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OBJECTIVE@#To study the effects of alcohol administration on benign prostate hyperplasia(BPH) and the reproductive toxicity during development of benign prostate hyperplasia.@*METHODS@#Seventy adult male Kunming mice were randomly divided into seven groups:control (group CON), negative control (group NC, injected subcutaneously with soybean oil, 25 mg/(kg·d), intragastric administration of distilled water, 7.5 ml/(kg·d)), alcohol for 7 and 21 days (group AL7 and AL21, intragastric administration with wine of 50% alcohol, 7.5 ml/(kg·d)), testosterone propionate for 7 and 21 days (group TP7 and TP21, injected subcutaneously with testosterone propionate, 25 mg/(kg·d)), testosterone propionate+alcohol for 7 days (group TP+AL7, injected subcutaneously with testosterone propionate, 25 mg/(kg·d), and intragastric administration with wine of 50% alcohol, 7.5 ml/(kg·d)),10 mice in each groups. Twenty-four hours after the last administration, mice were sacrificed. The indexes of prostate and testis and the parameters of sperm were determined in mice. The levels of free radicals, antioxidation and histopathological changes in testis and prostate were determined.@*RESULTS@#Compared with the control, TP7d group, AL7 and AL21d groups, the prostate coefficient of TP + AL7d group was increased significantly and the quantity and quality of sperm were decreased significantly (0.05).@*CONCLUSIONS@#The typical BPH state could be induced after 7-day treatment of testosterone propionate and alcohol. The testicular and sperm were damaged which enhanced the oxidative stress in reproductive system. The results indicated that alcohol could significantly promote the prostate hyperplasia induced by testosterone propionate in mice.
Subject(s)
Animals , Male , Mice , Plant Extracts , Prostatic Hyperplasia , Testosterone PropionateABSTRACT
AIM:To investigate the distribution and mechanism of M 1/M2 macrophages in inflammatory injury and repair process of renal tissues .METHODS: SD male rats ( n=45 ) were randomly divided into 2 parts: ischemia-reperfusion injury (IRI) and unilateral ureteral obstruction (UUO) renal injury.The rats with IRI were divided into sham operation group and operation groups (0, 6, 24, and 72 h after operation), and the rats with UUO were divided into sham operation group and operation groups (3, 7 and 14 d after operation).Automatic biochemical analyzer was used to detect serum levels of creatinine and urea nitrogen .The degree of renal injury in IRI group and UUO group were detected by HE staining.The expression of CD68 was examined by immunohistochemical staining .The levels of inducible nitric oxide syn-thase (iNOS), arginase-1 (Arg-1), transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α(TNF-α) were measured by ELISA.The polarizations of M1 ( CD68 +, F4/80 + and CD16/32 +) and M2 ( CD68 +, F4/80 +and CD206 +) macrophages were analyzed by flow cytometry .RESULTS:In IRI group, the infiltration of CD68 +macrophages and the degree of injury were increased with the prolongation of time in the renal tissues .At 24 h, the tissue injury and macrophage infiltration were the most serious , but then decreased .At 72 h, the tissue damage and CD68 +macrophage in-filtration were significantly reduced .In UUO group, obstructive injury was increased with the prolongation of time , and at 14 d, marked fibrous hyperplasia occurred .The infiltration of CD68 +macrophages at 7 d was the most serious , but then reduced at 14 d.Flow cytometry analysis showed that M 1 macrophages were the majority in the early stages of UUO and IRI, and the result of ELISA identified the higher level of iNOS .At the late stage of injury , the M1 macrophages were de-creased, while the M2 macrophages were increased with higher level of Arg-1.M1 macrophage-mediated early injury was due to the induction of TNF-αexpression, and M2 macrophage-mediated later recovery was due to enhancing TGF-β1 levels.CONCLUSION:The polarization of M1 and M2 macrophages is involved in the processes of UUO and IRI .M1 macrophages play a key role in early injury , and M2 macrophages contribute to the late stage of fibrotic repair .The polari-zation of macrophages during renal injury and repair provides a guiding significance for the clinical treatment .
