Breast cancer (BC) is a huge health threat for women worldwide. Although numerous microRNAs (miRNA) have been found to be aberrantly expressed in BC, the construction of a comprehensive miRNA-messenger RNA (mRNA) network is still needed.

Sorting nexins (SNX) are a family of PX domain-containing proteins with pivotal roles in trafficking and signaling. SNX-BARs, which also have a curvature-generating Bin/Amphiphysin/Rvs (BAR) domain, have membrane-remodeling functions, particularly at the endosome. The minimal PX-BAR module is a dimer mediated by BAR-BAR interactions. Many SNX-BAR proteins, however, additionally have low-complexity N-terminal regions of unknown function. Here, we present the cryo-EM structure of the full-length SNX-BAR Mvp1, which is an autoinhibited tetramer. The tetramer is a dimer of dimers, wherein the membrane-interacting BAR surfaces are sequestered and the PX lipid-binding sites are occluded. The N-terminal low-complexity region of Mvp1 is essential for tetramerization. Mvp1 lacking its N-terminus is dimeric and exhibits enhanced membrane association. Membrane binding and remodeling by Mvp1 therefore requires unmasking of the PX and BAR domain lipid-interacting surfaces. This work reveals a tetrameric configuration of a SNX-BAR protein that provides critical insight into SNX-BAR function and regulation.

DNA methylation plays a pivotal role in the development and progression of tumors. However, studies focused on the dynamic changes of DNA methylation in the development of hepatocellular carcinoma (HCC) are rare. To systematically illustrate the dynamic DNA methylation alternation from premalignant to early-stage liver cancer with the same genetic background, this study enrolled 5 HBV-related patients preceded with liver cirrhosis, pathologically identified as early-stage HCC with dysplastic nodules. Liver fibrosis tissues, dysplastic nodules and early HCC tissues from these patients were used to measure DNA methylation. Here, we report significant differences in the DNA methylation spectrum among the three types of tissues. In the early stage of HCC, DNA hypermethylation of tumor suppressor genes is predominant. Additionally, DNA hypermethylation in the early stage of HCC changes the binding ability of transcription factor TP53 to the promoter of tumor suppressor gene ZNF334, and inhibits the expression of ZNF334 at the transcription level. Furthermore, through a series of in vivo and in vitro experiments, we have clarified the exacerbation effect of tumor suppressor gene ZNF334 deletion in the occurrence of HCC. Combined with clinical data, we found that the overall survival and relapse-free survival of patients with high ZNF334 expression are significantly longer. Thus, we partly elucidated a sequential alternation of DNA methylation modification during the occurrence of HCC, and clarified the biological function and regulatory mechanism of the tumor suppressor gene ZNF334, which is regulated by related DNA methylation sites. Our study provides a new target and clinical evidence for the early diagnosis and sheds light on the precise treatment of liver cancer.

Kappa-opioid receptor (KOR) agonists are known for having opposite and/or different effects compared with Mu-opioid receptor (MOR) agonists. This study is aimed at clarifying the analgesic effect and tolerance of nalbuphine combined with morphine, and quantifying the mRNA and protein expression of spinal MOR and KOR in a mouse bone cancer pain (BCP) model treated with nalbuphine and morphine.

Adjunctive tirofiban administration in patients undergoing endovascular treatment (EVT) for acute large vessel occlusion (LVO) has been investigated in several studies. However, the findings are conflict. This study aimed to compare the effect of different administration pathways of tirofiban on patients undergoing EVT for acute LVO with intracranial atherosclerotic disease (ICAD).

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated a genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H = A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.

In contrast to the well-characterized gut microbiomes, the composition and function of the insect body-surface microbiotas are still elusive and highly underexplored. Here we report the dynamic features of the Drosophila melanogaster surface microbiomes. It was found that the microbiomes assembled on fly surfaces could defend insects against fungal parasitic infections. The substantial increase of bacterial loads occurred within 10 days of fly eclosion, especially the expansion of Gilliamella species. The culturable bacteria such as Lactiplantibacillus plantarum could effectively inhibit fungal spore germinations, and the gnotobiotic addition of the isolated bacteria could substantially delay fungal infection of axenic flies. We found that the fly tarsal segments were largely accumulated with bacterial cells, which could accelerate cell dispersal onto different body parts to deter fungal spore germinations. Our findings will facilitate future investigations of the surface microbiotas affecting insect physiologies.

The formylpeptide receptors (FPRs) mediate pattern recognition of formylated peptides derived from invading pathogens or mitochondria from dead host cells. They can also sense other structurally distinct native peptides and even lipid mediators to either promote or resolve inflammation. Pharmacological targeting of FPRs represents a novel therapeutic approach in treating inflammatory diseases. However, the molecular mechanisms underlying FPR ligand recognition are elusive. We report cryo-EM structures of Gi-coupled FPR1 and FPR2 bound to a formylpeptide and Gi-coupled FPR2 bound to two synthetic peptide and small-molecule agonists. Together with mutagenesis data, our structures reveal the molecular mechanism of formylpeptide recognition by FPRs and structural variations of FPR1 and FPR2 leading to their different ligand preferences. Structural analysis also suggests that diverse FPR agonists sample a conserved activation chamber at the bottom of ligand-binding pockets to activate FPRs. Our results provide a basis for rational drug design on FPRs.

