Developmental validation of an mRNA kit: A 5-dye multiplex assay designed for body-fluid identification.

Identifying the sources of biosamples found at crime scenes is crucial for forensic investigations. Among the markers used for body fluid identification (BFI), mRNA has emerged as a well-studied marker because of its high specificity and remarkable stability. Despite this potential, commercially available mRNA kits specifically designed for BFI are lacking. Therefore, we developed an mRNA kit that includes 21 specific mRNA markers of body fluids, along with three housekeeping genes for BFI, to identify four forensic-relevant fluids (blood, semen, saliva, and vaginal fluids). In this study, we tested 451 single-body-fluid samples, validated the universality of the mRNA kit, and obtained a gene expression profile. We performed the validation studies in triplicates and determined the sensitivity, specificity, stability, precision, and repeatability of the mRNA kit. The sensitivity of the kit was found to be 0.1 ng. Our validation process involved the examination of 59 RNA mixtures, 60 body fluids mixtures, and 20 casework samples, which further established the reliability of the kit. Furthermore, we constructed five classifiers that can handle single-body fluids and mixtures using this kit. The classifiers output possibility values and identify the specific body fluids of interest. Our results showed the reliability and suitability of the BFI kit, and the Random Forest classifier performed the best, with 94% precision. In conclusion, we developed an mRNA kit for BFI which can be a promising tool for forensic practice.

A Therapeutic DNA Vaccine Targeting HPV16 E7 in Combination with Anti-PD-1/PD-L1 Enhanced Tumor Regression and Cytotoxic Immune Responses.

Persistent infection of high-risk human papillomavirus (HPV) and the expression of E6 and E7 oncoproteins are the main causes of cervical cancer. Several prophylactic HPV vaccines are used in the clinic, but these vaccines have limited efficacy in patients already infected with HPV. Since HPV E7 is vital for tumor-specific immunity, developing a vaccine against HPV E7 is an attractive strategy for cervical cancer treatment. Here, we constructed an HPV16 E7 mutant that loses the ability to bind pRb while still eliciting a robust immune response. In order to build a therapeutic DNA vaccine, the E7 mutant was packaged in an adenovirus vector (Ad-E7) for efficient expression and enhanced immunogenicity of the vaccine. Our results showed that the Ad-E7 vaccine effectively inhibited tumor growth and increased the proportion of interferon-gamma (IFN-γ)-secreting CD8+ T cells in the spleen, and tumor-infiltrating lymphocytes in a mouse cervical cancer model was achieved by injecting with HPV16-E6/E7-expressing TC-1 cells subcutaneously. Combining the Ad-E7 vaccine with the PD-1/PD-L1 antibody blockade significantly improved the control of TC-1 tumors. Combination therapy elicited stronger cytotoxic T lymphocyte (CTL) responses, and IFN-γ secretion downregulated the proportion of Tregs and MDSCs significantly. The expressions of cancer-promoting factors, such as TNF-α, were also significantly down-regulated in the case of combination therapy. In addition, combination therapy inhibited the number of capillaries in tumor tissues and increased the thickness of the tumor capsule. Thus, Ad-E7 vaccination, in combination with an immune checkpoint blockade, may benefit patients with HPV16-associated cervical cancer.

Cl2 ⋠- Mediates Direct and Selective Conversion of Inert C(sp3 )-H Bonds into Aldehydes/Ketones.

Developing new reactive pathway to activate inert C(sp3 )-H bonds for valuable oxygenated products remains a challenge. We prepared a series of triazine conjugated organic polymers to photoactivate C-H into aldehyde/ketone via O2 →H2 O2 →⋅OH→Cl⋅→Cl2 ⋅- . Experiment results showed Cl2 ⋅- could successively activate C(sp3 )-H more effectively than Cl⋅ to generate unstable dichlorinated intermediates, increasing the kinetic rate ratio of dichlorination to monochlorination by a factor of 2,000 and thus breaking traditional dichlorination kinetic constraints. These active intermediates were hydrolyzed into aldehydes or ketones easily, when compared with typical stable dichlorinated complexes, avoiding chlorinated by-product generation. Moreover, an integrated two-phase system in an acid solution strengthened the Cl2 ⋅- mediated process and inhibited product overoxidation, where the conversion rate of toluene reached 16.94 mmol/g/h and the selectivity of benzaldehyde was 99.5 %. This work presents a facile and efficient approach for selective conversion of inert C(sp3 )-H bonds using Cl2 ⋅- .

