cGAS suppresses hepatocellular carcinoma independent of its cGAMP synthase activity.

Cyclic GMP-AMP synthase (cGAS) is a key innate immune sensor that recognizes cytosolic DNA to induce immune responses against invading pathogens. The role of cGAS is conventionally recognized as a nucleotidyltransferase to catalyze the synthesis of cGAMP upon recognition of cytosolic DNA, which leads to the activation of STING and production of type I/III interferon to fight against the pathogen. However, given that hepatocytes are lack of functional STING expression, it is intriguing to define the role of cGAS in hepatocellular carcinoma (HCC), the liver parenchymal cells derived malignancy. In this study, we revealed that cGAS was significantly downregulated in clinical HCC tissues, and its dysregulation contributed to the progression of HCC. We further identified cGAS as an immune tyrosine inhibitory motif (ITIM) containing protein, and demonstrated that cGAS inhibited the progression of HCC and increased the response of HCC to sorafenib treatment by suppressing PI3K/AKT/mTORC1 pathway in cellular and animal models. Mechanistically, cGAS recruits SH2-containing tyrosine phosphatase 1 (SHP1) via ITIM, and dephosphorylates p85 in phosphatidylinositol 3-kinase (PI3K), which leads to the suppression of AKT-mTORC1 pathway. Thus, cGAS is identified as a novel tumor suppressor in HCC via its function independent of its conventional role as cGAMP synthase, which indicates a novel therapeutic strategy for advanced HCC by modulating cGAS signaling.

MyD88 in myofibroblasts enhances nonalcoholic fatty liver disease-related hepatocarcinogenesis via promoting macrophage M2 polarization.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver diseases and has emerged as the leading factor in the pathogenesis of hepatocellular carcinoma (HCC). MyD88 contributes to the development of HCC. However, the underlying mechanism by which MyD88 in myofibroblasts regulates NAFLD-associated liver cancer development remains unknown. RESULTS: Myofibroblast MyD88-deficient (SMAMyD88-/-) mice were protected from diet-induced obesity and developed fewer and smaller liver tumors. MyD88 deficiency in myofibroblasts attenuated macrophage M2 polarization and fat accumulation in HCC tissues. Mechanistically, MyD88 signaling in myofibroblasts enhanced CCL9 secretion, thereby promoting macrophage M2 polarization. This process may depend on the CCR1 receptor and STAT6/ PPARß pathway. Furthermore, liver tumor growth was attenuated in mice treated with a CCR1 inhibitor. CCLl5 (homologous protein CCL9 in humans) expression was increased in myofibroblasts of HCC and was associated with shorter survival of patients with HCC. Thus, our results indicate that MyD88 in myofibroblasts promotes NAFLD-related HCC progression and may be a promising therapeutic target for HCC treatment. CONCLUSION: This study demonstrates that MyD88 in myofibroblasts can promote nonalcoholic fatty liver disease-related hepatocarcinogenesis by enhancing macrophage M2 polarization, which might provide a potential molecular therapeutic target for HCC.

Elucidating the molecular mechanism of water migration in myosin gels of Nemipterus virgatus during low pressure coupled with heat treatment.

The relationship between myosin denaturation, aggregation and water migration in Nemipterus virgatus myosin gels with different treatment processes under optimal low pressure coupled with heat treatment was investigated to clarify the molecular mechanism of water migration. With the different treatment processes, the proportion of bound water of the myosin gels increased significantly (P < 0.05). Denaturation of myosin S1 sub-fragments and α-helical unfolding during different treatment processes led to an increase in ß-sheets content. These promote increased exposure of Try residues and hydrophobic groups of myosin, formation of clathrate hydrates, and reduced mobility of bound water. Furthermore, hydrophobic interactions and disulfide bonds caused the head-head and head-hinge to coalesce into a 3D honeycomb network with greater fractal dimension, less lacunarity, smaller water hole diameter and more water holes. This increased the capillary pressure experienced by the bound water, causing immobile water to migrate towards the bound water. The present study may be necessary to improve the mechanism of water migration in protein gel systems and to promote the industrial application of high pressure processing technology in surimi-based foods.

