Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

由 Streptomyces netropsis 選殖活化轉麩氨醯胺酶之金屬蛋白酶基因並於大腸桿菌進行表現及特性分析

Transglutaminase (amine γ-glutaminyltransferase, EC 2.3.2.13, TGase) from Streptomyces has been shown to be secreted as zymogen and activation of proTGase is achieved by cleavage of the pro-peptide with endogenous proteases. The genes for two extracellular metalloproteases (mp I and mp II) from S. netropsis (Sn) were previously identified and pro-TGase activation activity of Sn MP I had been demonstrated. Sequence analysis revealed that Sn MP I is synthesized as a precursor with the N- and C-terminal propeptides. During the maturation, it is necessary for Sn MP I to remove the N- and C-terminal propeptides by autoprocessing to form an active enzyme. In this study, purification and characterization of the extracellular recombinant Sn MP I expressed in E. coli was carried out. Polyclonal antibodies raised against Sn MP I was prepared from immunized mice with purified recombinant protein overexpressed in E. coli. The active mature Sn MP I was purified through 50-70% ammonium sulfate fractionation, Ni2+-NTA affinity chromatography and DEAE-sepharose ion exchanger. SDS-PAGE analysis showed the mature Sn MP I was purified to homogeneity with a molecular weight of 46 kDa. However, non-specific processing during the maturation of Sn MP I was identified and no enzymatically active form of Sn MP I could be obtained when C-terminal propeptide deleted mutant of Sn MP I was expressed in E. coli. The enzymatic activity of Sn MP I was demonstrated by co-expression Sn MP I and proTGase genes in E. coli. Mature TGase with a specific activity of 20.25 U/mg was obtained from the enzyme purified from culture medium. N-terminal amino acid sequence analysis of the purified mature TGase revealed that the peptide bond between Phe (-4) and Ser (-5) of proTGase was hydrolyzed by mature Sn MP I. Furthermore, a third metalloprotease gene (Sn mp III) was cloned and sequenced from the constructed S. netropsis partial genomic library in this study. DNA sequence analysis revealed that Sn mp III contains 1,617 bp and encoded a polypeptide of 538 amino acids be with an estimated molecular weight of 55.9 kDa. The deduced amino acid sequence contains a typical HEXXH motif of metallopeptidase family and a GXXNEXXSD motif of thermolysin-like protease family. Expression and purification of the recombinant Sn MP III in E. coli are undergoing and the pro-TGase activation activity will be determined.由 Streptomyces 所 分 泌 之 轉 麩 氨 醯 胺 酶(Transglutaminase, amine γ-glutaminyltransferase, EC 2.3.2.13, TGase) 是以酶原形式被分泌至胞外，必須依賴胞外蛋白酶將其 propeptide 進行切除，方能活化成為具有催化活性之成熟態。 過去本實驗室已由 Streptomyces netropsis 中選殖出兩個胞外金屬蛋白酶基因(Sn mp I 及 Sn mp II)，並確認 Sn MP I 金屬蛋白酶具有活化 S. netropsis proTGase 之能力。胺基酸序列比對分析顯示，Sn MP I 初生成時為一含有 N 端及 C 端 propeptide 之前驅物，必須於其成熟過程中藉由自我切割之方式將自身 N 端及 C 端 propeptide 進行移除，方能成為一具有活性之酵素。本研究以E. coli 為宿主，純化重組 Sn MP I 並進一步探討其酵素特性，先利用完整型態之純化重組 Sn MP I 作為抗原，對小鼠進行免疫反應，獲得抗 Sn MP I 金屬蛋 白酶之多株抗體。於純化成熟態重組 Sn MP I 方面，將在 E. coli 表現含有重組 Sn MP I 之胞外培養液，經由 50~70% 飽和濃度之硫酸銨分割沉澱後，利用 Ni2+-NTA 親和性層析與 DEAE-sepharose 離子交換層析進行純化，可獲得成熟態 Sn MP I，根據 SDS-PAGE 分析顯示其分子量為 46 kDa。然而，若於 E. coli 表現去除 C 端 propeptide 之 Sn MP I，則發現其成熟過程中會出現非專一性自我切割現象，且無法獲得具有酵素活性之成熟態 Sn MP I。本研究亦於 E. coli 中共同表現 Sn MP I 與 Streptomyces proTGase，結果顯示胞外培養液中存在大 量 mature TGase，經純化後可獲得比活性為 20.25 U/mg 之 mature TGase。根據 mature TGase N 端胺基酸序列定序分析，得知 mature Sn MP I 之切割位於 propeptide 與 mature TGase 之間的 Ser (-5) 與 Phe (-4) 位置上。此外，本研究以構築部分基因庫方式由 S. netropsis 選殖出第三個金屬蛋白酶基因 (Sn mp III)，經由 DNA 序列分析顯示 Sn mp III 基因全長 1,617 bp ，可轉譯出 538 個胺基酸，其分子量推估為 55.9 kDa。推衍之 Sn MP III 胺基酸序列，含有典型金屬蛋白酶 家 族 所 具 有 之 HEXXH motif ， 以 及 thermolysin-like family 特 有 之 GXXNEXXSD motif。而關於 Sn MP III 金屬蛋白酶於 E. coli 中之表現與純化以及其活化 proTGase 之能力，尚待進一步探討。