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Karim, S.

Publications and source records attributed to Karim, S..

At least 19 recordsLinked to original sources

Elucidating the Design Space of Generative Models for Single-Cell Perturbation Prediction

Next-token prediction has produced predictable scaling in language, but the recipe presumes a sequence of tokens with a meaningful order. Single-cell RNA-seq counts have no natural gene ordering, so applying the recipe directly to raw expression fails under an ill-suited left-to-right bias. We instead ask whether a learned latent can supply the structure the recipe needs. We introduce ExpressionVAE (eVAE), a discrete-latent perturbation model that compresses each cell into a short sequence of discrete codes through a finite-scalar-quantization (FSQ) bottleneck and trains a perturbation-conditioned discrete prior over those codes. On Replogle and Parse 1M, eVAE sets a new state of the art on every distributional metric and leads on most cell-eval perturbation metrics, with Frechet distance and MMD2 roughly 3 to 20x lower than the strongest continuous-latent baseline. Swapping the prior between autoregressive and masked discrete diffusion leaves performance near-identical, isolating the gain to the discrete latent itself rather than the prior family. A decoder-head ablation then exposes a single design axis, the richness of the predictive distribution at inference, that splits the standard metrics into two groups, variance-sensitive and mean-sensitive, which move in opposite directions along the axis. Finally, on a held-out CRISPRi reversion benchmark of 1,732 perturbations under inflammatory cytokine stress, the frozen eVAE encoder outperforms UMAP and differential expression and matches scGPT on perturbation ranking at a fraction of the data.1 We release our code.2

bioinformatics↗

Discrete Diffusion for Single-Cell Gene Expression Modeling

AO_SCPLOWBSTRACTC_SCPLOWCurrent generative modeling of single-cell transcriptomics relies on continuous latent representations, transforming inherently discrete and sparse gene counts into continuous space. We propose Discrete Cell Models (DCM), a diffusion-based framework that learns cellular representations directly in the discrete domain. Our framework supports both unconditional and conditional generation, allowing for precise modeling of complex biological scenarios such as cell-type-specific transcriptional responses to genetic perturbations. We demonstrate that DCM scales effectively and achieves strong performance against current state-of-the-art methods, including scVI, CPA, STATE, scGPT, and scLDM. On the Dentate Gyrus benchmark, DCM achieves a 5-fold improvement in MMD2RBF and a nearly 2-fold improvement in W2 distance, over the leading continuous diffusion baseline (scLDM). On the conditional Replogle perturbation benchmark, DCM sets a new state of the art on W2 distance while remaining competitive on MMD2RBF. Together, these results establish discrete diffusion as a promising direction for foundational models of cellular biology.

cell biology↗

Development of Long-Term Human Adipocyte Organoids Manifesting Aging in Response to Intermittent Hypoxia

