The role of chlamydial inclusion membrane proteins in host-pathogen interaction and the development of novel methods for studying chlamydial biology

The majority of our modern understanding of bacterial pathogenesis is based on the strategy that involves screening bacterial genomes for the presence of the genes encoding pathogenic factors, and analysis of these genes via forward and reverse genetics. Chlamydiae represent a unique group of pathogenic bacteria in which it is not feasible to apply genetic approaches that are currently used for other organisms. The obligate intracellular nature of these organisms also makes transfection with foreign DNA and subsequent selection for potentially successful transformants very challenging. This thesis explores techniques that address the study of these organisms in the absence of a workable genetic system. The first goal of this study was to examine the role of a collection of unique chlamydial proteins, known as the Inc-proteins, in the host-pathogen interactions. Inc proteins are localized on the inclusion membrane, and are exposed to the cytosolic surface of this membrane. Such localization brings Inc proteins into direct contact with the cytoplasm of the host cell. To study the influence of Inc proteins on host cell biology we used the method of transient expression of genes from a eukaryotic expression plasmid, followed by the analysis of the resulting host cell phenotype. These experiments were conducted in both infected and uninfected cells. It was demonstrated that expression of Chlamydophila (Chlamydia) caviae IncA protects host cells from infection with C. caviae by blocking the development of chlamydiae inside of the host cells. This effect of C. caviae IncA was specific, because the expression of the homologous IncA protein from Chlamydia trachomatis has no effect on the development of C. trachomatis or C. caviae. Using the same approach it was shown that Inc proteins CT223, CT224 and CT225 from C. trachomatis each were capable of affecting the cell cycle of the host cells by blocking cytokinesis. This is significant, as other investigators have reported that infection with C. trachomatis leads to a similar phenotype. Transient expression of CT223, CT224, and CT225 affect host cell cytokinesis on the same manner as C. trachomatis infection. A statistically significant proportion of cells producing any of these three Inc proteins had a multinuclear phenotype and multiple centrosomes, indicating that cell division was blocked late in the cell cycle. This effect was specific for these particular Inc proteins and was observed both in murine and human cultured cells. Many scientists are trying to develop methods for chlamydial mutagenesis and transformation. However, these attempts have not yet been successful, and the delivery of DNA into chlamydiae is not the only obstacle. Even if chlamydiae are transformed, the successful transformants have to be effectively selected and segregated. To address the challenge of isolating individual candidate chlamydial transformants, we developed a novel technique for the rapid and productive separation of microbiological clones of chlamydiae. This was accomplished using a new method that is based on a unique feature of live chlamydiae to accumulate and retain the fluorescent dye C6-NBD-ceramide. This labeling results in clearly identifiable chlamydial inclusions, and thus, clearly identifiable infected cells. These labeled, infected, cells can then be separated from uninfected cells on a fluorescence activated cell sorter. The approach was used to isolate clonal populations of prototype strains from Chlamydia trachomatis, Chlamydia caviae, and Chlamydia suis. Recent clinical isolates were also successfully cloned. The procedure is simple and rapid, with single cloning cycles being completed 24h post-culture of a sample. As will be discussed, this technique has applications in both research and clinical settings

Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

<p>Abstract</p> <p>Background</p> <p>The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The <it>inc </it>gene <it>CT223 </it>is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested <it>C. trachomatis </it>serovars.</p> <p>Results</p> <p>A plasmid transfection approach was used to examine the function of the product of <it>CT223 </it>and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that <it>CT223</it>, and, to a lesser extent, adjacent <it>inc </it>genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells.</p> <p>Conclusion</p> <p>These studies suggest that certain Inc proteins block cytokinesis in <it>C. trachomatis</it>-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.</p

