Functional characterization of the novel centrosomal protein Nlp (ninein-like protein)

The centrosome is the major microtubule organizing centre (MTOC) in animal cells. Most microtubules (MTs) emanate from the centrosome, where gamma-tubulin ring complexes (gammaTuRCs) act as templates for MT nucleation. During interphase, the centrosome organizes a MT array that imparts shape and polarity to the cell and is essential for intracellular transport and positioning of organelles such as the Golgi apparatus. During mitosis, centrosomes ensure bipolarity and correct orientation of the spindle by forming the spindle poles. In order to switch from the interphasic to the mitotic state, the centrosome undergoes a structural reorganization, termed maturation, which is mainly characterized by an increase in MT nucleation activity. A full appreciation of how centrosomes contribute to cellular functions requires the isolation and characterization of unknown centrosome-associated molecules. Here we describe the identification and characterization of a novel centrosomal component, the human protein Nlp (ninein-like protein) related to the previously characterized MT-anchoring protein ninein. In the first part of the present thesis we describe the identification of Nlp as a novel centrosomal substrate of Polo-like kinase 1 (Plk1), an important regulator of mitosis whose activity is required for centrosome maturation. Nlp interacts with two distinct gammaTuRC components, gamma-tubulin and hGCP4, and stimulates MT nucleation. Plk1 phosphorylates Nlp and disrupts its centrosomal association. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites induces defects in mitotic spindle formation. We propose that Nlp acts as a gammaTuRC binding protein (GTBP), contributing to the MT nucleation activity of the centrosome during interphase. At the onset of mitosis, the displacement of Nlp from the centrosome triggered by Plk1 phosphorylation could represent an important step in the maturation process which allows the centrosome to switch from the interphasic to the mitotic state. Thus, we conclude that Nlp, as well as the related protein ninein, plays an important role in MT organization. However the function of these two proteins possibly diverged during evolution: whilst Nlp gained a more prominent role in MT nucleation, ninein became principally involved in MT anchoring. In the second part of this thesis we report the initial characterization of the molecular mechanisms underlying the ability of Nlp and ninein to induce the fragmentation of the Golgi apparatus when overexpressed in human cells. We show that the ability of these two centrosomal proteins to affect the organization of the Golgi clearly depends on their capacity to associate with the cytoplasmic dynein-dynactin complex, a molecular motor complex primarly involved in the maintainance of Golgi architecture. We propose that the excess of Nlp and ninein could induce the disruption of the Golgi apparatus by sequestering the dynein-dynactin complexes. Future investigations should be aimed at understanding whether the dissociation of the Golgi apparatus from the centrosome induced by the excess of Nlp and ninein could interfere with cell migration and cell polarization processes, which require a highly coordinated action of these two organelles. Cell migration and cell polarization represent critical events for immune responses as well as for embryonic development, invasive growth and metastasis. Thus, our findings raise the interesting possibility that an upregulation in the expression levels of structural centrosomal proteins could represent the molecular basis for developmental disorders and malfunctioning of the immune system and, on the other hand, modulate the acquisition of invasive properties by neoplastic cells

New diagnostics for tuberculosis: fulfilling patient needs first

An effective tuberculosis (TB) control programme requires early diagnosis and immediate initiation of treatment. Any delays in diagnosing TB not only impair a patient's prognosis, but also increase the risks of transmitting the disease within the community. Unfortunately, the most recent TB diagnostic tools still depend on high-infrastructure laboratories, making them poorly adapted for use in resource-limited settings. Additionally, existing tests show poor performance in diagnosing TB in children, people living with HIV/AIDS, and extrapulmonary forms of the disease. As a consequence, TB patients are still to date left with either fair access to poor diagnostics or poor access to fair diagnostics

New Approaches to Filling the Gap in Tuberculosis Drug Discovery

For the first time in decades, say the authors, there is a tuberculosis drug pipeline, but the paucity of candidates is still cause for alarm

Integrating pediatric TB services into child healthcare services in Africa: study protocol for the INPUT cluster-randomized stepped wedge trial

