See the Unseen: Red-Emissive Carbon Dots for Visualizing the Nucleolar Structures in Two Model Animals and In Vivo Drug Toxicity.

Nucleolus, which participates in many crucial cellular activities, is an ideal target for evaluating the state of a cell or an organism. Here, bright red-emissive carbon dots (termed CPCDs) with excitation-independent/polarity-dependent fluorescence emission are synthesized by a one-step hydrothermal reaction between congo red and p-phenylenediamine. The CPCDs can achieve wash-free, real-time, long-term, and high-quality nucleolus imaging in live cells, as well as in vivo imaging of two common model animals-zebrafish and Caenorhabditis elegans (C. elegans). Strikingly, CPCDs realize the nucleolus imaging of organs/flowing blood cells in zebrafish at a cellular level for the first time, and the superb nucleolus imaging of C. elegans suggests that the germ cells in the spermatheca probably have no intact nuclei. These previously unachieved imaging results of the cells/tissues/organs may guide the zebrafish-related studies and benefit the research of C. elegans development. More importantly, a novel strategy based on CPCDs for in vivo toxicity evaluation of materials/drugs (e.g., Ag+ ), which can visualize the otherwise unseen injuries in zebrafish, is developed. In conclusion, the CPCDs represent a robust tool for visualizing the structures and dynamic behaviors of live zebrafish and C. elegans, and may find important applications in cell biology and toxicology.

Structural Characterization, Functional Profiling, and Mechanism Study of Four Antimicrobial Peptides for Antibacterial and Anticancer Applications.

Antimicrobial peptides (AMPs) are potent compounds for treating bacterial infection and cancer, drawing ever-increasing interest. However, the function and mechanism of most AMPs remain to be explored. In this research, we focused on investigating the antibacterial and anticancer activities of four AMPs (Dhvar4, Lasioglossin-III, Macropin 1, and Temporin La) and the possible corresponding mechanisms. All four AMPs are cationic α-helical with moderate hydrophobicity and high helicity. They have broad-spectrum antibacterial capacities, among which the antibacterial activities of Dhvar4 and Temporin La are not as effective as Lasioglossin-III and Macropin 1. Macropin 1 exhibited the highest antibacterial effect with a pretty low minimal inhibitory concentration (MIC) of 2-8 µM. Meanwhile, Lasioglossin-III exhibited the strongest anticancer activities, displaying the IC50 of 26.36 µM for A549 and 7.75 µM for HepG2. Although Dhvar4 possessed the highest positive charge and entered the bacterial and animal cells in large amounts, it displayed the lowest bactericidal and anticancer activities which might be ascribed to its lowest hydrophobicity and thus the weakest cell membrane damage capability. It seems that the positive charge and cell internalization play a supporting rather than a determined role in antibacterial and anticancer activities of AMPs. All the four AMPs damaged the bacterial cell membrane with Macropin 1 damaging the cell membrane of Escherichia coli the most and Lasioglossin-III destroying the cell membrane of Staphylococcus aureus the worst. In addition, the animal cellular internalization of the four peptides was temperature-dependent and mainly mediated by caveolae-mediated endocytosis, and they were distributed in lysosomes once inside the cells. These findings expand our knowledge on the function and mechanism of AMPs, laying the fundamental theoretical basis for designing and engineering AMPs for infection and cancer treatment.

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

Rose Bengal-Derived Ultrabright Sulfur-Doped Carbon Dots for Fast Discrimination between Live and Dead Cells.

