In this study,we investigated how Oroxylum indicum leaf and fruit extracts affect the viability and migration of MCF-7 breast cancer cells and the mechanisms of action responsible for these effects.MCF-7 cells treated...In this study,we investigated how Oroxylum indicum leaf and fruit extracts affect the viability and migration of MCF-7 breast cancer cells and the mechanisms of action responsible for these effects.MCF-7 cells treated with the extracts were examined using the sulforhodamine B,colony formation and caspase 3 activity assays,and by Western blotting.O.indicum extracts were found to inhibit MCF-7 cell growth in a concentration-and time-dependent manner,with 48 h IC50 values of 57.02±2.85μg/mL and 131.3±19.2μg/mL for leaf and fruit extracts,respectively.Further,the O.indicum leaf extract caused a reduction in MCF-7 cell viability,induction of MCF-7 cell apoptosis and ROS formation,and an increase in caspase 3 activity.Also,the two extracts inhibited MCF-7 cell migration and reduced both MMP 9 and ICAMP1 gene expression and MMP9 protein expression.Additionally,O.indicum extracts greatly reduced expression of the cell cycle regulatory protein Rac1 in the mevalonate pathway.In summary,O.indicum leaf and fruit extracts reduce breast cancer cell growth,cell viability and cell migration.O.indicum constituents could,therefore,be useful for augmenting the activity of chemotherapeutic drugs employed to treat breast cancer.展开更多
Exposure to explosive shockwave often leads to blast-induced traumatic brain injury in military and civilian populations.Unprotected ears are most often damaged following exposure to blasts.Although there is an associ...Exposure to explosive shockwave often leads to blast-induced traumatic brain injury in military and civilian populations.Unprotected ears are most often damaged following exposure to blasts.Although there is an association between tympanic membrane perforation and TBI in blast exposure victims,little is known about how and to what extent blast energy is transmitted to the central nervous system via the external ear canal.The present study investigated whether exposure to blasts directed through the ear canal causes brain injury in LongEvans rats.Animals were exposed to a single blast(0–30 pounds per square inch(psi))through the ear canal,and brain injury was evaluated by histological and behavioral outcomes at multiple time-points.Blast exposure not only caused tympanic membrane perforation but also produced substantial neuropathological changes in the brain,including increased expression of c-Fos,induction of a profound chronic neuroinflammatory response,and apoptosis of neurons.The blast-induced injury was not limited only to the brainstem most proximal to the source of the blast,but also affected the forebrain including the hippocampus,amygdala and the habenula,which are all involved in cognitive functions.Indeed,the animals exhibited long-term neurological deficits,including signs of anxiety in open field tests 2 months following blast exposure,and impaired learning and memory in an 8-arm maze 12 months following blast exposure.These results suggest that the unprotected ear canal provides a locus for blast waves to cause TBI.This study was approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center(Animal protocol#0932 E,approval date:September 30,2016 and 0932 F,approval date:September 27,2019).展开更多
Objective To describe the epidemiologic,clinical,laboratory,and radiological characteristics and prognoses of COVID-19 confirmed patients in a single center in Beijing,China.Methods The study retrospectively included ...Objective To describe the epidemiologic,clinical,laboratory,and radiological characteristics and prognoses of COVID-19 confirmed patients in a single center in Beijing,China.Methods The study retrospectively included 19 patients with nucleic acid-confirmed SARS-CoV^2 infection at our hospital from January 20 to March 5,2020.The final follow-up date was March 14,2020.The epidemiologic and clinical information was obtained through direct communication with the patients or their family members.Laboratory results retrieved from medical records and radiological images were analyzed both qualitatively by two senior chest radiologists as well as quantitatively via an artificial intelligence software.Results We identified 5 family clusters(13/19,68.4%)from the study cohort.All cases had good clinical prognoses and were either mild(3/19)or moderate(16/19)clinical types.Fever(15/19,78.9%)and dry cough(11/19,57.9%)were common symptoms,Two patients received negative results for more than three consecutive viral nucleic acid tests.The longest interval between an initial CT abnormal finding and a confirmed diagnosis was 30 days.One patient's nucleic acid test turned positive on the follow-up examination after discharge.The presence of radiological abnormalities was non-specific for the diagnosis of COVID-19.Conclusions COVID-19 patients with mild or no clinical symptoms are common in Beijing,China.Radiological abnormalities are mostly non-specific and massive CT examinations for COVID-19 screening should be avoided.Analyses of the contact histories of diagnosed cases in combination with clinical,radiological and laboratory findings are crucial for the early detection of COVID-19.Close monitoring after discharge is also recommended.展开更多
