CsBPC2 is essential for cucumber survival under cold stress.

Cold stress affects the growth and development of cucumbers. Whether the BPC2 transcription factor participates in cold tolerance and its regulatory mechanism in plants have not been reported. Here, we used wild-type (WT) cucumber seedlings and two mutant Csbpc2 lines as materials. The underlying mechanisms were studied by determining the phenotype, physiological and biochemical indicators, and transcriptome after cold stress. The results showed that CsBPC2 knockout reduced cucumber cold tolerance by increasing the chilling injury index, relative electrical conductivity and malondialdehyde (MDA) content and decreasing antioxidant enzyme activity. We then conducted RNA sequencing (RNA-seq) to explore transcript-level changes in Csbpc2 mutants. A large number of differentially expressed genes (1032) were identified and found to be unique in Csbpc2 mutants. However, only 489 down-regulated genes related to the synthesis and transport of amino acids and vitamins were found to be enriched through GO analysis. Moreover, both RNA-seq and qPT-PCR techniques revealed that CsBPC2 knockout also decreased the expression of some key cold-responsive genes, such as CsICE1, CsCOR413IM2, CsBZR1 and CsBZR2. These results strongly suggested that CsBPC2 knockout not only affected cold function genes but also decreased the levels of some key metabolites under cold stress. In conclusion, this study reveals for the first time that CsBPC2 is essential for cold tolerance in cucumber and provides a reference for research on the biological function of BPC2 in other plants.

CsBPC2 is a key regulator of root growth and development.

BASIC PENTACYSTEINE (BPCs) transcription factors are important regulators of plant growth and development. However, the regulatory mechanism of BPC2 in roots remains unclear. In our previous study, we created Csbpc2 cucumber mutants by the CRISPR/Cas9 system, and our studies on the phenotype of Csbpc2 mutants showed that the root growth was inhibited compared with wide-type (WT). Moreover, the surface area, volume and number of roots decreased significantly, with root system architecture changing from dichotomous branching to herringbone branching. Compared with WT, the leaf growth of the Csbpc2 mutants was not affected. However, the palisade and spongy tissue were significantly thinner, which was not beneficial for photosynthesis. The metabolome of root exudates showed that compared with WT, amino acids and their derivatives were significantly decreased, and the enriched pathways were mainly regulated by amino acids and their derivatives, indicating that knockout of CsBPC2 mainly affected the amino acid content in root exudates. Importantly, transcriptome analysis showed that knockout of CsBPC2 mainly affected root gene expression. Knockout of CsBPC2 significantly reduced the gene expression of gibberellins synthesis. However, the expression of genes related to amino acid synthesis, nitrogen fixation and PSII-related photosynthesis increased significantly, which may be due to the effect of knocking out CsBPC2 on gibberellins synthesis, resulting in the inhibition of seedling growth, thus forming negative feedback regulation. Generally, we showed for the first time that BPC2 is a key regulator gene of root growth and development, laying the foundation for future mechanisms of BPC2 regulation in roots.

Astrocyte-Microglia Crosstalk: A Novel Target for the Treatment of Migraine.

Migraine is a pervasive neurologic disease closely related to neurogenic inflammation. The astrocytes and microglia in the central nervous system are vital in inducing neurogenic inflammation in migraine. Recently, it has been found that there may be a crosstalk phenomenon between microglia and astrocytes, which plays a crucial part in the pathology and treatment of Alzheimer's disease and other central nervous system diseases closely related to inflammation, thus becoming a novel hotspot in neuroimmune research. However, the role of the crosstalk between microglia and astrocytes in the pathogenesis and treatment of migraine is yet to be discussed. Based on the preliminary literature reports, we have reviewed relevant evidence of the crosstalk between microglia and astrocytes in the pathogenesis of migraine and summarized the crosstalk pathways, thereby hoping to provide novel ideas for future research and treatment.

The Analgesic Effect and Potential Mechanisms of Acupuncture for Migraine Rats: A Systematic Review and Meta-Analysis.

