Impact of Vitamin D Supplementation on Arterial Vasomotion, Stiffness and Endothelial Biomarkers in Chronic Kidney Disease Patients

Background: Cardiovascular events are frequent and vascular endothelial function is abnormal in patients with chronic kidney disease (CKD). We demonstrated endothelial dysfunction with vitamin D deficiency in CKD patients; however the impact of cholecalciferol supplementation on vascular stiffness and vasomotor function, endothelial and bone biomarkers in CKD patients with low 25-hydroxy vitamin D [25(OH)D] is unknown, which this study investigated. Methods: We assessed non-diabetic patients with CKD stage 3/4, age 17–80 years and serum 25(OH)D ,75 nmol/L. Brachial artery Flow Mediated Dilation (FMD), Pulse Wave Velocity (PWV), Augmentation Index (AI) and circulating blood biomarkers were evaluated at baseline and at 16 weeks. Oral 300,000 units cholecalciferol was administered at baseline and 8-weeks. Results: Clinical characteristics of 26 patients were: age 50614 (mean61SD) years, eGFR 41611 ml/min/1.73 m2, males 73%, dyslipidaemia 36%, smokers 23% and hypertensives 87%. At 16-week serum 25(OH)D and calcium increased (43616 to 84629 nmol/L, p,0.001 and 2.3760.09 to 2.4260.09 mmol/L; p = 0.004, respectively) and parathyroid hormone decreased (10.868.6 to 7.464.4; p = 0.001). FMD improved from 3.163.3% to 6.163.7%, p = 0.001. Endothelial biomarker concentrations decreased: E-Selectin from 566662123 to 525662058 pg/mL; p = 0.032, ICAM-1, 3.4560.01 to 3.1061.04 ng/mL; p = 0.038 and VCAM-1, 54633 to 42633 ng/mL; p = 0.006. eGFR, BP, PWV, AI, hsCRP, von Willebrand factor and Fibroblast Growth Factor-23, remained unchanged. Conclusion: This study demonstrates for the first time improvement of endothelial vasomotor and secretory functions with vitamin D in CKD patients without significant adverse effects on arterial stiffness, serum calcium or FGF-23. Trial Registration: ClinicalTrials.gov NCT0200571

Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum

A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch’s postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants

Nitrated α–Synuclein Immunity Accelerates Degeneration of Nigral Dopaminergic Neurons

The neuropathology of Parkinson's disease (PD) includes loss of dopaminergic neurons in the substantia nigra, nitrated alpha-synuclein (N-alpha-Syn) enriched intraneuronal inclusions or Lewy bodies and neuroinflammation. While the contribution of innate microglial inflammatory activities to disease are known, evidence for how adaptive immune mechanisms may affect the course of PD remains obscure. We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration.Nitrotyrosine (NT)-modified alpha-Syn was detected readily in cervical lymph nodes (CLN) from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Antigen-presenting cells within the CLN showed increased surface expression of major histocompatibility complex class II, initiating the molecular machinery necessary for efficient antigen presentation. MPTP-treated mice produced antibodies to native and nitrated alpha-Syn. Mice immunized with the NT-modified C-terminal tail fragment of alpha-Syn, but not native protein, generated robust T cell proliferative and pro-inflammatory secretory responses specific only for the modified antigen. T cells generated against the nitrated epitope do not respond to the unmodified protein. Mice deficient in T and B lymphocytes were resistant to MPTP-induced neurodegeneration. Transfer of T cells from mice immunized with N-alpha-Syn led to a robust neuroinflammatory response with accelerated dopaminergic cell loss.These data show that NT modifications within alpha-Syn, can bypass or break immunological tolerance and activate peripheral leukocytes in draining lymphoid tissue. A novel mechanism for disease is made in that NT modifications in alpha-Syn induce adaptive immune responses that exacerbate PD pathobiology. These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination

Background Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. Methods In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. Findings Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. Interpretation The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. Funding This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination

