Bu çalışma Çin Ulusal Doğa Bilimleri Vakfı (Grant No 21576279, 20906076) ve KRIBB Araştırma Girişimi Programı (KGM2211531) tarafından desteklenmiştir.

1. Bambu Hidrolizatının Hazırlanması <ol><li> 250 mL&#39;lik bir balon içinde% 10&#39;luk (g / g) nihai konsantrasyon elde etmek için 40 ml NaOH çözeltisine 5 gr bambu tozu ilave edin. 0.05 M&#39;lik artışlarla 0.05 M ila 0.50 M aralığında değişen bir dizi NaOH çözeltisi kullanın. <ol><li> Karışımı, 60 ° C&#39;de 60 dakika veya bir su banyosu içinde 100 ° C&#39;de inkübe edin. Otoklavda 121 ° C&#39;de inkübe edin. </li></ol></li><li> Kuluçka işleminden sonra, karışım oda sıcaklığında 4 saat bekletilir. Daha sonra süpernatanı çıkarın. 100 mL taze su ilave edin ve karıştırın. <ol><li> Süpernatanın pH değeri 6.8 olana kadar bu işlemi 5-6 kez tekrarlayın. Elde edilen katıyı, enzimatik hidrolizin bir sonraki basamağı için kullanın. </li></ol></li><li> Hidroliz için yukarıdaki karışımın 50 mL&#39;sini pH 5.0&#39;a% 98 H204 kullanarak ayarlayın. Ardından 0.5 mL 200 filtre kağıdı aktivitesi (FPU) / mL selülaz ekleyin. NOT: Selülazın bambuya nihai oranıOuld, 1 gr bambu başına 20 FPU olmalıdır. </li><li> Karışımı 50 ° C&#39;de bir çalkalayıcıda 36 saat inkübe edin. NOT: Hidrolizden sonra karışımda bir miktar katı kalacaktır. </li><li> 7,690 xg&#39;de 5 dakika santrifüj ederek açık bir hidrolizat elde edin. </li><li> Glikoz, ksiloz ve diğer kimyasalları bir kırma indisi detektörü ve bir fotodiyot dizi detektörü ile donatılmış bir yüksek basınçlı sıvı kromatograf sistemiyle ölçün. Bir HPX-87H sütununu (300 mm x 7.8 mm) ve 0.8 mL / dk&#39;lık bir akış hızında 5 mM H2SO4 çözeltisinin mobil fazı kullanın. </li><li> Büyük miktarda hidrolizat preparatı için 2 kg bambu tozu ve 18 L 0.25 M NaOH&#39;yi 30 L&#39;lik paslanmaz çelik bir depoda karıştırın. <ol><li> Depoyu 121 ° C&#39;de bir otoklav içinde 60 dakika inkübe edin. Kuluçkadan sonra, karışım 60 L&#39;lik plastik bir depoda 40 L su ile yıkanır. Bu yıkamayı 5 kez tekrarlayın. </li><li> Toplam yıkanmış bamboyu 20 L hacmine ayarlamak için su kullanın.% 98 H204 kullanarak karışımın pH değerini 5.0&#39;a ayarlayın. </li><li> Karışımı iyi dağıtılmasını sağlamak için mekanik bir karıştırıcı ile teçhiz edilmiş modifiye sallama suyu banyosunda karışımı enzimatik olarak hidrolize edin. 200 mL 200 FPU / mL selülaz ekleyin ve karışımı 50 ° C&#39;de 36 saat inkübe edin. </li><li> Hidrolizden sonra, karışımı 10 dakika boyunca 4.700 xg&#39;de santrifüjleyin. </li></ol></li></ol> 2. Wildtype K. pneumoniae ile 2,3-Butandiol Üretimi <ol><li> Orta hazırlık için çözücü olarak 3 L bambu hidrolizat kullanın. </li><li> 4 g / L mısır dik likörü, 2 g / L (NH4) 2S04, 6 g / LK2 HPO4, 3 g / L KH2PO4 ve 0.2 g / L MgS04 içeren bir fermantasyon ortamı hazırlayın. 