High-throughput mRNA sequencing of human placenta shows sex differences across gestation.

INTRODUCTION: Fetal sex affects fetal and maternal health outcomes in pregnancy, but this connection remains poorly understood. As the placenta is the route of fetomaternal communication and derives from the fetal genome, placental gene expression sex differences may explain these outcomes. OBJECTIVES: We utilized next generation sequencing to study the normal human placenta in both sexes in first and third trimester to generate a normative transcriptome based on sex and gestation. STUDY DESIGN: We analyzed 124 first trimester (T1, 59 female and 65 male) and 43 third trimester (T3, 18 female and 25 male) samples for sex differences within each trimester and sex-specific gestational differences. RESULTS: Placenta shows more significant sexual dimorphism in T1, with 94 T1 and 26 T3 differentially expressed genes (DEGs). The sex chromosomes contributed 60.6% of DEGs in T1 and 80.8% of DEGs in T3, excluding X/Y pseudoautosomal regions. There were 6 DEGs from the pseudoautosomal regions, only significant in T1 and all upregulated in males. The distribution of DEGs on the X chromosome suggests genes on Xp (the short arm) may be particularly important in placental sex differences. Dosage compensation analysis of X/Y homolog genes shows expression is primarily contributed by the X chromosome. In sex-specific analyses of first versus third trimester, there were 2815 DEGs common to both sexes upregulated in T1, and 3263 common DEGs upregulated in T3. There were 7 female-exclusive DEGs upregulated in T1, 15 female-exclusive DEGs upregulated in T3, 10 male-exclusive DEGs upregulated in T1, and 20 male-exclusive DEGs upregulated in T3. DISCUSSION: This is the largest cohort of placentas across gestation from healthy pregnancies defining the normative sex dimorphic gene expression and sex common, sex specific and sex exclusive gene expression across gestation. The first trimester has the most sexually dimorphic transcripts, and the majority were upregulated in females compared to males in both trimesters. The short arm of the X chromosome and the pseudoautosomal region is particularly critical in defining sex differences in the first trimester placenta. As pregnancy is a dynamic state, sex specific DEGs across gestation may contribute to sex dimorphic changes in overall outcomes.

Cellular Modeling of CLN6 with IPSC-derived Neurons and Glia.

Neuronal ceroid lipofuscinosis (NCL), type 6 (CLN6) is a neurodegenerative disorder associated with progressive neurodegeneration leading to dementia, seizures, and retinopathy. CLN6 encodes a resident-ER protein involved in trafficking lysosomal proteins to the Golgi. CLN6p deficiency results in lysosomal dysfunction and deposition of storage material comprised of Nile Red + lipids/proteolipids that include subunit C of the mitochondrial ATP synthase (SUBC). White matter involvement has been recently noted in several CLN6 animal models and several CLN6 subjects had neuroimaging was consistent with leukodystrophy. CLN6 patient-derived induced pluripotent stem cells (IPSCs) were generated from several of these subjects. IPSCs were differentiated into oligodendroglia or neurons using well-established small-molecule protocols. A doxycycline-inducible transgenic system expressing neurogenin-2 (the I3N-system) was also used to generate clonal IPSC-lines (I3N-IPSCs) that could be rapidly differentiated into neurons (I3N-neurons). All CLN6 IPSC-derived neural cell lines developed significant storage material, CLN6-I3N-neuron lines revealed significant Nile Red + and SUBC + storage within three and seven days of neuronal induction, respectively. CLN6-I3N-neurons had decreased tripeptidyl peptidase-1 activity, increased Golgi area, along with increased LAMP1 + in cell bodies and neurites. SUBC + signal co-localized with LAMP1 + signal. Bulk-transcriptomic evaluation of control- and CLN6-I3N-neurons identified >1300 differentially-expressed genes (DEGs) with Gene Ontogeny (GO) Enrichment and Canonical Pathway Analyses having significant changes in lysosomal, axonal, synaptic, and neuronal-apoptotic gene pathways. These findings indicate that CLN6-IPSCs and CLN6-I3N-IPSCs are appropriate cellular models for this disorder. These I3N-neuron models may be particularly valuable for developing therapeutic interventions with high-throughput drug screening assays and/or gene therapy.