Implementação eficiente de programas educacionais na universidade pedagógica

O estudo apresenta novas abordagens para a diversificação dos programas educacionais da universidade pedagógica, cujo objetivo é melhorar a qualidade da formação profissional de professores no contexto de conteúdos educacionais atualizados. Ele também analisa o estado atual da educação no Cazaquistão, descreve as conquistas e os problemas neste campo. O conteúdo dos programas educacionais proporciona uma formação de sucesso e torna os professores modernos competitivos no mercado de trabalho. Leva em consideração o desenvolvimento de competências-chave: comunicação interpessoal, liderança estratégica, prontidão para mudanças, atividades de projeto, colaboração, responsabilidade ética e profissional e gestão eficaz do tempo. As principais prioridades do desenvolvimento de programas educacionais são as seguintes: generalização das atividades acadêmicas e de pesquisa, desenvolvimento e implementação de programas educacionais baseados na orientação prática, interdisciplinaridade, oferta de treinamento em profundidade em línguas e criação de um ambiente educacional único. A estrutura de conteúdo, algoritmo de criação, condições e recursos para a implementação de programas educacionais são descritos. O objetivo deste artigo é definir e resolver o problema do desenvolvimento da educação

The cryogenic detector for cosmology observation

https://iopscience.iop.org/article/10.1088/1757-899X/502/1/012060/pdfThis paper will present the implementation of the Microwave Kinetic Inductance Detectors (MKID) for mm/submm astronomy purposes in our cryocooler at the Energetic Cosmos Laboratory (ECL). Authors have used the robust cryocooler 106 Shasta from HPD Company. The refrigerator can cool down the temperature to 30 millikelvin. In this report, authors describe our experiences about millikelvin refrigerator, implementation challenges of MKIDs in refrigerator and the evaluation results

Data Acquisition System for Microwave Kinetic Inductance Detectors

https://iopscience.iop.org/article/10.1088/1742-6596/1182/1/012006Energetic Cosmos Laboratory is developing Kinetic Inductance Detectors (MKID) for mm/submm astronomy purposes. In order to measure the instantaneous resonance frequency and dissipation of superconducting microresonators of the MKID arrays, we have developed a data acquisition system with emphasis on precision, readout speed and digital processing capabilities. We use IQ mixer to down convert the MKIDs signal in the range of 20-80 MHz, then digitize them by 250 MSPS ADC. Processed data at FPGA-Kintex 7 will be transferred to the PC for further analysis by the rate of 10 Gbit/sec. In this report, we describe the technical details and the algorithm we developed

A Chlamydia effector recruits CEP170 to reprogram host microtubule organization

The obligate intracellular bacterial pathogen Chlamydia trachomatis deploys virulence effectors to subvert host cell functions enabling its replication within a specialized membrane-bound compartment termed an inclusion. The control of the host cytoskeleton is critical for Chlamydia uptake, inclusion biogenesis and cell exit. Here we demonstrate how a Chlamydia effector rearranges the microtubule network by initiating organization of the microtubules at the inclusion surface. We identified an inclusion-localized effector sufficient to interfere with microtubule assembly that we term inclusion protein acting on microtubules (IPAM). We established that IPAM recruits and stimulates the centrosomal protein 170kDa (CEP170) to hijack the microtubule organizing functions of the host cell. We show that CEP170 is essential for chlamydial control of host microtubule assembly, and is required for inclusion morphogenesis and bacterial infectivity. Together, we demonstrate how a pathogen effector reprograms the host microtubule network to support its intracellular development

Discovering and Differentiating New and Emerging Clonal Populations of Chlamydia trachomatis with a Novel Shotgun Cell Culture Harvest Assay

This assay, coupled with ompA and 16S rRNA sequencing, characterized clonal populations of C. trachomatis

Клинико-лабораторные особенности COVID-19 у пациентов с сочетанием ВИЧ-инфекция + туберкулез

