell invasion through the suppression of connective tissue growth factor (CTGF). We therefore validate the anti-HCC effects of miR-212-3p through its ability to suppress CTGF and subsequent EMT.

The RNA binding protein neuro-oncological ventral antigen 1 (NOVA1) regulates IL-6 mRNA stability to enhance JAK2-STAT3 signaling in CRC.

The molecular mechanisms governing the metastasis of colorectal cancer (CRC) are incompletely understood. In the present study, we found NOVA1 to be expressed at higher levels in CRC cell lines and tissue samples, and this upregulation was positively correlated with TNM stage (p = 0.034), poor differentiation (p = 0.001), and lymph node metastasis (p = 0.008). Both overall survival (OS) and relapse-free survival (RFS) were both significantly decreased in patients with high NOVA1 expression relative to those with low expression. Through a multivariate analysis, we determined that NOVA1 independently predicted poor outcomes in those with CRC. In further functional studies, we found that NOVA1 expression controlled the proliferation and invasive characteristics of CRC cells via a mechanism wherein NOVA1 bound and stabilized the IL6 mRNA, enhancing IL-6/JAK2/STAT3 signaling to in turn upregulate matrix metalloproteinases (MMPs) 2, 7, and 9. NOVA1 therefore plays key functional roles in regulating CRC progression, and our results further indicate that it serve as a valuable prognostic biomarker and potentially a target for therapeutic treatment in individuals with CRC.

Clinical features and mismatch repair genes analyses of Chinese suspected hereditary non-polyposis colorectal cancer: a cost-effective screening strategy proposal.

China has the largest numbers of hereditary non-polyposis colorectal cancer (HNPCC) patients based on its population of 1.4 billion. However, the clinical data and mismatch repair (MMR) gene analyses have been limited. Here we performed microsatellite instability (MSI) and immunohistochemistry (IHC) analyses on a series of patients with a high-risk for HNPCC: 61 patients with family histories fulfilling Amsterdam criteria II (ACII-HNPCC) or suspected HNPCC criteria (S-HNPCC), and 106 early onset colorectal cancer (CRC) patients. Sixty late-onset CRC patients were used as control. Methylation of the hMLH1 promoter was analyzed on tumors lacking hMLH1 expression. MMR germ-line mutations were screened on patients with tumors classified as MSI-H/L or negative for IHC. We identified 27 germ-line MMR variants in the 167 patients with a high-risk for HNPCC while only one germ-line mutation in hMSH6 was found in the late-onset CRC group. Of those, 23 were pathogenic mutations. The high incidence of gastric and hepatobiliary cancers coupled with the increasing number of small families in China reduces the sensitivity (43.5%, 30.4%) and positive predictive value (PPV) (45.5%, 17.9%) of the ACII- or S-HNPCC criteria. MSI or IHC testing are highly sensitive in detecting pathogenic mutations (sensitivities = 91.3% and 95.6%, respectively), but the PPVs are quite low (25.6% and 27.8%, respectively). Considering that all 12 tumors with pathogenic mutations in hMLH1 also showed promoter unmethylation, the sensitivity of IHC in conjunction with hMLH1 promoter methylation analysis is not reduced, but the PPV was increased from 27.8% to 61.1%, and the total cost was greatly reduced.