Splicing Dysregulation Contributes to the Pathogenicity of Several F9 Exonic Point Variants

Background: Pre‐mRNA splicing is a complex process requiring the identification of donor site, acceptor site, and branch point site with an adjacent polypyrimidine tract sequence. Splicing is regulated by splicing regulatory elements (SREs) with both enhancer and suppressor functions. Variants located in exonic regions can impact splicing through dysregulation of native splice sites, SREs, and cryptic splice site activation. While splicing dysregulation is considered primary disease‐inducing mechanism of synonymous variants, its contribution toward disease phenotype of non‐synonymous variants is underappreciated. Methods: In this study, we analyzed 415 disease‐causing and 120 neutral F9 exonic point variants including both synonymous and non‐synonymous for their effect on splicing using a series of in silico splice site prediction tools, SRE prediction tools, and in vitro minigene assays. Results: The use of splice site and SRE prediction tools in tandem provided better prediction but were not always in agreement with the minigene assays. The net effect of splicing dysregulation caused by variants was context dependent. Minigene assays revealed that perturbed splicing can be found. Conclusion: Synonymous variants primarily cause disease phenotype via splicing dysregulation while additional mechanisms such as translation rate also play an important role. Splicing dysregulation is likely to contribute to the disease phenotype of several non‐synonymous variants

Elevated levels of antibodies against phosphatidylserine/prothrombin complex and/or cardiolipin associated with infection and recurrent purpura in a child: a forme fruste of antiphospholipid syndrome?

Antiphospholipid syndrome is an autoimmune disorder characterized by the occurrence of venous and arterial thrombosis, as well as morbidity in pregnancy, in the presence of anti-phospholipid antibodies. The diagnosis of antiphospholipid syndrome is usually established based on clinical and laboratory findings by strictly following the 2006 Sapporo classification. However, the diagnosis remains challenging owing to the ongoing debates on the serological criteria. We report a case we describe as forme fruste antiphospholipid syndrome in which these criteria were not fulfilled. Purpura appeared repeatedly in a female infant starting from the age of 6 months and following episodes of upper respiratory infections and vaccinations. The levels of anti-cardiolipin IgG antibodies and anti-phosphatidylserine/prothrombin complex antibodies were elevated in accordance with these events. Histopathological evaluation revealed multiple small vessel thrombi in the dermis and adipose tissue. After 2 weeks of treatment with aspirin and heparin, the cutaneous symptoms subsided. Infection has long been associated with antiphospholipid syndrome, and anti-phosphatidylserine/prothrombin antibodies are considered a new marker for the diagnosis of antiphospholipid syndrome. Forme fruste antiphospholipid syndrome should be considered even if the antiphospholipid syndrome diagnostic criteria are not completely fulfilled, especially in the presence of elevated levels of anti-phosphatidylserine/prothrombin antibodies and known preceding infections

簡便な成熟ブタ膵内分泌細胞分離法の検討

膵細胞移植に際しては,大量かつ高純度の膵内分泌細胞を得ることが重要である.本研究では,リンパ球分離溶液を用いた簡便な成熟ブタ膵内分泌細胞の分離法を検討した.屠殺場より入手した成熟ブタ膵を細切・攪拌した後,細胞懸濁液を遠心分離し単離肝細胞を収集した.これをリンパ球分離溶液であモノポリ分離溶液(mono-poly resolving medium: MPRM)を用いて,膵内分泌細胞を外分泌細胞および血管内皮細胞,血液細胞などより分離・精製した.得られた膵分泌細胞数を計測し,形態学的・機能的観察を行った.その結果,(1)得られた膵内分泌細胞数:3.40±1.32×10^5/gのうち,ジチゾン染色陽性細胞数は2.81±1.09×10^5/gで,純度は82.6±2.5%であった.(2)免疫組織化学染色では60%がB細胞であった.(3)電顕では,典型的な分泌顆粒を有するB細胞,A細胞が認められた.(4)グルコース負荷試験では,分離直後のインスリン分泌能低下は1週間の培養で改善し,また40日間の培養中インスリンの分泌が保たれた.成熟ブタ膵内分泌細胞径は,ヒトリンパ球径に類似しており,MPRMを用いた1回の遠心操作で,膵分泌細胞は明瞭に分離され安定した細胞数が得られた.また,膵内分泌細胞の構成は膵島におけるそれと類似しており,超微細構造も保たれていた.以上より,MPRMを用いた膵内分泌細胞の分離法は,簡便で安定した細胞数が得られ,その形態・機能とも良好で有用な方法であると考えられた.Adult pig pancreatic endocrine cells were harvested by auto-digestion without added enzymes. The isolated, crude cells were purified by Mono-poly resolving medium (MPRM). The purity of the harvested cells was determined by dithizone staining and the number of pancreatic endocrine cells was counted. A large number of the cells were stained red with dithizone and showed high viability and a good insulin secretory response to glucose stimulation. The average number of cells purified by MPRM was 3.40 ± 1.32 × 10^5 cells/g pancreas and the number of dithizone-stained cells was 2.81 ± 1.09 × 10^5 cells/g pancreas. The insulin secretion from the pancreatic endocrine cells was maintained throughout a 40-day observation period and high glucose stimulation induced an increase in insulin secretion from the cultured cells. In the cells purified by MPRM, light and electron microscopic studies showed the cells to be typical pancreatic endocrine cells. The present purification method using MPRM allowed us to obtain quickly a large amount of adult pig pancreatic endocrine cells from the unpurified preparations. This is useful for transplantation and biochemical or biological studies of adult pig pancreatic endocrine cells

