Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

RNase H1 facilitates recombinase recruitment by degrading DNA-RNA hybrids during meiosis.

DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.

Genome-wide mapping reveals R-loops associated with centromeric repeats in maize.

R-loops are stable chromatin structures comprising a DNA:RNA hybrid and a displaced single-stranded DNA. R-loops have been implicated in gene expression and chromatin structure, as well as in replication blocks and genome instability. Here, we conducted a genome-wide identification of R-loops and identified more than 700,000 R-loop peaks in the maize (Zea mays) genome. We found that sense R-loops were mainly enriched in promoters and transcription termination sites and relatively less enriched in gene bodies, which is different from the main gene-body localization of sense R-loops in Arabidopsis and Oryza sativa At the chromosome scale, maize R-loops were enriched in pericentromeric heterochromatin regions, and a significant portion of R-loops were derived from transposable elements. In centromeres, R-loops preferentially formed within the binding regions of the centromere-specific histone CENH3, and centromeric retrotransposons were strongly associated with R-loop formation. Furthermore, centromeric retrotransposon R-loops were observed by applying the single-molecule imaging technique of atomic force microscopy. These findings elucidate the fundamental character of R-loops in the maize genome and reveal the potential role of R-loops in centromeres.

Intragenic tRNA-promoted R-loops orchestrate transcription interference for plant oxidative stress responses.

Eukaryotic genomes are transcribed by at least three RNA polymerases, RNAPI, II, and III. Co-transcriptional R-loops play diverse roles in genome regulation and maintenance. However, little is known about how R-loops regulate transcription interference, the transcriptional event that is caused by different RNA polymerases transcribing the same genomic templates. Here, we established that the intragenic transfer RNA (tRNA) genes can promote sense R-loop enrichment (named intra-tR-loops) in Arabidopsis thaliana, and found that intra-tR-loops are decreased in an RNAPIII mutant, NUCLEAR RNA POLYMERASE C, SUBUNIT 7(nrpc7-1). NRPC7 is co-localized with RNAPIIS2P at intragenic tRNA genes and interferes with RNAPIIS2P elongation. Conversely, the binding of NRPC7 at intragenic tRNA genes is increased following inhibition of RNAPII elongation. The transcription of specific tRNA host genes is inhibited by RNAPIII, and the inhibition of tRNA host genes is intra-tR-loop dependent. Moreover, alleviating the inhibition of tRNAPro-induced intra-tR-loops on its host gene AtNUDX1 promotes oxidative stress tolerance in A. thaliana. Our work suggests intra-tR-loops regulate host gene expression by modulating RNA polymerases interference.

Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis.

Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.

Effects of Medium Additives on the Mycelial Growth and Polysaccharide Biosynthesis in Submerged Culture of Bjerkandera fumosa.

Medium additives have been shown to affect the synthesis of active products in fungi. This study investigated the effects of corn stalk, poplar sawdust, Tween-80, and oleic acid on mycelial biomass and physicochemical properties, as well as the bioactivity of polysaccharides, including exopolysaccharides (EPS) and intracellular polysaccharides (IPS), in the submerged culture of Bjerkandera fumosa. Results showed that the addition of corn stalk or poplar sawdust increased the production of EPS but decreased the production of IPS; Tween-80 had less effect on the production of EPS and IPS; and oleic acid stimulated polysaccharide production significantly. Polysaccharide property analysis showed that the addition of corn stalk or poplar sawdust promoted the production of high-molecular-weight components in polysaccharides and changed the monosaccharide composition of polysaccharides, as well as increased the mannose, glucuronic acid, and xylose contents of IPS. Tween-80 and oleic acid also changed the molecular weight distribution of polysaccharides but only slightly affected the composition of monosaccharides. The bioactivity assay indicated that the polysaccharides obtained by adding corn stalk possessed high hydroxyl radical scavenging and antitumor activities. The effect of poplar sawdust was slightly weaker than that of corn stalk. EPS and IPS obtained from a culture with Tween-80 and oleic acid possessed low antioxidant activity. Moreover, their antitumor activity was improved and lost, respectively. The results obtained in this work are useful for improving the understanding of the optimization and regulation of bioactive polysaccharide production in the submerged culture of B. fumosa.

