We managed general anesthesia for transcatheter aortic valve replacement (TAVR) under elective extracorporeal membrane oxygenation (ECMO) in a patient with aortic valve stenosis (AS) complicated with acute decompensated heart failure. The patient was an 87-year-old woman with acute heart failure due to severe AS who had been hospitalized. However, her low cardiac output did not improve, and weaned her off catecholamines was difficult, so semi-urgent TAVR was performed. Due to her acute decompensated heart failure complicated by low-left ventricular function, we decided electively to use ECMO for transfemoral TAVR to prevent hemodynamic collapse during induction of anesthesia and surgery, enabling the safe perioperative management of this patient under general anesthesia.
{"title":"Anesthetic Management of Transcatheter Aortic Valve Replacement under Extracorporeal Membrane Oxygenation in a Patient with Acute Decompensated Heart Failure: A Case Report.","authors":"Takuya Okada, Takuya Yoshida, Shohei Makino, Norihiko Obata, Satoshi Mizobuchi","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>We managed general anesthesia for transcatheter aortic valve replacement (TAVR) under elective extracorporeal membrane oxygenation (ECMO) in a patient with aortic valve stenosis (AS) complicated with acute decompensated heart failure. The patient was an 87-year-old woman with acute heart failure due to severe AS who had been hospitalized. However, her low cardiac output did not improve, and weaned her off catecholamines was difficult, so semi-urgent TAVR was performed. Due to her acute decompensated heart failure complicated by low-left ventricular function, we decided electively to use ECMO for transfemoral TAVR to prevent hemodynamic collapse during induction of anesthesia and surgery, enabling the safe perioperative management of this patient under general anesthesia.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 3","pages":"E90-E94"},"PeriodicalIF":0.0,"publicationDate":"2019-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012320/pdf/kobej-65-e90.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37619188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible.
Methods: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2.
Results: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing.
Conclusion: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.
背景:利用滤纸上的干血斑(DBS)样本DNA进行聚合酶链反应(PCR)分析是脊髓性肌萎缩症(SMA)新生儿筛查的一项关键技术。然而,从DBS中提取DNA非常耗时,而且从DBS中去除PCR抑制剂几乎是不可能的。方法:用DBS纸打孔圈PCR扩增5例SMA患者和5例对照的2个同源SMA相关基因SMN - 1和SMN2的外显子7。测试了两种DNA制备方法;DNA提取(将提取的DNA加入PCR管中)和非DNA提取(将打好的DBS环放入PCR管中)。在DNA聚合酶方面,比较了两种不同的酶;TaKaRa Ex Taq™和KOD FX Neo™。为检验PCR产物的诊断质量,采用DraI酶切法进行限制性内切片段长度多态性(RFLP)分析,区分SMN1和SMN2。结果:对提取的DNA进行PCR, TaKaRa Ex Taq™和KOD FX Neo™均能充分扩增,扩增效率无显著差异。在直接PCR中,使用KOD FX Neo™聚合酶可以获得充分的扩增,而使用TaKaRa Ex Taq™则没有扩增。KOD FX Neo™直接PCR产物的RFLP分析清楚地分离了SMN1和SMN2序列,并证明对照组中同时存在SMN1和SMN2,而SMA患者中仅存在SMN2,这表明KOD FX Neo™直接PCR产物具有足够的SMA诊断质量。结论:采用KOD FX NeoTM等DNA聚合酶进行直接PCR,避免了DBS DNA的提取过程,且在存在PCR抑制剂的情况下仍能精确扩增目标序列,具有在不久的将来广泛应用于SMA新生儿筛查的潜力。
{"title":"Newborn Screening for Spinal Muscular Atrophy: DNA Preparation from Dried Blood Spot and DNA Polymerase Selection in PCR.","authors":"Atsuko Takeuchi, Chisato Tode, Masayoshi Nishino, Yogik Onky Silvana Wijaya, Emma Tabe Eko Niba, Hiroyuki Awano, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Poh San Lai, Yoshihiro Bouike, Hisahide Nishio, Masakazu Shinohara","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible.</p><p><strong>Methods: </strong>Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2.</p><p><strong>Results: </strong>In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing.</p><p><strong>Conclusion: </strong>Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 3","pages":"E95-E99"},"PeriodicalIF":0.0,"publicationDate":"2019-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012323/pdf/kobej-65-e95.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37619189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Puguh Indrasetiawan, Chie Aoki-Utsubo, Muhammad Hanafi, Sri Hartati, Tutik Sri Wahyuni, Masanori Kameoka, Yoshihiko Yano, Hak Hotta, Yoshitake Hayashi
Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Current therapeutic drugs for chronic hepatitis B using pegylated interferons and nucleos(t)ide analogs have limited efficacy. Therefore, the development of novel and safe antivirals is required. Natural products including medicinal plants produce complex and structurally diverse compounds, some of which offer suitable targets for antiviral screening studies. In the present study, we screened various crude extracts from Indonesian plants for anti-HBV activity by determining their effects on the production of extracellular HBV DNA in Hep38.7-Tet cells and HBV entry onto a HBV-susceptible cell line, HepG2-NTCP, with the following results: (1) In Hep38.7-Tet cells, Cananga odorata exhibited the highest anti-HBV activity with a 50% inhibitory concentration (IC50) of 56.5 µg/ml and 50% cytotoxic concentration (CC50) of 540.2 µg/ml (Selectivity Index: 9.6). (2) The treatment of HepG2-NTCP cells with Cassia fistula, C. odorata, and Melastoma malabathricum at concentrations of 100 µg/ml lowered the levels of HBsAg production to 51.2%, 58.0%, and 40.1%, respectively, compared to untreated controls, and IC50 and CC50 values of C. odorata were 142.9 µg/ml and >400 µg/ml. In conclusion, the C. odorata extract could be a good candidate for the development of anti-HBV drugs.
{"title":"Antiviral Activity of Cananga odorata Against Hepatitis B Virus.","authors":"Puguh Indrasetiawan, Chie Aoki-Utsubo, Muhammad Hanafi, Sri Hartati, Tutik Sri Wahyuni, Masanori Kameoka, Yoshihiko Yano, Hak Hotta, Yoshitake Hayashi","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Current therapeutic drugs for chronic hepatitis B using pegylated interferons and nucleos(t)ide analogs have limited efficacy. Therefore, the development of novel and safe antivirals is required. Natural products including medicinal plants produce complex and structurally diverse compounds, some of which offer suitable targets for antiviral screening studies. In the present study, we screened various crude extracts from Indonesian plants for anti-HBV activity by determining their effects on the production of extracellular HBV DNA in Hep38.7-Tet cells and HBV entry onto a HBV-susceptible cell line, HepG2-NTCP, with the following results: (1) In Hep38.7-Tet cells, Cananga odorata exhibited the highest anti-HBV activity with a 50% inhibitory concentration (IC50) of 56.5 µg/ml and 50% cytotoxic concentration (CC50) of 540.2 µg/ml (Selectivity Index: 9.6). (2) The treatment of HepG2-NTCP cells with Cassia fistula, C. odorata, and Melastoma malabathricum at concentrations of 100 µg/ml lowered the levels of HBsAg production to 51.2%, 58.0%, and 40.1%, respectively, compared to untreated controls, and IC50 and CC50 values of C. odorata were 142.9 µg/ml and >400 µg/ml. In conclusion, the C. odorata extract could be a good candidate for the development of anti-HBV drugs.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 2","pages":"E71-E79"},"PeriodicalIF":0.0,"publicationDate":"2019-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012192/pdf/kobej-65-e71.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37557449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sung-Mi Park, Nao Saito, Soo Ryang Kim, Ikuko Miyawaki
The objective of this study was to clarify the lifestyle characteristics of patients with alcoholic liver disease (ALD) who were readmitted to the hospital, and to identify the background factors associated with these characteristics. This was a prospective observational study. Over a period of 3 months following hospital discharge, we conducted structured interviews to investigate the following five lifestyle characteristics based on our previous research: dietary intake, alcohol consumption or abstinence, psycho-emotional status, regularity of life habits, adherence to treatment. We also collected data on background factors from medical records and questionnaires. The analysis was performed using conceptual cluster matrices, with participants divided into two groups (at-home recovery and readmission). Lifestyle, health status, and background factors were compared between the two groups. Of the 34 patients with ALD recruited, 21 completed the one-month follow-up and were included in the analysis-14 patients were in the at-home recovery group and 7 in the readmission group. The at-home group's lifestyle was characterized by controlled alcohol consumption, but with maintenance of regular life and eating habits and adherence to treatment. In contrast, irregular eating habits (p=0.006) and the development of irregular life habits or the discontinuation of treatment very quickly after hospital discharge characterized the readmission group's lifestyle. Experiences of loss were a lifestyle-related background factor that was associated with readmission (p=0.017). Based on these findings, supporting patients with ALD in maintaining regular eating habits and taking experiences of loss into consideration would be important in avoiding readmission over the short-term.
