Branch Site Research Articles

Nail Reconstruction Using an Osteo-onychocutaneous Flap with the Dorsal Digital Artery of the Hallux as the Pedicle: Techniques and Anatomical Insights

Nail reconstruction with preserved pulp tissue can be challenging due to the use of oversized flaps and nail deformities often resulting from bone resorption. This study describes a novel technique using the dorsal digital artery of the hallux as a pedicle for an osteo-onychocutaneous flap. A preoperative handheld Doppler was used to identify the course of the dorsal digital artery, and intraoperatively, the flap was elevated without disturbing the plantar tissue, thereby minimizing the risk of foot complications. A prospective study was conducted on all partial toe transplantation cases between September 2020 and March 2023 to analyze the anatomical variations of the dorsal digital artery. Thirty-one patients were included in the study. The dorsal digital artery was successfully identified preoperatively and confirmed intraoperatively in all cases. It branched from the first dorsal metatarsal artery in 24 cases and from the plantar digital artery in seven cases. The branching location was found to be 24.3 ± 4.5mm proximal to the web in the former group and 3.9 ± 3.9mm (p < 0.001) in the latter group. The vascular diameter at the branching site was measured to be 0.8 ± 0.2mm, with no significant difference based on the location. Nail reconstruction procedures were successfully performed using this flap for defects in the thumb, index finger, and little finger. Stable nails were reconstructed in all cases with minimal foot complications. In conclusion, this technique offers a reliable approach to nail reconstruction, minimizing donor site morbidity and preserving natural nail morphology.

Relative Residence Time Can Account for Half of the Anatomical Variation in Fatty Streak Prevalence Within the Right Coronary Artery.

The patchy anatomical distribution of atherosclerosis has been attributed to variation in haemodynamic wall shear stress (WSS). The consensus is that low WSS and a high Oscillatory Shear Index (OSI) trigger the disease. We found that atherosclerosis at aortic branch sites correlates threefold better with transverse WSS (transWSS), a metric which quantifies multidirectional near-wall flow. Coronary artery disease has greater clinical significance than aortic disease but computation of WSS metrics is complicated by the substantial vessel motion occurring during each cardiac cycle. Here we present the first comparison of the distribution of atherosclerosis with WSS metrics computed for moving coronary arteries. Maps of WSS metrics were computed using dynamic geometries reconstructed from angiograms of ten non-stenosed human right coronary arteries (RCAs). They were compared with maps of fatty streak prevalence derived from a previous study of 1852 RCAs. Time average WSS (TAWSS), OSI, transWSS and the cross-flow index (CFI), a non-dimensional form of the transWSS, gave non-significant or significant but low spatial correlations with lesion prevalence. The highest correlation coefficient (0.71) was for the relative residence time (RRT), a metric that decreases with TAWSS and increases with OSI. The coefficient was not changed if RRT was calculated using CFI, which captures multidirectional WSS only, rather than OSI, which encompasses both multidirectional and oscillatory WSS. Contrary to our earlier findings in the aorta, low WSS in combination with highly multidirectional flow correlates best with lesion location in the RCA, explaining approximately half of its anatomical variation.

Branch site recognition by the spliceosome.

The spliceosome is a eukaryotic multimegadalton RNA-protein complex that removes introns from transcripts. The spliceosome ensures the selection of each exon-intron boundary through multiple recognition events. Initially, the 5' splice site (5' SS) and branch site (BS) are bound by the U1 small nuclear ribonucleoprotein (snRNP) and the U2 snRNP, respectively, while the 3' SS is mostly determined by proximity to the branch site. A large number of splicing factors recognize the splice sites and recruit the snRNPs before the stable binding of the snRNPs occurs by base-pairing the snRNA to the transcript. Fidelity of this process is crucial, as mutations in splicing factors and U2 snRNP components are associated with many diseases. In recent years, major advances have been made in understanding how splice sites are selected in Saccharomyces cerevisiae and humans. Here, I review and discuss the current understanding of the recognition of splice sites by the spliceosome with a focus on recognition and binding of the branch site by the U2 snRNP in humans.

KHSRP ameliorates acute liver failure by regulating pre-mRNA splicing through its interaction with SF3B1

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.

