Electrospun Scaffold Micro-Architecture Induces an Activated Transcriptional Phenotype within Tendon Fibroblasts

Biomaterial augmentation of surgically repaired rotator cuff tendon tears aims to improve the high failure rates (∼40%) of traditional repairs. Biomaterials that can alter cellular phenotypes through the provision of microscale topographical cues are now under development. We aimed to systematically evaluate the effect of topographic architecture on the cellular phenotype of fibroblasts from healthy and diseased tendons. Electrospun polydioxanone scaffolds with fiber diameters ranging from 300 to 4000 nm, in either a highly aligned or random configuration, were produced. Healthy tendon fibroblasts cultured for 7 days on scaffolds with highly aligned fibers demonstrated a distinctive elongated morphology, whilst those cultured on randomly configured fibers demonstrated a flattened and spread morphology. The effect of scaffold micro-architecture on the transcriptome of both healthy and diseased tendon fibroblasts was assessed with bulk RNA-seq. Both healthy (n = 3) and diseased tendon cells (n = 3) demonstrated a similar transcriptional response to architectural variants. Gene set enrichment analysis revealed that large diameter (≥2000 nm) aligned scaffolds induced an upregulation of genes involved in cellular replication and a downregulation of genes defining inflammatory responses and cell adhesion. Similarly, PDPN and CD248, markers of inflammatory or “activated” fibroblasts, were downregulated during culture of both healthy and diseased fibroblasts on aligned scaffolds with large (≥2000 nm) fiber diameters. In conclusion scaffold architectures resembling that of disordered type III collagen, typically present during the earlier phases of wound healing, resulted in tendon fibroblast activation. Conversely, scaffolds mimicking aligned diameter collagen I fibrils, present during tissue remodelling, did not activate tendon derived fibroblasts. This has implications for the design of scaffolds used during rotator cuff repair augmentation.

Toxoplasma ERK7 defends the apical complex from premature degradation

Accurate cellular replication balances the biogenesis and turnover of complex structures. Apicomplexan parasites such as Plasmodium and Toxoplasma replicate by forming daughter cells within an intact mother cell, creating additional challenges to ensuring fidelity of division. Critical to these parasites' infectivity is an intricate cytoskeleton structure called the apical complex. Before the daughter apical complex can be inserted into the plasma membrane, the maternal material must be turned over. We previously identified the kinase ERK7 as required for the maturation of the apical complex in Toxoplasma gondii. Here we define the Toxoplasma ERK7 interactome, and identify a putative E3 ligase, CSAR1, as the downstream effector responsible for the phenotype. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by a mislocalization from the parasite residual body to the maternal and daughter apical complexes. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

BK Polyomavirus Activates HSF1 Stimulating Human Kidney Hek293 Cell Proliferation

Objectives. Some DNA viruses, such as BKPyV, are capable of inducing neoplastic transformation in human tissues through still unclear mechanisms. The goal of this study is to investigate the carcinogenic potential of BK polyomavirus (BKPyV) in human embryonic kidney 293 (Hek293) cells, dissecting the molecular mechanism that determines the neoplastic transformation. Materials and Methods. BKPyV, isolated from urine samples of infected patients, was used to infect monolayers of Hek293 cells. Subsequently, intracellular redox changes, GSH/GSSH concentration by HPLC, and reactive oxygen/nitrogen species (ROS/RNS) production were monitored. Moreover, to understand the signaling pathway underlying the neoplastic transformation, the redox-sensitive HFS1-Hsp27 molecular axis was examined using the flavonoid quercetin and polishort hairpin RNA technologies. Results. The data obtained show that while BKPyV replication is closely linked to the transcription factor p53, the increase in Hek293 cell proliferation is due to the activation of the signaling pathway mediated by HSF1-Hsp27. In fact, its inhibition blocks viral replication and cell growth, respectively. Conclusions. The HSF1-Hsp27 signaling pathway is involved in BKPyV infection and cellular replication and its activation, which could be involved in cell transformation.