Obstructive sleep apnea (OSA) (and consequent intermittent hypoxia (IH)) is increasingly recognized as a driver of adipose tissue dysfunction, insulin resistance, and aging. However, current in vitro experimental models inadequately capture the long-term effects of IH on human adipocytes. Here, we report the development and optimization of a robust long-term human adipocyte organoid culture system that faithfully recapitulates IH-induced adipocyte aging in vitro. Human stromal vascular fraction (SVF) cells, isolated from subcutaneous abdominal fat biopsies, were embedded in Matrigel and seeded into Biofloat U-bottom 96-well plates. Using a 1:1 Matrigel-cell mixture and optimized seeding volumes (5-20 {micro}L), we established adipocyte organoids that formed within 10-12 days and remained viable with stable morphology for up to 90 days or more. Matrigel was essential for organoid integrity, while alternative matrices such as gelatin and low-melting agarose failed to support proper organoid formation. Subcutaneous preadipocyte medium with 10% FBS from ZenBio was superior to "Advanced/F12K" medium for adipogenic differentiation and long-term maintenance. To model OSA-related hypoxic stress, we exposed organoids to intermittent hypoxia using a programmable hypoxia chamber. IH treatment suppressed adipogenesis, as shown by reduced lipid accumulation, downregulation of adipogenic markers (e.g., PPAR{gamma}, adiponectin, FABP4), and smaller intracellular lipid droplets. Transmission electron microscopy (TEM) revealed IH-induced structural abnormalities, including ER fragmentation, mitochondrial disruption, nuclear enlargement, and heterochromatin formation, all of which are hallmarks of cellular aging. Furthermore, IH upregulated HIF1, H2AX, and aging-associated histone methylation markers (H3K9me3, H3K79me3, H4K20me3), as well as extracellular matrix remodeling proteins such as fibronectin and LOX. Insulin signaling was also impaired, evidenced by decreased phosphorylation of PI3K and AKT. Collectively, these results establish a reliable platform for long-term human adipocyte organoid culture and demonstrate its utility in modeling IH-induced adipocyte dysfunction and aging. This system offers a physiologically relevant tool for mechanistic studies and preclinical therapeutic screening targeting hypoxia-related metabolic disorders.

cell biology↗

Clodronate liposomes untangle the role of hemocytes in Apis mellifera response to temperature variation and microbial infection

IntroductionThe cellular immune response, mediated by hemocytes, is a fundamental component of honey bee (Apis mellifera) health. However, the specific contribution of hemocyte subtypes to resilience against combined stressors remains poorly characterized. This study investigated the effect of temperature and bacterial challenge on hemocyte abundance. We employed clodronate liposomes (CLD) for the first time in honey bees as a novel tool for the selective depletion of hemocytes to investigate this gap. MethodsFive-day-old (nurses) and fifteen-day-old (foragers) honey bees were treated with CLD, control liposomes, PBS, or left untreated, then exposed at either 32{degrees}C or 22{degrees}C and challenged with the gram-negative bacterium, Escherichia coli, or the gram-positive bacterium, Staphylococcus aureus. Survival, hemolymph volume, total hemocyte counts, and differential hemocyte counts were monitored over seven days from the start of exposure. ResultsThe CLD application demonstrated significant reductions in granulocyte and prohemocyte populations, indicating the highest vulnerability. A temperature drop to 22{degrees}C buffered the negative impact on survival of CLD-induced immunosuppression. While bacterial challenges universally reduced hemocyte counts, we found an age-dependent difference where nurses maintained significantly higher baseline total hemocyte counts than foragers. Furthermore, temperature did not affect overall total hemocyte counts in 5-day-old nurse bees, but in 15-day-old foragers, it significantly modulated the hemocyte response to bacterial infection. ConclusionOur findings show that hemocyte function is non-uniform, with specific subtypes being essential for overall resilience. The results highlight a previously underappreciated role for temperature as a key modulator of immune capacity, particularly in immunocompromised bees. The age-related differences in hemocyte abundance suggest a life-history trade-off that may prompt the increased vulnerability of honey bees as they age. This work establishes CLD as a powerful tool for insect immunology and sets a precedent for using precise immune manipulation to study host-pathogen-environment interactions.

zoology↗

Acute bee paralysis virus regulation of microRNA expression dynamics in the insect host (Apis mellifera) cell line, AmE-711

