msBERT-Promoter: a multi-scale ensemble predictor based on BERT pre-trained model for the two-stage prediction of DNA promoters and their strengths.

BACKGROUND: A promoter is a specific sequence in DNA that has transcriptional regulatory functions, playing a role in initiating gene expression. Identifying promoters and their strengths can provide valuable information related to human diseases. In recent years, computational methods have gained prominence as an effective means for identifying promoter, offering a more efficient alternative to labor-intensive biological approaches. RESULTS: In this study, a two-stage integrated predictor called "msBERT-Promoter" is proposed for identifying promoters and predicting their strengths. The model incorporates multi-scale sequence information through a tokenization strategy and fine-tunes the DNABERT model. Soft voting is then used to fuse the multi-scale information, effectively addressing the issue of insufficient DNA sequence information extraction in traditional models. To the best of our knowledge, this is the first time an integrated approach has been used in the DNABERT model for promoter identification and strength prediction. Our model achieves accuracy rates of 96.2% for promoter identification and 79.8% for promoter strength prediction, significantly outperforming existing methods. Furthermore, through attention mechanism analysis, we demonstrate that our model can effectively combine local and global sequence information, enhancing its interpretability. CONCLUSIONS: msBERT-Promoter provides an effective tool that successfully captures sequence-related attributes of DNA promoters and can accurately identify promoters and predict their strengths. This work paves a new path for the application of artificial intelligence in traditional biology.

Non-use of Contraception by Canadian Youth Aged 15 to 24: Findings From the 2009-2010 Canadian Community Health Survey.

OBJECTIVES: Non-use of contraception is an important contributor to unintended pregnancy. This study assessed non-use of contraception and its determinants among Canadian youth aged 15 to 24. METHODS: Data from the 2009-2010 Canadian Community Health Survey respondents aged 15 to 24 were used to identify non-users of contraception among heterosexual youth who had had intercourse within the previous 12 months, were not pregnant or sterilized, and felt it was important to avoid pregnancy. Sociodemographic, behavioural, and geographic factors were compared for non-users and users of contraception. RESULTS: Among youth at risk for unintended pregnancy, 15.5% were non-users of contraception. There were no differences between sexes. Across regions of Canada, Quebéc had the highest proportion of at-risk youth, but at-risk Quebéc youth were the least likely to be non-users (7.4%; CI 5.7%-9.0%) compared with at-risk youth in the Territories (28.3%; CI 21.6%-35.0%). In the multivariable analysis, aside from residence outside of Quebéc, younger age, lower income, Aboriginal identification (adjusted OR [aOR] 1.67; CI 1.18-2.37), and smoking (aOR 1.55; CI 1.24-1.92) were associated with non-use. Canadian-born youth (aOR 0.61; CI 0.39-0.96) and those enrolled in school (aOR 0.63; CI 0.50-0.81) were less likely to be non-users. CONCLUSION: The 15.5% of Canadian youth at risk for unintended pregnancy who were non-users of contraception represent an estimated 300 000 Canadian youth. Policies and programs to promote and support access to sexual health services and effective contraception with specific attention to supporting the needs of younger teens, Aboriginal youth, newcomers, low-income youth, and youth who are not in school are needed.

Designing dual inhibitors of Mdm2/MdmX: Unexpected coupling of water with gatekeeper Y100/99.

Mdm2 and MdmX share high structural similarity in their N-terminal domains, yet dual inhibitors are challenging to design due to differences in the conformations of the binding pockets, and notably of the proposed gatekeeper residue, Y100/99. Analysis of crystal structures and molecular dynamics (MD) simulations of complexes of Mdm2 and MdmX resulted in the identification of a water molecule with a long residence time that appears to be modulated by the conformation of Y100/99. These observations lead us to speculate that dual inhibitors either (i) stabilize both Mdm2 and MdmX with Y100/99 in the open conformation typically seen in complexes of Mdm2 with p53, or (ii) the dual inhibitors are agnostic to the conformation of Y100/99. The recently developed potent dual inhibitory stapled peptide Atsp7041 appears to be agnostic to the conformation of the gatekeeper residue. Proteins 2017; 85:1493-1506. © 2017 Wiley Periodicals, Inc.