{"title":"Lifestyle of Patients with Alcoholic Liver Disease and Factors Leading to Hospital Readmission: A Prospective Observational Study.","authors":"Sung-Mi Park, Nao Saito, Soo Ryang Kim, Ikuko Miyawaki","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The objective of this study was to clarify the lifestyle characteristics of patients with alcoholic liver disease (ALD) who were readmitted to the hospital, and to identify the background factors associated with these characteristics. This was a prospective observational study. Over a period of 3 months following hospital discharge, we conducted structured interviews to investigate the following five lifestyle characteristics based on our previous research: dietary intake, alcohol consumption or abstinence, psycho-emotional status, regularity of life habits, adherence to treatment. We also collected data on background factors from medical records and questionnaires. The analysis was performed using conceptual cluster matrices, with participants divided into two groups (at-home recovery and readmission). Lifestyle, health status, and background factors were compared between the two groups. Of the 34 patients with ALD recruited, 21 completed the one-month follow-up and were included in the analysis-14 patients were in the at-home recovery group and 7 in the readmission group. The at-home group's lifestyle was characterized by controlled alcohol consumption, but with maintenance of regular life and eating habits and adherence to treatment. In contrast, irregular eating habits (p=0.006) and the development of irregular life habits or the discontinuation of treatment very quickly after hospital discharge characterized the readmission group's lifestyle. Experiences of loss were a lifestyle-related background factor that was associated with readmission (p=0.017). Based on these findings, supporting patients with ALD in maintaining regular eating habits and taking experiences of loss into consideration would be important in avoiding readmission over the short-term.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 3","pages":"E80-E89"},"PeriodicalIF":0.0,"publicationDate":"2019-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012321/pdf/kobej-65-e80.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37618786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Expressive language development depends on anatomical factors, such as motor control of the tongue and oral cavity needed for vocalization, as well as cognitive aspects for comprehension and speech. The purpose of this study was to examine the differences in expressive language development between normal-birth-weight (NBW) infants and very-low-birth-weight (VLBW) infants in infancy using a formant analysis. We also examined the presence of differences between infants with a normal development and those with a high risk of autism spectrum disorder who were expected to exist among VLBW infants. The participants were 10 NBW infants and 10 VLBW infants 12-15 months of age whose speech had been recorded at intervals of approximately once every 3 months. The recorded speech signal was analyzed using a formant analysis, and changes due to age were observed. One NBW and 3 VLBW infants failed to pass the screening tests (CBCL and M-CHAT) at 24 months of age. The formant frequencies (F1 and F2) of the three groups of infants (NBW, VLBW and CBCL·M-CHAT non-passing infants) were scatter-plotted by age. For the NBW and VLBW infants, the area of the plot increased with age, but there was no significant expansion of the plot area for the CBCL·M-CHAT non-passing infants. The results showed no significant differences in expressive language development between NBW infants at 24 months old and VLBW infants at the corrected age. However, different language developmental patterns were observed in CBCL·M-CHAT non-passing infants, regardless of birth weight, suggesting the importance of screening by acoustic analyses.