BackgroundHoney bees (Apis mellifera) are essential pollinators that support global food production and economic stability. Their health and survival are threatened by diminishing floral resources, pesticide exposure, parasitic mites, and microbial and viral diseases. Among these stressors, viral infections are particularly challenging, often exacerbated by the parasitic mite, Varroa destructor, a competent vector of multiple honey bee viruses. Understanding the mechanisms underlying honey bee-virus interactions is critical for mitigating the negative impact of infections on colony health. One understudied aspect is the role of microRNAs (miRNAs) in viral pathogenesis and antiviral defense. miRNAs are short, non-coding RNAs produced by both hosts and pathogens that act as post-transcriptional regulators of gene expression and can influence host-pathogen dynamics during infection. In this study, we used a honey bee-derived cell line to test the hypothesis that viral infection perturbs honey bee- and viral-encoded miRNA expression. MethodsSmall RNA libraries from honey bee AmE-711 cells subjected to one of four treatments: media change only (uninfected), heat-killed Acute bee paralysis virus (ABPV), the viral mimic Poly(I:C), or infectious ABPV, were prepared using an Illumina Truseq kit. Sequencing data were analyzed using miRDeep2 and sRNAtoolbox to identify differentially expressed (DE) miRNAs, which were subsequently validated by RT-qPCR assay. ResultsSequencing yielded > 3.6 x 108 raw reads that were assigned to 12 small RNA libraries, from which, 481 unique miRNAs were identified. Moreover, 15 miRNAs were DE in ABPV-infected cells compared to uninfected cells: miR-2b-5p, miR-33-5p, miR-133-3p, miR-6001-3p, miR-996-3p, miR-965-3p, miR-125-5p, miR-13b-3p, miR-79-3p, miR-971-3p, miR-277-3p, miR-92c-5p, miR-6065-3p, miR-965-5p, and miR-3786-5p. We highlight some of the DE miRNAs identified in ABPV-infected cells that show regulatory effects in other systems in response to infection. ConclusionThis study identified miRNAs differentially expressed in ABPV-infected cells, suggesting roles in either antiviral defense or in promoting viral pathogenesis through suppression of host immune responses. These results provide a foundation for functional studies using honey bee cell lines to clarify the cellular mechanisms governing honey bee-virus interactions.

zoology↗

Processing of Genomic RNAs by Dicer in Bat Cells Limits SARS-CoV-2 Replication

Bats are reservoirs for numerous viruses that cause serious diseases in other animals and humans. Several mechanisms are proposed to contribute to the tolerance of bats to these pathogens. This study investigates the response of bat cells to double-stranded RNA generated by SARS-CoV-2 replication. Here, we found the involvement of Dicer in the processing of viral genomic RNAs during SARS-CoV-2 infection. Examining RNA sequencing of infected cells, small-interfering RNA (siRNA)-like fragments were found derived from viral RNAs. Depletion of Dicer showed a reduction in these RNAs and an increase in viral loads suggesting unlike other mammals, bats may use Dicer to limit viral replication. This prompted the exploration of key dsRNA sensors in bat cells. Our analysis showed significant upregulation of OAS1 and MX1 in response to dsRNA, while PKR levels remained low, suggesting alternative dsRNA-response mechanisms are present that eschew the common PKR-based system. These results further show how bats employ distinct strategies for antiviral defense that may contribute to tolerating viral infections. They suggest the involvement of Dicer in antiviral mechanisms in bats, a function not observed in other mammals. This highlights a mechanism for bat originating viruses to evolve features that in other animals could cause extreme antiviral responses such as is seen with SARS-CoV-2. GRAPHICAL SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/632425v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1755d68org.highwire.dtl.DTLVardef@475cb8org.highwire.dtl.DTLVardef@1185813org.highwire.dtl.DTLVardef@13e847a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Adipocyte Aldosterone in Obstructive Sleep Apnea