Effects of active vitamin D analogues on muscle strength and falls in elderly people: an updated meta-analysis.

Background: Elderly people are at high risk of falls due to decreased muscle strength. So far, there is currently no officially approved medication for treating muscle strength loss. The active vitamin D analogues are promising but inconsistent results have been reported in previous studies. The present study was to meta-analyze the effect of active vitamin D analogues on muscle strength and falls in elderly people. Methods: The protocol was registered with PROSPERO (record number: CRD42021266978). We searched two databases including PubMed and Cochrane Library up until August 2023. Risk ratio (RR) and standardized mean difference (SMD) with 95% confidence intervals (95% CI) were used to assess the effects of active vitamin D analogues on muscle strength or falls. Results: Regarding the effects of calcitriol (n= 1), alfacalcidol (n= 1) and eldecalcitol (n= 1) on falls, all included randomized controlled trials (RCT) recruited 771 participants. Regarding the effects of the effects of calcitriol (n= 4), alfacalcidol (n= 3) and eldecalcitol (n= 3) on muscle strength, all included RCTs recruited 2431 participants. The results showed that in the pooled analysis of three active vitamin D analogues, active vitamin D analogues reduced the risk of fall by 19%. Due to a lack of sufficient data, no separate subgroup analysis was conducted on the effect of each active vitamin D analogue on falls. In the pooled and separate analysis of active vitamin D analogues, no significant effects were found on global muscle, hand grip, and back extensor strength. However, a significant enhancement of quadriceps strength was observed in the pooled analysis and separate analysis of alfacalcidol and eldecalcitol. The separate subgroup analysis on the impact of calcitriol on the quadriceps strength was not performed due to the lack to sufficient data. The results of pooled and separate subgroup analysis of active vitamin D analogues with or without calcium supplementation showed that calcium supplementation did not affect the effect of vitamin D on muscle strength. Conclusions: The use of active vitamin D analogues does not improve global muscle, hand grip, and back extensor strength but improves quadriceps strength and reduces risk of falls in elderly population.

VDR is a potential prognostic biomarker and positively correlated with immune infiltration: a comprehensive pan-cancer analysis with experimental verification.

The vitamin D receptor (VDR) is a transcription factor that mediates a variety of biological functions of 1,25-dihydroxyvitamin D3. Although there is growing evidence of cytological and animal studies supporting the suppressive role of VDR in cancers, the conclusion is still controversial in human cancers and no systematic pan-cancer analysis of VDR is available. We explored the relationships between VDR expression and prognosis, immune infiltration, tumor microenvironment, or gene set enrichment analysis (GSEA) in 33 types of human cancers based on multiple public databases and R software. Meanwhile, the expression and role of VDR were experimentally validated in papillary thyroid cancer (PTC). VDR expression decreased in 8 types and increased in 12 types of cancer compared with normal tissues. Increased expression of VDR was associated with either good or poor prognosis in 13 cancer types. VDR expression was positively correlated with the infiltration of cancer-associated fibroblasts, macrophages, or neutrophils in 20, 12, and 10 cancer types respectively and this correlation was experimentally validated in PTC. Increased VDR expression was associated with increased percentage of stromal or immune components in tumor microenvironment (TME) in 24 cancer types. VDR positively and negatively correlated genes were enriched in immune cell function and energy metabolism pathways, respectively, in the top 9 highly lethal tumors. Additionally, VDR expression was increased in PTC and inhibited cell proliferation and migration. In conclusion, VDR is a potential prognostic biomarker and positively correlated with immune infiltration as well as stromal or immune components in TME in multiple human cancers.

Exploring Symptom Clusters in Chinese Patients with Diabetic Kidney Disease: A Network Analysis.

Purpose: The research on symptom management in patients with diabetic kidney disease (DKD) has shifted from separate symptoms to symptom clusters and networks recently. This study aimed to evaluate the unpleasant symptoms of DKD patients, and to investigate how these symptom clusters could affect patients. Methods: 408 DKD patients were recruited in this cross-sectional study. The symptoms of DKD patients were measured using the modified Dialysis Symptom Index. Network analysis was employed to evaluate the symptom network and the characteristics of individual nodes, while factor analysis was utilized to identify symptom clusters. Results: Blurred vision was the most prevalent symptom among DKD patients. The symptoms identified as the most distressing, severe, and frequent were light headache or dizziness, arteriovenous fistula/catheterization pain, and diarrhea, respectively. Five symptom clusters were obtained from factor analysis, and the most central symptom cluster in the entire symptom network was sexual dysfunction. Conclusion: This study identified five symptom clusters in Chinese DKD patients, with sexual dysfunction emerging as the most central cluster. These findings carry significant clinical implications, underscoring the necessity of assessing symptom clusters and their associations to enhance symptom management in DKD patients. Further research is essential to elucidate the underlying mechanisms of symptoms and to clarify the associations among symptoms in DKD patients across different disease trajectories or treatment modalities.

Nano-energy interference: A novel strategy for blunting tumor adaptation and metastasis.

Blunting the tumor's stress-sensing ability is an effective strategy for controlling tumor adaptive survival and metastasis. Here, we have designed a cyclically amplified nano-energy interference device based on lipid nanoparticles (LNP), focused on altering cellular energy metabolism. This innovative nano device efficiently targets and monitors the tumor's status while simultaneously inhibiting mitochondrial respiration, biogenesis and ribosome production. To this end, we first identified azelaic acid (AA), a binary acid capable of disrupting the mitochondrial respiratory chain. Upon encapsulation in LNP and linkage to mitochondrial-targeting molecules, this disruptive effect is further augmented. Consequently, tumors exhibit a substantial upregulation of the glycolytic pathway, intensifying their glucose demand and worsening the tumor's energy-deprived microenvironment. Then, the glucose analog, 2-Deoxy-D-glucose (2-DG), linked to the LNP, efficiently targets tumors and competitively inhibits the tumor's normal glucose uptake. The synergetic results of combining AA with 2-DG induce comprehensive energy deficiency within tumors, blocking the generation of energy-sensitive ribosomes. Ultimately, the disruption of both mitochondria and ribosomes depletes energy supply and new protein-generating capacity, weakening tumor's ability to adapt to environmental stress and thereby inhibiting growth and metastasis. Comprehensively, this nano-energy interference device, by controlling the tumor's stress-sensing ability, provides a novel therapeutic strategy for refractory tumors.

MicroRNA-214 promotes myogenic differentiation by facilitating exit from mitosis via down-regulation of proto-oncogene N-ras.

Vertebrate muscle differentiation is coordinated by an intricate network of transcription factors requiring proliferating myogenic precursors to withdraw irreversibly from the cell cycle. Recent studies have implicated a large number of microRNAs exerting another layer of control in many aspects of muscle differentiation. By annealing to short recognition sequences in the 3'-untranslated region, microRNAs attenuate target gene expression through translation repression or mRNA degradation. Here, we show that miR-214 promotes myogenic differentiation in mouse C2C12 myoblasts at a step preceding the induction of p21 and myogenin. Blocking miR-214 function with a 2'-O-methylated double-stranded inhibitor maintained C2C12 cells in the active cell cycle, thereby inhibiting the myogenic differentiation. By global gene expression profiling, we identified the proto-oncogene N-ras as one of miR-214 targets. Furthermore, manipulating the N-Ras level with small interfering RNA or adenovirus-mediated forced expression either augmented or attenuated the effect of miR-214, respectively. Thus, our data uncovered a novel microRNA-mediated mechanism that controls myogenic differentiation.

Vitamin D supplementation reduces the risk of fall in the vitamin D deficient elderly: An updated meta-analysis.