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AIM:To investigate the effect of dopamine ( DA) on the glutamate ( Glu)-uptake ability of astro-cytes, and the role of mammalian target of rapamycin (mTOR)-excitatory amino acid transporter 2(EAAT2) pathway in this process .METHODS:Extracellular Glu levels in DA-treated primary cortical astrocytes ( PCAs) were measured by a fluorimetric method .The relative expression of EAAT 2 and mTOR at mRNA and protein levels was measured by RT-qPCR and Western blot .PCAs stimulated with or without DA in the presence or absence of mTOR antagonist rapamycin or mTOR agonist MHY1485 were used to determine the expression of mTOR and EAAT 2, and Glu content in the culture supernatant was also measured.RESULTS: The expression of mTOR in DA-treated PCAs was decreased, the expression of EAAT2 was also decreased .Extracellular Glu levels of DA-treated PCAs were elevated significantly .When the PCAs were stimula-ted with DA in the presence of rapamycin , the expression of EAAT2 was decreased , and the levels of extracellular Glu was significantly increased.In the presence of MHY1485, the expression of EAAT2 was elevated, and significant decrease in the levels of extracellular Glu was also observed .CONCLUSION:DA interacts with mTOR-EAAT2 pathway to reduce the Glu-uptake ability of the astrocytes , and causes extracellular Glu accumulation , ultimately destroys the function of astro-cytes.
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AIM:To investigate the effect of dopamine ( DA) on the glutamate ( Glu)-uptake ability of astro-cytes, and the role of mammalian target of rapamycin (mTOR)-excitatory amino acid transporter 2(EAAT2) pathway in this process .METHODS:Extracellular Glu levels in DA-treated primary cortical astrocytes ( PCAs) were measured by a fluorimetric method .The relative expression of EAAT 2 and mTOR at mRNA and protein levels was measured by RT-qPCR and Western blot .PCAs stimulated with or without DA in the presence or absence of mTOR antagonist rapamycin or mTOR agonist MHY1485 were used to determine the expression of mTOR and EAAT 2, and Glu content in the culture supernatant was also measured.RESULTS: The expression of mTOR in DA-treated PCAs was decreased, the expression of EAAT2 was also decreased .Extracellular Glu levels of DA-treated PCAs were elevated significantly .When the PCAs were stimula-ted with DA in the presence of rapamycin , the expression of EAAT2 was decreased , and the levels of extracellular Glu was significantly increased.In the presence of MHY1485, the expression of EAAT2 was elevated, and significant decrease in the levels of extracellular Glu was also observed .CONCLUSION:DA interacts with mTOR-EAAT2 pathway to reduce the Glu-uptake ability of the astrocytes , and causes extracellular Glu accumulation , ultimately destroys the function of astro-cytes.
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<p><b>OBJECTIVE</b>To investigate the safety and effectiveness of neonatal ABO or Rh(D) by using compatible blood transfusion through retrospective analysis of data from cases received compatible blood transfusion and type matched blood transfusion.</p><p><b>METHODS</b>The clinical data of 26 cases of neonatal compatible blood transfusion in Chinese Nanchang area from January 2014 to October 2016 were collected, and 26 cases of neonatal type-matched blood transfusion were selected according to ratio of 1:1 cases. The efficiency and safety index of 26 patients compatible blood transfusion were compared with that of type-matched blood transfusion. The efficiency indexes included: patients' basic characteristics, red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Hct), and the safety indexes contain Hb level and indirect bilirubin (IBiL) value before and after blood transfusion, irregular antibody screening, direct antiglobulin test (DAT) results and the adverse reactions of blood transfusion.</p><p><b>RESULTS</b>The age, sex, days of hospitalization between compatible blood transfusion and type matched blood transfusion were not statistically significantly different (P>0.05). The Hb level before transfusion, blood transfusion volume and the increase of Hb, Hct and RBC were not statistically significantly different between two groups (P>0.05). The values of Hb, Hct and RBC in 2 groups significantly increased at the day 1 after blood transfusion (P<0.05). No blood transfusion adverse reaction occurred in 2 groups. The IBiL value significantly decreased in compatible blood transfusion patients at the day 1 after blood transfusion (P<0.05). No new irregular antibodies had been detected after transfusion in all patients, and the others' DAT and screening for irregular antibodies were negative except 22 patients with neonatal hemolysis. The values of Hb and IBiL statistically significantly differenence were not in 12 patients between 1d, 3d, 7d after blood transfusion (P>0.05).</p><p><b>CONCLUSION</b>The efficiency and safety between compatible blood transfusion and type matched blood transfusion are the same in neonatal blood transfusion. Compatible blood transfusion is a safe and effective in clinical blood transfusion.</p>
