Barriers and facilitators to mobile health and active surveillance use among older adults with skin disease.

BACKGROUND: The COVID-19 pandemic has accelerated the adoption of telemedicine, including teledermatology. Monitoring skin lesions using teledermatology may become increasingly important for several skin diseases, including low-risk skin cancers. The purpose of this study was to describe the key factors that could serve as barriers or facilitators to skin disease monitoring using mobile health technology (mHealth) in older adults. METHODS: Older adult dermatology patients 65 years or older and their caregivers who have seen a dermatologist in the last 18 months were interviewed and surveyed between December 2019 and July 2020. The purpose of these interviews was to better understand attitudes, beliefs and behaviours that could serve as barriers and facilitators to the use of mHealth and active surveillance to monitor low-risk skin cancers. RESULTS: A total of 33 interviews leading to 6022 unique excerpts yielded 8 factors, or themes, that could serve as barriers, facilitators or both to mHealth and active surveillance. We propose an integrated conceptual framework that highlights the interaction of these themes at both the patient and provider level, including care environment, support systems and personal values. DISCUSSION AND CONCLUSIONS: These preliminary findings reveal factors influencing patient acceptance of active surveillance in dermatology, such as changes to the patient-provider interaction and alignment with personal values. These factors were also found to influence adoption of mHealth interventions. Given such overlap, it is essential to address barriers and facilitators from both domains when designing a new dermatology active surveillance approach with novel mHealth technology. PATIENT OR PUBLIC CONTRIBUTION: The patients included in this study were participants during the data collection process. Members of the Stanford Healthcare and Denver Tech Dermatology health-care teams aided in the recruitment phase of the data collection process.

Development of a Drosophila melanogaster based model for the assessment of cadmium and mercury mediated renal tubular toxicity.

Xenobiotic mediated renal toxicity is one of the major health concerns to the organisms, including humans. New chemicals with nephrotoxic potential are continuously being added to the list of existing nephrotoxicants. To predict the nephrotoxicity of these new chemicals, reliable and cost-effective alternative animal models are required. It is a prerequisite for the identification and assessment of these compounds as potential nephrotoxicants to prevent renal toxicity in the exposed population. Drosophila melanogaster, a genetically tractable invertebrate animal model, has a renal system functionally analogous to humans. The Malpighian tubules (MTs) of D. melanogaster are similar to the tubular part of nephron of the human kidney. Besides, it recapitulates the renal toxicity hallmark with mammals when exposed to known nephrotoxicants. In this study, first instar larvae of D. melanogaster (Oregon R) were exposed to different concentrations of two well-known nephrotoxicants, cadmium (Cd) and mercury (Hg). Akin to higher organisms, Cd and Hg exposure to D. melanogaster produce similar phenotypes. MTs of exposed D. melanogaster larvae exhibited increased oxidative stress, activated cellular antioxidant defense mechanism, GSH depletion, increased cleaved caspase-3 expression, increased DEVDase activity and increased cell death. The functional status of MTs was assessed by fluid secretion rate (FSR), efflux activity of transporter protein, mitochondrial membrane potential (MMP), ATP level and expression of junctional protein (Dlg). All the phenotypes observed in MTs of D. melanogaster larvae recapitulate the phenotypes observed in higher organisms. Increased uric acid level, the hallmark of renal dysfunction, was also observed in exposed larvae. Taken together, the study suggests that MTs of D. melanogaster may be used as a functional model to evaluate xenobiotic mediated nephrotoxicity.

Development of a patient decision aid for the management of superficial basal cell carcinoma (BCC) in adults with a limited life expectancy.

BACKGROUND: Basal cell carcinoma (BCC) is a slow-growing, rarely lethal skin cancer that affects people 65 years or older. A range of treatment options exist for BCC, but there is little evidence available to guide patients and providers in selecting the best treatment options. OBJECTIVES: This study outlines the development of a patient decision aid (PDA) for low-risk BCC that can be used by patients and providers to assist in shared decision-making. METHODS: In accordance with the International Patient Decision Aids Standards (IPDAS) Collaboration framework, feedback from focus groups and semi-structured interviews with patients and providers, an initial prototype of the PDA was developed. This was tested using cognitive interviews and iteratively updated. RESULTS: We created eighteen different iterations using feedback from 24 patients and 34 providers. The key issues identified included: 1) Addressing fear of cancer; 2) Communicating risk and uncertainty; 3) Values clarification; and 4) Time lag to benefit. LIMITATIONS: The PDA does not include all possible treatment options and is currently paper based. CONCLUSIONS: Our PDA has been specifically adapted and designed to support patients with a limited life expectancy in making decisions about their low risk BCC together with their doctors.