Characterization of Effects of Different Tea Harvesting Seasons on Quality Components, Color and Sensory Quality of "Yinghong 9" and "Huangyu" Large-Leaf-Variety Black Tea.

Harvesting seasons are crucial for the physicochemical qualities of large-leaf-variety black tea. To investigate the effect of harvesting seasons on physicochemical qualities, the color and sensory characteristics of black tea produced from "Yinghong 9" (Yh) and its mutant "Huangyu" (Hy) leaves were analyzed. The results demonstrated that Hy had better chemical qualities and sensory characteristics, on average, such as a higher content of tea polyphenols, free amino acids, caffeine, galloylated catechins (GaCs) and non-galloylated catechins (NGaCs), while the hue of the tea brew (ΔE*ab and Δb*) increased, which meant that the tea brew was yellower and redder. Moreover, the data showed that the physicochemical qualities of SpHy (Hy processed in spring) were superior to those of SuHy (Hy processed in summer) and AuHy (Hy processed in autumn), and 92.6% of the total variance in PCA score plots effectively explained the separation of the physicochemical qualities of Yh and Hy processed in different harvesting seasons. In summary, Hy processed in spring was superior in its physicochemical qualities. The current results will provide scientific guidance for the production of high-quality large-leaf-variety black tea in South China.

UBR7 inhibits HCC tumorigenesis by targeting Keap1/Nrf2/Bach1/HK2 and glycolysis.

BACKGROUND: Glycolysis metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options. The ubiquitin-proteasome system facilitates the turnover of most intracellular proteins with E3 ligase conferring the target selection and specificity. Ubiquitin protein ligase E3 component N-recognin 7 (UBR7), among the least studied E3 ligases, recognizes its substrate through a plant homeodomain (PHD) finger. Here, we bring into focus on its suppressive role in glycolysis and HCC tumorigenesis, dependent on its E3 ubiquitin ligase activity toward monoubiquitination of histone H2B at lysine 120 (H2BK120ub). METHODS: In this study, we carried out high-throughput RNAi screening to identify epigenetic candidates in regulating lactic acid and investigated its possible roles in HCC progression. RESULTS: UBR7 loss promotes HCC tumorigenesis both in vitro and in vivo. UBR7 inhibits glycolysis by indirectly suppressing HK2 expression, a downstream target of Nrf2/Bach1 axis. Mechanically, UBR7 regulates H2BK120ub to bind to Keap1 promoter through H2BK120ub monoubiquitination, thereby modulating Keap1 expression and downstream Nrf2/Bach1/HK2 signaling. Pharmaceutical and genetic inhibition of glycolytic enzymes attenuate the promoting effect of UBR7 deficiency on tumor growth. In addition, methyltransferase ALKBH5, downregulated in HCC, regulated UBR7 expression in an m6A-dependent manner. CONCLUSIONS: These results collectively establish UBR7 as a critical negative regulator of aerobic glycolysis and HCC tumorigenesis through regulation of the Keap1/Nrf2/Bach1/HK2 axis, providing a potential clinical and therapeutic target for the HCC treatment.

LINC01468 drives NAFLD-HCC progression through CUL4A-linked degradation of SHIP2.

Accumulating evidence suggests that long noncoding RNAs (lncRNAs) are deregulated in hepatocellular carcinoma (HCC) and play a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the current understanding of the role of lncRNAs in NAFLD-associated HCC is limited. In this study, transcriptomic profiling analysis of three paired human liver samples from patients with NAFLD-driven HCC and adjacent samples showed that LINC01468 expression was significantly upregulated. In vitro and in vivo gain- and loss-of-function experiments showed that LINC01468 promotes the proliferation of HCC cells through lipogenesis. Mechanistically, LINC01468 binds SHIP2 and promotes cullin 4 A (CUL4A)-linked ubiquitin degradation, thereby activating the PI3K/AKT/mTOR signaling pathway, resulting in the promotion of de novo lipid biosynthesis and HCC progression. Importantly, the SHIP2 inhibitor reversed the sorafenib resistance induced by LINC01468 overexpression. Moreover, ALKBH5-mediated N6-methyladenosine (m6A) modification led to stabilization and upregulation of LINC01468 RNA. Taken together, the findings indicated a novel mechanism by which LINC01468-mediated lipogenesis promotes HCC progression through CUL4A-linked degradation of SHIP2. LINC01468 acts as a driver of HCC progression from NAFLD, highlights the potential of the LINC01468-SHIP2 axis as a therapeutic target for HCC.

mTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A.