{"title":"Study on the Language Formation Process of Very-Low-Birth-Weight Infants in Infancy Using a Formant Analysis.","authors":"Hidetaka Maebayashi, Tetsuya Takiguchi, Satoshi Takada","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Expressive language development depends on anatomical factors, such as motor control of the tongue and oral cavity needed for vocalization, as well as cognitive aspects for comprehension and speech. The purpose of this study was to examine the differences in expressive language development between normal-birth-weight (NBW) infants and very-low-birth-weight (VLBW) infants in infancy using a formant analysis. We also examined the presence of differences between infants with a normal development and those with a high risk of autism spectrum disorder who were expected to exist among VLBW infants. The participants were 10 NBW infants and 10 VLBW infants 12-15 months of age whose speech had been recorded at intervals of approximately once every 3 months. The recorded speech signal was analyzed using a formant analysis, and changes due to age were observed. One NBW and 3 VLBW infants failed to pass the screening tests (CBCL and M-CHAT) at 24 months of age. The formant frequencies (F1 and F2) of the three groups of infants (NBW, VLBW and CBCL·M-CHAT non-passing infants) were scatter-plotted by age. For the NBW and VLBW infants, the area of the plot increased with age, but there was no significant expansion of the plot area for the CBCL·M-CHAT non-passing infants. The results showed no significant differences in expressive language development between NBW infants at 24 months old and VLBW infants at the corrected age. However, different language developmental patterns were observed in CBCL·M-CHAT non-passing infants, regardless of birth weight, suggesting the importance of screening by acoustic analyses.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 2","pages":"E59-E70"},"PeriodicalIF":0.0,"publicationDate":"2019-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012195/pdf/kobej-65-e59.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37557448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Emma Tabe Eko Niba, Mawaddah Ar Rochmah, Nur Imma Fatimah Harahap, Hiroyuki Awano, Ichiro Morioka, Kazumoto Iijima, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Atsuko Takeuchi, Poh San Lai, Yoshihiro Bouike, Masafumi Matsuo, Hisahide Nishio, Masakazu Shinohara
Background: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time "modified competitive oligonucleotide priming"-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP.
Method: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide.
Results: The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMA patient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered.
Conclusion: In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.
{"title":"Spinal Muscular Atrophy: Advanced Version of Screening System with Real-Time mCOP-PCR and PCR-RFLP for SMN1 Deletion.","authors":"Emma Tabe Eko Niba, Mawaddah Ar Rochmah, Nur Imma Fatimah Harahap, Hiroyuki Awano, Ichiro Morioka, Kazumoto Iijima, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Atsuko Takeuchi, Poh San Lai, Yoshihiro Bouike, Masafumi Matsuo, Hisahide Nishio, Masakazu Shinohara","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time \"modified competitive oligonucleotide priming\"-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP.</p><p><strong>Method: </strong>DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide.</p><p><strong>Results: </strong>The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMA patient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered.</p><p><strong>Conclusion: </strong>In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 2","pages":"E49-E53"},"PeriodicalIF":0.0,"publicationDate":"2019-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012194/pdf/kobej-65-e49.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37557446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Emma Tabe Eko Niba, Mawaddah Ar Rochmah, Nur Imma Fatimah Harahap, Hiroyuki Awano, Ichiro Morioka, Kazumoto Iijima, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Atsuko Takeuchi, Poh San Lai, Yoshihiro Bouike, Masafumi Matsuo, Hisahide Nishio, Masakazu Shinohara
Background: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion, it is necessary to differentiate SMN1 from its highly homologous gene, SMN2. We developed a modified competitive oligonucleotide priming-PCR (mCOP-PCR) method using dried blood spot (DBS)-DNA, in which SMN1 and SMN2-specific PCR products are detected with gel-electrophoresis. Next, we added a targeted pre-amplification step prior to the mCOP-PCR step, to avoid unexpected, non-specific amplification. The pre-amplification step enabled us to combine mCOP-PCR and real-time PCR. In this study, we combined real-time mCOP-PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) to develop a new screening system for detection of SMN1 deletion.
Methods: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification), the products of which were then used as a template in the real-time PCR with mCOP-primer sets. To confirm the results, the pre-amplified products were subject to the second PCR-RFLP.
