Biology subjects

Lam, L.

Publications and source records attributed to Lam, L..

11 recordsLinked to original sources

bioRxiv2026

C19 trityl and silyl andrographolide analogs exhibit in vitro anticancer activity in murine cancer models

Andrographolide, a labdane diterpenoid isolated from A. paniculata has been investigated for activity in a broad panel of biological contexts, including its anti-inflammatory, antiviral, and anticancer properties. Previously, A-ring analogs of andrographolide bearing silyl and trityl ethers at C19 have demonstrated improved in vitro anticancer activity in human cancer cell lines, with putative activity through the Wnt1/{beta}-catenin signaling axis. In order to investigate the activity of this series of C19 andrographolide analogs, alongside a cyclic A-ring acetonide analog, as anticancer agents in murine cancer cell models, we evaluated antiproliferative activity of these compounds in B16F10 melanoma, CT26 colorectal adenocarcinoma, Neuro2a neuroblastoma, and 4T1 mammary ductal carcinoma cell lines, and found that most compounds exhibited potent micromolar level anticancer activity. Among these, all trityl ethers exhibited nearly indistinguishable IC50 values of 4-20 M, and generally exhibited greater potency compared to C19 silyl ether analogs. This anticancer activity was further validated through apoptosis flow cytometry, wherein we concluded that our compounds induced apoptosis to a greater extent than the parent compound, andrographolide. Further, we examine the role of modulation of canonical Wnt1/{beta}-catenin signaling in a series of co-delivery assays with CHIR-99021, a selective GSK-3{beta} kinase inhibitor, and MSAB, a selective {beta}-catenin degrader. Finally, we demonstrate that a selection of silyl ether analogs of andrographolide elicit cell cycle arrest by flow cytometry in a manner that is decoupled to in vitro potency trends. Altogether, we report the first in vitro evaluation of trityl and silyl ether analogs of andrographolide within murine adherent cancer cells.

cancer biology↗

bioRxiv2026

Gut microbiome-dependent IL-1 signaling is a mediator of ACVR1R206H-driven heterotopic ossification

Inflammatory diseases cause significant morbidity and mortality, but their pathobiology is often difficult to dissect due to complex genetic-environmental interactions. Genetic forms of heterotopic ossification, such as fibrodysplasia ossificans progressiva (FOP), reduce genetic variability, allowing careful dissection of non-genetic drivers of inflammation. While >95% of FOP patients harbor the ACVR1R206H mutation, patients exhibit significant variability in disease progression, suggesting a role of environmental drivers. Here, we identify the gut microbiome as a regulator of inflammation-driven HO in FOP. Metagenomic profiling of cohabitating FOP/unaffected sibling pairs revealed a pathogenic gut microbiome profile in FOP patients (Bray-Curtis, p < 0.05). In Pdgfr-Cre/Acvr1R206H (FOP) mice, gut microbiome ablation by antibiotics reduced spontaneous HO formation (47.4% reduction, p < 0.05) and reduced plasma IL-1 pathway activity. IL-1{beta} blockade in FOP mice suppressed trauma-induced HO formation. These findings identify a gut microbiome-IL-1-HO axis with modifiable targets for developing treatments for HO and related inflammatory conditions. One Sentence SummaryAntibiotic disruption of the gut microbiome reduces HO in FOP mice via an IL-1 mediated pathway.

immunology↗

bioRxiv2024

Resolving an unconventional non-photochemical quenching signature at the light-to-dark transition

Non-photochemical quenching (NPQ) protects photosynthetic organisms via diverse molecular players contributing at varying timescales. However, in the absence of one of the largest contributors to NPQ, energy-dependent quenching (qE), we observe an unusual but universal phenomenon: a transient increase in quenching in the dark following high light exposure. To mechanistically interrogate this light-to-dark (LtD) NPQ phenotype, we performed chlorophyll fluorescence lifetime snapshot measurements across a diverse array of Arabidopsis mutant backgrounds and chemical treatments. We found that the electrochemical gradient across the thylakoid membrane is essential for this phenomenon. Through analysis of higher-order Arabidopsis mutants, we also found that LtD NPQ is independent of the known forms of photoprotective NPQ, as well as the major and minor light-harvesting complexes (LHCII). Our results point to LtD NPQ as a photoinhibition (qI)-related, reaction center quenching with implications for photoprotection in fluctuating light.