BACKGROUND Mucoepidermoid carcinoma(MEC)is a rare malignancy of the head and neck;however,it accounts for a majority of the tumors of the salivary glands.This study used a national population-based registry to describ...BACKGROUND Mucoepidermoid carcinoma(MEC)is a rare malignancy of the head and neck;however,it accounts for a majority of the tumors of the salivary glands.This study used a national population-based registry to describe the pre-treatment and treatment-related prognostic factors that influence survival in patients with MEC of the major salivary glands.To our knowledge,this is the largest populationbased study examining predictors of both overall and cause-specific survival of MEC of the major salivary glands.AIM To identify prognostic factors influencing overall survival(OS)and cause-specific survival(CSS)of patients with MEC of the major salivary glands.METHODS We used the Surveillance,Epidemiology and End-Results Database of the National Cancer Institute to investigate a variety of factors that could influence survival of patients diagnosed with mucoepidermoid carcinoma of the major salivary glands.A total of 2210 patients diagnosed with MEC of the major salivary glands during the years of 1975-2016 were studied.The primary endpoints were OS and CSS.Cox regression analysis was used to perform univariate and multivariate analyses of clinical variables such as age at diagnosis,diagnosis year,sex,race,tumor size,stage,grade,treatment with or without surgical excision,and adjuvant radiotherapy treatment.RESULTS A total of 2210 patients diagnosed with MEC of the major salivary glands met inclusion criteria.In this study,95%of patients underwent surgical excision and 41%received adjuvant radiation therapy.Median OS time for Grade I,II,and III/IV was 401 mo(±48.25,95%CI),340 mo(±33.68,95%CI)and 55 mo(±11.05,95%CI),respectively.Univariate analysis revealed that lack of surgical excision was associated with decreased OS[hazard ratio(HR)4.26,P<0.0001]and that patients with localized disease had improved OS compared to both regional and distant disease(HR 3.07 and 6.96,respectively,P<0.0001).Additionally,univariate analysis demonstrated that male sex,age over 50 at diagnosis,Grade III tumors,and increasing tumor size were associated with worsened OS(P<0.0006).Univariate analysis of CSS similarly revealed that lack of surgical excision and Grade III carcinoma conferred decreased CSS(HR 4.37 and 5.44,respectively,P<0.0001).Multivariate analysis confirmed that increasing age,in 10-year age bands,advanced tumor stage,increasing tumor size,Grade III carcinoma,male sex,and lack of surgical excision were associated with a statistically significant decrease in OS and CSS(P<0.04).Of note,multivariate analysis revealed that the use of adjuvant radiation therapy was not associated with improved OS or CSS.CONCLUSION Multivariate analysis demonstrated increasing age,advanced tumor stage,increasing tumor size,Grade III carcinoma,male sex,and lack of surgical excision were associated with decreased OS and CSS(P<0.04).展开更多
In this study, the distribution of five Alzheimer's disease(AD)-related single nucleotide polymorphisms(SNPs) in the Han population was examined in combination with the evaluation of clinical cognition and brain p...In this study, the distribution of five Alzheimer's disease(AD)-related single nucleotide polymorphisms(SNPs) in the Han population was examined in combination with the evaluation of clinical cognition and brain pathological analysis. The associations among SNPs,clinical daily cognitive states, and postmortem neuropathological changes were analyzed in 110 human brains from the Chinese Academy of Medical Sciences/Peking Union Medical College(CAMS/PUMC) Human Brain Bank.APOE ?4(OR = 4.482, P = 0.004), the RS2305421 GG genotype(adjusted OR = 4.397, P = 0.015), and the RS10498633 GT genotype(adjusted OR = 2.375,P = 0.028) were associated with a higher score on the ABC(Ab plaque score, Braak NFT stage, and CERAD neuritic plaque score) dementia scale. These results advance our understanding of the pathogenesis of AD,the relationship between pathological diagnosis and clinical diagnosis, and the SNPs in the Han population for future research.展开更多