Aldosterone, a hormone synthesized by the adrenal glands, plays a pivotal role in regulating both blood pressure and electrolyte balance and may contribute to long term cardiovascular risk. Recent findings suggest that adipose tissue might serve as a source for aldosterone production and secretion. Patients suffering from obstructive sleep apnea (OSA) often exhibit elevated blood pressure levels and increased cardiovascular risk. Therefore, we sought to investigate whether increased aldosterone synthesis in fat tissue could contribute to the increase in hypertension and cardiovascular disease in OSA patients. To address this, we conducted a comparative analysis of adipocyte aldosterone level in OSA patients versus Age and BMI matched healthy individuals. Our data revealed no significant differences in aldosterone content between the two groups. Further examination involved measuring the expression of the key enzyme CYP11B2 responsible for aldosterone synthesis. We observed comparable expression levels of CYP11B2 in both the OSA and control groups. To corroborate these findings, we isolated stromal vascular fraction (SVF) from human fat tissues. The SVF cells were cultured in preadipocyte medium and subjected to normoxia and intermittent hypoxia (IH), mimicking OSA conditions as per established protocol. Subsequent analysis of aldosterone levels in cell lysates and conditioned medium revealed no significant differences. Similarly, in vitro examination of CYBP11B2 expression was not different in normoxic versus IH conditions. In conclusion, our study did not discern significant differences in adipocyte aldosterone production and secretion between patients with OSA and matched control subjects, nor in human preadipocytes under normoxic versus IH conditions.

molecular biology↗

Phytochemical and Therapeutic Evaluation of Ficus nervosa Leaves: Impact on Oxidative Stress, Analgesic, Diarrhea, and Blood Clotting in a Mice Model

BackgroundFicus nervosa (FN) leaves have been traditionally used in ethnomedicine for the treatment of various ailments, this study aimed to investigate the cold methanol extract of FN for its antioxidant, analgesic, antidiarrheal, and thrombolytic properties, along with preliminary phytochemical screening to identify key bioactive compounds and acute toxicity test to assess the safety profile of the extract. MethodsIn this study, we conducted an initial investigation to identify the major phytochemical groups present in the leaves of Ficus nervosa. Using conventional phytochemical screening methods on cold methanol extracts, we consistently identified phenols and flavonoids as the predominant bioactive compounds. Following this phytochemical characterization, we assessed the biological activities of the extracts across multiple models. The antioxidant capacity and free radical-scavenging ability of the leaves extracts were evaluated using the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Analgesic efficacy was assessed through acetic acid-induced writhing and electrically induced heat nociception tests. Antidiarrheal effects were examined using castor oil-induced diarrhea and an enteropooling assay in mice. To determine thrombolytic activity, we conducted a human blood clot lysis assay, while preclinical toxicity testing was conducted to evaluate the safety profile of the extracts. Statistical analysis was performed using one-way ANOVA with Duncans Multiple Range Test (DMRT) to validate the significance of the results. ResultsThe study of Antioxidant Potential, the ESFs (ethyl soluble fraction) antioxidant capacity, measured at 409.91 {+/-} 0.59 mg of ascorbic acid equivalent (AAE)/g in the phosphomolybdenum assay, signifies a strong free radical scavenging activity. This level of antioxidant action, alongside a potent free radical neutralizing concentration (IC of 9.99 {+/-} 0.41 g/mL), suggests that the ESF could effectively counteract oxidative stress. Analgesic activity at 500 mg/kg, FN extract produced a 68.05% suppression of writhing in mice, which was statistically significant (p<0.001) compared to standard morphines (74.05%) inhibition. The extract also significantly prolonged reaction latency to thermal-induced pain in hotplate model. The antidiarrheal activity showed 52.09% and 59.0% suppression of defecation, compared to standard loperamides 73.1%, with statistically significant p-values (p<0.04 and p<0.01). These effects, combined with the notable decrease in intestinal fluid accumulation and defecation frequency in enteropooling assays, indicate that FN extract might interact with intestinal motility or fluid secretion pathways, providing antidiarrheal benefits. Additionally, the chloroform-soluble fraction of FN exhibited moderate thrombolytic activity, achieving 39.79% clot lysis compared to streptokinase (69.52%). Although this thrombolytic potential is moderate, it indicates that the fraction contains compounds with some fibrinolytic properties, which could be useful as adjunctive agents in preventing thrombosis. ConclusionOverall, the cold methanol extract of FN leaves demonstrates the therapeutic potential in preclinical settings. Future research is warranted to isolate the specific bioactive compounds and explain their mechanisms of action to further support the development of new treatments and contributing to modern medicinal practices based on this plant leaves.