中文摘要……………………………………………………………………………i 英文摘要……………………………………………………………………………ii 縮寫字對照表………………………………………………………………………iii 前言…………………………………………………………………………………1 材料方法 I. 實驗材料 一、菌體與質體…………………………………………………………10 二、藥品…………………………………………………………………10 三、純化管柱……………………………………………………………10 四、酵素…………………………………………………………………11 五、培養基與緩衝液……………………………………………………11 六、引子…………………………………………………………………11 II. 實驗方法 一、E.coli 質體 DNA 小量製備………………………………………12 二、DNA 片段回收與純化……………………………………………12 三、DNA 黏合作用……………………………………………………13 四、E.coli 勝任細胞之製備……………………………………………13 五、熱休克轉形作用……………………………………………………13 六、蛋白質凝膠電泳 (SDS-PAGE) ……………………………………13 七、硝酸銀染色 (silver staining) ………………………………………14 八、西方墨點法 (western blot) ………………………………………14 九、聚合酶鏈鎖反應 (polymerase chain reaction) ……………………15 十、Streptomyces netropsis 染色體 DNA 萃取………………………15 十一、製備 S. netropsis 金屬蛋白酶基因之 DNA 探針………………16 十二、南方墨點雜交法 (southern hybridization) ………………………16 十三、 S. netropsis 染色體部分基因庫之構築…………………………17 十四、菌落雜交法 (colony hybridization) ………………………………17 十五、菌種保存……………………………………………………………18 十六、重組 S. netropsis 胞外金屬蛋白酶 MP I 之純化………………18 十七、胞外重組 S. netropsis 金屬蛋白酶之活性分析…………………19 十八、酵素活性染色 (zymography) ……………………………………19 十九、 S. netropsis 胞外金屬蛋白酶抗體製備…………………………20 二十、經 E. coli BL21(DE3)(pET26-SmPTGA-PMP)(pREP4-cm) 共同表 現所得之重組 Sm mature TGase 之純化………………………21 二十一、重組 S. mobaraensis mature TGase N 端胺基酸序列定序分析…22 二十二、 TGase 活性測定…………………………………………………23 結果 一、 S. netropsis MP I 胞外金屬蛋白酶抗體之製備…………………24 二、重組 S. netropsis 胞外金屬蛋白酶 Sn MP I 之純化與酵素 特性分析……………………………………………………………… 25 1. 重組 S. netropsis 胞外金屬蛋白酶 Sn MP I 之純化………… 25 2. S. netropsis MP I 最適反應 pH 值測試………………………26 3. Ca2+ 離子對 S. netrposis MP I 熱穩定度之影響分析………… 27 4. S. netrposis MP I 酵素專一性測試…………………………… 27 三、S. netrposis MP I 自我切割機制及其 C 端 propeptide 功能之探討…………………………………………………………28 1. 以液相層析串聯式質譜儀分析成熟態 S. netrposis MP I 之 C 端 胺基酸序列………………………………………………………28 2. pET26-PMP△C-pro 表現載體之構築…………………………28 3. E. coli BL21(DE3)(pET26-PMP△C-pro) 胞外蛋白之表現與純化………………………………………………………………. 29 四、於 E. coli 共同表現 S. netropsis MP I 與重組proTGase………… 30 1. 以雙質體形式於 E. coli 進行共同表現……………………… 30 (A) pTBHR-PMP (stop) 表現載體之構築……………………… 30 (B) 利用 E.coli BL21(DE3)(pET26-Shkpro-SkTGA)(pTBHR-PMP) 進行胞外重組 Sn TGase 之表現與純化………………… 30 (C) 利用 E.coli BL21(DE3)(pET26-SmPTGA)(pTBHR-PMP) 進行 胞外重組 Sm TGase 之表現與純化……………………… 31 2. 將 S. netropsis mp I 基因與 S. mobaraensis TGase 基因構築於同一個質體上於 E. coli進行共同表現…………………………… 31 (A) pET26-SmPTGA-PMP 表現載體之構築………………… 31 (B) pREP4-cm 表現載體之構築……………………………… 32 (C) 利用 E. coli BL21(DE3)(pET26-SmPTGA-PMP)(pREP4-cm) 進行胞外重組 Sn MP I 與 SmTGase 之共同表現與純化… 32 3. 以 Edman method 分析純化之 S. mobaraensis mature TGase N 端胺基酸序列………………………………………………………… 34 五、S. netropsis 胞外金屬蛋白酶 Sn mp III 基因選殖與表現分析…34 1. Southern blot 分析 S. netropsis 染色體 DNA……………………35 2. 部分染色體基因庫之構築及 S. netropsis mp III 基因篩選……35 3. S. netropsis mp III 基因序列比對及分析………………………36 4. pET26-MP III 表現質體之構築…………………………………37 5. 胞外重組 Sn MP III 之表現純化及活性測試…………………37 討論 一、 S. netropsis 胞外金屬蛋白酶抗體之製備…………………………29 二、重組 S. netropsis 胞外金屬蛋白酶 Sn MP I 之純化與酵素特性分析………………………………………………………40 三、S. netrposis MP I 自我切割機制及其 C 端 propeptide 功能之探討…………………………………………………………………12 四、於 E. coli 共同表現 S. netropsis MP I 與重組proTGase…………43 五、S. netropsis 胞外金屬蛋白酶 Sn mp III 基因選殖與表現分析……………………………………………………………44 參考文獻………………………………………………………………………………46 圖表……………………………………………………………………………………60 附錄……………………………………………………………………………………86 附錄一、S. netropsis metalloprotease I 及 metalloprotease III 胺基酸序列比較……………………………………………………………… 86 附錄二、S. netropsis metalloprotease II 及 metalloprotease III 胺基酸序列比較……………………………………………………………… 87 附錄三、去除 C terminal extension region 之 S. netropsis metalloprotease I 及 metalloprotease III 胺基酸序列比較…………………………… 88 附錄四、S. netropsis metalloprotease III 與不同 Streptomyces 菌株之金屬蛋白酶胺基酸序列比對………………………………………… 89 附錄五、S. netropsis metalloprotease I 與不同 Streptomyces 菌株之金屬蛋白酶胺基酸序列比對………………………………………… 91 附錄六、S. netropsis metalloprotease II 與不同 Streptomyces 菌株之金屬蛋白酶胺基酸序列比對………………………………………… 94 附錄七、培養基與緩衝溶液…………………………………………………… 9

