Analysis of the influence of host lifestyle (coffee consumption, drinking, and smoking) on Korean oral microbiome.

If a DNA sample collected in the field is old or degraded, short tandem repeat analysis is difficult to perform, a representative analysis method currently used for individual identification. Given that microorganisms exist everywhere and within the human body, in similar amounts to human cells, microbial analysis could be used to identify individuals even in cases in which human DNA-based identification is difficult. Research has demonstrated that the types of microorganisms within the human body differ depending on various internal or external factors, such as body part or bodily fluid type, lifestyle, geographical area of residence, sex, and age. In this study, we aimed to examine the relationship between lifestyle factors and the composition and diversity of the oral microbiome in individuals living in Korea. We collected 43 saliva samples from Korean individuals and analyzed the oral microbiome and its variations due to external factors, such as coffee consumption, drinking, and smoking. Linear discriminant analysis effect size revealed that Oribacterium, Campylobacter, and Megasphaera were abundant in coffee consumers, whereas Saccharimonadales, Clostridia, and Catonella were abundant in alcohol non-drinkers. We found increased levels of Stomatobaculum in the saliva of smokers, compared with that of non-smokers. Thus, our analysis revealed characteristic microorganisms for each parameter that was evaluated (coffee consumption, smoking, drinking). Consequently, our study provides insight into the oral microbiome in the Korean population and lays the foundation for developing the Korean Forensic Microbiome Database.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.

Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884 ng/µL and 23.767 ng/µL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.

Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain­RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Functional human IgA targets a conserved site on malaria sporozoites.

Immunoglobulin (Ig)A antibodies play a critical role in protection against mucosal pathogens. However, the role of serum IgA in immunity to nonmucosal pathogens, such as Plasmodium falciparum, is poorly characterized, despite being the second most abundant isotype in blood after IgG. Here, we investigated the circulating IgA response in humans to P. falciparum sporozoites that are injected into the skin by mosquitoes and migrate to the liver via the bloodstream to initiate malaria infection. We found that circulating IgA was induced in three independent sporozoite-exposed cohorts: individuals living in an endemic region in Mali, malaria-naïve individuals immunized intravenously with three large doses of irradiated sporozoites, and malaria-naïve individuals exposed to a single controlled mosquito bite infection. Mechanistically, we found evidence in an animal model that IgA responses were induced by sporozoites at dermal inoculation sites. From malaria-resistant individuals, we isolated several IgA monoclonal antibodies that reduced liver parasite burden in mice. One antibody, MAD2-6, bound to a conserved epitope in the amino terminus of the P. falciparum circumsporozoite protein, the dominant protein on the sporozoite surface. Crystal structures of this antibody revealed a unique mode of binding whereby two Fabs simultaneously bound either side of the target peptide. This study reveals a role for circulating IgA in malaria and identifies the amino terminus of the circumsporozoite protein as a target of functional antibodies.

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Commensal microbiota drive the functional diversification of colon macrophages.

Mononuclear phagocytes are a heterogeneous population of leukocytes essential for immune homeostasis that develop tissue-specific functions due to unique transcriptional programs driven by local microenvironmental cues. Single cell RNA sequencing (scRNA-seq) of colonic myeloid cells from specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice revealed extensive heterogeneity of both colon macrophages (MPs) and dendritic cells (DCs). Modeling of developmental pathways combined with inference of gene regulatory networks indicate two major trajectories from common CCR2+ precursors resulting in colon MP populations with unique transcription factors and downstream target genes. Compared to SPF mice, GF mice had decreased numbers of total colon MPs, as well as selective proportional decreases of two major CD11c+CD206intCD121b+ and CD11c-CD206hiCD121b- colon MP populations, whereas DC numbers and proportions were not different. Importantly, these two major colon MP populations were clearly distinct from other colon MP populations regarding their gene expression profile, localization within the lamina propria (LP) and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations at the molecular level and highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.

Mitochondria ubiquitin ligase, MARCH5 resolves hepatitis B virus X protein aggregates in the liver pathogenesis.

Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.

Defective IgA response to atypical intestinal commensals in IL-21 receptor deficiency reshapes immune cell homeostasis and mucosal immunity.

Despite studies indicating the effects of IL-21 signaling in intestinal inflammation, its roles in intestinal homeostasis and infection are not yet clear. Here, we report potent effects of commensal microbiota on the phenotypic manifestations of IL-21 receptor deficiency. IL-21 is produced highly in the small intestine and appears to be critical for mounting an IgA response against atypical commensals such as segmented filamentous bacteria and Helicobacter, but not to the majority of commensals. In the presence of these atypical commensals, IL-21R-deficient mice exhibit reduced numbers of germinal center and IgA+ B cells and expression of activation-induced cytidine deaminase in Peyer's patches as well as a significant decrease in small intestine IgA+ plasmablasts and plasma cells, leading to higher bacterial burdens and subsequent expansion of Th17 and Treg cells. These microbiota-mediated secondary changes in turn enhance T cell responses to an oral antigen and strikingly dampen Citrobacter rodentium-induced immunopathology, demonstrating a complex interplay between IL-21-mediated mucosal immunity, microbiota, and pathogens.

PKconverter: R package to convert the pharmacokinetic parameters.