BACKGROUND: Exploiting cancer metabolism during nutrient availability holds immense potential for the clinical and therapeutic benefits of hepatocellular carcinoma (HCC) patients. Dietary methionine is a metabolic dependence of cancer development, but how the signal transduction integrates methionine status to achieve the physiological demand of cancer cells remains unknown. METHODS: Low or high levels of dietary methionine was fed to mouse models with patient-derived xenograft or diethyl-nitrosamine induced liver cancer. RNA sequence and metabolomics were performed to reveal the profound effect of methionine restriction on gene expression and metabolite changes. Immunostaining, sphere formation assays, in vivo tumourigenicity, migration and self-renewal ability were conducted to demonstrate the efficacy of methionine restriction and sorafenib. RESULTS: We discovered that mTORC1-c-Myc-SIRT4 axis was abnormally regulated in a methionine-dependent manner and affected the HCC progression. c-Myc rewires methionine metabolism through TRIM32 mediated degradation of SIRT4, which regulates MAT2A activity by ADP-ribosylation on amino acid residue glutamic acid 111. MAT2A is a key enzyme to generate S-adenosylmethionine (SAM). Loss of SIRT4 activates MAT2A, thereby increasing SAM level and dynamically regulating gene expression, which triggers the high proliferation rate of tumour cells. SIRT4 exerts its tumour suppressive function with targeted therapy (sorafenib) by affecting methionine, redox and nucleotide metabolism. CONCLUSIONS: These findings establish a novel characterization of the signaling transduction and the metabolic consequences of dietary methionine restriction in malignant liver tissue of mice. mTORC1, c-Myc, SIRT4 and ADP ribosylation site of MAT2A are promising clinical and therapeutic targets for the HCC treatment.

Hepatocyte-Specific Smad4 Deficiency Alleviates Liver Fibrosis via the p38/p65 Pathway.

Liver fibrosis is a wound-healing response caused by the abnormal accumulation of extracellular matrix, which is produced by activated hepatic stellate cells (HSCs). Most studies have focused on the activated HSCs themselves in liver fibrosis, and whether hepatocytes can modulate the process of fibrosis is still unclear. Sma mothers against decapentaplegic homologue 4 (Smad4) is a key intracellular transcription mediator of transforming growth factor-ß (TGF-ß) during the development and progression of liver fibrosis. However, the role of hepatocyte Smad4 in the development of fibrosis is poorly elucidated. Here, to explore the functional role of hepatocyte Smad4 and the molecular mechanism in liver fibrosis, a CCl4-induced liver fibrosis model was established in mice with hepatocyte-specific Smad4 deletion (Smad4Δhep). We found that hepatocyte-specific Smad4 deficiency reduced liver inflammation and fibrosis, alleviated epithelial-mesenchymal transition, and inhibited hepatocyte proliferation and migration. Molecularly, Smad4 deletion in hepatocytes suppressed the expression of inhibitor of differentiation 1 (ID1) and the secretion of connective tissue growth factor (CTGF) of hepatocytes, which subsequently activated the p38 and p65 signaling pathways of HSCs in an epidermal growth factor receptor-dependent manner. Taken together, our results clearly demonstrate that the Smad4 expression in hepatocytes plays an important role in promoting liver fibrosis and could therefore be a promising target for future anti-fibrotic therapy.

PSMB5 overexpression is correlated with tumor proliferation and poor prognosis in hepatocellular carcinoma.

