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Although many studies revealed transcriptomic subtypes of HCC, concordance of the subtypes are not fully examined. We aim to examine a consensus of transcriptomic subtypes and correlate them with clinical outcomes.By integrating 16 previously established genomic signatures for HCC subtypes, we identified five clinically and molecularly distinct consensus subtypes. STM (STeM) is characterized by high stem cell features, vascular invasion, and poor prognosis. CIN (Chromosomal INstability) has moderate stem cell features, but high genomic instability and low immune activity. IMH (IMmune High) is characterized by high immune activity. BCM (Beta-Catenin with high Male predominance) is characterized by prominent β-catenin activation, low miRNA expression, hypomethylation, and high sensitivity to sorafenib. DLP (Differentiated and Low Proliferation) is differentiated with high hepatocyte nuclear factor 4A activity. We also developed and validated a robust predictor of consensus subtype with 100 genes and demonstrated that five subtypes were well conserved in patient-derived xenograft models and cell lines. By analyzing serum proteomic data from the same patients, we further identified potential serum biomarkers that can stratify patients into subtypes.Five HCC subtypes are correlated with genomic phenotypes and clinical outcomes and highly conserved in preclinical models, providing a framework for selecting the most appropriate models for preclinical studies.

Hepatocellular carcinoma (HCC) is a heterogeneous disease. Mouse models are commonly used as preclinical models to study hepatocarcinogenesis, but how well these models recapitulate molecular subtypes of human HCC is unclear. Here, integration of genomic signatures from molecularly and clinically defined human HCC (n = 11) and mouse models of HCC (n = 9) identified the mouse models that best resembled subtypes of human HCC and determined the clinical relevance of each model. Mst1/2 knockout (KO), Sav1 KO, and SV40 T antigen mouse models effectively recapitulated subtypes of human HCC with a poor prognosis, whereas the Myc transgenic model best resembled human HCCs with a more favorable prognosis. The Myc model was also associated with activation of β-catenin. E2f1, E2f1/Myc, E2f1/Tgfa, and diethylnitrosamine (DEN)-induced models were heterogeneous and were unequally split into poor and favorable prognoses. Mst1/2 KO and Sav1 KO models best resemble human HCC with hepatic stem cell characteristics. Applying a genomic predictor for immunotherapy, the six-gene IFNγ score, the Mst1/2 KO, Sav1 KO, SV40, and DEN models were predicted to be the least responsive to immunotherapy. Further analysis showed that elevated expression of immune-inhibitory genes (Cd276 and Nectin2/Pvrl2) in Mst1/2 KO, Sav1 KO, and SV40 models and decreased expression of immune stimulatory gene (Cd86) in the DEN model might be accountable for the lack of predictive response to immunotherapy.Implication: The current genomic approach identified the most relevant mouse models to human liver cancer and suggests immunotherapeutic potential for the treatment of specific subtypes. Mol Cancer Res; 16(11); 1713–23. ©2018 AACR.

S1. Correlation of prognostic probability and risk scores for mouse HCCs. S2. Glycolytic gene expression signature in mouse liver. S3. Glycolytic expression signature in mouse HCC tumors from 9 models. S4. Correlation of hepatic stem cell probability with SOH probability. S5. Expression of hepatic stem cell markers in 9 mouse models. S6. Expression of Krt-7 in Sav1 KO liver issues. S7. Expression of downstream target genes in mouse tumors. S8. Expression of Pdcd1 and Cd274 and their correlation with IFNG6 score in mouse HCCs in nine models. S9. Regulation of Cd276 by Yap1.

Upregulated or downregulated genes by PJA1 knockdown and TGF-β1 streatment in HepG2 cells

Characteristics of HCC patients with PJA1 genetic alterations

Supplementary Materials and Methods

Upregulated or downregulated genes in samples expressing a high level of PJA1

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFβ/SMAD signaling. SMAD3 and its adaptors, such as β2SP, are important mediators of TGFβ signaling and regulate gene expression to suppress stem cell–like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/β2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3+/−), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFβ1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFβ in liver carcinogenesis.

Supplementary Figures and legends

Gene numbers regulated by PJA1 knockdown and TGF-β1 treatment

Due to its advantages of spatial uniformity and ion energy control, a dual-frequency (DF) capacitive-coupled plasma is widely used in semiconductor etching and deposition processes. In low-pressure discharges, the mean free path of ions is longer than the sheath width, and the ion energy distribution function is sensitive to the driving voltage waveform. In this respect, it is necessary to use a particle-in-cell (PIC) simulation to observe ion movement according to the time-varying electric field in the sheath. This study uses a two-dimensional PIC simulation parallelized with a graphics processing unit to monitor the ion energy distribution and flux according to the DF voltage waveform. We suggested a method to control the ion energy through a phase-resolved ion energy distribution in the region, where the ion transit time is longer than the high-frequency period and shorter than the low-frequency period.

Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes in vitro and in vivo. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in Drosophila, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.

Autophagy is a catabolic cellular response to stress that has been liked to various human diseases. However, the precise involvement of autophagy in health and disease remains unclear. To explore the molecular mechanisms of autophagy, we investigated the effect of TMP21. We found that the down-regulation of TMP21 induced autophagy in SH-SY5Y cells. In addition, the enhanced autophagy observed upon TMP21 depletion was almost completely blocked in ATG5 knockout (KO) or ATG7-KO HeLa cells. Silencing of TMP21 in SH-SY5Y cells also increased the production of cellular reactive oxygen species (ROS). Accordingly, treatment with the ROS scavenger NAC suppressed autophagy activation as well as ROS production in TMP21-depleted cells. In addition, the inhibition of mTOR by treatment with Torin1 was mitigated in TMP21 overexpressing cells compared with that in control cells. Taken together, these results indicated that TMP21 could regulate autophagy by modulating ROS production and mTOR activation.

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-β (Aβ) production. Treatment with Aβ increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aβ: amyloid-β; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

Objectives Non-typhoid Salmonella infection is a major agent of food-borne outbreaks as well as individual cases worldwide. However, few studies on drug-resistant Salmonella strains, especially those recovered from young children, are available. Therefore, we determined the prevalence and characteristics of cephalosporin-resistant Salmonella isolates in the south-west region of Korea over a five-year period. Methods Non-duplicate Salmonella clinical isolates were recovered from diarrhoeagenic patient specimens at 12 hospitals in Gwangju, Korea between January 2014 and December 2018. Antimicrobial susceptibility testing and molecular features of cephalosporin-resistant isolates were determined. Results A total of 652 Salmonella isolates were collected and 48 cefotaxime-resistant Salmonella isolates (7.4%), that belonged to nine Salmonella serovars, were identified. These were S. Enteritidis, S. Typhimurium, S. I 4,[5],12:i:-, S. Virchow, S. Agona, S. Bareilly, S. Infantis, S. Newport, and S. Schleissheim. The prevalence rate increased from 5.3% in 2014 to 10.3% in 2018. S. Virchow (44.4%) showed significantly high resistant rate compared to the other serovars. PGFE genotyping revealed high genetic homogeneities among each Salmonella serovars, suggesting clonal dissemination of cephalosporin-resistant strains. Conclusions Progressive increases in carriage rates and the possibility of community outbreaks by cephalosporin-resistant Salmonella in young children may pose tangible public health threats.

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1β and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

BACKGROUND & AIMS: Late recurrence of hepatocellular carcinoma (HCC) is regarded as de novo HCC from chronic hepatitis. This study investigated clinicopathological and molecular factors to develop a nomogram for predicting late HCC recurrence (> 2 years after curative resection). METHODS: The training and validation cohorts included HCC patients with a major etiology of hepatitis B who underwent curative resection. Clinicopathological features including lobular and porto-periportal inflammatory activity, fibrosis, and liver cell change were evaluated. Proteins encoded by genes related to late recurrence were identified using a reverse phase protein array of 95 non-tumorous liver tissues. Immunoexpression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), plasminogen activator inhibitor-1, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and spleen tyrosine kinase (SYK) was measured. RESULTS: Late recurrence occurred in 74/402 (18%) and 47/243 (19%) in the training and validation cohorts, respectively. Cirrhosis, moderate/severe lobular inflammatory activity, and expression of pSTAT3, pERK1/2, and SYK proteins correlated to the gene signature of hepatocyte injury and regeneration were independently associated with late recurrence, with odds ratios (95% confidence intervals) of 2.0 (1.2–3.3), 21.1 (4.3–102.7), and 6.0 (2.1–17.7), respectively (p < 0.05 for all). A nomogram based on these variables (histological parameters and immunohistochemical marker combinations) showed high reliability in both the training and validation cohorts (Harrell’s C index: 0.701 and 0.716; 95% confidence intervals: 0.64–0.76 and 0.64–0.79, respectively). CONCLUSIONS: The combination of pSTAT3, pERK1/2, and SYK immunoexpression with high lobular inflammatory activity and cirrhosis (fibrosis) predicts late HCC recurrence.

