Derivation and validation of a nomogram incorporating modifiable lifestyle factors to predict development of colorectal adenomas after negative index colonoscopy.

This retrospective cohort study aimed to identify baseline patient characteristics involving modifiable lifestyle factors that are associated with the development of colorectal adenomas, and establish and validate a nomogram for risk predictions among high-risk populations with negative index colonoscopy. A total of 83,076 participants who underwent an index colonoscopy at the Tianjin Union Medical Center between 2004 and 2019 were collected. According to meticulous inclusion and exclusion criteria, 249 subjects were enrolled and categorized into the primary and validation cohorts. Based on the primary cohort, we utilized the LASSO-Cox regression and the univariate/multivariate Cox proportional hazards (Cox-PH) regression parallelly to select variables, and incorporated selected variables into two nomogram models established using the multivariate Cox-PH regression. Comparison of the Akaike information criterion and the area under the receiver operating characteristic curve of the two models demonstrated that the nomogram model constituted by four covariates retained by the LASSO-Cox regression, including baseline age, body mass index, physical activity and family history of colorectal cancer (CRC) in first-degree relatives, performed better at predicting adenoma-free survival probabilities. Further validation including the concordance index, calibration plots, decision curve analysis and Kaplan-Meier survival curves also revealed good predictive accuracy, discriminating ability, clinical utility and risk stratification capacity of the nomogram model. Our nomogram will assist high-risk individuals with negative index colonoscopy to prevent colorectal adenoma occurrence and CRC morbidity with improved cost-effectiveness.

Andrographolide sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the EGFR/AKT and PDGFRß/AKT signaling pathways.

BACKGROUND: Cetuximab, an inhibitor targeting EGFR, is widely applied in clinical management of colorectal cancer (CRC). Nevertheless, drug resistance induced by KRAS-mutations limits cetuximab's anti-cancer effectiveness. Furthermore, the persistent activation of EGFR-independent AKT is another significant factor in cetuximab resistance. Nevertheless, the mechanism that EGFR-independent AKT drives cetuximab resistance remains unclear. Thus, highlighting the need to optimize therapies to overcome cetuximab resistance and also to explore the underlying mechanism. PURPOSE: This work aimed to investigate whether and how andrographolide enhance the therapeutic efficacy of cetuximab in KRAS-mutant CRC cells by modulating AKT. METHODS: The viabilities of CRC cell lines were analyzed by CCK-8. The intracellular proteins phosphorylation levels were investigated by Human Phospho-kinase Antibody Array analysis. Knockdown and transfection of PDGFRß were used to evaluate the role of andrographolide on PDGFRß. The western blotting was used to investigate Wnt/ß-catenin pathways, PI3K/AKT, and EMT in KRAS-mutant CRC cells. The animal models including subcutaneous tumor and lung metastasis were performed to assess tumor response to therapy in vivo. RESULTS: Andrographolide was demonstrated to decrease the expression of PI3K and AKT through targeting PDGFRß and EGFR, and it enhanced cetuximab effect on KRAS-mutant CRC cells by this mechanism. Meanwhile, andrographolide helped cetuximab to inhibit Wnt/ß-catenin, CRC cell migration and reduced Vimentin expression, while increasing that of E-cadherin. Lastly, co-treatment with cetuximab and andrographolide reduced the growth of KRAS-mutant tumors and pulmonary metastases in vivo. CONCLUSIONS: Our findings suggest that andrographolide can overcome the KRAS-mutant CRC cells' resistance to cetuximab through inhibiting the EGFR/PI3K/AKT and PDGFRß /AKT signaling pathways. This research provided a possible theory that andrographolide sensitizes KRAS-mutant tumor to EGFR TKI.

Colonoscopy compliance and diagnostic yield in a large population-based colorectal cancer screening programme.