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Objective To explore the therapeutic effect and nursing points of different linked modes in hemodialysis combined with hemoperfusion on renal osteopathy disease in maintenance hemodialysis (MHD) patients.Methods Sixty-four MHD patients with renal osteopathy disease were enrolled in the study.They were divided into two groups by random digits table method:the front connection group and the back connection group with 32 cases each.Their clinical data,serum parameters and complications were recorded.The therapeutic effect of two groups were compared.Results The serum level of intact parathyroid hormone(iPTH) was (191.36 ± 53.96) ng/L in the front connection group,while (133.02 ± 38.63) ng/L in the back connection group,and there was significant difference,t=3.517,P＜0.05.The incidence of lower blood pressure and blood coagulation were 8.85％(51/576),6.94％(40/576) in the back connection group,while 14.93％(86/576),15.97％(92/576) in the front connection group,and there were significant differences,x2=10.149,23.136,P＜0.01.Conclusion The back linked mode in hemodialysis combined with hemoperfusion can obviously decrease iP:TH of MHD patients combined with renal osteopathy disease and has less complications,worth clinical promotion.
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<p><b>OBJECTIVE</b>To observe a turning performance in the rats excited by using a proper electrical stimuli of the barrel cortex region (BC), and the expression of choline acetyltransferase (ChAT) in the BC regions after electoral stimulation.</p><p><b>METHODS</b>SD rats were divided into three groups. The stimulation electrodes were surgically implanted into the bilateral BC regions in the control group and the experimental group rats. The experiment group post surgery for seven days was given the electrical impulses via connection with the electrodes for three times each day through consecutive three days. Three groups of the rats were killed and the brains were quickly removed for frozen sections and then performed with conventional HE and immunohistochemistry staining. And protein samples were prepared from brain and the hippocampus tissues of the three groups to detect the level of the ChAT protein by Western blot.</p><p><b>RESULTS</b>The experimental rats turn left or right when continuously stimulation in the bilateral BC regions with electric pulse. HE staining showed no significant damage around electrodes in the cerebral cortex. Compared with the control and blank groups, the ChAT positive rate in the brain section in the experimental rats was significantly high by immunohistochemistry assay; the level of the ChAT protein in the rats given the electrical stimulation increased.</p><p><b>CONCLUSION</b>Turnings performance of the rat could be initiated hy electrical stimuli in the BC region. Expression of ChAT is significantly higher in the BC regions of rat under electrical stimulation, suggesting that acetylcholine might be associated with signal transmission between senses and movement behavior in the nervous central system.</p>
Subject(s)
Animals , Rats , Acetylcholine , Metabolism , Cerebral Cortex , Metabolism , Choline O-Acetyltransferase , Metabolism , Electric Stimulation , Rats, Sprague-DawleyABSTRACT
The clinical data and routine biochemical parameters of 64 maintenance hemodialysis (MHD)patients and 20 controls were analyzed in the study.Serum cystatin C levels were detected by latex particle enhalice inununo-turbidimetry:and the cardial structure and function were assessed by echocardiography.As MHD time extended,the levels of semm cystatin C increased gradually,accompanied with high incidence of left ventricular hypertrophy(LVH).The LVH-positive patients had higher systolic blood pressure and left veHtricular mass index,and had higher serum cystatin C than tllose in LVH-negative patients.The serum cystatin C levels were positively correlated with left yentricular mass index(r=0.633,P<0.01)and systolic blood pressure(r:0.397,P<0.01).The results suggest that serum cystatin levels may be an influencing factor for long-term cardiacvascular complication in MHD patients.