plant biology↗

bioRxiv2024

Proteome asymmetry in mouse and human embryos before fate specification

Pre-patterning of the embryo, driven by spatially localized factors, is a common feature across several non-mammalian species1-4. However, mammals display regulative development and thus it was thought that blastomeres of the embryo do not show such pre-patterning, contributing randomly to the three lineages of the blastocyst: the epiblast, primitive endoderm and trophectoderm that will generate the new organism, the yolk sac and placenta respectively 4-6. Unexpectedly, early blastomeres of mouse and human embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts7-12. Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Here, by utilizing multiplexed and label-free single-cell proteomics by mass-spectrometry13, we show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins exhibiting strong functional enrichment for protein synthesis, transport, and degradation. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. These protein asymmetries increase at 4-cell stage. Intriguingly, halved mouse zygotes display asymmetric protein abundance that resembles alpha and beta blastomeres, suggesting differential proteome localization already within zygotes. We find that beta blastomeres give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres and that vegetal blastomeres, which are known to have a reduced developmental potential, are more likely to be alpha. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. To our knowledge, this is the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation.

developmental biology↗

bioRxiv2024

In vivo analysis of the relationship between CP26 and qE-type NPQ via higher-order Arabidopsis cp26 mutants

CP26 is a monomeric minor light-harvesting complex of PSII (LHCII) protein that connects major LHCII trimers to the PSII core in photosynthetic thylakoid membranes. Previous studies have proposed that CP26 is not only involved in light harvesting but could also be involved in non-photochemical quenching (NPQ). Here, we analyzed higher-order Arabidopsis cp26 mutants using biophysical and pharmacological approaches to investigate the nature of NPQ and its relationship to known NPQ regulators (PSII subunit S (PsbS), the xanthophyll-converting enzyme VDE and the pH gradient across the thylakoid membrane). Maximum PSII quantum efficiencies (Fv/Fm) and chlorophyll fluorescence lifetimes in the dark were significantly lower in cp26 mutants, confirming that CP26 deficiency leads to a sustained quenched state even in the absence of light. Destabilized PSII-LHCII supercomplexes as observed with native PAGE analysis are the likely cause for this pre-quenched state, without other antenna proteins being able to rescue this phenotype. Further analyses revealed that cp26 mutants exhibit modest (single mutant) to highly significant (double mutants) reductions in overall NPQ capacity, which do not directly rely on PsbS and VDE (although the effect is more pronounced when these qE components are altered) but depend on thylakoid lumen acidification and protonation of protein residues. Together, these results show that the NPQ component lacking in cp26 mutants acts independently of qE and qZ and is induced in a slower phase of NPQ induction that most likely relies on pH-dependent conformational changes.

plant biology↗

bioRxiv2023

Chlorophyll to Zeaxanthin Energy Transfer in Non-Photochemical Quenching: An Exciton Annihilation-free Transient Absorption Study

Zeaxanthin (Zea) is a key component in the energy-dependent, rapidly reversible, non-photochemical quenching process (qE) that regulates photosynthetic light harvesting. Previous transient absorption (TA) studies suggested that Zea can participate in direct quenching via Chlorophyll (Chl) to Zea energy transfer. However, the contamination of intrinsic exciton-exciton annihilation (EEA) makes the assignment of TA signal ambiguous. In this study, we present EEA-free TA data using Nicotiana benthamiana thylakoid membranes, including wild type and three NPQ mutants (npq1, npq4, and lut2) generated by CRISPR/Cas9 mutagenesis. Results show a strong correlation between excitation energy transfer from excited Chl Qy to Zea S1 and the xanthophyll cycle during qE activation. Notably, a Lut S1 signal is absent in the npq1 thylakoids which lack zeaxanthin. Additionally, the fifth-order response analysis shows a reduction in the exciton diffusion length (LD) from 55 {+/-} 5 nm to 38 {+/-} 3 nm under high light illumination, consistent with the reduced range of exciton motion being a key aspect of plants response to excess light.

