Phylogeny and Comparative Analysis for the Plastid Genomes of Five Tulipa (Liliaceae).

Species of Tulipa (Liliaceae) are of great horticultural importance and are distributed across Europe, North Africa, and Asia. The Tien Shan Mountain is one of the primary diversity centres of Tulipa, but the molecular studies of Tulipa species from this location are lacking. In our study, we assembled four Tulipa plastid genomes from the Tien Shan Mountains, T. altaica, T. iliensis, T. patens, and T. thianschanica, combined with the plastid genome of T. sylvestris to compare against other Liliaceae plastid genomes. We focussed on the species diversity and evolution of their plastid genomes. The five Tulipa plastid genomes proved highly similar in overall size (151,691-152,088 bp), structure, gene order, and content. With comparative analysis, we chose 7 mononucleotide SSRs from the Tulipa species that could be used in further population studies. Phylogenetic analyses based on 24 plastid genomes robustly supported the monophyly of Tulipa and the sister relationship between Tulipa and Amana, Erythronium. T. iliensis, T. thianschanica, and T. altaica were clustered together, and T. patens was clustered with T. sylvestris, with our results clearly demonstrating the relationships between these five Tulipa species. Our results provide a more comprehensive understanding of the phylogenomics and comparative genomics of Tulipa.

Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19.

Within a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 100 million people worldwide with a death toll over 2 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Å and 2.6 Å respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. The tightly closed conformation is stabilized by fatty acid and polysorbate 80 binding at the receptor binding domains (RBDs) and the N terminal domains (NTDs) respectively. Additionally, we identified an important pH switch in the WT S-Trimer that shows dramatic conformational change and accounts for its increased stability at lower pH. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.IMPORTANCEEffective vaccine against SARS-CoV-2 is critical to end the COVID-19 pandemic. Here, using Trimer-Tag technology, we are able to produce stable and large quantities of WT S-Trimer, a subunit vaccine candidate for COVID-19 with high safety and efficacy from animal and Phase 1 clinical trial studies. Cryo-EM structures of the S-Trimer subunit vaccine candidate show that it predominately adopts tightly closed pre-fusion state, and resembles that of the native and full-length spike in detergent, confirming its structural integrity. WT S-Trimer is currently being evaluated in global Phase 2/3 clinical trial. Combining with published structures of the S protein, we also propose a model to dissect the conformation change of the spike protein before receptor binding.

Comparative analysis of complete plastid genomes from Lilium lankongense Franchet and its closely related species and screening of Lilium-specific primers.

Lilium lankongense Franchet is a lily species found on the Qinghai-Tibet Plateau. It is pink with deep red spots, has a high ornamental value, and is used in hybrid breeding of horticultural lily varieties. We have insufficient knowledge of the genetic resources of L. lankongense and its phylogenetic relationships with related species. Recent molecular phylogenetic studies have shown a very close phylogenetic relationship between L. lankongense and the five species L. duchartrei, L. stewartianum, L. matangense, L. lophophorum, and L. nanum. However, molecular markers still lack sufficient signals for population-level research of the genus Lilium. We sequenced and compared the complete plastid sequences of L. lankongense and its five related species. The genomes ranged from 152,307 bp to 152,611 bp. There was a slight inconsistency detected in inverted repeat and single copy boundaries and there were 53 to 63 simple sequence repeats in the six species. Two of the 12 highly variable regions (trnC-petN and rpl32-trnL) were verified in 11 individuals and are promising for population-level studies. We used the complete sequence of 33 plastid genomes, the protein-coding region sequence, and the nuclear ITS sequence to reconstruct the phylogenetic tree of Lilium species. Our results showed that the plastid gene tree and nuclear gene tree were not completely congruent, which may be caused by hybridization, insufficient information contained in the nuclear ITS, or the small number of samples. The results of phylogenetic analysis based on plastid genomes indicated that the six Lilium species were closely related. Our study provides a preliminarily rebuilt backbone phylogeny that is significant for future molecular and morphological studies of Lilium.

S-Trimer, a COVID-19 subunit vaccine candidate, induces protective immunity in nonhuman primates.

SARS-CoV-2 is the underlying cause for the COVID-19 pandemic. Like most enveloped RNA viruses, SARS-CoV-2 uses a homotrimeric surface antigen to gain entry into host cells. Here we describe S-Trimer, a native-like trimeric subunit vaccine candidate for COVID-19 based on Trimer-Tag technology. Immunization of S-Trimer with either AS03 (oil-in-water emulsion) or CpG 1018 (TLR9 agonist) plus alum adjuvants induced high-level of neutralizing antibodies and Th1-biased cellular immune responses in animal models. Moreover, rhesus macaques immunized with adjuvanted S-Trimer were protected from SARS-CoV-2 challenge compared to vehicle controls, based on clinical observations and reduction of viral loads in lungs. Trimer-Tag may be an important platform technology for scalable production and rapid development of safe and effective subunit vaccines against current and future emerging RNA viruses.