Extracellular vesicles from bone marrow-derived mesenchymal stem cells protect against murine hepatic ischemia/reperfusion injury.

Hepatic ischemia/reperfusion injury (IRI) and associated inflammation contributes to liver dysfunction and complications after liver surgery and transplantation. Mesenchymal stem cells (MSCs) have been reported to reduce hepatic IRI because of their reparative immunomodulatory effects in injured tissues. Recent studies have highlighted beneficial effects of extracellular vesicles from mesenchymal stem cells (MSC-EV) on tissue injury. The effects of systemically administered mouse bone marrow-derived MSC-EV were evaluated in an experimental murine model of hepatic IRI induced by cross-clamping the hepatic artery and portal vein for 90 minutes followed by reperfusion for periods of up to 6 hours. Compared with controls, intravenous administration of MSC-EV 30 minutes prior to IRI dramatically reduced the extent of tissue necrosis, decreased caspase 3-positive and apoptotic cells, and reduced serum aminotransferase levels. MSC-EV increased hepatic messenger RNA (mRNA) expression of NACHT, LRR, and PYD domains-containing protein 12, and the chemokine (C-X-C motif) ligand 1, and reduced mRNA expression of several inflammatory cytokines such as interleukin 6 during IRI. MSC-EV increased cell viability and suppressed both oxidative injury and nuclear factor kappa B activity in murine hepatocytes in vitro. In conclusion, the administration of extracellular vesicles derived from bone marrow-derived MSCs may ameliorate hepatic IRI by reducing hepatic injury through modulation of the inflammatory response.Liver Transplantation 23 791-803 2017 AASLD.

Enhanced production and characterization of a solvent stable amylase from solvent tolerant Bacillus tequilensis RG-01: thermostable and surfactant resistant.

Ten bacterial strains isolated from the soil samples in the presence of cyclohexane were screened for amylase production. Among them, culture RG-01 was adjudged as the best amylase producer and was identified as Bacillus tequilensis from MTCC, Chandigarh. The isolate showed maximum amylase production (8100 U/mL) in the presence of starch, peptone, and Ca(2+) ions at 55°C pH 7.0 within 24 h of incubation. The enzyme was stable in the presence of n-dodecane, isooctane, n-decane, xylene, toluene, n-hexane, n-butanol, and cyclohexane, respectively. The presence of benzene, methanol, and ethanol marginally reduced the amylase stability, respectively. The enzyme was showed it 100% activity at 55°C and pH 7.0 with 119% and 127% stability at 55°C and pH 7.0, respectively. The enzyme was also stable in the presence of SDS, Tween-40, Tween-60, and Tween-80 (1%) and was found stimulatory effect, respectively. Only Triton-X-100 showed a moderate inhibitory effect (5%) on amylase activity. This isolate (Bacillus tequilensis RG-01) may be useful in several industrial applications owing to its thermotolerant and organic solvents and surfactants resistance characteristics.

Cancer stem cell in prostate cancer progression, metastasis and therapy resistance.

Prostate cancer (PCa) is the most diagnosed malignancy in the men worldwide. Cancer stem cells (CSCs) are the sub-population of cells present in the tumor which possess unique properties of self-renewal and multilineage differentiation thus thought to be major cause of therapy resistance, disease relapse, and mortality in several malignancies including PCa. CSCs have also been shown positive for the common stem cells markers such as ALDH EZH2, OCT4, SOX2, c-MYC, Nanog etc. Therefore, isolation and characterization of CSCs specific markers which may discriminate CSCs and normal stem cells are critical to selectively eliminate CSCs. Rapid advances in the field offers a theoretical explanation for many of the enduring uncertainties encompassing the etiology and an optimism for the identification of new stem-cell targets, development of reliable and efficient therapies in the future. The emerging reports have also provided unprecedented insights into CSCs plasticity, quiescence, renewal, and therapeutic response. In this review, we discuss the identification of PCa stem cells, their unique properties, stemness-driving pathways, new diagnostics, and therapeutic interventions.

