Incorporating temporal information during feature engineering bolsters emulation of spatio-temporal emergence.

MOTIVATION: Emergent biological dynamics derive from the evolution of lower-level spatial and temporal processes. A long-standing challenge for scientists and engineers is identifying simple low-level rules that give rise to complex higher-level dynamics. High-resolution biological data acquisition enables this identification and has evolved at a rapid pace for both experimental and computational approaches. Simultaneously harnessing the resolution and managing the expense of emerging technologies-e.g. live cell imaging, scRNAseq, agent-based models-requires a deeper understanding of how spatial and temporal axes impact biological systems. Effective emulation is a promising solution to manage the expense of increasingly complex high-resolution computational models. In this research, we focus on the emulation of a tumor microenvironment agent-based model to examine the relationship between spatial and temporal environment features, and emergent tumor properties. RESULTS: Despite significant feature engineering, we find limited predictive capacity of tumor properties from initial system representations. However, incorporating temporal information derived from intermediate simulation states dramatically improves the predictive performance of machine learning models. We train a deep-learning emulator on intermediate simulation states and observe promising enhancements over emulators trained solely on initial conditions. Our results underscore the importance of incorporating temporal information in the evaluation of spatio-temporal emergent behavior. Nevertheless, the emulators exhibit inconsistent performance, suggesting that the underlying model characterizes unique cell populations dynamics that are not easily replaced. AVAILABILITY AND IMPLEMENTATION: All source codes for the agent-based model, emulation, and analyses are publicly available at the corresponding DOIs: 10.5281/zenodo.10622155, 10.5281/zenodo.10611675, 10.5281/zenodo.10621244, respectively.

Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction.

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.

Urogenital development in Pallister-Hall syndrome is disrupted in a cell-lineage-specific manner by constitutive expression of GLI3 repressor.

Pallister-Hall syndrome (PHS) is a rare disorder caused by mutations in GLI3 that produce a transcriptional repressor (GLI3R). Individuals with PHS present with a variably penetrant variety of urogenital system malformations, including renal aplasia or hypoplasia, hydroureter, hydronephrosis or a common urogenital sinus. The embryologic mechanisms controlled by GLI3R that result in these pathologic phenotypes are undefined. We demonstrate that germline expression of GLI3R causes renal hypoplasia, associated with decreased nephron number, and hydroureter and hydronephrosis, caused by blind-ending ureters. Mice with obligate GLI3R expression also displayed duplication of the ureters that was caused by aberrant common nephric duct patterning and ureteric stalk outgrowth. These developmental abnormalities are associated with suppressed Hedgehog signaling activity in the cloaca and adjacent vesicular mesenchyme. Mice with conditional expression of GLI3R were utilized to identify lineage-specific effects of GLI3R. In the ureteric bud, GLI3R expression decreased branching morphogenesis. In Six2-positive nephrogenic progenitors, GLI3R decreased progenitor cell proliferation reducing the number of nephrogenic precursor structures. Using mutant mice with Gli3R and Gli3 null alleles, we demonstrate that urogenital system patterning and development is controlled by the levels of GLI3R and not by an absence of full-length GLI3. We conclude that the urogenital system phenotypes observed in PHS are caused by GLI3R-dependent perturbations in nephric duct patterning, renal branching morphogenesis and nephrogenic progenitor self-renewal.

Cre-dependent DNA recombination activates a STING-dependent innate immune response.

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies.

Automated Transition State Theory Calculations for High-Throughput Kinetics.

A scarcity of known chemical kinetic parameters leads to the use of many reaction rate estimates, which are not always sufficiently accurate, in the construction of detailed kinetic models. To reduce the reliance on these estimates and improve the accuracy of predictive kinetic models, we have developed a high-throughput, fully automated, reaction rate calculation method, AutoTST. The algorithm integrates automated saddle-point geometry search methods and a canonical transition state theory kinetics calculator. The automatically calculated reaction rates compare favorably to existing estimated rates. Comparison against high level theoretical calculations show the new automated method performs better than rate estimates when the estimate is made by a poor analogy. The method will improve by accounting for internal rotor contributions and by improving methods to determine molecular symmetry.

The mouse endometrium contains epithelial, endothelial and leucocyte populations expressing the stem cell marker telomerase reverse transcriptase.

