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Hepatoid adenocarcinoma of the stomach (HAS), a rare subtype of gastric cancer (GC), has a low incidence but a high mortality rate. Little is known about the molecular features of HAS. Here we applied whole-exome sequencing (WES) on 58 tumours and the matched normal controls from 54 HAS patients, transcriptome sequencing on 30 HAS tumours, and single-cell RNA sequencing (scRNA-seq) on one HAS tumour. Our results reveal that the adenocarcinomatous component and hepatocellular-like component of the same HAS tumour originate monoclonally, and HAS is likely to initiate from pluripotent precursor cells. HAS has high stemness and high methionine cycle activity compared to classical GC. Two genes in the methionine cycle, MAT2A, and AHCY are potential targets for HAS treatments. We provide the first integrative genomic profiles of HAS, which may facilitate its diagnosis, prognosis, and treatment.

The ability of some bacteria within a population to tolerate antibiotic treatment is often attributed to prolonged bacterial infection1-3. Unlike antibiotic resistance, which generally results from genetic mutations or plasmid transfer4,5, antibiotic tolerance usually refers to the phenomenon that a subgroup of cells can survive high dose antibiotic treatment as a result of phenotypic heterogeneity6,7. Previous studies mainly associate antibiotic tolerance with cell dormancy, by hypothesizing that the lethal effects of antibiotics are disabled due to the extremely slow metabolic and proliferation rates in dormant bacteria 8,9. However, less is known about how surviving bacteria subsequently escape from the dormant state and resuscitate, which is equally important for disease recurrence. Here we monitored the process of bacterial antibiotic tolerance and regrowth at the single-cell level, and found that each individual survival cell shows different ‘dormancy depth’, which in return regulates whether and when it can resume growth after removal of antibiotic. The persister cells are considered to be in shallow dormancy depth, while the viable but non-culturable cells (VBNC cells) are in deep dormancy depth. We further implemented time-lapse fluorescent imaging and biochemical analysis to establish that dynamic endogenous protein aggregation is an important indicator of bacterial dormancy depth. For cells to leave the dormant state and resuscitate, clearance of cellular protein aggregates and recovery of proteostasis are required. Through additional mutagenesis studies, we found the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the timeline (whether and when) for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

Although bacterial persistence was first observed in 1944, its underlying mechanism is just beginning to be understood and many fundamental questions remain. In this review, we summarize studies in order to chart the full map of bacterial persistence. Because persistence significantly contributes to disease recalcitrance, we also elucidate the probable relationships between bacterial persistence and prolonged chronic infections, with some comments on future research directions and therapeutic strategies.

Bacterial persisters are phenotypic variants of an isogenic cell population that can survive antibiotic treatment and resume growth after the antibiotics have been removed. Cell dormancy has long been considered the principle mechanism underlying persister formation. However, dormancy alone is insufficient to explain the full range of bacterial persistence. Our recent work revealed that in addition to 'passive defense' via dormancy, persister cells employ 'active defense' via enhanced efflux activity to expel drugs. This finding suggests that persisters combine two seemingly contradictory mechanisms to tolerate antibiotic attack. Here, we review the passive and active aspects of persister formation, discuss new insights into the process, and propose new techniques that can facilitate the study of bacterial persistence.

The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis., Author Summary Auxin regulates plant growth and development through auxin signaling, which begins with the interaction of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein co-receptor. Auxin binding to the co-receptor complex triggers ubiquitination and 26S proteasome degradation of Aux/IAA proteins, leading to a downstream signaling cascade that induces the expression of auxin-responsive genes. Auxin signaling is manipulated by plant pathogens to maximize their own multiplication, but the underlying mechanisms are poorly understood. Here we report that the P2 capsid protein encoded by Rice dwarf virus (RDV) sabotages auxin signaling by interacting with the rice Aux/IAA protein, OsIAA10, thereby shielding it from degradation and causing infected plants to display typical RDV symptoms including dwarfism, excessive tillering and stunted crown roots. Importantly, these symptoms are phenocopied by transgenic rice plants overexpressing OsIAA10 or its degradation-resistant mutant. Conversely, down-regulating OsIAA10 expression in rice led to milder RDV infection. Together these findings reveal a novel mechanism by which RDV reprograms auxin signaling, leading to enhanced viral infection.

Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.

Insect transmission is an essential process of infection for numerous plant and animal viruses. How an insect-transmissible plant virus enters an insect cell to initiate the infection cycle is poorly understood, especially for nonenveloped plant and animal viruses. The capsid protein P2 of rice dwarf virus (RDV), which is nonenveloped, is necessary for insect transmission. Here, we present evidence that P2 shares structural features with membrane-fusogenic proteins encoded by enveloped animal viruses. When RDV P2 was ectopically expressed and displayed on the surface of insect Spodoptera frugiperda cells, it induced membrane fusion characterized by syncytium formation at low pH. Mutational analyses identified the N-terminal and a heptad repeat as being critical for the membrane fusion-inducing activity. These results are corroborated with results from RDV-infected cells of the insect vector leafhopper. We propose that the RDV P2-induced membrane fusion plays a critical role in viral entry into insect cells. Our report that a plant viral protein can induce membrane fusion has broad significance in studying the mechanisms of virus entry into insect cells and insect transmission of nonenveloped plant and animal viruses.

Yeast two-hybrid and coimmunoprecipitation assays indicated that P8, an outer capsid protein of Rice dwarf phytoreovirus (RDV), interacts with rice glycolate oxidase (GOX), a typical enzyme of peroxisomes. Confocal immunofluorescence microscopy revealed that P8 was colocalized with GOX in peroxisomes. Time course analysis demonstrated that the localization of P8 in Spodoptera frugiperda cells changed from diffuse to discrete, punctuate inclusions during expression from 24 to 48h post inoculation. Coexpression of GOX with P8 may target P8 into peroxisomes, which serve as replication sites for a number of viruses. Therefore, we conclude that the interaction of P8 with the GOX of host cells leads to translocation of P8 into peroxisomes and we further propose that the interaction between P8 and GOX may play important roles in RDV targeting into the replication site of host cells. Our findings have broad significance in studying the mechanisms whereby viruses target appropriate replication sites and begin their replication.