Aberrant expression of members of the proteasome subunit beta (PSMB) family (including PSMB2, PSMB4, PSMB7 and PSMB8) has been reported in hepatocellular carcinoma (HCC). However the role of PSMB5 in HCC is unclear. To address this issue, we examined the expression of PSMB5 in HCC tissues using the The Cancer Genome Atlas, International Cancer Genome Consortium and Gene Expression Omnibus databases. A quantitative real-time PCR and immunohistochemistry were performed to validate the expression of PSMB5 in HCC. The survival mutation status and immune cell infiltration of PSMB5 were also evaluated in HCC. We then examined the effect of knocking down PSMB5 expression through RNA interference in the HCC cell line Huh7. High expression of PSMB5 was observed in HCC tissues and was associated with poor prognosis. PSMB5 expression and clinical characteristics were then incorporated to build a prognostic nomogram. We observed that PSMB5 expression was closely related to the abundance of B cells, CD4+ T cells, CD8+ T cells, dendritic cell macrophages and neutrophils. Moreover silencing of PSMB5 in Huh7 significantly suppressed cell proliferation and migration at the same time as increasing apoptosis. Inhibition of the phosphatidylinositol-3-kinase/Akt/mechanistic target of rapamycin pathway was observed after PSMB5 downregulation in Huh7 cells. Our findings suggest that PSMB5 may promote the proliferation of HCC cells by inactivating the phosphatidylinositol-3-kinase/Akt/mechanistic target of rapamycin signaling pathway and thus PSMB5 may have potential as a biomarker for diagnosis and prognosis of HCC.

Insight into the mechanism of optimal low-level pressure coupled with heat treatment to improve the gel properties of Nemipterus virgatus surimi combined with water migration.

The effect of optimal low-level pressure coupled with heat treatment (OPH treatment) on the gel properties and water migration of Nemipterus virgatus surimi was studied and compared with optimal high-pressure processing treatment (OP treatment) and traditional two-stage heat treatment (H treatment). Furthermore, the mechanism of OPH treatment in improving the gel properties were explored based on myosin. OPH treatment was found to be more conducive in improving the gel strength and water-holding capacity (WHC) of surimi gel than H or OP treatments. Moreover, OPH treatment induced an increase in the proportion of myosin ß-sheets and exposed more intramolecular Tyr residues as compared to the other two treatments, which promoted myosin to form large protein clusters through disulfide bonds and hydrophobic interactions, and a honeycomb three-dimensional network structure with larger fractal dimension, lower porosity, smaller water hole diameter, and a greater number of water holes, was obtained. These helped the OPH-induced surimi gel lock in more unfrozen bound water and immobile water, and ultimately rendered better gel properties.

MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization.

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl4-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAPMyD88-/- mice) or aHSCs (α-SMAMyD88-/- mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.

MyD88 in myofibroblasts regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation.

Hepatic stellate cells (HSCs) and cancer-associated fibroblasts (CAFs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). MyD88 controls the expression of several key modifier genes in liver tumorigenesis; however, whether and how MyD88 in myofibroblasts contributes to the development of fibrosis-associated liver cancer remains elusive. Here, we used an established hepatocarcinogenesis mouse model involving apparent liver fibrogenesis in which MyD88 was selectively depleted in myofibroblasts. Myofibroblast MyD88-deficient (Fib-MyD88 KO) mice developed significantly fewer and smaller liver tumor nodules. MyD88 deficiency in myofibroblasts attenuated liver fibrosis and aerobic glycolysis in hepatocellular carcinoma tissues. Mechanistically, MyD88 signaling in myofibroblasts increased the secretion of CCL20, which promoted aerobic glycolysis in cancer cells. This process was dependent on the CCR6 receptor and ERK/PKM2 signaling. Furthermore, liver tumor growth was greatly relieved when the mice were treated with a CCR6 inhibitor. Our data revealed a critical role for MyD88 in myofibroblasts in the promotion of hepatocellular carcinoma by affecting aerobic glycolysis in cancer cells and might provide a potential molecular therapeutic target for HCC. © 2021 The Pathological Society of Great Britain and Ireland.

MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs.

Chronic liver disease mediated by the activation of hepatic stellate cells (HSCs) leads to liver fibrosis. The signal adaptor MyD88 of Toll-like receptor (TLR) signaling is involved during the progression of liver fibrosis. However, the specific role of MyD88 in myeloid cells in liver fibrosis has not been thoroughly investigated. In this study, we used a carbon tetrachloride (CCl4)-induced mouse fibrosis model in which MyD88 was selectively depleted in myeloid cells. MyD88 deficiency in myeloid cells attenuated liver fibrosis in mice and decreased inflammatory cell infiltration. Furthermore, deficiency of MyD88 in macrophages inhibits the secretion of CXC motif chemokine 2 (CXCL2), which restrains the activation of HSCs characterized by NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation. Moreover, targeting CXCL2 by CXCR2 inhibitors attenuated the activation of HSCs and reduced liver fibrosis. Thus, MyD88 may represent a potential candidate target for the prevention and treatment of liver fibrosis.

Sanitation of tomatoes based on a combined approach of washing process and pulsed light in conjunction with selected disinfectants.

In this study, we evaluated the performance of a large-scale decontamination system based on a washing process in combination with pulsed light (PL) exposure and H2O2/chlorine. In order to identify optimum processing condition, we first evaluated the effect of single and combined PL treatments on the inactivation of Salmonella on grape tomatoes using a small sample size of 50 g. Two inoculation methods, spot and dip, were used to simulate different contamination scenarios and two wash water quality, clear tap water and turbid tap water with extremely high levels of organic load and soil, were used to represent clean and very dirty wash water. In general, the combined PL-Chlorine and PL-H2O2 treatments were more or as effective as chlorine washing in killing Salmonella on grape tomatoes and were able to keep residual Salmonella in wash water below the detection limit of 2 CFU/mL. The PL alone and combined PL-H2O2 treatments were chosen and further tested for their decontamination efficacy under turbid wash water condition using large sample sizes, 300, 1000 and 2000 g. Sample size did not negatively affect the single and combined PL treatments on the inactivation of Salmonella on grape tomatoes. The combined PL-H2O2 treatment in general showed better inactivation effect of Salmonella on tomatoes than the PL alone treatment. Additionally, the combined PL-H2O2 treatment reduced Salmonella in turbid wash water below the detection limit of 2 CFU/mL in the majority of cases. In conclusion, the combined PL-H2O2 treatment could potentially be used as an environmentally friendly alternative to chlorine washing for tomato decontamination and cleaning.

Strategies to enhance fresh produce decontamination using combined treatments of ultraviolet, washing and disinfectants.

This study investigated the effect of a water-assisted ultraviolet system (WUV; samples were treated by UV while being immersed in agitated water) on the inactivation of Salmonella on baby spinach, iceberg lettuce, blueberry, grape tomato, and baby-cut carrot. The Salmonella inactivation effect of the WUV system was tested in two scales, and three disinfectants, chlorine, peroxyacetic acid (PAA) and hydrogen peroxide (H2O2), were tested in combination with the system to see whether the Salmonella inactivation effect could be enhanced. The fresh produce samples were dip-inoculated with a Salmonella cocktail to final concentrations of 4.6-7.6 log CFU/g. To simulate the washing process in the industry, fresh produce extracts and/or silicon dioxide were added in the wash water to adjust chemical oxygen demand to ~2000 mg/L and turbidity to >60 NTU. In general, the decontamination efficacy of WUV treatments followed this order: Tomato > Carrot > Lettuce ≈ Blueberry > Spinach. In the small-scale study, WUV alone was able to achieve 0.9, 2.6, >3.6, 1.7, and 2.0 log CFU/g reductions of Salmonella on fresh produce for spinach, lettuce, tomato, blueberry, and carrot, respectively. For all fresh produce items, WUV combined with PAA could achieve significantly (P < 0.05) higher Salmonella reduction on fresh produce than chlorine wash and PAA wash. The WUV treatments combined with chlorine or PAA were able to keep residual Salmonella in wash water below the detection limit (2 CFU/mL) for almost all the replicates. Similar Salmonella reductions on fresh produce and in wash water were found in the large-scale study. Considering the decontamination efficacy on fresh produce, the ability to disinfect the wash water, and the cost, we recommend chlorine wash for baby spinach, WUV alone for grape tomato and WUV combined with PAA for iceberg lettuce, blueberry and baby-cut carrot.