OBJECTIVES: With the intention of providing a reference for secondary prevention, our study provides some insight on diagnostic yield of factors influencing compliance with colonoscopy and the presence of advanced adenomas (AA). METHODS: We conducted large-scale CRC screening among local Tianjin residents aged 40-75 years between 2012 and 2019. A high-risk factor questionnaire (HRFQ) was distributed to each participant, followed by the performance of a fecal immunochemical test (FIT). Participants who tested positively for any of these items were advised to undergo a colonoscopy. Relevant basic information was collected from participants during CRC screening, and the screening data were sorted and analysed. RESULTS: A total of 5,670,924 people participated in CRC screening by the end of 2019, including 275,708 people in the high-risk group, and 74,685 (27.1%) people who underwent colonoscopy. The results of the logistic regression model demonstrated that participants with a history of mucous bloody stool (OR = 8.20, 95% CI: 7.92, 8.50, p < 0.001), chronic diarrhea (OR = 5.73, 95% CI: 5.57, 5.89, p < 0.001), and higher level of education (OR = 1.87, 95% CI: 1.80, 1.93, p < 0.001) were more likely to comply with a colonoscopy. Several factors including age (70-75 years old:OR = 3.72, 95% CI: 2.71, 5.10, p < 0.001), and FIT( +) (OR = 1.65, 95% CI: 1.42,1.90, p < 0.001) were identified to be associated with the presence of AA. CONCLUSIONS: Increased compliance with colonoscopy is urgently needed. Our findings can inform the design of future effective large-scale population-based CRC screening programmes.

Marsdenia tenacissima enhances immune response of tumor infiltrating T lymphocytes to colorectal cancer.

Introduction: Tumor-infiltrating T lymphocytes in the tumor microenvironment are critical factors influencing the prognosis and chemotherapy outcomes. As a Chinese herbal medicine, Marsdenia tenacissima extract (MTE) has been widely used to treat cancer in China. Its immunoregulatory effects on tumor-associated macrophages is well known, but whether it regulates tumor-infiltrating T-cell functions remains unclear. Method: We collected 17 tumor samples from MTE-administered colorectal cancer patients, 13 of which showed upregulation of CD3+/CD8+ tumor-infiltrating T cells. Further in vitro and in vivo experiments were performed to investigate the regulatory effects of MTE on tumor-infiltrating T cells and immune escape of tumors. Results: Under single and co-culture conditions, MTE inhibited TGF-ß1 and PD-L1 expression in the colorectal cancer (CRC) cell lines HCT116 and LoVo. In Jurkat cells, MTE inhibited FOXP3 and IL-10 expression, increased IL-2 expression, but had no effect on PD-1 expression. These findings were confirmed in vitro using subcutaneous and colitis-associated CRC mouse models. MTE also increased the density of CD3+/CD8+ tumor-infiltrating T cells and exhibited considerable tumor-suppressive effects in these two tumor mouse models. Conclusions: Our findings suggested that MTE inhibits the immune escape of cancer cells, a precipitating factor increasing the immune response of T lymphocytes.

Platycodin D represses ß-catenin to suppress metastasis of cetuximab-treated KRAS wild-type colorectal cancer cells.

Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is extensively used for clinical therapy in KRAS wild-type colorectal cancer (CRC) patients. However, some patients still cannot get benefit from the therapy, because metastasis and resistance occur frequently after cetuximab treatment. New adjunctive therapy is urgently needed to suppress metastasis of cetuximab-treated CRC cells. In this study, we used two KRAS wild-type CRC cells, HT29 and CaCo2, to investigate whether platycodin D, a triterpenoid saponin isolated from Chinese medicinal herb Platycodon grandifloras, is able to suppress the metastasis of cetuximab-treated CRC. Label-free quantitative proteomics analyses showed that platycodin D but not cetuximab significantly inhibited expression of ß-catenin in both CRC cells, and suggested that platycodin D counteracted the inhibition effect of cetuximab on cell adherence and functioned in repressing cell migration and invasion. Western blot results showed that single platycodin D treatment or combined platycodin D and cetuximab enhanced inhibition effects on expressions of key genes in Wnt/ß-catenin signaling pathway, including ß-catenin, c-Myc, Cyclin D1 and MMP-7, compared to single cetuximab treatment. Scratch wound-healing and transwell assays showed that platycodin D combined with cetuximab suppressed migration and invasion of CRC cells, respectively. Pulmonary metastasis model of HT29 and CaCo2 in nu/nu nude mice consistently showed that combined treatment using platycodin D and cetuximab inhibited metastasis significantly in vivo. Our findings provide a potential strategy to inhibit CRC metastasis during cetuximab therapy by addition of platycodin D.

