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BACKGROUND: Poly L-lactic acid (PLLA) can stimulate fibrous tissue regeneration to exert a filling effect. However, severe inflammatory reactions and unsatisfactory effects remain a concern. OBJECTIVE: Herein, we describe the mechanism of action, efficacy, and safety of PLLA microspheres in suspension (PLLA-b-PEG/HA) for facial contouring and soft tissue augmentation. METHODS: PLLA-b-PEG/HA, ssynthesized by copolymerization with ethylene glycol, were suspended in hyaluronic acid (HA). Physiological verification was performed using scanning electron microscopy and X-ray computed tomography. PLLA-b-PEG/HA were subcutaneously injected into the dorsal region of 4-month-old rabbits. Ultrasound assessed volumetric capacity at 3 days and 1, 2, 4, and 12 weeks. The inflammatory response, collagen production, and HA degradation were evaluated. A retrospective case series of 10 patients who received PLLA-b-PEG/HA injections was conducted to assess long-term efficacy and safety. RESULTS: PLLA-b-PEG exhibited a spherical structure with a smooth surface (20-45 µm diameter). In rabbits, implant site volume increased within 4 weeks, gradually decreasing thereafter. Fibrous capsules, microvessel density, and new collagen fiber formation progressively increased at 4, 12, and 26 weeks after injection. Clinical data demonstrated significant improvements in face contouring at months 3 and 12 after injection. All patients showed improved internal contours based on the Global Aesthetic Improvement Scale. After 12 months, 90% of the patients retained good shaping and support effects with minimal adverse reactions. CONCLUSIONS: PLLA-b-PEG/HA demonstrated superior biocompatibility and facial regeneration potential, with outstanding dual collagen-stimulating properties. The clinical efficacy and safety of PLLA-b-PEG/HA have been validated and established as a promising therapeutic option.
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BACKGROUND: B cell exhaustion (BEX) refers to the impairment of normal B cell functions and decreased proliferation capability. However, the prognostic value of BEX-related genes in bladder cancer (BLCA) remains unclear. METHODS: BLCA cases from TCGA were used for training, while GSE5287, GSE13507, GSE31684, and GSE32894 cohorts from GEO were used for external validation. BEX-related genes were identified through literature retrieval, unsupervised clustering, and genomic difference detection. Gene pairing, LASSO, random forest, and Cox regression were employed to construct a predictive model. B cell samples from scRNAseqDB, GSE111636, and IMvigor210 were utilized to explore immunoprofiles and the predictive ability of the model in immunotherapeutic response. Additionally, 21 pairs of BLCA and paracarcinoma samples from Nanfang Hospital were used to re-confirm our findings through RT-qPCR, immunofluorescence, and flow cytometry. RESULTS: 39 BEX-related genes were identified. A 4-gene-pair signature was constructed and served as a reliable prognostic predictor across multiple datasets (pooled HR = 2.32; 95 % CI = 1.81-2.98). The signature reflected the BEX statuses of B cells (FDR < 0.05) and showed promise in evaluating immunotherapeutic sensitivity (P < 0.001). In the local cohort, CD52, TUBB6, and CAV1 were down-regulated in BLCA tissues, while TGFBI, UBE2L6, TINAGL1, and IL32 were up-regulated (all P < 0.05). Furthermore, the infiltration levels of CD19 + CD52 + and CD19 + TUBB6 + B cells in paracarcinoma samples were higher than those in BLCA samples (all P < 0.05). CONCLUSION: A BEX-related gene signature was developed to predict prognosis and immunotherapeutic sensitivity in BLCA, providing valuable guidance for personalized treatment.