BACKGROUND： Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor ne...BACKGROUND： Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor neuron axons is implicated in several debilitating disorders including hereditary spastic paraplegia （HSP）. Since the discovery of the first HSP gene, SPASTthat encodes spastin, over 70 distinct genetic loci associated with HSP have been identified. How the mutations of these functionally diverse genes result in axonal degeneration and why certain axons are affected in HSP remain largely unknown. The development of induced pluripotent stem cell （iPSC） technology has provided researchers an excellent resource to generate patient-specific human neurons to model human neuropathological processes including axonal defects. METHODS： In this article, we will first review the pathology and pathways affected in the common forms of liSP subtypes by searching the PubMed database. We will then summarize the findings and insights gained from studies using iPSC-based models, and discuss challenges and future directions. RESULTS： HSPs, a heterogeneous group of genetic neurodegenerative disorders, exhibit similar pathological changes that result from retrograde axonal degeneration of cortical motor neurons. Recently, iPSCs have been generated from several common forms of HSP including SPG4, SPG3A, and SPG11 patients. Neurons derived from HSP iPSCs exhibit impaired neurite outgrowth, increased axonal swellings, and reduced axonal transport, recapitulating disease-specific axonal defects. CONCLUSIONS： These patient-derived neurons offer a unique tool to study the pathogenic mechanisms and explore the treatments for rescuing axonal defects in HSP, as well as other diseases involving axonopathy.展开更多
Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide,but the molecular basis underlying its development remains unclear.An accumulating body of evidence supports gasdermin D(G...Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide,but the molecular basis underlying its development remains unclear.An accumulating body of evidence supports gasdermin D(GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases.However,there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis.In this work,we investigated the role of GSDMD-mediated pyroptosis in silicosis.Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression.Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica.Measurement of interleukin-1βrelease,lactic dehydrogenase activity,and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages.Additionally,we verified that both canonical(caspase-1-mediated)and non-canonical(caspase-4/5/11-mediated)signaling pathways mediated silica-induced pyroptosis activation,in vivo and in vitro.Notably,Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes,which highlighted the pivotal role of pyroptosis in this disease.Taken together,our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.展开更多
Dear Editor Research on neurodegenerative diseases is a hot topic worldwide[1],and MRI,genetics and epigenetics,and animal models have been commonly used.Considering species differences,research on human brain samples...Dear Editor Research on neurodegenerative diseases is a hot topic worldwide[1],and MRI,genetics and epigenetics,and animal models have been commonly used.Considering species differences,research on human brain samples is irreplaceable(2)Many countries and regions have built brain banks to collect and store brain tissue from donors.Samples provided by a brain bank can be used to study synaptic structure,protein,DNA,RNA[3,4],and lipids to illuminate the pathological mechanisms underlying neurodegenerative diseases.When brain tissue is used for gene expression research,the quantitative real-time polymerase chain reaction(RT-qPCR)is an accurate method,which simultaneously amplifies and quantifies the expression of target genes by measuring the intensity of fluorescence in each PCR cycle.展开更多
基金financially supported by Mahasarakham University(Fast Track 2019)。
文摘In this study,we investigated how Oroxylum indicum leaf and fruit extracts affect the viability and migration of MCF-7 breast cancer cells and the mechanisms of action responsible for these effects.MCF-7 cells treated with the extracts were examined using the sulforhodamine B,colony formation and caspase 3 activity assays,and by Western blotting.O.indicum extracts were found to inhibit MCF-7 cell growth in a concentration-and time-dependent manner,with 48 h IC50 values of 57.02±2.85μg/mL and 131.3±19.2μg/mL for leaf and fruit extracts,respectively.Further,the O.indicum leaf extract caused a reduction in MCF-7 cell viability,induction of MCF-7 cell apoptosis and ROS formation,and an increase in caspase 3 activity.Also,the two extracts inhibited MCF-7 cell migration and reduced both MMP 9 and ICAMP1 gene expression and MMP9 protein expression.Additionally,O.indicum extracts greatly reduced expression of the cell cycle regulatory protein Rac1 in the mevalonate pathway.In summary,O.indicum leaf and fruit extracts reduce breast cancer cell growth,cell viability and cell migration.O.indicum constituents could,therefore,be useful for augmenting the activity of chemotherapeutic drugs employed to treat breast cancer.