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<p><b>OBJECTIVE</b>To investigate the mRNA and protein expression of nucleostemin (NS) in human esophageal squamous cell carcinoma.</p><p><b>METHODS</b>The mRNA and protein expression of NS were detected in 31 mucosal atypical hyperplasia specimens, 62 esophageal squamous cell carcinoma specimens and the matched normal esophageal mucosa samples by RT-PCR and immunohistochemistry method, respectively.</p><p><b>RESULTS</b>The positive expression rate of NS protein in normal esophageal mucosa, atypical hyperplasia and esophageal squamous cell carcinoma was 17.7% (11/62), 41.9% (13/31) and 69.4% (43/62), respectively. There was a significant difference among the above three groups (chi2 = 33.676, P < 0.01). The expression levels of NS mRNA in esophageal squamous cell carcinoma (0.971 +/- 0.121) was significantly higher than that in the atypical hyperplasia (0.913 +/- 0.085) and also in the normal esophageal mucosa (0.866 +/- 0.103; F = 14.829, P < 0.01). The expression level of both NS protein and mRNA was positively correlated with histological grade, infiltration depth, and lymph node metastasis (P < 0.05), but not with age, gender or pathological type (P > 0.05).</p><p><b>CONCLUSION</b>Our results indicate that nucleostemin mRNA and protein are over-expressed in human esophageal squamous cell carcinoma, and it may be related with its oncogenesis.</p>
Subject(s)
Female , Humans , Male , Middle Aged , Carcinoma, Squamous Cell , Metabolism , Pathology , Carrier Proteins , Genetics , Esophageal Neoplasms , Metabolism , Pathology , Esophagus , Pathology , GTP-Binding Proteins , Gene Expression Regulation, Neoplastic , Hyperplasia , Lymphatic Metastasis , Mucous Membrane , Metabolism , Neoplasm Invasiveness , Neoplasm Staging , Nuclear Proteins , Genetics , Precancerous Conditions , Metabolism , Pathology , RNA, Messenger , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To screen effective sequences of small interfering RNA targeting MDR1 gene in human gastric cancer SGC7901/VCR cells.</p><p><b>METHODS</b>Four siRNAs (MDR1si326, MDR1si1513, MDR1si2631 and MDR1si3071) targeting MDR1 gene were designed and synthesized by in vitro transcription. The siRNA duplexes were used to transfect into the human gastric cancer SGC7901/VCR cells. The expression level of MDR1 mRNA and P-gp were detected by RT-PCR and Western blotting, respectively. The accumulation of intracellular adriamycin (ADR) was examined by flow cytometry and the cell sensitivity to ADR was demonstrated by MTT.</p><p><b>RESULTS</b>The SGC7901/VCR cells treated with 4 siRNAs led to reversal effect on multidrug resistance to different extents. Among the SGC7901/VCR cells treated by siRNAs for 48 h, the expression level of MDR1 mRNA in cells of MDR1si326 or MDR1si2631 group (0.42 +/- 0.07 or 0.49 +/- 0.02) was more decreased than that in cells of MDR1si1513 or MDR1si3071 group (P < 0.05). The accumulation of ADR in cells of MDR1si326 group was the most; in cells of MDR1si2631 group, more; in cells of MDR1si3071 group, lower and in cells of MDR1si1513 group, the lowest (P < 0.05). The relative reversal efficiency of cells of MDR1si2631 group to ADR was the highest and in cells of MDR1si326 group, higher (P < 0.05). There was no significant difference in the relative reversal efficiency between the cells of MDR1si1513 and MDR1si3071 groups (P > 0.05). The expression level of P-gp in cells of MDR1si326 group was the lowest among the SGC7901/VCR cells treated by siRNAs for 72 h.</p><p><b>CONCLUSION</b>The MDR1si326 with most, MDR1si2631 with more, MDR1si3071 with less and MDR1si1513 with least reversal effects on MDR1 gene mediated multidrug resistance were found in the human gastric cancer SGC7901/VCR cells.