Multilevel neurium-mimetic individualized graft via additive manufacturing for efficient tissue repair

Complicated peripheral nerve injuries or defects, especially at branching sites, remain a prominent clinical challenge after the application of different treatment strategies. Current nerve grafts fail to match the expected shape and size for delicate and precise branched nerve repair on a case-by-case basis, and there is a lack of geometrical and microscale regenerative navigation. In this study, we develop a sugar painting-inspired individualized multilevel epi-/peri-/endoneurium-mimetic device (SpinMed) to customize natural cues, featuring a selectively protective outer sheath and an instructive core, to support rapid vascular reconstruction and consequent efficient neurite extension along the defect area. The biomimetic perineurium dictates host-guest crosslinking in which new vessels secrete multimerin 1 binding to the fibroin filler surface as an anchor, contributing to the biological endoneurium that promotes Schwann cell homing and remyelination. SpinMed implantation into rat sciatic nerve defects yields a satisfactory outcome in terms of structural reconstruction, with sensory and locomotive function restoration. We further customize SpinMed grafts based on anatomy and digital imaging, achieving rapid repair of the nerve trunk and branches superior to that achieved by autografts and decellularized grafts in a specific beagle nerve defect model, with reliable biosafety. Overall, this intelligent art-inspired biomimetic design offers a facile way to customize sophisticated high-performance nerve grafts and holds great potential for application in translational regenerative medicine.

Molecular basis for the activation of human spliceosome

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5′-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.

Latent Dirichlet allocation mixture models for nucleotide sequence analysis.

Strings of nucleotides carrying biological information are typically described as sequence motifs represented by weight matrices or consensus sequences. However, many signals in DNA or RNA are recognized by multiple factors in temporal sequence, consist of distinct alternative motifs, or are best described by base composition. Here we apply the latent Dirichlet allocation (LDA) mixture model to nucleotide sequences. Using positions in an alignment of human or Drosophila splice sites as samples, we show that LDA readily identifies motifs, including such elusive cases as the intron branch site. Using whole sequences with positional k-mers as features, LDA can identify sequence subtypes enriched in long vs. short introns. LDA with bulk k-mers can reliably distinguish reading frame and species of origin in coding sequences from humans and Drosophila. We find that LDA is a useful model for describing heterogeneous signals, for assigning individual sequences to subtypes, and for identifying and characterizing sequences that do not fit recognized subtypes. Because LDA topic models are interpretable, they also aid the discovery of new motifs, even those present in a small fraction of samples. In summary, LDA can identify and characterize signals in nucleotide sequences, including candidate regulatory factors involved in biological processes.

SpliceAPP: an interactive web server to predict splicing errors arising from human mutations

BackgroundSplicing variants are a major class of pathogenic mutations, with their severity equivalent to nonsense mutations. However, redundant and degenerate splicing signals hinder functional assessments of sequence variations within introns, particularly at branch sites. We have established a massively parallel splicing assay to assess the impact on splicing of 11,191 disease-relevant variants. Based on the experimental results, we then applied regression-based methods to identify factors determining splicing decisions and their respective weights.ResultsOur statistical modeling is highly sensitive, accurately annotating the splicing defects of near-exon intronic variants, outperforming state-of-the-art predictive tools. We have incorporated the algorithm and branchpoint information into a web-based tool, SpliceAPP, to provide an interactive application. This user-friendly website allows users to upload any genetic variants with genome coordinates (e.g., chr15 74,687,208 A G), and the tool will output predictions for splicing error scores and evaluate the impact on nearby splice sites. Additionally, users can query branch site information within the region of interest.ConclusionsIn summary, SpliceAPP represents a pioneering approach to screening pathogenic intronic variants, contributing to the development of precision medicine. It also facilitates the annotation of splicing motifs. SpliceAPP is freely accessible using the link https://bc.imb.sinica.edu.tw/SpliceAPP. Source code can be downloaded at https://github.com/hsinnan75/SpliceAPP.

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5’ splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

Structural insights into the cross-exon to cross-intron spliceosome switch

Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2–5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5′ splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10–16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5′ splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.

The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes invivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Conserved intronic secondary structures with concealed branch sites regulate alternative splicing of poison exons.

Alternative splicing (AS) generates multiple RNA isoforms and increases the complexities of transcriptomes and proteomes. However, it remains unclear how RNA structures contribute to AS regulation. Here, we systematically search transcriptomes for secondary structures with concealed branch sites (BSs) in the alternatively spliced introns and predict thousands of them from six organisms, of which many are evolutionarily conserved. Intriguingly, a highly conserved stem-loop structure with concealed BSs is found in animal SF3B3 genes and colocalizes with a downstream poison exon (PE). Destabilization of this structure allows increased usage of the BSs and results in enhanced PE inclusion in human and Drosophila cells, leading to decreased expression of SF3B3. This structure is experimentally validated using an in-cell SHAPE-MaP assay. Through RNA interference screens of 28 RNA-binding proteins, we find that this stem-loop structure is sensitive to U2 factors. Furthermore, we find that SF3B3 also facilitates DNA repair and protects genome stability by enhancing interaction between ERCC6/CSB and arrested RNA polymerase II. Importantly, both Drosophila and human cells with the secondary structure mutated by genome editing exhibit altered DNA repair in vivo. This study provides a novel and common mechanism for AS regulation of PEs and reveals a physiological function of SF3B3 in DNA repair.