The R-Loop Atlas of Arabidopsis Development and Responses to Environmental Stimuli.

R-loops are a common chromatin feature with essential functions in multiple cellular processes and diseases. However, little is known about the dynamic patterns of R-loops in a given organism. Here, using our recently developed genome-wide R-loop profiling method, we generated a comprehensive atlas quantifying the R-loop patterns of Arabidopsis (Arabidopsis thaliana) in 53 samples during development and during responses to environmental stimuli. The R-loop patterns were fairly stable in plants at the vegetative stage and in response to different light spectra and other environmental stimuli. Notably, the R-loops showed turnover during the plant life cycle, with patterns switching between generations. Importantly, R-loop dynamics was not strongly associated with RNA abundance, indicating that the mechanisms regulating R-loop formation and RNA accumulation are independent. We also observed enrichment of R-loops in transcription factor binding regions, suggesting that R-loops could function as potential cis-transcriptional regulators. This study provides an overview of R-loop dynamics in Arabidopsis during development and stress responses, highlights the unique dynamics of R-loops in the flowering plant Arabidopsis, and lays the groundwork for elucidating the functions of R-loops.

The Arabidopsis Nodulin Homeobox Factor AtNDX Interacts with AtRING1A/B and Negatively Regulates Abscisic Acid Signaling.

The phytohormone abscisic acid (ABA) and the Polycomb group proteins have key roles in regulating plant growth and development; however, their interplay and underlying mechanisms are not fully understood. Here, we identified an Arabidopsis (Arabidopsis thaliana) nodulin homeobox (AtNDX) protein as a negative regulator in the ABA signaling pathway. AtNDX mutants are hypersensitive to ABA, as measured by inhibition of seed germination and root growth, and the expression of AtNDX is downregulated by ABA. AtNDX interacts with the Polycomb Repressive Complex1 (PRC1) core components AtRING1A and AtRING1B in vitro and in vivo, and together, they negatively regulate the expression levels of some ABA-responsive genes. We identified ABA-INSENSITIVE (ABI4) as a direct target of AtNDX. AtNDX directly binds the downstream region of ABI4 and deleting this region increases the ABA sensitivity of primary root growth. Furthermore, ABI4 mutations rescue the ABA-hypersensitive phenotypes of ndx mutants and ABI4-overexpressing plants are hypersensitive to ABA in primary root growth. Thus, our work reveals the critical functions of AtNDX and PRC1 in some ABA-mediated processes and their regulation of ABI4.

RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts.

Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.

Multifunctional Surface Construction for Long-Term Cycling Stability of Li-Rich Mn-Based Layered Oxide Cathode for Li-Ion Batteries.

Li-rich Mn-based layered oxides (LMLOs) are promising cathode material candidate for the next-generation Li-ion batteries (LIBs) of high energy density. However, the fast capacity fading and voltage decay as well as low Coulombic efficiency caused by irreversible oxygen release and phase transition during the electrochemical process hinder their practical application. To solve these problems, in the present study, a multifunctional surface construction involving a coating layer, spinel-layered heterostructure, and rich-in oxygen vacancies is successfully conducted by a facile thermal reduction of the LMLO particles with potassium borohydride (KBH4 ) as the reducing agent. The multifunctional surface structure plays synergistic effects on suppressing the interface side reaction, reducing the dissolution of transition metal, increasing electron conductivity and lithium diffusion rate. As a result, electrochemical performances of the LMLO cathode are effectively enhanced. With optimization of the addition of KBH4 , the electrode delivers a reversible capacity of 280 mAh g-1 at 0.1 C, which maintains after 100 cycles. The capacity retention with respect to the initial capacity is as high as 98% at 1 C after 400 cycles. The present work provides insights into designing a highly effective functional surface structure of LMLO cathode materials for high-performance LIBs.

Functional consequences of splicing of the antisense transcript COOLAIR on FLC transcription.

Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

The Taste-Masking Mechanism of Chitosan at the Molecular Level on Bitter Drugs of Alkaloids and Flavonoid Glycosides from Traditional Chinese Medicine.