Purpose: To assess the inhibitory effect of acupuncture on pain symptoms in migraine models, and to further summarize the potential mechanisms of acupuncture in regulating hyperalgesia in the treatment of migraine. Materials and Methods: Literature search in databases such as China National Knowledge Infrastructure (CNKI), PubMed, and Web of Science (WOS) etc. The quality was evaluated by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) bias risk assessment tool and Collaborative Approach to Meta-analysis and Review of Animal Data from Experimental Studies (CAMARADES) checklist. Meta-analyses were performed using Stata 17.0 software. Results: Twenty-one studies involving 489 animals were identified. The qualitative score ranged from 3 to 9 points. Facial mechanical withdrawal threshold (FMWT) and paw mechanical withdrawal threshold (PMWT) measured by Von Frey filaments were selected as major outcomes, and serum calcitonin gene-related peptide (CGRP) levels measured by ELISA were selected as secondary outcome. Meta-analysis results revealed that true acupuncture (TA) group significantly increased FMWT, PMWT and CGRP compared to model group. TA group showed superior effect in FMWT, PMWT relative to sham acupuncture (SA) group. Subgroup analysis results showed that high risk of bias scores may be responsible for the high heterogeneity of FMWT; additionally, CGRP analysis suggests that acupoint selection and blood collection sites may be sources of heterogeneity. In the treatment of migraine pain symptoms, the underlying mechanism of acupuncture treatment is either the regulation of hyperalgesia and neurotransmitters, or the reduction of inflammatory factors. Conclusion: The results indicate that TA treatment effectively increased the pain threshold and reduced hyperalgesia in migraine rats. In summary, our study highlights the potential of TA as an effective treatment for migraine, but further investigation is required to fully comprehend its mechanism of action and optimize its clinical application.

Bioequivalence Study of Miglitol Orally Disintegrating Tablets in Healthy Chinese Volunteers Under Fasting Condition Based on Pharmacodynamic and Pharmacokinetic Parameters.

To investigate the bioequivalence of miglitol orally disintegrating tablets in healthy Chinese volunteers based on pharmacodynamic (PD) and pharmacokinetic (PK) parameters. Additionally, the safety profile was estimated. Two randomized, open-label, single-dose, crossover trials were conducted under fasting conditions. In the PD trial (CTR20191811), 45 healthy volunteers were randomly divided into 3 groups in a 1:1:1 ratio and administered sucrose alone or coadministered with 50 mg of miglitol orally disintegrating tablet test or reference formulation/sucrose. In the PK trial (CTR20191696), 24 healthy volunteers were randomized (1:1) to receive the test or reference formulation (50 mg). Blood samples were collected at 15 and 17 sampling points per cycle in the PD and PK trials, respectively. Plasma miglitol and serum glucose concentrations were analyzed using a validated liquid chromatography-tandem mass spectrometry method. Serum insulin concentrations were measured using electrochemiluminescent immunoassay. Statistical analyses for the PD and PK parameters were subsequently performed. The volunteers' physical indicators were monitored and documented during the entire study to estimate drug safety. The PD and PK parameters of the two formulations were similar. The main PD and PK end points were both within the prespecified range of 80%-125%. The incidences of treatment-emergent adverse events (TEAEs) and drug-related TEAEs were similar between the test and reference formulation groups, and no serious TEAEs or deaths occurred during the 2 trials. These 2 formulations were demonstrated to be bioequivalent and well tolerated in healthy Chinese volunteers under fasting condition.

SEPPA-mAb: spatial epitope prediction of protein antigens for mAbs.

Identifying the exact epitope positions for a monoclonal antibody (mAb) is of critical importance yet highly challenging to the Ab design of biomedical research. Based on previous versions of SEPPA 3.0, we present SEPPA-mAb for the above purpose with high accuracy and low false positive rate (FPR), suitable for both experimental and modelled structures. In practice, SEPPA-mAb appended a fingerprints-based patch model to SEPPA 3.0, considering the structural and physic-chemical complementarity between a possible epitope patch and the complementarity-determining region of mAb and trained on 860 representative antigen-antibody complexes. On independent testing of 193 antigen-antibody pairs, SEPPA-mAb achieved an accuracy of 0.873 with an FPR of 0.097 in classifying epitope and non-epitope residues under the default threshold, while docking-based methods gave the best AUC of 0.691, and the top epitope prediction tool gave AUC of 0.730 with balanced accuracy of 0.635. A study on 36 independent HIV glycoproteins displayed a high accuracy of 0.918 and a low FPR of 0.058. Further testing illustrated outstanding robustness on new antigens and modelled antibodies. Being the first online tool predicting mAb-specific epitopes, SEPPA-mAb may help to discover new epitopes and design better mAbs for therapeutic and diagnostic purposes. SEPPA-mAb can be accessed at http://www.badd-cao.net/seppa-mab/.

