Scholarly Works, Fralin Life Sciences Institute

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Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis
Johnson, Zachary; Xu, Xiguang; Pacholec, Christina; Xie, Hehuang (Oxford University Press, 2022-03-31)
The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.
Egr2 Deletion in Autoimmune-Prone C57BL6/lpr Mice Suppresses the Expression of Methylation-Sensitive Dlk1-Dio3 Cluster MicroRNAs
Wang, Zuhang; Heid, Bettina; He, Jianlin; Xie, Hehuang; Reilly, Christopher M.; Dai, Rujuan; Ahmed, S. Ansar (Oxford University Press, 2023-12)
We previously demonstrated that the upregulation of microRNAs (miRNAs) at the genomic imprinted Dlk1-Dio3 locus in murine lupus is correlated with global DNA hypomethylation. We now report that the Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice is hypomethylated, linking it to increased Dlk1-Dio3 miRNA expression. We evaluated the gene expression of methylating enzymes, DNA methyltransferases (DNMTs), and demethylating ten-eleven translocation proteins (TETs) to elucidate the molecular basis of DNA hypomethylation in lupus CD4+ T cells. There was a significantly elevated expression of Dnmt1 and Dnmt3b, as well as Tet1 and Tet2, in CD4+ T cells of three different lupus-prone mouse strains compared to controls. These findings suggest that the hypomethylation of murine lupus CD4+ T cells is likely attributed to a TET-mediated active demethylation pathway. Moreover, we found that deletion of early growth response 2 (Egr2), a transcription factor gene in B6/lpr mice markedly reduced maternally expressed miRNA genes but not paternally expressed protein-coding genes at the Dlk1-Dio3 locus in CD4+ T cells. EGR2 has been shown to induce DNA demethylation by recruiting TETs. Surprisingly, we found that deleting Egr2 in B6/lpr mice induced more hypomethylated differentially methylated regions at either the whole-genome level or the Dlk1-Dio3 locus in CD4+ T cells. Although the role of methylation in EGR2-mediated regulation of Dlk1-Dio3 miRNAs is not readily apparent, these are the first data to show that in lupus, Egr2 regulates Dlk1-Dio3 miRNAs, which target major signaling pathways in autoimmunity. These data provide a new perspective on the role of upregulated EGR2 in lupus pathogenesis.
Dynamics of RNA m⁵C modification during brain development
Johnson, Zachary; Xu, Xiguang; Lin, Yu; Xie, Hehuang (Elsevier, 2023-05)
Post-transcriptional RNA modifications have been recognized as key regulators of neuronal differentiation and synapse development in the mammalian brain. While distinct sets of 5-methylcytosine (m5C) modified mRNAs have been detected in neuronal cells and brain tissues, no study has been performed to characterize methylated mRNA profiles in the developing brain. Here, together with regular RNA-seq, we performed transcriptome-wide bisulfite sequencing to compare RNA cytosine methylation patterns in neural stem cells (NSCs), cortical neuronal cultures, and brain tissues at three postnatal stages. Among 501 m5C sites identified, approximately 6% are consistently methylated across all five conditions. Compared to m5C sites identified in NSCs, 96% of them were hypermethylated in neurons and enriched for genes involved in positive transcriptional regulation and axon extension. In addition, brains at the early postnatal stage demonstrated substantial changes in both RNA cytosine methylation and gene expression of RNA cytosine methylation readers, writers, and erasers. Furthermore, differentially methylated transcripts were significantly enriched for genes regulating synaptic plasticity. Altogether, this study provides a brain epitranscriptomic dataset as a new resource and lays the foundation for further investigations into the role of RNA cytosine methylation during brain development.