Interaction between M. tuberculosis Lineage and Human Genetic Variants Reveals Novel Pathway Associations with Severity of TB

Tuberculosis (TB) remains a major public health threat globally, especially in sub-Saharan Africa. Both human and Mycobacterium tuberculosis (MTBC) genetic variation affect TB outcomes, but few studies have examined if and how the two genomes interact to affect disease. We hypothesize that long-term coexistence between human genomes and MTBC lineages modulates disease to affect its severity. We examined this hypothesis in our TB household contact study in Kampala, Uganda, in which we identified three MTBC lineages, of which one, L4.6-Uganda, is clearly derived and hence recent. We quantified TB severity using the Bandim TBscore and examined the interaction between MTBC lineage and human single-nucleotide polymorphisms (SNPs) genome-wide, in two independent cohorts of TB cases (n = 149 and n = 127). We found a significant interaction between an SNP in PPIAP2 and the Uganda lineage (combined p = 4 × 10−8). PPIAP2 is a pseudogene that is highly expressed in immune cells. Pathway and eQTL analyses indicated potential roles between coevolving SNPs and cellular replication and metabolism as well as platelet aggregation and coagulation. This finding provides further evidence that host–pathogen interactions affect clinical presentation differently than host and pathogen genetic variation independently, and that human–MTBC coevolution is likely to explain patterns of disease severity.

Interaction between M. tuberculosis Lineage and Human Genetic Variants Reveals Novel Pathway Associations With Severity of TB

Tuberculosis (TB) remains a major public health threat globally, especially in sub-Saharan Africa. Both human and Mycobacterium tuberculosis (MTBC) genetic variation affect TB outcomes, but few studies have examined if and how the two genomes interact to affect disease. We hypothesize that long-term coexistence between human genomes and MTBC lineages modulate disease to affect its severity. We examined this hypothesis in our TB household contact study in Kampala, Uganda, in which we identified 3 MTBC lineages of which one, L4.6-Uganda, is clearly derived and hence recent. We quantified TB severity using the Bandim TBscore and examined the interaction between MTBC lineage and human single nucleotide polymorphisms (SNPs) genome-wide, in two independent cohorts of TB cases (N=149 and N=127). We found a significant interaction between a SNP in PPIAP2 and the Uganda lineage (combined p=4x10-8). PPIAP2 is a pseudogene that is highly expressed in immune cells. Pathway and eQTL analyses indicated potential roles between coevolving SNPs and cellular replication and metabolism as well as platelet aggregation and coagulation. This finding provides further evidence that host-pathogen interactions affect clinical presentation differently than host and pathogen genetic variation independently, and that human-MTBC coevolution is likely to explain patterns of disease severity.

Dangerous Liaisons: Long-Term Replication with an Extrachromosomal HPV Genome

Papillomaviruses cause persistent, and usually self-limiting, infections in the mucosal and cutaneous surfaces of the host epithelium. However, in some cases, infection with an oncogenic HPV can lead to cancer. The viral genome is a small, double-stranded circular DNA molecule that is assembled into nucleosomes at all stages of infection. The viral minichromosome replicates at a low copy number in the nucleus of persistently infected cells using the cellular replication machinery. When the infected cells differentiate, the virus hijacks the host DNA damage and repair pathways to replicate viral DNA to a high copy number to generate progeny virions. This strategy is highly effective and requires a close association between viral and host chromatin, as well as cellular processes associated with DNA replication, repair, and transcription. However, this association can lead to accidental integration of the viral genome into host DNA, and under certain circumstances integration can promote oncogenesis. Here we describe the fate of viral DNA at each stage of the viral life cycle and how this might facilitate accidental integration and subsequent carcinogenesis.