Subject(s)
B-Lymphocytes , Immunotherapy , Urinary Bladder Neoplasms , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/immunology , Humans , Prognosis , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Male , Female , Gene Expression Profiling/methods , Transcriptome , Middle Aged , Immune System ExhaustionABSTRACT
In transdermal drug delivery system (TDDS) patches, achieving prolonged adhesion, high drug loading, and rapid drug release simultaneously presented a significant challenge. In this study, a PHT-SP-Cu2+ adhesive was synthesized using polyethylene glycol (PEG), hexamethylene diisocyanate (HDI), trimethylolpropane (TMP), and silk protein (SP) as functional monomers which were combined with Cu2+ to improve the adhesion, drug loading, and drug release of the patch. The structure of the adhesion chains and the formation of Cu2+-p-π conjugated network in PHT-SP-Cu2+ were characterized and elucidated using different characterization methods including FT-IR, 13C NMR, XPS, SEM imaging and thermodynamic evaluation. The formulation of pressure-sensitive adhesive (PSA) was optimized through comprehensive research on adhesion, mechanics, rheology, and surface energy. The formulation of 3 wt.% SP and 3 wt.% Cu2+ provided superior adhesion properties compared to commercial standards. Subsequently, the peel strength of PHT-SP-Cu2+ was 7.6 times higher than that of the commercially available adhesive DURO-TAK® 87-4098 in the porcine skin peel test. The adhesion test on human skin confirmed that PHT-SP-Cu2+ could adhere to the human body for more than six days. Moreover, the drug loading, in vitro release test and skin permeation test were investigated using ketoprofen as a model drug, and the results showed that PHT-SP-Cu2+ had the efficacy of improving drug compatibility, promoting drug release and enhancing skin permeation as a TDDS. Among them, the drug loading of PHT-SP-Cu2+ was increased by 6.25-fold compared with PHT, and in the in vivo pharmacokinetic analysis, the AUC was similarly increased by 19.22-fold. The mechanism of α-helix facilitated drug release was demonstrated by Flori-Hawkins interaction parameters, molecular dynamics simulations and FT-IR. Biosafety evaluations highlighted the superior skin cytocompatibility and safety of PHT-SP-Cu2+ for transdermal applications. These results would contribute to the development of TDDS patch adhesives with outstanding adhesion, drug loading and release efficiency. STATEMENT OF SIGNIFICANCE: A new adhesive, PHT-SP-Cu2+, was created for transdermal drug delivery patches. Polyethylene glycol, hexamethylene diisocyanate, trimethylolpropane, silk protein, and Cu2+ were used in synthesis. Characterization techniques confirmed the structure and Cu2+-p-π conjugated networks. Optimal formulation included 3 wt.% SP and 3 wt.% Cu2+, exhibiting superior adhesion. PHT-SP-Cu2+ showed 7.6 times higher peel strength than DURO-TAK® 87-4098 on porcine skin and adhered to human skin for over six days. It demonstrated a 6.25-fold increase in drug loading compared to PHT, with 19.22-fold higher AUC in vivo studies. α-helix facilitated drug release, proven by various analyses. PHT-SP-Cu2+ showed excellent cytocompatibility and safety for transdermal applications. This study contributes to developing efficient TDDS patches.
Subject(s)
Administration, Cutaneous , Drug Liberation , Silk , Tissue Adhesives , Animals , Tissue Adhesives/chemistry , Tissue Adhesives/pharmacology , Humans , Silk/chemistry , Drug Delivery Systems , Swine , Skin/metabolism , Skin/drug effects , Transdermal Patch , Copper/chemistry , Copper/pharmacokineticsABSTRACT
BACKGROUND: Female androgenetic alopecia (FAGA) is a condition that affects women and involves the gradual loss of terminal hair in specific areas of the scalp. The limited treatment options for FAGA necessitate the development of new strategies. This study aimed to evaluate the potential benefit of using a combination therapy composed of autologous platelet-rich plasma (PRP) and a non-cross-linked hyaluronic acid (HA) compound in the treatment of FAGA. METHODS: This was a retrospective, case-series study, which enrolled nine female patients with FAGA between September 2021 and December 2022. The non-cross-linked HA compound (Hearty®, Imeik Technology Development Co., Ltd.) and PRP were implanted into the areas of hair loss over four treatment sessions separated by 4-week intervals. Patients were monitored for overall improvement in their hair loss, hair count, treatment satisfaction, and adverse events at 1, 3, and 6 months follow-up. RESULTS: The improvement rates, subjectively evaluated by the study physician, were 88.89% at the 1-month and 100% at the 3-month follow-up, relative to baseline. Moreover, the quantitative evaluation results showed that the FAGA patients' hair density increased by 54.51% at the 1-month and by 77.25% at the 3-month follow-up. CONCLUSION: The combination of PRP and non-cross-linked HA compound appeared to be a certain positive effective procedure for FAGA without serious adverse event. We envisage that this work will contribute to the development of new treatment options for women suffering from this condition.
