In Vivo Exposure Pathways of Ambient Magnetite Nanoparticles Revealed by Machine Learning-Aided Single-Particle Mass Spectrometry.

Nanosized ultrafine particles (UFPs) from natural and anthropogenic sources are widespread and pose serious health risks when inhaled by humans. However, tracing the inhaled UFPs in vivo is extremely difficult, and the distribution, translocation, and metabolism of UFPs remain unclear. Here, we report a label-free, machine learning-aided single-particle inductively coupled plasma mass spectrometry (spICP-MS) approach for tracing the exposure pathways of airborne magnetite nanoparticles (MNPs), including external emission sources, and distribution and translocation in vivo using a mouse model. Our results provide quantitative analysis of different metabolic pathways in mice exposed to MNPs, revealing that the spleen serves as the primary site for MNP metabolism (84.4%), followed by the liver (11.4%). The translocation of inhaled UFPs across different organs alters their particle size. This work provides novel insights into the in vivo fate of UFPs as well as a versatile and powerful platform for nanotoxicology and risk assessment.

Dose-Dependent Effect on Plant Growth of Exposure to Metal-Organic Framework MIL-101(Cr).

With the increasing use of metal-organic frameworks (MOFs), they will inevitably enter the environment intentionally or unintentionally. However, the effects of MOFs on plant growth are poorly understood. Here, we investigated the effects of exposure of the rhizosphere to MOFs on plant growth. MIL-101(Cr) was selected as a research model due to its commercial availability and wide use. Soybean plants at the two-leaf stage were subjected to various durations (1-7 days) and concentrations (0-1000 mg/L) of exposure in hydroculture with a control group treated with ultrapure water. We found that MIL-101(Cr) had a positive effect on soybean growth at a lower dose (i.e., 200 mg/L); however, at higher doses (i.e., 500 and 1000 mg/L), it exhibited significant toxicity to plant growth, which is evidenced by leaf damage. To investigate the mechanism of this effect, we used Cr as an indicator to quantify, track, and image MIL-101(Cr) in the plant with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Results indicated that MIL-101(Cr) primarily accumulated in the cortex of roots (up to 40 times higher than that in stems), with limited translocation to stems and negligible presence in leaves and cotyledons. In addition, metabolomic analysis of soybeans indicated that low-dose MIL-101(Cr) could increase the sucrose content of soybean roots to promote plant growth, while a high dose could induce lipid oxidation in roots. This study provides valuable insights into the ecological toxicology of MOFs and underscores the importance of assessing their environmental impact for sustainable agricultural practices.

Research on the toxicological prognostic significance of age-related genes in endometrial cancer unveiling key factors in patient prognosis.

This study investigates the influence of aging-related genes on endometrial cancer, a prominent gynecological malignancy with rising incidence and mortality. By analyzing gene expression differences between cancerous and normal endometrial tissues, 42 aging-related genes were identified as differentially expressed. Utilizing the TCGA-UCEC sample, consensus clustering divided the samples into two molecular subgroups, Aging low and Aging high, based on their expression profiles. These subgroups showed distinct prognoses and survival rates, with the Aging high group associated with DNA repair and cell cycle pathways, and the Aging low group showing suppressed metabolic pathways and increased immune cell infiltration, suggesting a potential for better immunotherapy outcomes. Mutation analysis did not find significant differences in mutation frequencies between the groups, but a high Tumor Mutation Burden (TMB) correlated with better prognosis. A risk score model was also developed, showcasing significant prognostic power. Further analysis of the SIX1 gene revealed its overexpression in cancer cells. Drug sensitivity tests indicated that the low-risk group might respond better to chemotherapy. This research underscores the significance of aging-related genes in endometrial cancer, offering insights into their prognostic value and therapeutic potential, which could lead to personalized treatment approaches and enhanced patient management.

Deep Neural Network-Based Electron Microscopy Image Recognition for Source Distinguishing of Anthropogenic and Natural Magnetic Particles.

