Clinical application of pulse-gated non-enhanced rapid magnetic resonance imaging in the definitive diagnosis of aortic dissection

Abstract Objective: Aortic Dissection (AD) is one of the most fatal acute diseases in cardiovascular diseases, with rapid onset and progression and a high fatality rate. This study aims to investigate the clinical values of non-enhancement peripheral pulse-gating rapid magnetic resonance imaging in deterministic diagnosis of AD. Methods: Aorta magnetic resonance imaging was performed in 21 healthy volunteers at a 1.5t MR scanner sequences including cardiac-gated and peripheral pulse-gated True-FISP and HASTE were carried out separately. Acquisition Time (TA), Signal to Noise Ratio (SNR), Contrast Noise Ratio (CNR), and entirety of vessel wall blood flow artifacts were measured and compared. A total of 56 AD cases were displayed by non-enhancement peripheral pulse-gating fast MR imaging, and the results were compared with pathological findings or CTA of the aorta. The dissection rupture, tear film, true and false lumen, thrombosis, hydropericardium, and the main branches of AD were evaluated respectively. Results: There were no significant differences in SNR, CNR, entirety of the vessel wall, and blood flow artifact between cardiac-gated and peripheral pulse-gated fast MR imaging. Non-enhancement pulse-gated fast scanning takes less TA time. By the pulse-gated non-enhancement fast MR imaging, the dissection rupture, tear film, true and false cavity, thrombosis, hydropericardium, and the main branches of aortic dissection were shown clearly. Multi-planar and multi-angle scans helped to show the extent of entrapment rupture, whereas partial complex tears or bi-directional tears were slightly less well visualized. Conclusion: Non-enhancement peripheral pulse-gated rapid magnetic resonance imaging can be used for deterministic diagnosis of AD.

Monitoring and analysis of use of non-national immunization program vaccines in Fujian Province from 2014 to 2020 / 中国生物制品学杂志

@#Objective To monitor and analyze the use of non-national immunization program(nNIP) vaccines in Fujian Province from 2014 to 2020,and provide a reference for improving the management of nNIP vaccination service.Methods The nNIP vaccination data from 2014 to 2020 were extracted from the National Immunization Program Information System(NIPIS),and analyzed by descriptive epidemiological method.Results From 2014 to 2020,a total of 28 835 834 doses of nNIP vaccines were administered in Fujian Province,with an average of 1 015 doses per 10 000 population,and the varieties used increased from 22 to 28.The annual administered doses of nNIP vaccine,the proportion in total doses of all vaccines and the number of vaccinations per capita basically increased year by year.The most widely used varieties mainly include varicella attenuated live vaccine(VarV),hepatitis B(HepB) vaccine,rabies vaccine(RabV) for human use,haemophilus influenza type b(Hib) polysaccharide conjugate vaccine,influenza vaccine(InfV) and enterovirus 71(EV71)inactivated vaccine.The three vaccines with the highest replacement rates were hepatitis A vaccine,group A meningococcal vaccine and group A and C meningococcal vaccine,with the replacement rates of 22.42%,10.32% and 9.89%respectively.Conclusion There are more and more types and doses of nNIP vaccines used in Fujian Province,so it is necessary to further strengthen monitoring and management and improve vaccination service.

The Effect of Torsion of Blood Vessel after Stent Implantation on Mechanical State of Stent / 中国医疗器械杂志

OBJECTIVE@#In daily life, the movement of the neck will cause certain deformation of the blood vessel and the stent. This study explores the quantitative influence of the torsion deformation of the blood vessel on the mechanical properties of the stent.@*METHODS@#In the finite element simulation software Abaqus, the numerical simulation of the crimping and releasing process of the stent, the numerical simulation of the torsion process of the blood vessel with the stent, and the numerical simulation of the pressure loading process of the outer wall of the blood vessel were carried out.@*RESULTS@#After the stent was implanted, when a load was applied to the outer surface of the blood vessel wall, when the applied load did not change, as the torsion angle increased, the smallest cross-sectional area in the blood vessel decreased.@*CONCLUSIONS@#After the stent is placed, when the external load is fixed, the radial support capacity of the stent will decrease as the torsion angle increases.