plant biology↗

bioRxiv2021

The AdcACB/AdcAII system is Essential for Zinc Homeostasis and an Important Contributor of Enterococcus faecalis Virulence

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis, a major pathogen of nosocomial and community-associated infections, have not been investigated. Here, we show that E. faecalis encoded an ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to significant reduction in intracellular Zn under Zn-restricted conditions, heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo. Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance towards cell envelope-targeting antibiotics, which may be associated to altered fatty acid abundance and species. Lastly, we show that the AdcACB/AdcAII system contributes to E. faecalis virulence in an invertebrate (Galleria mellonella) infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is essential for the pathogenesis of E. faecalis indicating that the surface-associated AdcA and AdcAII lipoproteins are potential therapeutic targets.

microbiology↗

bioRxiv2021

Photoprotective qH in Arabidopsis occurs in the light-harvesting complex II trimer

Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as non-photochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants survival under abiotic stress. Here, we tested whether we could isolate photosynthetic subcomplexes (from plants in which qH was induced) that would remain in an energy-dissipative state. Interestingly chlorophyll fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein or lipid content of antenna with active or inactive qH, and no evident differences were detected. Finally, we investigated whether specific antenna subunits of the major antenna were required for qH but found it insensitive to trimer composition. Because qH can occur in the absence of specific xanthophylls, and no changes in pigments were detected, we propose that the energy-dissipative state reported here may stem from chlorophyll-chlorophyll excitonic interaction.

plant biology↗

bioRxiv2021

Engineered type six secretion systems deliver active exogenous effectors and Cre recombinase

Genetic editing has revolutionized biotechnology but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. Most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118 amino acid) delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implicates that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications ranging from studying T6SS effectors to genetic editing. ImportanceDelivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.

molecular biology↗

bioRxiv2020

BBrowser: Making single-cell data easily accessible

BioTurings BBrowser is a software solution that helps scientists effectively analyze single-cell omics data. It combines big data with big computation and modern data visualization to create a unique platform where scientists can interact and obtain important biological insights from the massive amounts of single-cell data. BBrowser has three main components: a curated single-cell database, a big-data analytics layer, and a data visualization module. BBrowser is available for download at: https://bioturing.com/bbrowser/download.

bioinformatics↗

bioRxiv2017

Epigenome-wide association in adipose tissue from the METSIM cohort identifies novel loci and the involvement of adipocytes and macrophages in diabetes traits

Most epigenome-wide association studies to date have been conducted in blood. However, metabolic syndrome is mediated by a dysregulation of adiposity and therefore it is critical to study adipose tissue in order to understand the effects of this syndrome on epigenomes. To determine if natural variation in DNA methylation was associated with metabolic syndrome traits, we profiled global methylation levels in subcutaneous abdominal adipose tissue. We measured association between 32 clinical traits related to diabetes and obesity in 201 people from the Metabolic Syndrome In Men cohort. We performed epigenome-wide association studies between DNA methylation levels and traits, and identified associations for 13 clinical traits in 21 loci. We prioritized candidate genes in these loci using eQTL, and identified 18 high confidence candidate genes, including known and novel genes associated with diabetes and obesity traits. Using methylation deconvolution, we examined which cell types may be mediating the associations, and concluded that most of the loci we identified were specific to adipocytes. We determined whether the abundance of cell types varies with metabolic traits, and found that macrophages increased in abundance with the severity of metabolic syndrome traits. Finally, we developed a DNA methylation based biomarker to assess type II diabetes risk in adipose tissue. In conclusion, our results demonstrate that profiling DNA methylation in adipose tissue is a powerful tool for understanding the molecular effects of metabolic syndrome on adipose tissue, and can be used in conjunction with traditional genetic analyses to further characterize this disorder.

genetics↗