Losartan Attenuates Insulin Resistance and Regulates Browning Phenomenon of White Adipose Tissue in ob/ob Mice

Insulin resistance (IR) is a villain role to the pathology of fatty liver diseases implicated in adipose tissue dysfunction, which is characterized by lipid droplets (LDs) accumulation and hypoxia-inducible factor 1α (HIF1α) related macrophage infiltration. HIF1α is required for its lipogenic actions in adipocytes, while and it regulates M1 and M2 polarization features of macrophages. Losartan has been shown to be an insulin sensitizer in obese states, actions involving in HIF1α signaling. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, GTT, ITT, and HOMA-IR were identified losartan alleviated IR signaling in obese mice. This alleviation may through inhibits HIF1α by suppressing STAT3-NF-κB signaling, which, in turn, revealed HIF1α-dependent decreases the angiogenesis pathway in adipose tissue, including regulation of VEGF and TGFβR2 levels. In white adipose tissue, a set of lipogenesis-related genes, Srebp1, Fas, and Scd-1 were markedly downregulated after losartan intervention, as well as reduced LDs size and LD-associated proteins, perilipin family proteins (PLINs) compared with obese mice. Losartan abolished macrophage infiltration with upregulation of M2 and inhibition of M1 macrophage markers in obese mice. Our data suggest that losartan attenuated obese-induced fatty liver, linked to alleviating inflammation in adipose tissues and a shift in M1/M2 macrophage balance. Furthermore, losartan might improve mitochondria biogenesis by upregulating SIRT1, PGC1α, UCP1, and mRNA of Tfam, Cd137, Tmem26, Ucp1 expression in white adipose tissue compared with the obese group. Taken together, losartan may improve IR and adipose dysfunction by inhibiting lipotoxicity and HIF1α pathways

Losartan Prevents Hepatic Steatosis and Macrophage Polarization by Inhibiting HIF-1α in a Murine Model of NAFLD