Suicidal ideation of freshmen in one university of Tianjin from 2013 to 2020 / 中国学校卫生

Objective@#To understand the current situation of suicidal ideation in college students of Tianjin, and to provide reference for suicide prevention, early warning and emergency intervention.@*Methods@#A total of 31 596 freshmen from one university in Tianjin were selected to participate in the mental health survey during 2013 to 2020.@*Results@#A total of 1 349 freshmen reported suicidal ideation. The incidence of suicidal ideation varied from 2.60% to 6.18% across the years (the overall average annual incidence of suicidal ideation was 4.27%). Significant differences were observed in the incidence of suicidal ideation among freshmen of different origin (4.11% in urban areas, 4.74% in rural areas; χ 2=6.01, P <0.01), whether the only children in the family (4.10% in only children, 4.60% in non only children; χ 2=4.38, P <0.01). During the past five years, the in depth interview showed that the proportion of those with suicidal attempt ranged from 17.09%-33.73% (the total annual average proportion of those with suicidal attempt was 24.84%). Results from electroencephalonquadrantography varied significantly between college students with and without suicidal ideation and those with and without suicidal attempt.@*Conclusion@#It is necessary to strengthen the screening of college students for suicidal ideation and to identify the risk of students with suicide attempt, so as to provide a better reference for the prevention and intervention of psychological crisis.

Research Progress of Pathology of Gout and Clinical Treatment with Traditional Chinese and Western Medicine / 中国实验方剂学杂志

With the improvement of people's living standard and the change of dietary structure, the prevalence of gout has increased gradually with the increased intake of protein, sugar and fat. There has been a positive correlation between gout and age, and the age of onset decreased gradually. The inflammation induced by sodium urate crystal is the pathological basis of gout, which activates innate immunity, releases many kinds of inflammatory mediators, such as interleukin(IL)-1β, IL-6 and tumor necrosis factor(TNF)-α, and then causes inflammatory cascade reaction and acute attacks, such as joint redness, swelling and heat pain. There is a spontaneous remission mechanism in gout. For one thing, macrophages reduce the stimulation of monosodium urate(MSU) through phagocytosis of MSU crystals as foreign bodies, for another, differentiated and mature macrophages secrete anti-inflammatory factor transforming growth factor(TGF)-β1, inhibit the expression of inflammatory factors and promote spontaneous relief of acute gout attack. In addition to the activation mechanism of intracellular signaling molecules associated with inflammatory response, the inflammatory mechanism of gout also involves complement activation, cell activation and other pathways. The complications caused by gout, such as cardiovascular system damage and joint destruction, are seriously harmful to human health. At present, western drugs, such as allopurinol and febuxostat, exert an effect in inhibiting xanthine oxidase. Benzimarone has effect in reducing renal absorption of uric acid and promoting uric acid excretion by inhibiting uric acid transporter 1(URAT1) and glucose transporter 9(GLUT9). Even Lesinurad and other medicines in current studies are based on the inhibition of uric acid re-absorption, but with adverse reactions that limit the clinical application. The treatment of gout with traditional Chinese medicine(TCM) has multi-target characteristics, with advantages in reducing uric acid, resisting inflammation and improving joint function and a high safety. It has been gradually popularized and applied in clinical treatment of gout. Therefore, it is a promising research direction to treat gout with TCM and western medicine based on the pathomechanism of gout.