The accuracy of the FIT in detecting advanced neoplasm is highest in young people aged 40 to 49 years: an analysis based on sex and age.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers and is associated with high incidence and mortality rates worldwide. CRC has caused a tremendous loss of human health and wealth. The incidence and mortality of colorectal carcinoma are increasing in young adults. Early cancer detection and prevention are made possible through screening. At present, the faecal immunochemical test (FIT) is a noninvasive method that can be used for the large-scale clinical screening of CRC status. Therefore, this study, based on CRC screening results in Tianjin from 2012 to 2020, was conducted to analyse the major differences in diagnostic performance parameters according to sex and age. METHODS: This study was based on 39,991 colonoscopies performed for individuals in the Tianjin CRC screening program from 2012 to 2020. Of these individuals, they had complete FIT and colonoscopy results. The differences in FIT results were analysed by sex and age. RESULTS: According to this study, males were generally more likely to develop advanced neoplasms (ANs) than females, and the prevalence increased with age. Males with negative FIT results were more likely to have advanced neoplasms than females with positive results. The accuracy of the FIT in detecting ANs in each age group was 54.9%, 45.5%, 48.6% and 49.5% in the 40-49, 50-59, 60-69, and ≥ 70 age groups, respectively. CONCLUSIONS: The FIT detected ANs with highest accuracy in the 40-49 age group. Our research can provide guidance to formulate CRC screening strategies.

The comparison of risk factors for colorectal neoplasms at different anatomical sites.

AIM: Both the clinical manifestation and molecular characteristics of colorectal cancer (CRC) vary according to the anatomical site. We explored the risk factors for four groups of colorectal neoplasms (CRN) at different anatomical sites. METHODS: We extracted data from the database of Tianjin Colorectal Cancer Screening Program from 2010 to 2020. According to the CRN anatomical sites, patients were divided into four groups: the proximal colon group, the distal colon group, the rectum group, and the multiple colorectal sites. Binary logistic regression analysis was used to explore the differences in risk factors of CRN at different anatomical sites. RESULTS: The numbers of patients with CRN in the proximal colon, distal colon, rectum, and multiple colorectal sites were 4023, 6920, 3657, and 7938, respectively. Male sex was associated with a higher risk from the proximal colon to the rectum. Advanced age and obesity were also significantly associated with overall colorectal CRN risk, but there were some differences between men and women. Smoking was associated with CRN risk only in the distal colon and rectum in both men and women. Frequent alcohol consumption and family history of CRC in first-degree relatives (FDRs) were associated with the risk of multisite colorectal CRN only in males. CONCLUSIONS: We observed differences in advanced age, obesity, smoking, alcohol consumption, and family history of colorectal cancer at different anatomical sites of colorectal neoplasms. These factors vary by gender.

Young Patients With Colorectal Cancer Have Higher Early Mortality but Better Long-Term Survival.