Independent and joint association of cord plasma pantothenate and cysteine levels with autism spectrum disorders and other neurodevelopmental disabilities in children born term and preterm
Raghavan, Ramkripa; Wang, Guoying; Hong, Xiumei; Pearson, Colleen; Xie, Hehuang; Adams, William G.; Augustyn, Marilyn; Wang, Xiaobin (2023-05-11)
Background: Pantothenate (vitamin B5) is a precursor for coenzyme A (CoA) synthesis, which serves as a cofactor for hundreds of metabolic reactions. Cysteine is an amino acid in the CoA synthesis pathway. To date, research on the combined role of early life pantothenate and cysteine levels in childhood neurodevelopmental disabilities is scarce. Objective: To study the association between cord pantothenate and cysteine levels and risk of autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD) and other developmental disabilities (DD) in children born term and preterm. Methods: The study sample (n = 996, 177 born preterm) derived from the Boston Birth Cohort included 416 neurotypical children, 87 ASD, 269 ADHD, and 224 other DD children, who were mutually exclusive. Participants were enrolled at birth and were followed up prospectively (from October 1, 1998, to June 30, 2018) at the Boston Medical Center. Cord blood sample was collected at birth. Plasma pantothenate and cysteine levels were measured using liquid chromatography-tandem mass spectrometry. Results: Higher cord pantothenate (≥50th percentile vs. <50th percentile) was associated with a greater risk of ASD (adjusted odds ratio [aOR]: 1.94, 95% confidence interval [CI]: 1.06, 3.55) and ADHD (aOR: 1.66, 95% CI: 1.14, 2.40), after adjusting for potential confounders. However, cord cysteine alone was not associated with risk of ASD, ADHD, or other DD. When considering the joint association, greater ASD risk was noted when both cord pantothenate and cysteine levels were elevated (≥50th percentile) (aOR: 3.11, 95% CI: 1.24, 7.79), when compared to children with low cord pantothenate (<50th percentile) and high cysteine. Even though preterm and higher pantothenate independently increased the ASD risk, the greatest risk was found in preterm children who also had elevated pantothenate (≥50th percentile), which was true for all three outcomes: ASD (aOR: 5.36, 95% CI: 2.09, 13.75), ADHD (aOR: 3.31, 95% CI: 1.78, 6.16), and other DD (aOR: 3.39, 95% CI: 1.85, 6.24). Conclusions: In this prospective birth cohort, we showed that higher cord pantothenate individually and in combination with higher cysteine or preterm birth were associated with increased risk of ASD and ADHD. More study is needed to explore this biologically plausible pathway.
Endothelial deletion of EPH receptor A4 alters single-cell profile and Tie2/Akap12 signaling to preserve blood-brain barrier integrity
Cash, Alison; de Jager, Caroline; Brickler, Thomas; Soliman, Eman; Ladner, Liliana; Kaloss, Alexandra M.; Zhu, Yumeng; Pridham, Kevin J.; Mills, Jatia; Ju, Jing; Basso, Erwin Kristobal Gudenschwager; Chen, Michael; Johnson, Zachary; Sotiropoulos, Yianni; Wang, Xia; Xie, Hehuang; Matson, John B.; Marvin, Eric A.; Theus, Michelle H. (National Academy of Sciences, 2023-10-10)
Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice promotes blood–brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.
Monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation
Navabpour, Shaghayegh; Farrell, Kayla; Kincaid, Shannon E.; Omar, Nour; Musaus, Madeline; Lin, Yu; Xie, Hehuang; Jarome, Timothy J. (Cold Spring Harbor Laboratory Press, 2024-03)
Posttranslational modification of histone proteins is critical for memory formation. Recently, we showed that monoubiquitination of histone H2B at lysine 120 (H2Bub) is critical for memory formation in the hippocampus. However, the transcriptome controlled by H2Bub remains unknown. Here, we found that fear conditioning in male rats increased or decreased the expression of 86 genes in the hippocampus but, surprisingly, siRNA-mediated knockdown of the H2Bub ligase, Rnf20, abolished changes in all but one of these genes. These findings suggest that monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation.