Deep learning models excel at image recognition of macroscopic objects, but their applications to nanoscale particles are limited. Here, we explored their potential for source-distinguishing environmental particles. Transmission electron microscopy (TEM) images can reveal distinguishable features in particle morphology from various sources, but cluttered foreground objects and scale variations pose challenges to visual recognition models. In this proof-of-concept work, we proposed a novel instance segmentation model named CoMask to tackle these issues with atmospheric magnetic particles, a key species of PM2.5. CoMask features a densely connected feature extraction module to excavate multiscale spatial cues at the single-particle level and enlarges the receptive field size for improved representation capability. We also employed a collaborative learning strategy to further improve performance. Compared with other state-of-the-art models, CoMask was competitive on benchmark and TEM data sets. The application of CoMask not only enables the source-distinguishing of magnetic particles but also opens up a new vista for machine learning applications.

Appearance of retinal arterial macroaneurysms in patients using swept-source optical coherence tomographic angiography.

BACKGROUND: Retinal arterial macroaneurysm (RAM) is a common clinical disease leading to vision loss in elderly individuals. The appropriate interpretation of swept-source optical coherence tomographic angiography (SS-OCTA), a noninvasive examination, is easy and convenient for detecting the status of RAMs and guiding treatment. METHODS: The objectives of this study were to describe the morphologic characteristics of RAMs using SS-OCTA and to observe whether there are differences in the morphologies of RAMs between SS-OCTA and fundus fluorescein angiography (FFA), before and after treatment. We retrospectively evaluated twenty-two eyes of 22 patients who were diagnosed with RAMs. All patients underwent a complete ophthalmologic examination, including a review of medical records, best-corrected visual acuity (BCVA), fundus photography, FFA and SS-OCTA. RAMs were recorded by SS-OCTA before any treatment or observation decisions were made. The morphologic findings of the RAMs on SS-OCTA were investigated. RESULTS: On SS-OCTA, RAMs can show local dilatation or an irregular linear blood flow signal, and the dilated cystic lumen may show thrombosis with a low reflection signal. After treatment, the shape of the RAMs will show reactive changes. The findings on SS-OCTA are not very consistent with those on FFA. CONCLUSIONS: The same RAM may have different manifestations on OCTA and FFA, and OCTA can more conveniently reflect the changes in blood flow signals and treatment response of RAMs.

Glycosyltransferase-Related Protein GtrA Is Essential for Localization of Type IX Secretion System Cargo Protein Cellulase Cel9A and Affects Cellulose Degradation in Cytophaga hutchinsonii.

The Gram-negative bacterium Cytophaga hutchinsonii digests cellulose through a novel cellulose degradation mechanism. It possesses the lately characterized type IX secretion system (T9SS). We recently discovered that N-glycosylation of the C-terminal domain (CTD) of a hypothetical T9SS substrate protein in the periplasmic space of C. hutchinsonii affects protein secretion and localization. In this study, green fluorescent protein (GFP)-CTDCel9A recombinant protein was found with increased molecular weight in the periplasm of C. hutchinsonii. Site-directed mutagenesis studies on the CTD of cellulase Cel9A demonstrated that asparagine residue 900 in the D-X-N-X-S motif is important for the processing of the recombinant protein. We found that the glycosyltransferase-related protein GtrA (CHU_0012) located in the cytoplasm of C. hutchinsonii is essential for outer membrane localization of the recombinant protein. The deletion of gtrA decreased the abundance of the outer membrane proteins and affected cellulose degradation by C. hutchinsonii. This study provided a link between the glycosylation system and cellulose degradation in C. hutchinsonii. IMPORTANCE N-Glycosylation systems are generally limited to some pathogenic bacteria in prokaryotes. The disruption of the N-glycosylation pathway is related to adherence, invasion, colonization, and other phenotypic characteristics. We recently found that the cellulolytic bacterium Cytophaga hutchinsonii also has an N-glycosylation system. The cellulose degradation mechanism of C. hutchinsonii is novel and mysterious; cellulases and other proteins on the cell surface are involved in utilizing cellulose. In this study, we identified an asparagine residue in the C-terminal domain of cellulase Cel9A that is necessary for the processing of the T9SS cargo protein. Moreover, the glycosyltransferase-related protein GtrA is essential for the localization of the GFP-CTDCel9A recombinant protein. Deletion of gtrA affected cellulose degradation and the abundance of outer membrane proteins. This study enriched the understanding of the N-glycosylation system in C. hutchinsonii and provided a link between N-glycosylation and cellulose degradation, which also expanded the role of the N-glycosylation system in bacteria.

