scholarly journals Cell-based assay for ciliopathy patients to improve accurate diagnosis using ALPACA

2021 ◽  
Author(s):  
Cenna Doornbos ◽  
Ronald van Beek ◽  
Ernie M. H. F. Bongers ◽  
Dorien Lugtenberg ◽  
Peter. H. M. Klaren ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Accurate Diagnosis ◽  
Clinical Heterogeneity ◽  
Healthy Individuals ◽  
Phenotypic Data ◽  
Functional Assays ◽  
Vertebrate Cell ◽  
Conclusive Diagnosis ◽  
Cilium Length

AbstractSkeletal ciliopathies are a group of disorders caused by dysfunction of the cilium, a small signaling organelle present on nearly every vertebrate cell. This group of disorders is marked by genetic and clinical heterogeneity, which complicates accurate diagnosis. In this study, we developed a robust, standardized immunofluorescence approach to accurately diagnose a subset of these disorders. Hereto we determined and compared the cilium phenotype of healthy individuals to patients from three different ciliopathy subgroups, using skin-derived fibroblasts. The cilium phenotype assay consists of three parameters; (1) ciliogenesis, based on the presence or absence of cilium markers, (2) cilium length, measured by the combined signal of an axonemal and a cilium membrane marker, and (3) retrograde intraflagellar transport (IFT), quantified by the area of the ciliary tip. Analysis of the cilium phenotypic data yielded comparable and reproducible results and in addition, displayed identifiable clusters for healthy individuals and two ciliopathy subgroups, i.e. ATD and CED. Our results illustrate that standardized analysis of the cilium phenotype can be used to discriminate between ciliopathy subgroups. Therefore, we believe that standardization of functional assays analyzing cilium phenotypic data can provide additional proof for conclusive diagnosis of ciliopathies, which is essential for routine diagnostic care.

scholarly journals Staphylococcus aureus Pneumonia: Preceding Influenza Infection Paves the Way for Low-Virulent Strains

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 734
Author(s):  
Stefanie Deinhardt-Emmer ◽  
Karoline Frieda Haupt ◽  
Marina Garcia-Moreno ◽  
Jennifer Geraci ◽  
Christina Forstner ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Genes ◽  
Influenza Infection ◽  
Severe Infection ◽  
Healthy Individuals ◽  
Functional Assays ◽  
Host Pathogen Interaction ◽  
Virulent Strains ◽  
Cell Culture Systems ◽  
Staphylococcus Aureus Pneumonia

Staphylococcus aureus is a facultative pathogenic bacterium that colonizes the nasopharyngeal area of healthy individuals, but can also induce severe infection, such as pneumonia. Pneumonia caused by mono- or superinfected S. aureus leads to high mortality rates. To establish an infection, S. aureus disposes of a wide variety of virulence factors, which can vary between clinical isolates. Our study aimed to characterize pneumonia isolates for their virulent capacity. For this, we analyzed isolates from colonization, pneumonia due to S. aureus, and pneumonia due to S. aureus/influenza virus co-infection. A total of 70 strains were analyzed for their virulence genes and the host–pathogen interaction was analyzed through functional assays in cell culture systems. Strains from pneumonia due to S. aureus mono-infection showed enhanced invasion and cytotoxicity against professional phagocytes than colonizing and co-infecting strains. This corresponded to the high presence of cytotoxic components in pneumonia strains. By contrast, strains obtained from co-infection did not exhibit these virulence characteristics and resembled strains from colonization, although they caused the highest mortality rate in patients. Taken together, our results underline the requirement of invasion and toxins to cause pneumonia due to S. aureus mono-infection, whereas in co-infection even low-virulent strains can severely aggravate pneumonia.

scholarly journals Single-cell transcriptomic analysis reveals disparate effector differentiation pathways in human Treg compartment

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuechen Luo ◽  
Changlu Xu ◽  
Bing Wang ◽  
Qing Niu ◽  
Xiuhua Su ◽  
...  
Keyword(s):  
Single Cell ◽  
Trajectory Analysis ◽  
Cell Receptor ◽  
Healthy Individuals ◽  
Graft Versus Host ◽  
Functional Assays ◽  
Hla Dr ◽  
Health And Disease ◽  
Differentiation Pathways ◽  
Undergo Stem Cell Transplantation

AbstractHuman FOXP3+ regulatory T (Treg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate Treg cells into naïve, activated, and effector stages, and resolve the HLA-DRhi, LIMS1hi, highly suppressive FOXP3hi, and highly proliferative MKI67hi effector subsets. Trajectory analysis assembles Treg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3hi and MKI67hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3hi and MKI67hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of Treg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of Treg complexity in health and disease.

scholarly journals Pitfalls and progress in adrenocortical carcinoma diagnosis: the utility of a multidisciplinary approach, immunohistochemistry and genomics

