Single-cell measurements are uniquely capable of characterizing cell-to-cell heterogeneity and have been used to explore the large diversity of cell types and physiological functions present in ...tissues and other complex cell assemblies. An intriguing application of single-cell proteomics is the characterization of proteome dynamics during biological transitions, like cellular differentiation or disease progression. Time-course experiments, which regularly take measurements during state transitions, rely on the ability to detect dynamic trajectories in a data series. However, in a single-cell proteomics experiment, cell-to-cell heterogeneity complicates the confident identification of proteome dynamics as measurement variability may be higher than expected. Therefore, a critical question for these experiments is how many data points need to be acquired during the time course to enable robust statistical analysis. We present here an analysis of the most important variables that affect statistical confidence in the detection of proteome dynamics: fold change, measurement variability, and the number of cells measured during the time course. Importantly, we show that datasets with less than 16 measurements across the time domain suffer from low accuracy and also have a high false-positive rate. We also demonstrate how to balance competing demands in experimental design to achieve a desired result.
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•Detecting temporal change in proteins depends on fold change and variability.•Replicate time courses improve reliability of detecting temporal dynamics.•Temporal experiments require a dense sampling of cells to track gradual transitions.•Time-course trajectory experiments require more samples than two-state comparisons.
Cellular development and disease progression are gradual transitions between phenotypic stages. Time-course measurements that explicitly measure this transition are important to discover proteome dynamics. Single-cell measurements are a powerful tool for understanding heterogeneity, especially during phenotypic transitions. Single-cell proteomics measurements are emerging as an available tool to characterize the cellular state. We created a statistical method that predicts the success of an experimental design for temporal dynamics.
The goal of proteomics is to identify and quantify the complete set of proteins in a biological sample. Single-cell proteomics specializes in the identification and quantitation of proteins for ...individual cells, often used to elucidate cellular heterogeneity. The significant reduction in ions introduced into the mass spectrometer for single-cell samples could impact the features of MS2 fragmentation spectra. As all peptide identification software tools have been developed on spectra from bulk samples and the associated ion-rich spectra, the potential for spectral features to change is of great interest. We characterize the differences between single-cell spectra and bulk spectra by examining three fundamental spectral features that are likely to affect peptide identification performance. All features show significant changes in single-cell spectra, including the loss of annotated fragment ions, blurring signal and background peaks due to diminishing ion intensity, and distinct fragmentation pattern, compared to bulk spectra. As each of these features is a foundational part of peptide identification algorithms, it is critical to adjust algorithms to compensate for these losses.
We report a case of chagasic encephalitis diagnosed by 28S rRNA sequencing. The diagnosis of chagasic encephalitis is challenging, given the broad differential diagnosis for central nervous system ...lesions in immunocompromised patients and low sensitivity of traditional diagnostics. Sequencing should be part of the diagnostic armamentarium for potential chagasic encephalitis.
Extranodal marginal zone B-cell lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT) that affects the ocular adnexa, also known as ocular adnexal MALT lymphomas (OAML), are low-grade lymphomas ...that mostly affect elderly individuals. This study was conducted to explore the genetic and microbial drivers of OMAL, and unique morphometric phenotypes associated with these mutations and infections.
In this study, we performed targeted deep sequencing of 8 OAML cases to identify its potential genetic and microbial drivers. We additionally performed computational digital image analysis of cases to determine if morphologic features corresponded to genetic mutations and disease biology.
We identified TBL1XR1 as recurrently mutated in OAML (4/8), and mutations in several other oncogenes, tumor suppressors, transcription regulators, and chromatin remodeling genes. Morphologically, OAML cases with mutations in TBL1XR1 showed lymphoma cells with significantly lower circularity and solidity by computational digital image analysis (p-value <0.0001). Additionally, cases of OAML with mutations in TBL1XR1 showed equivalent or increased vascular density compared to cases without mutations in TBL1XR1. Finally, we did not find any infectious microbial organisms associated with OAML.
Our study showed recurrent mutations in TBL1XR1 are associated with unique morphometric phenotypes in OMAL cases. Additionally, mutations in genes associated with the methylation status of histone 3, nuclear factor (NF)-κB pathway, and NOTCH pathway were enriched in OMAL cases. Our findings have biologic and clinical implications as mutations in TBL1XR1 and other genes have the potential to be used as markers for the diagnosis of OAML, and also demonstrate a specific biologic phenotypic manifestation of TBL1XR1 mutations.
Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors ...play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.
Recombinant adeno-associated virus (AAV)-mediated degeneration of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) has been observed in non-human primates (NHPs) following ...intravenous (IV) and intrathecal (IT) delivery. Administration of recombinant AAV encoding a human protein transgene via a single intra-cisterna magna (ICM) injection in New Zealand white rabbits resulted in histopathology changes very similar to NHPs: mononuclear cell infiltration, degeneration/necrosis of sensory neurons, and nerve fiber degeneration of sensory tracts in the spinal cord and of multiple nerves. AAV-associated clinical signs and incidence/severity of histologic findings indicated that rabbits were equally or more sensitive than NHPs to sensory neuron damage. Another study using human and rabbit transgene constructs of the same protein demonstrated comparable changes suggesting that the effects are not an immune response to the non-self protein transgene. Rabbit has not been characterized as a species for general toxicity testing of AAV gene therapies, but these studies suggest that it may be an alternative model to investigate mechanisms of AAV-mediated neurotoxicity and test novel AAV designs mitigating these adverse effects.