Structural optimization of siRNA conjugates for albumin binding achieves effective MCL1-directed cancer therapy

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.

Gas-Phase Ion/Ion Reactions to Enable Radical-Directed Dissociation of Fatty Acid Ions: Application to Localization of Methyl Branching.

Methyl branching on the carbon chains of fatty acids and fatty esters is among the structural variations encountered with fatty acids and fatty esters. Branching in fatty acid/ester chains is particularly prominent in bacterial species and, for example, in vernix caseosa and sebum. The distinction of branched chains from isomeric straight-chain species and the localization of branching can be challenging to determine by mass spectrometry (MS). Condensed-phase derivatization strategies, often used in conjunction with separations, are most commonly used to address the identification and characterization of branched fatty acids. In this work, a gas-phase ion/ion strategy is presented that obviates condensed-phase derivatization and introduces a radical site into fatty acid ions to facilitate radical-directed dissociation (RDD). The gas-phase approach is also directly amenable to fatty acid anions generated via collision-induced dissociation from lipid classes that contain fatty esters. Specifically, divalent magnesium complexes bound to two terpyridine ligands that each incorporate a ((2,2,6,6-tetramethyl-1-piperidine-1-yl)oxy) (TEMPO) moiety are used to charge-invert fatty acid anions. Following the facile loss of one of the ligands and the TEMPO group of the remaining ligand, a radical site is introduced into the complex. Subsequent collision-induced dissociation (CID) of the complex exhibits preferred cleavages that localize the site(s) of branching. The approach is illustrated with iso-, anteiso-, and isoprenoid branched-chain fatty acids and an intact glycerophospholipid and is applied to a mixture of branched- and straight-chain fatty acids derived from Bacillus subtilis.

Cortactin stabilizes actin branches by bridging activated Arp2/3 to its nucleated actin filament

Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes, including cell migration and membrane trafficking. Cortactin is important for actin branch stabilization, but the mechanism by which this occurs is unclear. Given this, we determined the structure of vertebrate cortactin-stabilized Arp2/3 actin branches using cryogenic electron microscopy. We find that cortactin interacts with the new daughter filament nucleated by the Arp2/3 complex at the branch site, rather than the initial mother actin filament. Cortactin preferentially binds activated Arp3. It also stabilizes the F-actin-like interface of activated Arp3 with the first actin subunit of the new filament, and its central repeats extend along successive daughter-filament subunits. The preference of cortactin for activated Arp3 explains its retention at the actin branch and accounts for its synergy with other nucleation-promoting factors in regulating branched actin network dynamics.

Textbook Outcomes Among Patients with Aneurysmal Subarachnoid Hemorrhage Following Endovascular Treatment.

The case fatality rate among patients with aneurysmal subarachnoid hemorrhage (aSAH) has decreased progressively, with numerous patients subjected to contemporary paradigms that minimize the use of agonizing therapeutic processes. The concept of the "Textbook Outcome" (TO), a composite outcome that highlights numerous favorable outcomes, was developed in the context of gastrointestinal tumor surgeries and expeditiously extended across diverse surgical spheres. The aim of this study was to explore the factors hindering the achievement of optimal prognoses in postinterventional aSAH patients, employ textbook outcomes, and establish predictive models. We conducted a retrospective review of data from 1270 aSAH patients who received endovascular treatment between 2012 and 2018. We delineated an exemplary TO within the aSAH domain, characterized by favorable clinical results, minimal complications, and the absence of retreatments. This TO-oriented approach is explained within the manuscript. The findings revealed that preoperative intraventricular hemorrhage (IVH), preoperative Hunt and Hess grade (H&H) ≥ 3, World Federation of Neurosurgical Societies (WFNS) grade ≥ 3, the presence of blebs on the aneurysm, aneurysms situated at branching sites, and non-stent-assisted endovascular intervention were the strongest risk factors for not achieving textbook outcomes (non-"Textbook Outcome" [N-TO]). Decision curve analysis and calibration analyses revealed strong concordance between the predictions of the N-TO nomogram model and the actual observations. Treatment Outcomes hold significant practical value in clinical studies of aSAH patients receiving endovascular treatment. The likelihood of N-TOs was predicted by IVH, H&H grade ≥ 3, WFNS grade ≥ 2, presence o f bleb on the aneurysm, and aneurysms located at branching sites.