The discrimination between dead and live cells is crucial for cell viability evaluation. Carbon dots (CDs), with advantages like simple and cost-effective synthesis, excellent biocompatibility, and high photostability, have shown potential for realizing selective live/dead cell staining. However, most of the developed CDs with the live/dead cell discrimination capacity usually have low photoluminescence quantum yields (PLQYs) and excitation wavelength-dependent fluorescence emission (which can cause fluorescence overlap with other fluorescent probes and make dual-color live/dead staining impossible), and hence, developing ultrabright CDs with excitation wavelength-independent fluorescence emission property for live/dead cell discrimination becomes an important task. Here, using a one-pot hydrothermal method, we prepared ultrasmall (∼1.6 nm), ultrabright (PLQY: ∼78%), and excitation wavelength-independent sulfur-doped carbon dots (termed S-CDs) using rose bengal and 1,4-dimercaptobenzene as raw materials and demonstrated that the S-CDs could rapidly (∼5 min) and accurately distinguish dead cells from live ones for almost all the cell types including bacterial, fungal, and animal cells in a wash-free manner. We confirmed that the S-CDs could rapidly pass through the dead cell surfaces to enter the interior of the dead cells, thus visualizing these dead cells. In contrast, the S-CDs could not enter the interior of live cells and thus could not stain these live cells. We further verified that the S-CDs presented better biocompatibility and higher photostability than the commercial live/dead staining dye propidium iodide, ensuring its bright application prospect in cell imaging and cell viability assessment. Overall, this work develops a type of CDs capable of realizing the live/dead cell discrimination of almost all the cell types (bacterial, fungal, and animal cells), which has seldom been achieved by other fluorescent nanoprobes.

M2 Macrophage-Derived Exosomes Facilitate HCC Metastasis by Transferring αM ß2 Integrin to Tumor Cells.

BACKGROUND AND AIMS: The development and progression of hepatocellular carcinoma (HCC) is dependent on its local microenvironment. Tumor-associated macrophages (TAMs) are deemed a key factor for the tumor microenvironment and attribute to contribute to tumor aggressiveness. However, the detailed mechanism underlying the pro-metastatic effect of TAMs on HCC remains undefined. APPROACH AND RESULTS: The present study proved that TAMs were enriched in HCC. TAMs were characterized by an M2-polarized phenotype and accelerated the migratory potential of HCC cells in vitro and in vivo. Furthermore, we found that M2-derived exosomes induced TAM-mediated pro-migratory activity. With the use of mass spectrometry, we identified that integrin, αM ß2 (CD11b/CD18), was notably specific and efficient in M2 macrophage-derived exosomes (M2 exos). Blocking either CD11b and/or CD18 elicited a significant decrease in M2 exos-mediated HCC cell metastasis. Mechanistically, M2 exos mediated an intercellular transfer of the CD11b/CD18, activating the matrix metalloproteinase-9 signaling pathway in recipient HCC cells to support tumor migration. CONCLUSIONS: Collectively, the exosome-mediated transfer of functional CD11b/CD18 protein from TAMs to tumor cells may have the potency to boost the migratory potential of HCC cells, thus providing insights into the mechanism of tumor metastasis.

Transmembrane transport process and endoplasmic reticulum function facilitate the role of gene cel1b in cellulase production of Trichoderma reesei.

BACKGROUND: A total of 11 ß-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each ß-glucosidase remained unknown. RESULTS: We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. CONCLUSIONS: Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the ß-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry.

Carbon dots for multicolor cell imaging and ultra-sensitive detection of multiple ions in living cells: One Stone for multiple Birds.

Given the significant impact of ions on environment pollution and human health, it is urgently needed to establish effective and convenient ion detection approaches, particularly in living cells. In this paper, we constructed multicolor N-doped-carbon dots (mPD-CDs) by facile one-step hydrothermal carbonization of m-phenylenediamine (mPD). mPD-CDs were successfully deployed for multicolor cellular imaging for animal cells, fungi, and bacteria in a wash-free way with high photostability and satisfactory biocompability. Moreover, mPD-CDs can be used as a fluorescent sensing probe for ultrasensitive detection of both iodide ion (I-) and typical heavy metals such as cadmium (Cd2+), copper (Cu2+), mercury (Hg2+), gadolinium (Gd3+), ferrous ion (Fe2+), Zinc (Zn2+), and ferric ion (Fe3+). This is the first report using CDs as optical sensing probe for the detection of Gd3+, and for detection of Fe3+ with fluorescence "turn on". More significantly, with these versatile and fascinating properties, we applied mPD-CDs for intracellular ion detection in living cells like Hep G2 and S. cerevisiae, and zebra fish. Altogether, mPD-CDs displayed great potential for multicolor cell imaging and the multiple ion detection in vitro and in vivo, presenting a promising strategy for in-situ ultrasensitive sensing of multiple metal ions in the environment and the biological systems.