plant biology↗

Methanol Crude Extract of Litsea Monopetala Leaves combats oxidative stress, clot formation, inflammation and stool frequency in Animal Model

Litsea monopetala (LM) leaves are used in traditional medicine systems in the South Asian region for treating ailments such as digestive issues, respiratory problems, and skin disorders. In this study, we first investigated the possible antioxidant, thrombolytic characteristics, analgesic, and antidiarrheal activity of the methanolic crude extract of LM leaves. To assess the antioxidant, thrombolytic, analgesic, and antidiarrheal activity of the crude extract we used DPPH free radical scavenging, clot lysis, acetic acid, and castor oil-induced animal model, respectively. Before applying the extract to the animal model, phytochemical screening was performed to estimate the bioactive compounds (e.g.- phenol, flavonoid, alkaloids, tannins, and others) present. The extract exhibited DPPH free radical scavenging features with an IC50 of 8.99 {micro}g/mL compared to ascorbic acid, an IC50 of 13.38 {micro}g/ml. At 500 mg/kg dose, the extract produced a significant decrease (67.05%) in the frequency of writhings while diclofenac sodium decreased by 74.15%. The extract also significantly (P<0.01) decreased the frequency of diarrhea when compared to the standard drug of loperamide (3mg/kg). Finally, we assessed the clot lysis function as representative of the thrombolytic activity of extracts in comparison to streptokinase and demonstrated clot lysis action (74.15% decrease). From a therapeutic implication, we suggest that crude extract of LMs leaves may contribute to the alternative or additive strategy to modulate conditions such as oxidative stress, clot formation, inflammation and diarrhea.

pharmacology and toxicology↗

Phytochemical screening, analgesic, anti-pyretic and antibacterial potentials of Litsea glutinosa (L) leaves extracts in vivo and in vitro technique

BackgroundLitsea glutinosa leaves (LG) have been used as traditional medicine to treat diseases since ancient times. This study aimed to investigate the cold methanol extract of LG for its analgesic, antipyretic and antibacterial activities, along with preliminary phytochemical screening and acute toxicity test. MethodsIn this study, we first investigated the major phytochemicals group present applying modern chromatographic technique HPLC in addition to conventional methods for Phyto screening in cold methanol extracts LG leaves. Both methods simultaneously demonstrated major bioactive compounds found in the extract were phenol, flavonoid. The purposive efficacy and toxicity were then assessed through preclinical testing ResultsIn hot plate method, the highest pain inhibitory activity was found at a dose of 500 mg/kg of crude extract (3.37{+/-} 0.31sec) which differed significantly (P <0.01 and P <0.001) with that of the standard drug morphine (6.47{+/-} 0.23 sec). The extract significantly prolonged reaction latency to thermal-induced pain in hotplate model. Analgesic activity at 500 mg/kg, LG extract produced a 70% suppression of writhing in mice, which was statistically significant (p<0.001) compared to standard morphines (77.5%) inhibition. In antipyretic activity assay, the crude extract showed notable reduction in body temperature (36.17 {+/-} 0.32{degrees}C) at dose of 300 mg/kg-body weight, when the standard (at dose 100 mg/kg-body weight) exerted (36.32 {+/-} 0.67{degrees}C) after 3 h of administration. In antibacterial studies, results showed that inhibition of bacterial growth at 400 g dose of each extract clearly inhibited growth of bacteria from 11 to 22 mm. The extractives carbon tetrachloride fraction, chloroform soluble fraction, ethyl acetate fraction demonstrated notably greater inhibitory zone widths (p < 0.05) against tested strains. ConclusionOverall, the cold methanol extract of LG leaves demonstrates the therapeutic potential in preclinical settings.

pharmacology and toxicology↗

Exploring Pharmacogenetic Factors Influencing Hydroxyurea Response in Tanzanian Sickle Cell Disease Patients: A Genomic Medicine Approach.