Cell Differentiation and Replication during Postnatal Development of the Murine First Molar

Various signaling molecular pathways are involved in odontogenesis to promote cellular replication and differentiation. Tooth formation is controlled mainly by epithelial–mesenchymal interactions. The aim of this work was to investigate how cellular replication and differentiation ensue during the formation of the murine first molar in postnatal ages until eruption, focusing on morphogenesis, odontoblast differentiation and cellular replication. Wild-type CD1 mice were examined from birth to weaning. Morphogenesis and interaction between developing epithelial and mesenchymal tissues were evaluated in hematoxylin–eosin and Gomori trichome stained sections. Immunohistochemistry for nestin, which mediates the differentiation of odontoblasts, especially their polarization and elongation, showed that this intermediate filament was apparent already at postnatal day P1 in the apical region of odontoblasts and progressed apically from cusp tips, while it was not present in epithelial tissues. The expression of nuclear antigen Ki-67 highlighted dividing cells in both epithelial and mesenchymal tissues at P1, while one week later they were restricted to the cementoenamel junction, guiding root elongation. The link between odontoblast maturation and cellular replication in the different tooth tissues is essential to understand the development of tooth shape and dimension, to outline mechanisms of tooth morphogenesis and possibly eruption.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Acquired hypolipoproteinemia and hemophagocytic lymphohistiocytosis: A previously unexplored association?

e19041 Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare disease that is the result of macrophage hyperactivation, leading to disordered cellular phagocytosis that is highly fatal if left untreated. The precise precipitating factor that leads to HLH is presently unknown. Secondary HLH is more common in adults and has numerous causes, including several autoimmune diseases, rheumatological disorders, cancers, and infections. These predisposing conditions are similar in that they are characterized by increased rates of cellular replication and often accompanied by significant oxidative stress. Lipoprotein changes in HLH have not been previously described. Methods: Between August 2018 and June 2020, three cases of secondary HLH were discovered to have profound hypolipoproteinemia with severely reduced total cholesterol, undetectable LDL-C, and extremely depressed HDL-C levels. As a result, a retrospective review of the University of Michigan HLH registry was performed, identifying cases of HLH where full lipid panels were performed as part of their diagnosis and/or treatment between 2012 and 2020. 18 total patients were identified with a confirmed diagnosis of HLH and who had a full lipid panel performed as a part of their diagnostic evaluation. Results: 100% of patients were found to have HDL-C less than 30 mg/dL, consistent with severe HDL-C deficiency, and 84% had HDL-C less than 20 mg/dL. Similarly, 74% of patients were found to have LDL-C < 100 mg/dL, 47% with LDL-C < 50 mg/dL, and 33% had undetectable LDL-C levels. Median total cholesterol was 124 mg/dL, median LDL-C was 35 mg/dL, and median HDL-C was 7 mg/dL. Notably, these reductions were not explained by the observed variability in hypertriglyceridemia, and marked hypertriglyceridemia > 500 mg/dL was not seen in 88% of patients, with a median triglyceride value of 279. Interestingly, one patient displayed an opposite phenotype with extreme hyperlipidemia, with total cholesterol of 727 mg/dL and LDL-C of 658 md/dL. Conclusions: Severe derangements in circulating lipoproteins appear to be a common finding within sHLH.These findings have not been previously described within the context of the disease. Herein, we provide a framework for hypothesizing why HLH may potentially occur in the setting of hypolipidemia. Furthermore, we provide an additional hypothesis that serves to justify why these changes may occur in an evolutionary context.

Persistent Human Papillomavirus Infection

Persistent infection with oncogenic human papillomavirus (HPV) types is responsible for ~5% of human cancers. The HPV infectious cycle can sustain long-term infection in stratified epithelia because viral DNA is maintained as low copy number extrachromosomal plasmids in the dividing basal cells of a lesion, while progeny viral genomes are amplified to large numbers in differentiated superficial cells. The viral E1 and E2 proteins initiate viral DNA replication and maintain and partition viral genomes, in concert with the cellular replication machinery. Additionally, the E5, E6, and E7 proteins are required to evade host immune responses and to produce a cellular environment that supports viral DNA replication. An unfortunate consequence of the manipulation of cellular proliferation and differentiation is that cells become at high risk for carcinogenesis.