Taste masking of traditional Chinese medicines (TCMs) containing multiple bitter components remains an important challenge. In this study, berberine (BER) in alkaloids and phillyrin (PHI) in flavonoid glycosides, which are common bitter components in traditional Chinese medicines, were selected as model drugs. Chitosan (CS) was used to mask their unfriendly taste. Firstly, from the molecular level, we explained the taste-masking mechanism of CS on those two bitter components in detail. Based on those taste-masking mechanisms, the bitter taste of a mixture of BER and PHI was easily masked by CS in this work. The physicochemical characterization results showed the taste-masking compounds formed by CS with BER (named as BER/CS) and PHI (named as PHI/CS) were uneven in appearance. The drug binding efficiency of BER/CS and PHI/CS was 50.15 ± 2.63% and 67.10 ± 2.52%, respectively. The results of DSC, XRD, FTIR and molecular simulation further indicated that CS mainly masks the bitter taste by disturbing the binding site of bitter drugs and bitter receptors in the oral cavity via forming hydrogen bonds between its hydroxyl or amine groups and the nucleophilic groups of BER and PHI. The taste-masking evaluation results by the electronic tongue test confirmed the excellent taste-masking effects on alkaloids, flavonoid glycosides or a mixture of the two kinds of bitter components. The in vitro release as well as in vivo pharmacokinetic results suggested that the taste-masked compounds in this work could achieve rapid drug release in the gastric acid environment and did not influence the in vivo pharmacokinetic results of the drug. The taste-masking method in this work may have potential for the taste masking of traditional Chinese medicine compounds containing multiple bitter components.

R-loop: The new genome regulatory element in plants.

An R-loop is a three-stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex, which was thought to be a rare by-product of transcription. However, recent genome-wide data have shown that R-loops are widespread and pervasive in a variety of genomes, and a growing body of experimental evidence indicates that R-loops have both beneficial and harmful effects on an organism. To maximize benefit and avoid harm, organisms have evolved several means by which they tightly regulate R-loop levels. Here, we summarize our current understanding of the biogenesis and effects of R-loops, the mechanisms that regulate them, and methods of R-loop profiling, reviewing recent research advances on R-loops in plants. Furthermore, we provide perspectives on future research directions for R-loop biology in plants, which might lead to a more comprehensive understanding of R-loop functions in plant genome regulation and contribute to future agricultural improvements.

Intronic heterochromatin prevents cryptic transcription initiation in Arabidopsis.

Intronic transposable elements (TEs) comprise a large proportion in eukaryotic genomes, but how they regulate the host genes remains to be explored. Our forward genetic screen disclosed the plant-specific RNA polymerases IV and V in suppressing intronic TE-mediated cryptic transcription initiation of a chimeric transcripts at FLC (FLCTE ). Initiation of FLCTE transcription is blocked by the locally formed intronic heterochromatin, which is directly associated with RNA Pol V to inhibit the entry of RNA Pol II and the occupancy of H3K4 methylation. Genome-wide Pol II Ser5p native elongation transcription sequencing revealed that a significant number of intronic heterochromatin-containing genes undergo this mechanism. This study sheds light on deeply understanding the function of intronic heterochromatin on host genes expression in eukaryotic genome.

RNase H1 Cooperates with DNA Gyrases to Restrict R-Loops and Maintain Genome Integrity in Arabidopsis Chloroplasts.

Maintaining organellar genome integrity is essential for eukaryotic cells, and many factors can threaten genome integrity. R-loops are DNA:RNA duplexes produced during transcription, with the nontemplated DNA forming a single-stranded region. R-loops function in the regulation of transcription, DNA replication, and DNA repair, but can also be susceptible to lesions that form double-stranded breaks and thus induce genome instability. From investigating the function of a plant chloroplast-localized R-loop removing enzyme AtRNH1C, we have found that it is responsible for plastid R-loop homeostasis, chloroplast genome instability, and development. Interactome analysis revealed that AtRNH1C associates with multiple chloroplast-localized DNA and RNA metabolism-related proteins, including the core DNA gyrases complex. The interaction between AtRNH1C and AtGyrases was critical for R-loop homeostasis in chloroplast and important to release the transcription-replication conflicts in the highly transcribed and replication originated cp-rDNA regions and thus to reduce the DNA damage. Our results reveal the plastid R-loop accumulation leads to chloroplast DNA instability and provide insight into the maintenance of genome integrity in chloroplasts, in which the evolutionarily conserved RNase H1 and DNA gyrase proteins are involved.

