Scholarly Works, Fralin Life Sciences Institute

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Spatial Transcriptomics and Single-Nucleus Multi-Omics Analysis Revealing the Impact of High Maternal Folic Acid Supplementation on Offspring Brain Development
Xu, Xiguang; Lin, Yu; Yin, Liduo; Serpa, Priscila da Silva; Conacher, Benjamin; Pacholec, Christina; Carvallo, Francisco; Hrubec, Terry; Farris, Shannon; Zimmerman, Kurt; Wang, Xiaobin; Xie, Hehuang (MDPI, 2024-11-07)
Background: Folate, an essential vitamin B9, is crucial for diverse biological processes, including neurogenesis. Folic acid (FA) supplementation during pregnancy is a standard practice for preventing neural tube defects (NTDs). However, concerns are growing over the potential risks of excessive maternal FA intake. Objectives/Methods: Here, we employed a mouse model and spatial transcriptomic and single-nucleus multi-omics approaches to investigate the impact of high maternal FA supplementation during the periconceptional period on offspring brain development. Results: Maternal high FA supplementation affected gene pathways linked to neurogenesis and neuronal axon myelination across multiple brain regions, as well as gene expression alterations related to learning and memory in thalamic and ventricular regions. Single-nucleus multi-omics analysis revealed that maturing excitatory neurons in the dentate gyrus (DG) are particularly vulnerable to high maternal FA intake, leading to aberrant gene expressions and chromatin accessibility in pathways governing ribosomal biogenesis critical for synaptic formation. Conclusions: Our findings provide new insights into specific brain regions, cell types, gene expressions and pathways that can be affected by maternal high FA supplementation.
Hybridization between Aedes aegypti and Aedes mascarensis mosquitoes leads to disruption of male sex determination
Liang, Jiangtao; Kang, Lin; Michalak, Pawel; Sharakhov, Igor V. (Springer Nature, 2024-07-22)
Understanding the sex determination pathway and its disruptions in mosquitoes is critical for the effective control of disease vectors through genetic manipulations based on sex separation. When male hybrids of Aedes aegypti females and Ae. mascarensis males are backcrossed to Ae. aegypti females, a portion of the backcross progeny manifests as males with abnormal sexual differentiation. We discovered a significant correlation between pupal abnormalities and the feminization of subsequent adults exemplified by the relative abundance of ovarian and testicular tissues. All intersex individuals were genetic males as they expressed a male determining factor, Nix. Further, our analysis of the sex-specific splicing of doublesex and fruitless transcripts demonstrated the presence of both male and female splice variants indicating that sex determination is disrupted. A comparative transcriptomic analysis revealed similar expression levels of most female-associated genes in reproductive organs and carcasses between intersexual males and normal females. Moreover, intersexes had largely normal gene expression in testes but significant gene downregulation in male accessory glands when compared with normal males. We conclude that evolving hybrid incompatibilities between Ae. aegypti and Ae. mascarensis involve disruption of sex determination and are accompanied by changes in gene expression associated with sexual differentiation.
Two Nested Inversions in the X Chromosome Differentiate the Dominant Malaria Vectors in Europe, Anopheles atroparvus and Anopheles messeae
Soboleva, Evgenia S.; Kirilenko, Kirill M.; Fedorova, Valentina S.; Kokhanenko, Alina A.; Artemov, Gleb N.; Sharakhov, Igor V. (MDPI, 2024-04-26)
The Maculipennis subgroup of malaria mosquitoes includes both dominant malaria vectors and non-vectors in Eurasia. Understanding the genetic factors, particularly chromosomal inversions, that differentiate Anopheles species can provide valuable insights for vector control strategies. Although autosomal inversions between the species in this subgroup have been characterized based on the chromosomal banding patterns, the number and positions of rearrangements in the X chromosome remain unclear due to the divergent banding patterns. Here, we identified two large X chromosomal inversions, approximately 13 Mb and 10 Mb in size, using fluorescence in situ hybridization. The inversion breakpoint regions were mapped by hybridizing 53 gene markers with polytene chromosomes of An. messeae. The DNA probes were designed based on gene sequences from the annotated An. atroparvus genome. The two nested inversions resulted in five syntenic blocks. Only two small syntenic blocks, which encompass 181 annotated genes in the An. atroparvus genome, changed their position and orientation in the X chromosome. The analysis of the An. atroparvus genome revealed an enrichment of gene ontology terms associated with immune system and mating behavior in the rearranged syntenic blocks. Additionally, the enrichment of DNA transposons was found in sequences homologous to three of the four breakpoint regions. This study demonstrates the successful application of the physical genome mapping approach to identify rearrangements that differentiate species in insects with polytene chromosomes.
