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Biology subjects

Pham, T.

Publications and source records attributed to Pham, T..

At least 55 records · Page 3Linked to original sources

High throughput PRIME editing screens identify functional DNA variants in the human genome

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and base-pair resolution manner remains challenging. Here, we develop a novel pooled prime editing screen method, PRIME, which can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated saturation mutagenesis. Next, we applied PRIME to functionally characterize 1,304 non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate PRIME capable of characterizing genetic variants at base-pair resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.

genomics↗

Single-cell T cell receptor sequencing of paired tissue and blood samples reveals clonal expansion of CD8+ effector T cells in patients with calcific aortic valve disease

Calcific aortic valve disease (CAVD) is a complex cardiovascular pathology, culminating in aortic stenosis, heart failure and premature mortality, with no comprehensive treatment strategy, except valve replacement. While T cells have been identified within the valve, their contribution to pathogenesis remains unclear. To elucidate the heterogenous phenotype of the immune populations present within patients with CAVD, deep phenotypic screens of paired valve and peripheral blood cells were conducted via flow cytometry (n=20) and immunohistochemistry (n=10). Following identification of a significant population of memory T cells; specifically, CD8+ T cells within the valve, single cell RNA sequencing and paired single T cell receptor sequencing was conducted on a further 4 patients on CD45+ CD3+, CD4+ or CD8+ T cells. Through unsupervised clustering, 7 T cell populations were identified within the blood and 10 identified within the valve. Tissue resident memory (TRM) T cells were detected for the first time within the valve, exhibiting a highly cytotoxic, activated, and terminally differentiated phenotype. This pan-pro-inflammatory signal was differentially identified in T cells originating from the valve, and not observed in the blood, indicative of an adaptive, local not-systemic inflammatory signature in CAVD patients. T cell receptor analysis identified hyperexpanded clones within the CD8+ T cell central memory (TCM) population, with TRM cells comprising the majority of large and medium clonal expansion within the entire T cell population. Clonal interaction network analysis demonstrated the greatest proportion of clones originating from CD8+ T cell effector memory (TEM) and CD4+ naive / TCM populations and ending in the CD8+ TRM and CD8+ TCM clusters, suggesting a clonal expansion and predicted trajectory of T cells towards a tissue resident, cytotoxic environment within the valve. CDR3 epitope predictive analysis identified 7 potential epitope targets, of which GALNT4 and CR1L have previously been implicated in a cardiovascular context as mediators of inflammation. Taken together, the data identified T cell sub-populations within the context of CAVD and further predicted possible epitopes responsible for the clonal expansion of the valvular T cells, which may be important for propagating inflammation in CAVD.

immunology↗

Exploring Nonlinear Dynamics In Brain Functionality Through Phase Portraits And Fuzzy Recurrence Plots

AO_SCPLOWBSTRACTC_SCPLOWMuch of the complexity and diversity found in nature is driven by nonlinear phenomena, and this holds true for the brain. Nonlinear dynamics theory has been successfully utilized in explaining brain functions from a biophysics standpoint, and the field of statistical physics continues to make substantial progress in understanding brain connectivity and function. This study delves into complex brain functional connectivity using biophysical nonlinear dynamics approaches. We aim to uncover hidden information in high-dimensional and nonlinear neural signals, with the hope of providing a useful tool for analyzing information transitions in functionally complex networks. By utilizing phase portraits and fuzzy recurrence plots, we investigated the latent information in the functional connectivity of complex brain networks. Our numerical experiments, which include synthetic linear dynamics neural time series and a biophysically realistic neural mass model, showed that phase portraits and fuzzy recurrence plots are highly sensitive to changes in neural dynamics and can also be used to predict functional connectivity based on structural connectivity. Furthermore, the results showed that phase trajectories of neuronal activity encode low-dimensional dynamics, and the geometric properties of the limit-cycle attractor formed by the phase portraits can be used to explain the neurodynamics. Additionally, our results showed that the phase portrait and fuzzy recurrence plots can be used as functional connectivity descriptors, and both metrics were able to capture and explain nonlinear dynamics behavior during specific cognitive tasks. In conclusion, our findings suggest that phase portraits and fuzzy recurrence plots could be highly effective as functional connectivity descriptors, providing valuable insights into nonlinear dynamics in the brain. SignificanceHere we report that phase trajectories and fuzzy recurrence plots can serve as descriptors of nonlinear functional dynamics networks. Both metrics are highly sensitive to variations in neural signals and are powerful tools for capturing distinct patterns from brain signals, making brain fingerprinting possible. This has significant implications for understanding brain state dynamics through phase trajectories and fuzzy recurrence plots, as both metrics are effective in identifying nonlinear dynamics patterns.