Results: The real-time mCOP-PCR separately amplified SMN1 and SMN2 exon7, and clearly demonstrated SMN1 deletion in an SMA patient. The results of the real-time mCOP-PCR using DBS-DNA were completely consistent with those of the first and second PCR-RFLP analysis.
Conclusion: In our new system for detection of SMN1 deletion, real-time mCOP-PCR rapidly proved the presence or absence of SMN1 and SMN2, and the results were easily tested by PCR-RFLP. This solid genotyping system will be useful for SMA screening.
{"title":"Spinal Muscular Atrophy: New Screening System with Real-Time mCOP-PCR and PCR-RFLP for SMN1 Deletion.","authors":"Emma Tabe Eko Niba, Mawaddah Ar Rochmah, Nur Imma Fatimah Harahap, Hiroyuki Awano, Ichiro Morioka, Kazumoto Iijima, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Atsuko Takeuchi, Poh San Lai, Yoshihiro Bouike, Masafumi Matsuo, Hisahide Nishio, Masakazu Shinohara","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion, it is necessary to differentiate SMN1 from its highly homologous gene, SMN2. We developed a modified competitive oligonucleotide priming-PCR (mCOP-PCR) method using dried blood spot (DBS)-DNA, in which SMN1 and SMN2-specific PCR products are detected with gel-electrophoresis. Next, we added a targeted pre-amplification step prior to the mCOP-PCR step, to avoid unexpected, non-specific amplification. The pre-amplification step enabled us to combine mCOP-PCR and real-time PCR. In this study, we combined real-time mCOP-PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) to develop a new screening system for detection of SMN1 deletion.</p><p><strong>Methods: </strong>DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification), the products of which were then used as a template in the real-time PCR with mCOP-primer sets. To confirm the results, the pre-amplified products were subject to the second PCR-RFLP.</p><p><strong>Results: </strong>The real-time mCOP-PCR separately amplified SMN1 and SMN2 exon7, and clearly demonstrated SMN1 deletion in an SMA patient. The results of the real-time mCOP-PCR using DBS-DNA were completely consistent with those of the first and second PCR-RFLP analysis.</p><p><strong>Conclusion: </strong>In our new system for detection of SMN1 deletion, real-time mCOP-PCR rapidly proved the presence or absence of SMN1 and SMN2, and the results were easily tested by PCR-RFLP. This solid genotyping system will be useful for SMA screening.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 2","pages":"E44-E48"},"PeriodicalIF":0.0,"publicationDate":"2019-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012196/pdf/kobej-65-e44.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37557445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yogik Onky Silvana Wijaya, Emma Tabe Eko Niba, Mawaddah Ar Rochmah, Nur Imma Fatimah Harahap, Hiroyuki Awano, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Atsuko Takeuchi, Poh San Lai, Yoshihiro Bouike, Hisahide Nishio, Masakazu Shinohara
Background: Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder caused by SMN1 gene deletion. SMA has been considered an incurable disease. However, a newly-developed antisense oligonucleotide drug, nusinersen, brings about a good outcome to SMA patients in the clinical trials. Now, a screening for SMA is required for early diagnosis and early treatment so as to give a better clinical outcome to the patients. We have invented a new technology, mCOP-PCR, for SMA screening using dried blood spot (DBS) on the filter paper. One of the problems encountered in SMA screening is poor quality and quantity of DNA extracted from DBS.
Methods: DNA was extracted from DBS of six individuals. Fresh blood DNA of each individual had already been genotyped using PCR/RFLP. The fragments including the sequence of SMN1/SMN2 exon 7 were pre-amplified with conventional PCR. To determine which pre-amplified product is a better template for the real-time mCOP-PCR, we did pre-amplification with a single PCR or pre-amplification with a nested PCR.
Results: The real-time mCOP-PCR using pre-amplified products with a single PCR brought about ambiguous results in some SMN1-carrying individuals. However, the results of real-time mCOP-PCR following pre-amplification with a nested PCR were completely matched with those of PCR-RFLP.
Conclusion: In our study on the real-time mCOP-PCR screening system for SMA, a nested PCR secured the DNA template quality and quantity, leading to unambiguous results of SMA screening.
