Meta-genome analysis of a newly enriched azo dyes detoxification halo-thermophilic bacterial consortium.

Treating textile wastewaters were always inhibited by its higher salt concentration and temperature. In this study, a halo-thermophilic bacterial consortium YM was enriched with ability to decolorize acid brilliant scarlet GR (ABS) at 55 °C and 10% salinity. Under optimum conditions of pH (8), temperature (55 °C), and salinity (10%), YM decolorized 97% of ABS under anaerobic conditions. Alteribacillus was identified to be the dominant genus in consortium YM. Consortium YM showed significant decolorization ability under a wide range of salinity (1%-10%), pH (7-9) and temperature (45 °C-60 °C). The degradation pathway of ABS was proposed by the combination of UV-vis spectral analysis, Fourier transform infrared (FTIR), gas chromatography mass spectrometric (GC-MS), and metagenomic analysis. Azoreductase, which was an important enzyme in decolorization process, was identified with great variation in the genome of consortium YM. Meanwhile, the metabolic intermediates after decolorization was identified with low biotoxicity by phytotoxicity tests. This study first identified that Alterbacillus play an important role in azo dye decolorization and degradation process under halo-thermophlic conditions and provided significant knowledge for azo dye decolorization and degradation process.

An actin-like protein PoARP9 involves in the regulation of development and cellulase and amylase expression in Penicillium oxalicum.

AIMS: In eukaryotic cells, chromatin remodelling complexes are essential for the accessibility of transcription factors to the specific regulating regions of downstream genes. Here, we identified an actin-like protein PoARP9 in cellulase production strain Penicillium oxalicum 114-2, which was an essential member of SWI/SNF complex. To investigate the physiological function of PoARP9 in transcriptional regulation, the coding gene Poarp9 was deleted in P. oxalicum 114-2. METHODS AND RESULTS: The absence of PoARP9 affected the colony growth on medium with glucose, cellulose or starch as sole carbon source. Meanwhile, the expression levels of major cellulase genes were all upregulated in ΔPoarp9 under the cellulase-inducing condition. In addition, the expression levels of amylase transcription activator AmyR as well as two major amylase genes were also increased in ΔPoarp9. CONCLUSIONS: These results demonstrated that chromatin remodelling affects the development and expression of cellulase and amylase in P. oxalicum. And the SWI/SNF complex member PoARP9 plays essential roles in these processes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided new insights into the regulation of cellulase and development in P. oxalicum. And the regulatory function of SWI/SNF complex member ARP9 towards cellulase and amylase expression in P. oxalicum was verified for the first time.

The Two-Steps Reaction Fluorescent Probe for the Selective Detection of Cysteine and Its Applications.

We reported the specific fluorescent probe (MC-BOD-XDS) with two-steps reaction based on monosulfanyl-coumarin-BODIPY for selective detection of cysteine, high activity sulfanyl-coumarin as the multiple reaction group instead of a group internal standard fluorophore. The reaction mechanism of MC-BOD-XDS for detecting cysteine was different from the reported probes about the nucleophilic aromatic substitution reaction (SNAr) of chlorinated BODIPY. The fluorescent color of MC-BOD-XDS changed from yellow to red, and then to orange. The linear calibration diagram showed that it can potentially be used for quantitatively detection of Cys. Its potential applications were demonstrated by employing it for detection of Cys in artificial urine and in fluorescent imaging in HeLa cells.

A dual-site multifunctional fluorescent probe for selective detection of endogenous H2O2 and SO2 derivatives based on ICT process and its bioimaging application.

In this paper, we reported a coumarin-based fluorescent probe for selective detection of H2O2/SO2 derivatives via ICT process. To the best of our knowledge, it was few reported with the same probe to enable visual detection of H2O2/SO2 derivatives in vivo and in vitro. H2O2 and SO32- were selectively sensed over other analytes, and the probe displayed 20-fold and 220-fold relative fluorescence intensity respectively, as well as the good linear relationship and the excellent detection limits of 2.7 * 103 nM and 19.3 nM. Furthermore, the probe was successfully used for fluorescence imaging of the HeLa cells and the mice to monitor exogenous and endogenous H2O2 and SO32-, suggesting its potential biomedical application for investigation and detection the intermediate indicator of oxidative stress in vitro and in vivo.

