DSpace community: 生物物理研究所

光學奈米流道應用於至十萬鹼基對之DNA極速尺寸分析;Ultrafast size profiling of 100 kilo-base paired DNA using optonanofluidic device

Tue, 04 Oct 2022 03:04:22 GMT

title: 光學奈米流道應用於至十萬鹼基對之DNA極速尺寸分析;Ultrafast size profiling of 100 kilo-base paired DNA using optonanofluidic device abstract: DNA 長度的量測在生物領域中是項非常重要的技術。例如DNA 指紋鑑定(DNA fingerprinting)、限制酶定位法(restriction mapping)、流行病學基因分型 (epidemiological genotyping)、次世代定序(next-generation sequencing)等等的生物技術中，都需要量測DNA 之長度。最傳統的DNA 長度量測方式為凝膠電泳法(Gel electrophoresis)。然而此方法只適用於量測長度50 kbps 以下之DNA。對於50 kbps 以上之DNA，則需要藉由週期性改變電場方向的方式，也就是脈衝場凝膠電泳法(Pulsed Field Gel Electrophoresis)，來完成DNA 長度的量測。然而脈衝場凝膠電泳的量測時間需要數小時至數天。此篇論文將展示一種新的DNA 長度量測技術。此技術結合奈米流道生物晶片以及單分子數位影像分析，目前已經可以於10~60 分鐘，量測長度最長 100 kbps左右之DNA。未來此技術將有潛力達到於30 分鐘內，量測1000 kbps 長度以上之DNA。;DNA sizing is one of the most crucial processes in molecular biology. It is important for processes in DNA fingerprinting, restriction mapping , epidemiological genotyping, and the growing utility of next-generation sequencing. In the past decades, DNA gel electrophporesis has been the main tool at lab-bench to separateDNA fragments; however, challenges persist when sizing DNA molecules up to 50 kbp. Although pulse-field gel electrophoresis (PFGE) can separate long DNA fragments up to mega-base pairs by the periodic change of the electric field direction, PFGE usually lasts from hours to days. Here, we provide a simple single-molecule based DNA profiling device and methodology with designated algorithm to achieve an ultrafast size profiling. Samples up to 100 kbp DNA molecules were efficiently sized into bands from 10 to 60 minutes. Our results establish the ability, far beyond the conventional gel electrophoresis, for easy and quick DNA sizing up to 100-base pairs in complex DNA samples. We expect our method can size DNA molecules up to mega-base pairs for less than 30 minutes.

團藻自我推進的非線性現象研究;A study of Nonlinear Phenonomena of Volvox self-propelling motion

Tue, 04 Oct 2022 03:04:14 GMT

title: 團藻自我推進的非線性現象研究;A study of Nonlinear Phenonomena of Volvox self-propelling motion abstract: 本實驗利用團藻的趨光性當作主動粒子，藉此研究團藻在黏滯液體中移動與阻塞現象。為了解團藻的基本性質，我們以光源驅使團藻移動，藉此計算團藻大小、軌跡與速度關係，並在不同黏度的培養液環境測量對團藻移動的影響。實驗結果發現，液體黏滯度越低時團藻速度越快，且有無光刺激的影響對團藻移動速度並無明顯差異。團藻半徑小時70μm速度與半徑正相關，但當團藻半徑大時70μm速度開始下降。在阻塞方面，我們改變玻璃針管管徑、不同濃度PVP混合培養液改變液體黏滯度，來研究主動粒子在管徑改變、不同流速所造成的阻塞現象。以PVP重量百分濃度4%為例：我們發現在管子頸部與顆粒尺寸比在4.6、6.0與8.0時阻塞的比例為100%、80%與15%。在棘輪部分，我們給予團藻各種持續的光刺激(線性、次方與指數)並觀察團藻分布情況。結果發現團藻會依光刺激的類型大略排列出匹配圖形。團藻在線性及次方亮度變化下，穩定度會比指數棘輪高。 ;This thesis uses volvox, with its phototaxis ability, as a self-driven particle system to study the motions and the phenomena of jamming in a viscous fluid. In order to measure the basic self-driven properties of volvox, the volvox is driven by light so as to find the relations between the size and the speed of volvox particles. Besides, we use fluid with different viscosity to study the viscous effects on their movement. Our experiment shows that the speed of volvox is negatively correlation with the viscosity of the fluid and their speed is independent of the intensity of the light source. The speed will reach a maximum when the radii of volvox particle are approximately 70μm. To study the phenomenon of jamming , we shrink the central part of a glass tube (the neck) to different diameters, and use volvox suspended in medium with different viscosities produced by mixing with different PVP concentrations. We find that when the concentration of PVP is 4% , the jamming ratio are 100%、80% and 15% when the neck-to-particle size ratio is 4.6, 6 and 8 respectively. We also generate various background light simulations (linear, square and exponential) to observe the distribution of volvox under these backgrounds. We find that volvox would have a spatial distribution according to the intensity of the light. The stability of volvox is higher in the linear and square spatial light variations than an exponential one.