Traditional Chinese medicine in treating ischemic stroke by modulating mitochondria: A comprehensive overview of experimental studies.

Ischemic stroke has been a prominent focus of scientific investigation owing to its high prevalence, complex pathogenesis, and difficulties in treatment. Mitochondria play an important role in cellular energy homeostasis and are involved in neuronal death following ischemic stroke. Hence, maintaining mitochondrial function is critical for neuronal survival and neurological improvement in ischemic stroke, and mitochondria are key therapeutic targets in cerebral stroke research. With the benefits of high efficacy, low cost, and high safety, traditional Chinese medicine (TCM) has great advantages in preventing and treating ischemic stroke. Accumulating studies have explored the effect of TCM in preventing and treating ischemic stroke from the perspective of regulating mitochondrial structure and function. In this review, we discuss the molecular mechanisms by which mitochondria are involved in ischemic stroke. Furthermore, we summarized the current advances in TCM in preventing and treating ischemic stroke by modulating mitochondria. We aimed to provide a new perspective and enlightenment for TCM in the prevention and treatment of ischemic stroke by modulating mitochondria.

Comprehensive Microbiome and Metabolome Analyses Reveal the Medicinal Components of Paeonia lactiflora.

Paeonia lactiflora Pall. is not only a traditional ornamental plant, but also an important medicinal plant. Currently, some P. lactiflora cultivars are used for ornamental purposes, but their potential medicinal value is ignored. To explore the medicinal potential of the ornamental varieties, the medicinal cultivar 'Hangbaishao' (HS) and the ornamental cultivar 'Zifengyu' (ZFY) were selected, and microbiome and metabolome analyses were performed to compare the composition of the endophytes and metabolites in the roots. The diversity and abundance of bacteria were not significantly different between HS and ZFY; however, the diversity and abundance of endophytic fungi in the ornamental cultivar ZFY were much higher than those in the medicinal cultivar HS. The flavonoids and phenolic acid contents of the ornamental cultivar ZFY were significantly higher than those of the medicinal cultivar HS, indicating that ZFY has medicinal value. The differences in root endophytes between HS and ZFY may lead to differences in phenolic acids and flavonoids. To explore the relationship between endophytes and the accumulation of phenolic acids and flavonoids, a joint analyses of the microbiome and metabolome were performed. The key bacterium, Ruminococcaceae bacterium GD7, led to the accumulation of phenolic acids and flavonoids in the ZFY. This study contributes to future research on the potential medicinal value of ornamental P. lactiflora and provides a new approach for realizing the 'dual use of medicine and appreciation' of P. lactiflora.

[Acupuncture relieves pain by inhibiting expression of Cx43 in astrocytes and release of interfe-ron-γ in neurons of trigeminal spinal nucleus in rats with migraine].