Increasing H2B Monoubiquitination Improves the Transcriptome and Memory in the Aged Hippocampus
Kincaid, Shannon; Setenet, Gueladouan; Preveza, Natalie J.; Arndt, Kaiser C.; Gwin, Phillip; Lin, Yu; Xie, Hehuang; Jarome, Timothy J. (Society for Neuroscience, 2025-04)
A decline in cognitive abilities is associated with the aging process, affecting nearly 33% of US adults over the age of 70, and is a risk factor for the development of dementia and Alzheimer's disease. Several studies have reported age-related alterations in the transcriptome in the hippocampus, a major site of memory storage that is among the first regions impacted with age, dementia, and Alzheimer's disease. However, much remains unknown about why these transcriptional changes exist in the aged hippocampus and how this impacts memory late in life. Here, we show that monoubiquitination of histone H2B (H2Bubi), an epigenetic mechanism recently reported to be major regulator of the epigenome and transcriptome during memory formation in the young adult brain, decreases with age in the hippocampus of male rats. In vivo CRISPR-dCas9-mediated upregulation of Rnf20, the only ubiquitin E3 ligase for H2B, in the hippocampus significantly improved memory retention in aged rats. Remarkably, RNA-seq analysis revealed that in addition to the 18 genes typically upregulated in the aged rat hippocampus following contextual fear conditioning, Rnf20 upregulation caused learning-related increases and decreases in 40 and 11 unique genes, respectively, suggesting that these 51 genes may be among those most critical for improving memory in advanced age. Together, these data suggest that H2B monoubiquitination is a significant regulator of age-related dysregulation of the transcriptome and impairments in memory.
Species Composition, Ecological Preferences, and Chromosomal Polymorphism of Malaria Mosquitoes of the Crimean Peninsula and the Black Sea Coast of the Caucasus
Moskaev, Anton V.; Bega, Anna G.; Brusentsov, Ilya I.; Naumenko, Anastasia N.; Karagodin, Dmitriy A.; Razumeiko, Vladimir N.; Andrianov, Boris V.; Goryacheva, Irina I.; Lee, Elizaveta Y.; Panov, Vladimir I.; Sharakhov, Igor V.; Sharakhova, Maria V.; Gordeev, Mikhail I. (MDPI, 2025-04-01)
In this study, we sampled malaria mosquito larvae in natural and artificial breeding places to study the geographical distribution, ecological preferences, and chromosomal variability of different species of the genus Anopheles in the territory of the Crimean Peninsula and the Black Sea coast of the Caucasus. Species were diagnosed using a combination of morphological, cytogenetic, and molecular markers. The ecological conditions of the larval habitats, such as dissolved oxygen content in the water, acidity, salinity, and temperature, were measured. Seven species of malaria mosquitoes were identified in the pool of 2229 individual mosquitoes collected at 56 breeding sites, including An. atroparvus, An. claviger, An. daciae (formerly identified as An. messeae s. l.), An. hyrcanus, An. maculipennis s. s., An. plumbeus, and An. melanoon. The previously recorded species of An. algeriensis, An. messeae s. s., An. sacharovi, and An. superpictus were not found in this study. Anopheles maculipennis was dominant in typical anophylogenic water bodies. Anopheles plumbeus, which used to breed mainly in tree holes in coastal forests, has spread to urban settlements along the Black Sea coast and breeds in artificial containers. Chromosomal polymorphism was studied and found in An. atroparvus and An. daciae populations. Differences in the chromosomal composition of An. daciae populations in Crimea and on the Black Sea coast of the Caucasus were revealed. The Crimean population had a low level of polymorphism in autosomal inversions. The data obtained in this study can be used to inform a better control of potential malaria vectors in the Black Sea coastal region.