使用指向性圓二色光譜與多片層X光繞射技術進行daptomycin與細胞膜作用之結構研究;Structural studies on the interaction between daptomycin and membranes by oriented circular dichroism and lamellar X-ray diffraction

Wed, 02 Sep 2020 07:28:40 GMT

title: 使用指向性圓二色光譜與多片層X光繞射技術進行daptomycin與細胞膜作用之結構研究;Structural studies on the interaction between daptomycin and membranes by oriented circular dichroism and lamellar X-ray diffraction abstract: 抗菌胜肽存在於動植物的免疫系統中，藉由直接與細胞膜作用來消滅入侵體內的病原體或微生物，而細胞膜的組成很難因基因突變發生變化，所以不易產生抗藥性，因此研究抗菌胜肽的抑菌機制對解決日益嚴重的抗藥性問題是一個重要的課題。 daptomycin為環脂肽，是獲得美國FDA認證的新型結構抗菌胜肽。它會與革蘭陽氏菌的細胞膜作用導致膜通透來殺死細胞，在有鈣離子存在與細胞膜上含有phosphatidylglycerol (PG)脂質分子的情況下才具有抑菌活性。儘管經過多年的臨床使用與研究，其具體的分子作用機制仍是未知的。此篇研究中，我們使用指向性圓二色光譜(Oriented Circular Dichroism, OCD)來測量daptomycin在細胞膜上的二級結構的變化，進一步決定其與細胞膜的結合狀態。再使用多片層X光繞射(Lamellar X-ray Diffraction, LXD)測量daptomycin與細胞膜結合後所產生的厚度變化。 OCD的結果顯示，在PG與鈣離子都存在的情況下，daptomycin的OCD光譜在232(nm)附近會有明顯翻轉，其光譜翻轉情形與daptomycin和鈣離子濃度有關。藉由分析其光譜翻轉，發現與細胞膜結合的daptomycin超過臨界濃度，daptomycin會改變其與細胞膜的結合狀態；且確認了daptomycin與鈣離子的化學劑量比例約為1到2之間。LXD的結果顯示，與daptomycin作用會使細胞膜變薄，而與鈣離子作用會使細胞膜變厚。在daptomycin、PG與鈣離子並存的情況下，細胞膜則會變厚，我們提出不同模型進行計算來解釋細胞膜厚度的變化，進一步討論daptomycin、鈣離子與細胞膜的作用。 ;Antimicrobial peptides are widely used by animals and plants in their innate immune systems to eliminate invading pathogens or microbes via directly targeting to their membranes. The composition of the membrane is difficult to be changed by gene mutation so that it is rare to exhibit antibiotic resistance. Therefore, studies on the antibacterial mechanism of antibacterial peptides is an key issue to solve the serious problem of antibiotic resistance. Daptomycin, a cyclic lipopeptide, represents a new structural class of the FDA approved antibiotics. It interacts with the cytoplasmic membranes of Gram-positive pathogens causing membrane permeabilization to kill cells. The antibiotic activity is calcium ion dependent and correlates with the targeted membrane’s content of phosphatidylglycerol (PG), otherwise its underlying molecular mechanism is so far unknown in despite of clinical usages and researches for many years. Here we used oriented circular dichroism (OCD) to probe the change of second structure to determine the binding states of daptomycin in membranes. Lamellar X-ray diffraction (LXD) was used to monitor the thickness change of membrane induced by daptomycin binding. In the coexistence of daptomycin, PG and calcium ions, the result shows that OCD spectra of daptomycin will be significantly reversed around 232 (nm) and the reversed spectra is correlated to the concentrations of daptomycin as well as calcium ions. Consequently, it indicates that the concentration of binding daptomycin excess a threshold, daptomycin will change its state. And the stoichiometric ratio of daptomycin to calcium was confirmed between 1 and 2. The LXD result shows that daptomycin induced membrane thinning and calcium ions induced membrane thickening, respectively. In the coexistence of daptomycin, PG and calcium ions, membrane thickening was observed. In this study, we attempt to propose different models to fit experimental data to clarify the change of membrane thickness in different concentrations of daptomycin and calcium ions. Finally, the daptomycin/calcium ion/membrane interaction will be discussed.

