Özel sayesinde HIV ile ilgili akış teşhis hizmetleri ulusal organizasyon için Güney Afrika Cumhuriyeti Ulusal Sağlık Laboratuvar Hizmetleri (NHLS) (http: \ \ www.nhls.ac.za ve http://www.plgcd4 net). Özellikli çalışmalar, Hematoloji ve Witwatersrand Üniversitesi Moleküler Tıp Anabilim Dalı&#39;nda yapılmıştır (Johannesburg, Güney Afrika Bölümü Başkanı, Prof. Wendy Stevens tarafından desteklenen). Yazarlar, ticari şirketler veya finans kurumları, bu yayın için herhangi bir hibe desteği aldık. Ifade edilen görüşler yazarlarına mutlaka Witwatersrand Üniversitesi ya da Güney Afrika NHLS Kurumları görüşlerini yansıtan.

<ol>
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Burada açıklanan CD4 yöntemi için uluslararası patent hakları, Güney Afrika NHLS ve şu anda BCI, Miami, Fl, ABD lisansı altında tutulur.

Son yıllarda hayır odaklı ve hibe tahsis programlar yüksek HIV yükü ile dünya kaynak sınırlı alanlarda mükemmel bazı klinik uygulamaların kurulmasına yardımcı olmuştur. SANAT programları ölümlerini azaltmada başarılı oldu ve şimdi yüksek CD4 başlatılan ediliyor 2,3 sayar. HIV özel bir aşı olmamasına rağmen, zaten bu yeni önlemler, HIV enfeksiyonunun 4,5 yayılmasını azalacak dair göstergeler vardır. Bizim Video biz belge ART hastaların izlenmesi, bu teknikler daha odaklı ve batı laboratuarları (Liste 1) yapılan rutin uygulamada gözlenen daha fazla maliyet-etkin hale olabilir. Afrika güdümlü Böyle bir yaklaşım, ART ile tedavi edilen HIV enfeksiyonu, kronik bir hastalık haline geliyor, bir defada, laboratuvar hizmetlerinin iyileştirilmesi desteklemek için gerekli olabilir. Böyle bir izleme ART ART 16 başlangıç ​​hakkında bir karar yardımcı olmak üzere keyfi gruba hasta yerleştirmek için CD4 sayımı gerçekleştirmek için kesin CD4 sayımı ve viral yük, ilgili teknoloji, daha zorlu bir görev gerektirir. Bu teşhis yardımı ile, yani CD8/CD38 testi kullanılarak,&gt; SANAT 16,19 hastaların% 60 VL-izleme ve maliyetlerini azaltmak için mümkün. VL ribaund ve / veya hasta uyumu eksikliği erken belirtileri ilaç almak için büyük olasılıkla CD4 sayısı 37, CD8/CD38 testi 16,17 ve vurgulamak için de önemlidir. Koordineli bir maliyet-etkin bir merkezi bir unsuru, operasyonun teknik ve tıbbi yönleri ile tanıdık uzman ve akademik danışmanlar tarafından desteklenen büyük ölçekli ulusal ya da bölgesel bir ağ durumu. Bu küçük merkezler için cesaret, eğitim ve kalite değerlendirmesi sağlar ve bu da &#39;nokta-bakım hizmetleri içerebilir Güney Afrika NHLS ağ faaliyetleri ve CD4-PLG.net eğitim programları 33, 36 olduğunu göstermiştir. Önemlisi, merkez ve bakım noktası laboratuvarlarının operasyonları tamamlayıcı niteliktedir. Eski bir maliyet-etkili, büyük ölçekli servis operasyonunun yanı sıra periferik laboratuarlar 33,38 temel / ileri eğitim veren çift bir rol olsa da, ikincisi için uygun olan HIV + hastalarda işe SANAT yayılmasını kontrol etmek için katkı taşra klinikleri de dahil olmak üzere daha geniş bir tabanı, ART. Noktası bakım teşhis çok daha pahalı olabilir ve otomatik olarak 38 doğruluğu garanti etmez, çünkü bazı dikkatli gereklidir. Bu nedenle, iki katmanlı bir sistem korumak için ve iki kol azalmaya izin vermeden iki katmanları arasındaki koordinasyonu kolaylaştırmak için mantıklı. Sonuç olarak, bulaşıcı hastalıkların klinik yönetimi tanı izleme alanında gerekli bir maliyet tasarrufu, önemli ölçüde geliştirilmiş kalite 13,39, daha pahalı ve daha iyi koordine modern teknoloji kullanan apt servisleri tarafından sağlanabilir. İyi organize olmuş ulusal (bölgesel) tanı hizmet dikkatli bir ön olmadan daha iyi bir senaryo bu zengin ülkelerden ithal edilen hemen hemen tüm hibe desteği en kısa sürede pahalı ve / veya denenmemiş yöntemleri üzerinde israf edilir kaçınarak 6-8 desteklenen olmalıdır davranarak yapıcı bir inceleme. Benzer şekilde, daha esnek bir tasarım, yerel doktorların ve sağlık personelinin tam katılımı ile yapılan klinik terapötik çalışmaların gereksiz yere sık sık ve / veya laboratuvar testleri 3 iddialı üzerinde azalmasına yol açacaktır . Bu politika, ART hastaların koordineli bir klinik / laboratuvar değerlendirmesi doğru teşhis sonuçlarının kullanımı ile daha dolgun bir şans daha vermek istiyorum. Bu akıllı teşhis aynı zamanda mevcut tanıdık bir akış sitometrik platformu 40 tüberküloz alanlarda ve lösemiler kaynak yoksul ülkelerde 8 öncülük olabilir.

