Post-crotonylation oxidation by a monooxygenase promotes acetyl-CoA synthetase degradation in Streptomyces roseosporus.

Protein post-translational modifications (PTMs) with various acyl groups play central roles in Streptomyces. But whether these acyl groups can be further modified, and the influences of these potential modifications on bacterial physiology have not been addressed. Here in Streptomyces roseosporus with rich crotonylation, a luciferase monooxygenase LimB is identified to elaborately regulate the crotonylation level, morphological development and antibiotic production by oxidation on the crotonyl groups of an acetyl-CoA synthetase Acs. This chemical modification on crotonylation leads to Acs degradation via the protease ClpP1/2 pathway and lowered intracellular crotonyl-CoA pool. Thus, we show that acyl groups after PTMs can be further modified, herein named post-PTM modification (PPM), and LimB is a PTM modifier to control the substrate protein turnover for cell development of Streptomyces. These findings expand our understanding of the complexity of chemical modifications on proteins for physiological regulation, and also suggest that PPM would be widespread.

Development of a native-locus dual reporter system for the efficient screening of the hyper-production of natural products in Streptomyces.

Streptomyces is renowned for its abundant production of bioactive secondary metabolites, but most of these natural products are produced in low yields. Traditional rational network refactoring is highly dependent on the comprehensive understanding of regulatory mechanisms and multiple manipulations of genome editing. Though random mutagenesis is fairly straightforward, it lacks a general and effective strategy for high throughput screening of the desired strains. Here in an antibiotic daptomycin producer S. roseosporus, we developed a dual-reporter system at the native locus of the daptomycin gene cluster. After elimination of three enzymes that potentially produce pigments by genome editing, a gene idgS encoding the indigoidine synthetase and a kanamycin resistant gene neo were integrated before and after the non-ribosomal peptidyl synthetase genes for daptomycin biosynthesis, respectively. After condition optimization of UV-induced mutagenesis, strains with hyper-resistance to kanamycin along with over-production of indigoidine were efficiently obtained after one round of mutagenesis and target screening based on the dual selection of the reporter system. Four mutant strains showed increased production of daptomycin from 1.4 to 6.4 folds, and significantly improved expression of the gene cluster. Our native-locus dual reporter system is efficient for targeting screening after random mutagenesis and would be widely applicable for the effective engineering of Streptomyces species and hyper-production of these invaluable natural products for pharmaceutical development.

Bacterial DNA on the skin surface overrepresents the viable skin microbiome.

The skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here, we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it.

Efficacy and Safety of Extracellular Matrix on Wound Healing After Picosecond Laser Therapy.

BACKGROUND: Extracellular matrix (ECM), a material with tissue repair function, is applied to treat various wounds. However, the role of ECM in facilitating wound healing after facial laser treatment remains elusive. OBJECTIVE: To assess the efficacy and safety of ECM in promoting wound healing after picosecond laser therapy (PLT). MATERIALS AND METHODS: Eighteen female subjects with benign pigmentation disorders were randomly assigned to the ECM (n = 9) and control groups (n = 9). After PLT, the ECM and control groups were treated with ECM and facial moisturizer in the first 7 days, respectively. The severity of erythema and edema was assessed using photographs. The duration of erythema, edema, scab shedding, postinflammatory hyperpigmentation incidence (PIH), and adverse events was documented in detail. RESULTS: Compared with the control group, the ECM group had a shorter duration of erythema, edema, and scab shedding after PLT (p < .01). A significantly decreased severity of erythema (p < .05) and edema (p < .01) was found in the ECM group versus the control group, respectively. The PIH incidence in the ECM group was lower than in controls, albeit without statistical significance. No serious adverse events were observed during the follow-up. CONCLUSION: Extracellular matrix is an effective and safe dressing for promoting wound healing after PLT.

Precision targeting of autoantigen-specific B cells in muscle-specific tyrosine kinase myasthenia gravis with chimeric autoantibody receptor T cells.

Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune disease that causes life-threatening muscle weakness due to anti-MuSK autoantibodies that disrupt neuromuscular junction signaling. To avoid chronic immunosuppression from current therapies, we engineered T cells to express a MuSK chimeric autoantibody receptor with CD137-CD3ζ signaling domains (MuSK-CAART) for precision targeting of B cells expressing anti-MuSK autoantibodies. MuSK-CAART demonstrated similar efficacy as anti-CD19 chimeric antigen receptor T cells for depletion of anti-MuSK B cells and retained cytolytic activity in the presence of soluble anti-MuSK antibodies. In an experimental autoimmune MG mouse model, MuSK-CAART reduced anti-MuSK IgG without decreasing B cells or total IgG levels, reflecting MuSK-specific B cell depletion. Specific off-target interactions of MuSK-CAART were not identified in vivo, in primary human cell screens or by high-throughput human membrane proteome array. These data contributed to an investigational new drug application and phase 1 clinical study design for MuSK-CAART for the treatment of MuSK autoantibody-positive MG.

Engineered bacteria for augmented in situ tumor vaccination.

In situ tumor vaccination has aroused tremendous interest with its capability for eliciting strong and systemic antitumor immune responses. Unlike traditional cancer vaccines, in situ tumor vaccination avoids the laborious process of tumor antigen identification and can modulate tumor immunosuppressive microenvironment at the same time. In recent years, bacteria have been used as both efficient tumor-targeted delivery vehicles and potent adjuvants. Regarding the rapid development in this area, in this review, we summarize recent advances in the application of bacteria for in situ cancer vaccination. We illustrate the mechanisms of bacteria as both efficient tumor immunogenic cell death inducers and tumor-targeted delivery platforms. Then we comprehensively review the engineering strategies for designing bacteria-based in situ vaccination, including chemical modification, nanotechnology, and genetic engineering. The current dilemma and future directions are discussed at the end of this review.

Coordination of Polyketide Release and Multiple Detoxification Pathways for Tolerable Production of Fungal Mycotoxins.

Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self-protection is necessary to avoid self-harm resulting from over-accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs are not well understood. Herein, in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin, we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over-produced, the non-enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for self-protection. These findings provided new insight into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.

Complex Interplay and Catalytic Versatility of Tailoring Enzymes for Efficient and Selective Biosynthesis of Fungal Mycotoxins.

Mycotoxins have substantial impacts on agricultural production and food preservation. Some have high similarities in bioactivity but subtle differences on structures from various fungal producers. Understanding of their complex cross-biosynthesis will provide new insights into enzyme functions and food safety. Here, based on structurally related mycotoxins, such as aurovertins, asteltoxin, and citreoviridin, we showed that methyltransferase (MT)-catalyzed methylation is required for efficient oxidation and polyketide stability. MTs have broad interactions with polyketide synthases and flavin-containing monooxygenases (FMOs), while MT AstB is required for FMO AstC functionality in vivo. FMOs have common catalysis on pyrone-polyene intermediates but different catalytic specificity and efficiency on oxidative intermediates for the selective production of more toxic and complex mycotoxins. Thus, the subtle protein interaction and elaborate versatile catalysis of biosynthetic enzymes contribute to the efficient and selective biosynthesis of these structure-related mycotoxins and provide the basis to re-evaluate and control mycotoxins for agricultural and food safety.

Immunotherapy for Pemphigus: Present and Future.

Pemphigus is a chronic and severe autoimmune bullous disease caused by autoantibodies targeting adhesion molecules between keratinocytes. It requires 2-3 years on average to manage the disease. To date, although Rituximab combined with short-term systemic glucocorticoids was accepted as first-line therapy, systemic glucocorticoids remain the primary therapeutic option for pemphigus patients, successfully decreasing morbidity and mortality from pemphigus. However, novel therapeutic strategies are desirable due to the low efficacy in some subset of patients and the long-term severe adverse effects of traditional therapies. Recently, immunotherapy has proved to be encouraging for disease control or cure. Based on the current understanding of the immune mechanisms of pemphigus, we review the immune targets and corresponding agents applied in practice or under clinical trials. The goals of the novel treatments are to improve the quality of life of pemphigus patients by improving efficacy and safety, minimizing side effects, achieving fast disease control, or curing the disease.

m4C DNA methylation regulates biosynthesis of daptomycin in Streptomyces roseosporus L30.