基金supported by the National Institutes of Health(NIH)grants R21 DC017293(to HZ,WZ),R01 DC018919(to HZ,WZ),AG050049(to FF),AG057842(to FF),P20GM104357(to FF,RJR),and HL138685(to RJR)。
文摘Exposure to explosive shockwave often leads to blast-induced traumatic brain injury in military and civilian populations.Unprotected ears are most often damaged following exposure to blasts.Although there is an association between tympanic membrane perforation and TBI in blast exposure victims,little is known about how and to what extent blast energy is transmitted to the central nervous system via the external ear canal.The present study investigated whether exposure to blasts directed through the ear canal causes brain injury in LongEvans rats.Animals were exposed to a single blast(0–30 pounds per square inch(psi))through the ear canal,and brain injury was evaluated by histological and behavioral outcomes at multiple time-points.Blast exposure not only caused tympanic membrane perforation but also produced substantial neuropathological changes in the brain,including increased expression of c-Fos,induction of a profound chronic neuroinflammatory response,and apoptosis of neurons.The blast-induced injury was not limited only to the brainstem most proximal to the source of the blast,but also affected the forebrain including the hippocampus,amygdala and the habenula,which are all involved in cognitive functions.Indeed,the animals exhibited long-term neurological deficits,including signs of anxiety in open field tests 2 months following blast exposure,and impaired learning and memory in an 8-arm maze 12 months following blast exposure.These results suggest that the unprotected ear canal provides a locus for blast waves to cause TBI.This study was approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center(Animal protocol#0932 E,approval date:September 30,2016 and 0932 F,approval date:September 27,2019).
基金This study has received funding from the Beijing Municipal Science and Technology Planning Project(Grant No.Z211100003521009).
文摘Objective To describe the epidemiologic,clinical,laboratory,and radiological characteristics and prognoses of COVID-19 confirmed patients in a single center in Beijing,China.Methods The study retrospectively included 19 patients with nucleic acid-confirmed SARS-CoV^2 infection at our hospital from January 20 to March 5,2020.The final follow-up date was March 14,2020.The epidemiologic and clinical information was obtained through direct communication with the patients or their family members.Laboratory results retrieved from medical records and radiological images were analyzed both qualitatively by two senior chest radiologists as well as quantitatively via an artificial intelligence software.Results We identified 5 family clusters(13/19,68.4%)from the study cohort.All cases had good clinical prognoses and were either mild(3/19)or moderate(16/19)clinical types.Fever(15/19,78.9%)and dry cough(11/19,57.9%)were common symptoms,Two patients received negative results for more than three consecutive viral nucleic acid tests.The longest interval between an initial CT abnormal finding and a confirmed diagnosis was 30 days.One patient's nucleic acid test turned positive on the follow-up examination after discharge.The presence of radiological abnormalities was non-specific for the diagnosis of COVID-19.Conclusions COVID-19 patients with mild or no clinical symptoms are common in Beijing,China.Radiological abnormalities are mostly non-specific and massive CT examinations for COVID-19 screening should be avoided.Analyses of the contact histories of diagnosed cases in combination with clinical,radiological and laboratory findings are crucial for the early detection of COVID-19.Close monitoring after discharge is also recommended.
