Biology subjects

Lai, M.

Publications and source records attributed to Lai, M..

26 records · Page 2Linked to original sources

bioRxiv2020

HIV-1 provirus excised by single CRISPR/Cas9 RNA guide persists in host cell and may be reactivated

Gene editing may be used to cut out the human immunodeficiency virus type-1 (HIV-1) provirus from the host cell genome and eradicate infection. Here, using cells acutely or latently infected by HIV and treated with long terminal repeat-targeting CRISPR/Cas9, we show that the excised HIV provirus persists for a few weeks and, by means of HIV Integrase, rearranges in circular molecules. Circularization and integration restore proviral transcriptional activity that is enhanced in the presence of exogenous Tat and Rev or tumor necrosis factor-, respectively, in acutely or latently infected cells. Although confirming that gene editing is a powerful tool to eradicate HIV infection, this work highlights that, to achieve this goal, the provirus has to be cleaved in several pieces and the infected cells treated with antiviral therapy before and after editing.

microbiology↗

bioRxiv2020

Genome-wide binding analysis of 195 DNA Binding Proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. In the past two decades since the sequencing of the human genome there has been a deluge of genome-wide experiments which have measured gene-expression and DNA binding events across numerous cell-types and tissues. Here we re-analyze ENCODE data in a highly reproducible manner by utilizing standardized analysis pipelines, containerization, and literate programming with Rmarkdown. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODEs goals in providing these reproducible data resources. This approach also revealed several new findings: (i) 1,362 promoters, termed reservoirs, have up to 111 different DNA binding-proteins localized on one promoter yet do not have any expression of steady-state RNA (ii) The human specific SVA repeat element may have been co-opted for enhancer regulation. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 - Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

genomics↗

bioRxiv2020

Infection Groups Differential (IGD) Score Reveals Infection Ability Difference between SARS-CoV-2 and Other Coronaviruses

The Corona Virus Disease 2019 (COVID-19) pandemic that began in late December 2019 has resulted in millions of cases diagnosed worldwide. Reports have shown that SARS-CoV-2 shows extremely higher infection rates than other coronaviruses. This study conducted a phylogenetics analysis of 91 representative coronaviruses and found that the functional spike protein of SARS-CoV-2, which interacts with the human receptor ACE2, is actually not undergoing distinct selection pressure compared to other coronaviruses. Furthermore, we define a new measurement, infection group differential (IGD) score, in assessing the infection ability of two human coronavirus groups. There are nine extremely high IGD (ehIGD) sites in the receptor-binding domain (RBD) out of 40 high IGD (hIGD) sites that exhibit a unique infection-related pattern from the haplotype network and docking energy comparison. These 40 hIGD sites are basically conserved among the SARS-CoV-2, i.e. there are only two hIGD sites mutated in four out of 1,058 samples, defined as rare-mutation hIGD (rhIGD) sites. In conclusion, ehIGD and rhIGD sites might be of great significance to the development of vaccines.

bioinformatics↗

bioRxiv2020

Activity-dependent tuning of intrinsic excitability in mouse and human neurogliaform cells

The ability to modulate the efficacy of synaptic communication between neurons constitutes an essential property critical for normal brain function. Animal models have proved invaluable in revealing a wealth of diverse cellular mechanisms underlying varied plasticity modes. However, to what extent these processes are mirrored in humans is largely uncharted thus questioning their relevance to human circuit function. In this study, we focus on neurogliaform cells, a specialized form of neuron that possess physiological features enabling them to impart a widespread inhibitory influence on neural activity. We demonstrate that this prominent neuronal subtype, embedded in both mouse and human neural circuits, undergo remarkably similar activity-dependent modulation manifesting as epochs of enhanced intrinsic excitability. In principle, these evolutionary conserved plasticity routes likely tune the extent of neurogliaform cell mediated inhibition thus constituting canonical circuit mechanisms relevant for human cognitive processing and behavior.

neuroscience↗

bioRxiv2019

Regeneration of dorsal spinal cord neurons after injury via in situ NeuroD1-mediated astrocyte-to-neuron conversion

Spinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimers disease by overexpressing a single neural transcription factor NeuroD1 via retroviruses. Here we demonstrate regeneration of dorsal spinal cord neurons from reactive astrocytes after SCI via adeno-associated virus (AAV), a more clinically relevant gene delivery system. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency ([~]95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate of conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model, allowing future studies of evaluating this reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift for spinal cord repair using in vivo astrocyte-to-neuron conversion technology to generate functional neurons in the grey matter.

neuroscience↗

bioRxiv2019

Neurogranin Stimulates Ca2+/calmodulin-dependent Kinase II by Inhibiting Calcineurin at Specific Calcium Spike Frequencies