neuroscience↗

Endothelial Knockdown of the Tumor Suppressor, WWOX, Increases Inflammation in Ventilator-Induced Lung Injury

BackgroundChronic cigarette smoke exposure downregulates lung expression of WWOX, an ARDS relevant tumor suppressor. Prior work has revealed a barrier protective function of WWOX during infectious models of ARDS. Proteomic analysis of WWOX-silenced lung endothelial cells suggest involvement of WWOX in protection against mechanical stretch-induced inflammation. MethodsProtein lysates from WWOX-silenced endothelial cells (ECs) were analyzed using tandem mass tag mass spectrometry (TMT-MS) to determine the differential expression status of the proteome compared to wild type ECs. WWOX-silenced ECs as well as those isolated from endothelial Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 hours). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. Dual silencing of WWOX and zyxin was achieved to determine the role of zyxin upregulation in IL-8 production following mechanical stretch and during WWOX knockdown. Control and EC Wwox KO mice were subjected to high tidal volume ventilation (VILI, 40ml/kg, 65 breath/min, 4hours). Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histologic assays. ResultsTMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1{beta}, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 MAPK phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion. ConclusionLoss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears to be dependent on upregulation of zyxin.

molecular biology↗

Intracellular Proteomics and Extracellular Vesiculomics as a Metric of Disease Recapitulation in 3D Bioprinted Aortic Valve Arrays

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD, due to the lack of: 1) appropriate experimental models that recapitulate this complex environment; and 2) benchmarking novel engineered AV-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, 3D-bioprinted into 96-well arrays. LC-MS/MS analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model vs. traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% vs. 70% of 2D proteins). Integration of cellular/vesicular datasets identified known and novel proteins ubiquitous to AV calcification. This study explores how 2D vs. 3D-bioengineered systems recapitulate unique aspects of human disease, positions multi-omics as a novel technique for the evaluation of high throughput-based bioengineered model systems and potentiates future drug discovery.

bioengineering↗

Endothelial cells secrete small extracellular vesicles bidirectionally containing distinct cargo to uniquely reprogram vascular cells in the circulation and vessel wall

Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions. Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall. Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1{beta} activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively. Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated. Non-standard Abbreviations and Acronyms O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/538787v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@8e62dforg.highwire.dtl.DTLVardef@1fd4adeorg.highwire.dtl.DTLVardef@3e4433org.highwire.dtl.DTLVardef@1efa1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract: Polarized endothelial extracellular vesicle communication with luminal and abluminal vascular cellsEndothelial cell small extracellular vesicle (EC-EV) release from apical (luminal) and basolateral (abluminal) surfaces in quiescence and after endothelial activation. Quiescent EC-EVs are depicted in blue (bright blue=apical, light blue=basolateral), while activated EC-EVs are depicted in red (bright red=apical, light red=basolateral). Luminal monocyte is represented in purple with upregulation of pro-inflammatory transcripts (bright purple) after uptake of activated EC-EVs from the apical surface, compared to uptake of quiescent apical EC-EVs (light purple). Basolateral EC-EVs are taken up by an abluminal resident smooth muscle cell depicted in yellow. Smooth muscle cell uptake of activated basolateral EC-EVs with upregulation of pro-inflammatory/pro-atherogenic transcripts (bright yellow), as compared to uptake of quiescent EC-EVs (light yellow).

cell biology↗

A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single- cell RNA-sequencing (scRNA-seq) dataset of [~]7 million cells profiled, and a spatially resolved transcriptomic dataset of [~]4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

neuroscience↗

Signature morpho-electric properties of diverse GABAergic interneurons in the human neocortex