Peptidoglycan in osteoarthritis synovial tissue is associated with joint inflammation
Holub, Meaghan N.; Wahhab, Amanda; Rouse, Joseph R.; Danner, Rebecca; Hackner, Lauren G.; Duris, Christine B.; McClune, Mecaila E.; Dressler, Jules M.; Strle, Klemen; Jutras, Brandon L.; Edelstein, Adam I.; Lochhead, Robert B. (2024-03-27)
Objectives: Peptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes. Methods: Intraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC) and immunofluorescence microscopy (IFM). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response. Results: A total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, and PG staining colocalized with markers of synovial macrophages and fibroblasts by IFM. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels. Conclusion: Peptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.
Risk of Excess Maternal Folic Acid Supplementation in Offspring
Xu, Xiguang; Zhang, Ziyu; Lin, Yu; Xie, Hehuang (MDPI, 2024-03-06)
Folate, also known as vitamin B9, facilitates the transfer of methyl groups among molecules, which is crucial for amino acid metabolism and nucleotide synthesis. Adequate maternal folate supplementation has been widely acknowledged for its pivotal role in promoting cell proliferation and preventing neural tube defects. However, in the post-fortification era, there has been a rising concern regarding an excess maternal intake of folic acid (FA), the synthetic form of folate. In this review, we focused on recent advancements in understanding the influence of excess maternal FA intake on offspring. For human studies, we summarized findings from clinical trials investigating the effects of periconceptional FA intake on neurodevelopment and molecular-level changes in offspring. For studies using mouse models, we compiled the impact of high maternal FA supplementation on gene expression and behavioral changes in offspring. In summary, excessive maternal folate intake could potentially have adverse effects on offspring. Overall, we highlighted concerns regarding elevated maternal folate status in the population, providing a comprehensive perspective on the potential adverse effects of excessive maternal FA supplementation on offspring.
SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice
Pavan, María Florencia; Bok, Marina; Betanzos San Juan, Rafael; Malito, Juan Pablo; Marcoppido, Gisela Ariana; Franco, Diego Rafael; Militelo, Daniela Ayelen; Schammas, Juan Manuel; Bari, Sara Elizabeth; Stone, William; López, Krisangel; Porier, Danielle LaBrie; Muller, John Anthony; Auguste, A. Jonathan; Yuan, Lijuan; Wigdorovitz, Andrés; Parreño, Viviana Gladys; Ibañez, Lorena Itat (MDPI, 2024-01-25)
Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.
Mathematical modeling of mechanosensitive reversal control in Myxococcus xanthus
Chen, Yirui; Topo, Elias J.; Nan, Beiyan; Chen, Jing (Frontiers, 2024-01-08)
Adjusting motility patterns according to environmental cues is important for bacterial survival. Myxococcus xanthus, a bacterium moving on surfaces by gliding and twitching mechanisms, modulates the reversal frequency of its front-back polarity in response to mechanical cues like substrate stiffness and cell-cell contact. In this study, we propose that M. xanthus’s gliding machinery senses environmental mechanical cues during force generation and modulates cell reversal accordingly. To examine our hypothesis, we expand an existing mathematical model for periodic polarity reversal in M. xanthus, incorporating the experimental data on the intracellular dynamics of the gliding machinery and the interaction between the gliding machinery and a key polarity regulator. The model successfully reproduces the dependence of cell reversal frequency on substrate stiffness observed in M. xanthus gliding. We further propose reversal control networks between the gliding and twitching motility machineries to explain the opposite reversal responses observed in wild type M. xanthus cells that possess both motility mechanisms. These results provide testable predictions for future experimental investigations. In conclusion, our model suggests that the gliding machinery in M. xanthus can function as a mechanosensor, which transduces mechanical cues into a cell reversal signal.