STUDY HYPOTHESIS: The mouse endometrium harbours stem/progenitor cells that express the stem cell marker mouse telomerase reverse transcriptase (mTert). STUDY FINDING: We used a mouse carrying a transgenic reporter for mTert promoter activity to identify rare endometrial populations of epithelial and endothelial cells that express mTert. WHAT IS KNOWN ALREADY: Stem/progenitor cells are hypothesized to be responsible for the remarkable regenerative capacity of the endometrium, but the lack of convenient endometrial stem/progenitor markers in the mouse has hampered investigations into the identity of these cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A mouse containing a green fluorescent protein (GFP) reporter under the control of the telomerase reverse transcriptase promoter (mTert-GFP) was used to identify potential stem/progenitor cells in the endometrium. mTert promoter activity was determined using fluorescence microscopy and flow cytometry to identify GFP(+) cells. GFP(+) cells were examined for epithelial, stromal, endothelial, leucocyte and proliferation markers and bromodeoxyuridine retention to determine their identity. The endometrium of ovariectomized mice was compared to that of intact cycling mice to establish the role of ovarian hormones in maintaining mTert-expressing cells. MAIN RESULTS AND THE ROLE OF CHANCE: We found that mTert-GFP is expressed by rare luminal and glandular epithelial cells (0.3% of epithelial cells by flow cytometry), rare CD45(-) cells in the stromal compartment (0.028 ± 0.010% of stromal cells by microscopy) and many CD45(+) leucocytes. Ovariectomy resulted in significant decrease of mTert-GFP(+) epithelial cells (P = 0.029 for luminal epithelium; P = 0.034 for glandular epithelium) and a decrease in the percentage of mTert-GFP(+) CD45(+) leucocytes in the stromal compartment (P = 0.015). However, CD45(-) mTert-GFP(+) cells in the stromal compartment were maintained in ovariectomized mice. This population is enriched for cells bearing the endothelial marker CD31 (10.3% of CD90(-) CD45(-) and 97.8% CD90(+) CD45(-) by flow cytometry). CD45(-) mTert-GFP(+) cells also immunostained for the endothelial marker von Willebrand factor. These results suggest that the endometrial epithelium and vasculature are foci of stem/progenitor activity and provide a system to investigate molecular mechanisms involved in endometrial regeneration and repair. LIMITATIONS, REASONS FOR CAUTION: The stem/progenitor activity of endometrial mTert-GFP(+) cells needs to be experimentally verified. WIDER IMPLICATIONS OF THE FINDINGS: The identification and characterization of mTert-expressing progenitor cells in the mouse will facilitate the identification of equivalent populations in the human endometrium that are likely to be involved in endometrial function, fertility and disease. LARGE-SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was funded by National Health and Medical Research Council (NHMRC) of Australia grants (1085435, C.E.G., J.A.D.), 1021127 (C.E.G.), NHMRC Senior Research Fellowship (1042298, C.E.G.), the Victorian Infrastructure Support Program, U.S. National Institutes of Health grant R01 DK084056 (D.T.B.) and the Harvard Stem Cell Institute (D.T.B.). The authors have no conflicts of interest to declare.

Interaction of smoothened with integrin-linked kinase in primary cilia mediates Hedgehog signalling.

Here we report that ILK localizes in the mouse primary cilium, a sensory organelle required for signalling by the Hedgehog (Hh) pathway. Genetic or pharmacological inhibition of ILK blocks ciliary accumulation of the Hh pathway effector smoothened (Smo) and suppresses the induction of Gli transcription factor mRNAs by SHh. Conditional deletion of ILK or Smo also inhibits SHh-driven activation of Gli2 in the embryonic mouse cerebellum. ILK regulation of Hh signalling probably requires the physical interaction of ILK and Smo in the cilium, and we also show selective cilia-associated interaction of ILK with ß-arrestin, a known mediator of Smo-dependent signalling.

Deletions in 16q24.2 are associated with autism spectrum disorder, intellectual disability and congenital renal malformation.