Evaluation of the installation and use of air purification equipments in primary schools in Beijing / 中国学校卫生

Objective@#To investigate the current situation of the installation and use of air purification equipment in primary schools in Beijing in order to provide data for school-based air pollution prevention and intervention.@*Methods@#A questionnaire survey was conducted in 50 primary schools in 6 districts in Beijing. Spearman correlation coefficient and Pearson correlation coefficient were used to test the correlation between installation rate of air purification equipment and per capita disposable income.@*Results@#The overall installation rate of air purifier or fresh air system in primary schools in Beijing was 62.0%, the overall installation rate of air purifier was 50.0% and the overall installation rate of fresh air system was 18.0%. Installation rate of fresh air system or air purifier was highly correlated with per capita disposable income (r=0.92, P=0.01).@*Conclusion@#The installation rate of air purification equipment in primary schools is relatively high, but parameters and purification types are not unified, and there is a lack of scientific guideline. Therefore, the installation and use of air purification equipment in schools should be further standardized and unified to protect the health of children and adolescents.

miR-425-5p suppresses tumorigenesis and DDP resistance in human-prostate cancer by targeting GSK3 β and inactivating the Wnt/β-catenin signaling pathway

In previous studies, we found interferon-a (IFN-a) could reduce protein levels of p11, 5-hydroxytryptamine receptor 1b(5-HT1b) and 5-hydroxytryptamine receptor 4 (5-HT4), but does not influence their messenger RNA levels in SH-sy5ycells. Thus, we investigated the post-transcriptional modulation of these molecules by IFN-a. SH-sy5y cells were treatedwith IFN-a, NH4Cl or MG132 alone or in combination, and then the protein levels of p11, 5-HT1b and 5-HT4 wereanalyzed by western blots. The regulatory effects of p11 on 5-HT1b and 5-HT4 were also determined in p11 knock-downcells. NH4Cl but not MG132 could reverse the protein level of p11 in IFN-a-treated SH-sy5y cells. MG132 could recoverthe protein levels of 5-HT1b and 5-HT4 in p11 knock-down cells. The down-regulation effects of IFN-a on p11, 5-HT1band 5-HT4 were associated with the lysosome and ubiquitin–proteasome-mediated pathways. p11 was identified as a potentregulator to modulate the ubiquitination of 5-HT1b and 5-HT4. Therefore, it could be potential target therapies in IFN-ainduced depression.

Family correlates of physical aggression among students in concentrated poverty-stricken minority areas / 中国学校卫生

Objective@#To explore family correlates of physical aggression among students in concentrated poverty-stricken minority areas.@*Methods@#By using cluster sampling, questionnaire survey was conducted among 2 960 students in Dongjing county, Wucun county and Babie village in Baise City. Information of general demographic characteristics and physical aggression was collected.@*Results@#Family economic status, family structure, family education system, family emotional atmosphere and household function showed significant effects on physical aggression(F/t=35.15, -5.78, 5.57, 9.54, 60.86, P<0.05).From multivariate logistic regression showed that family structure, parenting style, family emotional atmosphere and household function significantly accounted for variation of physical aggression, with one-unit improve of above indicators resulted in 0.11, 0.17, 0.17 and 0.36 decrease in physical aggression, respectively.@*Conclusion@#Parents should be encouraged to construct a harmonious family atmosphere, democratic parenting and well-functioning family, and cultivate positive attitude and outlook on life of their children through active life perspectives, aiming to reduce physical aggression and promote physical and mental well-being.

Effect of 27nt-miRNA on regulation of SM22α expression in vascular smooth muscle cells and its effect on cell viability, migration and phenotypic changes / 中国病理生理杂志

AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs).METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs.Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion.The cell viability was measured by MTT assay.The migration ability was detected by scratch assay.The mRNA and protein expression of SM22αwas determined by RT-PCR, immunocytochemical staining and Western blot.RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05) , the migration ability was increased (P<0.05) and the expression of SM22αat mRNA and protein level was decreased (P<0.05).Compared with negative control lentiviral group, the cell viability in 27ntmiRNA over-expression group was decreased (P<0.05) , the migration ability was decreased (P<0.05) , and the mRNA and protein expression of SM22αwas increased (P<0.05).While in anti-27nt-miRNA group, the cell viability was increased (P<0.05) , the migration ability was increased (P<0.05) , and the mRNA and protein expression of SM22αwas decreased (P<0.05).CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.

