Biology subjects

Chan, F.

Publications and source records attributed to Chan, F..

6 recordsLinked to original sources

bioRxiv2025

Inhibitory GABAergic neuron loss due to oxidative damage during ex vivo acute brain slice preparation influences genesis and dynamics of epileptiform activity

Ex vivo acute brain slice is a popular technique in neuroscience research. Since its inception five decades ago, many variations of the brain slice preparation method have emerged. While all variations are currently used by many labs throughout the world, no study has comprehensively examined the impact of these variation on the quality of the acute brain slice preparation. In this study, we comprehensively examined the effect of animal sacrifice methods (decapitation or transcardial perfusion) and cutting solution (normal or sucrose artificial cerebrospinal fluid) on brain slice preparation. Neuronal population was quantified by immunohistochemistry against various neuronal markers. Neuronal dynamics was evaluated by in vitro electrophysiology using two acute epilepsy models - zero-magnesium and 4-aminopyridine. To modulate the stress incurred in acute brain slice preparation, we administered antioxidants or HIF-1 inhibitor, chrysin, to the cutting solution. The method of brain slice preparation significantly affected the quality of the brain slice preparation. In general, the combination of transcardial perfusion and sucrose artificial cerebrospinal fluid produces the optimal brain slice preparation. This is evidenced by a preservation of inhibitory GABAergic neurons in slices prepared with this combination. We subsequently found that this loss of inhibitory GABAergic neurons significantly influenced the genesis and dynamics of induced acute epileptiform activity. The slices with preserved inhibition had less successful induction of acute epileptiform activity and a seizure-like event that is typical of those induced in brain with preserved inhibition. Finally, we found that loss of inhibitory GABAergic neurons during brain slice preparation is primarily due to oxidative damage. Limiting oxidative stress is an effective neuroprotection strategy to prevent loss of inhibition in brain slice preparation. In conclusion, consideration of brain slice preparation method is crucial in preservinginhibitory GABAergic neurons and the degree of inhibition in ex vivo acute brain slice preparation.

neuroscience↗

bioRxiv2023

The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation

engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal disks; small IDE-reporter transgenes are expressed in the anterior compartment, the opposite of what is expected. Our data show that the En protein binds to en IDEs, and we suggest that En directly represses IDE function. We identified two en IDEs, O and S. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from the endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data show that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic regulators. Further, the endogenous locus imparts a stability to en function not seen even in a large transgene, reflecting the importance of both positive and negative epigenetic influences that act over relatively large distances in chromatin. Author SummaryGenes that control development are often used at different times and places in a developing embryo. Transcription of these important genes must be tightly regulated; therefore, these genes often have large arrays of regulatory DNA. In Drosophila, discrete fragments of DNA (enhancers) can be identified that turn genes on in patterns in the early embryo. In cells where the genes are transcriptionally ON, there are active modifications on chromatin, setting later enhancers in a transcription-permission environment. In cells where the genes are OFF, repressive chromatin marks keep later enhancers inactive. In this paper we studied two late enhancers of the Drosophila en gene. We show that the correct activity of these enhancers is dependent on being next to other, earlier acting en enhancers. Our data also show that En can repress its own expression, likely directly by acting on these late enhancers. The chromatin-regulated activity of these en late enhancers is similar to what was described for a late enhancer of another Drosophila developmental gene, Ubx. We suggest that this mode of regulation is likely to be important for many late-acting developmental enhancers in many different organisms.

developmental biology↗

bioRxiv2022

Robust deep learning based protein sequence design using ProteinMPNN

While deep learning has revolutionized protein structure prediction, almost all experimentally characterized de novo protein designs have been generated using physically based approaches such as Rosetta. Here we describe a deep learning based protein sequence design method, ProteinMPNN, with outstanding performance in both in silico and experimental tests. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4%, compared to 32.9% for Rosetta. Incorporation of noise during training improves sequence recovery on protein structure models, and produces sequences which more robustly encode their structures as assessed using structure prediction algorithms. We demonstrate the broad utility and high accuracy of ProteinMPNN using X-ray crystallography, cryoEM and functional studies by rescuing previously failed designs, made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target binding proteins. One-sentence summaryA deep learning based protein sequence design method is described that is widely applicable to current design challenges and shows outstanding performance in both in silico and experimental tests.