Phosducin-like protein PoPlp1 impacts cellulase and amylase expression and development in Penicillium oxalicum via the G protein-cAMP signaling pathway.

In this study, a phosducin-like protein, PoPlp1, was identified and functionally studied in the cellulase-producing strain Penicillium oxalicum 114-2. PoPlp1 was proven to participate in several biological processes, including mycelium development, conidiation, and expression of cellulases and amylases. With deletion of Poplp1, morphology and development varied significantly in ΔPoplp1. Colony growth, glucose utilization, and the hydrolysis capability of starch and cellulose were limited, whereas conidiation was enhanced. Based on detection of the levels of expression of transcription factors involved in asexual development, we conjectured that PoPlp1 is involved in conidiation via the major factor BrlA. We explored the effect of PoPlp1 on cellulase and amylase expression and observed that cellulase and amylase activity and major gene transcription levels were all dramatically reduced in ΔPoplp1. Deletion of PoPlp1 caused a decrease in intracellular cAMP levels, and the cellulase gene expression level of ΔPoplp1 was restored to a certain extent through external addition of cAMP. These findings demonstrate that PoPlp1 may affect cellulase and amylase expression by regulating cAMP concentration. To comprehensively explore the mechanism of PoPlp1 in regulating multiple biological processes, we performed a comparative transcriptomic analysis between strains P. oxalicum 114-2 and ΔPoplp1. The major cellulase and amylase genes were all downregulated, congrent with the results of real-time quantitative polymerase chain reaction analysis. The genes involved in the G protein-cAMP signaling pathway, including several G-protein-coupled receptors, one regulator of G protein signaling, and two cAMP phosphodiesterases, were disrupted by deletion of PoPlp1. These results confirm the positive function of PoPlp1 in the G protein-cAMP signaling pathway. This functional analysis of PoPlp1 will be very beneficial for further study of the regulatory mechanisms of cellulase expression and other biological processes in P. oxalicum 114-2 via the G protein-cAMP signaling pathway.

Functional Study of cAMP-Dependent Protein Kinase A in Penicillium oxalicum.

Signaling pathways play a crucial role in regulating cellulase production. The pathway mediated by signaling proteins plays a crucial role in understanding how cellulase expression is regulated. In this study, using affinity purification of ClrB, we have identified sixteen proteins that potentially interact with ClrB. One of the proteins, the catalytic subunit of cAMP-dependent protein kinase A (PoPKA-C), is an important component of the cAMP/PKA signaling pathway. Knocking out PoPKA-C resulted in significant decreases in the growth, glucose utilization, and cellulose hydrolysis ability of the mutant strain. Furthermore, the cellulase activity and gene transcription levels were significantly reduced in the ΔPoPKA-C mutant, while the expression activity of CreA, a transcriptional regulator of carbon metabolism repression, was notably increased. Additionally, deletion of PoPKA-C also led to earlier timing of conidia production. The expression levels of key transcription factor genes stuA and brlA, which are involved in the production of the conidia, showed significant enhancement in the ΔPoPKA-C mutant. These findings highlight the involvement of PoPKA-C in mycelial development, conidiation, and the regulation of cellulase expression. The functional analysis of PoPKA-C provides insights into the mechanism of the cAMP/PKA signaling pathway in cellulase expression in filamentous fungi and has significant implications for the development of high-yielding cellulase strains.

Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism.

Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. It highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is relatively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we performed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2. AAVzk2 displayed a similar yield but divergent post-translational modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Overall, we provide a novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.

The Impact of Different Rose Bengal Formulations on Corneal Thickness and the Efficacy of Rose Bengal/Green Light Corneal Cross-linking in the Rabbit Eye.