Population pharmacokinetic analysis and modeling procedures typically require estimates of both population and individual pharmacokinetic parameters. However, only some of these parameters are contained in models and only parameters in the model can be estimated. In this paper, we introduce a new R package, PKconverter, to calculate pharmacokinetic parameters using the relationships among them. After fitting the model, other parameters can be calculated from the functional relationship among the parameters. PKconverter provides the functions to calculate whole parameters along with a Shiny application for converting the parameters. With this package, it is also possible to calculate the standard errors of the other parameters that are not in the model and estimate individual parameters simultaneously.

The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling.

Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5(+/-) mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5(H43W)), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions.

The role of type I interferons in intestinal infection, homeostasis, and inflammation.

Type I interferons are a widely expressed family of effector cytokines that promote innate antiviral and antibacterial immunity. Paradoxically, they can also suppress immune responses by driving production of anti-inflammatory cytokines, and dysregulation of these cytokines can contribute to host-mediated immunopathology and disease progression. Recent studies describe their anti-inflammatory role in intestinal inflammation and the locus containing IFNAR, a heterodimeric receptor for the type I interferons has been identified as a susceptibility region for human inflammatory bowel disease. This review focuses on the role of type I IFNs in the intestine in health and disease and their emerging role as immune modulators. Clear understanding of type I IFN-mediated immune responses may provide avenues for fine-tuning existing IFN treatment for infection and intestinal inflammation.

Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

Resistance to inhibitors of cholinesterase (Ric)-8A and Gαi contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps)34 activity.

Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gαi subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.

The loss of RGS protein-Gα(i2) interactions results in markedly impaired mouse neutrophil trafficking to inflammatory sites.

Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gα(i) subunits to exchange GDP for GTP. By limiting the duration that Gα(i) subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gα(i2) interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gα(i) activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.

The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells.

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.

Beta-agonist-associated reduction in RGS5 expression promotes airway smooth muscle hyper-responsiveness.

Although short-acting and long-acting inhaled ß(2)-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent ß(2)-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to ß(2)-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cß and myosin light chain). In contrast, ß-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5(-/-) mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive ß(2)-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.

Ric-8A and Gi alpha recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle.

In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein alpha (G alpha) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G alpha(i) function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G alpha(i), thus preventing its GEF activity for G alpha(i). Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G alpha(i) expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.

HBx targeting to mitochondria and ROS generation are necessary but insufficient for HBV-induced cyclooxygenase-2 expression.

Chronic inflammation can be a major risk factor for cancer development and may contribute to the high worldwide incidence of hepatocellular carcinoma (HCC). Cyclooxygenase-2 (COX-2) is known to be an important mediator of inflammatory responses; however, its link to hepatitis B virus (HBV)-mediated inflammatory responses has not been established. Here, we demonstrate that the expression of COX-2 mRNA and protein was significantly elevated in cells transfected by HBV replicon but not in cells transfected by HBV genome lacking the HBx gene. Notably, COX-2 induction was correlated with HBx's ability to increase reactive oxygen species (ROS) levels. Consistently with this, antioxidant treatment and ectopic expression of manganese superoxide dismutase or catalase completely abolished COX-2 induction. Interestingly, a mitochondria localization-defective mutant of HBx (HBx(Delta 68-117)) neither increased intracellular ROS levels nor induced COX-2 expression. HBx(68-117), which encodes only amino acids 68-117 and is sufficient for mitochondria localization, increased ROS levels but did not induce COX-2 expression. Similarly, HBx targeting to the outer membrane of mitochondria (Mito-HBx) increased ROS but also failed to increase COX-2 expression, suggesting that other cytoplasmic signaling pathways are involved in HBx-mediated COX-2 induction. Indeed, inhibition of cytoplasmic calcium signaling by cyclosporine A, blocking mitochondrial permeability transition pore, and herbimycin, and inhibition of calcium-dependent tyrosine kinase suppressed HBV-mediated COX-2 induction. Thus, the data indicate that both mitochondrial ROS and cytoplasmic calcium signaling are necessary for the COX-2 induction. Our studies revealed a pathophysiological link between HBV infection and hepatic inflammation, and this chain of events might contribute to early steps in HBV-associated liver carcinogenesis.

Depletion of BubR1 promotes premature centrosomal localization of cyclin B1 and accelerates mitotic entry.

The role of BubR1 has been established mainly in mitosis as an essential mitotic checkpoint protein although it is expressed throughout the cell cycle. To explore a possible role of BubR1 in regulating the G(2) phase of cell cycle, we have employed siRNA-mediated hBubR1 knockdown in HeLa cells. Here, we demonstrate that reducing BubR1 levels during the G(2) phase causes accelerated mitotic entry. As expected, BubR1 depletion leads to degradation of cyclin B(1) in the G(2) phase. Intriguingly, cyclin B(1) is prematurely targeted to centrosomes appearing at early G(2) phase in BubR1-depleted cells despite its low levels. This is in contrast to control cells where cyclin B(1) appears at the centrosomes in early prophase based on cell cycle-specific localization of CENP-F. Furthermore, cyclin B/Cdk1 kinase activity in early G(2) is aberrantly high in BubR1-depleted cells. Together, our results indicate that hBubR1 depletion triggers premature centrosomal localization of cyclin B(1) probably leading to premature mitotic entry. This study is the first to suggest a role of hBubR1 in controlling centrosome targeting of cyclin B(1) and timing of mitotic entry.