Biology subjects

Lam, C.

Publications and source records attributed to Lam, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Directed evolution of the Drosophila microbiome improves intestinal health and extends lifespan

The gut microbiome and its bacterially derived metabolites are known to affect many aspects of the host organisms health, including metabolism, immune response, intestinal inflammation, oxidative stress, and even lifespan. Because pathological changes in the gut microbiome and these functions are associated with aging, many have hypothesized that we could protect against aging by generating beneficial changes to the gut microbiome. Here, we directed evolution outside of the host (ex vivo) and generated a Drosophila gut microbiome resistant to paraquat, a toxin that causes oxidative stress. Compared to a control microbiome, this paraquat-resistant (PQR) microbiome transplanted back into the Drosophila gut endowed the host with multiple health benefits: increased resistance to dietary paraquat, reduced age-related pathologies in the gut, and extended lifespan. We identified the beneficial species of the PQR microbiome as Lactiplantibacillus plantarum and further identified mutations specific to lifespan-extending isolates linked to greater production of acetate. Directly feeding this short-chain fatty acid, acetate, to Drosophila was sufficient to recapitulate an extended lifespan, similar to that induced by gut colonization of PQR bacteria in the gut. These results serve as a proof of principle that increasing the resistance of the microbiome to oxidative stress via directed ex vivo evolution could serve as a therapeutic strategy to protect against aging.

physiology↗

bioRxiv2026

Proteomic analysis of human leptomeningeal matrisome identifies changes in Alzheimer's disease

The leptomeninges (arachnoid and pia) are the inner layers of the meninges that envelope the brain. They define the borders of the subarachnoid space (SAS) where cerebrospinal fluid (CSF) circulates. Leptomeninges act as the barrier tissue and contribute to fluid exchange and immune function of the brain. These processes are identified as key factors in the development of neurodegenerative diseases. The extracellular matrix (ECM) is a critical structural component of leptomeninges. However, it remains insufficiently characterized in health and disease. Here, we performed complete proteomic profiling of ECM proteins (matrisome) of human leptomeninges from individuals with Alzheimers disease (AD) and cognitively normal controls (CN) using sequential biochemical fractionation coupled with mass spectrometry. We resolved leptomeningeal matrisome based on the biochemical properties reflected by the protein solubility. This approach identified 211 ECM proteins in human leptomeninges and revealed disease-associated shifts in leptomeningeal solubility consistent with altered matrix organization and signaling. We found that ECM-linked regulators and secreted factors extracellular sulfatase SULF2, antithrombin-III (SERPINC1), secreted frizzled-related protein 3 (sFRP3) and integrin beta-5 (ITGB5), which is a receptor for fibronectin, were differentially expressed in AD leptomeninges based on the disease status. Seven ECM proteins were identified only in AD samples, and two ECM proteins in CN samples. Pathway enrichment implicated TGF-{beta} and Wnt-related signaling and coagulation as critical for leptomeningeal function. Our findings provide the first comprehensive proteomic characterization of the human leptomeningeal matrisome and establish a biochemical framework for investigating how meningeal matrix remodeling contributes to AD.

neuroscience↗

bioRxiv2025

PAMD-Ch17, a Polymeric Analog of Plerixafor, Induces Mitochondrial Dysfunction in T-ALL Cells Independent of CXCR4