Self-reporting and Biodegradable Thermosetting Solid Polymer Electrolyte.

To date, significant efforts have been dedicated to improve their ionic conductivity, thermal stability, and mechanical strength of solid polymer electrolytes (SPEs). However, direct monitoring of physical and chemical changes in SPEs is still lacking. Moreover, existing thermosetting SPEs are hardly degradable. Herein, by overcoming the limitation predicted by Flory theory, self-reporting and biodegradable thermosetting hyperbranched poly(β-amino ester)-based SPEs (HPAE-SPEs) are reported. HPAE is successfully synthesized through a well-controlled "A2+B4" Michael addition strategy and then crosslinked it in situ to produce HPAE-SPEs. The multiple tertiary aliphatic amines at the branching sites confer multicolour luminescence to HPAE-SPEs, enabling direct observation of its physical and chemical damage. After use, HPAE-SPEs can be rapidly hydrolysed into non-hazardous β-amino acids and polyols via self-catalysis. Optimized HPAE-SPE exhibits an ionic conductivity of 1.3×10-4 S/cm at 60 °C, a Na+ transference number ( ) of 0.67, a highly stable sodium plating-stripping behaviour and a low overpotential of ≈190 mV. This study establishes a new paradigm for developing SPEs by engineering multifunctional polymers. The self-reporting and biodegradable properties would greatly expand the scope of applications for SPEs.

Self‐reporting and Biodegradable Thermosetting Solid Polymer Electrolyte

AbstractTo date, significant efforts have been dedicated to improve their ionic conductivity, thermal stability, and mechanical strength of solid polymer electrolytes (SPEs). However, direct monitoring of physical and chemical changes in SPEs is still lacking. Moreover, existing thermosetting SPEs are hardly degradable. Herein, by overcoming the limitation predicted by Flory theory, self‐reporting and biodegradable thermosetting hyperbranched poly(β‐amino ester)‐based SPEs (HPAE‐SPEs) are reported. HPAE is successfully synthesized through a well‐controlled “A2+B4” Michael addition strategy and then crosslinked it in situ to produce HPAE‐SPEs. The multiple tertiary aliphatic amines at the branching sites confer multicolour luminescence to HPAE‐SPEs, enabling direct observation of its physical and chemical damage. After use, HPAE‐SPEs can be rapidly hydrolysed into non‐hazardous β‐amino acids and polyols via self‐catalysis. Optimized HPAE‐SPE exhibits an ionic conductivity of 1.3×10−4 S/cm at 60 °C, a Na+ transference number ( ) of 0.67, a highly stable sodium plating‐stripping behaviour and a low overpotential of ≈190 mV. This study establishes a new paradigm for developing SPEs by engineering multifunctional polymers. The self‐reporting and biodegradable properties would greatly expand the scope of applications for SPEs.

Structural insights into branch site proofreading by human spliceosome.

Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.

Patterns of diversity in stream macroinvertebrate communities in a low-gradient agricultural stream (Sydenham River, Ontario, Canada) were driven by surficial geology and catchment position

Abstract Freshwater biodiversity is increasingly at risk wherever land uses such as agriculture exert multiple stressors that degrade habitat quality. Although stream macroinvertebrates act as bioindicators for monitoring these impacts, their responses are context-specific: examining drivers of community composition is therefore important to understand the results of monitoring efforts. In a primarily agricultural landscape, 15 sites across the Sydenham River watershed, Ontario, Canada, were assessed for in-stream habitat quality and stream macroinvertebrate diversity. We predicted that community assemblage would be driven by differences in surficial geology across branch (east branch versus north branch) and catchment position (main stem versus tributary). We found that the main stem of the east branch was characterised by significantly higher proportions (P = 0.053) and abundance (P = 0.038) of Ephemeroptera–Plecoptera–Trichoptera (EPT) taxa than north branch sites were, and sites in the east and north branch tributaries were characterised by significantly lower Hilsenhoff Biotic Index (HBI) scores (P = 0.088). Redundancy analysis found that substrate size was the main driver of community composition, generating a model that described these patterns across branch and catchment position. Our findings suggest that EPT abundance and HBI scores were the variables that were most effective at revealing differences in stream communities due to agricultural impacts and that sediment size is an important driver of these patterns.