Intracellular Enzyme-Instructed Self-Assembly of Peptides (IEISAP) for Biomedical Applications.

Despite the remarkable significance and encouraging breakthroughs of intracellular enzyme-instructed self-assembly of peptides (IEISAP) in disease diagnosis and treatment, a comprehensive review that focuses on this topic is still desirable. In this article, we carefully review the advances in the applications of IEISAP, including the development of various bioimaging techniques, such as fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, positron-emission tomography imaging, radiation imaging, and multimodal imaging, which are successfully leveraged in visualizing cancer tissues and cells, bacteria, and enzyme activity. We also summarize the utilization of IEISAP in disease treatments, including anticancer, antibacterial, and antiinflammation applications, among others. We present the design, action modes, structures, properties, functions, and performance of IEISAP materials, such as nanofibers, nanoparticles, nanoaggregates, and hydrogels. Finally, we conclude with an outlook towards future developments of IEISAP materials for biomedical applications. It is believed that this review may foster the future development of IEISAP with better performance in the biomedical field.

Novel Type of Water-Soluble Photosensitizer from Trichoderma reesei for Photodynamic Inactivation of Gram-Positive Bacteria.

Antimicrobial photodynamic therapy (APDT) is a promising alternative to traditional antibiotics for the treatment of bacterial infections, which inactivates a broad spectrum of bacteria. However, many traditional photosensitizers (PSs) are hydrophobic with poor water solubility and easy aggregation. On the other hand, some light sources such as ultraviolet (UV) have poor penetration and high cytotoxicity. Both issues lead to undesired photodynamic therapy efficacy. To overcome these issues, we develop a novel water-soluble natural PS (sorbicillinoids) obtained by microbial fermentation using recombinant filamentous fungus Trichoderma reesei. Sorbicillinoids could effectively generate singlet oxygen (1O2) under UV light irradiation and ultimately display photoinactivation activity on Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus but not Gram-negative ones such as Escherichia coli and Proteus vulgaris. Sorbicillinoids were found to enter S. aureus but not E. coli. S. aureus treated with sorbicillinoids and UV light displayed high levels of intracellular reactive oxygen species (ROS), notable DNA photocleavage, and compromised cell semipermeability without overt cell membrane disruption, none of which was found in the treated E. coli. All these contribute to the sorbicillinoid-based photoinactivation of Gram-positive bacteria. Moreover, the dark toxicity and phototoxicity on mammalian cells or hemolysis activity of sorbicillinoids is negligible, showing its excellent biocompatibility. This study expands the utilization of UV light for surface sterilization to disinfection in solution. Therefore, sorbicillinoids, a type of secondary metabolite from fungus, have a promising future as a new PS for APDT using a nontoxic dose of UV irradiation.

Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii.

BACKGROUND: The ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out. RESULTS: The addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP. CONCLUSIONS: Four factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.

SUMO-specific protease 1 is critical for early lymphoid development through regulation of STAT5 activation.

Small ubiquitin-like modifier (SUMO) modification has emerged as an important regulatory mechanism during embryonic development. However, it is not known whether SUMOylation plays a role in the development of the immune system. Here, we show that SUMO-specific protease 1 (SENP1) is essential for the development of early T and B cells. STAT5, a key regulator of lymphoid development, is modified by SUMO-2 and is specifically regulated by SENP1. In the absence of SENP1, SUMO-2 modified STAT5 accumulates in early lymphoid precursors, resulting in a block in its acetylation and subsequent signaling. These results demonstrate a crucial role of SENP1 in the regulation of STAT5 activation during early lymphoid development.

Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer.

We evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I-IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis.

One-Step Synthesis of Epoxy Group-Terminated Organosilica Nanodots: A Versatile Nanoplatform for Imaging and Eliminating Multidrug-Resistant Bacteria and Their Biofilms.

Multidrug-resistant bacteria (MRB) and their biofilms, both of which develop high levels of drug tolerance, cause severe threats to global health. This study demonstrates that biocompatible fluorescent silicon-containing nanodots can be a multifunctional platform for simultaneously imaging and eliminating MRB and their biofilms. Ultrasmall epoxy group (oxirane)-functionalized organosilica nanodots (OSiNDs) with a high photoluminescence quantum yield of ≈31% are synthesized via a simple one-step hydrothermal treatment of an epoxy group-containing silane molecule, 3-glycidoxypropyltrimethoxysilane, and an organic dye, rose bengal. The resultant OSiNDs can be employed as a universal imaging reagent for visualizing various bacteria/biofilms, including MRB and their biofilms. Moreover, the epoxy group-terminated OSiNDs can be conjugated with amine-containing reagents only via the simple stirring of the mixtures at an elevated temperature (e.g., 60 °C) for several hours (e.g., 3 h) without the addition of activating reagents. The amine-containing antibiotic vancomycin (Van) can thus be easily conjugated with the OSiNDs, and the obtained OSiNDs-Van can successfully inhibit the growth of MRB and even eliminate their biofilms. Collectively, the present work may give new impetus to the development of novel antibacterial and anti-biofilm agents for overcoming the drug resistance of bacteria.

Glycol Chitosan: A Water-Soluble Polymer for Cell Imaging and Drug Delivery.

Glycol chitosan (GC), a water-soluble chitosan derivative with hydrophilic ethylene glycol branches, has both hydrophobic segments for the encapsulation of various drugs and reactive functional groups for facile chemical modifications. Over the past two decades, a variety of molecules have been physically encapsulated within or chemically conjugated with GC and its derivatives to construct a wide range of functional biomaterials. This review summarizes the recent advances of GC-based materials in cell surface labeling, multimodal tumor imaging, and encapsulation and delivery of drugs (including chemotherapeutics, photosensitizers, nucleic acids, and antimicrobial agents) for combating cancers and microbial infections. Besides, different strategies for GC modifications are also highlighted with the aim to shed light on how to endow GC and its derivatives with desirable properties for therapeutic purposes. In addition, we discuss both the promises and challenges of the GC-derived biomaterials.

Interactions between Surface-Immobilized Antimicrobial Peptides and Model Bacterial Cell Membranes.

Sum frequency generation (SFG) vibrational spectroscopy was used to study surface immobilization effects on the interactions between antimicrobial peptide cecropin P1 (CP1) and model cell membranes. While free CP1 in solution interacted with a model cell membrane composed of a phosphatidylglycerol (PG) bilayer, electrostatic interaction led to the attachment of CP1 molecules onto the PG surface and the hydrophobic domain in the lipid bilayer enabled the peptides to insert into the bilayer and form α-helices from random coil structures. While CP1 molecules immobilized on a self-assembled monolayer interacted with PG lipid vesicles, the intensity of the SFG peak for the peptide α-helix decreased as the PG vesicle concentration increased. It was believed that when surface-immobilized CP1 molecules interacted with lipid vesicles, they lay down on the surface or became random coils. When the immobilized CP1 interacted with a PG lipid monolayer on water, the strong interaction led to the lying-down orientation of all of the surface-immobilized peptides as well. Differently, no significant interactions between surface-immobilized CP1 with the mammalian cell membrane model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer were observed. Our results suggest that, instead of membrane insertion, the electrostatic interactions between the surface cationic charges of CP1 and anionic bacterial membranes may play an important role in the antimicrobial activity of the surface-immobilized CP1 peptide.