PAMD-Ch17 is a polymer composed of the CXCR4 inhibitor AMD3100/Plerixafor with a cholesterol modification. In previous work, we showed that PAMD-Ch17, but not AMD3100, induces cell death and differentiation in mouse Acute Myeloid Leukemia cells. To investigate the mechanism of PAMD-Ch17s novel anti-leukemic effects, we tested PAMD-Ch17 against a panel of human leukemia cell lines and found that PAMD-Ch17 is effective against a variety of acute leukemias, with T-ALL cell lines being highly sensitive. Surprisingly, CXCR4 knock out T-ALL cells were equally sensitive to PAMD-Ch17. Using a fluorescently tagged PAMD-Ch17, we found that the drug colocalized to the mitochondria. We also found that PAMD-Ch17 induced changes in expression of genes related to mitochondrial function, increased levels of mitochondrial superoxide, and decreased mitochondrial membrane potential. Using seahorse assays, we found that PAMD-Ch17 decreased baseline oxygen consumption, ATP production, and proton leakage. PAMD-Ch17 also decreased baseline extracellular acidification rate, indicating a decrease in overall metabolism. In mouse primary T-ALL but not healthy bone marrow cells, PAMD-Ch17 induced both mitochondrial superoxide and cell death. Using human bone marrow organoids, we found that PAMD-Ch17 induced mitochondrial superoxide and cell death in human primary T-ALL cells, but not in healthy stromal and hematopoietic cells. Collectively, our results indicate that PAMD-Ch17 has anti-leukemic effects against T-ALL cells but not healthy cells, likely mediated through a CXCR4 independent, mitochondrial based mechanism. These findings support further development of PAMDs as potential therapeutics for patients with T-ALL. KEY POINTSO_LIPAMD-Ch17, a polymeric drug based on AMD3100/Plerixafor, has novel anti-leukemic activities against T-ALL that are independent of CXCR4 inhibition. C_LIO_LIPAMD-Ch17 induces increased mitochondrial superoxide and cell death in primary ALL cells, but not healthy bone marrow cells. C_LI

cancer biology↗

bioRxiv2025

The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

Runt-related Transcription Factor 1 (RUNX1) is essential for definitive hematopoiesis and is among the most frequently mutated genes in leukemia. Previous work from our lab demonstrated that Histone Deacetylase 1 (HDAC1), a known RUNX1 partner, is unexpectedly required for active transcription suggesting a non-histone role for HDAC1 regulating components of the RUNX1 complex. Here, we use proteomics, genomics, and long-read transcriptomics to identify novel RUNX1 interacting partners and decipher their role in gene regulation and RNA splicing in leukemia cells. We demonstrate that Polypyrimidine Tract Binding Protein 1 (PTBP1) interacts with RUNX1 in an HDAC1 dependent manner. Chromatin profiling revealed extensive genome-wide overlap in sites occupied by RUNX1 and PTBP1, with significant enrichment at promoters of actively transcribed genes. Loss of PTBP1 in AML cells led to widespread alterations in RNA splicing and decreased expression of genes whose promoters are bound by both factors, including metabolic genes. In agreement with these findings, we found that loss of PTBP1 reduced glycolysis and glucose uptake and ultimately caused cell death. Based on our data, we propose that the interaction between RUNX1 and PTBP1 facilitates expression of metabolic proteins essential for leukemia cell growth and survival. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/654547v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@74e3b9org.highwire.dtl.DTLVardef@12a090org.highwire.dtl.DTLVardef@eb5a48org.highwire.dtl.DTLVardef@17465dc_HPS_FORMAT_FIGEXP M_FIG C_FIG KEY POINTSO_LIPTBP1 binds RUNX1 in a HDAC1-dependent manner and co-localizes to the promoters of target genes in leukemia cells. C_LIO_LILoss of PTBP1 decreases expression of key metabolic genes, resulting in decreased cell growth and glycolysis, increased sensitivity to chemotherapy, and cell death. C_LI

cancer biology↗

bioRxiv2025

Deep behavioral phenotyping tracks functional recovery following tibia fracture in mice