High-throughput mRNA-seq atlas of human placenta shows vast transcriptome remodeling from first to third trimester†.

The placenta, composed of chorionic villi, changes dramatically across gestation. Understanding differences in ongoing pregnancies are essential to identify the role of chorionic villi at specific times in gestation and develop biomarkers and prognostic indicators of maternal-fetal health. The normative mRNA profile is established using next-generation sequencing of 124 first trimester and 43 third trimester human placentas from ongoing healthy pregnancies. Stably expressed genes (SEGs) not different between trimesters and with low variability are identified. Differential expression analysis of first versus third trimester adjusted for fetal sex is performed, followed by a subanalysis with 23 matched pregnancies to control for subject variability using the same genetic and environmental background. Placenta expresses 14,979 polyadenylated genes above sequencing noise (transcripts per million > 0.66), with 10.7% SEGs across gestation. Differentially expressed genes (DEGs) account for 86.7% of genes in the full cohort [false discovery rate (FDR) < 0.05]. Fold changes highly correlate between the full cohort and subanalysis (Pearson = 0.98). At stricter thresholds (FDR < 0.001, fold change > 1.5), there remains 50.1% DEGs (3353 upregulated in first and 4155 upregulated in third trimester). This is the largest mRNA atlas of healthy human placenta across gestation, controlling for genetic and environmental factors, demonstrating substantial changes from first to third trimester in chorionic villi. Specific differences and SEGs may be used to understand the specific role of the chorionic villi throughout gestation and develop first trimester biomarkers of placental health that transpire across gestation, which can be used for future development of biomarkers for maternal-fetal health.

MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

High-throughput mRNA-seq atlas of human placenta shows vast transcriptome remodeling from first to third trimester.

Background: The placenta, composed of chorionic villi, changes dramatically across gestation. Understanding differences in ongoing pregnancies are essential to identify the role of chorionic villi at specific times in gestation and develop biomarkers and prognostic indicators of maternal- fetal health. Methods: The normative mRNA profile is established using next-generation sequencing of 124 first trimester and 43 third trimester human placentas from ongoing healthy pregnancies. Stably expressed genes not different between trimesters and with low variability are identified. Differential expression analysis of first versus third trimester adjusted for fetal sex is performed, followed by a subanalysis with 23 matched pregnancies to control for subject variability using the same genetic and environmental background. Results: Placenta expresses 14,979 mRNAs above sequencing noise (TPM>0.66), with 1,545 stably expressed genes across gestation. Differentially expressed genes account for 86.7% of genes in the full cohort (FDR<0.05). Fold changes highly correlate between the full cohort and subanalysis (Pearson = 0.98). At stricter thresholds (FDR<0.001, fold change>1.5), there are 6,941 differentially expressed protein coding genes (3,206 upregulated in first and 3,735 upregulated in third trimester). Conclusion: This is the largest mRNA atlas of healthy human placenta across gestation, controlling for genetic and environmental factors, demonstrating substantial changes from first to third trimester in chorionic villi. Specific differences and stably expressed genes may be used to understand the specific role of the chorionic villi throughout gestation and develop first trimester biomarkers of placental health that transpire across gestation, which can be used for future development of biomarkers in maternal-fetal disease.

Stacking the odds: Multiple sites for HSV-1 latency.

A hallmark of herpes simplex virus (HSV) infection is the establishment of latent virus in peripheral sensory ganglia of the latently infected host. We and others originally reported that the latency-associated transcript (LAT) is the only abundantly expressed viral gene in neurons within trigeminal ganglia (TG) of a latently infected host. Here, we investigated the possible contribution of various cells [i.e., B cells, dendritic cells (DCs), fibroblasts, glial cells, innate lymphoid cells (ILCs), macrophages, microglia, monocytes, natural killer cells, neurons, neutrophils, and T cells] isolated from TG of latently infected mice. Our results demonstrated that all of these cell types contain LAT, with DCs, neurons, and ILCs having the most LAT+ cells. These results suggest that HSV-1 can establish a quiescent/latent infection in a subset of nonneuronal cells, which enhances the chances that the virus will survive in its host.