Subject(s)
Hyaluronic Acid , Platelet-Rich Plasma , Humans , Female , Hyaluronic Acid/adverse effects , Retrospective Studies , Alopecia/drug therapy , Hair , Treatment OutcomeABSTRACT
BACKGROUND: Plasmodiophora brassicae is the causal agent of clubroot disease of cruciferous plants and one of the biggest threats to the rapeseed (Brassica napus) and brassica vegetable industry worldwide. DISEASE SYMPTOMS: In the advanced stages of clubroot disease wilting, stunting, yellowing, and redness are visible in the shoots. However, the typical symptoms of the disease are the presence of club-shaped galls in the roots of susceptible hosts that block the absorption of water and nutrients. HOST RANGE: Members of the family Brassicaceae are the primary host of the pathogen, although some members of the family, such as Bunias orientalis, Coronopus squamatus, and Raphanus sativus, have been identified as being consistently resistant to P. brassicae isolates with variable virulence profile. TAXONOMY: Class: Phytomyxea; Order: Plasmodiophorales; Family: Plasmodiophoraceae; Genus: Plasmodiophora; Species: Plasmodiophora brassicae (Woronin, 1877). DISTRIBUTION: Clubroot disease is spread worldwide, with reports from all continents except Antarctica. To date, clubroot disease has been reported in more than 80 countries. PATHOTYPING: Based on its virulence on different hosts, P. brassicae is classified into pathotypes or races. Five main pathotyping systems have been developed to understand the relationship between P. brassicae and its hosts. Nowadays, the Canadian clubroot differential is extensively used in Canada and has so far identified 36 different pathotypes based on the response of a set of 13 hosts. EFFECTORS AND RESISTANCE: After the identification and characterization of the clubroot pathogen SABATH-type methyltransferase PbBSMT, several other effectors have been characterized. However, no avirulence gene is known, hindering the functional characterization of the five intercellular nucleotide-binding (NB) site leucine-rich-repeat (LRR) receptors (NLRs) clubroot resistance genes validated to date. IMPORTANT LINK: Canola Council of Canada is constantly updating information about clubroot and P. brassicae as part of their Canola Encyclopedia: https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/. PHYTOSANITARY CATEGORIZATION: PLADBR: EPPO A2 list; Annex designation 9E.
Subject(s)
Brassica napus , Brassica , Plasmodiophorida , Plant Diseases , CanadaABSTRACT
Chilling stress is one of the major abiotic stresses which hinder seedling emergence and growth. Herein, we investigated the effects of chilling/low temperature stress on the morphological, physiological, and biochemical attributes of two silage corn genotypes during the seedling establishment phase. The experiment was conducted in a growth chamber, and silage corn seedlings of Yukon-R and A4177G-RIB were grown at optimum temperature up to V3 stage and then subjected to five temperature regimes (25 °C as control, 20 °C, 15 °C, 10 °C, and 5 °C) for 5 days. After the temperature treatment, the morphological, physiological, and biochemical parameters were recorded. Results indicated that temperatures of 15 °C and lower significantly affected seedling growth, photosynthesis system, reactive oxygen species (ROS) accumulation, and antioxidant enzyme activities. Changes in seedlings' growth parameters were in the order of 25 °C > 20 °C > 15 °C > 10 °C > 5 °C, irrespective of genotypes. The chlorophyll content, photosynthetic rate, and maximal photochemical efficiency of PS-II (Fv/Fm) were drastically decreased under chilling conditions. Moreover, chilling stress induced accumulation of hydrogen peroxide (H2O2)and malonaldehyde (MDA) contents. Increased proline content and enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxide (APX), were found to alleviate oxidative damage under chilling stress. However, the genotype of Yukon-R exhibited better adaption to chilling stress than A4177G3-RIB. Yukon-R showed significantly higher proline content and enzymatic antioxidant activities than A4177G3-RIB under severe chilling conditions (temperature ≤ 10 °C). Similarly, Yukon-R expressed low temperature-induced ROS accumulation. Furthermore, the interaction effects were found between temperature treatment and genotype on the ROS accumulation, proline content and antioxidant enzyme activities. In summary, the present study indicated that Yukon-R has shown better adaptation and resilience against chilling temperature stress, and therefore could be considered a potential candidate genotype to be grown in the boreal climate.