Calmodulin sits at the centre of molecular mechanisms underlying learning and memory. Its complex, and sometimes opposite influences, via the binding to various proteins, are yet to be fully understood. Calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) both bind open calmodulin, favouring Long-term potentiation (LTP) or depression (LTD) respectively. Neurogranin binds to the closed conformation of calmodulin and its impact on synaptic plasticity is less clear. We set up a mechanistic computational model based on allosteric principles to simulate calmodulin state transitions and its interaction with calcium ions and the three binding partners mentioned above. We simulated calcium spikes at various frequencies and show that neurogranin regulates synaptic plasticity along three modalities. At low spike frequencies, neurogranin inhibits the onset of LTD by limiting CaN activation. At intermediate frequencies, neurogranin limits LTP by precluding binding of CaMKII with calmodulin. Finally, at high spike frequencies, neurogranin promotes LTP by enhancing CaMKII autophosphorylation. While neurogranin might act as a calmodulin buffer, it does not significantly preclude the calmodulin opening by calcium. On the contrary, neurogranin synchronizes the opening of calmodulins two lobes and promotes their activation at specific frequencies, increasing the chance of CaMKII trans-autophosphorylation. Taken together, our study reveals dynamic regulatory roles played by neurogranin on synaptic plasticity, which provide mechanistic explanations to opposing experimental findings.\n\nAuthor SummaryHow our brains learn and remember things lies in the subtle changes of the strength with which brain cells connect to each other, the so-called synaptic plasticity. At the level of the recipient neuron, some of the information is encoded into patterns of intracellular calcium spikes. Calmodulin, a small bilobed protein which conformation is regulated by the binding of calcium ions, decodes these signals, and modulates the activity of specific binding partners.\n\nTwo key regulators, calcineurin and calcium/calmodulin-dependent protein kinase II, which respectively weaken or strengthen synaptic connections, bind both lobes of calmodulin in its open form, favoured by calcium. On the contrary, neurogranin binds preferentially to one lobe of calmodulin, in the closed form, unfavoured by calcium. It was thus initially suggested that it would inhibit calmodulin activation and decrease synaptic plasticity. However, past research showed that neurogranin sometimes actually enhances synaptic plasticity, though the mechanism was unclear.\n\nOur computational models showed that neurogranin synchronizes the activation of the two lobes of calmodulin, favouring opening at high frequency calcium spikes. By doing so, neurogranin increases the impact of calmodulin on calcium/calmodulin-dependent protein kinase II and reduces its effect on calcineurin, resulting in a strengthening of synaptic connections.

neuroscience↗

bioRxiv2017

Higher-order inter-chromosomal hubs shape 3-dimensional genome organization in the nucleus

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus of each cell. There are currently two distinct views of genome organization that are derived from different technologies. The first view, derived from genome-wide proximity ligation methods (e.g. Hi-C), suggests that genome organization is largely organized around chromosomes. The second view, derived from in situ imaging, suggests a central role for nuclear bodies. Yet, because microscopy and proximity-ligation methods measure different aspects of genome organization, these two views remain poorly reconciled and our overall understanding of how genomic DNA is organized within the nucleus remains incomplete. Here, we develop Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which moves away from proximity-ligation and enables genome-wide detection of higher-order DNA interactions within the nucleus. Using SPRITE, we recapitulate known genome structures identified by Hi-C and show that the contact frequencies measured by SPRITE strongly correlate with the 3-dimensional distances measured by microscopy. In addition to known structures, SPRITE identifies two major hubs of inter-chromosomal interactions that are spatially arranged around the nucleolus and nuclear speckles, respectively. We find that the majority of genomic regions exhibit preferential spatial association relative to one of these nuclear bodies, with regions that are highly transcribed by RNA Polymerase II organizing around nuclear speckles and transcriptionally inactive and centromere-proximal regions organizing around the nucleolus. Together, our results reconcile the two distinct pictures of nuclear structure and demonstrate that nuclear bodies act as inter-chromosomal hubs that shape the overall 3-dimensional packaging of genomic DNA in the nucleus.

genomics↗

bioRxiv2017

A Comparison Between Scalp- And Source-Reconstructed EEG Networks

EEG can be used to characterise functional networks using a variety of connectivity (FC) metrics. Unlike EEG source reconstruction, scalp analysis does not allow to make inferences about interacting regions, yet this latter approach has not been abandoned. Although the two approaches use different assumptions, conclusions drawn regarding the topology of the underlying networks should, ideally, not depend on the approach. The aim of the present work was to find an answer to the following questions: does scalp analysis provide a correct estimate of the network topology? how big are the distortions when using various pipelines in different experimental conditions? EEG recordings were analysed with amplitude- and phase-based metrics, founding a strong correlation for the global connectivity between scalp- and source-level. In contrast, network topology was only weakly correlated. The strongest correlations were obtained for MST leaf fraction, but only for FC metrics that limit the effects of volume conduction/signal leakage. These findings suggest that these effects alter the estimated EEG network organization, limiting the interpretation of results of scalp analysis. Finally, this study also suggests that the use of metrics that address the problem of zero lag correlations may give more reliable estimates of the underlying network topology.

neuroscience↗