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Biology subjects

Akella, S.

Publications and source records attributed to Akella, S..

5 recordsLinked to original sources

Efficient genetic code expansion tools enable in vivo study of lysine acetylation in non-model bacteria

Recent proteomic advancements have revealed widespread N{varepsilon}-lysine acetylation in pathways governing pathogenicity, metabolism, and antibiotic resistance in bacteria. The spontaneous, non-specific nature of this modification in prokaryotes obscures its biological role, necessitating prokaryotic specific in vivo interrogation systems. Genetic Code Expansion (GCE) offers a powerful method to investigate the roles and regulation dynamics of acetyl-lysine in vivo with the precise incorporation of a suite of non-canonical amino acids, including acetyl-lysine analogs. However, its use has been largely restricted to E. coli strains due to challenges associated with implementation and optimization of the technology in more diverse bacterial strains. Here, we present a bacterial host-agnostic, readily optimizable GCE platform designed to site-specifically incorporate non-canonical amino acids into target proteins within living bacteria. We further demonstrate the versatility of this technology by showcasing, for the first time, the successful incorporation of acetyl-lysine in a non-E. coli bacterium.

synthetic biology↗

Enhancing information extraction from field potentials in electrophysiology studies

Multi-channel recordings from the brain serve as the primary approach to address various mechanistic and behavioral questions about neural activity and the underlying circuitry. While electrodes can detect neural activity at a distance from their source generators, activity from other concurrently active neural sources is also volume conducted to the electrodes, thus creating linear dependencies among the channels. These dependencies pose challenges in discerning specific communication patterns between different neuronal populations, and their behavioral implications. In this study, we demonstrate the capability of a marked point process (MPP) representation of channel activity, focusing on oscillatory bursts, to curtail the effects of volume conduction noise. By characterizing the localized spectral information within oscillatory bursts, we achieved a[~] 45% reduction in channel correlations across three recording modalities of field potentials (electroencephalography, electrocorticography, and local field potentials). We further provide evidence that the implemented sparse representation preserves both behavioral and causal information in the signal. We illustrate our findings with two examples: 1) retention of finger-level movement information in field potentials recorded from humans, demonstrated using a simple online classifier, and 2) retention of top-down connectivity information between the prefrontal and motor cortices of behaving rats. Overall, our results underscore the novelty of using a marked point process representation of oscillatory bursts to concisely encode behavioral and connectivity information while attenuating the effects of volume conduction from the causal signal sources. Author summaryMulti-channel recordings from the brain are critical to understanding how different brain regions communicate and drive behavior. However, the electrodes that detect neural activity also pick up signals from other active neural sources, leading to misleading linear dependencies among the channels. In our study, we present a novel approach using a marked point process (MPP) representation of channel activity using oscillatory bursts, to minimize the interference caused by volume conduction noise. By isolating the spectral characteristics within these oscillatory bursts, we achieved a[~] 45% reduction in channel correlations across three different types of field potential recordings: electroencephalography (EEG), electrocorticography (ECoG), and local field potentials (LFPs). Importantly, our method not only reduces noise but also retains critical behavioral and causal information in the neural signals. Our findings reveal that a marked point process representation of oscillatory bursts provides a more precise and noise-resilient representation of neural activity. This advancement has significant implications for both understanding brain function and improving the accuracy of brain-machine interfaces.

neuroscience↗

Deciphering neuronal variability across states reveals dynamic sensory encoding

Influenced by factors such as brain states and behavior, neurons exhibit substantial response variability even to identical stimuli. Because these factors are non-stationary, they dynamically impact the fidelity of sensory processing. However, it remains unclear how their relative impact on neuronal variability evolves over time. To address this question, we designed an encoding model with latent states to partition visual cortical variability across three crucial categories of sources: internal brain dynamics, behavior, and external visual stimulus. Applying a hidden Markov model to the rhythmic patterns of cortical local field potentials, we consistently identified three distinct oscillation states. Each state revealed a unique variability profile and a consistent descending trend of stimulus modulation across the visual hierarchy. Regression models within each state revealed a dynamic composition of factors contributing to the observed spiking variability, with the primary influencing factor switching within seconds. In the state dominated by high-frequency oscillations, sensory inputs and behavior exerted the most influence on population dynamics. Conversely, internal brain activity explained most of the variance in the state dominated by low-frequency oscillations. This heterogeneity across states underscores the importance of partitioning variability over time, particularly when considering the dynamic influence of non-stationary factors on sensory processing.

neuroscience↗

Measurable fields to spikes causality and its dependence on cortical layer and area.

Distinct dynamics in different cortical layers are apparent in neuronal and local field potential (LFP) patterns, yet their associations in the context of laminar processing have been sparingly analyzed. Here, we study the laminar organization of spike-field causal flow within and across visual (V4) and frontal areas (PFC) of monkeys performing a visual task. Using an event-based quantification of LFPs and a directed information estimator, we found area and frequency specificity in the laminar organization of spike-field causal connectivity. Gamma bursts (40-80 Hz) in the superficial layers of V4 largely drove intralaminar spiking. These gamma influences also fed forward up the cortical hierarchy to modulate laminar spiking in PFC. In PFC, the direction of intralaminar information flow was from spikes [->] fields where these influences dually controlled top-down and bottom-up processing. Our results, enabled by innovative methodologies, emphasize the complexities of spike-field causal interactions amongst multiple brain areas and behavior.

neuroscience↗

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in [~]1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures. One sentence summaryCo-targeting two genes by co-electroporation of CRISPR/Cas9 RNPs and ssODN repair templates allows concomitant genome editing to create a selectable marker gene and to introduce precise modifications in another gene of interest.

genetics↗