INTRODUCTION: To define the prognosis of colorectal cancer (CRC) in young patients and to compare their postoperative treatment with that of older patients. METHODS: This multicenter study enrolled 5,457 patients with primary CRC who underwent surgical resection. The overall survival (OS), clinicopathologic characteristics, and postoperative treatment of 253 young patients aged 18-44 years and 5,204 older patients aged 44-80 years were analyzed. RESULTS: The OS rate was 77.1% for young and 74.2% for older patients (P = 0.348). Landmark analysis showed a significant difference in survival between young and older patients, with 63.8% of deaths among young patients being within 25 months of surgery compared with 42.4% among older patients (P = 0.002). Among those who survived more than 25 months, young patients had significantly better survival than older patients (P = 0.009). Multivariable analysis of young patients revealed that the tumor location, perineural invasion, and stage were associated with poor survival within 25 months; after this period, stage was the only prognostic marker. Young patients were more likely to receive chemotherapy, particularly multiagent regimens. For young patients, no significant difference in OS was found based on the chemotherapy regimen, regardless of disease stage (II, III, or IV, all P > 0.05). In addition, unlike in older patients, no difference in OS was found in young patients regardless of the drug regimen administered (all P > 0.05). DISCUSSION: Young-onset CRC may have a unique disease biology that warrants further research and therapy development.

Platycodin D sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the PI3K/Akt signaling pathway.

Cetuximab is a monoclonal antibody against epidermal growth factor receptor that blocks downstream signaling pathways of receptor tyrosine kinases, including Ras/Raf/MAPK and PI3K/Akt, thereby inhibiting tumor cell proliferation and inducing cancer cell apoptosis. Owing to KRAS mutations, the effectiveness of cetuximab is usually limited by intrinsic drug resistance. Continuous activation of the PI3K/Akt signaling pathway is another reason for cetuximab resistance. Platycodin-D, a bioactive compound isolated from the Chinese herb Platycodon grandiflorum, regulates Akt in different trends based on tissue types. To investigate whether platycodin-D can sensitize KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the PI3K/Akt signaling pathway, HCT116 and LoVo cells were treated with cetuximab and platycodin-D. LY294002 and SC79 were used to regulate Akt to further evaluate whether platycodin-D sensitizes cells to cetuximab by inhibiting Akt. Our results confirmed that platycodin-D increased the cytotoxic effects of cetuximab, including inhibition of growth, migration, and invasion, via downregulation of PI3K and Akt phosphorylation in HCT116 and LoVo cells both in vitro and in vivo. Given these data, platycodin-D may sensitize KRAS-mutant colorectal cancer cells to cetuximab via inhibition of the PI3K/Akt signaling pathway.

Connexin 43 contributes to the sensitization of colorectal cancer cells to photodynamic therapy through Akt inhibition.

BACKGROUND: Photodynamic therapy could be one approach to treat colorectal cancer though resistance leads to failure of therapy. Akt activation is a cellular survival response to photodynamic therapy and is also a reason for resistance. Thus, inhibition of Akt is a strategy to decrease resistance. Akt interacts with connexin 43, another protein involved in photodynamic therapy resistance. Connexin 43 is widely expressed in different human tissues and has a complex role in tumor development. However, the mechanism of inhibition of Akt by connexin 43 that sensitizes colorectal cancer cells to photodynamic therapy needs further investigation. METHODS: In this study, two colorectal cancer cells with low phosphorylated connexin 43 level were used to explore this mechanism. LY294002 was used as an Akt inhibitor, and connexin 43-pCMV3 was transfected into cells to increase connexin 43 expression. RESULTS: Akt and connexin 43 inhibit each other in both colorectal cancer cell lines. In vitro and in vivo experiments showed that LY294002 and connexin 43 transfection sensitized cells to hematoporphyrin-Photodynamic therapy. LY294002 increased the sensitivity of cells to photodynamic therapy with a pronounced effect in cells with high expression levels of connexin 43. CONCLUSIONS: Connexin 43 should be considered an important factor in increasing the phototoxicity of photodynamic therapy in colorectal cancer through Akt inhibition.

Dynamics of cooperative cargo transport by two elastically coupled kinesin motors.

