Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.

ESCRTs Cooperate with a Selective Autophagy Receptor to Mediate Vacuolar Targeting of Soluble Cargos.

Autophagy transports cytosolic materials into lysosomes/vacuoles either in bulk or selectively. Selective autophagy requires cargo receptor proteins, which usually link cargos to the macroautophagy machinery composed of core autophagy-related (Atg) proteins. Here, we show that fission yeast Nbr1, a homolog of mammalian autophagy receptor NBR1, interacts with and facilitates the transport of two cytosolic hydrolases into vacuoles, in a way reminiscent of the budding yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy. We term this pathway Nbr1-mediated vacuolar targeting (NVT). Surprisingly, unlike the Cvt pathway, the NVT pathway does not require core Atg proteins. Instead, it depends on the endosomal sorting complexes required for transport (ESCRTs). NVT components colocalize with ESCRTs at multivesicular bodies (MVBs) and rely on ubiquitination for their transport. Our findings demonstrate the ability of ESCRTs to mediate highly selective autophagy of soluble cargos, and suggest an unexpected mechanistic versatility of autophagy receptors.

CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques.

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a ß-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.

Subfrontal recurrence after cerebellar medulloblastoma resection without local relapse: case-based update.

OBJECTIVE: This report detailed four cases of tumor recurrence in the subfrontal region after cerebellar medulloblastoma resection without local relapse and explored the causes of recurrence. In addition, a case-based update and insight into the entity is attempted. METHODS: All four patients received cerebellar medulloblastoma resection and postoperative radiotherapy. They were admitted to our hospital when they were found to have a recurrent tumor in the subfrontal region of the anterior skull base. All four patients received re-resection of the tumor, which was confirmed to be recurrent medulloblastoma by postoperative pathological results. RESULTS: All patients received local radiotherapy and temozolomide chemotherapy after recurrent tumor resection. They all died due to multiple organ failure resulting from tumor metastasis to other sites or tumor regrowth within 2 years after the second operation. CONCLUSION: Medulloblastoma metastasize to the subfrontal region and develop a homogenous recurrence is rare. Underdosage of radiation, a gravity-related sanctuary effect, surgical position, and perioperative hydrocephalus management might be factors contributing to this supratentorial meningeal recurrence. A better prevention of tumor recurrence might be achieved by extensive microsurgical tumor resection in the initial operation and by minimizing the need for a permanent V-P shunt in the treatment of perioperative hydrocephalus as well as by administering full-dose radiotherapy to the region of the cribriform plate in the subfrontal area.

Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2.

Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3' endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5' endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases.

Increasing the Depth of Mass-Spectrometry-Based Structural Analysis of Protein Complexes through the Use of Multiple Cross-Linkers.

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS(3) and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins. In order to find the cross-linkers that can best complement the performance of BS(3) and DSS, we tested seven additional cross-linkers that either have different spacer arm structures or that target different amino acids (BS(2)G, EGS, AMAS, GMBS, Sulfo-GMBS, EDC, and TFCS). Using BSA, aldolase, the yeast H/ACA protein complex, and E. coli 70S ribosomes, we showed that, in terms of providing structural information not obtained through the use of BS(3) and DSS, EGS and Sulfo-GMBS worked better than the other cross-linkers that we tested. EGS generated a large number of cross-links not seen with the other amine-specific cross-linkers, possibly due to its hydrophilic spacer arm. We demonstrate that incorporating the cross-links contributed by the EGS and amine-sulfhydryl cross-linkers greatly increased the accuracy of Rosetta in docking the structure of the yeast H/ACA protein complex. Given the improved depth of useful information it can provide, we suggest that the multilinker CXMS approach should be used routinely when the amount of a sample permits.

[Intervention effects of Jiaotai pills on PCPA-induced insomnia in rats].

To elucidate the intervention effects of Jiaotai pills(JTP) on p-chlorophenylalanine (PCPA)-induced insomnia in rats and its underlying mechanism, the insomnia model was established by single intraperitoneal injection with PCPA in rats. The locomotor activity of rats was observed, and the levels of nerve growth factor(NGF) in hypothalamus, hippocampus, prefrontal cortex and serum of rats were determined by using ELISA. Moreover, a proton nuclear magnetic resonance(¹H-NMR)-based metabonomic approach was developed to profile insomnia-related metabolites in rat serum and hippocampus and analyze the intervention effects of JTP on changes in underlying biomarkers related to locomotor activity, NGF and insomnia. According to the results, JTP could significantly suppress the locomotor activity of insomnia rats, and increase the NGF levels in hypothalamus, hippocampus, prefrontal cortex and serum of rats with insomnia. The disturbed metabolic state associated with PCPA-induced insomnia in rat serum and hippocampus could be intervened by JTP. Meanwhile, six and five potential biomarkers related to insomnia in rat serum and hippocampus were reversed by administration of JTP. In conclusion, the current study demonstrated that JTP had protective effects against PCPA-induced insomnia in rats, which was probably correlated with regulation of NGF level and metabolism of amino acids, lipids and choline.