A Type IX Secretion System Substrate Involved in Crystalline Cellulose Degradation by Affecting Crucial Cellulose Binding Proteins in Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an abundant soil cellulolytic bacterium that uses a unique cellulose degradation mechanism different from those that involve free cellulases or cellulosomes. Though several proteins have been identified as important for cellulose degradation, the mechanism used by C. hutchinsonii to digest crystalline cellulose remains a mystery. In this study, chu_0922 was identified by insertional mutation and gene deletion as an important gene locus indispensable for crystalline cellulose utilization. Deletion of chu_0922 resulted in defects in crystalline cellulose utilization. The Δ0922 mutant completely lost the ability to grow on crystalline cellulose, even with extended incubation, and selectively utilized the amorphous region of cellulose, leading to increased crystallinity. As a protein secreted by the type IX secretion system (T9SS), CHU_0922 was found to be located on the outer membrane, and the outer membrane localization of CHU_0922 relied on the T9SS. Comparative analysis of the outer membrane proteins revealed that the abundance of several cellulose-binding proteins, including CHU_1276, CHU_1277, and CHU_1279, was reduced in the Δ0922 mutant. Further study showed that CHU_0922 is crucial for the full expression of the gene cluster containing chu_1276, chu_1277, chu_1278, chu_1279, and chu_1280 (cel9C), which is essential for cellulose utilization. Moreover, CHU_0922 is required for the cell surface localization of CHU_3220, a cellulose-binding protein that is essential for crystalline cellulose utilization. Our study provides insights into the complex system that C. hutchinsonii uses to degrade crystalline cellulose. IMPORTANCE The widespread aerobic cellulolytic bacterium Cytophaga hutchinsonii, belonging to the phylum Bacteroidetes, utilizes a novel mechanism to degrade crystalline cellulose. No genes encoding proteins specialized in loosening or disruption the crystalline structure of cellulose were identified in the genome of C. hutchinsonii, except for chu_3220 and chu_1557. The crystalline cellulose degradation mechanism remains enigmatic. This study identified a new gene locus, chu_0922, encoding a typical T9SS substrate that is essential for crystalline cellulose degradation. Notably, CHU_0922 is crucial for the normal transcription of chu_1276, chu_1277, chu_1278, chu_1279, and chu_1280 (cel9C), which play important roles in the degradation of cellulose. Moreover, CHU_0922 participates in the cell surface localization of CHU_3220. These results demonstrated that CHU_0922 plays a key role in the crystalline cellulose degradation network. Our study will promote the uncovering of the novel cellulose utilization mechanism of C. hutchinsonii.

N-Glycosylation of a Cargo Protein C-Terminal Domain Recognized by the Type IX Secretion System in Cytophaga hutchinsonii Affects Protein Secretion and Localization.

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes. It digests crystalline cellulose with an unknown mechanism and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of the CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with the CTD from CHU_2708. The CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTDCHU_2708 fusion protein was found to be glycosylated in the periplasm, with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide-N-glycosidase F, which can hydrolyze N-linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTDCHU_2708 suggested that N-glycosylation occurred on the CTD. CTD N-glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii. Glycosyltransferase-encoding gene chu_3842, a homologous gene of Campylobacter jejuni pglA, was found to participate in the N-glycosylation of C. hutchinsonii. Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided evidence that the CTD as the signal of T9SS was N-glycosylated in the periplasm of C. hutchinsonii. IMPORTANCE The bacterial N-glycosylation system has previously been found only in several species of Proteobacteria and Campylobacterota, and the role of N-linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell contact cellulose degradation mechanism, and many cell surface proteins, including cellulases, are secreted by the T9SS. In this study, we found that C. hutchinsonii, a member of the phylum Bacteroidetes, has an N-glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N-glycosylation of C. hutchinsonii proteins and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N-glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of the CTD appears to play an important role in affecting T9SS substrate transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N-glycosylation in bacteria.

Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues.

BACKGROUND: The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass. RESULTS: In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5' truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (- 640 to - 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (- 821 to - 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger ß-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively. CONCLUSIONS: The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Internal Exposure and Distribution of Airborne Fine Particles in the Human Body: Methodology, Current Understandings, and Research Needs.

Exposure to airborne fine particles (PM2.5, particulate matter with aerodynamic diameter <2.5 µm) severely threatens global human health. Understanding the distribution and processes of inhaled PM2.5 in the human body is crucial to clarify the causal links between PM2.5 pollution and diseases. In contrast to extensive research on the emission and formation of PM2.5 in the ambient environment, reports about the occurrence and fate of PM2.5 in humans are still limited, although many studies have focused on the exposure and adverse effects of PM2.5 with animal models. It has been shown that PM2.5, especially ultrafine particles (UFPs), have the potential to go across different biological barriers and translocate into different human organs (i.e., blood circulation, brain, heart, pleural cavity, and placenta). In this Perspective, we summarize the factors affecting the internal exposure of PM2.5 and the relevant analytical methodology and review current knowledge about the exposure pathways and distribution of PM2.5 in humans. We also discuss the research challenges and call for more studies on the identification and characterization of key toxic species of PM2.5, quantification of internal exposure doses in the general population, and further clarification of translocation, metabolism, and clearance pathways of PM2.5 in the human body. In this way, it is possible to develop toxicity-based air quality standards instead of the currently used mass-based standards.

Some novel features of strong promoters discovered in Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5' rapid amplification of cDNA ends (5'RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31 bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter Pchu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter Pchu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120 kDa in C. hutchinsonii. The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii.Key points• The conserved structure of strong promoter of C. hutchinsonii was elucidated.• Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered.• A new method for induced expression of cellulase in vivo was established.• Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii.

Continuous production of fructooligosaccharides by recycling of the thermal-stable ß-fructofuranosidase produced by Aspergillus niger.

OBJECTIVE: To achieve continuous production of fructooligosaccharides (FOS) by recycling of the mycelial cells containing the thermal-stable ß-fructofuranosidase in Aspergillus niger without immobilization. RESULTS: The thermal-stable ß-fructofuranosidase FopA-V1 was successfully expressed in A. niger ATCC 20611 under the control of the constitutive promoter PgpdA. The engineered A. niger strain FV1-11 produced the ß-fructofuranosidase with improved thermostability, which remained 91.2% of initial activity at 50 °C for 30 h. Then its mycelial ß-fructofuranosidase was recycled for the synthesis of FOS. It was found that the enzyme still had 79.3% of initial activity after being reused for six consecutive cycles, whereas only 62.3% ß-fructofuranosidase activity was detected in the parental strain ATCC 20611. Meanwhile, the FOS yield of FV1-11 after six consecutive cycles reached 57.1% (w/w), but only 51.0% FOS yield was detected in ATCC 20611. CONCLUSIONS: The thermal-stable ß-fructofuranosidase produced by A. niger can be recycled to achieve continuous synthesis of FOS with high efficiency, providing a powerful and economical strategy for the industrial production of FOS.

Extracellular protease production regulated by nitrogen and carbon sources in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is an important producer of industrial enzymes, and possesses abundant extracellular protease genes based on the genome sequence data. However, the production of extracellular proteases remains poorly understood. Here, protease production was extensively investigated on different carbon (glucose and lactose) and nitrogen sources ((NH4 )2 SO4 , NaNO3 , peptone, and corn steep liquor). It was found that protease production was dominantly regulated by nitrogen sources. Organic nitrogen sources were beneficial for protease production, while the preferred nitrogen source (NH4 )2 SO4 inhibited the expression of proteases. As for carbon sources, lactose was a more effective inducer than glucose for protease production. The protease activity was further examined by protease inhibitors, which suggested that protease activity was predominantly inhibited by phenylmethanesulfonyl fluoride (PMSF) and slightly suppressed by ethylenediaminetetraacetic acid (EDTA). Moreover, proteomic analysis revealed a total of 29 extracellular proteases, including 13 serine proteases, 6 aspartic proteases, and 10 metalloproteases. In addition, seven proteases were found to be present among all conditions. These results showed the regulatory profile of extracellular protease production in Trichoderma reesei grown on various carbon and nitrogen sources, which will facilitate the development of T. reesei to be an effective workhorse for enzyme or high-value protein production in industry.