IntroductionAn estimated 178 million fractures occur worldwide each year, with lower limb fractures in particular showing a high incidence of poor healing, and these often lead to reduced mobility and chronic pain. Bone healing and the ability to bear weight are closely tied to the mechanical stability of the fracture site. Although fracture stabilization is a well-established factor modulating the rate and extent of bone repair, there is a notable gap in non-destructive technologies that can rapidly and objectively quantify functional recovery in preclinical settings. We consider this to be a significant limiting factor in translational studies directed at improving fracture healing. Here, we describe a novel behavioral phenotyping approach that enables rapid quantification of post-fracture weightbearing and kinematic metrics in freely behaving mice. Our goal is to identify and characterize metrics most indicative of fracture-induced behavioral impairment and to use these metrics to quantify how functional recovery is altered in mice with pin stabilized versus non-stabilized fractures. We also use this approach to explore whether sex is a significant contributor to functional recovery. MethodsMale and female adult C57BL6/J received a mid-shaft tibial fracture that was either left unstabilized or fixed with an intramedullary pin. Non-fractured naive mice served as controls. Behavioral recordings of freely moving mice were acquired prior to fracture and then throughout the time course of healing, from 5 to 35 days post fracture (DPF). To track mice and analyze changes in paw pressure and kinematic behaviors after fracture, we then applied a novel machine learning-enabled behavioral phenotyping analysis. ResultsIn this study, we demonstrate that severity of the behavioral phenotype is more significant in mice with unstabilized fractures when compared to mice with pin-stabilized fractures. Pin stabilization generally allowed increased weightbearing and produced smaller changes in kinematic metrics. Interestingly, we observed only minor sex specific differences in fracture-induced behavioral impairments and recovery. Our analysis also revealed that functional recovery is more complex than is a set of individual parameters viewed in isolation. In fact, unique behavioral parameters identified different time windows for functional recovery. Therefore, we developed a comprehensive, unified graph theoretic metric of fracture recovery that encompasses all behavioral parameters quantified. Using this unified metric, we confirmed the increased severity of the fracture phenotype in unstabilized versus pin stabilized mice and identified a clear time window of functional recovery, for both fracture groups. DiscussionOur findings demonstrate how this novel comprehensive behavioral phenotyping approach, which combines machine learning and graph theory, makes it possible to rapidly quantify longitudinal changes in mice after fracture. This approach enables us to determine functional recovery patterns based on a unified behavioral metric of healing. Our data and methodology form a foundation for future mechanistic experiments focused on understanding biological or mechanical variables that influence functional healing and will also enable more rapid testing of various strategies to accelerate bone healing.

animal behavior and cognition↗

bioRxiv2025

Phenethylaminylation: Preliminary In Vitro Evidence for the Covalent Transamidation of Psychedelic Phenethylamines to Glial Proteins using 3,5-Dimethoxy-4-(2-Propynyloxy)-Phenethylamine as a Model Compound

Psychedelics are well known for their ability to produce profoundly altered states of consciousness. But, more importantly, the effects of psychedelics can influence neurobehavioral changes that last well after these acute subjective effects end. This phenomenon is currently being leveraged in the development of psychedelic-assisted psychotherapies for the treatment of multiple neuropsychiatric disorders. The cellular and molecular mechanisms by which single doses of psychedelics are able to mediate long-term cognitive changes are an active area of research. We hypothesize that psychedelics contribute to long term changes in cellular state by covalently modifying proteins. This post-translational modification by psychedelics is possible through the transglutaminase-mediated transamidation of their amine termini to glutamine carboxamide residues. Here, we synthesize and utilize a propargylated analogue of mescaline - the classic serotonergic psychedelic phenethylamine found in cacti species - to identify putative protein targets of psychedelic modifications through the use of click-chemistry in a primary human astrocyte cell culture model. Our preliminary findings indicate that a diverse array of glial proteins may be substrates for transglutaminase 2-mediated monoaminylation by our model phenethylamine ("phenethylaminylation"). Based on these points, we speculatively highlight new directions for the study of this putative noncanonical psychedelic activity.

molecular biology↗

bioRxiv2024

Programming the elongation of mammalian cell aggregates with synthetic gene circuits