A fine balance among key biophysical factors is required for recovery of bipolar mitotic spindle from monopolar and multipolar abnormalities
Li, Xiaochu; Bloomfield, Mathew; Bridgeland, Alexandra; Cimini, Daniela; Chen, Jing (American Society for Cell Biology, 2023-06-21)
During mitosis, equal partitioning of chromosomes into two daughter cells requires assembly of a bipolar mitotic spindle. Because the spindle poles are each organized by a centrosome in animal cells, centrosome defects can lead to monopolar or multipolar spindles. However, the cell can effectively recover the bipolar spindle by separating the centrosomes in monopolar spindles and clustering them in multipolar spindles. To interrogate how a cell can separate and cluster centrosomes as needed to form a bipolar spindle, we developed a biophysical model, based on experimental data, which uses effective potential energies to describe key mechanical forces driving centrosome movements during spindle assembly. Our model identified general biophysical factors crucial for robust bipolarization of spindles that start as monopolar or multipolar. These factors include appropriate force fluctuation between centrosomes, balance between repulsive and attractive forces between centrosomes, exclusion of the centrosomes from the cell center, proper cell size and geometry, and a limited centrosome number. Consistently, we found experimentally that bipolar centrosome clustering is promoted as mitotic cell aspect ratio and volume decrease in tetraploid cancer cells. Our model provides mechanistic explanations for many more experimental phenomena and a useful theoretical framework for future studies of spindle assembly.
Mathematical analysis of robustness of oscillations in models of the mammalian circadian clock
Yao, Xiangyu; Heidebrecht, Benjamin L.; Chen, Jing; Tyson, John J. (Public Library of Science, 2022-03-18)
Circadian rhythms in a wide range of organisms are mediated by molecular mechanisms based on transcription-translation feedback. In this paper, we use bifurcation theory to explore mathematical models of genetic oscillators, based on Kim & Forger’s interpretation of the circadian clock in mammals. At the core of their models is a negative feedback loop whereby PER proteins (PER1 and PER2) bind to and inhibit their transcriptional activator, BMAL1. For oscillations to occur, the dissociation constant of the PER:BMAL1 complex, Kbd, must be ≤ 0.04 nM, which is orders of magnitude smaller than a reasonable expectation of 1–10 nM for this protein complex. We relax this constraint by two modifications to Kim & Forger’s ‘single negative feedback’ (SNF) model: first, by introducing a multistep reaction chain for posttranscriptional modifications of Per mRNA and posttranslational phosphorylations of PER, and second, by replacing the first-order rate law for degradation of PER in the nucleus by a Michaelis-Menten rate law. These modifications increase the maximum allowable Kbd to ~2 nM. In a third modification, we consider an alternative rate law for gene transcription to resolve an unrealistically large rate of Per2 transcription at very low concentrations of BMAL1. Additionally, we studied extensions of the SNF model to include a second negative feedback loop (involving REV-ERB) and a supplementary positive feedback loop (involving ROR). Contrary to Kim & Forger’s observations of these extended models, we find that, with our modifications, the supplementary positive feedback loop makes the oscillations more robust than observed in the models with one or two negative feedback loops. However, all three models are similarly robust when accounting for circadian rhythms (~24 h period) with Kbd ≥ 1 nM. Our results provide testable predictions for future experimental studies.
Anti-inflammatory cytokine stimulation of HMC3 cells: Proteome dataset
Ahuja, Shreya; Lazar, Iulia M. (Elsevier, 2023-07-20)
The immunoprotective functions of microglia in the brain are mediated by the inflammatory M1 phenotype. This phenotype is challenged by anti-inflammatory cytokines which polarize the microglia cells to an immunosuppressive M2 phenotype, a trait that is often exploited by cancer cells to evade immune recognition and promote tumor growth. Investigating the molecular determinants of this behavior is crucial for advancing the understanding of the mechanisms that cancer cells use to escape immune attack. In this article, we describe liquid chromatography (LC)-mass spectrometry (MS)/proteomic data acquired with an EASY-nanoLC 1200-Q ExactiveTM OrbitrapTM mass spectrometer that reflect the response of human microglia cells (HMC3) to stimulation with potential cancer-released anti-inflammatory cytokines known to be key players in promoting tumorigenesis in the brain (IL-4, IL-13, IL-10, TGFB and MCP-1). The MS files were processed with the Proteome Discoverer v.2.4 software package. The cell culture conditions, the sample preparation protocols, the MS acquisition parameters, and the data processing approach are described in detail. The RAW and processed MS files associated with this work were deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the dataset identifiers PXD023163 and PXD023166, and the analyzed data in the Mendeley Data cloud-based repository with DOI 10.17632/fvhw2zwt5d.1. The biological interpretation of the data can be accessed in the research article “Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-inflammatory Cytokines” (Shreya Ahuja and Iulia M. Lazar, Frontiers in Immunology 2021 [1]). The proteome data described in this article will benefit researchers who are either interested in re-processing the data with alternative search engines and filtering criteria, and/or exploring the data in more depth to advance the understanding of cancer progression and the discovery of novel biomarkers or drug targets.
Mitotic checkpoint gene expression is tuned by codon usage bias
Esposito, Eric; Weidemann, Douglas E.; Rogers, Jessie M.; Morton, Claire M.; Baybay, Erod Keaton; Chen, Jing; Hauf, Silke (Wiley, 2022-08-01)
The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half-lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1+ mRNA has a short half-life despite a higher frequency of optimal codons, and despite the lack of known RNA-destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co-translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine-tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.
The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes
Ryazansky, Sergei S.; Chen, Chujia; Potters, Mark; Naumenko, Anastasia N.; Lukyanchikova, Varvara; Masri, Reem A.; Brusentsov, Ilya I.; Karagodin, Dmitriy A.; Yurchenko, Andrey A.; dos Anjos, Vitor L.; Haba, Yuki; Rose, Noah H.; Hoffman, Jinna; Guo, Rong; Menna, Theresa; Kelley, Melissa; Ferrill, Emily; Schultz, Karen E.; Qi, Yumin; Sharma, Atashi; Deschamps, Stéphane; Llaca, Victor; Mao, Chunhong; Murphy, Terence D.; Baricheva, Elina M.; Emrich, Scott; Fritz, Megan L.; Benoit, Joshua B.; Sharakhov, Igor V.; McBride, Carolyn S.; Tu, Zhijian; Sharakhova, Maria V. (2024-01-25)
Background: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. Methods: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. Results: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. Conclusion: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.
Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation
Farrell, Kayla; Auerbach, Aubrey; Musaus, Madeline; Navabpour, Shaghayegh; Liu, Catherine; Lin, Yu; Xie, Hehuang; Jarome, Timothy J. (Society for Neuroscience, 2024-01)
Memory formation requires coordinated control of gene expression, protein synthesis, and ubiquitin–proteasome system (UPS)-mediated protein degradation. The catalytic component of the UPS, the 26S proteasome, contains a 20S catalytic core surrounded by two 19S regulatory caps, and phosphorylation of the 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Recently, RPT6 was also shown to act outside the proteasome where it has a transcription factor-like role in the hippocampus during memory formation. However, little is known about the proteasome-independent function of “free” RPT6 in the brain or during memory formation and whether phosphorylation of S120 is required for this transcriptional control function. Here, we used RNA-sequencing along with novel genetic approaches and biochemical, molecular, and behavioral assays to test the hypothesis that pRPT6-S120 functions independently of the proteasome to bind DNA and regulate gene expression during memory formation. RNA-sequencing following siRNA-mediated knockdown of free RPT6 revealed 46 gene targets in the dorsal hippocampus of male rats following fear conditioning, where RPT6 was involved in transcriptional activation and repression. Through CRISPR-dCas9-mediated artificial placement of RPT6 at a target gene, we found that RPT6 DNA binding alone may be important for altering gene expression following learning. Further, CRISPR-dCas13-mediated conversion of S120 to glycine on RPT6 revealed that phosphorylation at S120 is necessary for RPT6 to bind DNA and properly regulate transcription during memory formation. Together, we reveal a novel function for phosphorylation of RPT6 in controlling gene transcription during memory formation.
Sex linked behavioral and hippocampal transcriptomic changes in mice with cell-type specific Egr1 loss
Swilley, Cody; Lin, Yu; Zheng, Yuze; Xu, Xiguang; Liu, Min; Jarome, Timothy J.; Hodes, Georgia E.; Xie, Hehuang (Frontiers, 2023-10-19)
The transcription factor EGR1 is instrumental in numerous neurological processes, encompassing learning and memory as well as the reaction to stress. Egr1 complete knockout mice demonstrate decreased depressive or anxiety-like behavior and impaired performance in spatial learning and memory. Nevertheless, the specific functions of Egr1 in distinct cell types have been largely underexplored. In this study, we cataloged the behavioral and transcriptomic character of Nestin-Cre mediated Egr1 conditional knockout (Egr1cKO) mice together with their controls. Although the conditional knockout did not change nociceptive or anxiety responses, it triggered changes in female exploratory activity during anxiety testing. Hippocampus-dependent spatial learning in the object location task was unaffected, but female Egr1cKO mice did exhibit poorer retention during testing on a contextual fear conditioning task compared to males. RNA-seq data analyses revealed that the presence of the floxed Egr1 cassette or Nestin-Cre driver alone exerts a subtle influence on hippocampal gene expression. The sex-related differences were amplified in Nestin-Cre mediated Egr1 conditional knockout mice and female mice are more sensitive to the loss of Egr1 gene. Differentially expressed genes resulted from the loss of Egr1 in neuronal cell lineage were significantly associated with the regulation of Wnt signaling pathway, extracellular matrix, and axon guidance. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in neuronal cell lineage have distinct impacts on hippocampal gene expression in a sex-specific manner.
Genomic analysis of two phlebotomine sand fly vectors of leishmania from the new and old World
Labbe, Frederic; Abdeladhim, Maha; Abrudan, Jenica; Araki, Alejandra Saori; Araujo, Ricardo N.; Arensburger, Peter; Benoit, Joshua B.; Brazil, Reginaldo Pecanha; Bruno, Rafaela V.; Rivas, Gustavo Bueno da Silva D. S.; de Abreu, Vinicius Carvalho; Charamis, Jason; Coutinho-Abreu, Iliano V.; da Costa-Latge, Samara G.; Darby, Alistair; Dillon, Viv M.; Emrich, Scott J.; Fernandez-Medina, Daniela; Gontijo, Nelder Figueiredo; Flanley, Catherine M.; Gatherer, Derek; Genta, Fernando A.; Gesing, Sandra; Giraldo-Calderon, Gloria I.; Gomes, Bruno; Aguiar, Eric Roberto Guimaraes Rocha; Hamilton, James GC C.; Hamarsheh, Omar; Hawksworth, Mallory; Hendershot, Jacob M.; Hickner, Paul V.; Imler, Jean-Luc; Ioannidis, Panagiotis; Jennings, Emily C.; Kamhawi, Shaden; Karageorgiou, Charikleia; Kennedy, Ryan C.; Krueger, Andreas; Latorre-Estivalis, Jose M.; Ligoxygakis, Petros; Meireles-Filho, Antonio Carlos A.; Minx, Patrick; Miranda, Jose Carlos; Montague, Michael J.; Nowling, Ronald J.; Oliveira, Fabiano; Ortigao-Farias, Joao; Pavan, Marcio G.; Pereira, Marcos Horacio; Pitaluga, Andre Nobrega; Olmo, Roenick Proveti; Ramalho-Ortigao, Marcelo; Ribeiro, Jose MC C.; Rosendale, Andrew J.; Sant'Anna, Mauricio RV V.; Scherer, Steven E.; Secundino, Nagila FC C.; Shoue, Douglas A.; Moraes, Caroline da Silva D. S.; Gesto, Joao Silveira Moledo; Souza, Nataly Araujo; Syed, Zainulabueddin; Tadros, Samuel; Teles-de-Freitas, Rayane; Telleria, Erich L.; Tomlinson, Chad; Traub-Cseko, Yara M.; Marques, Joao Trindade; Tu, Zhijian; Unger, Maria F.; Valenzuela, Jesus; Ferreira, Flavia; de Oliveira, Karla PV V.; Vigoder, Felipe M.; Vontas, John; Wang, Lihui; Weedall, Gareth D.; Zhioua, Elyes; Richards, Stephen; Warren, Wesley C.; Waterhouse, Robert M.; Dillon, Rod J.; McDowell, Mary Ann (Public Library of Science, 2023-04-12)
Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.
Expression of anti-chikungunya single-domain antibodies in transgenic Aedes aegypti reduces vector competence for chikungunya virus and Mayaro virus
Webb, Emily M.; Compton, Austin; Rai, Pallavi; Chuong, Christina; Paulson, Sally L.; Tu, Zhijian; Weger-Lucarelli, James (Frontiers, 2023-06-12)
Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.
Exploring the immunogenicity of an insect-specific virus vectored Zika vaccine candidate
Tanelus, Manette; López, Krisangel; Smith, Shaan; Muller, John A.; Porier, Danielle L.; Auguste, Dawn I.; Stone, William B.; Paulson, Sally L.; Auguste, A. Jonathan (Springer, 2023-12-01)
Zika virus (ZIKV) is an important re-emerging flavivirus that presents a significant threat to human health worldwide. Despite its importance, no vaccines are approved for use in humans. Insect-specific flaviviruses (ISFVs) have recently garnered attention as an antigen presentation platform for vaccine development and diagnostic applications. Here, we further explore the safety, immunogenicity, and efficacy of a chimeric ISFV-Zika vaccine candidate, designated Aripo-Zika (ARPV/ZIKV). Our results show a near-linear relationship between increased dose and immunogenicity, with 1011 genome copies (i.e., 108 focus forming units) being the minimum dose required for protection from ZIKV-induced morbidity and mortality in mice. Including boosters did not significantly increase the short-term efficacy of ARPV/ZIKV-vaccinated mice. We also show that weanling mice derived from ARPV/ZIKV-vaccinated dams were completely protected from ZIKV-induced morbidity and mortality upon challenge, suggesting efficient transfer of maternally-derived protective antibodies. Finally, in vitro coinfection studies of ZIKV with Aripo virus (ARPV) and ARPV/ZIKV in African green monkey kidney cells (i.e., Vero-76) showed that ARPV and ARPV/ZIKV remain incapable of replication in vertebrate cells, despite the presence of active ZIKV replication. Altogether, our data continue to support ISFV-based vaccines, and specifically the ARPV backbone is a safe, immunogenic and effective vaccine strategy for flaviviruses.
High-Frequency Dielectrophoresis Reveals That Distinct Bio-Electric Signatures of Colorectal Cancer Cells Depend on Ploidy and Nuclear Volume
Duncan, Josie L.; Bloomfield, Mathew; Swami, Nathan; Cimini, Daniela; Davalos, Rafael V. (MDPI, 2023-09-01)
Aneuploidy, or an incorrect chromosome number, is ubiquitous among cancers. Whole-genome duplication, resulting in tetraploidy, often occurs during the evolution of aneuploid tumors. Cancers that evolve through a tetraploid intermediate tend to be highly aneuploid and are associated with poor patient prognosis. The identification and enrichment of tetraploid cells from mixed populations is necessary to understand the role these cells play in cancer progression. Dielectrophoresis (DEP), a label-free electrokinetic technique, can distinguish cells based on their intracellular properties when stimulated above 10 MHz, but DEP has not been shown to distinguish tetraploid and/or aneuploid cancer cells from mixed tumor cell populations. Here, we used high-frequency DEP to distinguish cell subpopulations that differ in ploidy and nuclear size under flow conditions. We used impedance analysis to quantify the level of voltage decay at high frequencies and its impact on the DEP force acting on the cell. High-frequency DEP distinguished diploid cells from tetraploid clones due to their size and intracellular composition at frequencies above 40 MHz. Our findings demonstrate that high-frequency DEP can be a useful tool for identifying and distinguishing subpopulations with nuclear differences to determine their roles in disease progression.
The minimal intrinsic stochasticity of constitutively expressed eukaryotic genes is sub-Poissonian
Weidemann, Douglas E.; Holehouse, James; Singh, Abhyudai; Grima, Ramon; Hauf, Silke (American Association for the Advancement of Science, 2023-08-09)
Gene expression inherently gives rise to stochastic variation (“noise”) in the production of gene products. Minimizing noise is crucial for ensuring reliable cellular functions. However, noise cannot be suppressed below a certain intrinsic limit. For constitutively expressed genes, this limit is typically assumed to be Poissonian noise, wherein the variance in mRNA numbers is equal to their mean. Here, we demonstrate that several cell division genes in fission yeast exhibit mRNA variances significantly below this limit. The reduced variance can be explained by a gene expression model incorporating multiple transcription and mRNA degradation steps. Notably, in this sub-Poissonian regime, distinct from Poissonian or super-Poissonian regimes, cytoplasmic noise is effectively suppressed through a higher mRNA export rate. Our findings redefine the lower limit of eukaryotic gene expression noise and uncover molecular requirements for achieving ultralow noise, which is expected to be important for vital cellular functions.
Sex-Linked Growth Disorder and Aberrant Pituitary Gene Expression in Nestin-Cre-Mediated Egr1 Conditional Knockout Mice
Swilley, Cody; Lin, Yu; Zheng, Yuze; Xu, Xiguang; Liu, Min; Zimmerman, Kurt; Xie, Hehuang (MDPI, 2023-07-06)
Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.

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