Deuterium NMR Study of the effect of 7- dehydrocholesterol on the POPE Membranes

Wed, 02 Sep 2020 07:28:16 GMT

title: Deuterium NMR Study of the effect of 7- dehydrocholesterol on the POPE Membranes abstract: 固醇在生物膜的組成與功能中扮演了重要的角色。在含有膽固醇的人造細胞膜研究中發現，將膽固醇加入脂質膜中後會形成sterol-rich以及sterol-poor的區塊。一般認為這些處於liquid-ordered態的sterol-rich區塊為細胞膜上的脂筏。在本論文中，我們研究7- dehydrocholesterol (7DHC)對於POPE-d31膜在物理性質方面的影響。本研究使用氘核磁共振 (2H NMR) 來測量由POPE-d31和7- dehydrocholesterol (7DHC) 所組成的人造細胞膜。將不同濃度的7DHC樣品在不同溫度下量測已取得核磁共振光譜。光譜的M1值呈現出7DHC使膠態POPE脂膜變得無序，並使液晶態的脂膜變得有序。透過NMR光譜取得之溫度對7DHC濃度的POPE-d31脂質膜相圖顯示出膠態-液晶態共存與液晶態-inverted hexagonal態兩種兩相共存的區域，其中液晶態同時被發現在兩種兩相共存區域內。隨著7DHC濃度增加，液晶態從無序轉變成有序。這證明了POPE脂膜在含有7DHC時存在liquid-ordered 態。然而，在此系統中兩種液態相並沒有被觀測到同時存在。另一方面，7DHC 促進liquid-ordered態在膠態POPE脂膜中的形成。最後，高濃度與低濃度的7DHC對液晶態相變至inverted hexagonal態的影響有明顯的差異。;Sterols play important roles in membrane organization and functions. Model membranes containing cholesterol reveal that the addition of cholesterol in lipid membranes promotes sterol-rich, liquid-ordered-phase domains. In this work, we investigate the effect of 7- dehydrocholesterol (7DHC) on the physical properties of 1-palmitoyl(d31)-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE-d31) membranes using deuterium nuclear magnetic resonance. 2H NMR spectra are taken as a function of temperature and 7DHC concentration. The spectral first moments show that 7DHC disorders the gel phase and order the liquid-crystalline phase of POPE membranes. The phase diagram, constructed from the NMR spectra exhibits gel-liquid crystalline and liquid crystalline-inverted hexagonal phase coexistence regions. One liquid crystalline phase is observed in between these two two-phase regions. The liquid crystalline phase evolves from the liquid-disordered phase to the liquid-ordered phase as the 7DHC concentration increases. This is the first demonstration that liquid-ordered phase exists in POPE membranes containing 7DHC. However, liquid-liquid phase coexistence is not observed in this system. On the other hand, 7DHC promotes the liquid-ordered phase in the gel-phase POPE membranes. Finally, the liquid crystalline-to-inverted iii hexagonal phase transition exhibits dramatically different 7DHC concentration dependence at low and high concentrations.