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Antibiotics, Antineoplastic , Metabolism , Pharmacology , Antineoplastic Agents, Phytogenic , Pharmacology , Cell Line, Tumor , Doxorubicin , Metabolism , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Genes, MDR , RNA, Messenger , Genetics , RNA, Small Interfering , Pharmacology , Stomach Neoplasms , Metabolism , Pathology , Transfection , Vincristine , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of hTERT antisense oligodeoxynucleotide (ASODN) on the oncogenicity and the inductive apoptosis of HL-60 cells.</p><p><b>METHODS</b>Apoptosis of HL-60 cells was detected by flow cytometry (FCM) and agarose gel electrophoresis. Both treated and untreated HL-60 cells were collected and transplanted into 5 BALB/c nude mice respectively, the formation of transplanted neoplasm and its morphologic change were observed. After the transplanted neoplasms were uniform with the ameliorated method in another 10 BALB/c nude mice, they were divided into 2 groups and injected ASODN and PBS into the neoplasm respectively. Seven days later, the tumor were measured, its morphology were observed, and the apoptotic cells were detected with a TUNEL kit.</p><p><b>RESULTS</b>After 72 h treatment there were DNA ladders and early apoptosis peak in hTERT ASODN treated HL-60 cells but was none in SODN treated and blank control cells. In tumor formation experiment, neoplasms were formed in ASODN treated group at 16-17 d and untreated group at 12-13 d. Neoplasm was formed in 2 of 5 ASODN treated mice and 4 of 5 untreated mice respectively. In untreated mice tumor tissues were rich in blood vasa and stromal tissue compared with that in ASODN treated mice. In tumor therapy experiment, before treatment, there was no difference in the average neoplasm physical volume between ASODN treated group [(100.9 +/- 24.6) mm3] and PBS treated group [(98.4 +/- 23.1) mm3] (P > 0.05). After treatment, the neoplasm volume in ASODN treated group [(422.7 +/- 326.4) mm3] was smaller than that in PBS treated group [(786.4 +/- 357.6) mm3] (P < 0.05). Histologically, there were many apoptosis cells in ASODN treated group, but was seldom seen in PBS treated group. The TUNEL positive cells in ASODN treated group were much more than that in PBS treated group (P < 0.05).</p><p><b>CONCLUSION</b>The hTERT ASODN induces apoptosis of HL-60 cells in vitro, reduces the tumor formation in BALB/c nude mice and inhibits the growth of the transplanted neoplasm.</p>
Subject(s)
Animals , Humans , Mice , Apoptosis , HL-60 Cells , Mice, Inbred BALB C , Mice, Nude , Oligodeoxyribonucleotides, Antisense , Pharmacology , Telomerase , Genetics , Transfection , Xenograft Model Antitumor AssaysABSTRACT
In order to investigate the mechanism of acute lymphoblastic leukemic cell malignant proliferation, the expressions of hMSH2 mRNA and mutation P53 (mtP53) protein in bone marrow cells of de novo acute lymphoblastic leukemia (ALL) were determined by in situ hybridization and immunocytochemical methods. The results showed the that percentage of positive cell with hMSH2 mRNA expression was (32.88 +/- 11.46)% in the de novo ALL group and (64.22 +/- 8.51)% in the control group. The percentage of positive cell with mtP53 protein expression was (29.25 +/- 9.45)% in the de novo ALL group, and (12.63 +/- 6.66)% in the control group. There was a significant negative correlation between the positive percentages of hMSH2 mRNA expression and mtP53 protein expression (r = -0.45, P < 0.05). It is concluded that defective MSH2 mRNA expression plays an important role in the pathogenesis of acute lymphoblastic leukemia, mtP53 protein mutation plays an important role in the development of acute lymphoblastic leukemia, the hMSH2 mRNA defect can lead to accumulation of the mutant P53 protein in acute lymphoblastic leukemia, and both jointly promote the pathogenesis of ALL.