Human cortical interneurons have been challenging to study due to high diversity and lack of mature brain tissue platforms and genetic targeting tools. We employed rapid GABAergic neuron viral labeling plus unbiased Patch-seq sampling in brain slices to define the signature morpho-electric properties of GABAergic neurons in the human neocortex. Viral targeting greatly facilitated sampling of the SST subclass, including primate specialized double bouquet cells which mapped to two SST transcriptomic types. Multimodal analysis uncovered an SST neuron type with properties inconsistent with original subclass assignment; we instead propose reclassification into PVALB subclass. Our findings provide novel insights about functional properties of human cortical GABAergic neuron subclasses and types and highlight the essential role of multimodal annotation for refinement of emerging transcriptomic cell type taxonomies. One Sentence SummaryViral genetic labeling of GABAergic neurons in human ex vivo brain slices paired with Patch-seq recording yields an in-depth functional annotation of human cortical interneuron subclasses and types and highlights the essential role of multimodal functional annotation for refinement of emerging transcriptomic cell type taxonomies.

neuroscience↗

Transcriptomic cytoarchitecture reveals principles of human neocortex organization

Variation in cortical cytoarchitecture is the basis for histology-based definition of cortical areas, such as Brodmann areas. Single cell transcriptomics enables higher-resolution characterization of cell types in human cortex, which we used to revisit the idea of the canonical cortical microcircuit and to understand functional areal specialization. Deeply sampled single nucleus RNA-sequencing of eight cortical areas spanning cortical structural variation showed highly consistent cellular makeup for 24 coarse cell subclasses. However, proportions of excitatory neuron subclasses varied strikingly, reflecting differences in intra- and extracortical connectivity across primary sensorimotor and association cortices. Astrocytes and oligodendrocytes also showed differences in laminar organization across areas. Primary visual cortex showed dramatically different organization, including major differences in the ratios of excitatory to inhibitory neurons, expansion of layer 4 excitatory neuron types and specialized inhibitory neurons. Finally, gene expression variation in conserved neuron subclasses predicts differences in synaptic function across areas. Together these results provide a refined cellular and molecular characterization of human cortical cytoarchitecture that reflects functional connectivity and predicts areal specialization.

neuroscience↗

Inter-individual variation in human cortical cell type abundance and expression

Single cell transcriptomic studies have identified a conserved set of neocortical cell types from small post-mortem cohorts. We extend these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in middle temporal gyrus, but with varied abundances and gene expression signatures across donors, particularly in deep layer glutamatergic neurons. A minority of variance is explainable by known factors including donor identity and small contributions from age, sex, ancestry, and disease state. Genomic variation was significantly associated with variable expression of 150-250 genes for most cell types. Thus, human individuals display a highly consistent cellular makeup, but with significant variation reflecting donor characteristics, disease condition, and genetic regulation. One-Sentence SummaryInter-individual variation in human cortex is greatest for deep layer excitatory neurons and largely unexplainable by known factors.

neuroscience↗

Comparative transcriptomics reveals human-specific cortical features

Humans have unique cognitive abilities among primates, including language, but their molecular, cellular, and circuit substrates are poorly understood. We used comparative single nucleus transcriptomics in adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets from the middle temporal gyrus (MTG) to understand human-specific features of cellular and molecular organization. Human, chimpanzee, and gorilla MTG showed highly similar cell type composition and laminar organization, and a large shift in proportions of deep layer intratelencephalic-projecting neurons compared to macaque and marmoset. Species differences in gene expression generally mirrored evolutionary distance and were seen in all cell types, although chimpanzees were more similar to gorillas than humans, consistent with faster divergence along the human lineage. Microglia, astrocytes, and oligodendrocytes showed accelerated gene expression changes compared to neurons or oligodendrocyte precursor cells, indicating either relaxed evolutionary constraints or positive selection in these cell types. Only a few hundred genes showed human-specific patterning in all or specific cell types, and were significantly enriched near human accelerated regions (HARs) and conserved deletions (hCONDELS) and in cell adhesion and intercellular signaling pathways. These results suggest that relatively few cellular and molecular changes uniquely define adult human cortical structure, particularly by affecting circuit connectivity and glial cell function.