[Analysis of effects of arthroscopic treatment for recurrent patellar dislocation by lateral patellar retinacular release outside the synovial bursa of knee joint combined with reconstruction of medial patellofemoral ligament].

OBJECTIVE: To introduce an arthroscopic technique in managing recurrent dislocation of the patella and its clinical results. METHODS: Sixteen patients with recurrent patellar dislocation were reviewed, including 3 males and 13 females. The average age was 17.6 years old (ranged from 14 to 32 years). The patients suffering from patellar sub-luxation averaged 18.5 months (ranged from 6 to 23 months)before operation. These patients were treated with lateral patellar retinacular release outside the synovial bursa of knee joint and medial patellofemoral ligament reconstruction using the semitendinosus tendon free autograft. The Lyshohm scores before and after operation were used to evaluate outcomes at the final follow-up. RESULTS: All the patients were followed up, and the duration ranged from 6 to 48 months, with an average of 12 months. There was no recurrence. The Q angle decreased from (16.4 ± 3.7)° to (10.1 ± 1.4)°; insall index decreased from 1.37 ± 0.25 to 1.28 ± 0.23; congruence angle decreased from (21.3 ± 2.6)° to (5.86 ± 2.23)°; Lysholm score improved from 76.1 ± 5.2 to 89.8 ± 4.1 at 6 months after operation. CONCLUSION: Compared with conventional procedure, arthroscopic surgery for recurrent dislocation of the patella achieves excellent outcomes with minimum invasion.

Inactivation of a norovirus by high-pressure processing.

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20 degrees C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log(10) PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5 degrees C; a 5-min pressure treatment of 350 MPa at 30 degrees C inactivated 1.15 log(10) PFU of virus, while the same treatment at 5 degrees C resulted in a reduction of 5.56 log(10) PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5 degrees C and 20 degrees C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5 degrees C was sufficient to inactivate 4.05 log(10) PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.

Characterization of a spontaneous, pressure-tolerant Listeria monocytogenes Scott A ctsR deletion mutant.

A spontaneous, pressure-tolerant mutant of Listeria monocytogenes Scott A, designated 2-1, was isolated after several rounds of pressure treatments at 500 MPa for 10 min. Mutant 2-1 was almost 100,000-fold more resistant than the wild type to a pressure of 350 MPa, and about 100-fold more resistant to 450 MPa when pressurized in growth medium. Approximately ten times more mutant cells than wild-type cells survived a 20-min exposure to 55 degrees C, and the mutant appears also to be more resistant to 0.2% H(2)O(2), although the difference could not be confirmed statistically. About 10 times more wild-type than mutant cells survived exposure to growth medium adjusted to pH 2.5 with HCl. The mutant is about 16-fold more sensitive to nisin than the wild type. Mutant 2-1 is non-motile, produces hemolytic activity, is able to grow in fetal calf serum as well as the wild type, and exhibits a lower level of invasiveness of human ileocecal adenocarcinoma cells than the wild type. The mutation in strain 2-1 is a deletion in the ctsR gene that results in the predicted production of truncated CtsR of 20 amino acids compared to a CtsR of 152 amino acids in the wild type. With the exception of its response to pH and possibly also to H(2)O(2), mutant 2-1 shares most of the phenotypes of the previously described ctsR mutant, AK01. The isolation of another spontaneous, pressure-resistant ctsR mutant confirms the central role of this regulatory gene in pressure tolerance of L. monocytogenes. Although such mutants appear of lesser concern to human health then the wild type, current detection methods for Listeria monocytogenes are not able to distinguish between these mutants and wildtype cells.