Fruit sphere microenvironments and berry phenolic content of Cabernet Sauvignon (Vitis vinifera L.) cultivated under rain-shelter systems with coloured plastic film

Abstract Rain-shelter cultivation has been proven an important cultivation method for grape-plantings in continental monsoon climate zones, of which white plastic films are the most common shelter material. However, while this method and material reduces the occurrence of the disease, it can also decrease the grape berry quality. Five colours (including red, yellow, blue, purple, and white) of plastic films were covered above Cabernet Sauvignon (Vitis vinifera L.) grapevine rows before veraison. Rain-shelter cultivation reduced air temperature, wind speed, and total solar radiation and enhanced relative humidity in the fruit sphere of grapevines. For each particular colour plastic film, the irradiance of its corresponding spectrum band in the canopy of vines was higher than with other colour plastic films. Meanwhile, the blue plastic film treatment significantly promoted the accumulation of total phenolics, anthocyanins, flavonoids, tannins, and phenolic acids more than the other colours of plastic films. Blue plastic films are more beneficial for berry quality promotion of wine grapes, especially Cabernet Sauvignon, under rain-shelter cultivation in continental monsoon climate zones.

Cloning and expression analysis of sesquiterpene synthase gene in Dendrobium officinale / 中草药

Objective To clone the full-length cDNA encoding sesquiterpene synthase (SES) gene from Dendrobium officinale (DoSES), then to analyze the expression difference in different tissues and expression patterns of DoSES induced by methyl jasmonate (MeJA). Methods RACE was used to clone the full length cDNA of DoSES. Bioinformatics analysis was carried out by using related software and online website. Then, the expression patterns of DoSES were studied by quantitative real-time PCR (qRT-PCR). Results The DoSES gene was obtained (GenBank accession number KX278311), and the full-length cDNA was 1 890 bp, encoding the protein of 520 amino acids. DoSES had highest homology ( ≈ 60%) with SES proteins from other plants. Tissue expression analysis showed that DoSES had the highest expression in the stems, followed by leaves, flowers, protocorm and roots. The results of qRT-PCR analysis suggested that DoSES could be induced by MeJA. Conclusion The cDNA encoding DoSES was cloned. The research results provide basic materials for further function alanalysis of this gene in the future.

Gap junctions enhance the antiproliferative effect by transfer of microRNAs in glioma cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the permeability of gap junction composed of connexin 43 (Cx43) for microRNAs (miRNAs) and the impact of gap junction-mediated transfer of miRNAs in glioma U87 cells. METHODS Co-culture assay demonstrated the transmission of miRNAs through gap junction channel into adjacent cells. U87 cells were labeled with green fluorescein protein (GFP) as receivers and cells were transfected miRNAs as donors. Receiver cells and donor cells were mixed together in a ratio of 1:1. After 12 h co-culture, cells were separated using a BD influx flow cytometer based on the GFP labeled. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied detect to the expressions of miRNAs and Cx43 mRNA. Western blotting was performed to detect the protein expressions of Cx43 and GFP in U87 cells. CCK-8 assay is used to detect cell growth. RESULTS Co-culture assays demonstrated miR-34a could transfer between U87 cells. The role of the contact independent could also transfer of miR-34a. Gap junctions inhibitor (CBX and 18-α-GA) showed lower miR-34a expression than co-culture group, whereas gap junctions enhancer (RA and Galanglin) enhanced miR-34a expression. Knockdown of Cx43 could significantly decrease the transferring of miR-34a between U87 cells. Different length of miRNAs (miR-1827, miR-144, miR-203a and miR-1183) were similar to the expression of miR-34a between U87 cells. Additionally, we demonstrated that gap junctions mediate the effect of antiproliferation mediated by miR- 34a in U87 cells. The functional inhibition of gap junctions using either siRNA or inhibition eliminated the miR- 34a mediated antiproliferation, whereas the enhancement of gap junctions treatment augmented this miR34a-mediated antiproliferation. CONCLUSION Our study demonstrates that gap junction composed of Cx43-mediated transfer miRNAs in different length of nucleotides and gap junction-mediated transfer of miR-34a enhance the antiproliferative effect in glioma U87 cells.