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces, the global regulation based on epigenetic modification is not well explored. N4-methylcytosine (m4C), an abundant epigenetic marker in Actinomycetes' genome, but its regulatory mechanism remains unclear. In this study, we identify a m4C methyltransferase (SroLm3) in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary metabolism. Notably, three BGCs in ΔsroLm3 strain exhibited decreased expression compared to wild type. In-frame deletion of sroLm3 in S.roseosporus L30 further revealed its role in enhancing daptomycin production. In summary, we characterized a m4C methyltransferase, revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment, and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C methylation. Our research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.

High-Frequency Ultrasound for Long-term Safety Assessment of Poly-L-Lactic Acid Facial Filler.

BACKGROUND: Injectable poly- l -lactic acid (PLLA) is a new type of biodegradable dermal filler that has been utilized for soft tissue filling. However, there is no convenient and reliable method to assess the long-term safety of PLLA filler. OBJECTIVE: To assess the long-term safety of PLLA injection into nasolabial folds by high-frequency ultrasound and to select the ultrasonic probes with the most appropriate frequency. MATERIALS AND METHODS: After a 30-month PLLA injection into the deep dermis of the nasolabial fold, subjects were examined by high-frequency ultrasound with the 20 MHz and 50 MHz probes. RESULTS: Twenty subjects with nasolabial fold contour deficiency were enrolled in this study. After a 30-month PLLA injection in nasolabial folds, PLLA degraded entirely in 16 subjects (16/20, 80%), and abnormal echo in the skin was observed in 4 subjects (4/20, 20%) caused by undegraded PLLA microparticles, PLLA microparticles deposition, fibrous nodules, and granuloma. The 20-MHz probe is more appropriate than the 50-MHz probe for evaluating the adverse effects of PLLA injection. CONCLUSION: High-frequency ultrasound is a rapid, reliable, and noninvasive method to monitor the degradation condition of PLLA and the formation of papules and nodules associated with PLLA injection.

Photodynamic therapy in the treatment of rosacea: A systematic review.

OBJECTIVE: To systematically review the efficacy of photodynamic therapy (PDT) in the treatment of rosacea. METHODS: PubMed, Embase, and Cochrane Library databases were searched for articles published by February 5, 2022, using "photodynamic therapy" and "rosacea" as the keywords. RESULTS: Nine studies were included in the review. The number of patients varied from 1 to 30 in each study, with ages ranging from 18 to 76 years. Methyl aminolevulinate (MAL) and aminolevulinic acid (ALA) were used as the photosensitizer, and red light, blue light, intense pulsed light (IPL), long-pulsed dye laser (LPDL), pulsed dye laser (PDL), and tungsten lamp were used as the light or laser source. The follow-up time ranged from one month to 25 months. Most of the studies showed a satisfactory clinical response, and the side effects were tolerant and temporary. CONCLUSION: Current studies have provided preliminary evidence that PDT is an efficient and safe therapy in treating rosacea. However, rigorous randomized control trials (RCTs) with a larger sample size and longer follow-up time are warranted to verify the curative effects of PDT in treating rosacea and explore the most appropriate treatment schedule.

IL-13 Genetic Susceptibility to Bullous Pemphigoid: A Potential Target for Treatment and a Prognostic Marker.