A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii.

Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCECytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

Cytophaga hutchinsonii gldN, Encoding a Core Component of the Type IX Secretion System, Is Essential for Ion Assimilation, Cellulose Degradation, and Cell Motility.

The type IX secretion system (T9SS), which is involved in pathogenicity, motility, and utilization of complex biopolymers, is a novel protein secretion system confined to the phylum BacteroidetesCytophaga hutchinsonii, a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes, can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of chu_0174 (gldN) was obtained using PY6 medium supplemented with Stanier salts. GldN was verified to be a core component of C. hutchinsonii T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the ΔgldN mutant showed significant growth defects in Ca2+- and Mg2+-deficient media. These growth defects could be relieved by the addition of Ca2+ or Mg2+ The intracellular concentrations of Ca2+ and Mg2+ were markedly reduced in ΔgldN These results demonstrated that GldN is essential for the acquisition of trace amounts of Ca2+ and Mg2+, especially for Ca2+ Moreover, an outer membrane efflux protein, CHU_2807, which was decreased in abundance on the outer membrane of ΔgldN, is essential for normal growth in PY6 medium. The reduced intracellular accumulation of Ca2+ and Mg2+ in the Δ2807 mutant indicated that CHU_2807 is involved in the uptake of trace amounts of Ca2+ and Mg2+ This study provides insights into the role of T9SS in metal ion assimilation in C. hutchinsoniiIMPORTANCE The widespread Gram-negative bacterium Cytophaga hutchinsonii uses a novel but poorly understood strategy to utilize crystalline cellulose. Recent studies showed that a T9SS exists in C. hutchinsonii and is involved in cellulose degradation and motility. However, the main components of the C. hutchinsonii T9SS and their functions are still unclear. Our study characterized the function of GldN, which is a core component of the T9SS. GldN was proved to play vital roles in cellulose degradation and cell motility. Notably, GldN is essential for the acquisition of Ca2+ and Mg2+ ions under Ca2+- and Mg2+-deficient conditions, revealing a link between the T9SS and the metal ion transport system. The outer membrane abundance of CHU_2807, which is essential for Ca2+ and Mg2+ uptake in PY6 medium, was affected by the deletion of GldN. This study demonstrated that the C. hutchinsonii T9SS has extensive functions, including cellulose degradation, motility, and metal ion assimilation, and contributes to further understanding of the function of the T9SS in the phylum Bacteroidetes.

FOXM1 modulates docetaxel resistance in prostate cancer by regulating KIF20A.

BACKGROUND: Docetaxel resistance affects prognosis in advanced prostate cancer (PCa). The precise mechanisms remain unclear. Transcription factor Forkhead box M1 (FOXM1), which participates in cell proliferation and cell cycle progression, has been reported to affect the sensitivity of chemotherapy. This study explores the role of FOXM1 in PCa docetaxel resistance and its association with kinesin family member 20 A (KIF20A), which is known to promote therapeutic resistance in some cancers. METHODS: We monitored cell growth using MTT and colony formation assays, and cell apoptosis and cell cycle progression using flow cytometry. Wound-healing and transwell assays were used to detect cell invasion and migration. mRNA and protein expression were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. We monitored FOXM1 binding to the KIF20A promoter using a ChIP assay. Tumorigenicity in nude mice was used to assess in vivo tumorigenicity. RESULTS: FOXM1 knockdown induced cell apoptosis and G2/M cell cycle arrest, suppressing cell migration and invasion in docetaxel-resistant PCa cell lines (DU145-DR and VCaP-DR). Exogenous FOXM1 overexpression was found in their parental cells. Specific FOXM1 inhibitor thiostrepton significantly weakened docetaxel resistance in vitro and in vivo. We also found that FOXM1 and KIF20A exhibited consistent and highly correlated overexpression in PCa cells and tissues. FOXM1 also regulated KIF20A expression at the transcriptional level by acting directly on a Forkhead response element (FHRE) in its promoter. KIF20A overexpression could partially reverse the effect on cell proliferation, cell cycle proteins (cyclinA2, cyclinD1 and cyclinE1) and apoptosis protein (bcl-2 and PARP) of FOXM1 depletion. CONCLUSIONS: Our findings indicate that highly expressed FOXM1 may help promote docetaxel resistance by inducing KIF20A expression, providing insight into novel chemotherapeutic strategies for combatting PCa docetaxel resistance.