comprehensive &amp; cost effective laboratory monitoring of hiv/aids: an african role model

- Biz, ekonomik büyük ölçekli tanı testi bir Güney Afrika rol modeli temsil apt bir laboratuvar hizmeti tarafından anti-retroviral tedavi (ART) yardım ile ilgili video sunmak. Ucuz SANAT düşük gelirli ülkelerde HIV seropozitif hastaların hayatta kalma umutları dönüştürdü ama ART ve ne pahasına olursa olsun laboratuar izlenmesi için bir ihtiyaç olup olmadığını şüpheler vardır durumlarda patoloji hizmetlerinin genel kalitesini hala çok düşük. Uygun bir yanıt, daha iyi bir koordinasyon ve Batı ülkelerinde görülen daha sıkı iç kalite değerlendirmesi ile ekonomik ses hizmetleri kurmak için. Witwatersrand Üniversitesi, Johannesburg, Güney Afrika, Ulusal Sağlık Laboratuvar Hizmetleri (NHLS SA) yerde fotoğraflanmış bu video, 2-renkli orijinal CD4-CD45 PanLeucoGating stratejisi (PLG) genişleyen bu koordineli bir düzeni sağlar. Böylece video sunumu altı modül tek bir tüp içinde, hematolojik, immunolojik ve viroloji ilgili testler birleştirmek için 4 renkli akım sitometri testinin basitliği ortaya. Bu video modülleri şunlardır: (i) set-up araçların, (ii) numune hazırlama, (iii) mutlak sayıları ve boncuk sayısı oranı ile her bir örnek için izleme kalitesi test; beyaz hücre için (iv) heamatological CD45 testi sayımı ve diferansiyel, (v) CD4 sayıları, ve (vi) CD8 aktivasyon + CD38 gösterge ile ölçülen T hücrelerinin, viral yük ile ilgili parametre. Potansiyel maliyet tasarrufu ile dikkat çekicidir. Bu düzenleme, bir NHLS-SA ağ içinde diğer laboratuarlara teknoloji transferi ile de, Batı laboratuarlarda uygulanan bu daha sıkı kalite kontrol ile günde 800-1000 testleri yerine fizibilite için bir örnektir. Uzman danışmanlar, laboratuar, modern tıp teknolojisi tanıtan hakkında karar verme görevi taşıyan yöneticiler ve politika yapıcılar, sık sık bir konumda iş yükünün büyük yüklerin büyük bölgesel laboratuarlarda gerçekleştirilen son teknik detayları görmek için değildir. Dolayısıyla bu video bu yeni gelişmelerin ayrıntıları gösterir.

Watch this Scientific Journal Video about Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model at JoVE.com

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

HIV / AIDS tedavisi için anti-retroviral tedavi Güney Afrika&#39;da büyük bir ölçekte izlenir. Flow sitometri, hematoloji (CD45), immünoloji (CD4) ve CD38 tahlil bağlantılı viral yük için birleştirilmiştir. NHLS-SA laboratuarları, Johannesburg kaydedildi, kaynak sınırlı teşhis için rol modelleri olarak hizmet veren bu modern yöntemleri, artan yerel iç kalite kontrolü ile maliyet-etkin.

HIV / AIDS Kapsamlı ve Maliyet Etkin Laboratuvar Kontrolü: Afrikalı bir Rol Modeli

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

comprehensive-cost-effective-laboratory-monitoring-hivaids-an-african

Research

JoVE Journal

Immunology and Infection

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

16.6K Görüntüleme.  National Health Laboratory Services (NHLS-SA). HIV / AIDS tedavisi için anti-retroviral tedavi Güney Afrika'da büyük bir ölçekte izlenir. Flow sitometri, hematoloji (CD45), immünoloji (CD4) ve CD38 tahlil bağlantılı viral yük için birleştirilmiştir. NHLS-SA laboratuarları, Johannesburg kaydedildi, kaynak sınırlı teşhis için rol modelleri olarak hizmet veren bu modern yöntemleri, artan yerel iç kalite kontrolü ile maliyet-etkin.

Video: Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Analitik Oral Biyofilmler Kapsamlı biyokimyasal, yapısal ve Transcriptome Değerlendirme Aracı-box mutans Streptokok tarafından aracılığı ile

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

İmmünoloji ve Enfeksiyon

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

HIV-1 tropizm genotipik Çıkarım kullanarak Nüfus tabanlı Sıralama V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Özgül İnsan ve Hayvan Virüsler Mikrobiyal Kaynak Takip için maliyet-etkin Yöntemi

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host hastalığı indüksiyonu ve<em&gt; In Vivo</emBir MHC-eşlemeli murin Model Kullanma&gt; T Hücre İzleme

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

bir (A) Güvenlik Önlemleri ve Çalışma Usul BSL-4 Laboratuvar: 1. Biyogüvenlik Seviye 4 Suit Laboratuvar Suite Giriş ve Çıkış İşlemleri

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

İnsan Periferik Kan immünfenotipik Analizi Antikor Kokteyller Hazırlama A Yarı-otomatik Yaklaşım

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Biyoparlaklık Görüntüleme Afrika Trypanosomiasis Geç Evre Enfeksiyon Algılama için

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Modüle c-di-GMP sinyal İşte Küçük Moleküller Tarama için Yüksek verim Kur kurulması<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

ve Cefoperazone-tedavi edilmiş fare modeli klinik olarak ilgili<em&gt; Clostridium difficile</em&gt; Gerilme R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Bir IL-8 Geçici Transgenize Edilmiş Fare Modeli<emIn vivo</em&gt; İnflamatuvar Yanıtların Uzun Vadeli İzlenmesi

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...