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Being a macronutrient, phosphorus (P) is the backbone to complete the growth cycle of plants. However, because of low mobility and high fixation, P becomes the least available nutrient in podzolic soils; hence, enhancing phosphorus use efficiency (PUE) can play an important role in different cropping systems/crop production practices to meet ever-increasing demands in food, fiber, and fuel. Additionally, the rapidly decreasing mineral phosphate rocks/stocks forced to explore alternative resources and methods to enhance PUE either through improved seed P reserves and their remobilization, P acquisition efficiency (PAE), or plant's internal P utilization efficiency (IPUE) or both for sustainable P management strategies. The objective of this review article is to explore and document important domains to enhance PUE in crop plants grown on Podzol in a boreal agroecosystem. We have discussed P availabilities in podzolic soils, root architecture and morphology, root exudates, phosphate transporters and their role in P uptake, different contributors to enhance PAE and IPUE, and strategies to improve plant PUE in crops grown on podzolic soils deficient in P and acidic in nature.
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BACKGROUND: Ku70 participates in several pathological processes through mediating repair of DNA double-strand breaks. Our previous study has identified a highly conserved long noncoding RNA cardiac ischemia reperfusion associated Ku70 interacting lncRNA (CIRKIL) that was upregulated in myocardial infarction. The study aims to investigate whether CIRKIL regulates myocardial ischemia/reperfusion (I/R) through binding to Ku70. METHODS: CIRKIL transgenic and knockout mice were subjected to 45-minute ischemia and 24-hour reperfusion to establish myocardial I/R model. RNA pull-down and RNA immunoprecipitation assay were used to detect the interaction between CIRKIL and Ku70. RESULTS: The expression of CIRKIL was increased in I/R myocardium and H2O2-treated cardiomyocytes. Overexpression of CIRKIL increased the expression of γH2A.X, a specific marker of DNA double-strand breaks and aggravated cardiomyocyte apoptosis, whereas knockdown of CIRKIL produced the opposite changes. Transgenic overexpression of CIRKIL aggravated cardiac dysfunction, enlarged infarct area, and worsened cardiomyocyte damage in I/R mice. Knockout of CIRKIL alleviated myocardial I/R injury. Mechanistically, CIRKIL directly bound to Ku70 to subsequently decrease nuclear translocation of Ku70 and impair DNA double-strand breaks repair. Concurrent overexpression of Ku70 mitigated CIRKIL overexpression-induced myocardial I/R injury. Furthermore, knockdown of human CIRKIL significantly suppressed cell damage induced by H2O2 in adult human ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: CIRKIL is a detrimental factor in I/R injury acting via regulating nuclear translocation of Ku70 and DNA double-strand breaks repair. Thus, CIRKIL might be considered as a novel molecular target for the treatment of cardiac conditions associated with I/R injury.
Subject(s)
Coronary Artery Disease , Induced Pluripotent Stem Cells , Myocardial Infarction , Myocardial Ischemia , Myocardial Reperfusion Injury , RNA, Long Noncoding , Animals , Apoptosis , Coronary Artery Disease/metabolism , DNA/metabolism , Humans , Hydrogen Peroxide/toxicity , Induced Pluripotent Stem Cells/metabolism , Mice , Mice, Knockout , Myocardial Infarction/metabolism , Myocardial Ischemia/metabolism , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , ReperfusionABSTRACT
Purpose: Cardiomyocyte senescence is associated with a progressive decline in cardiac physiological function and the risk of cardiovascular events. lncRNA H19 (H19), a well-known long noncoding RNA (lncRNA), is involved in the pathophysiological process of multiple cardiovascular disease such as heart failure, cardiac ischemia and fibrosis. However, the role of H19 in cardiomyocyte senescence remains to be further explored. Methods: Senescence-associated ß-galactosidases (SA-ß-gal) staining was used to detect cardiomyocyte senescence. Western blot, qRT-PCR and luciferase reporter assay were employed to evaluate the role of H19 in cardiomyocyte senescence and its underling molecular mechanism. Results: H19 level was significantly increased in high glucose-induced senescence cardiomyocytes and aged mouse hearts. Overexpression of H19 enhanced the number of SA-ß-gal-positive cells, and the expression of senescence-related proteins p53 and p21, whereas H19 knockdown exerted the opposite effects. Mechanistically, H19 was demonstrated as a competing endogenous RNA (ceRNA) for microRNA-19a (miR-19a): H19 overexpression downregulated miR-19a level, while H19 knockdown upregulated miR-19a. The expression of SOSC1 was dramatically increased in senescence cardiomyocytes and aged mouse hearts. Further experiments identified SOCS1 as a downstream target of miR-19a. H19 upregulated SOCS1 expression and activated the p53/p21 pathway by targeting miR-19a, thus promoting the cardiomyocytes senescence. Conclusion: Our results show that H19 is a pro-senescence lncRNA in cardiomyocytes acting as a ceRNA to target the miR-19a/SOCS1/p53/p21 pathway. Our research reveals a molecular mechanism of cardiomyocyte senescence regulation and provides a novel target of the therapy for senescence-associated cardiac diseases.