Increasing the number of Latino persons with dementia who consent to brain donation (BD) upon death is an important public health goal that has not yet been realized. This study identified the need ...for culturally sensitive materials to answer questions and support the decision-making process for the family.
Information about existing rates of BD was obtained from the Alzheimer's Disease Centers. Several methods of data collection (query NACC database, contacting Centers, focus groups, online survey, assessing current protocol and materials) were used to give the needed background to create culturally appropriate BD materials.
A decision was made that a brochure for undecided enrollees would be beneficial to discuss BD with family members. For those needing further details, a step-by-step handout would provide additional information.
Through team collaboration and engagement of others in the community who work with Latinos with dementia, we believe this process allowed us to successfully create culturally appropriate informational materials that address a sensitive topic for Hispanic/Latino families.
Brain tissue is needed to further knowledge about underlying biological mechanism of neurodegenerative diseases, however it is a sensitive topic. Materials assist with family discussion and facilitate the family's follow-through with BD.
One hallmark of human aging is increased brain inflammation represented by glial activation. With age, there is also diminished function of the adaptive immune system, and modest decreases in ...circulating B- and T-lymphocytes. Lymphocytes traffic through the human brain and reside there in small numbers, but it is unknown how this changes with age. Thus we investigated whether B- and T-lymphocyte numbers change with age in the normal human brain. We examined 16 human subjects in a pilot study and then 40 human subjects from a single brain bank, ranging in age from 44–96 years old, using rigorous criteria for defining neuropathological changes due to age alone. We immunostained post-mortem cortical tissue for B- and T-lymphocytes using antibodies to CD20 and CD3, respectively. We quantified cell density and made a qualitative assessment of cell location in cortical brain sections, and reviewed prior studies. We report that density and location of both B- and T-lymphocytes do not change with age in the normal human cortex. Solitary B-lymphocytes were found equally in intravascular, perivascular, and parenchymal locations, while T-lymphocytes appeared primarily in perivascular clusters. Thus, any change in number or location of lymphocytes in an aging brain may indicate disease rather than normal aging.
Retinal hemorrhages are an important sequela of fatal head trauma. The accurate pathologic diagnosis of retinal hemorrhages has critical implications for determination of the manner of death.
We ...describe an autolytic postmortem histologic artifact of eosinophilic Müller cell foot process swelling that mimics a nerve fiber layer hemorrhage. From April 24, 2012, through November 11, 2014, we conducted postmortem examination of the eyes of 23 infants and children who were referred to our institution for possible nonaccidental head trauma. A focal artifact of Müller cell foot process swelling was identified in most patients (16 of 23) up to 4 years of age. Three infants, all of whom were younger than 3 months, demonstrated diffusely swollen Müller cell foot processes with intensely eosinophilic cytoplasm that mimicked erythrocytes of nerve fiber layer hemorrhages. The difference in the mean age between patients with diffuse eosinophilic artifacts (1.7 months) and patients with only a multifocal, focal, or absent artifact (13.3 months) was 11.6 months (95% CI, 6.5-16.7 months). Glycophorin C immunohistochemical analysis was useful to differentiate this artifact from nerve fiber layer hemorrhage.
Our case review demonstrates an artifact of eosinophilic Müller cell foot processes swelling in postmortem examination of young infant eyes, a potential pitfall in the diagnosis of retinal hemorrhages. Our findings have important implications for the diagnosis of retinal hemorrhages in potential cases of nonaccidental head injury.
Mutations in leucine-rich repeat kinase 2 (LRRK2), which are associated with autosomal dominant Parkinson's disease, elicit progressive dendrite degeneration in neurons. We hypothesized that synaptic ...dysregulation contributes to mutant LRRK2-induced dendritic injury. We performed in vitro whole-cell voltage clamp studies of glutamatergic receptor agonist responses and glutamatergic synaptic activity in cultured rat cortical neurons expressing full-length wild-type and mutant forms of LRRK2. Expression of the pathogenic G2019S or R1441C LRRK2 mutants resulted in larger whole-cell current responses to direct application of AMPA and NMDA receptor agonists. In addition, mutant LRRK2-expressing neurons exhibited an increased frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in conjunction with increased excitatory synapse density as assessed by immunofluorescence for PSD95 and VGLUT1. Mutant LRRK2-expressing neurons showed enhanced vulnerability to acute synaptic glutamate stress. Furthermore, treatment with the NMDA receptor antagonist memantine significantly protected against subsequent losses in dendrite length and branching complexity. These data demonstrate an early association between mutant LRRK2 and increased excitatory synapse activity, implicating an excitotoxic contribution to mutant LRRK2 induced dendrite degeneration.
•Neurons expressing mutant LRRK2 exhibit increased glutamatergic excitability.•G2019S or R1441C, but not kinase dead, LRRK2 increases excitatory synapse density.•The NMDA receptor antagonist memantine reduces mutant LRRK2-induced dendrite retraction.•Altered calcium flux may function upstream of other known effects of mutant LRRK2.•Synaptic dysfunction represents an early feature of mutant LRRK2 toxicity.
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