biophysics↗

bioRxiv2021

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa

The P23H mutation in rhodopsin (Rho), the visual pigment protein in rod photoreceptor neurons, is the most common genetic cause of autosomal dominant retinitis pigmentosa (adRP), a retinal disease that causes blindness. Despite multiple studies in animal models, the subcellular details of the fate of misfolded mutant Rho in rod photoreceptors have not been completely defined. We generated a new mouse model of adRP, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized there, but mutant P23H-Rho protein was specifically mislocalized in the inner segments of rods. Despite this cellular phenotype, the P23HhRhoRFP heterozygous mice exhibited only slowly progressing retinal degeneration; in ERG recordings, scotopic a-wave amplitudes were reduced by 24% and 26% at 30 days and 90 days respectively, and the corresponding scotopic b-waves by 18% and 24%. Outer nuclear layer thickness was still 80% of WT at 90 days, but at 364 days had declined to 40% of WT. Transmission electron microscopy revealed greatly expanded membrane lamellae in the inner segment, and by fluorescence imaging, we determined that the mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. TUNEL staining revealed a slow pace of cell death involving chromosomal endonucleolytic degradation. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases in levels of messages encoding the proteins BiP, CHOP, ATF6, XBP1, PERK, Eif2 and Derlin-1, but a decreased level of total Rhodopsin (mouse + human) mRNA levels. The decline in the rate of cell death after an initial burst suggests that P23HhRhoRFP mutant rods undergo an adaptative process that prolongs survival despite gross P23HhRhoRFP protein accumulation in the ER. Because of its slowly progressing nature, and easy visualization of the mutant protein, the P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.

neuroscience↗

bioRxiv2021

Biochemical Barriers on the Path to Ocean Anoxia?

The kinetics of microbial respiration suggest that, if excess organic matter is present, oxygen should fall to nanomolar levels, in the range of the Michaelis-Menten constants (Km). Yet even in many biologically productive coastal regions, lowest observed O2 concentrations often remain several orders of magnitude higher than respiratory Km values. We propose the Hypoxic Barrier Hypothesis (HBH) to explain this apparent discrepancy. The HBH postulates that oxidative enzymes involved in organic matter catabolism are kinetically limited by O2 at concentrations far higher than the thresholds for respiration. We found support for the HBH in a meta-analysis of 1137 O2 Km values reported in the literature: the median value for terminal respiratory oxidases was 350 nM, but for other oxidase types the median value was 67 M. The HBH directs our attention to the kinetic properties of an important class of oxygen-dependent reactions that could help explain the trajectories of ocean ecosystems experiencing O2 stress. IMPORTANCEDeclining ocean oxygen associated with global warming and climate change is impacting marine ecosystems across scales from microscopic planktonic communities to global fisheries. We report a fundamental dichotomy in the affinity of enzymes for oxygen. The importance of this observation has yet to be fully assessed, but it is predicted to impact the rate at which organic matter is oxidized in hypoxic ecosystems, and the types of organic matter that accumulate. Competition between intracellular enzymes for oxygen may also have impacted microbial strategies of adaptation to suboxia.

microbiology↗

bioRxiv2021

The scaffold-dependent function of RIPK1 in dendritic cells promotes injury-induced colitis

Receptor interacting protein kinase 1 (RIPK1) is a cytosolic multidomain protein that controls cell life and death. While RIPK1 promotes cell death through its kinase activity, it also functions as a scaffold protein to promote cell survival by inhibiting FADD-caspase 8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis. This pro-survival function is highlighted by excess cell death and a perinatal lethality in Ripk1-/- mice. Recently, loss of function mutation of RIPK1 was found in patients with immunodeficiency and inflammatory bowel diseases. Hematopoietic stem cell transplantation restored not only immunodeficiency but also intestinal inflammatory pathology, indicating that RIPK1 in hematopoietic cells is critical to maintain intestinal immune homeostasis. Here, we generated dendritic cell (DC)-specific Ripk1-/- mice in a genetic background with loss of RIPK1 kinase activity and found that the mice developed spontaneous colonic inflammation characterized by increased neutrophil infiltration. In addition, these mice were highly resistant to injury-induced colitis. The increased neutrophil infiltration in the colon and the resistance to colitis were restored by dual inactivation of RIPK3 and FADD, but not by inhibition of RIPK3, MLKL, or ZBP1 alone. Altogether, these results reveal a scaffold activity-dependent role of RIPK1 in protecting colonic DCs from apoptotic insults and maintenance of colonic immune homeostasis.

immunology↗