2.5 M NaOH kullanarak ortamın pH değerini nötr yapın. Fermantasyon için 3 L otoklava fermantasyon ortamı içeren 5 L&#39;lik bir biyoreaktör kullanın. </li><li> K. pneumoniae&#39;yi kullanın-80 ° C düşük sıcaklıkta buzdolabında saklanan hücreler. </li><li> Tohum kültürü için, 200 rpm&#39;de çalkalayarak, gece boyunca 37 ° C&#39;de 50 mL Luria-Bertani (LB) ortamı içeren 250 mL&#39;lik bir şişeyi inkübe edin. 10 g / L tripton, 5 g / L maya özütü ve 10 g / L NaCl içeren LB ortamı kullanın. NOT: Tohum kültürünün hücre yoğunluğu OD2&#39;ye (600 nm&#39;de optik yoğunluk) ulaşmalıdır. </li><li> Biyoreaktörde tohum kültürünün 50 mL&#39;lik bir şişesini aşılayın. Kültürü pH 6.0 ve 37 ° C&#39;de muhafaza edin. Mikroerobik bir durum oluşturan hava takviye hızı 2 L / dk ve çalkalama hızı 250 dev / dak. Kullanın. </li><li> Fermantasyon sırasında her 2 saatte bir 5 mL numuneler toplayın ve et suyu 4&#39;ündeki kimyasal konsantrasyonları belirlemek için yüksek basınçlı sıvı kromatografisiyle analiz edin. <ol><li> MFCS / DA kullanarak biyoreaktöre eklenen alkali&#39;yi izleyin. NOT: Organik asitler fermantasyon prosesinde,NaOH, kültür pH&#39;sını sabit tutmak için eklenir. Eklenen alkali hacmi, üretilen asit miktarını temsil eder. Alkali eklenen hattın plato seviyesine ulaşmasıyla, karbon kaynağı tükenmesi veya hücre ölümü yüzünden süreç sona erer. </li></ol></li></ol> 3. K. pneumoniae&#39;nin budC Mutant tarafından R- asetoin üretimi. <ol><li> 4 g / L mısır dik likörü, 2 g / L (NH4) 2 SO4, 3 g / L sodyum asetat, 0.4 g / L KCI ve 0.1 g / L MgS04 içeren asetoin üretimi için bir ortam hazırlayın. </li><li> R- asetoin üretimi için K. pneumoniae-ΔbudC kullanın. 2.4-2.5. Adımları tekrarlayın. NOT: budC 2,3-bütandiol dehidrojenaz kodlar. K. pneumoniae-ΔbudC 2,3-bütandiol dehidrogenaz aktivitesini kaybeder ve 2,3-bütandiol yerine R- asetoin üretir. </li><li> Kültürü pH 6.0 ve 37 ° C&#39;de muhafaza edin. Hava takviye oranı 4 L / dk ve bir agitatio kullanın450 rpm orana, aerobik bir durum. NOT: Oksijen takviyesi, 2,3-butandiol üretiminden daha yüksektir. </li><li> Örnekleri 2.6. Adımdaki gibi deneyin. </li></ol> 4. K. pneumoniae&#39;nin budA Mutant tarafından 2-Ketoglukonik Asit Üretimi <ol><li> 2-ketoglukonik asit üretimi için, R- asetoin üretimiyle aynı ortamı kullanın. </li><li> 2-ketoglukonik asit üretimi için K. pneumoniae-ΔbudA kullanın. 2.4-2.5. Adımları tekrarlayın. NOT: budA, α-asetolaktat dekarboksilaz kodlar. K. pneumoniae-ΔbudA α-asetolaktat dekarboksilaz aktivitesini kaybeder ve bir substrat olarak glikoz kullanarak 2-ketoglukonik asit üretir. NOT: 2-ketoglukonik asit sentezi asidik koşullara bağlı bir prosestir. 2-ketoglukonik asit üretimi için iki aşamalı fermantasyon geliştirilmiştir 10 ; İlk aşamada, tohum kültürü biyoreaktöre inoküle edilir. </li><lI&gt; Kültürü pH 7.0 ve 37 ° C&#39;de muhafaza edin. Hava takviye oranı 4 L / dk ve çalkalama hızı 500 rpm kullanın. NOT: Bu koşullardaki hücreler çok hızlı büyür (hücre yoğunluğu yaklaşık 4 saat içinde OD 7&#39;ye ulaşır). Ardından fermantasyon, kültür pH&#39;sının 5.0&#39;a düştüğü ikinci aşamaya doğru ilerlemektedir. Asit eklenmez; Kültürde üretilen organik asitler doğal olarak pH düşüşüne neden olurlar. Bu asidik koşullarda, hücre büyümesi durur, ancak 2-ketoglukonik asit sentezlenir ve suda birikir. </li><li> Örnekleri 2.6. Adımdaki gibi deneyin. Bambu hidrolizatını karbon kaynağı olarak kullanmak. NOT: Ortamdaki glikoz, 2-ketoglukonik asite dönüştürülür ve ksiloz, ksilonik asit haline dönüştürülür. Böylece, 2-ketoglukonik asit ve ksilonik asitten oluşan bir karışım elde edilir. </li></ol>