Intracellular transport is performed often by multiple motor proteins bound to the same cargo. Here, we study theoretically collective transport of the cargo by two kinesin motors. We propose that the motor has only the elastic interaction with the cargo via the linker connecting them and has no interaction with another motor. With parameters values for single motors from the available single-molecule data, we show that at linker's elastic strength [Formula: see text] pN/nm the theoretical data of both velocity and run length of the two-motor assembly under no load are identical to the available experimental data. The run length distribution is single exponential. The single-motor-bound state of the assembly dominates the transport. Both the force dependence of the velocity of the cargo driven by single load-bearing motor and that by two load-bearing motors in the assembly are consistent with the experimental data. The stall force of the assembly is larger than the sum of stall forces of two uncoupled motors. Moreover, we predict that the stall force increases with the increase of K and becomes saturated at large K, with the saturated value being 1.5-fold larger than the sum of stall forces of the two uncoupled motors.

Investigating role of conformational changes of microtubule in regulating its binding affinity to kinesin by all-atom molecular dynamics simulation.

Changes of affinity of kinesin head to microtubule regulated by changes in the nucleotide state are essential to processive movement of kinesin on microtubule. Here, using all-atom molecular dynamics simulations we show that besides the nucleotide state, large conformational changes of microtubule-tubulin heterodimers induced by strong interaction with the head in strongly binding state are also indispensable to regulate the affinity of the head to the tubulin. In strongly binding state the high affinity of the head to microtubule arises largely from mutual conformational changes of the microtubule and head induced by the specific interaction between them via an induced-fit mechanism. Moreover, the ADP-head has a much weaker affinity to the local microtubule-tubulin, whose conformation is largely altered by the interaction with the head in strongly binding state, than to other unperturbed tubulins. This indicates that upon Pi release the ADP-head temporarily has a much weaker affinity to the local tubulin than to other tubulins.

Involvement of G-triplex and G-hairpin in the multi-pathway folding of human telomeric G-quadruplex.

G-quadruplex (G4) can be formed by G-rich DNA sequences that are widely distributed throughout the human genome. Although G-triplex and G-hairpin have been proposed as G4 folding intermediates, their formation still requires further investigation by experiments. Here, we employed single-molecule FRET to characterize the folding dynamics of G4 from human telomeric sequence. First, we observed four states during G4 folding initially assigned to be anti-parallel G4, G-triplex, G-hairpin and unfolded ssDNA. Then we constructed putative intra-strand G-triplex, G-hairpin structures and confirmed their existences in both NaCl and KCl. Further studies revealed those structures are going through dynamic transitions between different states and show relatively weak dependence on cations, unlike G4. Based on those results and molecular dynamics simulations, we proposed a multi-pathway folding mechanism for human telomeric G4. The present work may shed new light on our current understanding about the existence and stability of G4 intermediate states.

Dynamics of bridge helix bending in RNA polymerase II.

One of critical issues for RNA polymerase is how the enzyme translocates along the DNA substrate during transcription elongation cycle. Comparisons of the structure of RNA polymerase II (Pol II) with that of bacterial enzyme have suggested that the transition of the bridge helix (BH) from straight to flipped-out conformations facilitates the translocation of upstream DNA-RNA hybrid. However, the flipped-out conformation of BH in Pol II has not been observed up to now and the detailed mechanism of how the BH facilitating upstream hybrid translocation still remains obscure. Here we use all-atom molecular dynamics simulations to study the transition dynamics of BH in Pol II. Two different flipped-out conformations (termed as F1 and F2) are derived from our simulation trajectories, both of which could contribute to upstream hybrid translocation. In particular, the structure of BH in F2 conformation shows nearly identical to that observed in free bacterial enzyme, showing the existence of the flipped-out conformation in Pol II. Analysis of hydrogen bonds and salt bridge formed intra BH in different conformations indicates that the flipped-out conformations are more unstable than the straight conformation. Moreover, a detailed understanding of how the transition of BH conformations facilitating upstream hybrid translocation is given. Proteins 2017; 85:614-629. © 2016 Wiley Periodicals, Inc.