A key goal of synthetic morphogenesis is the identification and implementation of methods to control morphogenesis. One line of research is the use of synthetic genetic circuits guiding the self-organization of cell ensembles. This approach has led to several recent successes, including control of cellular rearrangements in 3D via control of cell-cell adhesion by user-designed artificial genetic circuits. However, the methods employed to reach such achievements can still be optimized along three lines: identification of circuits happens by hand, 3D structures are spherical, and effectors are limited to cell-cell adhesion. Here we show the identification, in a computational framework, of genetic circuits for volumetric axial elongation via control of proliferation, tissue fluidity, and cell-cell signaling. We then seek to implement this design in mammalian cell aggregates in vitro. We start by identifying effectors to control tissue growth and fluidity in vitro. We then combine these new modules to construct complete circuits that control cell behaviors of interest in space and time, resulting in measurable tissue deformation along an axis that depends on the engineered signaling modules. Finally, we contextualize in vitro and in silico implementations within a unified morphospace to suggest further elaboration of this initial family of circuits towards more robust programmed axial elongation. These results and integrated in vitro/in silico pipeline demonstrate a promising method for designing, screening, and implementing synthetic genetic circuits of morphogenesis, opening the way to the programming of various user-defined tissue shapes.

synthetic biology↗

bioRxiv2024

Investigating the consequences of chronic short sleep for metabolism and survival of oxidative stress

In previous work, we found that short sleep caused sensitivity to oxidative stress; here we set out to characterize the physiological state of a diverse group of chronically short-sleeping mutants during hyperoxia as an acute oxidative stress. Using RNA-sequencing analysis, we found that short-sleeping mutants had a normal transcriptional oxidative stress response relative to controls. In both short-sleeping mutants and controls, hyperoxia led to downregulation of glycolytic genes and upregulation of genes involved in fatty acid metabolism, reminiscent of metabolic shifts during sleep. We hypothesized that short-sleeping mutants may be sensitive to hyperoxia because of defects in metabolism. Consistent with this, short-sleeping mutants were sensitive to starvation. Using metabolomics, we identified a pattern of low levels of long chain fatty acids and lysophospholipids in short-sleeping mutants relative to controls during hyperoxia, suggesting a defect in lipid metabolism. Though short-sleeping mutants did not have common defects in many aspects of lipid metabolism (basal fat stores, usage kinetics during hyperoxia, respiration rates, and cuticular hydrocarbon profiles), they were all sensitive to dehydration, suggesting a general defect in cuticular hydrocarbons, which protect against dehydration. To test the bi-directionality of sleep and lipid metabolism, we tested flies with both diet-induced obesity and genetic obesity. Flies with diet-induced obesity had no sleep or oxidative stress phenotype; in contrast, the lipid metabolic mutant, brummer, slept significantly more than controls but was sensitive to oxidative stress. Previously, all short sleepers tested were sensitive and all long sleepers resistant to oxidative stress. brummer mutants, the first exceptions to this rule, lack a key enzyme required to mobilize fat stores, suggesting that a defect in accessing lipid stores can cause sensitivity to oxidative stress. Taken together, we found that short-sleeping mutants have many phenotypes in common: sensitivity to oxidative stress, starvation, dehydration, and defects in lipid metabolites. These results argue against a specific role for sleep as an antioxidant and suggest the possibility that lipid metabolic defects underlie the sensitivity to oxidative stress of short-sleeping mutants.

physiology↗

bioRxiv2024

Loss of the Inv(16) Oncogene CBFB::MYH11 Eliminates Leukemia from the Blood and Spleen, but not the Bone Marrow

Inversion of chromosome 16 [inv(16)] generates the fusion gene CBFB::MYH11 (CM) and is one of the most common chromosomal rearrangements in Acute Myeloid Leukemia (AML). Expression of CM is required for leukemia initiation. Patients with inv(16) at diagnosis invariably have the rearrangement at relapse, leading to the assumption that CM is also required after leukemic transformation. However, a role for CM in leukemia maintenance has yet to be shown experimentally. To address this, we used an inducible CM knockdown (KD) mouse model and found that decreased CM eliminated leukemia cells from the peripheral blood and spleen, but not the bone marrow, despite all populations exhibiting significantly decreased CM mRNA and protein. The surviving CM KD cells in the bone marrow showed decreased apoptosis and proliferation, and increased expression of autophagy related genes. Surprisingly, with prolonged KD of CM, [~]40% of mice re-established disease despite maintaining decreased CM. Our work indicates that CM is required for leukemia survival in the spleen and peripheral blood, but in the bone marrow CM KD leukemia cells can survive and re-establish disease independent of the fusion protein. These findings imply that targeting CM alone has potential to reduce leukemic burden but not cure the disease.