Data sharing statement This is an Open Access article under the CC BY 4.0 license The ISARIC4C protocol, data sharing and publication policy are available at https://isaric4c.net. ISARIC4C's Independent Data and Material Access Committee welcomes applications for access to data and materials (https://isaric4c.net). The PHOSP-COVID protocol, consent form, definition and derivation of clinical characteristics and outcomes, training materials, regulatory documents, information about requests for data access, and other relevant study materials are available online: https://phosp.org/resource/. Access to these materials can be granted by contacting [email protected] and [email protected]. All data used in this study is available within ODAP and accessible under reasonable request. Data access criteria and information about how to request access is available online: https://phosp.org/resource/. If criteria are met and a request is made, access can be gained by signing the eDRIS user agreement.Supplementary data are available online at https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(22)00584-9/fulltext#supplementaryMaterial .Copyright © 2022 The Author(s). Background: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. Methods: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. Findings: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. Interpretation: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. Funding: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript.This work is supported by the following grants: The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute for Health and Care Research (grant references: MR/V027859/1 and COV0319). ISARIC4C is supported by grants from the National Institute for Health and Care Research (award CO-CIN-01) and the Medical Research Council (grant MC_PC_19059) Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research (grant reference: C18616/A25153). Other grants which have supported this work include: the UK Coronavirus Immunology Consortium [funder reference:1257927], the Imperial Biomedical Research Centre (NIHR Imperial BRC, grant IS-BRC-1215-20013), the Health Protection Research Unit (HPRU) in Respiratory Infections at Imperial College London and NIHR HPRU in Emerging and Zoonotic Infections at University of Liverpool, both in partnership with Public Health England, [NIHR award 200907], Wellcome Trust and Department for International Development [215091/Z/18/Z], Health Data Research UK (HDR UK) [grant code: 2021.0155], Medical Research Council [grant code: MC_UU_12014/12], and NIHR Clinical Research Network for providing infrastructure support for this research. FL is supported by an MRC clinical training fellowship [award MR/W000970/1]. LPH is supported by Oxford NIHR Biomedical Research Centre. AART is supported by a BHF Intermediate Clinical Fellowship (FS/18/13/33281). SLRJ receives support from UKRI, GCRF, Rosetrees Trust, BHIVA, EDCTP, Globvac. JDC has grants from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Gilead Sciences, Grifols, Novartis and Insmed. RAE holds a NIHR Clinician Scientist Fellowship (CS-2016-16-020). AH is currently supported by UK Research and Innovation. NIHR and NIHR Manchester BRC. BR receives support from BHF Oxford Centre of Research Excellence, NIHR Oxford BRC and MRC. SJD is funded by an NIHR Global Research Professorship [NIHR300791]. DW is supported by an NIHR Advanced Fellowship. AH has received support from MRC and the Coronavirus Immunology Consortium (MR/V028448/1). LVW has received support from UKRI, GSK/Asthma + Lung UK and NIHR for this study. MGS has received support from NIHR UK, MRC UK and Health Protection Research Unit in Emerging & Zoonotic Infections, University of Liverpool. JKB is supported by the Wellcome Trust (223164/Z/21/Z) and UKRI (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1, and MC_PC_20029). PJMO is supported by a NIHR Senior Investigator Award [award 201385]. LT is supported by the Wellcome Trust [clinical career development fellowship grant number 205228/Z/16/Z], the Centre of Excellence in Infectious Diseases Research (CEIDR) and the Alder Hey Charity