基金Supported by Community Cancer Fund in Spokane,Washington,United States.
文摘BACKGROUND Mucoepidermoid carcinoma(MEC)is a rare malignancy of the head and neck;however,it accounts for a majority of the tumors of the salivary glands.This study used a national population-based registry to describe the pre-treatment and treatment-related prognostic factors that influence survival in patients with MEC of the major salivary glands.To our knowledge,this is the largest populationbased study examining predictors of both overall and cause-specific survival of MEC of the major salivary glands.AIM To identify prognostic factors influencing overall survival(OS)and cause-specific survival(CSS)of patients with MEC of the major salivary glands.METHODS We used the Surveillance,Epidemiology and End-Results Database of the National Cancer Institute to investigate a variety of factors that could influence survival of patients diagnosed with mucoepidermoid carcinoma of the major salivary glands.A total of 2210 patients diagnosed with MEC of the major salivary glands during the years of 1975-2016 were studied.The primary endpoints were OS and CSS.Cox regression analysis was used to perform univariate and multivariate analyses of clinical variables such as age at diagnosis,diagnosis year,sex,race,tumor size,stage,grade,treatment with or without surgical excision,and adjuvant radiotherapy treatment.RESULTS A total of 2210 patients diagnosed with MEC of the major salivary glands met inclusion criteria.In this study,95%of patients underwent surgical excision and 41%received adjuvant radiation therapy.Median OS time for Grade I,II,and III/IV was 401 mo(±48.25,95%CI),340 mo(±33.68,95%CI)and 55 mo(±11.05,95%CI),respectively.Univariate analysis revealed that lack of surgical excision was associated with decreased OS[hazard ratio(HR)4.26,P<0.0001]and that patients with localized disease had improved OS compared to both regional and distant disease(HR 3.07 and 6.96,respectively,P<0.0001).Additionally,univariate analysis demonstrated that male sex,age over 50 at diagnosis,Grade III tumors,and increasing tumor size were associated with worsened OS(P<0.0006).Univariate analysis of CSS similarly revealed that lack of surgical excision and Grade III carcinoma conferred decreased CSS(HR 4.37 and 5.44,respectively,P<0.0001).Multivariate analysis confirmed that increasing age,in 10-year age bands,advanced tumor stage,increasing tumor size,Grade III carcinoma,male sex,and lack of surgical excision were associated with a statistically significant decrease in OS and CSS(P<0.04).Of note,multivariate analysis revealed that the use of adjuvant radiation therapy was not associated with improved OS or CSS.CONCLUSION Multivariate analysis demonstrated increasing age,advanced tumor stage,increasing tumor size,Grade III carcinoma,male sex,and lack of surgical excision were associated with decreased OS and CSS(P<0.04).
基金supported by grants from the National Natural Science Foundation of China(81271239,81771205,and 91632113)the Institute of Basic Medical Sciences/Chinese Academy of Medical Sciences(CAMS)Dean’s Fund(2011RC01)+1 种基金the CAMS Innovation Fund for Medical Sciences(2016-I2M-1004)the Natural Science Foundation and Major Basic Research Program of Shanghai Municipality,China(16JC1420500 and 16JC1420502)
文摘In this study, the distribution of five Alzheimer's disease(AD)-related single nucleotide polymorphisms(SNPs) in the Han population was examined in combination with the evaluation of clinical cognition and brain pathological analysis. The associations among SNPs,clinical daily cognitive states, and postmortem neuropathological changes were analyzed in 110 human brains from the Chinese Academy of Medical Sciences/Peking Union Medical College(CAMS/PUMC) Human Brain Bank.APOE ?4(OR = 4.482, P = 0.004), the RS2305421 GG genotype(adjusted OR = 4.397, P = 0.015), and the RS10498633 GT genotype(adjusted OR = 2.375,P = 0.028) were associated with a higher score on the ABC(Ab plaque score, Braak NFT stage, and CERAD neuritic plaque score) dementia scale. These results advance our understanding of the pathogenesis of AD,the relationship between pathological diagnosis and clinical diagnosis, and the SNPs in the Han population for future research.