The hunger hormone ghrelin activates the ghrelin receptor GHSR to stimulate food intake and growth hormone secretion and regulate reward signaling. Acylation of ghrelin at Ser3 is required for its agonistic action on GHSR. Synthetic agonists of GHSR are under clinical evaluation for disorders related to appetite and growth hormone dysregulation. Here, we report high-resolution cryo-EM structures of the GHSR-Gi signaling complex with ghrelin and the non-peptide agonist ibutamoren as an investigational new drug. Our structures together with mutagenesis data reveal the molecular basis for the binding of ghrelin and ibutamoren. Structural comparison suggests a salt bridge and an aromatic cluster near the agonist-binding pocket as important structural motifs in receptor activation. Notable structural variations of the Gi and GHSR coupling are observed in our cryo-EM analysis. Our results provide a framework for understanding GHSR signaling and developing new GHSR agonist drugs.

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.

Billions of doses of coronavirus disease 2019 (COVID-19) vaccines have been administered and several cases of thrombocytopenia with thrombosis syndrome (TTS) have been reported after the administration of adenoviral vector vaccines. However, the effects of an inactivated COVID-19 vaccine, CoronaVac, on coagulation are not well understood.

Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome.

We aimed to identify quantitative trait loci (QTL) for secondary traits related to grain yield (GY) in two BC1F2:3 backcross populations (LPSpop and DTPpop) under well-watered (4 environments; WW) and drought stressed (6; DS) conditions to facilitate breeding efforts towards drought tolerant maize. GY reached 5.6 and 5.8 t/ha under WW in the LPSpop and the DTPpop, respectively. Under DS, grain yield was reduced by 65% (LPSpop) to 59% (DTPpop) relative to WW. GY was strongly associated with the normalized vegetative index (NDVI; r ranging from 0.61 to 0.96) across environmental conditions and with an early flowering under drought stressed conditions (r ranging from -0.18 to -0.25) indicative of the importance of early vigor and drought escape for GY. Out of the 105 detected QTL, 53 were overdominant indicative of strong heterosis. For 14 out of 18 detected vigor QTL, as well as for eight flowering time QTL the trait increasing allele was derived from CML491. Collocations of early vigor QTL with QTL for stay green (bin 2.02, WW, LPSpop; 2.07, DS, DTPpop), the number of ears per plant (bins 2.02, 2.05, WW, LPSpop; 5.02, DS, LPSpop) and GY (bin 2.07, WW, DTPpop; 5.04, WW, LPSpop), reinforce the importance of the observed correlations. LOD scores for early vigor QTL in these bins ranged from 2.2 to 11.25 explaining 4.6 (additivity: +0.28) to 19.9% (additivity: +0.49) of the observed phenotypic variance. A strong flowering QTL was detected in bin 2.06 across populations and environmental conditions explaining 26-31.3% of the observed phenotypic variation (LOD: 13-17; additivity: 0.1-0.6d). Improving drought tolerance while at the same time maintaining yield potential could be achieved by combining alleles conferring early vigor from the recurrent parent with alleles advancing flowering from the donor. Additionally bin 8.06 (DTPpop) harbored a QTL for GY under WW (additivity: 0.27 t/ha) and DS (additivity: 0.58 t/ha). R2 ranged from 0 (DTPpop, WW) to 26.54% (LPSpop, DS) for NDVI, 18.6 (LPSpop, WW) to 42.45% (LPSpop, DS) for anthesis and from 0 (DTPpop, DS) to 24.83% (LPSpop, WW) for GY. Lines out-yielding the best check by 32.5% (DTPpop, WW) to 60% (DTPpop, DS) for all population-by-irrigation treatment combination (except LPSpop, WW) identified are immediately available for the use by breeders.

Hand, foot, and mouth disease (HFMD) has caused major public health concerns worldwide, and has become one of the leading causes of children death. China is the most serious epidemic area with a total of 3,419,149 reported cases just from 2008 to 2010, and its different geographic areas might have different spatial epidemiology characteristics at different spatial-temporal scale levels. We conducted spatial and spatial-temporal epidemiology analysis to HFMD at county level in Shandong Province, China.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Lung cancer is the leading cause of cancer-related mortality around the world. This malignancy has a 5-year survival rate of 21%, because most of the patients are diagnosed in the middle or late stage of the disease when local metastasis and tumor invasion have already progressed. Therefore, the investigation of the pathogenesis of lung cancer is an issue of crucial importance. MicroRNAs (miRNAs) seem to be involved in the evolution and development of lung cancer. MicroRNA-608 is likely to be downregulated in lung cancer tissues. Regarding this, the current study involved the determination of the fundamental mechanism of microRNA-608 in the development of lung cancer. Based on the results of quantitative reverse transcription polymerase chain reaction (RT-qPCR), the expression level of microRNA-608 was downregulated in 40 lung cancer tissues, compared to that in the adjacent normal tissues. The results of dual-luciferase reporter assay revealed that bromodomain-containing protein 4 (BRD4) was the direct target of microRNA-608. Accordingly, the lung cancer tissues had an elevated expression level of BRD4, in contrast to the adjacent normal tissues. The results of Cell Counting Kit 8 assay demonstrated that the high expression of microRNA-608 notably restrained lung cancer cell proliferation. The scratch wound and transwell assays showed that the upregulation of microRNA-608 suppressed the migration and invasion of lung cancer cells. Finally, the western blot assay showed that in the microRNA-608 mimics group, the expression levels of BRD4, p-JAK2, p-STATA3, CD44, and MMP9 were significantly decreased, compared with those in the negative control miRNA mimics group. Our results indicate that high expression of microRNA-608 inhibits the proliferation, migration, and invasion of lung cancer cells by targeting BRD4 via the JAK2/STAT3 pathway.

Long noncoding RNA PMS2L2 can inhibit inflammation induced by LPS, while LPS plays an important role in sepsis, indicating the possible involvement of PMS2L2 in sepsis.