Hypoxia and hepatosteatosis microenvironments are fundamental traits of nonalcoholic fatty liver disease (NAFLD). Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with NAFLD, whereas the route and regulation of lipid droplets (LDs) and macrophage polarization related to systemic inflammation in NAFLD is unknown. Losartan is an angiotensin II receptor antagonist, that approved portal hypertension and related HIF-1α pathways in hepatic injury models. Here, we show that losartan in a murine model of NAFLD significantly decreased hepatic de novo lipogenesis (DNL) as well as suppressed lipid droplets (LDs), LD-associated proteins, perilipins (PLINs), and cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector (CIDE) family in liver and epididymal white adipose tissues (EWAT) of ob/ob mice. Obesity-mediated macrophage M1 activation was also required for HIF-1α expression in the liver and EWAT of ob/ob mice. Administration of losartan significantly diminishes obesity-enhanced macrophage M1 activation and suppresses hepatosteatosis. Moreover, HIF-1α-mediated mitochondrial dysfunction was reversed in ob/ob mice treated with losartan. Together, the regulation of HIF-1α controls LDs protein expression and macrophage polarization, which highlights a potential target for losartan in NAFLD

Curated incidence of lysosomal storage diseases from the Taiwan Biobank

Abstract Lysosomal storage diseases (LSDs) are a group of metabolic disorders resulting from a deficiency in one of the lysosomal hydrolases. Most LSDs are inherited in an autosomal or X-linked recessive manner. As LSDs are rare, their true incidence in Taiwan remains unknown. In this study, we used high-coverage whole-genome sequencing data from 1,495 Taiwanese individuals obtained from the Taiwan Biobank. We found 3826 variants in 71 genes responsible for autosomal recessive LSDs. We first excluded benign variants by allele frequency and other criteria. As a result, 270 variants were considered disease-causing. We curated these variants using published guidelines from the American College of Medical Genetics and Genomics (ACMG). Our results revealed a combined incidence rate of 13 per 100,000 (conservative estimation by pathologic and likely pathogenic variants; 95% CI 6.92-22.23) to 94 per 100,000 (extended estimation by the inclusion of variants of unknown significance; 95% CI 75.96–115.03) among 71 autosomal recessive disease-associated genes. The conservative estimations were similar to those in published clinical data. No disease-causing mutations were found for 18 other diseases; thus, these diseases are likely extremely rare in Taiwan. The study results are important for designing screening and treatment methods for LSDs in Taiwan and demonstrate the importance of mutation curation to avoid overestimating disease incidences from genomic data

DYNC1H1 variant associated with epilepsy: Expanding the phenotypic spectrum

DYNC1H1 variants are associated with peripheral neuronal dysfunction and brain morphology abnormalities resulting in neurodevelopmental delay. However, few studies have focused on the association between DYNC1H1 variants and epilepsy. Herein, we report a case of drug-resistant focal epilepsy associated with a pathogenic variant of DYNC1H1. We further summarized the clinical, genetic, and neuroimaging characteristics of patients with DYNC1H1 variant–associated epilepsy from the relevant literature. This report expands the phenotypic spectrum of DYNC1H1-related disorder to include early-onset epilepsy, which is frequently associated with neurodevelopmental delay and intellectual disability, malformations of cortical development, and neuromuscular, ophthalmic, and orthopedic involvement

Frequency and spectrum of actionable pathogenic secondary findings in Taiwanese exomes

Abstract Background Exome sequencing has recently become more readily available, and more information about incidental findings has been disclosed. However, data from East Asia are scarce. We studied the application of exome sequencing to the identification of pathogenic/likely pathogenic variants in the ACMG 59 gene list and the frequency of these variants in the Taiwanese population. Methods This study screened 161 Taiwanese exomes for variants from the ACMG 59 gene list. The identified variants were reviewed based on information from different databases and the available literature and classified according to the ACMG standard guidelines. Results We identified seven pathogenic/likely pathogenic variants in eight individuals, with five participants with autosomal recessive variants in one allele and three participants with autosomal dominant variants. Approximately 1.86% (3/161) of the Taiwanese individuals had a reportable pathogenic/likely pathogenic variant as determined by whole‐exome sequencing (WES), which was comparable to the proportions published previously in other countries. We further investigated the high carrier rate of rare variants in the ATP7B gene, which might indicate a founder effect in our population. Conclusion This study was the first to provide Taiwanese population data of incidental findings and emphasized a high carrier rate of candidate pathogenic/likely pathogenic variants in the ATP7B gene

A saturated map of common genetic variants associated with human height

Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40–50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes1. Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel2) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10–20% (14–24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries