Biology subjects

Kramer, L.

Publications and source records attributed to Kramer, L..

12 recordsLinked to original sources

bioRxiv2025

Conserved and Lineage-Specific Roles of KEA-Mediated Ion Homeostasis in Chlamydomonas

In Arabidopsis thaliana, seamless plastid gene expression and development depend on finely balanced ion homeostasis across the inner envelope (IE) membrane, maintained by the K/H antiporters AtKEA1/2. To assess whether these functions are retained across mono- and polyplastidic representatives of the green lineage, we studied CrKEA1, the sole IE KEA homolog in the unicellular alga Chlamydomonas reinhardtii. Using CRISPR/Cas9, we generated a Cr-kea1 knockout mutant that exhibits impaired photoautotrophic growth, chloroplast deformation, and photoinhibition. Transcriptomics revealed strong induction of ribosome biogenesis genes and reduced abundance of transcripts associated with cell and plastid division. Further RNA analyses confirmed defects in stromal rRNA maturation of Cr-kea1, paralleling observations from Arabidopsis kea1kea2 mutants. Expression of CrKEA1 in Arabidopsis rescued growth and rRNA maturation in At-kea1kea2, demonstrating functional continuity after the ancient divergence between the two lineages. Cross-species transcriptomic comparisons further revealed that IE KEA loss elicits both shared and species-specific transcriptional responses: PhANG repression was conserved between algae and plants, whereas activation of the chloroplast unfolded protein response (cpUPR) and reduced expression of genes tied to cell-cycle and plastid fission occurred only in Chlamydomonas. Single-cell time-lapse imaging confirmed that Cr-kea1 exhibits an increased frequency of aberrant cytokinesis, unequal division, and division failure. Our findings demonstrate that while IE KEA transporters fulfill conserved roles in maintaining the conditions for plastid gene expression, their integration into broader cellular networks has diverged between unicellular chlorophytes and embryophytes (land plants). This underscores a lineage-dependent tuning of plastid-nucleus communication shaped by organismal complexity and plastid number. One-sentence summaryDisruption of KEA-mediated chloroplast ion homeostasis in Chlamydomonas reinhardtii reveals conserved and lineage-specific control of plastid rRNA processing and cell cycle progression.

plant biology↗

bioRxiv2025

Overcoming lentiviral delivery limitations in hard-to-transduce suspension cells for genome-wide CRISPR screening

Lentiviral vectors are a cornerstone delivery modality of biomedical research, renowned for their ability to stably integrate genetic material into the host genome, enabling sustained transgene expression and long-term genetic manipulation. These properties make them indispensable tools in functional genomics and genome engineering, particularly for delivering molecular components in high-throughput CRISPR screening, a powerful approach for uncovering the genetic basis of complex cellular mechanisms and phenotypes. However, challenges such as lentiviral-induced recombination, unpredictable integration profiles, and variable susceptibility of target cells to transduction can introduce noise and compromise experimental outcomes. In this study, we selected two suspension-adapted mammalian cell lines, Chinese Hamster Ovary cells CHO-K1 and Human Embryonic Kidney cells HEK293-6E, due to their widespread use in recombinant protein production. Recognizing the influence of intrinsic cell line properties and transduction methodology, we compared two distinct procedures: spinoculation and static transduction. By implementing a two-step static transduction protocol, we achieved significantly higher transduction efficiencies while minimizing cellular stress, streamlining workflows, and eliminating scalability limitations inherent to large-scale lentiviral applications like genome-wide CRISPR screens. To further characterize the variation in lentiviral integration, we used droplet digital PCR (ddPCR) to quantify copy number variation (CNV) both at the pooled population level and within individual clonal isolates. This comprehensive analysis underscores the robustness of our optimized protocol in enhancing transduction efficiency in difficult-to-transduce suspension cell lines. It further emphasizes the importance of carefully modulating infection rates to limit multiple integrations, ensuring the accuracy and consistency required for large-scale functional genomics applications.

molecular biology↗

bioRxiv2025

A genome-scale CRISPR deletion screen in Chinese Hamster Ovary cells reveals essential regions of the coding and non-coding genome

The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90% of the genome. This unannotated "dark matter" includes regulatory elements and other, poorly understood or characterized functionality of the genome that may be potentially critical for cell survival. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO survival, many of which currently lack gene annotation or known function. For these regions we present annotation status, transcriptional activity as well as annotated chromatin states such as enhancers. Selected regions, specifically those that were negative for all the above, were individually deleted for confirmation. This work sheds a novel light on a substantial part of the mammalian genome which is traditionally difficult to investigate and therefore, neglected.

cell biology↗

bioRxiv2024

Multi-Niche Human Bone Marrow On-A-Chip for Studying the Interactions of Adoptive CAR-T Cell Therapies with Multiple Myeloma

Multiple myeloma (MM), a cancer of bone marrow plasma cells, is the second-most common hematological malignancy. However, despite immunotherapies like chimeric antigen receptor (CAR)-T cells, relapse is nearly universal. The bone marrow (BM) microenvironment influences how MM cells survive, proliferate, and resist treatment. Yet, it is unclear which BM niches give rise to MM pathophysiology. Here, we present a 3D microvascularized culture system, which models the endosteal and perivascular bone marrow niches, allowing us to study MM-stroma interactions in the BM niche and model responses to therapeutic CAR-T cells. We demonstrated the prolonged survival of cell line-based and patient-derived multiple myeloma cells within our in vitro system and successfully flowed in donor-matched CAR-T cells. We then measured T cell survival, differentiation, and cytotoxicity against MM cells using a variety of analysis techniques. Our MM-on-a-chip system could elucidate the role of the BM microenvironment in MM survival and therapeutic evasion and inform the rational design of next-generation therapeutics. TEASERA multiple myeloma model can study why the disease is still challenging to treat despite options that work well in other cancers.

cancer biology↗

bioRxiv2023

Multi-Omics Analysis Of Antiviral Interactions Of Elizabethkingia anophelis And Zika Virus

BackgroundThe microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Elizabethkingia anophelis, originally isolated from the midgut of Anopheles gambiae, has drawn much attention due to its close association with Aedes and Anopheles mosquitoes, primary vectors of dengue virus and malaria parasites, respectively. E. anophelis possesses a broad-spectrum antiviral phenotype, yet a gap in knowledge regarding the mechanistic basis of its interaction with viruses exists. Methodology/Principal findingsTo further understand the antiviral interactions between E. anophelis and Zika virus (ZIKV), we utilized a non-targeted multi-omics approach, analyzing lipids, proteins, and metabolites of cell monolayers co-infected with ZIKV and E. anophelis. We further assessed the gene expression of ZIKV when cultured in the presence of E. anophelis. ZIKV cultured in the presence of E. anophelis resulted in an attenuated replicative fitness and unproductive virus infection. Further, in this treatment, we observed lower levels of the nonstructural protein 5 (NS5) and RNA-directed RNA polymerase (RdRp) protein. Lastly, a significant decrease in arginine levels, an essential requirement for viral replication and progression of viral infection was observed. Conclusions/SignificanceThis study provides insights into the molecular basis of E. anophelis antiviral phenotype. These findings improve our knowledge of how microbes and viruses interact to impact viral replication. In the future, our findings can be utilized to unravel the mechanism behind the antiviral phenotype of E. anophelis, and this can help develop novel paradigms for viral therapeutics. AUTHOR SUMMARYZika is a re-emerging disease and is endemic in many regions of sub-Saharan Africa, Asia and Latin America. It remains a major public health threat and lacks FDA-approved therapeutics or vaccines, hence the urgent need for the identification of alternative approaches that limit the transmission of the pathogens by its primary vector, Aedes spp. The microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Flavobacteria dominates the mosquito midgut including Elizabethkingia, which is a gram-negative bacillus prevalent in Aedes and Anopheles species of mosquitoes. E. anophelis, a poorly studied midgut microbe, has a broad-spectrum antiviral phenotype, yet the mechanism of its antiviral action is unknown. In this study, we have identified several pathways as well as Zika virus proteins perturbed when the Zika virus is cultivated in the presence of E. anophelis. Our findings do not only provide insights into microbial, virus interaction but could be harnessed to develop novel antiviral tools.

microbiology↗

bioRxiv2023

ATP-release pannexin channels are gated by lysophospholipids

In addition to its role as cellular energy currency, adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of membrane proteins that form heptameric large-pore channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Secretomics experiments reveal that lysophospholipid-activated pannexin 1 leads to the release of not only ATP but also other signaling metabolites, such as 5-methylthioadenosine, which is important for immunomodulation. We also demonstrate that lysophospholipids activate endogenous pannexin 1 in human monocytes, leading to the release of IL-1{beta} through inflammasome activation. Our results provide a connection between lipid metabolism and purinergic signaling, both of which play major roles in immune responses.

biophysics↗

bioRxiv2023

A biophysical rationale for the selective inhibition of PTP1B over TCPTP by nonpolar terpenoids

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including type 2 diabetes, obesity, and cancer. However, a high degree of structural similarity between the catalytic domains of these enzymes has made the development of selective pharmacological inhibitors an enormous challenge. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over TCPTP, two PTPs with high sequence conservation. Here, we use molecular modeling with experimental validation to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations indicate that PTP1B and TCPTP contain a conserved h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically influential WPD loop, which it links to the L-11 loop and 3 and 7 helices--the C-terminal side of the catalytic domain. Terpenoid binding to either of two proximal allosteric sites--an site and a {beta} site--can disrupt the allosteric network. Interestingly, binding to the site forms a stable complex with only PTP1B; in TCPTP, where two charged residues disfavor binding at the site, the terpenoids bind to the {beta} site, which is conserved between the two proteins. Our findings indicate that minor amino acid differences at the poorly conserved site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate, more broadly, how minor differences in the conservation of neighboring--yet functionally similar--allosteric sites can have very different implications for inhibitor selectivity.

bioengineering↗

bioRxiv2021

Cortical Spheroid Model for Studying the Effects of Ischemic Brain Injury

Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are costeffective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics. Significance StatementThose who survive after suffering a stroke often have long-term cognitive or physical disabilities. There is currently only one available therapeutic, tissue plasminogen activator (tPA), and it must be administered within a few hours after the onset of stroke. As stroke prevalence increases with our aging population, there is a growing need for therapies to mitigate or reverse the resulting brain damage. Three-dimensional (3D) culture systems have the potential to screen novel therapeutics more reliably than traditional in vitro models. Here we present a novel 3D cortical spheroid ischemia model which replicates many of the characteristics of stroke and has the potential to be an effective tool in therapeutic development.

neuroscience↗

bioRxiv2021

Revealing how variations in antibody repertoires correlate with vaccine responses

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold a key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated dataset, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study revealed that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

immunology↗

bioRxiv2021

Challenges of γ9δ2TCR affinity maturation when using phage display

BackgroundOver the past years we showed that the efficacy of {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) depends on the functional avidity of the {gamma}9{delta}2TCR. We hypothesized that functional avidity mediated through {gamma}9{delta}2TCR in the TEG format could be further enhanced by increasing affinity of the {gamma}9{delta}2TCR. MethodsWe attempted to overcome limited affinity of natural occurring {gamma}9{delta}2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains. ConclusionAffinity maturation of {gamma}9{delta}2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of {gamma}9{delta}2TCR constructs in E.coli which is a prerequisite for successful phage display. The underlying reason for this lack of expression was the instability of the single chain (sc)TCR format. Expression of scTCR formats in HEK293F cells yielded only 15-20% correctly folded scTCR.

immunology↗

bioRxiv2019

Zika virus infection and temperature significantly impact Aedes albopictus gut microbiota

Elizabethkingia anophelis has been the cause of four outbreaks with significant morbidity and mortality. Its transmission routes remain unknown and no point source of infection has been identified. Here we show that E. anophelis can be found in the saliva of Aedes mosquitoes, suggesting the novel possibility of vector-borne transmission of this bacterium. We additionally characterized diverse microbial communities in Aedes midguts, salivary glands and saliva. To the best of our knowledge, this represents the first description of the microbiome of Aedes saliva. Further, we demonstrate that increased abundance of E. anophelis is associated with decreased susceptibility and replication of Zika virus (ZIKV) in the midgut of Aedes mosquitoes, suggesting a novel transmission barrier for arboviruses transmitted by Aedes mosquitoes. Together, these results demonstrate the complex relationships between the mosquito, the midgut microbial community and arboviruses and offer insights into the epidemiology and control of emerging bacterial and viral pathogens. Author SummaryElizabethkingia anophelis has in the recent past caused outbreaks different parts of the world resulting both in morbidity and mortality. Until now, to the best of our knowledge, no study has been able to demonstrate that this bacterium can be transmitted by mosquitoes. We have demonstrated for the first time that Elizabethkingia anophelis is present in the saliva of both infected and non-infected Aedes mosquitoes. Further, we have shown that it confers an inhibitory effect on Zika virus establishment in the midguts of Aedes mosquitoes. Together, these results potentially display the potential for vector borne transmission of E. anophelis as well as a novel transmission barrier of ZIKV. Lastly, we have for the first time characterized salivary microbes of Aedes mosquitoes necessitating the investigation of the impact of salivary microbes in severity of disease in vertebrate hosts.

microbiology↗

bioRxiv2018

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

Dengue virus (DENV), an arthropod-borne (\"arbovirus\") virus causing a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome, proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Unexpectedly, mosquitoes and primates have distinct preferences when expressing their genes by translation, e.g. members of these taxa show taxonomic group-specific intolerance to certain codon pairs. This is called \"codon pair bias\". By necessity, arboviruses evolved to delicately balance this fundamental difference in their ORFs. Using the mosquito-borne human pathogen DENV we have undone the evolutionarily conserved genomic balance in its ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV raised concerns of gain-of-function, namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell cultures and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control an arboviruss host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

microbiology↗