Serum metabolomic profiling in patients with Alzheimer disease and amnestic mild cognitive impairment by GC/MS.

The aim of this study was to characterize the serum metabolic profiles of patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (AMCI) using metabolomics based on gas chromatography-mass spectrometry (GC/MS). Serum samples were collected from patients with AD (n = 30) and AMCI (n = 32), and normal healthy controls (NOR, n = 40). Metabolite profiles were performed with GC/MS in conjunction with multivariate statistical analysis, and possible biomarker metabolites were identified. Thirty-one kinds of endogenous metabolites could be identified simultaneously. Eleven components were chosen as biomarker metabolites between AD and NOR groups, and these metabolites were closely related to seven biological pathways: arginine and proline metabolism, phenylalanine metabolism, ß-alanine metabolism, primary bile acid synthesis, glutathione metabolism, starch and sucrose metabolism, and steroid hormone biosynthesis. Meanwhile, 10 components were chosen as biomarker metabolites between AMCI and NOR groups and seven biological pathways were closely related: arginine and proline metabolism, phenylalanine metabolism, citrate cycle, alanine, aspartate and glutamate metabolism, taurine and hypotaurine metabolism, starch and sucrose metabolism, and steroid hormone biosynthesis. Our study distinguished serum metabotypes between AD, AMCI and NOR patients successfully. The implementation of this metabolomic strategy may help to develop biochemical insight into the metabolic alterations in AD/AMCI and will be helpful for the further understanding of pathogenesis.

Genome-Wide Identification of R2R3-MYB Transcription Factors Regulating Secondary Cell Wall Thickening in Cotton Fiber Development.

MYB proteins represent one of the largest transcription factor (TF) families in plants, some of which act as key transcriptional regulators of secondary cell wall (SCW) biosynthesis. Cotton (Gossypium hirsutum) fiber is thought to be an ideal single-cell model to study cell elongation and SCW biosynthesis. However, little knowledge regarding the TFs controlling fiber SCW biosynthesis, particularly for R2R3-MYBs is known. By far, no comprehensive genome-wide analysis of the secondary wall-associated R2R3-MYBs has been reported in cultivated tetraploid upland cotton. In this study, we identified 419 R2R3-MYB genes by systematically examining the cotton genome. A combination of phylogenetic, RNA-seq and co-expression analyses indicated that 36 R2R3-MYBs were either preferentially or highly expressed in 20 day post anthesis (dpa) fibers and are putative SCW regulators. Among these MYB genes, 22 MYBs are homologs of known SCW MYB proteins and the other 14 MYBs are novel proteins without prior reported SCW biosynthesis-related functions. Finally, we highlighted on the roles of two MYBs named GhMYB46_D13 and GhMYB46_D9, both of which displayed the highest expression in 20 dpa fibers. Expression of GhMYB46_D13 or GhMYB46_D9 individually in Arabidopsis resulted in ectopic SCW deposition in transgenic plants. Furthermore, both GhMYB46_D13 and GhMYB46_D9 were able to activate the cotton fiber SCW cellulose synthase gene promoters. Thus, we have identified 36 R2R3-MYBs as potential SCW regulators in cotton fibers that represent strong candidates for further functional studies during fiber development and SCW thickening.

Cleavage of potassium channel Kv2.1 by BACE2 reduces neuronal apoptosis.