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

Data availability: This is an open access article under the CC BY 4.0 license. The PHOSP-COVID protocol, consent form, definition and derivation of clinical characteristics and outcomes, training materials, regulatory documents, information about requests for data access, and other relevant study materials are available online at ref. 76. Access to these materials can be granted by contacting [email protected] and [email protected]. The ISARIC4C protocol, data sharing and publication policy are available at https://isaric4c.net. ISARIC4C’s Independent Data and Material Access Committee welcomes applications for access to data and materials (https://isaric4c.net). The datasets used in the study contain extensive clinical information at an individual level that prevent them from being deposited in an public depository due to data protection policies of the study. Study data can only be accessed via the ODAP, a protected research environment. All data used in this study are available within ODAP and accessible under reasonable request. Data access criteria and information about how to request access is available online at ref. 76. If criteria are met and a request is made, access can be gained by signing the eDRIS user agreement.Code availability: Code was written within the ODAP, using R v4.2.0 and publicly available packages (‘data.table v1.14.2’, ‘EnvStats v2.7.0’, ‘tidyverse v1.3.2’, ‘lme4 v1.1-32’, ‘caret v6.0-93’, ‘glmnet v4.1-6’, ‘mdatools v0.14.0’, ‘ggpubbr v0.4.0’, ‘ggplot2 v3.3.6’, ‘bootnet v1.5.6’ and ‘qgraph v1.9.8’ packages). No new algorithms or functions were created and code used in-built functions in listed packages available on CRAN. The code used to generate data and to analyze data is publicly available at https://github.com/isaric4c/wiki/wiki/ISARIC; https://github.com/SurgicalInformatics/cocin_cc and https://github.com/ClaudiaEfstath/PHOSP_Olink_NatImm.One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials.This research used data assets made available by ODAP as part of the Data and Connectivity National Core Study, led by Health Data Research UK in partnership with the Office for National Statistics and funded by UK Research and Innovation (grant ref. MC_PC_20058). This work is supported by the following grants: the PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research (NIHR; grant references MR/V027859/1 and COV0319). ISARIC4C is supported by grants from the National Institute for Health and Care Research (award CO-CIN-01) and the MRC (grant MC_PC_19059) Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research (grant reference C18616/A25153). Other grants that have supported this work include the UK Coronavirus Immunology Consortium (funder reference 1257927), the Imperial Biomedical Research Centre (NIHR Imperial BRC, grant IS-BRC-1215-20013), the Health Protection Research Unit in Respiratory Infections at Imperial College London and NIHR Health Protection Research Unit in Emerging and Zoonotic Infections at University of Liverpool, both in partnership with Public Health England, (NIHR award 200907), Wellcome Trust and Department for International Development (215091/Z/18/Z), Health Data Research UK (grant code 2021.0155), MRC (grant code MC_UU_12014/12) and NIHR Clinical Research Network for providing infrastructure support for this research. We also acknowledge the support of the MRC EMINENT Network (MR/R502121/1), which is cofunded by GSK, the Comprehensive Local Research Networks, the MRC HIC-Vac network (MR/R005982/1) and the RSV Consortium in Europe Horizon 2020 Framework Grant 116019. F.L. is supported by an MRC clinical training fellowship (award MR/W000970/1). C.E. is funded by NIHR (grant P91258-4). L.-P.H. is supported by Oxford NIHR Biomedical Research Centre. A.A.R.T. is supported by a British Heart Foundation (BHF) Intermediate Clinical Fellowship (FS/18/13/33281). S.L.R.-J. receives support from UK Research and Innovation (UKRI), Global Challenges Research Fund (GCRF), Rosetrees Trust, British HIV association (BHIVA), European & Developing Countries Clinical Trials Partnership (EDCTP) and Globvac. J.D.C. has grants from AstraZeneca, Boehringer Ingelheim, GSK, Gilead Sciences, Grifols, Novartis and Insmed. R.A.E. holds a NIHR Clinician Scientist Fellowship (CS-2016-16-020). A. Horsley is currently supported by UK Research and Innovation, NIHR and NIHR Manchester BRC. B.R. receives support from BHF Oxford Centre of Research Excellence, NIHR Oxford BRC and MRC. D.G.W. is supported by an NIHR Advanced Fellowship. A. Ho has received support from MRC and for the Coronavirus Immunology Consortium (MR/V028448/1). L.T. is supported by the US Food and Drug Administration Medical Countermeasures Initiative contract 75F40120C00085 and the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections (NIHR200907) at the University of Liverpool in partnership with UK Health Security Agency (UK-HSA), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford. L.V.W. has received support from UKRI, GSK/Asthma and Lung UK and NIHR for this study. M.G.S. has received support from NIHR UK, MRC UK and Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool. J.K.B. is supported by the Wellcome Trust (223164/Z/21/Z) and UKRI (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1 and MC_PC_20029). The funders were not involved in the study design, interpretation of data or writing of this manuscript. The views expressed are those of the authors and not necessarily those of the Department of Health and Social Care (DHSC), the Department for International Development (DID), NIHR, MRC, the Wellcome Trust, UK-HSA, the National Health Service or the Department of Health. P.J.M.O. is supported by a NIHR Senior Investigator Award (award 201385). We thank all the participants and their families. We thank the many research administrators, health-care and social-care professionals who contributed to setting up and delivering the PHOSP-COVID study at all of the 65 NHS trusts/health boards and 25 research institutions across the United Kingdom, as well as those who contributed to setting up and delivering the ISARIC4C study at 305 NHS trusts/health boards. We also thank all the supporting staff at the NIHR Clinical Research Network, Health Research Authority, Research Ethics Committee, Department of Health and Social Care, Public Health Scotland and Public Health England. We thank K. Holmes at the NIHR Office for Clinical Research Infrastructure for her support in coordinating the charities group. The PHOSP-COVID industry framework was formed to provide advice and support in commercial discussions, and we thank the Association of the British Pharmaceutical Industry as well the NIHR Office for Clinical Research Infrastructure for coordinating this. We are very grateful to all the charities that have provided insight to the study: Action Pulmonary Fibrosis, Alzheimer’s Research UK, Asthma and Lung UK, British Heart Foundation, Diabetes UK, Cystic Fibrosis Trust, Kidney Research UK, MQ Mental Health, Muscular Dystrophy UK, Stroke Association Blood Cancer UK, McPin Foundations and Versus Arthritis. We thank the NIHR Leicester Biomedical Research Centre patient and public involvement group and Long Covid Support. We also thank G. Khandaker and D. C. Newcomb who provided valuable feedback on this work. Extended Data Fig. 10 was created using Biorender