A complex role of Arabidopsis CDKD;3 in meiotic progression and cytokinesis.

Meiosis is a specialized cell division that halves the number of chromosomes in two consecutive rounds of chromosome segregation. In angiosperm plants is meiosis followed by mitotic divisions to form rudimentary haploid gametophytes. In Arabidopsis, termination of meiosis and transition to gametophytic development are governed by TDM1 and SMG7 that mediate inhibition of translation. Mutants deficient in this mechanism do not form tetrads but instead undergo multiple cycles of aberrant nuclear divisions that are likely caused by the failure to downregulate cyclin dependent kinases during meiotic exit. A suppressor screen to identify genes that contribute to meiotic exit uncovered a mutation in cyclin-dependent kinase D;3 (CDKD;3) that alleviates meiotic defects in smg7 deficient plants. The CDKD;3 deficiency prevents aberrant meiotic divisions observed in smg7 mutants or delays their onset after initiation of cytokinesis, which permits formation of functional microspores. Although CDKD;3 acts as an activator of cyclin-dependent kinase A;1 (CDKA;1), the main cyclin dependent kinase that regulates meiosis, cdkd;3 mutation appears to promote meiotic exit independently of CDKA;1. Furthermore, analysis of CDKD;3 interactome revealed enrichment for proteins implicated in cytokinesis, suggesting a more complex function of CDKD;3 in cell cycle regulation.

Hsp27 over expression protect against cadmium induced nephrotoxicity in Drosophila melanogaster.

Cadmium (Cd) exposure to the animals including humans is reported as nephrotoxic compounds i.e., disturbing redox status (increase oxidative stress), mitochondrial dysfunction, renal cell death and altered transporters in the renal system. Hsp27 (a small heat shock protein) has been shown as one of the modulators in the renal dysfunction and increased against the Cd induced toxicity. However, no studies are reported on the genetic modulation of stress protein against the Cd-induced nephrotoxicity. The current study aimed to examine the protective role of hsp27 overexpression against the Cd-induced nephrotoxicity using Drosophila melanogaster as an animal model. D. melanogaster renal system includes nephrocytes and Malpighian tubules (MTs) that show the functional similarity with mammalian kidney nephron. Overexpression of the hsp27 was found to reduce the Cd induced oxidative stress, rescue cell death in MTs of Cd exposed D. melanogaster larvae. The rescued GSH level, NADPH level and glucose 6 phosphate dehydrogenase (G6PD) activity were also observed in the MTs of the Cd exposed organism. Function (efflux activity and fluid secretion rate) of the MTs was restored in Cd exposed hsp27 overexpressed larvae. Further, results were confirmed by restored brush border microvilli density and reduced uric acid level. Tissue specific knockdown of hsp27 developed Cd like phenotypes in MTs and the phenotypes enhanced in Cd exposed condition. The present study clearly shows the role of hsp27 overexpression in restoration of the MTs function and protection against the Cd induced renal toxicity.

Genotypic Analysis of COL4A1 Gene in Diabetic Nephropathy and Type 2 Diabetes Mellitus Patients: A Comparative Genetic Study.

Diabetic nephropathy (DN) is specified by microalbuminuria, glomerular lesions, and renal fibrosis leading to end-stage renal disease. The pathophysiology of DN is multifactorial as a result of gene-environment interaction. Clinical studies suggested that gene mutations affect various pathways involved in DN, including extracellular matrix (ECM). During chronic hyperglycemia, collagen type-4-mediated ECM overproduction occurs, leading to renal fibrosis and DN development. In this study, COL4A1 gene variant rs605143 (G/A) was analyzed in diabetes and DN patients from the study population. We genotyped 386 study subjects, comprising 120 type 2 diabetes mellitus (T2DM) patients, 120 DN, and 146 healthy controls. All study subjects were analyzed for biochemical assays by commercially available kits and genotypic analysis by polymerase chain reaction-restriction fragment length polymorphism and confirmed by Sanger sequencing. Statistical analyses were done using SPSS and GraphPad. Anthroclinicopathological parameters showed a significant association between T2DM and DN. Genotype AA of COL4A1 gene variant rs605143 (G/A) showed a significant association with T2DM and DN compared with controls with 5.87- and 8.01-folds risk, respectively. Mutant allele A also significantly associated with T2DM and DN independently compared with healthy controls with 2.29- and 2.81-time risk in the study population. This study's findings suggested that COL4A1 gene variant rs605143 (G/A) can be used as predictive biomarkers for T2DM and DN independently. However, this gene variant needs to be analyzed in a large sample to explore the shared genetic association between T2DM and DN.