INTRODUCTION: Vitamin D supplementation has been widely recommended to prevent falls. However, considerable controversy exists regarding the association of such supplementation and fall risk. Previous meta-analyses yielded inconsistent results because of differences in the baseline of 25-hydroxyvitamin D [25(OH)D] and dose of vitamin D and use of vitamin D or in combination with calcium in different studies. Furthermore, some studies published recently were not included in the previous meta-analyses. Therefore, an updated and comprehensive meta-analysis is warranted. METHODS: We systematically searched several literature databases including PubMed and the Embase from inception to September 2020. The protocol for this meta-analysis was registered with PROSPERO (CRD42021226380). Randomized clinical trials (RCTs) reporting the effect of vitamin D supplementation alone or with calcium on fall incidence were selected from studies. Qualitative and quantitative information was extracted; the random-effects model was conducted to pool the data for fall; statistical heterogeneity was assessed using the I2 test and potential for publication bias was assessed qualitatively by a visual estimate of the funnel plot and quantitatively by calculation of the Begg's test and the Egger's test. RESULTS: Of the citations retrieved, 31 eligible studies involving 57 867 participants met inclusion criteria, reporting 17 623 falls. A total of 21 RCTs of vitamin D alone and 10 RCTs of vitamin D plus calcium were included in the meta-analysis. The meta-analysis of 21 RCTs (51 984 participants) of vitamin D supplementation alone (daily or intermittent doses of 400-60 000 IU) did not show a reduced risk of falls (The risk ratio [RR] 1.00, 95% confidence intervals [CI] 0.95 to 1.05) compared to placebo or no treatment. Subgroup analyses showed that the baseline of serum 25(OH)D concentration less than 50 nmol/L resulted in a reduction of fall risk (RR 0.77, 95% CI 0.61 to 0.98). In contrast, the meta-analysis of 10 RCTs (5883 participants) of combined supplementation of vitamin D (daily doses of 700-1000 IU) and calcium (daily doses of 1000-1200 mg) showed a 12% reduction in the risk of fall (RR 0.88, 95% CI 0.80 to 0.97). CONCLUSIONS: The combination of vitamin D and calcium have beneficial effects on prevention falls in old adults. Although vitamin D supplementation alone has no effect on fall risk in old adults with 25(OH)D levels higher than 50 nmol/L, vitamin D supplementation alone does have a benefit on prevention of falls in old adults with 25(OH)D levels lower than 50 nmol/L.

A Novel Combined Conjugate Therapeutic Cancer Vaccine, Recombinant EGF-CRM197, in Patients With Advanced Solid Tumors: A Phase I Clinical Study.

INTRODUCTION: The therapeutic cancer vaccine recombinant Epidermal Growth Factor (EGF)-CRM197 is a novel combined conjugate EGF with CRM197 as a carrier protein. Immunization with the EGF-CRM197 vaccine can induce high levels of neutralizing anti-EGF antibodies that inhibit EGF/EGFR signaling and thereby suppress growth of tumors that rely on this signaling pathway. Herein, we characterize the humoral immune responses elicited by the recombinant EGF-CRM197 vaccine in patients with advanced solid tumors in a phase I clinical trial and assess the safety, tolerability, and immunogenicity of this vaccine (CTR20190473). METHODS: A total of 16 subjects were enrolled in this study. Under 6 + 3 design, patients in each dosing cohort were administrated subcutaneously at a dosage of 0.4 mg, 0.8 mg, and 1.6 mg, respectively. The patients received vaccinations for immune induction (once a week for 4 consecutive weeks) and booster vaccinations (once every 4 weeks). Safety evaluation was performed 1 week after the immune induction. Booster vaccination was given until the occurrence of disease progression, intolerance, withdrawal of informed consent by the patient, or negative result of anti-EGF test after two booster vaccinations. RESULTS: Vaccination with EGF-CRM197 is safe and well-tolerated in patients with advanced solid tumors. Adverse reactions at the injection site were the most common adverse events (AEs) in recipients. No severe adverse reactions post vaccination were observed in the present study. Vaccinated patients developed a robust neutralizing antibody response triggered by EGF-CRM197 that significantly reduced the levels of EGF in serum. For lung cancer patients who were super good antibody responders (sGAR) to EGF-CRM197, the median progress-free survival (PFS) was 4.83 months, significantly longer than that of the good antibody responder (GAR) patients with lung cancer whose median PFS was 2.10 months (P=0.0018). The median overall survival (OS) of GAR lung cancer patients was 10.67 months while the OS) for sGAR lung cancer patients was not reached until analysis was performed. The median follow-up of the sGAR lung cancer patients was 14.6 months. CONCLUSION: Our study demonstrates that the recombinant EGF-CRM197 therapeutic cancer vaccine can induce a good immune response in patients with advanced solid tumors and is safe and well tolerated, which ensures further clinical development of the vaccine for extending the survival time of EGF-CRM197 sensitive patients with advanced solid tumors. CLINICAL TRIAL REGISTRATION: http://www.chinadrugtrials.org.cn, identifier CTR20190473, EGF-CRM197.

