Biology subjects

Gopalakrishnan, A.

Publications and source records attributed to Gopalakrishnan, A..

3 recordsLinked to original sources

bioRxiv2026

Multi-step femtosecond laser-fabricated membranes for regulated migration of biomolecules and cells

Organ-on-chip (OoC) systems enable the recapitulation of key structural and functional characteristics of human tissues within controlled micro-engineered environments. In mechanically active tissues such as musculoskeletal, cardiac, and vascular systems, the incorporation of dynamic physical forces is essential for replicating the biomechanical cues governing cellular morphology and functional responses in-vivo. Without such stimuli, OoC models may fail to capture physiologically relevant tissue behaviors. Porous and semi-permeable membranes are critical components of OoCs, facilitating selective transport of nutrients, gases, and signaling molecules between cellular compartments to support biologically accurate barrier replication. Hence, fabrication strategies that permit precise modulation of membrane permeability are desirable to accommodate for the varying needs in pore size and porosity across organ systems. This study presents a two-stage fabrication process for stretchable, microporous polydimethylsiloxane (PDMS) membranes using femtosecond (fs-) pulse laser drilling. The laser-ablated pores exhibit a characteristic conical morphology, with diameters tapering from the laser entry to exit point. By modulating laser power and number of pulses, 6-15 m exit-end pore diameters were achieved in 50 m thick PDMS films. The membranes demonstrated strong mechanical resilience, with a 5-12% reduction in Youngs modulus after 500 cycles of strain loading. Furthermore, membranes fabricated at lower laser powers exhibited superior retention of elasticity, highlighting the influence of processing parameters on mechanical behavior. Cytocompatibility and permeability assessments confirmed that the membranes supported sustained cell viability and proliferation over at least three days. In size-restricted membrane pore geometries, cellular migration was constrained without any inhibition of biomolecular transport. This selective permeability is critical in multilayer OoC architectures, where a balance between biomolecular diffusion and cellular compartmentalization is necessary to preserve distinct tissue interfaces and functional organization. This work presents fs-laser micro-drilling as a robust and tunable fabrication strategy for producing mechanically resilient, selectively permeable PDMS membranes for physiologically relevant OoC applications.

bioengineering↗

bioRxiv2026

Vgll2 and Tead1 Govern Generation of Mouse and Human Hypothalamic Hypocretin (Orexin) Neurons

The hypothalamic hypocretin (Hcrt; aka orexin) neuropeptide neurons are crucial for wakefulness and their malfunction results in narcolepsy; a devastating disorder without current cures. Transplantation of mouse Hcrt cells into mouse narcolepsy models is pointing to cell therapy as a potential treatment for narcolepsy. However, progress in this area requires decoding the developmental pathways generating human HCRT neurons and a deeper understanding of their diversity and physiology. Here, we identify the hypothalamic progenitor domain generating mouse Hcrt neurons and propose a conserved mouse/human genetic cascade driving Hcrt specification. This cascade involves the Vgll2 and Tead1 transcription factor partners, which we find are crucial for mouse Hcrt neuron generation. Conversely, co-misexpression of VGLL2 and TEAD1 in human stem cell derived hypothalamic organoids can induce HCRT neuron differentiation. These findings provide an important element for the development of human induced HCRT neurons for physiological studies and for cell therapy in narcolepsy.

neuroscience↗

bioRxiv2024

Chromatin remodeling with combined FACT and BET inhibition disrupts oncogenic transcription in Diffuse Midline Glioma

Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, significantly extending survival in three independent orthotopic PDX models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility, synergizing with BET inhibition to cause broad transcriptional collapse, silencing several key oncogenes including MYC, PDGFRA, MDM4 and SOX2, as well as causing alterations to the splicing landscape. Notably, this combination also elicited immune-related effects, including activation of the interferon response and antigen presentation mechanisms in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting with spatial transcriptomics. Altogether, our data highlights the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

cancer biology↗