Patient Perspectives of Health System Barriers to Accessing Care for Hidradenitis Suppurativa: A Qualitative Study.

Importance: Patient-perceived barriers to hidradenitis suppurativa (HS) care are poorly understood. Understanding health care barriers is a critical first step toward improving care for this population. Objective: To characterize the health care experiences of people living with HS, including perceived barriers and facilitators to health care access, and to elucidate potential associations among these barriers and facilitators, health care access, and disease activity. Design, Setting, and Participants: In this qualitative study, an inductive thematic analysis was conducted on 45 in-depth, 60- to 90-minute semistructured interviews of 45 people with HS from diverse sociodemographic backgrounds that took place between March and April 2020. Individuals were eligible if they could speak English, were 18 years or older, and were diagnosed with HS. A diagnosis of HS was confirmed through physician diagnosis or through self-reported, affirmative response to the validated screening question, "Do you experience boils in your armpits or groin that recur at least every six months?" Main Outcomes and Measures: Interviews were audio recorded and transcribed verbatim. A modified grounded theory approach was used to develop the codebook, which investigators used for inductive thematic analysis. Results: Among the 45 participants included, the median (IQR) age was 37 (16) years, 33 (73%) were female, and 22 (49%) were White. There were 6 interrelated themes associated with participant-perceived barriers to accessing HS care: (1) bidirectional associations of disease activity and employment, (2) association of employment with health care coverage, (3) association of health care coverage with costs and perceived access to care, (4) association of costs with access to patient-centered care, (5) health care professional attitudes and knowledge influence patient-centered care and perceived access to care and disease activity, and (6) health system characteristics influence patient-centered care and associated costs, perceived access to care, and disease activity. Conclusions and Relevance: This qualitative study highlights themes that generate a conceptual model for understanding barriers that may act synergistically to limit health care access and influence disease activity. The disease activity of HS may be reduced when cycle elements are optimized. This study also highlights areas for future investigations and potential systems-level changes to improve access to patient-centered HS care.

Meiotic exit in Arabidopsis is driven by P-body-mediated inhibition of translation.

Meiosis, at the transition between diploid and haploid life cycle phases, is accompanied by reprograming of cell division machinery and followed by a transition back to mitosis. We show that, in Arabidopsis, this transition is driven by inhibition of translation, achieved by a mechanism that involves processing bodies (P-bodies). During the second meiotic division, the meiosis-specific protein THREE-DIVISION MUTANT 1 (TDM1) is incorporated into P-bodies through interaction with SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA 7 (SMG7). TDM1 attracts eIF4F, the main translation initiation complex, temporarily sequestering it in P-bodies and inhibiting translation. The failure of tdm1 mutants to terminate meiosis can be overcome by chemical inhibition of translation. We propose that TDM1-containing P-bodies down-regulate expression of meiotic transcripts to facilitate transition of cell fates to postmeiotic gametophyte differentiation.

Microwave photo-oxidation with diverse oxidants for Congo red degradation: effect of oxidants, degradation pathway and economic analysis.