ABSTRACT
Long non-coding RNAs (lncRNAs) account for a large proportion of genomic transcripts and are critical regulators in various cardiac diseases. Though lncRNAs have been reported to participate in the process of diverse cardiac diseases, the contribution of lncRNAs in cardiac fibrosis remains to be fully elucidated. Here, we identified a novel anti-fibrotic lncRNA, SAIL (scaffold attachment factor B interacting lncRNA). SAIL was reduced in cardiac fibrotic tissue and activated cardiac fibroblasts. Gain- and loss-of-function studies showed that knockdown of SAIL promoted proliferation and collagen production of cardiac fibroblasts with or without TGF-ß1 (transforming growth factor beta1) treatment, while overexpression of SAIL did the opposite. In mouse cardiac fibrosis induced by myocardial infarction, knockdown of SAIL exacerbated, whereas overexpression of SAIL alleviated cardiac fibrosis. Mechanically, SAIL inhibited the fibrotic process by directly binding with SAFB via 23 conserved nucleotide sequences, which in turn blocked the access of SAFB to RNA pol II (RNA polymerase II) and reduced the transcription of fibrosis-related genes. Intriguingly, the human conserved fragment of SAIL (hSAIL) significantly suppressed the proliferation and collagen production of human cardiac fibroblasts. Our findings demonstrate that SAIL regulates cardiac fibrosis by regulating SAFB-mediated transcription of fibrotic related genes. Both SAIL and SAFB hold the potential to become novel therapeutic targets for cardiac fibrosis.
Subject(s)
Cell Proliferation , Collagen/metabolism , Fibroblasts/metabolism , Myocardial Infarction/metabolism , Myocytes, Cardiac/metabolism , RNA, Long Noncoding/metabolism , Animals , Cells, Cultured , DNA-Binding Proteins/metabolism , Disease Models, Animal , Fibroblasts/pathology , Fibrosis , Male , Mice, Inbred C57BL , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocytes, Cardiac/pathology , RNA Polymerase II/metabolism , RNA, Long Noncoding/genetics , RNA-Binding Proteins/metabolism , Transcription, GeneticABSTRACT
Cardiac fibrosis is characterized by the activation of cardiac fibroblasts and accumulation of extracellular matrix. METTL3, a component of methyltransferase complex, participates in multiple biological processes associated with mammalian development and disease progression. However, the role of METTL3 in cardiac fibrosis is still unknown. We performed fibroblasts activation with TGF-ß1 (20 ng/mL) in vitro and established in vivo mouse models with lentivirus to assess the effects of METTL3 on cardiac fibroblasts proliferation and collagen formation. Methylated RNA immunoprecipitation (MeRIP) was used to define the potential fibrosis-regulated gene. The expression level of METTL3 was increased in cardiac fibrotic tissue of mice with chronic myocardial infarction and cultured cardiac fibroblats (CFs) treated with TGF-ß1. Enforced expression of METTL3 promoted proliferation and fibroblast-to-myofibroblast transition and collagens accumulation, while silence of METTL3 did the opposite. Silence of METTL3 by lentivirus carrying METTL3 siRNA markedly alleviated cardiac fibrosis in MI mice. Transcriptome and N6-methyladenosine (m6 A) profiling analyses revealed that the expression and m6 A level of collagen-related genes were altered after silence of METTL3. METTL3-mediated m6 A modification is critical for the development of cardiac fibrosis, providing a molecular target for manipulating fibrosis and the associated cardiac diseases.