Insights into phylogeny, age and evolution of Allium (Amaryllidaceae) based on the whole plastome sequences.

BACKGROUND AND AIMS: The genus Allium L., one of the largest monocotyledonous genera and one that includes many economically important crops with nutritional and medicinal value, has been the focus of classification or phylogeny studies for centuries. Recent studies suggested that the genus can be divided into 15 subgenera and 72 sections, which were further classified into three evolutionary lineages. However, the phylogenetic relationships reconstructed by one or two loci showed weaker support, especially for the third evolutionary lineage, which might not show the species relationships very clearly and could hinder further adaptive and evolutionary study. METHODS: In this study, a total of 39 complete chloroplast genomes of Allium (covering 12 Allium subgenera) were collected, and combining these with 125 species of plastomes from 19 other families of monocots, we reconstructed the phylogeny of the genus Allium, estimated the origin and divergence time of the three evolutionary lineages and investigated the adaptive evolution in this genus and related families. RESULTS: Our phylogenetic analysis confirmed the monophyly and three evolutionary lineages of Allium, while new species relationships were detected within the third evolutionary lineage. The divergence time of the three evolutionary lineages was estimated to be in the early Eocene to the middle Miocene, and numerous positive selected genes (PSGs) and PSGs with high average Ka/Ks values were found in Allium species. CONCLUSIONS: Our results detected a well-supported phylogenetic relationship of Allium. The PSGs and PSGs with high Ka/Ks values, as well as diversified morphologies, complicated chromosome characteristics and unique reproductive modes may play important roles in the adaptation and evolution of Allium species. This is the first study that conducted phylogenetic and evolutionary analyses on the genus Allium combined with the plastome and morphological and cytological data. We hope that this study can contribute to further analysis of Allium for other researchers.

Author Correction: Improvement of Pharmacokinetic Profile of TRAIL via Trimer-Tag Enhances its Antitumor Activity in vivo.

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Improvement of Pharmacokinetic Profile of TRAIL via Trimer-Tag Enhances its Antitumor Activity in vivo.

TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) has long been considered a tantalizing target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors DR4 or DR5. Despite initial promise, both recombinant human TRAIL (native TRAIL) and dimeric DR4/DR5 agonist monoclonal antibodies (mAbs) failed in multiple human clinical trials. Here we show that in-frame fusion of human C-propeptide of α1(I) collagen (Trimer-Tag) to the C-terminus of mature human TRAIL leads to a disulfide bond-linked homotrimer which can be expressed at high levels as a secreted protein from CHO cells. The resulting TRAIL-Trimer not only retains similar bioactivity and receptor binding kinetics as native TRAIL in vitro which are 4-5 orders of magnitude superior to that of dimeric TRAIL-Fc, but also manifests more favorable pharmacokinetic and antitumor pharmacodynamic profiles in vivo than that of native TRAIL. Taken together, this work provides direct evidence for the in vivo antitumor efficacy of TRAIL being proportional to systemic drug exposure and suggests that the previous clinical failures may have been due to rapid systemic clearance of native TRAIL and poor apoptosis-inducing potency of dimeric agonist mAbs despite their long serum half-lives.

Cationic Polystyrene Resolves Nonalcoholic Steatohepatitis, Obesity, and Metabolic Disorders by Promoting Eubiosis of Gut Microbiota and Decreasing Endotoxemia.

A pandemic of metabolic diseases, consisting of type 2 diabetes, nonalcoholic fatty liver disease, and obesity, has imposed critical challenges for societies worldwide, prompting investigation of underlying mechanisms and exploration of low-cost and effective treatment. In this report, we demonstrate that metabolic disorders in mice generated by feeding with a high-fat diet without dietary vitamin D can be prevented by oral administration of polycationic amine resin. Oral administration of cholestyramine, but not the control uncharged polystyrene, was able to sequester negatively charged bacterial endotoxin in the gut, leading to 1) reduced plasma endotoxin levels, 2) resolved systemic inflammation and hepatic steatohepatitis, and 3) improved insulin sensitivity. Gut dysbiosis, characterized as an increase of the phylum Firmicutes and a decrease of Bacteroidetes and Akkermansia muciniphila, was fully corrected by cholestyramine, indicating that the negatively charged components in the gut are critical for the dysbiosis. Furthermore, fecal bacteria transplant, derived from cholestyramine-treated animals, was sufficient to antagonize the metabolic disorders of the recipient mice. These results indicate that the negatively charged components produced by dysbiosis are critical for biogenesis of metabolic disorders and also show a potential application of cationic polystyrene to treat metabolic disorders through promoting gut eubiosis.