文摘BACKGROUND： Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor neuron axons is implicated in several debilitating disorders including hereditary spastic paraplegia （HSP）. Since the discovery of the first HSP gene, SPASTthat encodes spastin, over 70 distinct genetic loci associated with HSP have been identified. How the mutations of these functionally diverse genes result in axonal degeneration and why certain axons are affected in HSP remain largely unknown. The development of induced pluripotent stem cell （iPSC） technology has provided researchers an excellent resource to generate patient-specific human neurons to model human neuropathological processes including axonal defects. METHODS： In this article, we will first review the pathology and pathways affected in the common forms of liSP subtypes by searching the PubMed database. We will then summarize the findings and insights gained from studies using iPSC-based models, and discuss challenges and future directions. RESULTS： HSPs, a heterogeneous group of genetic neurodegenerative disorders, exhibit similar pathological changes that result from retrograde axonal degeneration of cortical motor neurons. Recently, iPSCs have been generated from several common forms of HSP including SPG4, SPG3A, and SPG11 patients. Neurons derived from HSP iPSCs exhibit impaired neurite outgrowth, increased axonal swellings, and reduced axonal transport, recapitulating disease-specific axonal defects. CONCLUSIONS： These patient-derived neurons offer a unique tool to study the pathogenic mechanisms and explore the treatments for rescuing axonal defects in HSP, as well as other diseases involving axonopathy.
基金supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(CIFMSNos.2021-1-I2M-049 and 2018-I2M-1-001,China)+1 种基金the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(Nos.2019RC330001 and 2021RC310002,China)National Natural Science Foundation of China(No.82090010)。
文摘Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide,but the molecular basis underlying its development remains unclear.An accumulating body of evidence supports gasdermin D(GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases.However,there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis.In this work,we investigated the role of GSDMD-mediated pyroptosis in silicosis.Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression.Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica.Measurement of interleukin-1βrelease,lactic dehydrogenase activity,and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages.Additionally,we verified that both canonical(caspase-1-mediated)and non-canonical(caspase-4/5/11-mediated)signaling pathways mediated silica-induced pyroptosis activation,in vivo and in vitro.Notably,Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes,which highlighted the pivotal role of pyroptosis in this disease.Taken together,our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.
基金supported by grants from the National Natural Science Foundation of China (81271239, 81771205, and 91632113)the Natural Science Foundation and Major Basic Research Program of Shanghai Municipality, China (16JC1420500 and 16JC1420502)+1 种基金the CAMS Innovation Fund for Medical Sciences (2017-I2M-3-008)sponsored by the China Human Brain Bank Consortium
文摘Dear Editor Research on neurodegenerative diseases is a hot topic worldwide[1],and MRI,genetics and epigenetics,and animal models have been commonly used.Considering species differences,research on human brain samples is irreplaceable(2)Many countries and regions have built brain banks to collect and store brain tissue from donors.Samples provided by a brain bank can be used to study synaptic structure,protein,DNA,RNA[3,4],and lipids to illuminate the pathological mechanisms underlying neurodegenerative diseases.When brain tissue is used for gene expression research,the quantitative real-time polymerase chain reaction(RT-qPCR)is an accurate method,which simultaneously amplifies and quantifies the expression of target genes by measuring the intensity of fluorescence in each PCR cycle.