<ol>
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Yazarların açıklayacak bir şeyi yok.

K. pneumoniae , Enterobacteriaceae familyasında Klebsiella cinsine aittir. K. pneumoniae , toprak, bitki örtüsü ve su gibi doğal ortamlarda yaygın olarak bulunur 14 . Bu araştırmada kullanılan vahşi K. pneumoniae suşu topraktan izole edilmiş ve 1,3-propandiol üretimi için kullanılmıştır. K. pneumoniae ve bu türün mutantları, farklı koşullar altında birçok kimyasal üretir. Kültür pH ve ha...

Klebsiella pneumoniae , hem aerobik hem de anaerobik koşullar altında iyi yetişen bir bakteridir. K. pneumoniae , birçok kimyasal üretmek için kullanılan önemli bir endüstriyel mikroorganizma. 1,3-propandiol, polimetilen tereftalatın sentezlenmesi için esas olarak bir monomer olarak kullanılan değerli bir kimyasal maddedir. Polimetilen terefitalat, 1,2-propandiol, butandiol veya etilen glikol 1&#39;den daha iyi özelliklere sahip, biyolojik olarak parçalanabilen bir polyesterdir. 1,3-propandiol, K. pneumoniae tarafından oksijen ile sınırlandırılmış koşullar altında substrat olarak gliserol kullanılarak üretilir. 2,3-butandiol ve türevleri, plastik, solvent üretimi ve sentetik kauçuk alanında uygulamalara sahiptir ve biyoyakıt olarak kullanılma potansiyeline sahiptir 3 . Glukoz, substrat olarak, 2,3-bütandiol, vahşi tipli 4 suşunun ana metabolitidir. 2,3-bütandiol sentezlenirPiruvattan alınmıştır. İlk olarak, iki moleküllü piruvat, α-asetolaktat üretmek üzere yoğunlaşır; Bu reaksiyon α-asetolaktat sintazı ile katalize edilir. A-asetolaktat daha sonra a-asetolaktat dekarboksilaz ile asetoine dönüştürülür. R- asetoin, butandiol dehidrojenaz ile katalize edildiğinde 2,3-bütandiole indirgenebilir. K. pneumoniae için uygun etkili bir gen değiştirme yöntemi araştırılmış ve birçok mutant 5 , 6 , 7 inşa edilmiştir. 2,3-butandiol dehidrogenaz aktivitesini kaybeden bir budC mutantı, kültür besiyerinde yüksek seviyelerde asetoin biriktirir. Acetoin, gıda lezzetini arttırmak için katkı maddesi olarak kullanılır 8 . Α-asetolaktat dekarboksilaz kodlayan budA mutasyona uğradığında, 2-ketoglukonik asit suda birikir. 2-ketoglukonik asit, eritorbik asit (izoasorbik asit) sentezi için kullanılır;, Gıda endüstrisinde kullanılan bir antioksidan 9 . 2-ketoglukonik asit glikoz oksidasyon yolağının bir ara ürünüdür; Periplazmik boşlukta bulunan bu yolda glikoz, glukonik asite okside edilir ve daha sonra 2-ketoglukonik aside oksitlenir. Periplazda üretilen glukonik asit ve 2-ketoglukonik asit, daha ileri metabolizma için sitoplazmaya taşınabilir. 