簡便な成熟ブタ膵内分泌細胞分離法の検討

膵細胞移植に際しては,大量かつ高純度の膵内分泌細胞を得ることが重要である.本研究では,リンパ球分離溶液を用いた簡便な成熟ブタ膵内分泌細胞の分離法を検討した.屠殺場より入手した成熟ブタ膵を細切・攪拌した後,細胞懸濁液を遠心分離し単離肝細胞を収集した.これをリンパ球分離溶液であモノポリ分離溶液(mono-poly resolving medium: MPRM)を用いて,膵内分泌細胞を外分泌細胞および血管内皮細胞,血液細胞などより分離・精製した.得られた膵分泌細胞数を計測し,形態学的・機能的観察を行った.その結果,(1)得られた膵内分泌細胞数:3.40±1.32×10^5/gのうち,ジチゾン染色陽性細胞数は2.81±1.09×10^5/gで,純度は82.6±2.5%であった.(2)免疫組織化学染色では60%がB細胞であった.(3)電顕では,典型的な分泌顆粒を有するB細胞,A細胞が認められた.(4)グルコース負荷試験では,分離直後のインスリン分泌能低下は1週間の培養で改善し,また40日間の培養中インスリンの分泌が保たれた.成熟ブタ膵内分泌細胞径は,ヒトリンパ球径に類似しており,MPRMを用いた1回の遠心操作で,膵分泌細胞は明瞭に分離され安定した細胞数が得られた.また,膵内分泌細胞の構成は膵島におけるそれと類似しており,超微細構造も保たれていた.以上より,MPRMを用いた膵内分泌細胞の分離法は,簡便で安定した細胞数が得られ,その形態・機能とも良好で有用な方法であると考えられた.Adult pig pancreatic endocrine cells were harvested by auto-digestion without added enzymes. The isolated, crude cells were purified by Mono-poly resolving medium (MPRM). The purity of the harvested cells was determined by dithizone staining and the number of pancreatic endocrine cells was counted. A large number of the cells were stained red with dithizone and showed high viability and a good insulin secretory response to glucose stimulation. The average number of cells purified by MPRM was 3.40 ± 1.32 × 10^5 cells/g pancreas and the number of dithizone-stained cells was 2.81 ± 1.09 × 10^5 cells/g pancreas. The insulin secretion from the pancreatic endocrine cells was maintained throughout a 40-day observation period and high glucose stimulation induced an increase in insulin secretion from the cultured cells. In the cells purified by MPRM, light and electron microscopic studies showed the cells to be typical pancreatic endocrine cells. The present purification method using MPRM allowed us to obtain quickly a large amount of adult pig pancreatic endocrine cells from the unpurified preparations. This is useful for transplantation and biochemical or biological studies of adult pig pancreatic endocrine cells

Recent advances in (therapeutic protein) drug development [version 1; referees: 2 approved]

Therapeutic protein drugs are an important class of medicines serving patients most in need of novel therapies. Recently approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, autoimmunity/inflammation, exposure to infectious agents, and genetic disorders. The latest advances in protein-engineering technologies have allowed drug developers and manufacturers to fine-tune and exploit desirable functional characteristics of proteins of interest while maintaining (and in some cases enhancing) product safety or efficacy or both. In this review, we highlight the emerging trends and approaches in protein drug development by using examples of therapeutic proteins approved by the U.S. Food and Drug Administration over the previous five years (2011–2016, namely January 1, 2011, through August 31, 2016)

Splicing Dysregulation Contributes to the Pathogenicity of Several F9 Exonic Point Variants

Background: Pre‐mRNA splicing is a complex process requiring the identification of donor site, acceptor site, and branch point site with an adjacent polypyrimidine tract sequence. Splicing is regulated by splicing regulatory elements (SREs) with both enhancer and suppressor functions. Variants located in exonic regions can impact splicing through dysregulation of native splice sites, SREs, and cryptic splice site activation. While splicing dysregulation is considered primary disease‐inducing mechanism of synonymous variants, its contribution toward disease phenotype of non‐synonymous variants is underappreciated. Methods: In this study, we analyzed 415 disease‐causing and 120 neutral F9 exonic point variants including both synonymous and non‐synonymous for their effect on splicing using a series of in silico splice site prediction tools, SRE prediction tools, and in vitro minigene assays. Results: The use of splice site and SRE prediction tools in tandem provided better prediction but were not always in agreement with the minigene assays. The net effect of splicing dysregulation caused by variants was context dependent. Minigene assays revealed that perturbed splicing can be found. Conclusion: Synonymous variants primarily cause disease phenotype via splicing dysregulation while additional mechanisms such as translation rate also play an important role. Splicing dysregulation is likely to contribute to the disease phenotype of several non‐synonymous variants

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches