Biology subjects

Asghar, A.

Publications and source records attributed to Asghar, A..

4 recordsLinked to original sources

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2025

Structural Validation of the Intermediate Leptomeningeal Layer in the Human Central Nervous System

Traditionally, the human central nervous system (CNS) is described as having three meningeal layers, from outer to inner: dura mater, arachnoid mater, and pia mater. The arachnoid and pia mater are called the leptomeninges, and the space between them is filled with cerebrospinal fluid (CSF). Using gross dissection, light microscopy, and ultrastructural analysis of fresh postmortem and cadaveric CNS specimens spanning fetal to adult ages (N=61), we demonstrate a fibrocellular intermediate leptomeningeal layer (ILL) from the cortex to the caudal end of the spinal cord. The ILL divides the subarachnoid space (SAS) into two distinct structural compartments, through which vessels and nerves pass. The ILL shows unique structural features, such as dips into the brains sulci and fissures, as a double-fold membrane that bears intra-layer trabeculae, carries vessels, and forms the perivascular sheath. Moreover, throughout the CNS, it appears to be a non-sieved barrier, characterized by the presence of tight and adherens junctions. ILL, predominantly in the spinal cord, contains macrophage-like cells, indicating its layer-specific immune properties. The ILL warrants recognition as a distinct human meningeal layer with potential barrier and immune functions. SignificanceO_ST_ABSAn Intermediate Leptomeningeal Layer encloses the Central Nervous System in HumansC_ST_ABSThe integrated analysis of our macroscopic, microscopic, and ultrastructural study provides robust support for an intermediate leptomeningeal layer (ILL) in the subarachnoid space (SAS) of the human central nervous system (CNS) along the entire neural axis. The ILL is a fibrocellular macroscopic structure, with restricted permeability, that divides the cerebrospinal fluid (CSF)-filled SAS into two distinct structural compartments. Uniquely, ILL revealed the presence of cells with macrophage-like properties, suggesting a possible role in immune surveillance. The ILL may redefine the established concept of protective coverings of CNS, CSF circulation dynamics, and the role of leptomeninges in health and disease, including drug delivery.

neuroscience↗

bioRxiv2025

LANA-Dependent Transcription-Replication Conflicts and R-Loops at the Terminal Repeats (TR) Correlate with KSHV Episome Maintenance

Transcription-replication conflicts frequently occur at repetitive DNA elements involved in genome maintenance functions. The KSHV terminal repeats (TR) function as the viral episome maintenance element when bound by the viral encoded nuclear antigen LANA. Here, we show that transcription-replication conflicts occur at or near LANA binding sites in the TR. We show by proximity ligation assay (PLA) that PCNA and RNAPII colocalize with LANA-nuclear bodies (LANA-NBs). Using DNA-RNA-IP (DRIP) assays with S9.6 antibody, we demonstrate that R-loops form at the TR. We find that these R-loops are also associated with histone H3pS10 a marker for R-loops associated with transcription-replication conflicts. Inhibitors of RNA polymerase eliminated LANA binding to the TR, along with the loss of R-loops and activation associated histone modifications, and the accumulation of heterochromatic marks. We show that LANA can induce all of these features on a plasmid containing 8, but not 2 copies of the TR, correlating strongly with episome maintenance function. Taken together, our study indicates that LANA induces histone modifications associated with RNA and DNA polymerase activity and the formation of R-loops that correlate with episome maintenance function. These findings provide new insights into mechanisms of KSHV episome maintenance during latency and more generally for genome maintenance of repetitive DNA. ImportanceKSHV latent infection is responsible for Kaposis Sarcoma (KS) and Pleural Effusion Lymphoma (PEL). KSHV latency and persistence depends on LANA binding to the terminal repeats (TR). We show that LANA binding promotes the formation of R-loops associated with transcription-replication conflicts and histone H3pS10 at the KSHV terminal repeats. These epigenetic features depend on active RNA polymerase at the TR and correlate strongly with KSHV episome maintenance function. The findings suggest a novel mechanism of chromatin structural maintenance dependent on LANA binding at the TR during KSHV latency.

molecular biology↗

bioRxiv2024

Aligning with the predecessors and counterarguments: A systematic review of the anatomical correlates for the newly discovered meningeal layer in the existing literature

A recent study reported the existence of a subarachnoid lymphatic-like membrane (SLYM) -an intermediate leptomeningeal layer between the arachnoid and pia mater in mice and human brains - dividing the subarachnoid space (SAS) into two functional compartments. Despite being a macroscopic structure, how it missed detection in previous studies is surprising. We systematically reviewed the published reports in animals and humans to explore whether prior descriptions of this meningeal layer have existed. An electronic search was conducted in PubMed/Medline, EMBASE, Google Scholar, Science Direct, and Web of Science databases using combinations of MeSH terms and keywords with Boolean operators from inception until 31st Dec 2023. We found at least eight studies that provided structural evidence of an intermediate leptomeningeal layer in the brain or spinal cord. However, unequivocal descriptions for this layer all along the central nervous system were scarce. Obscured names were used to describe it, i.e., the epipial layer, intermediate meningeal layer, intermediate lamella, and outer pial layer. Its microscopic/ultrastructural details closely resembled the SLYM. Further, we examined the counterarguments in current literature that are skeptical of this layers existence. Considering the significant physiological/clinical implications, exploring further structural and functional details of the new meningeal layer is a need of the hour.

neuroscience↗