Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).

Electrospun PVB/AVE NMs as mask filter layer for win-win effects of filtration and antibacterial activity.

The COVID-19 pandemic has caused serious social and public health problems. In the field of personal protection, the facial masks can prevent infectious respiratory diseases, safeguard human health, and promote public safety. Herein, we focused on preparing a core filter layer for masks using electrospun polyvinyl butyral/apocynum venetum extract nanofibrous membranes (PVB/AVE NMs), with durable interception efficiency and antibacterial properties. In the spinning solution, AVE acted as a salt to improve electrical conductivity, and achieve long-lasting interception efficiency with adjustable pore size. It also played the role of an antibacterial agent in PVB/AVE NMs to achieve win-win effects. The hydrophobicity of PVB-AVE-6% was 120.9° whereas its filterability reached 98.3% when the pressure drop resistance was 142 Pa. PVB-AVE-6% exhibited intriguing properties with great antibacterial rates of 99.38% and 98.96% against S. aureus and E. coli, respectively. After a prolonged usability test of 8 h, the filtration efficiency of the PVB/AVE masks remained stable at over 97.7%. Furthermore, the antibacterial rates of the PVB/AVE masks on S. aureus and E. coli were 96.87% and 96.20% respectively, after using for 2 d. These results indicate that PVB/AVE NMs improve the protective performance of ordinary disposable masks, which has certain application in air filtration.

AID function in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation of immunoglobulin (Ig) gene variable regions and class switch recombination (CSR) of Ig heavy chain constant regions. Two decades of intensive research has greatly expanded our knowledge of how AID functions in peripheral B cells to optimize antibody responses against infections, while maintaining tight regulation of AID to restrain its activity to protect B cell genomic integrity. The many exciting recent advances in the field include: 1) the first description of AID's molecular structure, 2) remarkable advances in high throughput approaches that precisely track AID targeting genome-wide, and 3) the discovery that the cohesion-mediate loop extrusion mechanism [initially discovered in V(D)J recombination studies] also governs AID-medicated CSR. These advances have significantly advanced our understanding of AID's biochemical properties in vitro and AID's function and regulation in vivo. This mini review will discuss these recent discoveries and outline the challenges and questions that remain to be addressed.

Current insights into the mechanism of mammalian immunoglobulin class switch recombination.

Immunoglobulin (Ig) class switch recombination (CSR) is the gene rearrangement process by which B lymphocytes change the Ig heavy chain constant region to permit a switch of Ig isotype from IgM to IgG, IgA, or IgE. At the DNA level, CSR occurs via generation and joining of DNA double strand breaks (DSBs) at intronic switch regions located just upstream of each of the heavy chain constant regions. Activation-induced deaminase (AID), a B cell specific enzyme, catalyzes cytosine deaminations (converting cytosines to uracils) as the initial DNA lesions that eventually lead to DSBs and CSR. Progress on AID structure integrates very well with knowledge about Ig class switch region nucleic acid structures that are supported by functional studies. It is an ideal time to review what is known about the mechanism of Ig CSR and its relation to somatic hypermutation. There have been many comprehensive reviews on various aspects of the CSR reaction and regulation of AID expression and activity. This review is focused on the relation between AID and switch region nucleic acid structures, with a particular emphasis on R-loops.

Exploration of human xylosyltransferase for chemoenzymatic synthesis of proteoglycan linkage region.

Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature, PGs are highly challenging synthetic targets due to the difficulties in integrating the sulfated glycans with the peptide backbone. To expedite the synthesis, herein, the utility of human xylosyltransferase I (XT-I), the enzyme responsible for initiating PG synthesis, has been explored. XT-I was found to be capable of efficiently installing the xylose unit onto a variety of peptide structures on mg scales. Furthermore, an unnatural sugar, i.e., 6-azidoglucose can be transferred by XT-I introducing a reactive handle onto the glycopeptide for selective functionalization. XT-I can be coupled with ß-4-galactosyl transferase-7 for one pot synthesis of glycopeptides bearing galactose-xylose disaccharide, paving the way toward efficient chemoenzymatic synthesis of PG glycopeptides and glycoproteins.

Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination.

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

Application of seasonal freeze-thaw to pretreat raw material for accelerating green waste composting.

The recalcitrance of green waste, caused by its high lignocellulose content, is a technical challenge for accelerating green waste composting. However, because lignocellulose degradation in litter (similar to green waste) can be promoted during the freeze-thaw season, and the composting is difficult to implement in this period (due to the low temperature); seasonal freeze-thaw was intended to be used as a pretreatment strategy for the existing technical challenge in the winter of cold regions. In this process, green waste was pretreated with seasonal freeze-thaw to enhance its lignocellulose degradation for subsequent composting. To verify this assumption, two strategies for the pretreatment were used: the green waste was either drenched or immersed in water during the freeze-thaw season, and the effects on subsequent composting were evaluated. The results demonstrated that both strategies can significantly promote the mineralization of TOC (total organic carbon, by 2.73%-8.01% compared with the control, the following comparisons were all based on the control), TN (total nitrogen, by 0.21%-0.52%), and lignocellulose (lignin degradation was promoted by 3.52%-3.73%, cellulose degradation was promoted by 13.23%-14.26%) during composting and that the synthesis of humus was also enhanced (by 19.19%-21.43%). Furthermore, since the loss of NH4+N and NO3-N was significantly less in the drenched treatment than in the immersed treatment (by 9.15% for the loss of NH4+N and 7.66% for the loss of NO3-N), drenching the green waste during the freeze-thaw season might be a better strategy than immersing for nitrogen conservation. An additional advantage of drenching compared to immersing is water conservation.

The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends.

The evidence that ATM affects resolution of RAG-induced DNA double-strand breaks is profuse and unequivocal; moreover, it is clear that the RAG complex itself cooperates (in an undetermined way) with ATM to facilitate repair of these double-strand breaks by the classical nonhomologous end-joining pathway. The mechanistic basis for the cooperation between ATM and the RAG complex has not been defined, although proposed models invoke ATM and RAG2's C terminus in maintaining the RAG postcleavage complex. In this study, we show that ATM reduces the rate of both coding and signal joining in a robust episomal assay; we suggest that this is the result of increased stability of the postcleavage complex. ATM's ability to inhibit VDJ joining requires its enzymatic activity. The noncore C termini of both RAG1 and RAG2 are also required for ATM's capacity to limit signal (but not coding) joining. Moreover, potential phosphorylation targets within the C terminus of RAG2 are also required for ATM's capacity to limit signal joining. These data suggest a model whereby the RAG signal end complex is stabilized by phosphorylation of RAG2 by ATM.

Class-Switch Recombination in the Absence of the IgH 3' Regulatory Region.

The ∼28-kb 3' regulatory region (3'RR), which is located at the most distal 3' region of the Ig H chain locus, has multiple regulatory functions that control IgH expression, class-switch recombination (CSR), and somatic hypermutation. In this article, we report that deletion of the entire 3'RR in a mouse B cell line that is capable of robust cytokine-dependent CSR to IgA results in reduced, but not abolished, CSR. These data suggest that 3'RR is not absolutely required for CSR and, thus, is not essential for targeting activation-induced cytidine deaminase to S regions, as was suggested. Moreover, replacing 3'RR with a DNA fragment including only its four DNase I hypersensitive sites (lacking the large spacer regions) restores CSR to a level equivalent to or even higher than in wild-type cells, suggesting that the four hypersensitive sites contain most of the CSR-promoting functions of 3'RR. Stimulated cells express abundant germline transcripts, with the presence or absence of 3'RR, providing evidence that 3'RR has a role in promoting CSR that is unique from enhancing S region transcription.

Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase ß in vivo.

Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase ß (Polß) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polß-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polß-DPC in vivo. In a dose-dependent fashion, Polß-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polß-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polß-DPC. Nonoxidative agents did not generate Polß-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polß-DPC depended on the Ape1 AP endonuclease, which generates the Polß lyase substrate, and they required the essential lysine-72 in the Polß lyase active site. Oxidative Polß-DPC had an unexpectedly short half-life (∼ 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polß than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polß-DPC and the biological pressure to repair them.