cancer biology↗

bioRxiv2024

scRNA-seq reveals transcriptional dynamics of Encephalitozoon intestinalis parasites in human macrophages

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Dissemination to other organ systems is also observed, and is potentially facilitated by macrophages. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the host and parasite during infection. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The parasite transcriptome reveals large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication and dissemination. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.

microbiology↗

bioRxiv2024

CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair

CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our in vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.

molecular biology↗

bioRxiv2023

Design and Mathematical Analysis of Synthetic Inhibitory Circuits that Program Self-Organizing Multicellular Structures

Bottom-up approaches are becoming increasingly popular for studying multicellular self-organization and development. In contrast to the classic top-down approach, where parts of the organization/developmental process are broken to understand the process, the goal is to build the process to understand it. For example, synthetic circuits have been built to understand how cell-cell communication and differential adhesion can drive multicellular development. The majority of current bottom-up efforts focus on using activatory circuits to engineer and understand development, but efforts with inhibitory circuits have been minimal. Yet, inhibitory circuits are ubiquitous and vital to native developmental processes. Thus, inhibitory circuits are a crucial yet poorly studied facet of bottom-up multicellular development. To demonstrate the potential of inhibitory circuits for building and developing multicellular structures, I designed several synthetic inhibitory circuits that combine engineered cell-cell communication and differential adhesion. Using a previously validated in silico framework, I examine the capability of these circuits for synthetic development. I show that the designed inhibitory circuits can build a variety of patterned, self-organized structures and even morphological oscillations. These results support that inhibitory circuits can be powerful tools for building, studying, and understanding developmental processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=136 HEIGHT=200 SRC="FIGDIR/small/567649v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@138d0eborg.highwire.dtl.DTLVardef@11c3487org.highwire.dtl.DTLVardef@bc99fforg.highwire.dtl.DTLVardef@224837_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine

AimsTo explore viral evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) are as effective at neutralising the SARS-CoV-2 variant of concern (VOC) Delta (B 1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. MethodsUsing a live-virus SARS-CoV-2 neutralisation assay in Vero E6 cells we determined neutralising antibody titres (nAbT) in 14 participants (vaccine-naive (n=2) and post-second dose of BNT162b2 vaccination (n=12), median age 45 years [IQR 29-65], median time after second dose = 21 days [IQR 19-28] against three SARS-CoV-2 strains: wild-type, Beta and Delta. The determination of nAbT was performed by visual inspection of cytopathic effect (CPE) and in-house quantitative reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 hrs, negative and positive controls underwent genome sequencing using the Respiratory Viral Oligo Panel version 2 (RVOP) (Illumina Inc. (San Diego, United States of America)) viral enrichment and short read sequencing using (Illumina Inc.San Diego, United States of America)(Figure 1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/558921v1_fig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f04650org.highwire.dtl.DTLVardef@1986fceorg.highwire.dtl.DTLVardef@d47ab9org.highwire.dtl.DTLVardef@1c524d5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Outline of virus neutralisation assays of emerging SARS-CoV-2 variants of concern C_FIG ResultsThere was a significant reduction in nAbT observed against the Delta and Beta VOC compared with wild-type, 4.4-fold (p = >0.0006) and 2.3-fold (p = 0.0140), respectively (Figure 2). Neutralizing antibodies were not detected in one vaccinated immunosuppressed participant nor the vaccine-naive participants (n=2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARS-CoV-2 infection 12 months prior (Table S1). Limited consensus level mutations occurred in the SARS-CoV-2 genome of any lineage during in vitro neutralisation, however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S) and membrane protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/558921v1_fig2.gif" ALT="Figure 2"> View larger version (18K): org.highwire.dtl.DTLVardef@1f51ee8org.highwire.dtl.DTLVardef@1c03b61org.highwire.dtl.DTLVardef@17a77d2org.highwire.dtl.DTLVardef@15084f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2:C_FLOATNO Neutralising Antibody Titres Figure description: Differences in neutralising antibody titres (nAbT) against the Wildtype, Beta and Delta SARS-CoV-2 lineages median of 21 days (IQR 19-28) after receiving the second dose of Pfizer-BioNTech (BNT162b2) vaccine measured by visual inspection of cytopathic effect (CPE) with confirmation of viral replication by SARS-CoV-2 by inhouse reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR). Results are reported in the box-whiskers plots as medians and upper and lower quartiles. There was a significant fold reduction in nAbT observed between both the Delta (M = 4.4, SD = 2), t(11) =-4.9, p = .00059, and Beta (M=2.3, SD=2) t(11) =-3 p = .01397 compared with wild-type. There was also a significant fold reduction in nAbT between Beta (M = 2.6, SD = 1.4), t(11) =-2.5, p = .02897 and Delta. Key: Delta - Delta (B.1.617.2) lineage; Beta - Beta (B.1.351) lineage; Wildtype - Wildtype (A.2.2) lineage; C_FIG DiscussionSignificant reductions in nAbT post-vaccination were identified, with Delta demonstrating a 4.4-fold reduction. The reduction in nAbT for the VOC Beta has been previously documented, however, limited data is available on vaccine evasion for the Delta VOC, the predominant strain currently circulating worldwide at the time. Studies in high incidence countries may not be applicable to low incidence settings such as Australia as nAbT may be significantly higher in vaccine recipients previously infected with SARS-CoV-2, as seen in our cohort. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 variants on vaccine effectiveness as mutational profiles in the sub-consensus genome could indicate increases in transmissibility, virulence or allow the development of antiviral resistance.

microbiology↗

bioRxiv2023

A laboratory framework for ongoing optimisation of amplification based genomic surveillance programs

Constantly evolving viral populations affect the specificity of primers and quality of genomic surveillance. This study presents a framework for continuous optimisation of sequencing efficiency for public health surveillance based on the ongoing evolution of the COVID-19 pandemic. SARS-CoV-2 genomic clustering capacity based on three amplification based whole genome sequencing schemes was assessed using decreasing thresholds of genome coverage and measured against epidemiologically linked cases. Overall genome coverage depth and individual amplicon depth were used to calculate an amplification efficiency metric. Significant loss of genome coverage over time was documented which was recovered by optimisation of primer pooling or implementation of new primer sets. A minimum of 95% genome coverage was required to cluster 94% of epidemiologically defined SARS-CoV-2 transmission events. Clustering resolution fell to 70% when only 85% of genome coverage was achieved. The framework presented in this study can provide public health genomic surveillance programs a systematic process to ensure an agile and effective laboratory response during rapidly evolving viral outbreaks.

genomics↗

bioRxiv2023

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen E. intestinalis

Microsporidia are an early-diverging group of fungal pathogens that infect a wide range of hosts. Several microsporidian species infect humans, and infections can lead to fatal disease in immunocompromised individuals. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on metabolites from their hosts for successful replication and development. Our knowledge of how microsporidian parasites develop inside the host remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has thus far relied largely on 2D TEM images and light microscopy. Here, we use serial block face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting microsporidian, Encephalitozoon intestinalis, within host cells. We track the development of E. intestinalis through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in each developing spore. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Together, our data provide insights into parasite development, polar tube assembly, and microsporidia-induced mitochondrial remodeling in the host cell.