The present study investigated the efficacy of microwave photo-oxidation (MWPO) process with two oxidants i.e. persulphate (PS) and hydrogen peroxide (H2O2) for degradation of Congo red (CR). The result indicated a CR degradation efficiency of 98% and 96.8% with PS and H2O2, respectively, in 30 min of reaction with corresponding PS dosage of 50 mg/L and H2O2 dosage of 180 mg/L. The COD removal efficiency with the two oxidants were 97.7% and 94.9%, respectively. Higher dosages of oxidant and CR reduced the efficiency of the process in both the cases due to self-quenching. Effect of pH and initial CR concentration on CR removal efficiency also has been studied. Degradation of CR followed pseudo-first-order kinetics with a removal rate constant of 0.12/min and 0.09/min, respectively, with PS and H2O2. The main mechanism of CR degradation was cleavage of the benzene-benzene bond, cleavage of benzene-N bond and hydroxylation. Economic analysis of the MWPO process indicated an energy consumption of 18.3 kWh/g of CR removal and 18.4 kWh/g of COD removal. The process was effective in the rapid degradation and mineralization of high concentration of CR within 30 min.

High sucrose diet induces morphological, structural and functional impairments in the renal tubules of Drosophila melanogaster: A model for studying type-2 diabetes mediated renal tubular dysfunction.

Continuous feeding of high dietary sugar is strongly associated with type 2 diabetes (T2D) and its secondary complications. Diabetic nephropathy (DN) is a major secondary complication that leads to glomerular and renal tubular dysfunction. The present study is aimed to investigate the effects of chronic exposure of high sugar diet (HSD) on renal tubules. Malpighian tubules (MTs), a renal organ of Drosophila, were used as a model in the study. Feeding of HSD develops T2D condition in Drosophila. The MTs showed structural abnormalities in 20 days of HSD fed flies. Impaired insulin signaling, oxidative stress, enhanced levels of AGE-RAGE and induction of apoptosis were observed in the MTs of these flies. Further, altered expression of transporters, enhanced uric acid level and reduced fluid secretion rate confirmed the impaired function of MTs in these flies. RNA-seq and RT-PCR analyses in the MTs of HSD fed-and control-flies revealed the altered expression of candidate genes that regulate several important pathways including extracellular matrix (ECM), advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE), transforming growth factor ß (TGF-ß), galactose, starch and sucrose metabolism that are well known mediators of renal tubular dysfunction in DN patients. Disruption of insulin signaling in the MTs also causes renal tubular dysfunction similar to HSD fed flies. Overall, the study suggests that phenotypes observed in the MTs of HSD fed flies recapitulate several hallmarks of renal tubular dysfunction in DN patients. Therefore, we conclude that MTs of HSD fed flies may be used for deciphering the underlying mechanisms of T2D mediated renal tubular dysfunction.

Multiplexed Detection and Quantitation of Extracellular Vesicle RNA Expression Using NanoString.

Several different types of RNA molecules such as microRNAs (miRNAs) have been detected within extracellular vesicles in the circulation. The detection and potential utility of these as disease biomarkers requires the ability to detect their presence with adequate sensitivity and to quantitate their expression. The potential for circulating miRNA to serve as biomarkers can be evaluated through their detection in association with specific disease states. Multiplexed detection of several miRNA simultaneously can be useful for discovery studies. We describe the analysis of miRNA from biological fluids like plasma and serum using the Nanostring nCounter platform. Assays can be used to quantitate the expression of miRNA using direct detection based on hybridization to target specific color-coded probes followed by counting each color-coded barcode digitally.

Functional Characterization of SMG7 Paralogs in Arabidopsis thaliana.

SMG7 proteins are evolutionary conserved across eukaryotes and primarily known for their function in nonsense mediated RNA decay (NMD). In contrast to other NMD factors, SMG7 proteins underwent independent expansions during evolution indicating their propensity to adopt novel functions. Here we characterized SMG7 and SMG7-like (SMG7L) paralogs in Arabidopsis thaliana. SMG7 retained its role in NMD and additionally appears to have acquired another function in meiosis. We inactivated SMG7 by CRISPR/Cas9 mutagenesis and showed that, in contrast to our previous report, SMG7 is not an essential gene in Arabidopsis. Furthermore, our data indicate that the N-terminal phosphoserine-binding domain is required for both NMD and meiosis. Phenotypic analysis of SMG7 and SMG7L double mutants did not indicate any functional redundancy between the two genes, suggesting neofunctionalization of SMG7L. Finally, protein sequence comparison together with a phenotyping of T-DNA insertion mutants identified several conserved regions specific for SMG7 that may underlie its role in NMD and meiosis. This information provides a framework for deciphering the non-canonical functions of SMG7-family proteins.