The role of G-density in switch region repeats for immunoglobulin class switch recombination.

The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.

The histone methyltransferase MMSET regulates class switch recombination.

Wolf-Hirschhorn syndrome (WHS) is a genetic disease with characteristic facial features and developmental disorders. Of interest, loss of the MMSET gene (also known as WHSC1) is considered to be responsible for the core phenotypes of this disease. Patients with WHS also display Ab deficiency, although the underlying cause of this deficiency is unclear. Recent studies suggest that the histone methyltransferase activity of MMSET plays an important role in the DNA damage response by facilitating the recruitment of 53BP1 to sites of DNA damage. We hypothesize that MMSET also regulates class switch recombination (CSR) through its effect on 53BP1. In this study, we show that MMSET indeed plays an important role in CSR through its histone methyltransferase activity. Knocking down MMSET expression impaired 53BP1 recruitment as well as the germline transcription of the Igh switch regions, resulting in defective CSR but no effect on cell growth and viability. These results suggest that defective CSR caused by MMSET deficiency could be a cause of Ab deficiency in WHS patients.

X-ray repair cross-complementing protein 1 (XRCC1) deficiency enhances class switch recombination and is permissive for alternative end joining.

DNA double-strand breaks (DSBs) are essential intermediates in Ig gene rearrangements: V(D)J and class switch recombination (CSR). In contrast to V(D)J recombination, which is almost exclusively dependent on nonhomologous end joining (NHEJ), CSR can occur in NHEJ-deficient cells via a poorly understand backup pathway (or pathways) often termed alternative end joining (A-EJ). Recently, several components of the single-strand DNA break (SSB) repair machinery, including XRCC1, have been implicated in A-EJ. To determine its role in A-EJ and CSR, Xrcc1 was deleted by targeted mutation in the CSR proficient mouse B-cell line, CH12F3. Here we demonstrate that XRCC1 deficiency slightly increases the efficiency of CSR. More importantly, Lig4 and XRCC1 double-deficient cells switch as efficiently as Lig4-deficient cells, clearly indicating that XRCC1 is dispensable for A-EJ in CH12F3 cells during CSR.

Overlapping activation-induced cytidine deaminase hotspot motifs in Ig class-switch recombination.

Ig class-switch recombination (CSR) is directed by the long and repetitive switch regions and requires activation-induced cytidine deaminase (AID). One of the conserved switch-region sequence motifs (AGCT) is a preferred site for AID-mediated DNA-cytosine deamination. By using somatic gene targeting and recombinase-mediated cassette exchange, we established a cell line-based CSR assay that allows manipulation of switch sequences at the endogenous locus. We show that AGCT is only one of a family of four WGCW motifs in the switch region that can facilitate CSR. We go on to show that it is the overlap of AID hotspots at WGCW sites on the top and bottom strands that is critical. This finding leads to a much clearer model for the difference between CSR and somatic hypermutation.

A Germinal Center Checkpoint of AIRE in B Cells Limits Antibody Diversification.

In response to antigens, B cells undergo affinity maturation and class switching mediated by activation-induced cytidine deaminase (AID) in germinal centers (GCs) of secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of GC antibody diversification is of fundamental importance but not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in GC B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.

A high-yielding strain of indole-3-acetic acid isolated from food waste compost: metabolic pathways, optimization of fermentation conditions, and application.