BACKGROUND: The contribution of copy-number variation (CNV) to disease has been highlighted with the widespread adoption of array-based comparative genomic hybridisation (aCGH) and microarray technology. Contiguous gene deletions involving ANKRD11 in 16q24.3 are associated with autism spectrum disorder (ASD) and intellectual disability (ID), while 16q24.1 deletions affecting FOXF1 are associated with congenital renal malformations, alveolar capillary dysplasia, and various other abnormalities. The disease associations of deletions in the intervening region, 16q24.2, have only been defined to a limited extent. AIM: To determine whether deletions affecting 16q24.2 are correlated with congenital anomalies. METHODS: 35 individuals, each having a deletion in 16q24.2, were characterised clinically and by aCGH and/or SNP-genotyping microarray. RESULTS: Several of the 35 16q24.2 deletions identified here closely abut or overlap the coding regions of FOXF1 and ANKRD11, two genes that have been previously associated with the disease. 25 patients were reported to have ASD/ID, and three were found to have bilateral hydronephrosis. 14 of the deletions associated with ASD/ID overlap the coding regions of FBXO31 and MAP1LC3B. These same genes and two others, C16orf95 and ZCCHC14, are also included in the area of minimal overlap of the three deletions associated with hydronephrosis. CONCLUSIONS: Our data highlight 16q24.2 as a region of interest for ASD, ID and congenital renal malformations. These conditions are associated, albeit without complete penetrance, with deletions affecting C16orf95, ZCCHC14, MAP1LC3B and FBXO31. The function of each gene in development and disease warrants further investigation.

A Semiautomated Proteomics and Phosphoproteomics Protocol for the Identification of Novel Therapeutic Targets and Predictive Biomarkers in In Vivo Xenograft Models of Pediatric Cancers.

Genomic profiling has identified therapeutic targets for precision treatment of certain cancers, but many patients lack actionable mutations. Additional omics approaches, like proteomics and phosphoproteomics, are essential for comprehensive mapping of cancer-associated molecular phenotypes. In vivo models, such as cell line and patient-derived xenografts (PDX), offer valuable insights into cancer biology and treatment strategies.This chapter presents a semiautomated high-throughput workflow for integrated proteomics and phosphoproteomics analysis on the Kingfish platform coupled with MagReSyn® Zr-IMAC HP. It enhances protein extraction from in vivo xenograft samples and provides better insights into cancers with poor prognosis. The approach successfully identified over 11,000 unique phosphosites and ~6000 proteins in SJSA-1 pediatric osteosarcoma xenografts, demonstrating its efficacy. This workflow is a valuable tool for studying tumor biology and developing precision oncology strategies.

Visualizing renal primary cilia.

Renal primary cilia are microscopic sensory organelles found on the apical surface of epithelial cells of the nephron and collecting duct. They are based upon a microtubular cytoskeleton, bounded by a specialized membrane, and contain an array of proteins that facilitate their assembly, maintenance and function. Cilium-based signalling is important for the control of epithelial differentiation and has been implicated in the pathogenesis of various cystic kidney diseases and in renal repair. As such, visualizing renal primary cilia and understanding their composition has become an essential component of many studies of inherited kidney disease and mechanisms of epithelial regeneration. Primary cilia were initially identified in the kidney using electron microscopy and this remains a useful technique for the high resolution examination of these organelles. New reagents and techniques now also allow the structure and composition of primary cilia to be analysed in detail using fluorescence microscopy. Primary cilia can be imaged in situ in sections of kidney, and many renal-derived cell lines produce primary cilia in culture providing a simplified and accessible system in which to investigate these organelles. Here we outline microscopy-based techniques commonly used for studying renal primary cilia.

The in silico lab: Improving academic code using lessons from biology.

"Good code" is often regarded as a nebulous, impractical ideal. Common best practices toward improving code quality can be inaccessible to those without a rigorous computer science or software engineering background, contributing to a gap between advancing scientific research and FAIR practices. We seek to equip researchers with the necessary background and context to tackle the challenge of improving code quality in computational biology research using analogies from biology to synthesize why certain best practices are critical for advancing computational research. Improving code quality requires active stewardship; we encourage researchers to deliberately adopt and share practices that ensure reusability, repeatability, and reproducibility.