PURPOSE: To examine central corneal thickness (CCT) changes during in vivo rose bengal-green light corneal cross-linking (RG-CXL) and compare the CXL efficacy of different rose bengal formulations. METHODS: After epithelium removal, the right eyes of rabbits were immersed in rose bengal solution prepared by different solvents (water, phosphate buffered saline, dextran, and hydroxypropyl methylcellulos [HPMC]) for 2 or 20 minutes, then the rose bengal distribution in the corneal stroma was analyzed by confocal fluorescence detection. During the RG-CXL process, the CCT was measured at seven time points. The left eyes served as the untreated control group. Corneal enzymatic resistance and corneal biomechanics were tested to compare the RG-CXL efficacy. RESULTS: The rose bengal infiltration depths were 120 and 200 µm for the 2- and 20-minute groups, respectively. CCT increased significantly after infiltration, then decreased significantly in the first 200 seconds of irradiation and decreased slowly for the next 400 seconds. The CCT of the 20-minute groups was significantly thicker than that of the 2-minute groups (P < .0001). All RG-CXL treatments improved the corneal enzymatic resistance and corneal biomechanics, with the effects being greater in the 20-minute groups. The inclusion of 1.1% HPMC in the rose bengal formulation helped to maintain CCT during irradiation while not affecting either the infiltration of rose bengal or the efficacy of RG-CXL. CONCLUSIONS: Within the range studied, RG-CXL efficacy increased with infiltration time. The incorporation of a 20-minute infiltration of 0.1% rose bengal-1.1% HPMC into the RG-CXL procedure may further improve the safety of the treatment and its prospects for clinical use. [J Refract Surg. 2022;38(7):450-458.].

Engineering Hibiscus-Like Riboflavin/ZIF-8 Microsphere Composites to Enhance Transepithelial Corneal Cross-Linking.

Riboflavin-5-phosphate (RF) is the most commonly used photosensitizer in corneal cross-linking (CXL), but its hydrophilicity and negative charge limit its penetration through the corneal epithelium into the stroma. To enhance the corneal permeability of RF and promote its efficacy in the treatment of keratoconus, novel hibiscus-like RF@ZIF-8 microsphere composites [6RF@ZIF-8 NF (nanoflake)] are prepared using ZIF-8 nanomaterials as carriers, which are characterized by their hydrophobicity, positive potential, biocompatibility, high loading capacities, and large surface areas. Both hematoxylin and eosin endothelial staining and TUNEL assays demonstrate excellent biocompatibility of 6RF@ZIF-8 NF. In in vivo studies, the 6RF@ZIF-8 NF displayed excellent corneal permeation, and outstanding transepithelial CXL (TE-CXL) efficacy, slightly better than the conventional CXL protocol. Furthermore, the special hibiscus-like structures of 6RF@ZIF-8 NF meant that it has better TE-CXL efficacy than that of 6RF@ZIF-8 NP (nanoparticles) due to the larger contact area with the epithelium and the shorter RF release passage. These results suggest that the 6RF@ZIF-8 NF are promising for transepithelial corneal cross-linking, avoiding the need for epithelial debridement.

A Homeodomain-Containing Transcriptional Factor PoHtf1 Regulated the Development and Cellulase Expression in Penicillium oxalicum.

Homeodomain-containing transcription factors (Htfs) play important roles in animals, fungi, and plants during some developmental processes. Here, a homeodomain-containing transcription factor PoHtf1 was functionally characterized in the cellulase-producing fungi Penicillium oxalicum 114-2. PoHtf1 was shown to participate in colony growth and conidiation through regulating the expression of its downstream transcription factor BrlA, the key regulator of conidiation in P. oxalicum 114-2. Additionally, PoHtf1 inhibited the expression of the major cellulase genes by coordinated regulation of cellulolytic regulators CreA, AmyR, ClrB, and XlnR. Furthermore, transcriptome analysis showed that PoHtf1 participated in the secondary metabolism including the pathway synthesizing conidial yellow pigment. These data show that PoHtf1 mediates the complex transcriptional-regulatory network cascade between developmental processes and cellulolytic gene expression in P. oxalicum 114-2. Our results should assist the development of strategies for the metabolic engineering of mutants for applications in the enzymatic hydrolysis for biochemical production.

Conotoxin αD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA, and is probably part of the binding site of this toxin. These results together suggest that αD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of αD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.