Two-dimensional metal-organic frameworks as bifunctional electrocatalysts for the oxygen evolution reaction and oxygen reduction reaction (OER/ORR): a theoretical study.

Effective bifunctional catalysts are needed for the two main processes in metal-air batteries (oxygen evolution reaction and oxygen reduction reaction (OER/ORR)) to increase efficiency. Herein, we systematically investigate the stability, electronic structure, and catalytic performance of the OER/ORR of two-dimensional (2D) conducting metal-organic frameworks (MOFs) M3(C6Se6)2 (M = Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zr, Nb, Mo, Ru, Rh, Pd, Ir, and Pt) by first-principles calculations. The results show that Co3(C6Se6)2 has an overpotential of 0.51 V and 0.3 V for the OER and ORR, respectively, and Rh3(C6Se6)2 has an overpotential of 0.53 V and 0.29 V for the OER and ORR, respectively, which are very promising bifunctional catalysts. In addition, Ir3(C6Se6)2 is a very promising ORR catalyst with a low overpotential of 0.34 V. Volcano plots and contour maps of OER/ORR activity versus intermediate adsorption strength were established to describe the activity trend of M3(C6Se6)2 based on the relationship of adsorbed intermediates. Furthermore, the d-band center theory and crystal orbital Hamilton populations (COHPs) were used to relate the OER/ORR activity to the d-electrons of the central metal. Our study not only provides a novel bifunctional electrocatalyst but also provides some references for other 2D MOFs as electrocatalysts.

Direct observation of cation diffusion driven surface reconstruction at van der Waals gaps.

Weak interlayer van der Waals (vdW) bonding has significant impact on the surface/interface structure, electronic properties, and transport properties of vdW layered materials. Unraveling the complex atomistic dynamics and structural evolution at vdW surfaces is therefore critical for the design and synthesis of the next-generation vdW layered materials. Here, we show that Ge/Bi cation diffusion along the vdW gap in layered GeBi2Te4 (GBT) can be directly observed using in situ heating scanning transmission electron microscopy (STEM). The cation concentration variation during diffusion was correlated with the local Te6 octahedron distortion based on a quantitative analysis of the atomic column intensity and position in time-elapsed STEM images. The in-plane cation diffusion leads to out-of-plane surface etching through complex structural evolutions involving the formation and propagation of a non-centrosymmetric GeTe2 triple layer surface reconstruction on fresh vdW surfaces, and GBT subsurface reconstruction from a septuple layer to a quintuple layer. Our results provide atomistic insight into the cation diffusion and surface reconstruction in vdW layered materials.

SIRT2 promotes the viability, invasion and metastasis of osteosarcoma cells by inhibiting the degradation of Snail.

Osteosarcomas (OS) are highly metastatic and usually lead to poor outcomes. Epithelial-mesenchymal transition (EMT) is reported to be a critical event in metastasis. SIRT2 exerts dual functions in many different tumors. However, the underlying molecular mechanisms of SIRT2 in osteosarcoma cell metastasis and the question of whether SIRT2 regulates EMT have not been fully explored. In this study, we confirmed that SIRT2 was highly-expressed in human osteosarcoma MG63 and Saos-2 cell lines. The viability, migration and invasion of osteosarcoma cells were inhibited by knockdown of SIRT2 and were enhanced by overexpression of SIRT2. Moreover, SIRT2 positively regulated EMT and upregulated the protein levels of the mesenchymal markers N-cadherin and Vimentin and the levels of MMP2 and MMP9. A xenograft mouse model showed that SIRT2 knockdown in osteosarcoma cells led to reduced tumor growth, decreased expression of mesenchymal markers and impaired lung and liver metastasis in vivo. Furthermore, we showed that SIRT2 interacted with and upregulated the protein level of the EMT-associated transcription factor Snail. SIRT2 inhibited Snail degradation via its deacetylase activity. Knockdown of Snail abrogated the promoting effects of SIRT2 on migration and invasion of osteosarcoma cells. In conclusion, SIRT2 plays a crucial role in osteosarcoma metastasis by inhibiting Snail degradation and may serve as a novel therapeutic target to manage osteosarcoma.