MYC drives platinum resistant SCLC that is overcome by the dual PI3K-HDAC inhibitor fimepinostat.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with an appalling overall survival of less than 5% (Zimmerman et al. J Thor Oncol 14:768-83, 2019). Patients typically respond to front line platinum-based doublet chemotherapy, but almost universally relapse with drug resistant disease. Elevated MYC expression is common in SCLC and has been associated with platinum resistance. This study evaluates the capacity of MYC to drive platinum resistance and through screening identifies a drug capable of reducing MYC expression and overcoming resistance. METHODS: Elevated MYC expression following the acquisition of platinum resistance in vitro and in vivo was assessed. Moreover, the capacity of enforced MYC expression to drive platinum resistance was defined in SCLC cell lines and in a genetically engineered mouse model that expresses MYC specifically in lung tumors. High throughput drug screening was used to identify drugs able to kill MYC-expressing, platinum resistant cell lines. The capacity of this drug to treat SCLC was defined in vivo in both transplant models using cell lines and patient derived xenografts and in combination with platinum and etoposide chemotherapy in an autochthonous mouse model of platinum resistant SCLC. RESULTS: MYC expression is elevated following the acquisition of platinum resistance and constitutively high MYC expression drives platinum resistance in vitro and in vivo. We show that fimepinostat decreases MYC expression and that it is an effective single agent treatment for SCLC in vitro and in vivo. Indeed, fimepinostat is as effective as platinum-etoposide treatment in vivo. Importantly, when combined with platinum and etoposide, fimepinostat achieves a significant increase in survival. CONCLUSIONS: MYC is a potent driver of platinum resistance in SCLC that is effectively treated with fimepinostat.

Ligand-dependent hedgehog signaling maintains an undifferentiated, malignant osteosarcoma phenotype.

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.

Implementation of DNA Methylation Array Profiling in Pediatric Central Nervous System Tumors: The AIM BRAIN Project: An Australian and New Zealand Children's Haematology/Oncology Group Study.

DNA methylation array profiling for classifying pediatric central nervous system (CNS) tumors is a valuable adjunct to histopathology. However, unbiased prospective and interlaboratory validation studies have been lacking. The AIM BRAIN diagnostic trial involving 11 pediatric cancer centers in Australia and New Zealand was designed to test the feasibility of routine clinical testing and ran in parallel with the Molecular Neuropathology 2.0 (MNP2.0) study at Deutsches Krebsforschungszentrum (German Cancer Research Center). CNS tumors from 269 pediatric patients were prospectively tested on Illumina EPIC arrays, including 104 cases co-enrolled on MNP2.0. Using MNP classifier versions 11b4 and 12.5, we report classifications with a probability score ≥0.90 in 176 of 265 (66.4%) and 213 of 269 (79.2%) cases, respectively. Significant diagnostic information was obtained in 130 of 176 (74%) for 11b4, and 12 of 174 (7%) classifications were discordant with histopathology. Cases prospectively co-enrolled on MNP2.0 gave concordant classifications (99%) and score thresholds (93%), demonstrating excellent test reproducibility and sensitivity. Overall, DNA methylation profiling is a robust single workflow technique with an acceptable diagnostic yield that is considerably enhanced by the extensive subgroup and copy number profile information generated by the platform. The platform has excellent test reproducibility and sensitivity and contributes significantly to CNS tumor diagnosis.

Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies.

Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.

ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.

ß-catenin causes renal dysplasia via upregulation of Tgfß2 and Dkk1.

Renal dysplasia, defined by defective ureteric branching morphogenesis and nephrogenesis, is the major cause of renal failure in infants and children. Here, we define a pathogenic role for a ß-catenin-activated genetic pathway in murine renal dysplasia. Stabilization of ß-catenin in the ureteric cell lineage before the onset of kidney development increased ß-catenin levels and caused renal aplasia or severe hypodysplasia. Analysis of gene expression in the dysplastic tissue identified downregulation of genes required for ureteric branching and upregulation of Tgfß2 and Dkk1. Treatment of wild-type kidney explants with TGFß2 or DKK1 generated morphogenetic phenotypes strikingly similar to those observed in mutant kidney tissue. Stabilization of ß-catenin after the onset of kidney development also caused dysplasia and upregulation of Tgfß2 and Dkk1 in the epithelium. Together, these results demonstrate that elevation of ß-catenin levels during kidney development causes dysplasia.

Fighting fire with fire: deploying complexity in computational modeling to effectively characterize complex biological systems.

Computational modeling empowers systems biologists to interrogate and understand increasingly complex biological phenomena, and the growing suite of computational approach presents both opportunities and challenges. Choosing the right computational approaches to address a given question requires managing a model's complexity, balancing goals and limitations including interpretability, data resolution, and computational cost. Excess model complexity can diminish the utility for building understanding, while excess simplicity can render the model insufficient for addressing the questions of interest. Using systems immunology as a case study, we review how different model design strategies uniquely manage complexity, ending with a consideration of composite models, which combine the benefits of individual paradigms but present additional challenges arising from added layers of complexity. We anticipate that considering general model design challenges and potential solutions through the lens of complexity will foster enhanced collaboration among computational and experimental researchers.

Distinctive molecular features of regenerative stem cells in the damaged male germline.

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.