Spatially resolved transcriptomics reveals genes associated with the vulnerability of middle temporal gyrus in Alzheimer's disease.

Human middle temporal gyrus (MTG) is a vulnerable brain region in early Alzheimer's disease (AD), but little is known about the molecular mechanisms underlying this regional vulnerability. Here we utilize the 10 × Visium platform to define the spatial transcriptomic profile in both AD and control (CT) MTG. We identify unique marker genes for cortical layers and the white matter, and layer-specific differentially expressed genes (DEGs) in human AD compared to CT. Deconvolution of the Visium spots showcases the significant difference in particular cell types among cortical layers and the white matter. Gene co-expression analyses reveal eight gene modules, four of which have significantly altered co-expression patterns in the presence of AD pathology. The co-expression patterns of hub genes and enriched pathways in the presence of AD pathology indicate an important role of cell-cell-communications among microglia, oligodendrocytes, astrocytes, and neurons, which may contribute to the cellular and regional vulnerability in early AD. Using single-molecule fluorescent in situ hybridization, we validated the cell-type-specific expression of three novel DEGs (e.g., KIF5A, PAQR6, and SLC1A3) and eleven previously reported DEGs associated with AD pathology (i.e., amyloid beta plaques and intraneuronal neurofibrillary tangles or neuropil threads) at the single cell level. Our results may contribute to the understanding of the complex architecture and neuronal and glial response to AD pathology of this vulnerable brain region.

Sex differences in microRNA expression in first and third trimester human placenta†.

Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be due to sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing identified miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n = 113) and parturition (n = 47). Sequencing analysis identified 986 expressed mature miRNAs from female and male placentae at first and third trimester (baseMean>10). Of these, 11 sexually dimorphic (FDR < 0.05) miRNAs were identified in the first and 4 in the third trimester, all upregulated in females, including miR-361-5p, significant in both trimesters. Sex-specific analyses across gestation identified 677 differentially expressed (DE) miRNAs at FDR < 0.05 and baseMean>10, with 508 DE miRNAs in common between female-specific and male-specific analysis (269 upregulated in first trimester, 239 upregulated in third trimester). Of those, miR-4483 had the highest fold changes across gestation. There were 62.5% more female exclusive differences with fold change>2 across gestation than male exclusive (52 miRNAs vs 32 miRNAs), indicating miRNA expression across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex-independent and sex-specific placenta miRNA atlas across gestation, which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences.

NLRP3 Inflammasome Mediates Immune-Stromal Interactions in Vasculitis.

[Figure: see text].

High-throughput miRNA sequencing of the human placenta: expression throughout gestation.

Aim: To understand miRNA changes across gestation in healthy human placentae. This is essential before miRNAs can be used as biomarkers or prognostic indicators during pregnancy. Materials & methods: Using next-generation sequencing, we characterize the normative human placenta miRNome in first (n = 113) and third trimester (n = 47). Results & conclusion: There are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (p ≥ 0.05, fold change ≤2) and 180 significantly different (false discovery rate <0.05, fold change >2). Of placenta-specific miRNA clusters, chromosome 14 miRNA cluster decreases across gestation and chromosome 19 miRNA cluster is overall highly expressed. Chromosome 13 clusters are upregulated in first trimester. This work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae.

Lay abstract The human body produces miRNAs which affect the expression of genes and proteins. This study uses next-generation sequencing to identify the miRNA profile of first and third trimester human placentae using a large cohort (n = 113 first trimester; n = 47 third trimester). All pregnancies resulted in healthy babies. We identify miRNAs with significantly different expression between first and third trimester, as well as stably expressed miRNAs. This work provides a baseline for future studies which may use miRNAs to monitor maternal­fetal health throughout pregnancy.

HLA class I-associated expansion of TRBV11-2 T cells in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vß chains results in Vß skewing, whereby T cells with specific Vß chains and diverse antigen specificity are overrepresented in the T cell receptor (TCR) repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCRß variable gene 11-2 (TRBV11-2), with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and complementarity-determining region 3 (CDR3) length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion shared HLA class I alleles A02, B35, and C04, indicating what we believe is a novel mechanism for CDR3-independent T cell expansion. In silico modeling indicated that polyacidic residues in the Vß chain encoded by TRBV11-2 (Vß21.3) strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

Identification of a unique TCR repertoire, consistent with a superantigen selection process in Children with Multi-system Inflammatory Syndrome.

Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares many clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. The superantigen specificity for binding different Vß-chains results in Vß-skewing, whereby T cells with specific Vß-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Βeta Variable gene (TRBV)11-2. Furthermore, TRBV11-2 skewing was remarkably correlated with MIS-C severity and serum cytokine levels. Further analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanding TRBV11-2 clones revealed extensive junctional diversity, indicating a superantigen-mediated selection process for TRBV expansion. In silico modelling indicates that polyacidic residues in TCR Vß11-2 engage in strong interactions with the superantigen-like motif of SARS-CoV-2 spike glycoprotein. Overall, our data indicate that the immune response in MIS-C is consistent with superantigenic activation.

Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements.

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.

Tianshengyuan-1 (TSY-1) regulates cellular Telomerase activity by methylation of TERT promoter.

Telomere and Telomerase have recently been explored as anti-aging and anti-cancer drug targets with only limited success. Previously we showed that the Chinese herbal medicine Tianshengyuan-1 (TSY-1), an agent used to treat bone marrow deficiency, has a profound effect on stimulating Telomerase activity in hematopoietic cells. Here, the mechanism of TSY-1 on cellular Telomerase activity was further investigated using HL60, a promyelocytic leukemia cell line, normal peripheral blood mononuclear cells, and CD34+ hematopoietic stem cells derived from umbilical cord blood. TSY-1 increases Telomerase activity in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells with innately low Telomerase activity but decreases Telomerase activity in HL60 cells with high intrinsic Telomerase activity, both in a dose-response manner. Gene profiling analysis identified Telomerase reverse transcriptase (TERT) as the potential target gene associated with the TSY-1 effect, which was verified by both RT-PCR and western blot analysis. The ß-galactosidase reporter staining assay showed that the effect of TSY-1 on Telomerase activity correlates with cell senescence. TSY-1 induced hypomethylation within TERT core promoter in HL60 cells but induced hypermethylation within TERT core promoter in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells. TSY-1 appears to affect the Telomerase activity in different cell lines differently and the effect is associated with TERT expression, possibly via the methylation of TERT promoter.

Maternal HIV infection influences the microbiome of HIV-uninfected infants.

More than 1 million HIV-exposed, uninfected infants are born annually to HIV-positive mothers worldwide. This growing population of infants experiences twice the mortality of HIV-unexposed infants. We found that although there were very few differences seen in the microbiomes of mothers with and without HIV infection, maternal HIV infection was associated with changes in the microbiome of HIV-exposed, uninfected infants. Furthermore, we observed that human breast milk oligosaccharides were associated with bacterial species in the infant microbiome. The disruption of the infant's microbiome associated with maternal HIV infection may contribute to the increased morbidity and mortality of HIV-exposed, uninfected infants.

Screening cell mechanotype by parallel microfiltration.

Cell mechanical phenotype or 'mechanotype' is emerging as a valuable label-free biomarker. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and cancer progression. Here we describe a simple and scalable technique to measure cell mechanotype: this parallel microfiltration assay enables multiple samples to be simultaneously measured by driving cell suspensions through porous membranes. To validate the method, we compare the filtration of untransformed and HRas(V12)-transformed murine ovary cells and find significantly increased deformability of the transformed cells. Inducing epithelial-to-mesenchymal transition (EMT) in human ovarian cancer cells by overexpression of key transcription factors (Snail, Slug, Zeb1) or by acquiring drug resistance produces a similar increase in deformability. Mechanistically, we show that EMT-mediated changes in epithelial (loss of E-Cadherin) and mesenchymal markers (vimentin induction) correlate with altered mechanotype. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application.

The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells.