Optimal numbers of residues in linkers of DNA polymerase I, T7 primase and DNA polymerase IV.

DNA polymerase I (PolI), T7 primase and DNA polymerase IV (Dpo4) have a common feature in their structures that the two main domains are connected by an unstructured polypeptide linker. To perform their specific enzymatic activities, the enzymes are required to rearrange the position and orientation of one domain relative to the other into an active mode. Here, we show that the three enzymes share the same mechanism of the transition from the inert to active modes and use the minimum numbers of residues in their linkers to achieve the most efficient transitions. The transition time to the finally active mode is sensitively dependent on the stretched length of the linker in the finally active mode while is insensitive to the position and orientation in the initially inert state. Moreover, we find that for any enzyme whose two domains are connected by an unstructured flexible linker, the stretched length (L) of the linker in the finally active mode and the optimal number (Nopt) of the residues in the linker satisfy relation L ≈ αNopt, with α = 0.24-0.27 nm being a constant insensitive to the system.

A Gene Expression Profiling of Early Rice Stamen Development that Reveals Inhibition of Photosynthetic Genes by OsMADS58.

Stamen is a unique plant organ wherein germ cells or microsporocytes that commit to meiosis are initiated from somatic cells during its early developmental process. While genes determining stamen identity are known according to the ABC model of floral development, little information is available on how these genes affect germ cell initiation. By using the Affymetrix GeneChip Rice Genome Array to assess 51 279 transcripts, we established a dynamic gene expression profile (GEP) of the early developmental process of rice (Oryza sativa) stamen. Systematic analysis of the GEP data revealed novel expression patterns of some developmentally important genes including meiosis-, tapetum-, and phytohormone-related genes. Following the finding that a substantial amount of nuclear genes encoding photosynthetic proteins are expressed at the low levels in early rice stamen, through the ChIP-seq analysis we found that a C-class MADS box protein, OsMADS58, binds many nuclear-encoded genes participated in photosystem and light reactions and the expression levels of most of them are increased when expression of OsMADS58 is downregulated in the osmads58 mutant. Furthermore, more pro-chloroplasts are observed and increased signals of reactive oxygen species are detected in the osmads58 mutant anthers. These findings implicate a novel link between stamen identity determination and hypoxia status establishment.

Insights into Pseudomonas aeruginosa ATCC9027 resistance to isothiazolones through proteomics.

Although many explanations have been proposed for drug resistance to isothiazolones, the scope of cellular and physiological changes associated with this resistance remains unclear. In this study, comparative proteomic profiles of Pseudomonas aeruginosa ATCC9027 (WT) and an induced strain of Pa-R, which showed resistance to Kathon (a type of isothiazolone), were characterized using two-dimensional electrophoresis and matrix-assisted desorption/ionization time-of-flight mass spectroscopy. The results showed that a total of 16 proteins were successfully identified, among which 5 proteins were upregulated and 11 proteins were found to be repressed in Pa-R. At the same time, there were 14 proteins that contributed to metabolic processes, 1 protein (ATP-binding component of ATP-binding cassette [ABC] transporter) was the cellular component, and 1 protein (LolA) exhibited a transporter activity. The respective gene expression patterns of all the identified proteins in both Pa-R and WT were also evaluated by quantitative real-time polymerase chain reaction and shown to consistently correlate with those deduced from the proteomic results. Moreover, the resistant levels of Pa-R and WT could be affected by temperature and pH. Additionally, Pa-R exhibited coresistance and cross-resistance to other types of antimicrobial agents. Our results suggest that the resistant levels of P. aeruginosa to isothiazolones could be affected by extracellular factors and the resistance features are a complex system.

Antibacterial activity and kinetics of Litsea cubeba oil on Escherichia coli.