Use of a Hollow Fiber Bioreactor to Collect Extracellular Vesicles from Cells in Culture.

Current approaches for collection of extracellular vesicles (EV) are based on classical cell culture media production. This involves collection from cells grown in flasks, and can require multiple rounds of centrifugation or filtration, followed by ultracentrifugation or density gradient centrifugation. There are several limitations of these approaches, for example, they require a large input volume, the yield and concentration is low, and the process is time consuming. Most cell cultures require the use of fetal bovine serum which contains a large amount of endogenous EV that can contaminate isolations of cell-derived EVs. The use of cell cultures within a hollow fiber bioreactor could address many of these limitations and produce a continuous source of highly concentrated EVs without contamination from serum EVs, and that are suitable for downstream applications.

Extracellular vesicle therapeutics for liver disease.

Extracellular vesicles (EVs) are endogenous nanoparticles that play important roles in intercellular communication. Unmodified and engineered EVs can be utilized for therapeutic purposes. For instance, mesenchymal stem cell (MSC)-derived EVs have shown promise for tissue repair, while drug-loaded EVs have the potential to be used for cancer treatment. The liver is an ideal target for EV therapy due to the intrinsic regenerative capacity of hepatic tissue and the tropism of systemically injected nanovesicles for this organ. This review will give an overview of the potential of EV therapeutics in liver disease. Specifically, the mechanisms by which MSC-EVs induce liver repair will be covered. Moreover, the use of drug-loaded EVs for the treatment of hepatocellular carcinoma will also be discussed. Although there are several challenges associated with the clinical translation of EVs, these biological nanoparticles represent a promising new therapeutic modality for liver disease.

Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during susceptible and resistance responses.

Root-knot nematodes (RKNs, Meloidogyne incognita) are economically important endoparasites with a wide host range. We used a comprehensive transcriptomic approach to investigate the expression of both tomato and RKN genes in tomato roots at five infection time intervals from susceptible plants and two infection time intervals from resistant plants, grown under soil conditions. Differentially expressed genes during susceptible (1827, tomato; 462, RKN) and resistance (25, tomato; 160, RKN) interactions were identified. In susceptible responses, tomato genes involved in cell wall structure, development, primary and secondary metabolite, and defence signalling pathways, together with RKN genes involved in host parasitism, development and defence, are discussed. In resistance responses, tomato genes involved in secondary metabolite and hormone-mediated defence responses, together with RKN genes involved in starvation stress-induced apoptosis, are discussed. In addition, 40 novel differentially expressed RKN genes encoding secretory proteins were identified. Our findings provide novel insights into the temporal regulation of genes involved in various biological processes from tomato and RKN simultaneously during susceptible and resistance responses, and reveal the involvement of a complex network of biosynthetic pathways during disease development.

Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction.

Root-knot nematodes (RKNs, Meloidogyne spp.) are the most damaging plant parasites causing severe losses to crop production. The present study reports genome-wide identification and characterization of both tomato and RKN miRNAs simultaneously from RKN-infected susceptible tomato roots using high-throughput sequencing technique. RNAseq data from 11 small RNA libraries derived from 5 disease development stages identified 281 novel miRNAs of tomato in addition to 52 conserved and 4 variants of conserved miRNAs. Additionally, the same set of RNAseq data identified 38 conserved and 290 novel RKN miRNAs. Both tomato and RKN miRNAs showed differential expression at 5 stages of disease development based on digital expression profiles. In tomato, further validation through qRT-PCR confirmed that majority of miRNAs were significantly upregulated during susceptible response whereas downregulated during resistance response. The predicted targets of 8 conserved and 1 novel miRNAs were validated through 5'RLM-RACE. A negative correlation between expression profiles of a few conserved miRNAs (miR156, miR159, miR164 and miR396) and their targets (SBP, GAMYB-like, NAC and GRF1 transcription factor) was confirmed. A novel Sly_miRNA996 also showed a negative correlation with its target MYB-like transcription factor. These results indicate that the conserved and novel tomato miRNAs are involved in regulating developmental changes in host root during RKN infection. In RKN, the targets of conserved miRNAs were also predicted and a few of their predicted target genes are known to be involved in nematode parasitism. Further, the potential roles of both tomato and RKN miRNAs have been discussed.