Changes in cell stiffness (Young's modulus, E), as measured via Atomic Force Microscopy (AFM), is a newly recognized characteristic of cancer cells and may play a role in platinum drug resistance of ovarian cancers. We previously showed that, compared to their syngeneic cisplatin-sensitive counterpart, cisplatin-resistant ovarian cancer cells are stiffer, and this cell stiffness was dependent on actin polymerization and presence of stress fibers. Here, we measured the correlation between Young's modulus (via AFM measurements on live, non-apoptotic cells in physiological buffer) and cisplatin-sensitivity (IC50 as determined via the XTT cell viability assay) in a panel of nine ovarian cancer cell lines representing a range of cisplatin sensitivities. We found that cisplatin-sensitive cells had a lower Young's modulus, compared to cisplatin-resistant cells and resistant cells had a cytoskeleton composed of long actin stress fibers. As Rho GTPase mediates stress fiber formation, we examined the role of Rho GTPase in cell stiffness and platinum resistance. Rho inhibition decreased cell stiffness in cisplatin-resistant CP70 cells and increased their cisplatin sensitivity while Rho activation increased cell stiffness in cisplatin-sensitive A2780 cells and decreased their cisplatin sensitivity. Based on changes in cell stiffness, IC50 and cellular actin stress fiber organization in CP70 and A2780 cells, our findings reveal a direct role of Rho mediated actin remodeling mechanism in cisplatin resistance of ovarian cancer cells. These findings suggest the potential applicability of cell mechanical phenotyping as a model for determining sensitivity of ovarian cancer cells that could have major implications in ovarian cancer diagnosis and personalized medicine.

Effect of PI3K/Akt pathway inhibition-mediated G1 arrest on chemosensitization in ovarian cancer cells.

BACKGROUND: Pharmacological inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway prevents G(1) cell cycle progression into S, resulting in G(1) accumulation. The hypothesis that this arrest might negatively impact on chemotherapeutic agents primarily effective in S, G(2) or M-phase was investigated. MATERIALS AND METHODS: Inhibition of PI3K/Akt pathway signaling via LY294002 and Akti-1/2 was demonstrated by immunoblotting. Cell cycle progression was determined by flow cytometric analysis. Cell proliferation was assayed using the XTT cell viability assay. The Chou and Talalay median effect principal was used to evaluate drug interaction. RESULTS: In SKOV3 and IGROV1 human ovarian cancer cells, LY294002 and Akti-1/2 increased the percentage of cells in G(1) and reversed the cell cycle effects of cisplatin, paclitaxel, gemcitabine and topotecan. Pathway blockade synergistically enhanced the cytotoxicity of cisplatin and paclitaxel, but antagonized gemcitabine and topotecan effects. CONCLUSION: Pharmacological PI3K/Akt inhibition antagonizes the efficacy of chemotherapeutic agents primarily effective in the S or G(2)-phase of the cell cycle.

Correlative nanomechanical profiling with super-resolution F-actin imaging reveals novel insights into mechanisms of cisplatin resistance in ovarian cancer cells.

The exact molecular mechanisms of ovarian cancer platinum resistance are not well understood, and biomarkers to reliably predict ovarian cancer resistance to platinum and other chemotherapeutic agents are lacking. Biomechanics of cisplatin-treated ovarian cancer cells were measured quantitatively at nanoscale level using atomic force microscopy. We demonstrate that cisplatin modulates the cellular nanomechanics of ovarian cancer cells; sensitive cells show dose-dependent increase in cell stiffness, which is effected by disrupting the F-actin polymerization. In contrast, resistant cells show no significant changes in cell stiffness upon cisplatin treatment. Further, stimulated emission depletion, an emerging super-resolution microscopy, shows that at the molecular level, F-actin is indeed remodeled considerably in cisplatin-sensitive and cisplatin-resistant cells. These findings reveal a direct role of the actin remodeling mechanism in cisplatin resistance of ovarian cancer cells, suggesting potential future applications of nanomechanical profiling as a marker for cancer drug sensitivity. FROM THE CLINICAL EDITOR: In this paper, nanomechanical profiling and an emerging super-resolution microscopy method was utilized to decipher the mechanisms of cisplatin resistance in ovarian cancer cells, paving the way to future studies of this and similar other problems with drug resistance in cancer biology.