Biology subjects

Haas, K. M.

Publications and source records attributed to Haas, K. M..

7 recordsLinked to original sources

bioRxiv2026

Targeting skin ageing at the source: a novel cellular reprogramming approach using natural extract combinations

Cellular senescence is a driver of skin ageing that leads to structural deterioration and aesthetic changes. This study evaluated a novel senomorphic strategy, using synergistic combinations of natural small molecules to target RNA splicing regulation via upstream ERK/AKT signaling pathways. Nature-identical synthetic molecules, and corresponding extracts were assessed for cytotoxicity, impact on senescent cell load, proliferation, DNA damage load and SASP markers in naturally aged human primary skin cells. A topical, simple formulation was evaluated for impact on senescence and skin structure using 3D human ex vivo full-thickness skin explants exposed to 1.5 J/cm2 UVC irradiation. Combination of H. lupulus and G. biloba extracts produced a dose-dependent senomorphic impact of up to 45% in aged dermal fibroblasts and 10% in aged human primary keratinocytes (p [≤] 0.0001 & 0.001), without inducing cell proliferation. A 50% decrease in DNA damage load was also observed (p [≤] 0.0001). Qualitatively, the simple formulation mitigated UV-induced epidermal hyperplasia, restored collagen fiber organization, and reduced markers of senescence and inflammation. Targeting cellular senescence is an emerging traction point to address the root biological causes of skin ageing. Targeting the splicing dysregulation/senescence axis using combinations of natural plant extracts may provide a novel mode of action to reprogram senescent cells for repair and protection of skin structure and function.

cell biology↗

bioRxiv2026

LANA-dependent Interaction of Host Factors DAXX and BRD4 Impact KSHV Lytic Replication

Kaposis Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposis Sarcoma (KS). KSHV alternates between latent and lytic phases. Latency is marked by limited viral gene expression and absence of virion production, whereas the lytic phase is characterized by the expression of all viral genes in a temporally and sequentially regulated cascade of immediate-early, early, and late genes, culminating in viral genome replication and infectious virion production. Both KSHV latent and lytic viral phases contribute to its pathogenesis and the development of KS. KSHV proteins hijack host proteins, rewiring host cellular processes and signaling pathways, and modulating host gene expression. KSHV establishes latency through stable interactions between viral proteins and host factors, with the Latency Associated Nuclear Antigen (LANA) serving as a key regulator that interacts with host machinery to maintain the viral episome and regulate viral replication. However, the consequences for host proteins binding viral proteins, including changes in their interaction networks and their impact on viral replication, remains poorly understood. Here, using Immunoprecipitation Mass Spectrometry (IP-MS), we identified and validated the host death domain-associated protein (DAXX) and DNA ligase 3 (LIG3) as major LANA-associated factors and discovered a LANA-dependent recruitment of Bromodomain-containing protein 4 (BRD4) to DAXX. This remodeling of the DAXX interactome guided functional analyses demonstrating roles for DAXX and BRD4 in supporting KSHV infection. Functional studies show that siRNA knockdown of DAXX or BRD4 in iSLK.BAC16 cells, followed by lytic reactivation, significantly induced viral gene transcription and protein expression of KSHV lytic genes ORF45, ORF59, ORF26, and K8.1. Conversely, siRNA knockdown of LIG3 reduced the transcription and protein expression of KSHV lytic genes. Viral genome replication and infectious virion production were elevated upon knockdown of DAXX or BRD4 and reduced after the knockdown of LIG3. Additionally, chemical inhibition of BRD4 activity by the drug JQ1 in iSLK.BAC16 cells, followed by lytic reactivation, resulted in elevated KSHV lytic gene expression, genome replication, and infectious virus production. Together, these data suggest that both DAXX and BRD4 host genes contribute to KSHV latency maintenance, while LIG3 is required for lytic reactivation. Understanding the functional significance of these LANA-interacting host factors in regulating KSHV infection is critical for identifying therapeutic targets and developing potential treatment strategies.

microbiology↗

bioRxiv2025

Divergent roles for complement components C3 and C4 in controlling Klebsiella pneumoniae gut colonization and systemic dissemination

Klebsiella pneumoniae is an escalating public health threat driven by the emergence of antibiotic-resistant and hyper-encapsulated strains that spread systemically from the gut. The immune defenses preventing gut colonization and dissemination remain poorly defined. Herein, we uncover distinct and context-dependent roles for complement proteins C3 and C4 in host defense following K. pneumoniae infection. Following gut colonization, C3 and C4 levels rise significantly. In addition to inducing alternative pathway-mediated C3b deposition on K. pneumoniae grown under gut-relevant conditions, C3 is critical for recruiting myeloid cells to the gut, promoting local opsonophagocytosis, and preventing lethal systemic spread. Depletion of systemic C3 reveals mucosal-derived C3 controls K. pneumoniae GI colonization, whereas systemic C3 is essential for limiting fatal dissemination. In contrast, C4 is dispensable for controlling GI colonization, dissemination, and myeloid recruitment under conditions of natural acquisition. However, C4 becomes critical for controlling GI burden and systemic disease following antibiotic-induced dysbiosis and supercolonization with antibiotic-resistant K. pneumoniae. Notably, mice deficient in CD21/35--a receptor for cleaved C3 and C4 fragments important for B cell activation and antigen retention--exhibit a defect similar to C4-/- mice, with significantly increased GI burden under antibiotic-induced supercolonization, suggesting distinct complement-dependent pathways are involved in mucosal protection. Collectively, these findings reveal a dual-layered immune strategy: C3-driven opsonophagocytosis is critical for controlling colonization and dissemination under baseline conditions, while C4 and CD21/35 become indispensable following antibiotic-induced supercolonization. This work advances our understanding of complement-dependent mucosal immune protection and identifies potential targets for preventing gut-to-bloodstream transition of this pathogen.