The objective: to study clinical and laboratory specific parameters of the COVID-19 course in patients with TB/HIV co-infection, to analyze changes in the lungs caused by COVID-19 in patients with pulmonary tuberculosis and concurrent HIV infection.Subjects and Methods. 68 HIV-infected patients aged 18-66 years old were included in the study, they all were admitted to the Novosibirsk State Regional Clinical Tuberculosis Hospital, Novosibirsk, with confirmed COVID-19 from May 2020 to May 2021. Clinical manifestations, CD4 count and HIV RNA level, and the presence of secondary infections were assessed.Results. In patients with severe and profound immunodeficiency, coronavirus infection was more often mild to moderate, and in immunocompetent patients – moderate to severe. However, patients with profound immunodeficiency were at greater risk of an adverse outcome due to lymphohematogenic progression of tuberculosis and concurrent opportunistic infections.Conclusion. When diagnosing and treating COVID-19 in patients with comorbid HIV infection and tuberculosis, it is important to consider the form of tuberculosis and CD4+ count.Цель исследования: изучение клинических и лабораторных особенностей течения COVID-19 у пациентов с сочетанием туберкулеза и ВИЧ-инфекции, анализ изменений в легких, вызванных COVID-19, у пациентов с туберкулезом легких на фоне ВИЧ-инфекции.Материалы и методы. В исследование включено 68 пациентов с ВИЧ-инфекцией в возрасте от 18 до 66 лет, госпитализированных в ГБУЗ НСО «Государственная областная Новосибирская клиническая туберкулезная больница» г. Новосибирска с подтвержденным COVID-19 в период с мая 2020 г. по май 2021 г. Изучены клинические проявления, содержание СD4-лимфоцитов и РНК ВИЧ, наличие вторичных инфекций.Результаты. У пациентов с выраженным и глубоким иммунодефицитом коронавирусная инфекция чаще протекала в легкой и среднетяжелой форме, а у иммунокомпетентных – в среднетяжелой и тяжелой форме. Однако пациенты с глубоким иммунодефицитом были подвержены бо́льшему риску неблагоприятного исхода в связи с лимфагематогенным прогрессированием туберкулеза и наличием оппортунистических инфекций.Заключение. При диагностике и лечении COVID-19 у пациентов с сочетанной патологией ‒ ВИЧ-инфекция и туберкулез – важно учитывать форму туберкулезного процесса и уровень CD4+ Т-лимфоцитов

The role of simultaneous treatment of chronic generalized parodonitis in the period of decompensation of type II diabetes

The results of examination of patients with the diabetes of type II in the phase of decompensation of severe degree who are and who are not being treated of chronic generalized periodontitis with severe degree of intensity are studied in the article. On the basis of clinical and laboratory tests of mixed saliva and blood some data were obtained that allow to judge about the faster recovery and selection of insulin for short and prolonged effect in smaller doses when curing patients suffering simultaneously from diabetes of type II and chronic generalized periodontitis.В статье рассмотрены результаты обследования пациентов с сахарным диабетом II типа в фазе декомпенсации тяжелой степени одновременно получающих и не получающих лечение хронического генерализованного пародонтита тяжелой степени выраженности. На основании клинико-лабораторных показателей смешанной слюны и крови получены данные позволяющие утверждать о более быстрых сроках реабилитации и подбора инсулинов короткого и продленного действий в меньших дозировках у пациентов с сахарным диабетом II типа одновременно проходящих лечение хронического генерализованного пародонтита

Characterization and intracellular localization of putative Chlamydia pneumoniae effector proteins

We here describe four proteins of Chlamydia pneumoniae, which might play a role in host-pathogen interaction. The hypothetical bacterial proteins CPn0708 and CPn0712 were detected in Chlamydia pneumoniae-infected host cells by indirect immunofluorescence tests with polyclonal antisera raised against the respective proteins. While CPn0708 was localized within the inclusion body, CPn0712 was identified in the inclusion membrane and in the surrounding host cell cytosol. CPn0712 colocalizes with actin, indicating its possible interaction with components of the cytoskeleton. Investigations on CPn0809 and CPn1020, two Chlamydia pneumoniae proteins previously described to be secreted into the host cell cytosol, revealed colocalization with calnexin, a marker for the ER. Neither CPn0712, CPn0809 nor CPn1020 were able to inhibit host cell apoptosis. Furthermore, transient expression of CPn0712, CPn0809 and CPn1020 by the host cell itself had no effect on subsequent infection with Chlamydia pneumoniae. However, microarray analysis of CPn0712-expressing host cells revealed six host cell genes which were regulated as in host cells infected with Chlamydia pneumoniae, indicating the principal usefulness of heterologous expression to study the effect of Chlamydia pneumoniae proteins on host cell modulation