The installation and operation of geothermal systems increased due to the expectation of good cooling and heating performance due to stable heat source temperatures. In actual geothermal system operations, heat source temperature rises or falls due to an imbalance of heating and cooling energy usage. Problems of source side temperature result in reduced geothermal system performance. The purpose of this study is to develop hybrid geothermal system operation technology to stabilize temperature and improve system performance by utilizing auxiliary heat source system. The auxiliary heat source system is operated by comparing the performance when operating the geothermal heat pump system alone and the performance when operating the hybrid geothermal heat pump system. The performance of a hybrid geothermal system is determined by the circulating water temperature of the geothermal system and the circulating water temperature of the auxiliary heat source system. Hybrid geothermal heat pump system performance is predicted through numerical analysis and collection of hybrid geothermal system performance data at various temperature ranges through field test. An operating method was developed using the predicted performance as the changeover operating point of the hybrid geothermal heat pump system. When applying the development and operation technology, it handled about 11% more load than the existing geothermal system operation. The addition of an auxiliary heat source increases the initial investment cost compared to the existing geothermal system, but decreases energy consumption, confirming that the initial investment cost of 15.3 years is recovered.

AT-rich interactive domain 1A (ARID1A) is one of the most frequently mutated genes in hepatocellular carcinoma (HCC), but its clinical significance is not clarified. We aimed to evaluate the clinical significance of low ARID1A expression in HCC. By analyzing the gene expression data of liver from Arid1a-knockout mice, hepatic Arid1a-specific gene expression signature was identified (p &lt, 0.05 and 0.5-fold difference). From this signature, a prediction model was developed to identify tissues lacking Arid1a activity and was applied to gene expression data from three independent cohorts of HCC patients to stratify patients according to ARID1A activity. The molecular features associated with loss of ARID1A were analyzed using The Cancer Genome Atlas (TCGA) multi-platform data, and Ingenuity Pathway Analysis (IPA) was done to uncover potential signaling pathways associated with ARID1A loss. ARID1A inactivation was clinically associated with poor prognosis in all three independent cohorts and was consistently related to poor prognosis subtypes of previously reported gene signatures (highly proliferative, hepatic stem cell, silence of Hippo pathway, and high recurrence signatures). Immune activity, indicated by significantly lower IFNG6 and cytolytic activity scores and enrichment of regulatory T-cell composition, was lower in the ARID1A-low subtype than ARID1A-high subtype. Ingenuity pathway analysis revealed that direct upstream transcription regulators of the ARID1A signature were genes associated with cell cycle, including E2F group, CCND1, and MYC, while tumor suppressors such as TP53, SMAD3, and CTNNB1 were significantly inhibited. ARID1A plays an important role in immune activity and regulating multiple genes involved in HCC development. Low-ARID1A subtype was associated with poor clinical outcome and suggests the possibility of ARID1A as a prognostic biomarker in HCC patients.

AT-rich interactive domain 1A (ARID1A) is one of the most frequently mutated genes in hepatocellular carcinoma (HCC), but its clinical significance is not clarified. We aimed to evaluate the clinical significance of low ARID1A expression in HCC. By analyzing the gene expression data of liver from Arid1a-knockout mice, hepatic Arid1a-specific gene expression signature was identified (p < 0.05 and 0.5-fold difference). From this signature, a prediction model was developed to identify tissues lacking Arid1a activity and was applied to gene expression data from three independent cohorts of HCC patients to stratify patients according to ARID1A activity. The molecular features associated with loss of ARID1A were analyzed using The Cancer Genome Atlas (TCGA) multi-platform data, and Ingenuity Pathway Analysis (IPA) was done to uncover potential signaling pathways associated with ARID1A loss. ARID1A inactivation was clinically associated with poor prognosis in all three independent cohorts and was consistently related to poor prognosis subtypes of previously reported gene signatures (highly proliferative, hepatic stem cell, silence of Hippo pathway, and high recurrence signatures). Immune activity, indicated by significantly lower IFNG6 and cytolytic activity scores and enrichment of regulatory T-cell composition, was lower in the ARID1A-low subtype than ARID1A-high subtype. Ingenuity pathway analysis revealed that direct upstream transcription regulators of the ARID1A signature were genes associated with cell cycle, including E2F group, CCND1, and MYC, while tumor suppressors such as TP53, SMAD3, and CTNNB1 were significantly inhibited. ARID1A plays an important role in immune activity and regulating multiple genes involved in HCC development. Low-ARID1A subtype was associated with poor clinical outcome and suggests the possibility of ARID1A as a prognostic biomarker in HCC patients.