Potassium channel Kv2.1 regulates potassium current in cortical neurons and potassium efflux is necessary for cell apoptosis. As a major component of delayed rectifier current potassium channels, Kv2.1 forms clusters in the membrane of hippocampal neurons. BACE2 is an aspartyl protease to cleave APP to prevent the generation of Aß, a central component of neuritic plaques in Alzheimer's brain. We now identified Kv2.1 as a novel substrate of BACE2. We found that BACE2 cleaved Kv2.1 at Thr376, Ala717, and Ser769 sites and disrupted Kv2.1 clustering on cell membrane, resulting in decreased Ik of Kv2.1 and a hyperpolarizing shift in primary neurons. Furthermore, we discovered that the BACE2-cleaved Kv2.1 forms, Kv2.1-1-375, Kv2.1-1-716, and Kv2.1-1-768, depressed the delayed rectifier Ik surge and reduced neuronal apoptosis. Our study suggests that BACE2 plays a neuroprotective role by cleavage of Kv2.1 to prevent the outward potassium currents, a potential new target for Alzheimer's treatment.

Intracellular tracking of drug release from pH-sensitive polymeric nanoparticles via FRET for synergistic chemo-photodynamic therapy.

BACKGROUND: Synergistic therapy of tumor is a promising way in curing cancer and in order to achieve effective tumor therapy with real-time drug release monitoring, dynamic cellular imaging and antitumor activity. RESULTS: In this work, a polymeric nanoparticle with Forster resonance energy transfer (FRET) effect and chemo-photodynamic properties was fabricated as the drug vehicle. An amphiphilic polymer of cyclo(RGDfCSH) (cRGD)-poly(ethylene glycol) (PEG)-Poly(L-histidine) (PH)-poly(ε-caprolactone) (PCL)-Protoporphyrin (Por)-acting as both a photosensitizer for photodynamic therapy (PDT) and absorption of acceptor in FRET was synthesized and self-assembled into polymeric nanoparticles with epirubicin (EPI)-acting as an antitumor drug for chemotherapy and fluorescence of donor in FRET. Spherical EPI-loaded nanoparticles with the average size of 150 ± 2.4 nm was procured with negatively charged surface, pH sensitivity and high drug loading content (14.9 ± 1.5%). The cellular uptake of EPI-loaded cRGD-PEG-PH-PCL-Por was monitored in real time by the FRET effect between EPI and cRGD-PEG-PH-PCL-Por. The polymeric nanoparticles combined PDT and chemotherapy showed significant anticancer activity both in vitro (IC50 = 0.47 µg/mL) and better therapeutic efficacy than that of free EPI in vivo. CONCLUSIONS: This work provided a versatile strategy to fabricate nanoassemblies for intracellular tracking of drug release and synergistic chemo-photodynamic therapy.

A nickel nanoparticle/nafion-graphene oxide modified screen-printed electrode for amperometric determination of chemical oxygen demand.

A nickel nanoparticle/nafion-graphene oxide (NiNP/Nf-GO) modified screen-printed electrode (SPE) was developed for rapid and environmentally friendly electrochemical determination of chemical oxygen demand (COD). The morphology and the electrochemical performance of the SPEs with different surface modifications were investigated by scanning electron microscopy, electrochemical impedance spectroscopy, amperometry, and cyclic voltammetry, respectively. Interestingly, incorporation of graphene oxide as supporting materials to the NiNP/Nf-GO modified SPE enables high catalyst loading and electrode contact, leading to excellent electrocatalytic oxidation ability. A flow detection system was constructed based the newly designed NiNP/Nf-GO modified SPE with USB connection, a 3D-printed thin-layer flow cell (TLFC), and a peristaltic pump. The flow detection system showed an excellent performance for COD analysis with a linear detection range of 0.1~400 mg L-1 and a lower detection limit of 0.05 mg L-1 with an oxidation potential of 0.45 V. The system was further applied to determine the COD in surface water samples. The results were consistent with those obtained by using the standard method (ISO 6060). Graphical abstract A novel nickel nanoparticle/nafion-graphene oxide (NiNP/Nf-GO) modified screen-printed electrode (SPE) with excellent electrocatalytic oxidation ability was designed and fabricated. This electrode with USB connection was applied in a flow detection system equipped with a 3D-printed thin-layer flow cell and a peristaltic pump for environmentally friendly electrochemical determination of chemical oxygen demand.