Non-junctional Cx32 mediates anti-apoptotic and pro-tumor effects via epidermal growth factor receptor in human cervical cancer cells / 中国药理学与毒理学杂志

ABSTRACT:OBJECTIVE To investigate the role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx). METHODS We analyze the expression of Cx26, Cx30, Cx32 and Cx43 in human specimens consisting of: Normal cervix (n=78), CaCx FIGO stage Ⅰ (n=148), CaCx FIGO stage Ⅱ (n=165). InCaCx cell lines, Hela- Cx32 (induced expression by doxycycline), C- 33A (endogenously express Cx32) and siHa (transiently transfected plasmid with Cx32), we detected the role of Cx32 against tostreptonigrin/cisplatin-induced apopotosisin presence or absence of functional GJ through using GJ inhibitors or low density cultural.Furtherly, we observed the relativity of Cx32 and EGFR expression in human specimens. Also, we detected the role of EGFR signaling pathway in the process of Cx32 anti-apoptosis through suppressed EGFR expression by inhibitors or siRNA sequences in cell lines. RESULTS We firstly demonstrated the expression of Cx32 was highly upregulated and accumulated in cytoplasm in the CaCx specimens, and the degree of upregulation correlated with advanced FIGO stages. Thus,in three human cervical cell lines, Cx32 was shown to suppress apoptosis when GJ formation is inhibited. No matter in cases of CaCx or cell lines, Cx32 expression was highly correlated with expression of EGFR and the EGFR pathway is an essential component of the Cx32-induced anti-apoptotic effect. CONCLUSION Cx32, traditionally tumor suppressive protein, was shown to be tumor protective against chemotherapy through EGFR pathway in a GJ-independent way.

Cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the underlying mechanism of drug resistance to cisplatin and increasing the sensitivity to therapeutic drugs are key steps towards the improved treatment of patients with ovarian cancer. Gap junction (GJ) and connexin (Cx) are closely related to tumor formation, but the relationship between cisplatin resistance and GJ or Cx are undetermined. METHODS We established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP over an 11-month period, with the concentration of cisplatin gradually increasing from 0.5 g·L-1 to 16 g·L-1. To explore the effect of GJ in the process of cisplatin resistance, we investigated GJ using a parachute dyecoupling assay in A2780- RI(1.2), A2780- RI(1.7), A2780- RI(2.9), A2780- RI(4.3) and A2780- CDDP cells. We further explored whether the Cxs responsible for GJ were related to cisplatin resistance. In A2780- RI(1.2), A2780-RI(1.7), A2780-RI(2.9), A2780-RI(4.3) and A2780-CDDP, we used q-PCR to analyze the levels of Cx43, Cx40, Cx37, and Cx32. To confirm the effect of Cx32 on cisplatin resistance, we knocked down Cx32 in A2780-CDDP cells with siRNA-Cx32. As GJ was decreased whereas Cx32 expression was elevated during the cisplatin resistance process, it drove us to explore the underlying mechanism. To resolve this issue, we extracted membrane-bound and cytoplasmic proteins from A2780 and A2780-CDDP cells. RESULTS Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression. Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment. Furthermore, although Cx32 expression was increased in A2780-CDDP cells, it was more localized to the cytoplasm rather than in the membrane, and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment. CONCLUSION In summary, Cx32 is involved in cisplatin resistance, and cytoplasmic Cx32 plays an important role in chemoresistance.

STAT3 mutations correlated with hyper-IgE syndrome lead to blockage of IL-6/STAT3 signalling pathway.