Sickle cell disease (SCD) continues to pose a significant public health challenge, particularly in sub-Saharan Africa. Despite its discovery over a century ago, the progress in developing and accessing effective interventions has been notably restricted. Currently, hydroxyurea stands as the primary drug in widespread use, and has been associated with elevated levels of fetal hemoglobin (HbF) and enhanced clinical outcomes. Notably, a substantial proportion, up to 30%, of patients do not exhibit a positive response to hydroxyurea treatment. There is compelling evidence suggesting that genetic factors play a crucial role in influencing the effectiveness of hydroxyurea. In this study, we present findings on the investigation of genetic variants influencing hydroxyurea response in 13 genetic loci associated with HbF synthesis and hydroxyurea drug metabolism focusing on MYB, HBB, HBG1, HBG2, BCL11A, KLF10, HAO2, NOS1, ARG2, SAR1A, CYP2C9, CYP2E1. We report remarkable genetic associations with CYP2C9, CYP2E1, KLF10, BCL11A, ARG2, HBG1, SAR1A, MYB, and NOS1 loci with hydroxyurea response. We also highlight associated pathways enrichment and gene-gene interactions analysis in the context of hydroxyurea treatment response.

genomics↗

Analysis of Amblyomma americanum microRNAs in response to Ehrlichia chaffeensis infection and their potential role in vectorial capacity

BackgroundMicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the hosts miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod hosts miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum. MethodsSmall RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. ResultsThe sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. ConclusionsThe current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.

systems biology↗

Bisphenol A and its Analogues Alter Appetite Control in Zebrafish

The regulation of appetite is of growing interest due to the significant rise in global obesity rates. Hunger and satiety are controlled by two hormones with functional activity in the brain; leptin, which is produced in adipocytes and suppresses food intake, and ghrelin, which is produced and released mainly by the stomach and functions as an appetite-stimulatory signal. In this study, zebrafish-based in vivo assays were used to examine whether BPA and five of its analogues, BPAF, BPE, BPC, BPC-CL, and BPS affect appetite regulation. The effect of bisphenol exposure on eating behavior was first examined. Four to six days old zebrafish larvae were exposed to a concentration range of the bisphenols and 17{beta}-estradiol, followed by being fed a stained egg yolk powder at day six. After an hour of feeding, the feed in the gut was imaged by microscopy. Quantitative PCR was used to analyze the gene expression of leptin and ghrelin, as well as eleven other genes involved in appetite control. Exposures to BPA, BPAF, BPE, BPC, BPC-Cl and BPS, resulted in increased amounts of feed in the gut of the larvae in a concentration dependent manner. The qPCR results suggested that leptin mRNA expression was downregulated with the increasing concentrations of BPA, BPAF and BPC-Cl, whereas ghrelin mRNA expression was upregulated. The expression of several additional anorexigenic genes were downregulated by BPAF and BPC-Cl exposure, whereas orexigenic genes were upregulated. In conclusion, bisphenol exposures resulted in an increased eating behavior in zebrafish larvae, which correlated to increased mRNA expression of appetite-stimulatory genes and decreased expression of satiety-inducing genes. In addition, the results suggest that zebrafish larvae can be used to efficiently assess obesogenic capacity of environmental pollutants.

pharmacology and toxicology↗

Identification of Alpha-Gal conjugated Lipids in Saliva of Lone-Star Tick (Amblyomma americanum)