Examination of the moderating effect of race on the relationship between Vitamin D status and COVID-19 test positivity using propensity score methods
Crandell, Ian; Rockwell, Michelle S.; Whitehead, Phyllis B.; Carter, Kimberly Ferren; Hanlon, Alexandra L. (Journal of the American College of Nutrition, 2021-09-02)
Introduction: With a well-established role in inflammation and immune function, vitamin D status has emerged as a potential factor for coronavirus disease-2019 (COVID-19). Objective: The purpose of this study was to evaluate the moderating effect of race on the relationship between vitamin D status and the risk of COVID-19 test positivity, and to compare propensity score (PS) model results to those obtained from classical bivariate and multivariable models, which have primarily comprised the literature to date. Methods: Electronic health record (EHR) data from TriNetX (unmatched n = 21,629; matched n = 16,602) were used to investigate the effect of vitamin D status, as measured by 25-hydroxyvitamin D [25(OH)D], on the odds of experiencing a positive COVID-19 test using multivariable logistic regression models with and without PS methodology. Results: Having normal (≥ 30 ng/mL) versus inadequate 25(OH)D (< 30 ng/mL) was not associated with COVID-19 positivity overall (OR = 0.913, p = 0.18), in White individuals (OR = 0.920, p = 0.31), or in Black individuals (OR = 1.006, p = 0.96). When 25(OH)D was analyzed on a continuum, a 10 ng/mL increase in 25(OH)D lowered the odds of having a positive COVID-19 test overall (OR = 0.949, p = 0.003) and among White (OR = 0.935, p = 0.003), but not Black individuals (OR = 0.994, p = 0.75). Conclusions: Models which use weighting and matching methods resulted in smaller estimated effect sizes than models which do not use weighting or matching. These findings suggest a minimal protective effect of vitamin D status on COVID-19 test positivity in White individuals and no protective effect in Black individuals.
Evaluation of ebony as a potential selectable marker for genetic sexing in Aedes aegypti
Nikolouli, Katerina; Compton, Austin; Tu, Zhijian J.; Bourtzis, Kostas (2025-02-25)
Background: Aedes aegypti is expected to invade previously unoccupied areas, mainly due to the climate change, the increase in travel and trade activities and the continuous transformation of the rural environment into urban areas. The sterile insect technique (SIT), which relies on the mass production and release of sterile males, is an environmentally friendly approach that can be applied for population control of Ae. aegypti. SIT programs can be greatly benefited by a genetic sexing strain (GSS) and a reliable sex sorting system to minimize any accidental female release. Visually detectable or conditionally lethal selectable markers can be used for the development of new GSSs. In this study, we evaluated the suitability and competence of a mutant Ae. aegypti ebony strain for the development of a new GSS. The ebony gene is known to be involved in the pigmentation pathway of several dipteran insects, including Ae. aegypti. Methods: An ebony gene knockout was developed though CRISPR/Cas9 mutagenesis. G0 individuals with the desired phenotype were crossed, and progeny were screened in every generation. PCR and sequencing were performed using gDNA from a pulled leg to determine the mutant genotype. Quality control tests, including pupae and adult recovery rates, male sex ratio and fecundity, were applied to the ebony mutant line to determine whether the mutation confers any fitness cost. Results: An Ae. aegypti ebony knockout mutant carrying a 5-bp deletion was obtained, which presented darker head and siphon phenotypes at the larval stage. However, genetic analysis revealed that this ebony mutation results in incomplete penetrance and variable expressivity. The establishment of a pure ebony mutant line was not possible because of the fitness costs conferred by the mutation. Conclusions: In this study, the adequacy and suitability of the ebony gene as a selectable marker for the development of a GSS in Ae. aegypti were assessed. Despite its clear phenotype early in larval development, the homozygous mutant line presented phenotypic inconsistency and loss of fertility. These drawbacks clearly indicate that this particular mutation is not suitable for the development of a new GSS. Nonetheless, it cannot be excluded that a different mutation will lead to a different expression and penetrance profile and a viable homozygous mutant line.