Phytochemical investigation, antioxidant and wound healing activities of Citrullus colocynthis (bitter apple)

Objective: To undertake metabolite profiling of various plant parts of Citrullus colocynthis, and assess antioxidant and wound healing activities of fractions for therapeutical applications. Methods: Extracts from leaves, stem, root, fruit pulp and seeds were analyzed using gas chromatography-mass spectrometry and high performance liquid chromatography. Variation in antioxidant potential was assayed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. The extract with highest antioxidant potential was subjected on in-vivo wound healing activity using excision wound model. Results: Metabolite profiling of Citrullus colocynthis identified 70 chemically diverse metabolites from different plant parts by using a combination of GC-MS and HPLC. Concentration of colocynthin, a principal active secondary metabolite, ranged from 3.15 mg/g dry weight to 242.00 mg/g dry weight, the lowest being in leaves and highest in fruit pulp. DPPH radical scavenging activity of free radical (IC50) ranged from 196.44 μg/mL in fruit pulp to 413.33 μg/mL in leaves tissues. Significant wound contraction and increase in hydroxyproline content of granulation tissue were observed with ointment formulated from methanolic extract of fruit pulp. Conclusions: The study indicates that the methanol extract of Citrullus colocynthis fruit pulp when applied topically may promote wound contraction in rat model attributable to the accumulation of colocynthin. The high quantity of colocynthin (242.00 mg/g dry weight) and substantial concentration of 2,4-di-tert butyl phenol (3.2%), squalene (4.2%) and δ -tocopherol (2.5%) make this plant to provide new opportunities for development of medicinal, nutraceutical and dietary supplements with optimized functionality

Comprehensive metabolite profiling in distinct chemotypes of <i>Commiphora wightii</i>

<p><i>Commiphora wightii</i> (Arn.) Bhandari, known as guggul, produces a medicinally important gum resin which is used extensively by Ayurvedic physicians to treat various ailments. However, most of the studies on <i>C. wightii</i> have been limited to its gum resin. Comprehensive metabolic profiling of leaves, stem and gum resin samples was undertaken to analyse aqueous and non-aqueous metabolites from three distinct chemotypes (NBRI-101, NBRI-102 and NBRI-103) shortlisted from different agro-climatic zones. GC-MS, HPLC and NMR spectroscopy were used for comprehensive metabolomics. Multivariate analysis showed characteristic variation in quinic and citric acids, <i>myo</i>-inositol and glycine (aqueous metabolites) and 2,6-di-<i>tert</i>-butyl-phenol, <i>trans</i>-farnesol and guggulsterones (non-aqueous metabolites) amongst the three chemotypes. Quinic acid, citric acid and <i>myo</i>-ionositol were detected in substantial quantities from leaves and stem samples which provide opportunities for novel nutraceutical and pharmaceutical formulations. Quinic acid, from the leaves, was identified as a marker metabolite for early selection of high guggulsterones-yielding cultivars.</p