immunology↗

bioRxiv2025

Pam3CSK4 as a Cross-Species Adjuvant for Polysaccharide Vaccines: Efficacy in Humanized Mouse and Non-Human Primate Models

Polysaccharide-based vaccines are critical for preventing bacterial infections, yet their efficacy is often limited by weak antibody responses. Unfortunately, efficacious adjuvants for licensed native polysaccharide vaccines are lacking. The TLR4 agonist, monophosphoryl lipid A (MPL), significantly increases antibody responses to capsular polysaccharides in mice via B cell-intrinsic TLR4 and MyD88-dependent signaling. However, due to the lack of TLR4-driven adjuvant effects on polysaccharide-specific responses in non-human primates and the limited responsiveness of human B cells to TLR4 agonists, we sought to identify alternative MyD88-activating TLR agonists that could serve as suitable adjuvants to enhance humoral responses to polysaccharide vaccines in humans. In vitro assays revealed the TLR1/2 agonist Pam3CSK4 synergized with strong BCR crosslinking to optimally enhance both mouse and human B cell activation and antibody secretion. In vivo, Pam3CSK4 alone had no effect, but when paired with a squalene-based emulsion significantly increased polysaccharide-specific antibodies in both immunocompetent and PBMC-humanized mice that proved highly protective against lethal pneumococcal infections. Although a dual TLR2-7 agonist showed similar potent in vitro activity, it failed to enhance polysaccharide-specific IgG responses in vivo, mirroring the antagonistic effects observed when TLR2 and TLR7 agonists were combined both in vitro and in vivo. By contrast, inclusion of Pam3CSK4 in an adjuvant containing MPL, synthetic cord factor, and squalene emulsion further augmented protective polysaccharide-specific antibody responses in mice and rescued adjuvant effects in non-human primates. These findings reveal Pam3CSK4-containing formulations as promising adjuvants for native polysaccharide vaccines, with strong translational potential to enhance humoral immunity in humans. One Sentence SummaryPam3CSK4-based adjuvants show promise for boosting protective antibody responses to polysaccharide vaccines.

immunology↗

bioRxiv2024

Preventing Inappropriate Signals Pre- and Post-Ligand Perception by a Toggle-Switch Mechanism of ERECTA

Dynamic control of signaling events requires swift regulation of receptors at an active state. By focusing on Arabidopsis ERECTA (ER) receptor kinase, which perceives peptide ligands to control multiple developmental processes, we report a mechanism preventing inappropriate receptor activity. The ER C-terminal tail (ER_CT) functions as an autoinhibitory domain: its removal confers higher kinase activity and hyperactivity during inflorescence and stomatal development. ER_CT is required for the binding of a receptor kinase inhibitor, BKI1, and two U-box E3 ligases PUB30 and PUB31 that inactivate activated ER. We further identify ER_CT as a phosphodomain trans-phosphorylated by the co-receptor BAK1. The phosphorylation impacts the tail structure, likely releasing from autoinhibition. The phosphonull version enhances BKI1 association, whereas the phosphomimetic version promotes PUB30/31 association. Thus, ER_CT acts as an off-on-off toggle switch, facilitating the release of BKI1 inhibition, enabling signal activation, and swiftly turning over the receptors afterwards. Our results elucidate a mechanism fine-tuning receptor signaling via a phosphoswitch module, keeping the receptor at a low basal state and ensuring the robust yet transient activation upon ligand perception. SignificanceCells perceive and process external signals through their cell-surface receptors, whose activity must be tightly maintained to prevent the spread of misinformation. How do plant cells prevent the inappropriate receptor activity? We identify a structural module within the C-terminal tail of the ERECTA (ER_CT), that inhibits the receptor pre- and post-signal activation. The ER_CT comprises of a linker and an -helix. Before activation, ER_CT is autoinhibitory and associates with an inhibitory protein. Ligand perception triggers the transphosphorylation of ER_CT by the co-receptor, which then recruits a degradation machinery to swiftly turn over the activated receptor. Thus, we reveal an off-on-off toggle switch mechanism that finely adjusts the activity of the plant receptor, enabling the precise control over cell signaling.

plant biology↗

bioRxiv2022

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

Understanding molecular mechanisms of cellular pathways requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography and subtomogram averaging provided 6.0 [A] resolution reconstructions of axonemal microtubules and their associated proteins. The well-resolved secondary and tertiary structures allowed us to computationally match, in an unbiased manner, novel densities in our 3D reconstruction maps with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105 and SPACA9 as novel microtubule inner proteins that form an extensive network crosslinking the lumen of microtubule and existing proteins. Additional biochemical and mass spectrometry analyses helped validate potential candidates. The novel axonemal sperm structures identified by this approach form an extensive interaction network within the lumen of microtubules, suggesting they have a role in the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella.

biophysics↗

bioRxiv2020

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

genetics↗