2-ketoglukonik asit birikimi asidik koşullara bağlıdır ve yüksek hava desteğiyle 2-ketoglukonik asit üretimi 10 tercih edilir . Gad tarafından kodlanan Gluconate dehydrogenase , Glukonik asitin 2-ketolgukonik asite dönüşmesini katalize eder. K. pneumoniae&#39;nin gad mutantı, 2-ketoglukonik asit yerine yüksek seviyede glükonik asit üretti ve bu süreç asidik koşullara da bağlı. Glukonik asit, toplu bir organik asittir ve çimento özelliklerini artırmak için bir katkı maddesi olarak kullanılır 11. Glikoz oksidasyonu, glikoz dehidrojenaz ile katalize edilir. Xylose de glikoz dehidrojenaz için uygun bir substrattır. Ksiloz, bir substrat olarak kullanıldığında, K. pneumoniae , ksilonik asit 12&#39;yi üretir. Biyokütle hammadde olarak kimyasal üretim, biyoteknolojide sıcak bir konudur 13 . Biyokütlenin ana bileşenleri selüloz, hemiselüloz ve lignin&#39;dir. Bununla birlikte, bu makromoleküler bileşikler çoğu mikroorganizma ( K. pneumoniae dahil) tarafından doğrudan katabolize edilemez. Biyokütledeki selüloz ve hemiselülozlar, glikoz ve ksiloz&#39;a hidrolize edilmeli ve daha sonra mikroorganizmalar tarafından kullanılabilmelidir. Lignoselülozlarda lignin varlığı, enzimler tarafından biyokütle hidrolizlenmesini önleyen koruyucu bir bariyer oluşturur. Bu nedenle, lignin ve hemiselülozları uzaklaştıran ve selülozun kristal yapısını azaltan bir ön işleme işlemi, biyokütle kullanımı sırasında mikrofondanroorganisms. Birçok ön-muamele yöntemi geliştirilmiştir: asit, alkalin, amonyak ve buhar ön-muameleleri sık görülür. Bambu tropik ve subtropikal bölgelerde bol miktarda bulunur ve önemli bir biyokütle kaynağıdır. Burada, bambu hidrolizatının hazırlanması ve kimyasal üretiminin bambu hidrolizat kullanılarak sunulması

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">autoclave</td>
			<td style="width:136px;">SANYO</td>
			<td align="right" style="width:119px;">3780</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">bioreactor</td>
			<td style="width:136px;">Sartorius stedim biotech</td>
			<td style="width:119px;">Bostat Aplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agitator</td>
			<td style="width:136px;">IKA</td>
			<td style="width:119px;">RW 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath shaker</td>
			<td style="width:136px;">Zhicheng</td>
			<td style="width:119px;">ZWY-110X50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">high performance liquid chromatograph system</td>
			<td style="width:136px;">Shimadzu Corp</td>
			<td style="width:119px;">20AVP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">centrifuge</td>
			<td style="width:136px;">Hitachi</td>
			<td style="width:119px;">CR22G III</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bamboo powder</td>
			<td style="width:136px;">purchased from Zhejiang Province, China</td>
			<td style="width:119px;"></td>
			<td>mesh number, 50</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">cellulase</td>
			<td style="width:136px;">Youtell Biochemical, Shandong, China</td>
			<td style="width:119px;"></td>
			<td>200 PFU/ml</td>
		</tr>
	</tbody>
</table>