microbiology↗

bioRxiv2023

Genomic markers of drug resistance in Mycobacterium tuberculosis populations with minority variants

Minority variants of Mycobacterium tuberculosis harbouring mutations conferring resistance can become dominant populations during tuberculosis (TB) treatment, leading to treatment failure. Our understanding of drug resistant within-host sub-populations and the frequency of resistance conferring mutations in minority variants remains limited. M. tuberculosis sequences recovered from liquid cultures of culture-confirmed TB cases notified between January 2017 and December 2021 in New South Wales, Australia were examined. Potential drug resistance conferring minority variants were identified using LoFreq, and mixed populations of different M. tuberculosis strains ([≥]100 SNPs apart) were examined using QuantTB. A total of 1831 routinely sequenced M. tuberculosis strains were included in the analysis. Drug resistance conferring minority variants were detected in 3.5% (65/1831) of sequenced cultures; 84.6% (55/65) had majority strains that were drug susceptible and 15.4% (10/65) had majority strains that were drug resistant. Minority variants with high confidence drug resistance conferring mutations were 1.5 times more common when the majority strains were drug resistant. Mixed M. tuberculosis strain populations were documented in 10.0% (183/1831) of specimens. Minority variants with high confidence drug resistance conferring mutations were more frequently detected in mixed M. tuberculosis strain populations (2.7%, 5/183) than in single strain populations (0.6%, 10/1648; p=0.01). Drug resistant minority variants require careful monitoring in settings that implement routine M. tuberculosis sequencing. The frequency with which drug resistant minority variants are detected is influenced by selective culture methods and culture-independent sequencing should provide a more accurate picture.

microbiology↗

bioRxiv2023

Design and Mathematical Analysis of Activating Amplifiers that Expand Synthetic Juxtacrine Receptor Control

The ability to control mammalian cells such that they self-organize or enact therapeutic effects as desired has incredible implications. Not only would it further our understanding of native processes such as development and the immune response, but it would also have powerful applications in medical fields such as regenerative medicine and immunotherapy. This control is typically obtained by synthetic circuits that use synthetic receptors, but control remains incomplete. For example, the synthetic juxtacrine receptors (SJRs) are widely used as they are fully modular and enable spatial control, but they have limited gene expression amplification and temporal control. I therefore designed transcription factor based amplifiers that amplify gene expression and enable unidirectional temporal control by prolonging duration of target gene expression. Using an in silico framework for SJR signaling, I combined these amplifiers with SJRs and show that these SJR amplifier circuits can improve the quality of self-organization and direct different spatiotemporal patterning. I then show that these circuits can improve chimeric antigen receptor (CAR) T cell tumor killing against heterogenous and homogenous antigen expression tumors. These amplifiers are flexible tools that improve control over SJR based circuits and have both basic and therapeutic applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=151 HEIGHT=200 SRC="FIGDIR/small/532861v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@4e29f5org.highwire.dtl.DTLVardef@1a4991corg.highwire.dtl.DTLVardef@1400650org.highwire.dtl.DTLVardef@5e95cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Protein-protein interactions in the Mla lipid transport system probed by computational structure prediction and deep mutational scanning

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from various external stressors, such as antibiotics. The Mla transport system is implicated in the Maintenance of outer membrane Lipid Asymmetry and is thought to mediate retrograde phospholipid transport across the cell envelope. This system uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex, via a periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA, but the underlying protein-protein interactions that facilitate lipid transfer are not well understood. Here, we take an unbiased deep mutational scanning approach to map the fitness landscape of MlaC, which provides insights into important functional sites. Combining this analysis with AlphaFold2 structure predictions and binding experiments, we map the MlaC-MlaA and MlaC-MlaD protein-protein interfaces. Our results suggest that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-electron microscopy (cryo-EM) maps of MlaC bound to MlaFEDB suggest that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions. These data lead us to a model for MlaC interaction with its binding partners and insights into lipid transfer steps that underlie phospholipid transport between the inner and outer membranes.

biophysics↗