OBJECTIVE: To observe the effect of acupuncture on the expression of connexin 43 (Cx43), glial fibrillary acidic protein (GFAP), interferon-γ (IFN-γ) in the trigeminal spinal nucleus (TNC) of rats with migraine, so as to explore its mechanisms underlying amelioration of migraine. METHODS: A total of 44 SD rats were randomly divided into control, model, acu-puncture, and sham acupuncture groups (n=11 in each group). Acupuncture was applied to bilateral "Shuaigu"(GB8) and "Yanglingquan"(GB34) or non-acupoint Ⅰ (the spot about 10 mm superior to the iliac spine and 20 mm lateral to the post-median line) and non-acupoint Ⅱ (behind the iliac spine, the ending-spot of the posterior superior iliac spine at the muscles) on both sides for 20 min, once daily for 9 days. Paw withdrawal latency (mechanical pain threshold,PWMT) and thermal tail flick latency (TFL) were measured using a VonFrey detector and photothermal tail pain meter, respectively. The content of IFN-γ of TNC tissue was detected by ELISA. The expression levels of Cx43 and IFN-γ proteins of TNC tissue were detected by Western blot. The immunofluorescence dual labeling method was used to detect the positive expression of GFAP and Cx43, IFN-γR and NeuN in TNC tissue, for displaying the activity of Cx43 in astrocytes and IFN-γ in neurons, respectively. RESULTS: Compared with the control group, both PWMT and TFL at 3, 5, 7 and 9 days after modeling were significantly decreased (P<0.01), while the expression of Cx43 and IFN-γ proteins, the immunofluorescence intensity of GFAP, Cx43, IFN-γR, and the content of IFN-γ were considerably up-regulated in the model group (P<0.01). In comparison with the model group, both PWMT and TFL at 3, 5, 7 and 9 days after modeling were obviously increased (P<0.01), whereas the expression of Cx43 and IFN-γ proteins, the immunofluorescence intensity of GFAP, Cx43, IFN-γR, and the content of IFN-γ in the acupuncture group, as well as the protein expression of IFN-γR in the sham acupuncture group were also remarkably decreased (P<0.05, P<0.01). The effect of acupuncture was significantly superior to that of sham acupuncture in down-regulating the expression of Cx43 and IFN-γ proteins, and the immunofluorescence intensity of GFAP, Cx43, and IFN-γR (P<0.05, P<0.01). Immunofluorescence dual labeling outcomes showed that in the model group, a large number of GFAP and Cx43 co-expressed astrocytes were found, and the cell body and protrusion of GFAP-labelled astrocytes were evidently increased, and Cx43 was mainly expressed on the surface of astrocyte membrane and the protrusion site, and the proportion of IFN-γR and NeuN co-expressing neurons in the model group was significantly increased, suggesting an activation of astrocytes and neurons after modeling. Whereas in the acupuncture group, the bright green clustered particles on the cell membrane and protrusion of astrocytes, and the proportion of IFN-γR and NeuN co-expressing neurons were significantly reduced, suggesting a suppression of activities of Cx43, astrocytes and neurons and IFN-γ release from TNC after acupuncture intervention. CONCLUSION: Acupuncture can relieve the pain response in rats with migraine, which may be associa-ted with its functions in inhibiting the expression of Cx43 and activation of astrocytes and neurons, and reducing release of pro-inflammatory factor IFN-γ in TNC.

Proteome and phosphoproteome analysis of 2,4-epibrassinolide-mediated cold stress response in cucumber seedlings.

The 2, 4-epibrassinolide (EBR) significantly increased plants cold tolerance. However, mechanisms of EBR in regulating cold tolerance in phosphoproteome and proteome levels have not been reported. The mechanism of EBR regulating cold response in cucumber was studied by multiple omics analysis. In this study, phosphoproteome analysis showed that cucumber responded to cold stress through multi-site serine phosphorylation, while EBR further upregulated single-site phosphorylation for most of cold-responsive phosphoproteins. Association analysis of the proteome and phosphoproteome revealed that EBR reprogrammed proteins in response to cold stress by negatively regulating protein phosphorylation and protein content, and phosphorylation negatively regulated protein content in cucumber. Further functional enrichment analysis of proteome and phosphoproteome showed that cucumber mainly upregulated phosphoproteins related to spliceosome, nucleotide binding and photosynthetic pathways in response to cold stress. However, different from the EBR regulation in omics level, hypergeometric analysis showed that EBR further upregulated 16 cold-up-responsive phosphoproteins participated photosynthetic and nucleotide binding pathways in response to cold stress, suggested their important function in cold tolerance. Analysis of cold-responsive transcription factors (TFs) by correlation between proteome and phosphoproteome showed that cucumber regulated eight class TFs may through protein phosphorylation under cold stress. Further combined with cold-related transcriptome found that cucumber phosphorylated eight class TFs, and mainly through targeting major hormone signal genes by bZIP TFs in response to cold stress, while EBR further increased these bZIP TFs (CsABI5.2 and CsABI5.5) phosphorylation level. In conclusion, the EBR mediated schematic of molecule response mechanisms in cucumber under cold stress was proposed.

Theoretical Evaluation of Potential Cytotoxicity of Graphene Quantum Dot to Adsorbed DNA.