production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock

Bambu önemli bir biyokütle ve bambu hidrolizatı Klebsiella pneumoniae tarafından kimyasal üretim için ham madde olarak kullanılır. Burada, bambu tozu NaOH ile ön işleme tabi tutuldu ve nötr bir pH&#39;a yıkandı. Önceden işlemden geçirilen bambu tozuya 30 g / L glikoz ve 15 g / L ksiloz içeren hidrolizatı oluşturmak için selülaz ilave edildi ve kimyasal üretim için bir ortam hazırlamak için karbon kaynağı olarak kullanıldı. Mikroerobik koşullar altında kültürlendiğinde, yabani tip K. pneumoniae tarafından 12.7 g / L 2,3-butandiol üretilmiştir. Aerobik koşullarda, 13.0 g / L R- asetoin, K. pneumoniae&#39;nin budC mutantı tarafından üretildi. 25.5 g / L 2-ketoglukonik asit ve 13.6 g / L xylonic asit karışımı, yüksek hava takviyesi ile iki aşamalı, pH kontrollü bir fermantasyonda K. pneumoniae budA mutantı tarafından üretildi. Fermantasyonun ilk aşamasında, kültür, nötr bir pH&#39;da muhafaza edildi; Hücre büyümesinden sonra, fermentasyonN ikinci aşamaya geçildi, bu sırada kültürün asidik kalmasına izin verildi.

Bu protokolde, bambu alkali kullanılarak ön işleme tabi tutuldu. Optimum inkübasyon parametreleri (121 ° C ve 0.25 M NaOH sıcaklıkları) Şekil 1 ve 2&#39;de belirlendi . Önceden işlemden geçirilmiş bambu, enzimatik olarak hidrolize edildi ve hidrolizatta elde edilen glikoz ve ksiloz konsantrasyonları ölçüldü. Daha yüksek sıcaklık şeker üretimini tercih etti, bu nedenle optimal sıcaklık olarak 121 ° C seçildi. Hidrolizatta üreti...

Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bambu tozu NaOH ile ön-muamele edildi ve enzimatik olarak hidrolize edildi. Bambu hidrolizatı, 2,3-butandiol, R- asetoin, 2-ketoglukonik asit ve Kylbsiella pneumoniae tarafından ksilonik asit üretimi için hammadde olarak kullanıldı.

Kimyasalların Üretimi<emKlebsiella pneumoniae</em&gt; Bambu Hidrolizatını Hammadde Olarak Kullanma

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

production-chemicals-klebsiella-pneumoniae-using-bamboo-hydrolysate

Research

JoVE Journal

Bioengineering

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

8.9K Görüntüleme.  Chinese Academy of Sciences.  Bambu tozu NaOH ile ön-muamele edildi ve enzimatik olarak hidrolize edildi. Bambu hidrolizatı, 2,3-butandiol, R- asetoin, 2-ketoglukonik asit ve Kylbsiella pneumoniae tarafından ksilonik asit üretimi için hammadde olarak kullanıldı. 

Video: Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Microwell Tabaklar kullanma büyüklüğü kontrollü Tümör küremsi Büyük Sayılar üretimi

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Biyomühendislik

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Bir Vaka Çalışması Olarak Tütün Geçici Protein Expression: Deneyler Yaklaşımı Tasarım Kullanma Karmaşık Sistemler Karakterizasyonu

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Üretim ve Monovalent Kuantum Noktalarının Hedefleme

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Yeniden İnşa İnsan Kornea gibi Epitelyal (RHCE) Doku Modeli kullanılarak Göz tahriş Kimyasalların Tehlike Tanımlaması Göz tahrişi Testi (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Sıvılardaki Darbeli Lazer-Ablasyonuyla Metal Nanopartikül Üretimi: Nanopartiküllerin Antibakteriyel Özelliklerini İncelemek İçin Bir Alet

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Otomatik Microbioreactor teknolojisini kullanarak Contegra Protein üretim duruma getirilmesi için genel iletişim kuralı

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

Üretim CRISPR/Cas9 kompleks Pronuclear enjeksiyonla genetiği altın Suriye hamster

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nano-formülasyonları endotoksin tespiti Limulus Amoebocyte Lysate (LAL) deneyleri kullanarak

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Lysolipid İçeren Sıcaklığa Duyarlı Lipozomların Mikroakışkan Üretimi

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenovirüs Üretimi İçin Verimli Bir Yöntem

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...