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Introduction Osteoarthritis (OA) is a common joint disease that can cause gradual disability among the aging population. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor that regulates the expression of phase II antioxidant enzymes that provide protection against oxidative stress and tissue damage. The use of histone deacetylase inhibitors (HDACi) has emerged as a potential therapeutic strategy for various diseases. They have displayed chondroprotective effects in various animal models of arthritis. Previous studies have established that Nrf2 acetylation enhances Nrf2 functions. Here we explore the role of Nrf2 in the development of OA and the involvement of Nrf2 acetylation in HDACi protection of OA. Methods Two OA models—monosodium iodoacetate (MIA) articular injection and destabilization of the medial meniscus (DMM)—were used with wild-type (WT) and Nrf2-knockout (Nrf2-KO) mice to demonstrate the role of Nrf2 in OA progression. A pan-HDACi, trichostatin A (TSA), was administered to examine the effectiveness of HDACi on protection from cartilage damage. The histological sections were scored. The expression of OA-associated matrix metalloproteinases (MMPs) 1, 3, and 13 and proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were assayed. The effectiveness of HDACi on OA protection was compared between WT and Nrf2-KO mice. Results Nrf2-KO mice displayed more severe cartilage damage in both the MIA and DMM models. TSA promoted the induction of Nrf2 downstream proteins in SW1353 chondrosarcoma cells and in mouse joint tissues. TSA also reduced the expression of OA-associated proteins MMP1, MMP3, and MMP13 and proinflammatory cytokines TNF-α, IL-1β, and IL-6. TSA markedly reduced the cartilage damage in both OA models but offered no significant protection in Nrf2-KO mice. Conclusions Nrf2 has a major chondroprotective role in progression of OA and is a critical molecule in HDACi-mediated OA protection.

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.

Plant cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR 4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature. Plant cryptochromes are regulated by blue-light dependent phosphorylation. Here the authors map the in vivo phosphorylation sites of Arabidopsis cryptochrome 2 and identify four closely related kinases that act to both activate and destabilize the receptor in response to blue light.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.

HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope-knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3' RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3'-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.

The zinc finger antiviral protein (ZAP) is a mammalian host restriction factor that inhibits the replication of a variety of RNA viruses, including retroviruses, alphaviruses and filoviruses, through interaction with the ZAP-responsive elements (ZRE) in viral RNA, and recruiting the exosome to degrade RNA substrate. Hepatitis B virus (HBV) is a pararetrovirus that replicates its genomic DNA via reverse transcription of a viral pregenomic (pg) RNA precursor. Here, we demonstrate that the two isoforms of human ZAP (hZAP-L and -S) inhibit HBV replication in human hepatocyte-derived cells through posttranscriptional down-regulation of viral pgRNA. Mechanistically, the zinc finger motif-containing N-terminus of hZAP is responsible for the reduction of HBV RNA, and the integrity of the four zinc finger motifs is essential for ZAP to bind to HBV RNA and fulfill its antiviral function. The ZRE sequences conferring the susceptibility of viral RNA to ZAP-mediated RNA decay were mapped to the terminal redundant region (nt 1820-1918) of HBV pgRNA. In agreement with its role as a host restriction factor and as an innate immune mediator for HBV infection, ZAP was upregulated in cultured primary human hepatocytes and hepatocyte-derived cells upon IFN-α treatment or IPS-1 activation, and in the livers of hepatitis B patients during immune active phase. Knock down of ZAP expression increased the level of HBV RNA and partially attenuated the antiviral effect elicited by IPS-1 in cell cultures. In summary, we demonstrated that ZAP is an intrinsic host antiviral factor with activity against HBV through down-regulation of viral RNA, and that ZAP plays a role in the innate control of HBV replication. Our findings thus shed light on virus-host interaction, viral pathogenesis, and antiviral approaches.