Background Tuberculosis is among the top-10 causes of mortality in children with more than 1 million children suffering from TB disease annually worldwide. The main challenge in young children is the difficulty in establishing an accurate diagnosis of active TB. The INPUT study is a stepped-wedge cluster-randomized intervention study aiming to assess the effectiveness of integrating TB services into child healthcare services on TB diagnosis capacities in children under 5 years of age. Methods Two strategies will be compared: i) The standard of care, offering pediatric TB services based on national standard of care; ii) The intervention, with pediatric TB services integrated into child healthcare services: it consists of a package of training, supportive supervision, job aids, and logistical support to the integration of TB screening and diagnosis activities into pediatric services. The design is a cluster-randomized stepped-wedge of 12 study clusters in Cameroon and Kenya. The sites start enrolling participants under standard-of-care and will transition to the intervention at randomly assigned time points. We enroll children aged less than 5 years with a presumptive diagnosis of TB after obtaining caregiver written informed consent. The participants are followed through TB diagnosis and treatment, with clinical information prospectively abstracted from their medical records. The primary outcome is the proportion of TB cases diagnosed among children < 5 years old attending the child healthcare services. Secondary outcomes include: number of children screened for presumptive active TB; diagnosed; initiated on TB treatment; and completing treatment. We will also assess the cost-effectiveness of the intervention, its acceptability among health care providers and users, and fidelity of implementation. Discussion Study enrolments started in May 2019, enrolments will be completed in October 2020 and follow up will be completed by June 2021. The study findings will be disseminated to national, regional and international audiences and will inform innovative approaches to integration of TB screening, diagnosis, and treatment initiation into child health care services.publishedVersio

Paediatric tuberculosis - new advances to close persistent gaps

Young children are most vulnerable to develop severe forms of tuberculosis (TB) and are over-represented among TB deaths. Almost all children estimated to have died from TB were never diagnosed or offered TB treatment. Improved access to TB preventive treatment (TPT) requires major upscaling of household contact investigation with allocation of adequate resources. Symptom-based screening is often discouraged in adults for fear of generating drug resistance, if TB cases are missed. However, the situation in vulnerable young children is different, as they present minimal risk of drug resistance generation. Further, the perceived need for additional diagnostic evaluation presents a major barrier to TPT access and underlies general reluctance to consider pragmatic decentralised models of care. Widespread roll-out of Xpert MTB/RIF Ultra® represents an opportunity for improved case detection in young children, but attaining full impact will require the use of non-sputum specimens. The new Fujifilm SILVAMP TB LAM® urine assay demonstrated good diagnostic accuracy in HIV-positive and malnourished children, but further validation is required. Given the limited accuracy of all available tests and the excellent tolerance of TB drugs in children, the global community may have to accept some over-treatment if we want to close the persistent case detection gap in young children

Tuberculosis Diagnostics and Biomarkers: Needs, Challenges, Recent Advances, and Opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostic

Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics

Phosphorylation of Nlp by Plk1 negatively regulates its dynein-dynactin-dependent targeting to the centrosome

When cells enter mitosis the microtubule (MT) network undergoes a profound rearrangement, in part due to alterations in the MT nucleating and anchoring properties of the centrosome. Ninein and the ninein-like protein (Nlp) are centrosomal proteins involved in MT organisation in interphase cells. We show that the overexpression of these two proteins induces the fragmentation of the Golgi, and causes lysosomes to disperse toward the cell periphery. The ability of Nlp and ninein to perturb the cytoplasmic distribution of these organelles depends on their ability to interact with the dynein-dynactin motor complex. Our data also indicate that dynactin is required for the targeting of Nlp and ninein to the centrosome. Furthermore, phosphorylation of Nlp by the polo-like kinase 1 (Plk1) negatively regulates its association with dynactin. These findings uncover a mechanism through which Plk1 helps to coordinate changes in MT organisation with cell cycle progression, by controlling the dynein-dynactin-dependent transport of centrosomal proteins