Improving the Phototherapeutic Efficiencies of Molecular and Nanoscale Materials by Targeting Mitochondria.

Mitochondria-targeted cancer phototherapy (PT), which works by delivering photoresponsive agents specifically to mitochondria, is a powerful strategy to improve the phototherapeutic efficiency of anticancer treatments. Mitochondria play an essential role in cellular apoptosis, and are relevant to the chemoresistance of cancer cells. Furthermore, mitochondria are a major player in many cellular processes and are highly sensitive to hyperthermia and reactive oxygen species. Therefore, mitochondria serve as excellent locations for organelle-targeted phototherapy. In this review, we focus on the recent advances of mitochondria-targeting materials for mitochondria-specific PT. The combination of mitochondria-targeted PT with other anticancer strategies is also summarized. In addition, we discuss both the challenges currently faced by mitochondria-based cancer PT and the promises it holds.

Simultaneous label-free screening of G-quadruplex active ligands from natural medicine via a microfluidic chip electrophoresis-based energy transfer multi-biosensor strategy.

Rapid screening of active compounds plays a crucial role in the research and application of complex natural medicines. Herein, a new method of simultaneous label-free multi-drug screening based on a selective aptamer-carboxyfluorescein/graphene oxide energy transfer optical sensor combined with microfluidic chip electrophoretic separation is reported. In this study, seven traditional Chinese medicinal monomers were chosen as targets for the screening of G-quadruplex ligands. The screening results of the G-quadruplex active ligands, including daidzein, berberine hydrochloride, jatrorrhizine hydrochloride, and fangchinoline, and non-active ligands, including geniposide and oxymatrine, were consistent with those reported in literature. Moreover, one new potential G4DNA active drug, jujuboside A, was identified. Molecular simulation of the interaction between G4DNA and drugs was also carried out using HyperChem and AutoDock to verify the results of the experimental screening. It further demonstrated the reliability of our strategy. This novel separation and concentration based multi-sensing strategy provides a simple, rapid, and sensitive tool for simultaneous multi-drug screening, which is very meaningful for drug screening and bio-interaction analysis.

A ß-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production.

BACKGROUND: The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low ß-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although ß-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear. RESULTS: The T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular ß-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 ß-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed. CONCLUSIONS: The T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular ß-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of ß-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.

Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

Differential expression of SUMO-specific protease 7 variants regulates epithelial-mesenchymal transition.

Two Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) variants are naturally expressed in breast epithelia. Breast cancer (BCa) onset down-regulates the short SENP7 splice variant (SENP7S) and enhances the long transcript (SENP7L). Here, we show that SENP7L induction promotes gene expression profiles that favor aberrant proliferation and initiate epithelial-mesenchymal transition (EMT). SENP7L exhibits an interaction domain for the epigenetic remodeler heterochromatin protein 1 α (HP1α) and isopeptidase activity against SUMO-modified HP1α. Loss of this interaction domain, as observed with SENP7S, favors HP1α SUMOylation. SUMOylated HP1α is enriched at E2F-responsive and mesenchymal gene promoters, silences transcription of these genes, and promotes cellular senescence. Elevated SENP7L renders HP1α hypo-SUMOylated, which relieves transcriptional repression of the same genes and concurrently decreases transcription of epithelial-promoting genes via an HP1α-independent mechanism. Consequently, SENP7L levels correlate with EMT, motility, and invasiveness of BCa cells. Stable knockdown of elevated SENP7L levels lessens the dissemination of highly metastatic BCa cells to the lungs from primary implantation sites in in vivo studies. Thus, differential splicing of the SENP7 regulates either tumor suppression or progression.