{"title":"Nested PCR Amplification Secures DNA Template Quality and Quantity in Real-time mCOP-PCR Screening for SMA.","authors":"Yogik Onky Silvana Wijaya, Emma Tabe Eko Niba, Mawaddah Ar Rochmah, Nur Imma Fatimah Harahap, Hiroyuki Awano, Yasuhiro Takeshima, Toshio Saito, Kayoko Saito, Atsuko Takeuchi, Poh San Lai, Yoshihiro Bouike, Hisahide Nishio, Masakazu Shinohara","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder caused by SMN1 gene deletion. SMA has been considered an incurable disease. However, a newly-developed antisense oligonucleotide drug, nusinersen, brings about a good outcome to SMA patients in the clinical trials. Now, a screening for SMA is required for early diagnosis and early treatment so as to give a better clinical outcome to the patients. We have invented a new technology, mCOP-PCR, for SMA screening using dried blood spot (DBS) on the filter paper. One of the problems encountered in SMA screening is poor quality and quantity of DNA extracted from DBS.</p><p><strong>Methods: </strong>DNA was extracted from DBS of six individuals. Fresh blood DNA of each individual had already been genotyped using PCR/RFLP. The fragments including the sequence of SMN1/SMN2 exon 7 were pre-amplified with conventional PCR. To determine which pre-amplified product is a better template for the real-time mCOP-PCR, we did pre-amplification with a single PCR or pre-amplification with a nested PCR.</p><p><strong>Results: </strong>The real-time mCOP-PCR using pre-amplified products with a single PCR brought about ambiguous results in some SMN1-carrying individuals. However, the results of real-time mCOP-PCR following pre-amplification with a nested PCR were completely matched with those of PCR-RFLP.</p><p><strong>Conclusion: </strong>In our study on the real-time mCOP-PCR screening system for SMA, a nested PCR secured the DNA template quality and quantity, leading to unambiguous results of SMA screening.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"65 2","pages":"E54-E58"},"PeriodicalIF":0.0,"publicationDate":"2019-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012193/pdf/kobej-65-e54.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37557447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Several companies in Japan introduced early working conditions (including recommendations on early morning work and prohibitions on nighttime overtime work) to decrease the number of long working hours at night. Nevertheless, individuals possess their own chronotype, i.e., their behavioral timing preference-be it morning or evening-that is associated with worker health. The purpose of this study was to investigate the influence of chronotype and working conditions on sleep and health related quality of life (HRQOL) using 126 daytime office workers who were classified as morning or evening type by their Morningness-Eveningness Questionnaire scores. We then compared morning and evening type workers' sleep variables (sleep onset/offset time and total sleep time), sleep quality (using the Japanese version of the Pittsburgh Sleep Quality Index), and HRQOL scores. Additionally, we compared the same sleep variables, sleep quality, and HRQOL scores of each chronotype category of worker under early and normal working conditions. As the results, evening type workers had late sleep onset/offset time, poor sleep quality, and low HRQOL (role-social component) compared to morning type workers. Furthermore, the evening type workers under early working conditions had earlier sleep onset/offset time and poorer sleep quality compared to those workers under normal working conditions. These results suggest that evening type workers in general have poor sleep and low HRQOL and those same workers under early working conditions, in particular, are associated with poor sleep quality. Therefore, in order to optimize worker health, we suggest that working conditions should be taken account of individual chronotypes.