The oncogenic receptor tyrosine kinase AXL is overexpressed in cancer and plays an important role in carcinomas of multiple organs. However, the mechanisms of AXL overexpression in cancer remain unclear. In this study, using HEK293T, Panc-1, and Panc-28 cells and samples of human pancreatic intraepithelial neoplasia (PanIN), along with several biochemical approaches and immunofluorescence microscopy analyses, we sought to investigate the mechanisms that regulate AXL over-expression in pancreatic ductal adenocarcinoma (PDAC). We found that AXL interacts with hematopoietic progenitor kinase 1 (HPK1) and demonstrate that HPK1 down-regulates AXL and decreases its half-life. The HPK1-mediated AXL degradation was inhibited by the endocytic pathway inhibitors leupeptin, bafilomycin A1, and monensin. HPK1 accelerated the movement of AXL from the plasma membrane to endosomes in pancreatic cancer cells treated with the AXL ligand growth arrest-specific 6 (GAS6). Moreover, HPK1 increased the binding of AXL to the Cbl proto-oncogene (c-Cbl); promoted AXL ubiquitination; decreased AXL-mediated signaling, including phospho-AKT and phospho-ERK signaling; and decreased the invasion capability of PDAC cells. Importantly, we show that AXL expression inversely correlates with HPK1 expression in human PanINs and that patients whose tumors have low HPK1 and high AXL expression levels have shorter survival than those with low AXL or high HPK1 expression (p < 0.001). Our results suggest that HPK1 is a tumor suppressor that targets AXL for degradation via the endocytic pathway. HPK1 loss of function may contribute to AXL overexpression and thereby enhance AXL-dependent downstream signaling and tumor invasion in PDAC.

Cadaverine is used for the synthesis of the novel bio-polyamides 54, 56, and 510. Here, we examine the feasibility of using a lysine decarboxylase (LdcC) from Escherichia coli for high-level production of cadaverine. After sequential optimization of whole-cell biotransformation conditions, recombinant E. coli-overexpressing LdcC (EcLdcC) could produce 1.0 M cadaverine from 1.2 M crude L-lysine solution after 9 h. EcLdcC retained a higher cadaverine yield after being reused 10 times at acidic and alkaline pH values than that of a recombinant E. coli strain overexpressing an inducible lysine decarboxylase (CadA), a conventional cadaverine producer (90 vs. 51% at pH 6 and 55 vs. 15% at pH 8). This study reveals that EcLdcC is a promising whole-cell biocatalyst for the bio-based production of cadaverine from industrial grade L-lysine in comparison to EcCadA.

Summary Transcriptional coactivator with PDZ‐binding motif (TAZ) directly interacts with transcription factors and regulates their transcriptional activity. Extensive functional studies have shown that TAZ plays critical regulatory roles in stem cell proliferation, differentiation, and survival and also modulates the development of organs such as the lung, kidney, heart, and bone. Despite the importance of TAZ in stem cell maintenance, TAZ function has not yet been evaluated in spermatogenic stem cells of the male reproductive system. Here, we investigated the expression and functions of TAZ in mouse testis. TAZ was expressed in spermatogenic stem cells; however, its deficiency caused significant structural abnormalities, including atrophied tubules, widened interstitial space, and abnormal Leydig cell expansion, thereby resulting in lowered sperm counts and impaired fertility. Furthermore, TAZ deficiency increased the level of apoptosis and senescence in spermatogenic cells and Leydig cells upon aging. The expression of senescence‐associated β‐galactosidase (SA‐βgal), secretory phenotypes, and cyclin‐dependent kinase inhibitors (p16, p19, and p21) significantly increased in the absence of TAZ. TAZ downregulation in testicular cells further increased SA‐βgal and p21 expression induced by oxidative stress, whereas TAZ overexpression decreased p21 induction and prevented senescence. Mechanistic studies showed that TAZ suppressed DNA‐binding activity of p53 through a direct interaction and thus attenuated p53‐induced p21 gene transcription. Our results suggested that TAZ may suppress apoptosis and premature senescence in spermatogenic cells by inhibiting the p53‐p21 signaling pathway, thus playing important roles in the maintenance and control of reproductive function.

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFβ/SMAD signaling. SMAD3 and its adaptors, such as β2SP, are important mediators of TGFβ signaling and regulate gene expression to suppress stem cell–like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/β2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3+/−), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFβ1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients. Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFβ in liver carcinogenesis.

Traditionally, medicine has held that some human body sites are sterile and that the introduction of microbes to these sites results in infections. This paradigm shifted significantly with the discovery of the human microbiome and acceptance of these commensal microbes living across the body. However, the central nervous system (CNS) is still believed by many to be sterile in healthy people. Using culture-independent methods, we examined the virome of cerebrospinal fluid (CSF) from a cohort of mostly healthy human subjects. We identified a community of DNA viruses, most of which were identified as bacteriophages. Compared to other human specimen types, CSF viromes were not ecologically distinct. There was a high alpha diversity cluster that included feces, saliva, and urine, and a low alpha diversity cluster that included CSF, body fluids, plasma, and breast milk. The high diversity cluster included specimens known to have many bacteria, while other specimens traditionally assumed to be sterile formed the low diversity cluster. There was an abundance of viruses shared among CSF, breast milk, plasma, and body fluids, while each generally shared less with urine, feces, and saliva. These shared viruses ranged across different virus families, indicating that similarities between these viromes represent more than just a single shared virus family. By identifying a virome in the CSF of mostly healthy individuals, it is now less likely that any human body site is devoid of microbes, which further highlights the need to decipher the role that viral communities may play in human health.