neuroscience↗

The RAM signaling pathway links morphology, thermotolerance, and CO2 tolerance in the global fungal pathogen Cryptococcus neoformans

The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO2-tolerant clinical strain, we found mutations leading to CO2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and Regulator of Ace2 and Morphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans, transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1{Delta} in C. neoformans that partially rescued defects in CO2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1, which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1{Delta} partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.

microbiology↗

Measurement of Cervical Neuronal Activity during Stress Challenge Using Novel Flexible Adhesive Surface Electrodes

This study introduces a flexible, adhesive-integrated electrode array that was developed to enable non-invasive monitoring of cervical nerve activity. The device uses silver-silver chloride as the electrode material of choice and combines it with a novel electrode array consisting of a customized biopotential data acquisition unit and integrated graphical user interface (GUI) for visualization of real-time monitoring. Preliminary testing demonstrated this novel electrode design can achieve a high signal to noise ratio during cervical neural recordings. To demonstrate the capability of the surface electrodes to detect changes in cervical neuronal activity, the cold-pressor test (CPT) and a timed respiratory challenge were employed as stressors to the autonomic nervous system. This sensor system recording, a new novel technique, was termed Cervical Electroneurography (CEN). By applying a custom spike sorting algorithm to the novel electrode measurements, neural activity was classified in two ways: 1) pre-to-post CPT, and 2) during a timed respiratory challenge. Unique to this work: 1) rostral to caudal channel position-specific (cephalad to caudal) firing patterns and 2) cross challenge biotype-specific change in average CEN firing, were observed with both CPT and the timed respiratory challenge. Future work is planned to develop an ambulatory CEN recording device that could provide immediate notification of autonomic nervous system activity changes that might indicate autonomic dysregulation in healthy subjects and clinical disease states.

physiology↗

DectiSomes: Glycan Targeting of Liposomal Drugs Improves the Treatment of Disseminated Candidiasis

Candida albicans causes life-threatening disseminated candidiasis. Individuals at greatest risk have weakened immune systems. An outer cell wall, exopolysaccharide matrix, and biofilm rich in oligoglucans and oligomannans help Candida spp. evade host defenses. Even after antifungal drug treatment the one-year mortality rate exceeds 25%. Undoubtedly there is room to improve antifungal drug performance. The mammalian C-type lectin pathogen receptors Dectin-1 and Dectin-2 bind to fungal oligoglucans and oligomannans, respectively. We previously coated amphotericin B-loaded liposomes, AmB-LLs, pegylated analogs of AmBisome, with the ligand binding domains of these two Dectins. DectiSomes, DEC1-AmB-LLs and DEC2-AmB-LLs, showed two distinct patterns of binding to the exopolysaccharide matrix surrounding C. albicans hyphae grown in vitro, while untargeted AmB-LLs did not bind. DectiSomes were preferentially associated with fungal colonies in the kidneys. In a neutropenic mouse model of candidiasis, DEC1-AmB-LLs and DEC2-AmB-LLs delivering only one dose of 0.2 mg/kg AmB significantly reduced the kidney fungal burden several fold relative to AmB-LLs, based on either colony forming units (P= 0.013 to 8.8 x 10-5) or quantitative PCR of fungal rRNA ITS (P= 5.5x10-5 to 3.0x10-10). DEC1-AmB-LLs and DEC2-AmB-LLs significantly increased the percent of surviving mice relative to AmB-LLs. Dectin-2 targeted anidulafungin loaded liposomes and AmBisomes, DEC2-AFG-LLs and DEC2-AmBisome reduced fungal burden in the kidneys several fold over their untargeted counterparts (P=7.8x10-5 and 0.0020, respectively). The data herein suggest that targeting of a variety of antifungal drugs to fungal glycans may achieve lower safer effective doses and improve drug efficacy against a variety of invasive fungal infections.

microbiology↗

Connectivity and Synaptic Physiology in the Mouse and Human Cortex

To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

neuroscience↗

Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2'for viral fusion/entry: indirect role for TMPRSS2