Fuzhenghuayu Decoction ameliorates hepatic fibrosis by attenuating experimental sinusoidal capillarization and liver angiogenesis.

Fuzhenghuayu (FZHY) is a compound extracted from natural plants. Its anti-fibrotic effect has been confirmed in experimental and clinical studies. However, precise effects and underlying mechanisms of FZHY in liver angiogenesis largely remain understood. In this study, we investigated the effects of FZHY on sinusoidal capillarization and angiogenesis with mice challenged for Carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN), in vitro human hepatic sinusoidal endothelial cells (HHSEC) and Human Umbilical Vein Endothelial Cell (HUVEC) 3D fibrin gel model. Besides its anti-fibrotic effect, FZHY ameliorated CCl4 and DMN-induced sinusoidal capillarization, angiogenesis and expression of angiogenesis-associated factors, i.e. CD31, VEGF, VEGF receptor II, phosphor-ERK and HIF-1α. Consistent with the findings based on animal models, inhibitory effects of FZHY on capillarization and angiogenesis were further confirmed in HHSEC and the HUVEC 3D fibrin gel model, respectively. These data suggest that FZHY ameliorates not only liver fibrosis but also vessel remodeling in experimental models. Therefore, FZHY might be a potentially useful drug to treat liver cirrhosis in clinical practice.

Mesenchymal Stem Cells: Allogeneic MSC May Be Immunosuppressive but Autologous MSC Are Dysfunctional in Lupus Patients.

Mesenchymal stem cells (MSCs) have a potently immunosuppressive capacity in both innate and adaptive immune responses. Consequently, MSCs transplantation has emerged as a potential beneficial therapy for autoimmune diseases even though the mechanisms underlying the immunomodulatory activity of MSCs is incompletely understood. Transplanted MSCs from healthy individuals with no known history of autoimmune disease are immunosuppressive in systemic lupus erythematosus (SLE) patients and can ameliorate SLE disease symptoms in those same patients. In contrast, autologous MSCs from SLE patients are not immunosuppressive and do not ameliorate disease symptoms. Recent studies have shown that MSCs from SLE patients are dysfunctional in both proliferation and immunoregulation and phenotypically senescent. The senescent phenotype has been attributed to multiple genes and signaling pathways. In this review, we focus on the possible mechanisms for the defective phenotype and function of MSCs from SLE patients and summarize recent research on MSCs in autoimmune diseases.

[The transcriptional regulation study on human delta globin gene with CAAT box C-->T point mutation in its promoter].

OBJECTIVE: To study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter. METHODS: Human delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR. RESULTS: The expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box. CONCLUSION: The defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.

Effect of Yinxieling decoction on PASI, TNF-α and IL-8 in patients with psoriasis vulgaris.

OBJECTIVE: To study the effect of Yinxieling decoction on PASI, TNF-α and IL-8 in patients with psoriasis vulgaris. METHODS: A total of 120 cases of psoriasis vulgaris were divided into 4 groups according to syndrome differentiation of TCM and randomized controlled method: wind heat syndrome group (group A), blood stasis syndrome group (group B), blood dryness syndrome group (group C) and control group (group D) (n=30 per group). Patients in observation groups were treated with Yinxieling decoction, while patients in control group were treated by placebo for 8 weeks. Levels of TNF-α and IL-8 were determined before treatment, 4 and 8 weeks after treatment. psoriasis area and severity index score was also performed before and after treatment. RESULTS: psoriasis area and severity index score and serum level of TNF-α, IL-8 were significantly decreased in all groups. The decrease in three observation groups was more significant (P<0.05 or P<0.01), and the decrease in wind heat syndrome group was the most significant (P<0.01). psoriasis area and severity index was positively correlated with TNF-α and IL-8, respectively (P<0.05). CONCLUSIONS: Yinxieling decoction has therapeutical effect on psoriasis vulgaris via regulating TNF-α and IL-8.