{"title":"The Influence of Chronotype and Working Condition on Sleep Status and Health Related Quality of Life of Daytime Office Workers.","authors":"Yuh Sasawaki, Hideyuki Shiotani","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Several companies in Japan introduced early working conditions (including recommendations on early morning work and prohibitions on nighttime overtime work) to decrease the number of long working hours at night. Nevertheless, individuals possess their own chronotype, i.e., their behavioral timing preference-be it morning or evening-that is associated with worker health. The purpose of this study was to investigate the influence of chronotype and working conditions on sleep and health related quality of life (HRQOL) using 126 daytime office workers who were classified as morning or evening type by their Morningness-Eveningness Questionnaire scores. We then compared morning and evening type workers' sleep variables (sleep onset/offset time and total sleep time), sleep quality (using the Japanese version of the Pittsburgh Sleep Quality Index), and HRQOL scores. Additionally, we compared the same sleep variables, sleep quality, and HRQOL scores of each chronotype category of worker under early and normal working conditions. As the results, evening type workers had late sleep onset/offset time, poor sleep quality, and low HRQOL (role-social component) compared to morning type workers. Furthermore, the evening type workers under early working conditions had earlier sleep onset/offset time and poorer sleep quality compared to those workers under normal working conditions. These results suggest that evening type workers in general have poor sleep and low HRQOL and those same workers under early working conditions, in particular, are associated with poor sleep quality. Therefore, in order to optimize worker health, we suggest that working conditions should be taken account of individual chronotypes.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"64 5","pages":"E189-E196"},"PeriodicalIF":0.0,"publicationDate":"2019-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668588/pdf/kobej-64-e189.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37318857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Purpose: This study assessed the symptoms, treatment, social systems use, and perception of living conditions of patients with young-onset Parkinson's disease (YOPD), and investigated the support needed by them.
Method: Among the 252 people who completed our questionnaire, we defined YOPD patients as those diagnosed as young onset or those with onset at ≤40 years. The data were compared with others.
Results: There were 24 patients with YOPD (9.5%) (average age: 61.7 years), with an average disease duration 6.4 years longer (p < 0.01) and time until diagnosis 0.7 years longer (p < 0.1) than those of other patients. This group took 1.6 times more types of medicines, and time to their next appointment was 8.6 days shorter than that of other patients (p < 0.05). Patients with YOPD had more pulsive walking and more sweating (p < 0.05), and more motor fluctuation (p < 0.1). More patients with YOPD had a physical disability certificate but felt they were not obtaining the required services (p < 0.05). 45.0% of the YOPD group wanted to work more, more used information and communication equipment (p < 0.05), and more felt their medications were adequate (p < 0.1).
Conclusions: Increased awareness of YOPD is needed. YOPD patients have motor fluctuation because of the longer disease duration. Thus, the support of doctors and nurses, and frequent examination visits, are indispensable for controlling symptoms to achieve middle age developmental tasks. Increased support for care-giving, leisure-time activities, and work is also necessary and may help maintain the desire to work in this group.
{"title":"Investigation of the Treatment and Living Assistance Needed by Patients with Young-Onset Parkinson's Disease.","authors":"Yumi Iwasa, Izumi Saito, Chieko Fujii","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Purpose: </strong>This study assessed the symptoms, treatment, social systems use, and perception of living conditions of patients with young-onset Parkinson's disease (YOPD), and investigated the support needed by them.</p><p><strong>Method: </strong>Among the 252 people who completed our questionnaire, we defined YOPD patients as those diagnosed as young onset or those with onset at ≤40 years. The data were compared with others.</p><p><strong>Results: </strong>There were 24 patients with YOPD (9.5%) (average age: 61.7 years), with an average disease duration 6.4 years longer (p < 0.01) and time until diagnosis 0.7 years longer (p < 0.1) than those of other patients. This group took 1.6 times more types of medicines, and time to their next appointment was 8.6 days shorter than that of other patients (p < 0.05). Patients with YOPD had more pulsive walking and more sweating (p < 0.05), and more motor fluctuation (p < 0.1). More patients with YOPD had a physical disability certificate but felt they were not obtaining the required services (p < 0.05). 45.0% of the YOPD group wanted to work more, more used information and communication equipment (p < 0.05), and more felt their medications were adequate (p < 0.1).</p><p><strong>Conclusions: </strong>Increased awareness of YOPD is needed. YOPD patients have motor fluctuation because of the longer disease duration. Thus, the support of doctors and nurses, and frequent examination visits, are indispensable for controlling symptoms to achieve middle age developmental tasks. Increased support for care-giving, leisure-time activities, and work is also necessary and may help maintain the desire to work in this group.</p>","PeriodicalId":39560,"journal":{"name":"Kobe Journal of Medical Sciences","volume":"64 5","pages":"E180-E188"},"PeriodicalIF":0.0,"publicationDate":"2019-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668587/pdf/kobej-64-e180.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37318856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}