Identification of cross-linked peptides from complex samples.

We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein-interaction studies.

Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4.

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.

Interventional chemoembolization for the treatment of severe ulcerative bleeding caused by advanced breast cancer: A report of two cases.

Local ulcerative cutaneous hemorrhage resulting from breast cancer profoundly effects the quality of life of patients, at times even posing a threat to life. While early diagnosis rates of breast cancer have shown improvement, some patients may present at an advanced stage upon consultation. Presently, there is no standardized treatment approach for these patients. In this context, the present study presented two case studies detailing the use of interventional embolization chemotherapy for addressing severe local ulcerative hemorrhage associated with breast cancer. Post-treatment, there was a notable amelioration in the mammary ulceration among the patients, an elevated hemoglobin level compared with baseline and a consequent enhancement in their overall quality of life. These cases may serve as valuable references for the management of such clinical situations.

MAS-Net:Multi-modal Assistant Segmentation Network For Lumbar Intervertebral Disc.

Objective.Despite advancements in medical imaging technology, the diagnosis and positioning of lumbar disc diseases still heavily rely on the expertise and experience of medical professionals. This process is often time-consuming, labor-intensive, and susceptible to subjective factors. Achieving automatic positioning and segmentation of lumbar intervertebral disc (LID) is the first and critical step in intelligent diagnosis of lumbar disc diseases. However, due to the complexity of the vertebral body and the ambiguity of the soft tissue boundaries of the LID, accurate and intelligent segmentation of LIDs remains challenging. The study aims to accurately and intelligently segment and locate LIDs by fully utilizing multi-modal lumbar magnetic resonance Images (MRIs).Approach.A novel multi-modal assistant segmentation network (MAS-Net) is proposed in this paper. The architecture consists of four key components: the multi-branch fusion encoder (MBFE), the cross-modality correlation evaluation (CMCE), the channel fusion transformer (CFT), and the selective Kernel (SK) based decoder. The MBFE module captures and integrates various modal features, while the CMCE module facilitates the fusion process between the MBFE and decoder. The CFT module selectively guides the flow of information between the MBFE and decoder and effectively utilizes skip connections from multiple layers. The SK module computes the significance of each channel using global pooling operations and applies weights to the input feature maps to improve the models recognition of important features.Main results.The proposed MAS-Net achieved a dice coefficient of 93.08% on IVD3Seg and 93.22% on DualModalDisc dataset, outperforming the current state-of-the-art network, accurately segmenting the LIDs, and generating a 3D model that can precisely display the LIDs.Significance.MAS-Net automates the diagnostics process and addresses challenges faced by doctors. Simplifying and enhancing the clarity of visual representation, multi-modal MRI allows for better information complementation and LIDs segmentation. By successfully integrating data from various modalities, the accuracy of LID segmentation is improved.

Physicochemical properties of different crystal forms of manganese dioxide prepared by a liquid phase method and their quantitative evaluation in capacitor and battery materials.

Although there are many studies on the preparation and electrochemical properties of the different crystal forms of manganese dioxide, there are few studies on their preparation by a liquid phase method and the influence of their physical and chemical properties on their electrochemical performance. In this paper, five crystal forms of manganese dioxide were prepared by using manganese sulfate as a manganese source and the difference of their physical and chemical properties was studied by phase morphology, specific surface area, pore size, pore volume, particle size and surface structure. The different crystal forms of manganese dioxide were prepared as electrode materials, and their specific capacitance composition was obtained by performing CV and EIS in a three-electrode system, introducing kinetic calculation and analyzing the principle of electrolyte ions in the electrode reaction process. The results show that δ-MnO2 has the largest specific capacitance due to its layered crystal structure, large specific surface area, abundant structural oxygen vacancies and interlayer bound water, and its capacity is mainly controlled by capacitance. Although the tunnel of the γ-MnO2 crystal structure is small, its large specific surface area, large pore volume and small particle size make it have a specific capacitance that is only inferior to δ-MnO2, and the diffusion contribution in the capacity accounts for nearly half, indicating it also has the characteristics of battery materials. α-MnO2 has a larger crystal tunnel structure, but its capacity is lower due to the smaller specific surface area and less structural oxygen vacancies. ε-MnO2 has a lower specific capacitance is not only the same disadvantage as α-MnO2, but also the disorder of its crystal structure. The tunnel size of ß-MnO2 is not conducive to the interpenetration of electrolyte ions, but its high oxygen vacancy concentration makes its contribution of capacitance control obvious. EIS data shows that δ-MnO2 has the smallest charge transfer impedance and bulk diffusion impedance, while the two impedances of γ-MnO2 were the largest, which shows that its capacity performance has great potential for improvement. Combined with the calculation of electrode reaction kinetics and the performance test of five crystal capacitors and batteries, it is shown that δ-MnO2 is more suitable for capacitors and γ-MnO2 is more suitable for batteries.

Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4.

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.

The nuclear Argonaute HRDE-1 directs target gene re-localization and shuttles to nuage to promote small RNA-mediated inherited silencing.

Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that induction of heterochromatin silencing in C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei. Tethering the nuclear Argonaute WAGO-9/HRDE-1 induces heterochromatin formation and independently induces small RNA amplification. Consistent with this finding, HRDE-1, while predominantly nuclear, also localizes to peri-nuclear nuage domains, where amplification is thought to occur. Tethering a heterochromatin-silencing factor, NRDE-2, induces heterochromatin formation, which subsequently causes de novo synthesis of HRDE-1 guide RNAs. HRDE-1 then acts to further amplify small RNAs that load on downstream Argonautes. These findings suggest that HRDE-1 plays a dual role, acting upstream to initiate heterochromatin silencing and downstream to stimulate a new cycle of small RNA amplification, thus establishing a self-enforcing mechanism that propagates gene silencing to future generations.

Artesunate ameliorates irinotecan-induced intestinal injury by suppressing cellular senescence and significantly enhances anti-tumor activity.

Irinotecan (CPT-11) is a topoisomerase I inhibitor that was approved for cancer treatment in 1994. To date, this natural product derivative remains the world's leading antitumor drug. However, the clinical application of irinotecan is limited due to its side effects, the most troubling of which is intestinal toxicity. In addition, irinotecan has certain toxicity to cells and even causes cellular senescence. Committed to developing alternatives to prevent these adverse reactions, we evaluated the activity of artesunate, which has never been tested in this regard despite its biological potential. Irinotecan accelerated the process of aging in vivo and in vitro, and we found that this was mainly caused by activating mTOR signaling targets. Artesunate inhibited the activity of mTOR, thereby alleviating the aging process. Our study found that artesunate treatment improved irinotecan-induced intestinal inflammation by reducing the levels of TNF-α, IL1, and IL6; reducing inflammatory infiltration of the colonic ileum in mice; and preventing irinotecan-induced intestinal damage by reducing weight loss and improving intestinal length. In addition, in mouse xenograft tumor models, artesunate and irinotecan significantly inhibited tumor growth in mice.

Comprehensive metabolomics and lipidomics profiling uncovering neuroprotective effects of Ginkgo biloba L. leaf extract on Alzheimer's disease.

Introduction: Ginkgo biloba L. leaf extract (GBLE) has been reported to be effective for alleviating cognitive and memory impairment in Alzheimer's disease (AD). Nevertheless, the potential mechanism remains unclear. Herein, this study aimed to explore the neuroprotective effects of GBLE on AD and elaborate the underlying therapeutic mechanism. Methods: Donepezil, the most widely prescribed drug for AD, was used as a positive control. An integrated metabolomics and lipidomics approach was adopted to characterize plasma metabolic phenotype of APP/PS1 double transgenic mice and describe the metabolomic and lipidomic fingerprint changes after GBLE intervention. The Morris water maze test and immunohistochemistry were applied to evaluate the efficacy of GBLE. Results: As a result, administration of GBLE significantly improved the cognitive function and alleviated amyloid beta (Aß) deposition in APP/PS1 mice, showing similar effects to donepezil. Significant alterations were observed in metabolic signatures of APP/PS1 mice compared with wild type (WT) mice by metabolomic analysis. A total of 60 markedly altered differential metabolites were identified, including 28 lipid and lipid-like molecules, 13 organic acids and derivatives, 11 organic nitrogen compounds, and 8 other compounds, indicative of significant changes in lipid metabolism of AD. Further lipidomic profiling showed that the differential expressed lipid metabolites between APP/PS1 and WT mice mainly consisted of phosphatidylcholines, lysophosphatidylcholines, triglycerides, and ceramides. Taking together all the data, the plasma metabolic signature of APP/PS1 mice was primarily characterized by disrupted sphingolipid metabolism, glycerophospholipid metabolism, glycerolipid metabolism, and amino acid metabolism. Most of the disordered metabolites were ameliorated after GBLE treatment, 19 metabolites and 24 lipids of which were significantly reversely regulated (adjusted-p<0.05), which were considered as potential therapeutic targets of GBLE on AD. The response of APP/PS1 mice to GBLE was similar to that of donepezil, which significantly reversed the levels of 23 disturbed metabolites and 30 lipids. Discussion: Our data suggested that lipid metabolism was dramatically perturbed in the plasma of APP/PS1 mice, and GBLE might exert its neuroprotective effects by restoring lipid metabolic balance. This work provided a basis for better understanding the potential pathogenesis of AD and shed new light on the therapeutic mechanism of GBLE in the treatment of AD.

3-Bromo-5-meth-oxy-4-(4-methyl-piperidin-1-yl)furan-2(5H)-one.

There are two molecules in the asymmetric unit of title compound, C(11)H(16)BrNO(3), which was obtained via the tandem Michael addition-elimination reaction of 3,4-dibromo-5-meth-oxy-furan-2(5H)-one and 4-methyl-piperidine in the presence of potassium fluoride. The furan-one rings are approximately planar [maximum atomic deviations of 0.026 (2) and 0.015 (2) Å, respectively]. The packing is stabilized by weak inter-molecular C-H⋯O and C-H⋯Br inter-actions.