Alpha-Gal Syndrome (AGS) is a delayed allergic reaction triggered by IgE antibodies targeting galactose--1,3-galactose (-gal), prevalent in red meat. Its global significance has increased, with over 450,000 estimated cases in the United States alone. AGS is linked to tick bites, causing sensitization and elevated -gal specific IgE levels. However, the precise mechanisms and tick intrinsic factors contributing to AGS development post-tick bites remain unclear. This study aims to characterize the alphagal conjugated lipid antigens in Amblyomma americanum (Am. americanum) salivary glands and saliva. Nanospray ionization mass spectrometry (NSI-MS) analysis revealed the identification of -gal bound lipid antigens in Am. americanum saliva. Additionally, activation of basophils by isolated alpha-gal bound lipids and proteins provides evidence of their antigenic capabilities.

immunology↗

Tick bite-induced Alpha-Gal Syndrome and Immunologic Responses in an Alpha-Gal Deficient Murine Model

IntroductionAlpha-Gal Syndrome (AGS) is a delayed allergic reaction due to specific IgE antibodies targeting galactose--1,3-galactose (-gal), a carbohydrate found in red meat. This condition has gained significant attention globally due to its increasing prevalence, with more than 450,000 cases estimated in the United States alone. Previous research has established a connection between AGS and tick bites, which sensitize individuals to -gal antigens and elevate the levels of -gal specific IgE. However, the precise mechanism by which tick bites influence the hosts immune system and contribute to the development of AGS remains poorly understood. This study investigates various factors related to ticks and the host associated with the development of AGS following a tick bite, using mice with a targeted disruption of alpha-1,3-galactosyltransferase (AGKO) as a model organism. MethodsLone-star tick (Amblyomma americanum) and gulf-coast tick (Amblyomma maculatum) nymphs were used to sensitize AGKO mice, followed by pork meat challenge. Tick bite site biopsies from sensitized and non-sensitized mice were subjected to mRNA gene expression analysis to assess the host immune response. Antibody responses in sensitized mice were also determined. ResultsOur results showed a significant increase in the titer of total IgE, IgG1, and -gal IgG1 antibodies in the lone-star tick-sensitized AGKO mice compared to the gulf-coast tick-sensitized mice. Pork challenge in Am. americanum -sensitized mice led to a decline in body temperature after the meat challenge. Gene expression analysis revealed that Am. americanum bites direct mouse immunity toward Th2 and facilitate host sensitization to the -gal antigen, while Am. maculatum did not. ConclusionThis study supports the hypothesis that specific tick species may increase the risk of developing -gal-specific IgE and hypersensitivity reactions or AGS, thereby providing opportunities for future research on the mechanistic role of tick and host-related factors in AGS development.

immunology↗

Ticks without borders: Microbial communities of immature Neotropical tick species parasitizing migratory landbirds along northern Gulf of Mexico