Effect of pharmacological intervention on MIP-1α, MIP-1ß and MCP-1 expression in patients with psoriasis vulgaris.

OBJECTIVE: To detect the expression level of macrophage inflammatory protein-1 (MIP-1)α, MIP-1ß and monocyte chemoattractant protein-1 (MCP-1) in with psoriasis vulgaris and explore the role in the pathogenesis of psoriasis vulgaris. METHODS: The level of MIP-1α, MIP-1ß and MCP-1 in peripheral blood from 50 patients with psoriasis vulgaris and 50 normal controls were measured by enzyme linked immunosorbent assay. The correlation with psoriasis area and severity index (PASI) was analyzed. The level of MIP-1α, MIP-1ß and MCP-1 was compared between psoriasis vulgaris patients at active stage and resting stage. And the change in MIP-1α, MIP-1ß and MCP-1 before and after therapy was also observed. RESULTS: The content of MIP-1α, MIP-1ß and MCP-1 in patients with psoriasis vulgaris was (1342.78 ± 210.30), (175.28 ± 28.18) and (266.86 ± 32.75) ng/L, respectively, significantly higher than those in control group (P<0.05). The expression level of MIP-1α, MIP-1ß and MCP-1 in peripheral blood of patients with psoriasis vulgaris was positively correlated with PASI (P<0.01). After acitretin therapy, expression level of MIP-1α, MIP-1ß and MCP-1 in peripheral blood of patients with psoriasis vulgaris was significantly decreased. CONCLUSIONS: Chemokine factor MIP-1α, MIP-1ß and MCP-1 may be involved in the pathogenesis of psoriasis vulgaris.

[Expression of Oct-4 transcription factor in squamous cell skin carcinoma and seborrheic keratosis].

OBJECTIVE: To detect the expression of stem cell transcription factor Oct-4 in squamous cell carcinoma (SCC) and seborrheic keratosis (SK) and its association with cancer stem cells. METHODS: Immunohistochemistry was used to detect Oct-4 expression in 35 SCC cases, 21 SK cases and 15 normal control skin tissues. RESULTS: Oct-4 expression was negative in normal skin and showed a significant difference between SCC and SK tissues (P<0.05). CONCLUSION: The Oct-4-positive cells in SCC and SK are probably tumor stem cells. Oct-4 expression may provide an important evidence for isolation and identification of human SCC and SK stem cells.

[Ouabain-induced apoptosis of Jurkat cells correlates with activation of caspase-3 and regulation of Bcl-2 gene family].

The study was aimed to explore the apoptosis effect of ouabain on Jurkat cells and its mechanism. MTT method was used to observe the inhibitory effect of ouabain on Jurkat cell proliferation. Apoptosis was detected by using flow cytometry (FCM) and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method (TUNEL). The protein expressions of Bax, Bcl-2 and active subunits of caspase-3 were measured by Western blot. Activities of caspase-3 were determined by colorimetry method. The results showed that ouabain could induce apoptosis of Jurkat cells, the expression of Bax protein in process of cell apoptosis, caspase-3 activity of Jurkat cells were remarkably enhanced after ouabain treatment. It is concluded that ouabain may induce apoptosis of Jurkat cells due to the activation of caspase-3 resulting from regulation of Bax protein and Bcl-2 gene expressions.

[MAPK signal pathway plays a role in proliferation of Jurkat cells induced by ouabain].

The object was to study the effect of ouabain on Jurkat cells and its possible mechanism. The effect of ouabain of low concentration on Jurkat cells was confirmed by MTT, while c-myc gene transcription was measured by RT-PCR, and the phosphorylation of MAPK (ERK1/2) as well as the expression of c-myc gene was tested by Western blot respectively. The results showed that ouabain at low concentration could induce the proliferation of Jurkat in a time-and dose-dependent manner. At the same time, the phosphorylation of MAPK (ERK1/2) and the expression of c-myc gene was enhanced. In conclusion, ouabain stimulates the intracellular MAPK signal pathway by acting on the Na, K-ATPase, and thus induce the proliferation of Jurkat cells, in which the regulation of c-myc gene expression may be involved.