As a zero-dimensional (0D) nanomaterial, graphene quantum dot (GQD) has a unique physical structure and electrochemical properties, which has been widely used in biomedical fields, such as bioimaging, biosensor, drug delivery, etc. Its biological safety and potential cytotoxicity to human and animal cells have become a growing concern in recent years. In particular, the potential DNA structure damage caused by GQD is of great importance but still obscure. In this study, molecular dynamics (MD) simulation was used to investigate the adsorption behavior and the structural changes of single-stranded (ssDNA) and double-stranded DNA (dsDNA) on the surfaces of GQDs with different sizes and oxidation. Our results showed that ssDNA can strongly adsorb and lay flat on the surface of GQDs and graphene oxide quantum dots (GOQDs), whereas dsDNA was preferentially oriented vertically on both surfaces. With the increase of GQDs size, more structural change of adsorbed ssDNA and dsDNA could be found, while the size effect of GOQD on the structure of ssDNA and dsDNA is not significant. These findings may help to improve the understanding of GQD biocompatibility and potential applications of GQD in the biomedical field.

Photosynthesis Mediated by RBOH-Dependent Signaling Is Essential for Cold Stress Memory.

Cold tolerance is improved by cold stress acclimation (CS-ACC), and the cold tolerance level is 'remembered' by plants. However, the underlying signaling mechanisms remain largely unknown. Here, the CS memory mechanism was studied by bioinformation, plant physiological and photosynthetic parameters, and gene expression. We found that CS-ACC induced the acquisition of CS memory and enhanced the maintenance of acquired cold tolerance (MACT) in cucumber seedlings. The H2O2 content and NADPH oxidase activity encoded by CsRBOH was maintained at higher levels during recovery after CS-ACC and inhibition of RBOH-dependent signaling after CS-ACC resulted in a decrease in the H2O2 content, NADPH oxidase activity, and MACT. CsRBOH2, 3, 4, and 5 showed high expression during recovery after CS-ACC. Many BZR-binding sites were identified in memory-responsive CsRBOHs promoters, and CsBZR1 and 3 showed high expression during recovery after CS-ACC. Inhibition of RBOH-dependent signaling or brassinosteroids affected the maintenance of the expression of these memory-responsive CsRBOHs and CsBZRs. The photosynthetic efficiency (PE) decreased but then increased with the prolonged recovery after CS-ACC, and was higher than the control at 48 h of recovery; however, inhibition of RBOH-dependent signaling resulted in a lower PE. Further etiolated seedlings experiments showed that a photosynthetic capacity was necessary for CS memory. Therefore, photosynthesis mediated by RBOH-dependent signaling is essential for CS memory.

HIT 2.0: an enhanced platform for Herbal Ingredients' Targets.

Literature-described targets of herbal ingredients have been explored to facilitate the mechanistic study of herbs, as well as the new drug discovery. Though several databases provided similar information, the majority of them are limited to literatures before 2010 and need to be updated urgently. HIT 2.0 was here constructed as the latest curated dataset focusing on Herbal Ingredients' Targets covering PubMed literatures 2000-2020. Currently, HIT 2.0 hosts 10 031 compound-target activity pairs with quality indicators between 2208 targets and 1237 ingredients from more than 1250 reputable herbs. The molecular targets cover those genes/proteins being directly/indirectly activated/inhibited, protein binders, and enzymes substrates or products. Also included are those genes regulated under the treatment of individual ingredient. Crosslinks were made to databases of TTD, DrugBank, KEGG, PDB, UniProt, Pfam, NCBI, TCM-ID and others. More importantly, HIT enables automatic Target-mining and My-target curation from daily released PubMed literatures. Thus, users can retrieve and download the latest abstracts containing potential targets for interested compounds, even for those not yet covered in HIT. Further, users can log into 'My-target' system, to curate personal target-profiling on line based on retrieved abstracts. HIT can be accessible at http://hit2.badd-cao.net.

SAS: A Platform of Spike Antigenicity for SARS-CoV-2.