The long-distance, seasonal migrations of birds make them an effective ecological bridge for the movement of ticks. The introduction of exotic tick species to new geographical regions can lead to the emergence of novel tick-borne pathogens or the re-emergence of previously eradicated ones. This study assessed the prevalence of exotic tick species parasitizing resident, short-distance, and long-distance songbirds during spring and autumn at stopover sites in the northern Gulf of Mexico using the mitochondrial 12S rDNA gene. Birds were captured for tick collection from six different sites from late August to early November in both 2018 and 2019. The highest number of ticks were collected in the 2019 season. Most ticks were collected off the Yellow-breasted Chat (Icteria virens) and Common Yellowthroat (Geothlypis trichas), and 54% of the total ticks collected were from Grand Chenier, LA. A high throughput 16S ribosomal RNA sequencing approach was followed to characterize the microbial communities and identify pathogenic microbes in all tick samples. Tick microbial communities, diversity, and community structure were determined using quantitative insight into microbial ecology (QIIME). The sparse correlations for compositional data (SparCC) approach was then used to construct microbial network maps and infer microbial correlations. A total of 421 individual ticks in the genera Amblyomma, Haemaphysalis, and Ixodes were recorded from 28 songbird species, of which Amblyomma and Amblyomma longirostre was the most abundant tick genus and species, respectively. Microbial profiles showed that Proteobacteria was the most abundant phylum. The most abundant bacteria include the pathogenic Rickettsia and endosymbiont Francisella, Candidatus Midichloria, and Spiroplasma. BLAST analysis and phylogenetic reconstruction of the Rickettsia sequences revealed the highest similarities to pathogenic spotted and non-spotted fever groups, including R. buchneri, R. conorii, R. prowazekii, R. bellii, R. australis, R. parkeri, R. monacensis, and R. monteiroi. Permutation multivariate analysis of variance revealed that the relative abundance of Francisella and Rickettsia drives microbial patterns across the tick genera. We also observed a higher percentage of positive correlations in microbe-microbe interactions among members of the microbial communities. Network analysis suggested a negative correlation between a) Francisella and Rickettsia and, b) Francisella and Cutibacterium. Lastly, mapping the distributions of bird species parasitized during spring migrations highlighted geographic hotspots where migratory songbirds could disperse ticks and their pathogens at stopover sites or upon arrival to their breeding grounds, the latter showing means dispersal distances from 421-5003 kilometers. These findings strongly highlight the potential role of migratory birds in the epidemiology of tick-borne pathogens. O_FIG O_LINKSMALLFIG WIDTH=99 HEIGHT=200 SRC="FIGDIR/small/563347v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@5b100borg.highwire.dtl.DTLVardef@7a59f3org.highwire.dtl.DTLVardef@a78ff3org.highwire.dtl.DTLVardef@dbce25_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract:C_FLOATNO Overview of the experimental approach for bird collection and characterization of neotropical ticks microbiome. A) Birds migrating through the Mississippi flyway into the United States were trapped and captured for identification and tick collection and B) immature developmental stages of feeding ticks infesting the birds were carefully removed and stored for morphological and molecular identification of tick species. C) Tick DNA was used for the PCR amplification of arthropods mitochondrial 12S rDNA and amplicons were sequenced and compared to deposited datasets in NCBI database. D) Microbial DNA was isolated using V3-V4 16S primers and unique barcodes attached to amplified 16S sequences prior to Illumina MiSeq sequences. E) Distribution and contribution of birds to tick infestation, and distribution and community profiles of sequenced microbial communities. C_FIG

zoology↗

Tick Innate Immune Responses to Hematophagy and Ehrlichia Infection at Single-Cell Resolution

Ticks rely on robust cellular and humoral immune responses to control microbial infection. However, several aspects of the ticks innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses toward microbes. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminate their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission. This study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. Our data exhibit the identification of twelve and nineteen distinct hemocyte populations, respectively, from uninfected and Ehrlichia-infected ticks. Our results show a significant increase of clusters representing granulocyte and oenocytoids populations with Ehrlichia infection. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy) and tick-microbial interactions. SignificanceThe immune response of ticks plays a crucial role in their ability to survive and transmit pathogens. Hemocytes, the primary immune cells in arthropods, are key mediators of ticks immune defense and provide deep insights into immune responses to microbial infection. However, tick hemocytes cellular complexity and heterogeneity have posed challenges for comprehensive characterization. This study employed single-cell RNA sequencing (scRNA-seq) to profile tick hemocytes and elucidate their transcriptional diversity. We identified distinct subpopulations of hemocytes and characterized their unique gene expression profiles. We observed significant variation in immune-related gene expression between hemocyte subpopulations during hematophagy and in response to Ehrlichia infection, suggesting specialized functional roles. Our analysis revealed potential marker genes associated with specific hemocyte functions. Our study provides the first comprehensive single-cell atlas of tick hemocytes, shedding light on these cells molecular diversity and immune functions. The findings enhance our understanding of tick-host interactions and offer a step toward understanding arthropod immunobiology.

immunology↗

co-opts phagocytic hemocytes for systemic dissemination in the Lone Star tick, .

Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissues-associated hemocytes. We demonstrate E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

immunology↗