Of all the causes identified for the disease hyper-immunoglobulinemia E syndrome (HIES), a homozygous mutation in tyrosine kinase2 (TYK2) and heterozygous mutations in STAT3 are implicated the defects in Jak/STAT signalling pathway in the pathogenesis of HIES. Mutations of STAT3 have been frequently clinically identified in autosomaldominant (AD) HIES patients’ cells, and therefore, the genotype of STAT3 has been associated with the phenotype of HIES. Here, we conducted studies on the functional loss of the seven specific STAT3 mutations correlated with ADHIES. Using STAT3-null human colon carcinoma cell line A4 cells, we generated seven mutants of STAT3 bearing single mutations clinically identified in AD-HIES patients’ cells and studied the functional loss of these mutants in IL- 6-Jak/STAT3 signalling pathway. Our results show that five STAT3 mutants bearing mutations in the DNA-binding domain maintain the phosphorylation of Tyr705 and the ability of dimerization while the other two with mutations in SH2 domain are devoid of the phosphorylation of Try705 and abrogate the dimerization in response to IL-6. The phosphorylation of Ser727 in these mutants shows diversity in response to IL-6. These mutations eventually converge on the abnormalities of the IL-6/Gp130/Jak2-mediated STAT3 transactivation on target genes, indicative of the dysregulation of JAK/STAT signalling present in HIES.

Risk analysis of upper gastrointestinal bleeding in elderly patients with aspirin and clopidogrel co-therapy / 中国基层医药

ObjectiveTo explore the current occurrence of upper gastrointestinal bleeding in elderly patients in our hospital and its possible risk factors and treatment program.MethodsAn observational prospective analysis of medical records of 758 consecutive patients was performed.Clinical presentation,etiology and treatment were evaluated.The follow-up ended on the dates of a first occurrence of UGIB,stopping co-therapy,death or 6 months after admitted in the group.Results426 male and 332 female were admitted in the group,UGIB occurred in 48 patients.PPI,H2RA,gastric mucosa protectant plus PPI,gastric mucosa protectant plus H2RA and control were prescribed in 108,240,70,102,238 patients respectively.There were statistics difference in each group compared with control in bleeding rate.ConclusionThe occurrence of UGIB associated with A + C co-therapy in elderly was 6.3％.Co-prescription with PPI and gastric mucosa protectant was associated with a lower risk.

Redifferentiation of human hepatoma cells (SMMC-7721) induced by two new highly oxygenated bisabolane-type sesquiterpenes.

Bisabolane-type sesquiterpenes are a class of biologically active compounds that has antitumour,antifungal, antibacterial,antioxidant and antivenom properties.We investigated the effect of two new highly oxygenated bisabolane-type sesquiterpenes (HOBS)isolated from Cremanthodium discoideum (C.discoideum) on tumour cells. Our results showed that HOBS induced morphological differentiation and reduced microvilli formation on the cell surface in SMMC-7721 cells.Flow cytometry analysis demonstrated that HOBS could induce cell-cycle arrest in the G1 phase. Moreover,HOBS was able to increase tyrosine-alpha ketoglutarate transaminase activity,decrease alpha- foetoprotein level and gamma-glutamyl transferase activity. In addition,we found that HOBS inhibited the anchorage- independent growth of SMMC-7721 cells in a dose-dependent manner.Taken together,all the above observations indicate that HOBS might be able to normalize malignant SMMC-7721 cells by inhibiting cell proliferation and inducing redifferentiation.

Clinical analysis of prognostic factors for patients with gastrointestinal stromal tumors / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the prognostic factors for patients with gastrointestinal stromal tumors (GIST).</p><p><b>METHODS</b>From 2000 to 2003, clinical data of 41 cases with GIST were reviewed retrospectively. The clinicopathologic diagnosis was determined by immunochemistry. The relationships of the prognosis with mitotic counts, tumor size and location,range of tumor resection were analyzed.</p><p><b>RESULTS</b>The patients with GIST had pathological section of high expression in CD117, CD34 and vimentin (92.7%, 82.9%, 78%, respectively). Patients with tumor location in intestine, tumor size > 5 cm,mitotic counts > 5/50HPF, incomplete resection had poorer outcome, compared with those with tumor location in stomach and colon,tumor size < or = 5 cm,mitotic counts < or = 5/50HPF and complete resection (all P< 0.05).</p><p><b>CONCLUSION</b>Complete gross resection can improve prognosis for patients with GIST. Tumors with mitotic counts > or = 5/50HPF, tumor size more than 5 cm and tumor location in intestine are poor prognostic factors.</p>