2022 ◽  
Vol 2022 ◽  
Author(s):  
Ray Wang ◽  
Benjamin Solomon ◽  
Stephen J Luen ◽  
Owen W.J. Prall ◽  
Christine Khoo ◽  
...  
Keyword(s):  
Adrenocortical Carcinoma ◽  
Clinical Signs ◽  
Accurate Diagnosis ◽  
List Type ◽  
Clinical Heterogeneity ◽  
Diagnostic Challenge ◽  
Diagnosis And Prognosis ◽  
Prognostic Utility ◽  
Rare Malignancy ◽  
Adrenocortical Carcinomas

Summary Adrenocortical carcinoma is a rare disease with poor prognosis whose clinical heterogeneity can at times present a challenge to accurate and timely diagnosis. We present the case of a patient who presented with extensive pulmonary lesions, mediastinal and hilar lymphadenopathy and an adrenal mass in whom the oncological diagnosis was initially uncertain. Through the use of immunohistochemistry, biochemistry and genomic testing, an accurate diagnosis of adrenocortical carcinoma was ultimately made which resulted in more directed treatment being administered. The use of multidisciplinary input and genomics to aid in diagnosis and prognosis of adrenocortical carcinoma is discussed. Learning points Adrenocortical carcinomas can present a diagnostic challenge to clinicians given it is a rare malignancy with significant clinical heterogeneity. Specialist multidisciplinary team input is vital in the diagnosis and management of adrenocortical carcinomas. Hormonal testing is recommended in the diagnostic workup of adrenal masses, even in the absence of overt clinical signs/symptoms of hormone excess. Immunostaining for the highly sensitive and specific steroidogenic factor-1 is vital for accurate diagnosis. Genomics can provide prognostic utility in management of adrenocortical carcinoma.

scholarly journals Discrepant molecular and clinical diagnoses in Beckwith-Wiedemann and Silver-Russell syndromes

2019 ◽  
Vol 101 ◽  
Author(s):  
Deborah J.G. Mackay ◽  
Jet Bliek ◽  
Maria Paola Lombardi ◽  
Silvia Russo ◽  
Luciano Calzari ◽  
...  
Keyword(s):  
Clinical Diagnosis ◽  
Accurate Diagnosis ◽  
Molecular Testing ◽  
Clinical Heterogeneity ◽  
Imprinting Disorders ◽  
Molecular Alterations ◽  
Clinical Diagnoses ◽  
Silver Russell Syndrome

AbstractBeckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are two imprinting disorders associated with opposite molecular alterations in the 11p15.5 imprinting centres. Their clinical diagnosis is confirmed by molecular testing in 50–70% of patients. The authors from different reference centres for BWS and SRS have identified single patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis. These patients clinically do not fit the characteristic phenotypes of SRS or BWS, but illustrate their clinical heterogeneity. Thus, comprehensive molecular testing is essential for accurate diagnosis and appropriate management, to avoid premature clinical diagnosis and anxiety for the families.

scholarly journals LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena

2019 ◽  
Author(s):  
Yu-Yang Jiang ◽  
Wolfgang Maier ◽  
Ralf Baumeister ◽  
Gregory Minevich ◽  
Ewa Joachimiak ◽  
...  
Keyword(s):  
Steady State ◽  
Total Internal Reflection ◽  
Feedback Loop ◽  
Intraflagellar Transport ◽  
Binding Motif ◽  
Loss Of Function ◽  
C Elegans ◽  
Extracellular Signals ◽  
Total Internal Reflection Microscopy ◽  
Cilium Length

AbstractThe length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. In Tetrahymena, a loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their total number per cell. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated the IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. A forward genetic screen identified a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. A loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. We propose that the total LF4/MOK activity per cilium is dependent on both its activation by an upstream CDK-related kinase and cilium length. Previous studies showed that the rate of assembly is high in growing cilia and decreases as cilia elongate to achieve the steady-state length. We propose that in a longer cilium, the IFT components, which travel from the base to the tip, are subjected to a higher dose of inhibition by the uniformly distributed LF4/MOK. Thus, in a feedback loop, LF4/MOK may translate cilium length into proportional inhibition of IFT, to balance the rates of assembly and disassembly at steady-state.Author summaryCilia are conserved organelles that generate motility and mediate vital sensory functions, including olfaction and vision. Cilia that are either too short or too long fail to generate proper forces or responses to extracellular signals. Several cilia-based diseases (ciliopathies) are associated with defects in cilia length. Here we use the multiciliated model protist Tetrahymena, to study a conserved protein kinase whose activity shortens cilia, LF4/MOK. We find that cells lacking an LF4/MOK kinase of Tetrahymena, LF4A, have excessively long, but also fewer cilia. We show that LF4A decreases the intraflagellar transport, a motility that shuttles ciliary precursors from the cilium base to the tip. Live imaging revealed that LF4A is distributed along cilium length and remains mostly immobile, likely due to its anchoring to ciliary microtubules. We proposed that in longer cilia, the intraflagellar transport machinery is exposed to a higher dose of inhibition by LF4A, which could decrease the rate of cilium assembly, to balance the rate of cilium disassembly in mature cilia that maintain stable length.

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