Isolation may select for earlier and higher peak viral load but shorter duration in SARS-CoV-2 evolution.

During the COVID-19 pandemic, human behavior change as a result of nonpharmaceutical interventions such as isolation may have induced directional selection for viral evolution. By combining previously published empirical clinical data analysis and multi-level mathematical modeling, we find that the SARS-CoV-2 variants selected for as the virus evolved from the pre-Alpha to the Delta variant had earlier and higher peak in viral load dynamics but a shorter duration of infection. Selection for increased transmissibility shapes the viral load dynamics, and the isolation measure is likely to be a driver of these evolutionary transitions. In addition, we show that a decreased incubation period and an increased proportion of asymptomatic infection are also positively selected for as SARS-CoV-2 mutated to adapt to human behavior (i.e., Omicron variants). The quantitative information and predictions we present here can guide future responses in the potential arms race between pandemic interventions and viral evolution.

Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair.

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.

Evaluation of COVID-19 epidemic outbreak caused by temporal contact-increase in South Korea.

OBJECTIVES: On March 15, 2020, 61.3% of the confirmed cases of COVID-19 infection in South Korea are associated with the worship service that was organized on February 9 in the Shincheonji Church of Jesus in Daegu. We aim to evaluate the effects of mass infection in South Korea and assess the preventive control intervention. METHOD: Using openly available data of daily cumulative confirmed cases and deaths, the basic and effective reproduction numbers was estimated using a modified susceptible-exposed-infected-recovered-type epidemic model. RESULTS: The basic reproduction number was estimated to be R0=1.77. The effective reproduction number increased approximately 20 times after the mass infections from the 31 st patient, which was confirmed on February 9 in the Shincheonji Church of Jesus, Daegu. However, the effective reproduction number decreased to less than unity after February 28 owing to the implementation of high-level preventive control interventions in South Korea, coupled with voluntary prevention actions by citizens. CONCLUSION: Preventive action and control intervention were successfully established in South Korea.

MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes.

Deubiquitinating enzymes (DUBs) remove ubiquitin (Ub) from Ub-conjugated substrates to regulate the functional outcome of ubiquitylation. Here we report the discovery of a new family of DUBs, which we have named MINDY (motif interacting with Ub-containing novel DUB family). Found in all eukaryotes, MINDY-family DUBs are highly selective at cleaving K48-linked polyUb, a signal that targets proteins for degradation. We identify the catalytic activity to be encoded within a previously unannotated domain, the crystal structure of which reveals a distinct protein fold with no homology to any of the known DUBs. The crystal structure of MINDY-1 (also known as FAM63A) in complex with propargylated Ub reveals conformational changes that realign the active site for catalysis. MINDY-1 prefers cleaving long polyUb chains and works by trimming chains from the distal end. Collectively, our results reveal a new family of DUBs that may have specialized roles in regulating proteostasis.

Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations.

Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6-AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.

AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity.

CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

Phosphorylation and ubiquitination-dependent degradation of CABIN1 releases p53 for transactivation upon genotoxic stress.

CABIN1 acts as a negative regulator of p53 by keeping p53 in an inactive state on chromatin. Genotoxic stress causes rapid dissociation of CABIN1 and activation of p53. However, its molecular mechanism is still unknown. Here, we reveal the phosphorylation- and ubiquitination-dependent degradation of CABIN1 upon DNA damage, releasing p53 for transcriptional activation. The DNA-damage-signaling kinases, ATM and CHK2, phosphorylate CABIN1 and increase the degradation of CABIN1 protein. Knockdown or overexpression of these kinases influences the stability of CABIN1 protein showing that their activity is critical for degradation of CABIN1. Additionally, CABIN1 was found to undergo ubiquitin-dependent proteasomal degradation mediated by the CRL4DDB2 ubiquitin ligase complex. Both phosphorylation and ubiquitination of CABIN1 appear to be relevant for controlling the level of CABIN1 protein upon genotoxic stress.

O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network.

O-linked-N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of diverse cellular processes, but its role in embryonic stem cells (ESCs) and pluripotency has not been investigated. Here we show that O-GlcNAcylation directly regulates core components of the pluripotency network. Blocking O-GlcNAcylation disrupts ESC self-renewal and reprogramming of somatic cells to induced pluripotent stem cells. The core reprogramming factors Oct4 and Sox2 are O-GlcNAcylated in ESCs, but the O-GlcNAc modification is rapidly removed upon differentiation. O-GlcNAc modification of threonine 228 in Oct4 regulates Oct4 transcriptional activity and is important for inducing many pluripotency-related genes, including Klf2, Klf5, Nr5a2, Tbx3, and Tcl1. A T228A point mutation that eliminates this O-GlcNAc modification reduces the capacity of Oct4 to maintain ESC self-renewal and reprogram somatic cells. Overall, our study makes a direct connection between O-GlcNAcylation of key regulatory transcription factors and the activity of the pluripotency network.

Cabin1 restrains p53 activity on chromatin.

The tumor suppressor p53 has been proposed to bind target promoters upon genotoxic stress. However, recent evidence shows that p53 occupies some target promoters without such stress, suggesting that a negative regulator might render p53 transcriptionally inactive on these promoters. Here we show that calcineurin binding protein 1 (Cabin1) is a negative regulator of p53. Downregulation of Cabin1 induces activation of a subset of p53 target genes. Cabin1 physically interacts with p53 on these target promoters and represses p53 transcriptional activity in the absence of genotoxic stress, by regulating histone modification and p53 acetylation marks. Knockdown of Cabin1 retards cell growth and promotes cell death after DNA damage in a p53-dependent manner. Thus, Cabin1 inhibits p53 function on chromatin in the quiescent state; the presence of inactive p53 on some promoters might allow a prompt response upon DNA damage.

Fermented guava leaf extract inhibits LPS-induced COX-2 and iNOS expression in Mouse macrophage cells by inhibition of transcription factor NF-kappaB.

The goal of this study was to elucidate the antiinflammatory activities of Psidium guajava L. (guava) leaf. To improve the functionality of guava leaf, it was fermented with Phellinus linteus mycelia, Lactobacillus plantarum and Saccharomyces cerevisiae. The ethanol extract from fermented guava leaf inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Western blot analysis showed that fermented guava leaf extract decreased LPS-induced inducible nitric oxide synthase (iNOS) and the cyclooxygenase-2 (COX-2) protein level in RAW 264.7 cells. To investigate the mechanism involved, the study examined the effect of fermented guava leaf extract on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. Fermented guava leaf extract significantly inhibited LPS-induced NF-kappaB transcriptional activity. Immunochemical analysis revealed that fermented guava leaf extract suppressed LPS-induced degradation of I-kappaBalpha. Taken together, the data indicate that fermented guava leaf extract is involved in the inhibition of iNOS and COX-2 via the down-regulation of NF-kappaB pathway, revealing a partial molecular basis for the antiinflammatory properties of fermented guava leaf extract.

Petalonia improves glucose homeostasis in streptozotocin-induced diabetic mice.

The anti-diabetic potential of Petalonia binghamiae extract (PBE) was evaluated in vivo. Dietary administration of PBE to streptozotocin (STZ)-induced diabetic mice significantly lowered blood glucose levels and improved glucose tolerance. The mode of action by which PBE attenuated diabetes was investigated in vitro using 3T3-L1 cells. PBE treatment stimulated 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation. At the molecular level, peroxisome proliferator-activated receptor gamma (PPARgamma) and terminal marker protein aP2, as well as the mRNA of GLUT4 were up-regulated by PBE. In mature adipocytes, PBE significantly stimulated the uptake of glucose and the expression of insulin receptor substrate-1 (IRS-1). Furthermore, PBE increased PPARgamma luciferase reporter gene activity in COS-1 cells. Taken together, these results suggest that the in vivo anti-diabetic effect of PBE is mediated by both insulin-like and insulin-sensitizing actions in adipocytes.