The spike (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic "priming" at PRRAR685{downarrow} into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at a S2 site for viral entry. In vitro, Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2 processing by Furin at KPSKR815{downarrow} was strongly enhanced by ACE2, but not for the optimized S2 KRRKR815{downarrow} mutant (S2), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin (BOS) and TMPRSS2 (Camostat). Consistently, these inhibitors potently reduce infectious viral titer and cytopathic effects, an outcome enhanced when both compounds were combined. Quantitative analyses of cell-to-cell fusion and spike processing revealed the key importance of the Furin sites for syncytia formation. Our assays showed that TMPRSS2 enhances fusion and proteolysis at S2 in the absence of cleavage at S1/S2, an effect that is linked to ACE2 shedding by TMPRSS2. Overall, our results indicate that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and in promoting viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCESARS-CoV-2 is the etiological agent of COVID-19 that resulted in >5 million deaths. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein is cleaved at two sites: S1/S2 and S2. Cleavage at S1/S2, induces a conformational change favoring the recognition of ACE2. The S2 cleavage is critical for cell-to-cell fusion and virus entry into host cells. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a non-toxic Furin inhibitor with a TMPRSS2 inhibitor could significantly reduce viral entry in lung cells, as evidenced by an average synergistic [~]95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

biochemistry↗

Human INCL fibroblasts display abnormal mitochondrial and lysosomal networks and heightened susceptibility to ROS-induced cell death

Infantile Neuronal Ceroid Lipofuscinosis (INCL) is a pediatric neurodegenerative disorder characterized by progressive retinal and central nervous system deterioration during infancy. This lysosomal storage disorder results from a deficiency in the Palmitoyl Protein Thioesterase 1 (PPT1) enzyme - a lysosomal hydrolase which cleaves fatty acid chains such as palmitate from lipid-modified proteins. In the absence of PPT1 activity, these proteins fail to be degraded, leading to the accumulation of autofluorescence storage material in the lysosome. The underlying molecular mechanisms leading to INCL pathology remain poorly understood. A role for oxidative stress has been postulated, yet little evidence has been reported to support this possibility. Here we present a comprehensive cellular characterization of human PPT1-deficient fibroblast cells harboring Met1Ile and Tyr247His compound heterozygous mutations. We detected autofluorescence storage material and observed distinct organellar abnormalities of the lysosomal and mitochondrial structures, which supported previous postulations about the role of ER, mitochondria and oxidative stress in INCL. An increase in the number of lysosomal structures was found in INCL patient fibroblasts, which suggested an upregulation of lysosomal biogenesis, and an association with endoplasmic reticulum stress response. The mitochondrial network also displayed abnormal spherical punctate morphology instead of normal elongated tubules with extensive branching, supporting the involvement of mitochondrial and oxidative stress in INCL cell death. Autofluorescence accumulation and lysosomal pathologies can be mitigated in the presence of conditioned wild type media suggesting that a partial restoration via passive introduction of the enzyme into the cellular environment may be possible. We also demonstrated, for the first time, that human INCL fibroblasts have a heightened susceptibility to exogenous reactive oxygen species (ROS)-induced cell death, which suggested an elevated basal level of endogenous ROS in the mutant cell. Collectively, these findings support the role of intracellular organellar networks in INCL pathology, possibly due to oxidative stress.

cell biology↗

Right lateralized posterior parietal theta-gamma coupling during sustained attention in mice

Sustained attention is supported by circuits in the frontoparietal attention network. In human and primate studies, the right posterior parietal cortex (PPC) shows dominance for sustained attention, and phase-amplitude coupling (PAC) throughout the frontoparietal network correlates with performance on attention tasks. Here we evaluate oscillatory dynamics of bilateral PPC in mice during the 5-Choice Serial Reaction Time Task (5-CSRTT). Right PPC theta-gamma PAC (TG-PAC) and gamma power were independently elevated to a greater degree than the left PPC during the period prior to a correct response and were significantly correlated with accuracy in both simple and difficult tasks. Greater task difficulty was also associated with greater hemispheric asymmetry in TG-PAC, favoring the right PPC. These findings highlight the engagement of PPC with sustained attention in mice, reflected by increases in TG-PAC and gamma power, with maximal expression in the right hemisphere.

neuroscience↗