Background: Bullous pemphigoid (BP) is a senile chronic autoimmune bullous skin disease with a high relapse rate, which significantly impairs patients' quality of life and contributes to disease mortality. This observational case-control study explores the gene polymorphisms of cytokines and their clinical significance in Chinese patients with BP. Methods: IL-1α (rs1800587), IL-1ß (rs16944, rs1143627, rs1143634), IL-4 (rs2243250), IL-6 (rs1800795), IL-10 (rs1800896, rs1800871, rs1800872), IL-13 (rs1800925, rs20541), TNF-α (rs1799964, rs1800630, rs1799724, rs361525), IFN-γ (rs1799964, rs1800630, rs361525, rs1800629, rs4248160, rs1800750), and TGF-ß1 (rs2317130, rs1800469, rs4803457) genes were genotyped in the healthy controls and BP patients, respectively. Expression of these cytokines in serum was measured. Medical profiles of patients, including baseline characteristics and prognosis, were statistically analyzed. Results: We found that IL-1 ß and IL-13 concentrations were higher in the BP patients' sera compared to those in the controls. For IL-13, significant differences were found in the nucleotide ratio/genotype/haploid frequency/haplotype, respectively. IL-13 (rs20541, rs1800925) is related to gender, and the IL-13 genotype was significantly associated with recurrence. Conclusions: BP is associated with IL-13 gene polymorphism and IL-13 concentration is elevated in blood circulation in patients with BP. Our results support that IL-13 is relevant in the pathogenesis of BP, suggesting that IL-13 could potentially represent a promising target for BP therapy and a prognostic marker.

A target and efficient synthetic strategy for structural and bioactivity optimization of a fungal natural product.

Drugs have been largely inspired from natural products, while enzymes underlying their biosynthesis have enabled complex structures and diverse bioactivities. Nevertheless, the high enzyme specificity and limited in vivo precursor types have restricted the natural product reservoir, but Nature has imprinted natural products with active sites, which can be readily modified by chemosynthesis with various functional groups for more favorable druggability. Here in the less exploited fungal natural products, we introduced CtvA, a polyketide synthase for a mycotoxin citreoviridin biosynthesis in Aspergillus, into an endophytic fungus Calcarisporium arbuscula to expand tetrahydrofuran (THF) into a dioxabicyclo-octane (DBO) ring moiety based on versatility and promiscuity of the aurovertin biosynthetic enzyme. Alternative acylations on the hydroxyl groups essential for cell toxicity by chemosynthesis produced compounds with improved anti-tumor activities and pharmacokinetics. Thus, we showed an effective strategic way to optimize the fungal natural product efficiently for more promising drug development.

Fine-Tuning Cas9 Activity with a Cognate Inhibitor AcrIIA4 to Improve Genome Editing in Streptomyces.

Efficient enabling technology is required for synthetic biology in Streptomyces due to its natural product reservoir. Though the CRISPR-Cas9 system is powerful for genome editing in this genus, the proposed Cas9 toxicity has limited its application. Here on the basis of previous inducible Cas9 expression at the transcriptional and translational levels coupled with atpD overexpression, a Cas9 cognate inhibitor AcrIIA4 was further introduced to fine-tune the Cas9 activity. In both laboratory and industrial Streptomyces species, we showed that, compared to the constitutively expressed Cas9, incorporating AcrIIA4 increased the conjugation efficiency from 700- to 7000-fold before induction, while a comparable 65%-90% editing efficiency was obtained even on multiple loci for simultaneous deletion after Cas9 expression was induced, along with no significant off-targets. Thus, AcrIIA4 could be a modulator to control Cas9 activity to significantly improve genome editing, and this new toolkit would be widely adaptable and fasten genetic engineering in Streptomyces.

Discovery of a Potential Liver Fibrosis Inhibitor from a Mushroom Endophytic Fungus by Genome Mining of a Silent Biosynthetic Gene Cluster.

Liver fibrosis has accounted for liver diseases and overall mortality, but no relevant drug has been developed. Filamentous fungi are important resources of natural products for pharmaceutical development. Calcarisporium arbuscula is a mushroom endophytic fungus, which primarily produces aurovertins. Here, in an aurovertin null-production mutant, one silent gene cluster (mca17) was activated by overexpression of a pathway-specific zinc finger transcriptional regulator, and a tetramic acid-type compound (1, MCA17-1) was identified. Along with detailed structural characterization, its biosynthesis was proposed to be produced from the core PKS-NRPS hybrid enzyme. Moreover, 1 suppressed the activation of LX-2 upon transforming growth factor-ß (TGF-ß) challenge and had stronger bioactivity than the positive control obeticholic acid (OCA) against liver fibrosis. Our work suggested that this engineered fungus could be a producer of 1 for promising pharmaceutical development, and alternatively, it would be developed as a mushroom ingredient in dietary therapy to prevent liver fibrosis.