Cathepsin H-Mediated Degradation of HDAC4 for Matrix Metalloproteinase Expression in Hepatic Stellate Cells: Implications of Epigenetic Suppression of Matrix Metalloproteinases in Fibrosis through Stabilization of Class IIa Histone Deacetylases.

In three-dimensional extracellular matrix, mesenchymal cells including hepatic stellate cells (HSCs) gain the ability to express matrix metalloproteinases (MMPs) on injury signals. In contrast, in myofibroblastic HSCs in fibrotic liver, many MMP genes are silenced into an epigenetically nonpermissive state. The mechanism by which the three-dimensional extracellular matrix confers the MMP genes into an epigenetically permissive state has not been well characterized. In continuation of previous work, we show here that the up-regulation of MMP genes is mediated through degradation of class IIa histone deacetylases (HDACs) by certain cysteine cathepsins (Cts). In three-dimensional extracellular matrix culture, CtsH, among other cysteine cathepsins, was up-regulated and localized as puncta in the nuclear and cytoplasmic compartments in a complex with HDAC4 for its degradation. Conversely, along with HSC trans-differentiation, CtsH and CtsL were progressively down-regulated, whereas HDAC4 was concurrently stabilized. The inhibition of cysteine cathepsins by specific proteinase inhibitors or chloroquine, which raises cellular pH, restored HDAC4. Recombinant CtsH could break down HDAC4 in the transfected cells and in vitro at acidic pH. In human cirrhotic liver, activated HSCs express high levels of class IIa HDACs but little CtsH. We propose that cysteine cathepsin-mediated degradation of class IIa HDACs plays a key role in the modulation of MMP expression/suppression and HSC functions in tissue injury and fibrosis.

Vitamin D Signaling through Induction of Paneth Cell Defensins Maintains Gut Microbiota and Improves Metabolic Disorders and Hepatic Steatosis in Animal Models.

Metabolic syndrome (MetS), characterized as obesity, insulin resistance, and non-alcoholic fatty liver diseases (NAFLD), is associated with vitamin D insufficiency/deficiency in epidemiological studies, while the underlying mechanism is poorly addressed. On the other hand, disorder of gut microbiota, namely dysbiosis, is known to cause MetS and NAFLD. It is also known that systemic inflammation blocks insulin signaling pathways, leading to insulin resistance and glucose intolerance, which are the driving force for hepatic steatosis. Vitamin D receptor (VDR) is highly expressed in the ileum of the small intestine, which prompted us to test a hypothesis that vitamin D signaling may determine the enterotype of gut microbiota through regulating the intestinal interface. Here, we demonstrate that high-fat-diet feeding (HFD) is necessary but not sufficient, while additional vitamin D deficiency (VDD) as a second hit is needed, to induce robust insulin resistance and fatty liver. Under the two hits (HFD+VDD), the Paneth cell-specific alpha-defensins including α-defensin 5 (DEFA5), MMP7 which activates the pro-defensins, as well as tight junction genes, and MUC2 are all suppressed in the ileum, resulting in mucosal collapse, increased gut permeability, dysbiosis, endotoxemia, systemic inflammation which underlie insulin resistance and hepatic steatosis. Moreover, under the vitamin D deficient high fat feeding (HFD+VDD), Helicobacter hepaticus, a known murine hepatic-pathogen, is substantially amplified in the ileum, while Akkermansia muciniphila, a beneficial symbiotic, is diminished. Likewise, the VD receptor (VDR) knockout mice exhibit similar phenotypes, showing down regulation of alpha-defensins and MMP7 in the ileum, increased Helicobacter hepaticus and suppressed Akkermansia muciniphila. Remarkably, oral administration of DEFA5 restored eubiosys, showing suppression of Helicobacter hepaticus and increase of Akkermansia muciniphila in association with resolving metabolic disorders and fatty liver in the HFD+VDD mice. An in vitro analysis showed that DEFA5 peptide could directly suppress Helicobacter hepaticus. Thus, the results of this study reveal critical roles of a vitamin D/VDR axis in optimal expression of defensins and tight junction genes in support of intestinal integrity and eubiosis to suppress NAFLD and metabolic disorders.