Glycation inhibitory activity and the identification of an active compound in Plantago asiatica extract.

The glycation reaction involves a series of non-enzymatic reactions between the carbonyl group on reducing sugars and the amino group on proteins leading to the formation of advanced glycation end-products (AGEs), which are acknowledged to be involved in the pathogenesis of diabetic and aging-related complications. Consequently, the development of AGE inhibitors is considered to have therapeutic potential in patients with diabetes or age-related diseases. The preliminary results showed that a methanol extract (PAE) of Plantago asiatica, which is traditionally used as a folk medicine in Asian countries to treat fever, cough, wound etc., had strong glycation inhibitory activity. The effects of the extract on AGE fluorescence were dose-dependent, reaching 41% inhibition at 0.1 microg/mL of extract. The purified principle from PAE was identified as plantamajoside. As well as antioxidant activities, in vitro glycation inhibitory activities with 10 and 25 mm plantamajoside were higher than those with 10 and 25 mm aminoguanidine. The results demonstrate that PAE and plantamajoside had significant effects on in vitro AGE formation, and the glycation inhibitory activity and antioxidant activity of plantamajoside were comparable to those obtained using millimolar concentrations of the standard antiglycation agent aminoguanidine, and the antioxidant ascorbate, respectively.

Nobiletin from citrus fruit peel inhibits the DNA-binding activity of NF-kappaB and ROS production in LPS-activated RAW 264.7 cells.

Post-treatment with nobiletin (5,6,7,8,3',4'-hexamethoxy flavone), which was purified from the fruit peel of Citrus sunki Hort. ex Tanaka, at concentration 6-50 microM significantly suppressed NF-kappaB transcriptional activation, NO and PGE(2) production, and iNOS and COX-2 protein expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Nobiletin inhibited neither LPS-induced phosphorylation/degradation of inhibitory kappaB-alpha nor LPS-induced nuclear translocation of NF-kappaB. However, it interrupted the DNA-binding activity of activated NF-kappaB. As reactive oxygen species (ROS) are also known to regulate the activation of NF-kappaB, we tested the effect of nobiletin on LPS-induced ROS generation. Nobiletin significantly inhibited LPS-induced intracellular ROS production in RAW 264.7 cells. These results suggest that nobiletin may exert an anti-inflammatory effect through the interruption of NF-kappaB DNA-binding activity and the suppression of ROS generation.

Involvement of extracellular signal-regulated kinase in nobiletin-induced melanogenesis in murine B16/F10 melanoma cells.

Nobiletin contributes to pharmacological activities such as anti-cancer and anti-inflammatory effects, but little is known about its effect on melanogenesis. In this study, we found that nobiletin increased melanin content and tyrosinase activity in murine B16/F10 melanoma cells. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK) pathway with U0216 resulted in inhibition of nobiletin-induced melanin synthesis and tyrosinase expression.

Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits.

We investigated the correlation between the flavonoid content and NO production inhibitory activity of fruit peel extracts using 20 citrus plants. The contents of seven flavonoids (naringin, naringenin, hesperidin, hesperetin, rutin, nobiletin, and tangeretin) were determined by HPLC analysis. Each citrus peel extract varied in flavonoid content, but the contents of nobiletin and tangeretin, which were contained in all 20 fruit peels, showed a positive and significant correlation with each other (r=0.879, p<0.0005 for immature fruit peels; r=0.858, p<0.0005 for mature fruit peels). All citrus peel extracts dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. This inhibitory effect was significantly and positively correlated with the content of nobiletin and tangeretin. Nobiletin showed a more potent NO production inhibitory activity (IC50=26.5 microM) compared to tangeretin (IC50=136.6 microM). This result supports the premise that nobiletin-rich citrus may provide protection against disease resulting from excessive NO production.