A Cell Factory of a Fungicolous Fungus Calcarisporiumarbuscula for Efficient Production of Natural Products.

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus Calcarisporiumarbuscula NRRL 3705 predominantly produces the highly reduced polyketide-type mycotoxins aurovertins. Here after evaluation of the aurovertin-null mutant ΔaurA as an efficient host, we further screened two strong promoters aurBp and A07068p based on RNA-Seq, and successfully activated an endogenous gene cluster from C. arbuscula as well as three additional exogenous BGCs from other fungi to produce polyketide-type natural products. Thus, we showed an efficient expression system from the fungicolous fungus C. arbuscula, which will be highly beneficial and complementary to the conventional Aspergillus and Penicillium fungal cell factories, and provides a useful toolkit for genome-wide mining of bioactive natural products from fungicolous fungi.

Analysis of the autoimmune response against BP180 in patients with Alzheimer's disease.

BACKGROUND: Current epidemiological studies suggest a significant correlation between Alzheimer's disease (AD) and bullous pemphigoid (BP). However, the autoimmune response against BP180 in patients with AD has not been fully understood. investigated. METHODS: We randomly enrolled 48 patients with AD and 50 sex- and age-matched healthy controls. We detected the presence/absence and the level of anti-BP180/BP230 immunoglobulin G (IgG) autoantibodies in the patients' serum to determine whether said antibodies possess reactivity against the BP180 protein in the human brain and skin. RESULTS: The enzyme-linked immunosorbent assay (ELISA) results revealed that the positive rate of anti-BP180 autoantibodies in patients with AD (23/48, 47.9%) was significantly higher than that in controls (4/50, 8.0%; P<0.0001). These ELISA-positive patients were further examined through immunoblotting. Sera from nine patients with AD (9/23, 39.1%) and one control (1/4, 25.0%) reacted with human cutaneous recombinant full-length BP180 and BP180-noncollagenous 16A (NC16A). Sera from 11 (11/23, 47.8%) patients with AD reacted with a 180-kDa protein from the human brain extract, but none of the controls' sera recognized the corresponding protein band. The majority of the patients in the anti-BP180-positive AD group were men (14/23, 60.9%) who were older (74.0 years) compared with those in the control group (6/25, 24.0%; P<0.05) (72.2 years; P<0.01). CONCLUSIONS: Anti-BP180 autoantibodies are present in AD and recognize human recombinant full-length BP180 and a 180-kDa protein from the human brain extract, suggesting that BP180 is a shared autoantigen in AD and BP and may help clarify the mechanism to explain why a high risk of BP exists in AD. Elderly male patients with AD are significantly more likely to develop BP180 serum autoreactivity compared with other patients with AD.

Detection of underlying dementia in bullous pemphigoid patients using cognitive evaluation tests: a multicenter case-control study.

BACKGROUND: Preliminary observation in clinical practice showed that subjective neurocognitive complaints are relatively common in bullous pemphigoid (BP) patients. Yet, little has been done to investigate the neurocognitive status in BP. METHODS: This is a multicenter observational case-control study comprised of 61 BP patients and 65 matched control subjects from 3 medical centers in China from 2014 to 2019. To evaluate the cognitive function between BP patients and matched controls, all the subjects finished the mini-mental state examination (MMSE) and montreal cognitive assessment (MoCA). RESULTS: The overall scores were significantly lower in BP than in controls (PMoCA<0.001). The percentage of patients screened positive for cognitive impairment in the BP group was significantly higher than in the control group (PMMSE<0.001, PMoCA<0.001). On multivariate analysis, BP was associated with increased odds of cognitive impairment by MMSE and MoCA (PMMSE=0.017, PMoCA=0.007). CONCLUSIONS: BP patients had decreased cognitive abilities and were at a higher risk of cognitive impairment. The evaluation of cognitive impairment may be warranted for BP patients in clinical practice for early diagnosis and therapy of dementia.