Proteomic insights into breast cancer response to brain cell-secreted factors
Ahuja, Shreya; Lazar, Iuliana M. (Springer, 2024-08-21)
The most devastating feature of cancer cells is their ability to metastasize to distant sites in the body. HER2 + and TN breast cancers frequently metastasize to the brain and stay potentially dormant for years until favorable conditions support their proliferation. The sheltered and delicate nature of the brain prevents, however, early disease detection and effective delivery of therapeutic drugs. Moreover, the challenges associated with the acquisition of brain biopsies add compounding difficulties to exploring the mechanistic aspects of tumor development. To provide insights into the determinants of cancer cell behavior at the brain metastatic site, this study was aimed at exploring the early response of HER2 + breast cancer cells (SKBR3) to factors present in the brain perivascular niche. The neural microenvironment was simulated by using the secretome of a set of brain cells that come first in contact with the cancer cells upon crossing the blood brain barrier, i.e., endothelial cells, astrocytes, and microglia. Cytokine microarrays were used to investigate the secretome mediators of intercellular communication, and proteomic technologies for assessing the changes in the behavior of cancer cells upon exposure to the brain cell-secreted factors. The cytokines detected in the brain secretomes were supportive of inflammatory conditions, while the SKBR3 cells secreted numerous cancer-promoting growth factors that were either absent or present in lower abundance in the brain cell cultures, indicating that upon exposure the SKBR3 cells may have been deprived of favorable conditions for optimal growth. Altogether, the results suggest that the exposure of SKBR3 cells to the brain cell-secreted factors altered their growth potential and drove them toward a state of quiescence, with broader overall outcomes that affected cellular metabolism, adhesion and immune response processes. The findings of this study underscore the key role played by the neural niche in shaping the behavior of metastasized cancer cells, provide insights into the cellular cross-talk that may lead cancer cells into dormancy, and highlight novel opportunities for the development of metastatic breast cancer therapeutic strategies.
Proteomic assessment of SKBR3/HER2+ breast cancer cellular response to Lapatinib and investigational Ipatasertib kinase inhibitors
Karcini, Arba; Mercier, Nicole R.; Lazar, Iuliana M. (Frontiers, 2024-08-29)
Introduction: Modern cancer treatment strategies aim at achieving cancer remission by using targeted and personalized therapies, as well as harnessing the power of the immune system to recognize and eradicate the cancer cells. To overcome a relatively short-lived response due to resistance to the administered drugs, combination therapies have been pursued. Objective: The objective of this study was to use high-throughput data generation technologies such as mass spectrometry and proteomics to investigate the broader implications, and to expand the outlook, of such therapeutic approaches. Specifically, we investigated the systems-level response of a breast cancer cell line model to a mixture of kinase inhibitors that has not been adopted yet as a standard therapeutic regime. Methods: Two critical pathways that sustain the growth and survival of cancer cells, EGFR and PI3K/AKT, were inhibited in SKBR3/HER2+ breast cancer cells with Lapatinib (Tyr kinase inhibitor) and Ipatasertib (Ser/Thr kinase inhibitor), and the landscape of the affected biological processes was investigated with proteomic technologies. Results: Over 800 proteins matched by three unique peptide sequences were affected by exposing the cells to the drugs. The work corroborated the anti-proliferative activity of Lapatinib and Ipatasertib and uncovered a range of impacted cancer-supportive hallmark processes, among which immune response, adhesion, and migration emerged as particularly relevant to the ability of drugs to effectively suppress the proliferation and dissemination of cancer cells. Changes in the expression of key cancer drivers such as oncogenes, tumor suppressors, EMT and angiogenesis regulators underscored the inhibitory effectiveness of drugs on cancer proliferation. The supplementation of Lapatinib with Ipatasertib further affected additional transcription factors and proteins involved in gene expression, trafficking, DNA repair, and development of multidrug resistance. Furthermore, over fifty of the impacted proteins represent approved or investigational targets in the DrugBank database, which through their protein-protein interaction networks can inform the selection of effective therapeutic partners. Conclusion: Altogether, the exposure of SKBR3/HER2+ cells to Lapatinib and Ipatasertib kinase inhibitors uncovered a broad plethora of yet untapped opportunities that can be further explored for enhancing the anti-cancer effects of each drug as well as of many other multi-drug therapies that target the EGFR/ERBB2 and PI3K/AKT pathways.