Food waste is a potential resource to prepare microbial fertilizer. However, functional microorganisms derived from the food waste compost (FWC) are relatively lacking. We have isolated, identified, characterized and optimized a high-yielding indole-3-acetic acid (IAA) strain from FWC and further evaluated its growth promoting effect on plants. A IAA high-yielding strain, Providencia sp.Y, with an initial IAA yield of 139.98 mg L-1, was obtained through high-throughput screening, and identified by 16S rRNA gene sequence. The novel strain Y may simultaneously involve the following three pathways from L-tryptophan to IAA, which were identified using liquid chromatography-tandem mass spectrometry: (1) L-tryptophan-indole-3-ethanol-indole-3-acetaldehyde-indole-3-acetic acid; (2) L-tryptophan-1-hydroxy-indole-3-ethanol-indole-3-acetic acid; (3) L-tryptophan-indole-3-acetamide-indole-3-acetic acid. The most suitable comprehensive conditions for IAA production, which were optimized by single factor experiment, were: culture time 12 h, inoculation amount 2% (v/v), NaCl concentration 4% (w/v), culture temperature 25℃, initial pH = 5, and L-tryptophan concentration 3.0 g L-1. The yield of IAA after optimization was increased by 590.48%, from 139.98 mg L-1 (before optimization) to 966.54 mg L-1. Diluted 200-fold microbial suspension could significantly improve the growth of pakchoi seedlings. The seedling plant height, root length, leaf width, leaf length, and fresh weight with microbial suspension increased by 17.39%, 107.35%, 77.98%, 37.75%, and 215.38%, respectively, compared with those without microbial suspension. The increase was greater than that of commercial bacterial agents. In conclusion, this isolated strain can be used as an economical microbial inoculant and provides a new germplasm resource for developing microbial fertilizers.

Reuse of composted food waste from rural China as vermicomposting substrate: effects on earthworms, associated microorganisms, and economic benefits.

ABSTRACTAerobic composting of food waste (FW) from rural China using a composting device results in a substantial financial burden on the government. This study aimed to assess the feasibility of mitigating this cost using vermicomposting of composted FW. The specific aims were to elucidate the effects of composted FW on earthworm growth and reproduction, reveal the changes in the physical and chemical properties of earthworm casts during vermicomposting, identify the microbial community structure associated with vermicomposting, and perform a financial analysis based on the yield of earthworms and earthworm casts. Mixing composted FW and mature cow dung in an equal ratio achieved the highest earthworm reproduction rate, where 100 adult earthworms produced 567 juvenile earthworms and 252 cocoons in 40 d. Earthworms reduce salt content of vermicomposting substrates by assimilating Na+ and promoting humification by transforming humin into humic and fulvic acid, thus producing earthworm casts with a high generation index > 80%. When composted FW was added to a vermicomposting substrate, a distinctive microbial community structure with alkaliphilic, halophilic, and lignocellulolytic microorganisms dominated the microflora. The dominant bacterial species was Saccharopolyspora rectivirgula, and the dominant fungal species changed from Kernia nitida to Coprinopsis scobicola. Furthermore, microbial genes for refractory organic matter and fat degradation were observed in Vibrio cholerae, Kernia nitida, and Coprinopsis scobicola. Financial analysis showed that vermicomposting has the potential to reduce the cost associated with FW disposal from $ 57 to $ 18/t.

Evaluation of a data-driven intelligent waste classification system for scientific management of garbage recycling in a Chinese community.

Waste classification management is effective in addressing the increasing waste output and continuous deterioration of environmental conditions. The waste classification behaviour of resident is an important basis for managers to collect and allocate resources. Traditional analysis methods, such as questionnaire, have limitations considering the complexity of individual behaviour. An intelligent waste classification system (IWCS) was applied and studied in a community for 1 year. Time-based data analysis framework was constructed to describe the residents' waste sorting behaviour and evaluate the IWCS. The results showed that residents preferred to use face recognition than other modes of identification. The ratio of waste delivery frequency was 18.34% in the morning and 81.66% in the evening, respectively. The optimal time windows of disposing wastes were from 6:55 to 9:05 in the morning and from 18:05 to 20:55 in the evening which can avoid crowding. The percentage of accuracy of waste disposal increased gradually in a year. The amount of waste disposal was largest on every Sunday. The average accuracy was more than 94% based on monthly data, but the number of participating residents decreased gradually. Therefore, the study demonstrates that IWCS is a potential platform for increasing the accuracy and efficiency of waste disposal and can promote regulations implementation.