Subject(s)
Gene Silencing , Methyltransferases/genetics , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardium/pathology , Myofibroblasts/metabolism , Animals , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cells, Cultured , Collagen/genetics , Collagen/metabolism , Disease Models, Animal , Fibrosis , Male , Mice , Mice, Inbred C57BL , Myofibroblasts/drug effects , Signal Transduction/drug effects , Signal Transduction/genetics , Transcriptome , Transfection , Transforming Growth Factor beta1/pharmacology , Up-Regulation/geneticsABSTRACT
A photoredox-catalyzed regio- and stereoselective trifluoroethylation reaction of enamides using commercially available 2,2,2-trifluoroethyl iodide as trifluoroethylating agents has been developed, furnishing geometrically defined and synthetically and physiochemically pivotal ß-trifluoroethylated enamides bearing a diverse range of functional groups.
ABSTRACT
Root membrane lipids are important biomolecules determining plant's ability to adapt to different growing environmental or climatic conditions. Herein, we demonstrate the potential use of root membrane lipids as biomarkers to discriminate silage-corn genotypes based on herbicide and insect/pest resistance genetic traits when cultivated on podzolic soils under short growing and moderately warm summer season in boreal climate. Lipids in root membranes of field grown silage-corn genotypes were previously quantified at crop maturity by ultra-high-performance liquid chromatography-hydrophilic interaction chromatography-heated electrospray ionization mass spectrometry. The lipid identified and quantified in silage-corn roots were phospholipids, glycolipids and sphingolipids. Following hierarchical cluster analysis, three groups of membrane lipids were observed to be very effective in segregating the five silage-corn genotypes. The first group consisted of hexosylceramide (HexCer), phosphatidylcholine (PC) and phosphatidylinositol (PI). The second group consisted of lysophosphatidic acid (LPA16:0) and lysophosphatidylcholine (LPC16:0), while the third group consisted of 37 molecular species from observed lipids (phospholipids, glycolipids, sphingolipids). Partial least squares-discriminant analysis (PLS-DA) based on 37 membrane lipid species, as well as principal component analysis using the variables important in projection derived from the PLS-DA segregated the five silage-corn genotypes into three groups according to their pesticide/herbicide resistant traits. This study is second to none using root lipidomics in discriminating different silage-corn genotypes based on their herbicide and insect/pest resistance genetic traits for cultivation in boreal climates. The segregated genotypes possess three different genetic traits for herbicide and insect/pest resistance including VT Double Pro (VT2P), VT Triple Pro Roundup Ready (VT3P/RR) and Roundup Ready-2 corn (RR2). These findings demonstrate that root membrane lipids could serve as appropriate chemical biosignatures to identify silage-corn genotypes based on herbicide and insect/pest resistance genetic traits suitable for cultivation in boreal climates.
Subject(s)
Silage , Zea mays , Biomarkers , Genotype , Membrane Lipids , Soil , Zea mays/geneticsABSTRACT
The aim of this study was to investigate the effects of a volcanic rock porous carrier (VRPC) on sludge reduction, pollutant removal, and microbial community structure in an anaerobic side-stream reactor (ASSR). Three lab-scale membrane bioreactors (MBRs), including an anoxic-oxic MBR, which served as the control (C-MBR), an ASSR-coupled MBR (A-MBR), and an A-MBR filled with VRPC (FA-MBR) were stably and simultaneously operated for 120 days. The effect of the three reactors on the removal of chemical oxygen demand (COD) was almost negligible (all greater than 95%), but the average removal efficiency of ammonium nitrogen, total nitrogen, and total phosphorus was significantly improved by the insertion of an ASSR, especially when the ASSR was filled with VRPC. Finally, A-MBR and FA-MBR achieved 16.2% and 26.4% sludge reduction rates, with observed sludge yields of 0.124 and 0.109 g mixed liquid suspended solids/g COD, respectively. Illumina MiSeq sequencing revealed that microbial diversity and richness were highest in the VRPC, indicating that a large number of microorganisms formed on the carrier surface in the form of a biofilm. Abundant denitrifying bacteria (Azospira, Comamonadaceae_unclassified, and Flavobacterium) were immobilized on the carrier biofilm, which contributed to increased nitrogen removal. The addition of a VRPC to the ASSR successfully immobilized abundant hydrolytic, fermentative, and slow-growing microorganisms, which all contributed to reductions in sludge yield.