Since the outbreak of SARS-CoV-2, antigenicity concerns continue to linger with emerging mutants. As recent variants have shown decreased reactivity to previously determined monoclonal antibodies (mAbs) or sera, monitoring the antigenicity change of circulating mutants is urgently needed for vaccine effectiveness. Currently, antigenic comparison is mainly carried out by immuno-binding assays. Yet, an online predicting system is highly desirable to complement the targeted experimental tests from the perspective of time and cost. Here, we provided a platform of SAS (Spike protein Antigenicity for SARS-CoV-2), enabling predicting the resistant effect of emerging variants and the dynamic coverage of SARS-CoV-2 antibodies among circulating strains. When being compared to experimental results, SAS prediction obtained the consistency of 100% on 8 mAb-binding tests with detailed epitope covering mutational sites, and 80.3% on 223 anti-serum tests. Moreover, on the latest South Africa escaping strain (B.1.351), SAS predicted a significant resistance to reference strain at multiple mutated epitopes, agreeing well with the vaccine evaluation results. SAS enables auto-updating from GISAID, and the current version collects 867K GISAID strains, 15.4K unique spike (S) variants, and 28 validated and predicted epitope regions that include 339 antigenic sites. Together with the targeted immune-binding experiments, SAS may be helpful to reduce the experimental searching space, indicate the emergence and expansion of antigenic variants, and suggest the dynamic coverage of representative mAbs/vaccines among the latest circulating strains. SAS can be accessed at https://www.biosino.org/sas.

Rank-in: enabling integrative analysis across microarray and RNA-seq for cancer.

Though transcriptomics technologies evolve rapidly in the past decades, integrative analysis of mixed data between microarray and RNA-seq remains challenging due to the inherent variability difference between them. Here, Rank-In was proposed to correct the nonbiological effects across the two technologies, enabling freely blended data for consolidated analysis. Rank-In was rigorously validated via the public cell and tissue samples tested by both technologies. On the two reference samples of the SEQC project, Rank-In not only perfectly classified the 44 profiles but also achieved the best accuracy of 0.9 on predicting TaqMan-validated DEGs. More importantly, on 327 Glioblastoma (GBM) profiles and 248, 523 heterogeneous colon cancer profiles respectively, only Rank-In can successfully discriminate every single cancer profile from normal controls, while the others cannot. Further on different sizes of mixed seq-array GBM profiles, Rank-In can robustly reproduce a median range of DEG overlapping from 0.74 to 0.83 among top genes, whereas the others never exceed 0.72. Being the first effective method enabling mixed data of cross-technology analysis, Rank-In welcomes hybrid of array and seq profiles for integrative study on large/small, paired/unpaired and balanced/imbalanced samples, opening possibility to reduce sampling space of clinical cancer patients. Rank-In can be accessed at http://www.badd-cao.net/rank-in/index.html.

The Evolution Pathway of Ammonia-Oxidizing Archaea Shaped by Major Geological Events.

Primordial nitrification processes have been studied extensively using geochemical approaches, but the biological origination of nitrification remains unclear. Ammonia-oxidizing archaea (AOA) are widely distributed nitrifiers and implement the rate-limiting step in nitrification. They are hypothesized to have been important players in the global nitrogen cycle in Earth's early history. We performed systematic phylogenomic and marker gene analyses to elucidate the diversification timeline of AOA evolution. Our results suggested that the AOA ancestor experienced terrestrial geothermal environments at ∼1,165 Ma (1,928-880 Ma), and gradually evolved into mesophilic soil at ∼652 Ma (767-554 Ma) before diversifying into marine settings at ∼509 Ma (629-412 Ma) and later into shallow and deep oceans, respectively. Corroborated by geochemical evidence and modeling, the timing of key diversification nodes can be linked to the global magmatism and glaciation associated with the assembly and breakup of the supercontinent Rodinia, and the later oxygenation of the deep ocean. Results of this integrated study shed light on the geological forces that may have shaped the evolutionary pathways of the AOA, which played an important role in the ancient global nitrogen cycle.

Molecular dynamics study on the adsorption and release of doxorubicin by chitosan-decorated graphene.

The safe and effective delivery of anticancer drug molecules (e.g., doxorubicin [DOX]) into target sites is of great significance in cancer therapy. Recently, considerable attention has been devoted to non-covalently functionalized graphene as a potential anticancer delivery material. Herein, molecular dynamics simulations were performed to investigate the interaction mechanism between DOX and chitosan-decorated graphene with atomic details at the molecular level. The results demonstrated that the controllable loading and release of DOX by chitosan-decorated graphene may be achieved by adjusting the solution pH (the protonation state of chitosan) and the concentration of both DOX and chitosan molecules. In particular, the bare surface of graphene can be controlled by the aggregation and dispersion of chitosan, which further affects the adsorption and release of DOX molecules.