Identification of a cell-surface protein involved in glucose assimilation and disruption of the crystalline region of cellulose by Cytophaga hutchinsonii.

The crystalline region of cellulose is the main barrier to the utilization of crystalline cellulose. Cytophaga hutchinsonii actively digests the crystalline region of cellulose by an unknown mechanism. Transposon mutagenesis was done to identify a novel gene locus chu_1557, which is required for efficient disruption of the crystalline region of cellulose, and the absence of CHU_1557 resulted in decreased glucose assimilation efficiency. The defect of the mutant in the disruption of the crystalline region of cellulose was partially retained by additional glucose or pre-culturing the mutant in a low glucose concentration medium which could improve its glucose absorption efficiency. These results suggested that extracellular glucose has important roles in the disruption of crystalline cellulose by C. hutchinsonii. Further study showed that the expression of an outer membrane protein CHU_3732 was downregulated by the absence of CHU_1557 in a low glucose concentration medium. CHU_3732 was involved in uptake of glucose and its expression was induced by a low concentration of glucose. CHU_3732 was predicted to be a porin, so we inferred that it may work as a glucose transport channel in the outer membrane. Based on these results, we deduced that CHU_1557 played a role in the process of glucose assimilation and its disruption affected the expression of other proteins related to glucose transportation such as CHU_3732, and then affected the cell growth in a low glucose concentration medium and disruption of the crystalline region of cellulose.

Effects of the histone-like protein HU on cellulose degradation and biofilm formation of Cytophaga hutchinsonii.

Cytophaga hutchinsonii, belonging to Bacteroidetes, is speculated to use a novel cell-contact mode to digest cellulose. In this study, we identified a histone-like protein HU, CHU_2750, in C. hutchinsonii, whose transcription could be induced by crystalline but not amorphous cellulose. We constructed a CHU_2750-deleted mutant and expressed CHU_2750 in Escherichia coli to study the gene's functions. Our results showed that although the deletion of CHU_2750 was not lethal to C. hutchinsonii, the mutant displayed an abnormal filamentous morphology, loose nucleoid, and obvious defects in the degradation of crystalline cellulose and cell motility. Further study indicated that the mutant displayed significantly decreased cell surface and intracellular endoglucanase activities but with ß-glucosidase activities similar to the wild-type strain. Analyses by real-time quantitative PCR revealed that the transcription levels of many genes involved in cellulose degradation and/or cell motility were significantly downregulated in the mutant. In addition, we found that CHU_2750 was important for biofilm formation of C. hutchinsonii. The main extracellular components of the biofilm were analyzed, and the results showed that the mutant yielded significantly less exopolysaccharide but more extracellular DNA and protein than the wild-type strain. Collectively, our findings demonstrated that CHU_2750 is important for cellulose degradation, cell motility, and biofilm formation of C. hutchinsonii by modulating transcription of certain related genes, and it is the first identified transcriptional regulator in these processes of C. hutchinsonii. Our study shed more light on the mechanisms of cellulose degradation, cell motility, and biofilm formation by C. hutchinsonii.

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3 Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220 Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE: Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii.

Expression and characterization of a glucose-tolerant ß-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and ß-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a ß-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most ß-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10-2, 5.6 × 10-2, and 5.3 × 10-2 mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and ß-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.