Litsea cubeba oil is extracted from the fresh fruits of Litsea cubeba by distillation. In this study, its chemical constituents, antibacterial activity, kinetics and effects against Escherichia coli were studied. Its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were both 0.125% (v/v) by toxic food method. Moreover, the antibacterial kinetic curves indicated 0.0625% (v/v) of litsea cubeba oil was able to prolong the growth lag phase of E. coli cells to approximate 12 hours while 0.125% (v/v) of litsea cubeba oil was able to kill the cells completely. Furthermore, transmission electron microscope (TEM) observation showed most E. coli cells treated with 0.125% (v/v) of litsea cubeba oil were killed or destroyed severely within 2 hours. The litsea cubeba oil might penetrate and destroy the outer and inner membrane of E. coli cells. Thus many holes and gaps were observed on the damaged cells, which led to their death eventually. The antibacterial effects of litsea cubeba oil mainly attributed to the presence of aldehydes, which accounted for approximately 70% in its whole components analyzed by GC/MS. Based on the antimicrobial properties, litsea cubeba oil would have a broad application in the antimicrobial industry.

Antifungal effect and mechanism of garlic oil on Penicillium funiculosum.

Garlic oil is a kind of fungicide, but little is known about its antifungal effects and mechanism. In this study, the chemical constituents, antifungal activity, and effects of garlic oil were studied with Penicillium funiculosum as a model strain. Results showed that the minimum fungicidal concentrations (MFCs, v/v) were 0.125 and 0.0313 % in agar medium and broth medium, respectively, suggesting that the garlic oil had a strong antifungal activity. The main ingredients of garlic oil were identified as sulfides, mainly including disulfides (36 %), trisulfides (32 %) and monosulfides (29 %) by gas chromatograph-mass spectrometer (GC/MS), which were estimated as the dominant antifungal factors. The observation results by transmission electron microscope (TEM) and scanning electron microscope (SEM) indicated that garlic oil could firstly penetrate into hyphae cells and even their organelles, and then destroy the cellular structure, finally leading to the leakage of both cytoplasm and macromolecules. Further proteomic analysis displayed garlic oil was able to induce a stimulated or weakened expression of some key proteins for physiological metabolism. Therefore, our study proved that garlic oil can work multiple sites of the hyphae of P. funiculosum to cause their death. The high antifungal effects of garlic oil makes it a broad application prospect in antifungal industries.

Efficacy of metal ions and isothiazolones in inhibiting Enterobacter cloacae BF-17 biofilm formation.

Enterobacter cloacae is a nosocomial pathogen. The E. cloacae strain BF-17, with a high capacity for biofilm formation, was screened and identified from industrially contaminated samples, carried out in our laboratory. To develop an efficient strategy to deal with biofilms, we investigated the effects of metal ions, including Na⁺, K⁺, Ca⁺, Mg⁺, Cu⁺, and Mn⁺, and 3 isothiazolones, on elimination of E. cloacae BF-17 biofilm formation by using a 0.1% crystal violet staining method. The results revealed that higher concentrations of Na⁺ or K⁺ significantly inhibited E. cloacae BF-17 biofilm development. Meanwhile, Ca²âº and Mn²âº stimulated biofilm formation at low concentration but exhibited a negative effect at high concentration. Moreover, biofilm formation decreased with increasing concentration of Mg²âº and Cu²âº. The isothiazolones Kathon (14%), 1,2-benzisothiazolin-3-one (11%), and 2-methyl-4-isothiazolin-3-one (10%) stimulated initial biofilm formation but not planktonic growth at low concentrations and displayed inhibitory effects on both biofilm formation and planktonic growth at higher concentrations. Unfortunately, the 3 isothiazolones exerted negligible effects on preformed or fully mature biofilms. Our findings suggest that Na⁺, K⁺, Mg²âº, and isothiazolones could be used to prevent and eliminate E. cloacae BF-17 biofilms.