The exceptionally rich fossil record available for the equid family has provided textbook examples of macroevolutionary changes. Horses, asses, and zebras represent three extant subgenera of Equus lineage, while the Sussemionus subgenus is another remarkable Equus lineage ranging from North America to Ethiopia in the Pleistocene. We sequenced 26 archaeological specimens from Northern China in the Holocene that could be assigned morphologically and genetically to Equus ovodovi , a species representative of Sussemionus . We present the first high-quality complete genome of the Sussemionus lineage, which was sequenced to 13.4× depth of coverage. Radiocarbon dating demonstrates that this lineage survived until ~3500 years ago, despite continued demographic collapse during the Last Glacial Maximum and the great human expansion in East Asia. We also confirmed the Equus phylogenetic tree and found that Sussemionus diverged from the ancestor of non-caballine equids ~2.3–2.7 million years ago and possibly remained affected by secondary gene flow post-divergence. We found that the small genetic diversity, rather than enhanced inbreeding, limited the species’ chances of survival. Our work adds to the growing literature illustrating how ancient DNA can inform on extinction dynamics and the long-term resilience of species surviving in cryptic population pockets.

Chickens represent by far the most important poultry species, yet the number, locations, and timings of their domestication have remained controversial for more than a century. Here we report ancient mitochondrial DNA sequences from the earliest archaeological chicken bones from China, dating back to ∼10,000 B.P. The results clearly show that all investigated bones, including the oldest from the Nanzhuangtou site, are derived from the genus Gallus , rather than any other related genus, such as Phasianus . Our analyses also suggest that northern China represents one region of the earliest chicken domestication, possibly dating as early as 10,000 y B.P. Similar to the evidence from pig domestication, our results suggest that these early domesticated chickens contributed to the gene pool of modern chicken populations. Moreover, our results support the idea that multiple members of the genus Gallus , specifically Gallus gallus and Gallus sonneratii contributed to the gene pool of the modern domestic chicken. Our results provide further support for the growing evidence of an early mixed agricultural complex in northern China. Significance Ancient DNA analysis is a powerful tool to reveal the geographical origins of domesticated species. Here we obtained ancient mtDNA sequences from the earliest archaeological chicken bones from northern China as early as 10,000 y ago. Combined analyses of our ancient sequences with a large dataset of published modern and ancient chicken mtDNA sequences suggest that northern China was likely one of several regions of chicken domestication and provide further insights into the process of human-mediated spread of chickens across the globe. Our results not only suggest that the oldest archaeological chicken bones recovered so far are indeed from ancestors of domestic chickens, but also provide further evidence for one of the earliest, mixed agricultural complexes in the world.

Goats are one of the most widespread farmed animals across the world; however, their migration route to East Asia and local evolutionary history remain poorly understood. Here, we sequenced 27 ancient Chinese goat genomes dating from the Late Neolithic period to the Iron Age. We found close genetic affinities between ancient and modern Chinese goats, demonstrating their genetic continuity. We found that Chinese goats originated from the eastern regions around the Fertile Crescent, and we estimated that the ancestors of Chinese goats diverged from this population in the Chalcolithic period. Modern Chinese goats were divided into a northern and a southern group, coinciding with the most prominent climatic division in China, and two genes related to hair follicle development, FGF5 and EDA2R, were highly divergent between these populations. We identified a likely causal de novo deletion near FGF5 in northern Chinese goats that increased to high frequency over time, whereas EDA2R harbored standing variation dating to the Neolithic. Our findings add to our understanding of the genetic composition and local evolutionary process of Chinese goats.

Mold flux serves the crucial metallurgical function of absorbing inclusions, directly impacting the smoothness of the casting process as well as the cast slab quality. In this study, the dissolution behavior and mechanism of TiO 2 and TiN inclusions in molten CaO–SiO 2– B 2 O 3 -based fluorine-free mold flux were explored by in situ single hot thermocouple technology combined with X-ray photoelectron spectroscopy. The results showed that TiO 2 inclusions are effectively dissolved by the molten slag within 76 s, during which the original octahedral [TiO 6 ] 8− structures are destroyed and convert to the networker tetrahedral [TiO 4 ] 4− structures. However, the dissolution rate is much lower for TiN inclusions than for TiO 2 inclusions. This can be attributed to the fact that the TiN particles need to be oxidized and then dissolved in the molten slag to form tetrahedral [TiO 4 ] 4− and octahedral [TiO 6 ] 8− structures during the TiN inclusion dissolution process, which is accompanied by the generation of a large amount of N 2 gas. Moreover, CaTiO 3 crystals tend to nucleate and grow on bubble surfaces with sufficient octahedral [TiO 6 ] 8− structures and Ca 2+ ions, eventually resulting in the molten slag being in a solid–liquid mixed state.