Diversity of unique, nonmycorrhizal endophytic fungi in cultivated Phalaenopsis orchids: A pilot study
Watkinson, Jonathan I.; Winkel, Brenda S. J. (Wiley, 2024-05-17)
Orchids comprise one of the largest, most diverse, and most broadly distributed families of flowering plants and contribute significantly to habitat biodiversity. One key aspect of orchid growth and development is the formation of mycorrhizal symbioses with compatible endophytic fungi, which are maintained throughout the life of the plant. Substantial efforts to identify the fungi that form mycorrhizal symbioses across a range of orchid species have often also uncovered numerous nonmycorrhizal, endophytic fungi. These fungi could also have significant effects on orchid growth and development and are beginning to be analyzed more closely, particularly in wild species. The role of endophytic fungi in the production, distribution, and continued growth by the hobbyist of orchids is not known. As an initial step toward characterizing nonmycorrhizal endophytic fungi associated with cultivated orchids, we undertook a survey of fungi residing within roots of Phalaenopsis plants growing in home environments. Sequence analysis of ITS regions amplified from total DNA isolated from roots allowed rapid identification of endophytic fungi to the class level and may offer a useful initial screening method for beneficial species, for example, in horticultural settings. ITS-PCR sequences subsequently obtained from individual fungi cultured from surface-sterilized orchid roots corroborated the findings of the initial screen, while also providing a more complete characterization of the array of fungal taxa that were present. Although lower in diversity than has been reported for orchids growing in the wild, these endophytes have the potential to substantially enhance the growth and disease resistance of horticultural orchids.
Geostatistical Maps Of Malaria Patients And Their Correlation With Distribution Indicators In Southern Iran
Shabanipoor, Mehdi; Kamali, Maryam; Raz, Abbasali; Dayer, Mohammad Saaid; Sharakhov, Igor V. (2024-04-12)
Malaria is a disease caused by the Plasmodium parasite, with a prevalent distribution among people in Africa and some Asian countries. Migration of people can play a significant role in the spread of malaria. This study aims to develop and investigate the geostatistical maps of malaria patients and their correlation with distribution indicators for disease management in the years 2016-2020 in southern Iran. Malaria patient data was obtained from the Ministry of Health and drawn as geostatistical maps. To achieve full understanding and alignment of the malaria patients’ distribution patterns, five indices (Taylor, Iwao, Morisita, variance-to-mean ratio, and kappa) were used. The results showed that the aggregated distribution wave obtained from the geostatistical map in 2016, 2017, and 2018 were 2, 3, and 2, respectively. The Taylor index indicated a cumulative pattern due to higher sensitivity to the clustering of distribution in 2016 and 2017, with Taylor coefficients of 1.02 and 1.07, respectively. The Iwao coefficient showed random distribution of malaria patients. Furthermore, the alignment of maps with Morisita, variance-to-mean ratio, and kappa indices indicated that in 2016 and 2017, 15% and 20% of patients were cumulatively clustered, while 85% to 80% were randomly distributed in southern Iran. The distribution pattern of malaria patients in 2019 and 2020 was random according to all indices and geostatistical maps. In general, it can be concluded that the distribution of malaria patients in southern Iran does not show a critical phase. However, considering the migration of individuals from neighboring countries, passive and active surveillance should be carried out simultaneously to identify patients in subsequent years.
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.

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