Subject(s)
Adult , Female , Humans , Male , Gene Expression Regulation, Neoplastic , Immunohistochemistry , MutS Homolog 2 Protein , Genetics , Mutant Proteins , Mutation , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Genetics , RNA, Messenger , Genetics , Tumor Suppressor Protein p53 , GeneticsABSTRACT
To investigate the effects of c-myc antisense oligodeoxynucleotide (ASODN) on the telomerase activity and the induction of apoptosis in HL-60 cells, and to explore the relationship between the telomerase activity and the expression of c-myc gene in HL-60 cells, after treatment by c-myc ASODN, the expression of c-myc was detected by RT-PCR, the apoptosis, cell cycle were detected with agarose gel electrophoresis and flow cytomety, and the telomerase activity was determined with TRAP-ELISA. The results showed that after blocking c-myc gene with ASODN for 72 hours, it is obvious that the expression of c-myc gene was inhibited. The percentage of S phase HL-60 cells decreased from 55.6% to 30%, the early apoptosis peak appeared (the percentage of apoptosis cells were 25.2%) and the DNA ladders were shown. OD(450 - 690) were 2.648 +/- 0.42, 2.324 +/- 0.36, 2.162 +/- 0.38, 1.952 +/- 0.14, 1.805 +/- 0.40, 1.616 +/- 0.41 and 2.466 +/- 0.29, respectively, as the cells were treated with 0, 1, 2, 3, 4, 5 micromol/L ASODN and 5 micromol/L SODN for 72 hours. The difference was significant when compared 3, 4, 5 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 1, 2 micromol/L ASODN and 5 micromol/L SODN groups with 0 micromol/L ASODN group (P > 0.05). It is concluded that the c-myc gene ASODN may induce the apoptosis of cells, inhibit cells from G(1) phase into S phase and regulate the telomerase activity down in HL-60 cells by blocking the expression of c-myc gene.
Subject(s)
Humans , Apoptosis , Dose-Response Relationship, Drug , Flow Cytometry , HL-60 Cells , Oligonucleotides, Antisense , Genetics , Pharmacology , Proto-Oncogene Proteins c-myc , Genetics , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Telomerase , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To detect methylation in promoter region of hMSH2 gene in esophageal cancer.</p><p><b>METHODS</b>Specimens of cancer and normal tissues freshly removed from 32 cases of esophageal cancer patients without previous radiotherapy, chemotherapy or other treatment were preserved at -80 degrees C within 30 min. Methylation specific PCR (MSP) was used to detect methylation of mismatch repair gene (MMR) hMSH2 in promoter region in esophageal cancer and normal esophageal tissues.</p><p><b>RESULTS</b>The frequencies of methylation of hMSH2 gene in promoter region of cancer and normal esophageal tissues were 32.4% (11/32) and 0/30 (0%), respectively, and significant difference was found between the two groups (P < 0.01). The frequency of methylation in elder patients (> or = 70 years old) was significantly higher than that in younger patients (< 70 years old) (P < 0.05). Methylation was less frequently found in grade I-II (18.2%) than in grade III-IV (70.0%) (P < 0.05).</p><p><b>CONCLUSION</b>Methylation of hMSH2 gene in promoter region is related to patients' age and histopathological grade of the esophageal cancer.</p>
Subject(s)
Aged , Female , Humans , Male , Base Pair Mismatch , Carcinoma, Squamous Cell , Genetics , Pathology , DNA Methylation , Esophageal Neoplasms , Genetics , Pathology , Gene Expression Regulation, Neoplastic , MutS Homolog 2 Protein , Genetics , Promoter Regions, Genetic , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the association between attention-deficit hyperactivity disorder (ADHD) in Han Chinese children and Val158Met polymorphism of catechol-O-methyltransferase (COMT) gene caused by the missense mutation of G158A in exon 4.</p><p><b>METHODS</b>By using polymerase chain reaction-restriction fragment length polymorphisms the Val158Met polymorphism of COMT gene was tested in 117 children with the diagnosis of ADHD as defined by DSM-IV and in 105 healthy controls living in Shanghai.</p><p><b>RESULTS</b>The frequencies of A allele were 25.21% and 23.81% in the ADHD group and the health controls respectively, which showed no significant difference between the two groups (Chi2=0.5197, P>0.05). There was also no significant difference in the distribution of all genotypes of COMT gene between the ADHD patients and the controls (P>0.05).</p><p><b>CONCLUSION</b>It was suggested that for the Han Chinese children with ADHD in this study, there was no association between ADHD and Val158Met polymorphism of COMT gene.</p>