Highly Potassiophilic Graphdiyne Skeletons Decorated with Cu Quantum Dots Enable Dendrite-Free Potassium-Metal Anodes.

Employing an Al foil current collector at the potassium anode side is an ideal choice to entail low-cost and high-energy potassium-metal batteries (PMBs). Nevertheless, the poor affinity between the potassium and the planar Al can cause uneven K plating/stripping and, hence, an undermined anode performance, which remains a significant challenge to be addressed. Herein, a nitrogen-doped carbon@graphdiyne (NC@GDY)-modified Al current collector affording potassiophilic properties is proposed, which simultaneously suppresses the dendrite growth and prolongs the lifespan of K anodes. The thin and light modification layer (7 µm thick, with a mass loading of 500 µg cm-2 ) is fabricated by directly growing GDY nanosheets interspersed with Cu quantum dots on NC polyhedron templates. As a result, symmetric cell tests reveal that the K@NC@GDY-Al electrode exhibits an unprecedented cycle life of over 2400 h at a 40% depth of discharge. Even at an 80% depth of discharge, the cell can still sustain for 850 h. When paired with a potassium Prussian blue cathode, the thus-assembled full cell demonstrates comparable capacity and rate performance with state-of-the-art PMBs.

A cosmopolitan fungal pathogen of dicots adopts an endophytic lifestyle on cereal crops and protects them from major fungal diseases.

Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to help safeguard such resources for increasing human populations on a global scale. Here, we find that Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, rice, barley, maize, and oat, providing protection against Fusarium head blight, stripe rust, and rice blast. Protection is also provided by disabled S. sclerotiorum strains harboring a hypovirulence virus. The disabled strain DT-8 promoted wheat yields by 4-18% in the field and consistently reduced Fusarium disease by 40-60% across multiple field trials. We term the host-dependent trophism of S. sclerotiorum, destructively pathogenic or mutualistically endophytic, as schizotrophism. As a biotroph, S. sclerotiorum modified the expression of wheat genes involved in disease resistance and photosynthesis and increased the level of IAA. Our study shows that a broad-spectrum pathogen of one group of plants may be employed as a biocontrol agent in a different group of plants where they can be utilized as beneficial microorganisms while avoiding the risk of in-field release of pathogens. Our study also raises provocative questions about the potential role of schizotrophic endophytes in natural ecosystems.

ORF Ι of Mycovirus SsNSRV-1 is Associated with Debilitating Symptoms of Sclerotinia sclerotiorum.

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (-) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum. In this study, functional analysis of Open Reading Frame Ι (ORF Ι) of SsNSRV-1 was performed. The integration and expression of ORF Ι led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF Ι were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF Ι were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF Ι could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF Ι may be a potential gene used for the prevention of S. sclerotiorum.

Correction to: Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells.

Following the publication of this article [1], the authors noted the following errors.

Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells.

BACKGROUND: The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. RESULTS: Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. CONCLUSIONS: Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

MAPKK Inhibitor U0126 Inhibits Plasmodiophora brassicae Development.

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAK1/PbMAK3. The PbMAKKK7-PbMAKK3-PbMAK1/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity.

Endosphere microbiome comparison between symptomatic and asymptomatic roots of Brassica napus infected with Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.

Transcriptome Analysis of Arabidopsis thaliana in Response to Plasmodiophora brassicae during Early Infection.

Clubroot disease is a serious threat to cruciferous plants worldwide, especially to oilseed rape. However, knowledge on pathogenic molecular mechanisms and host interaction is limited. We presume that the recognition between Arabidopsis thaliana and Plasmodiophora brassicae occurs at the early stage of infection and within a relatively short period. In this study, we demonstrated changes on gene expression and pathways in A. thaliana during early infection with P. brassicae using transcriptome analysis. We identified 1,903 and 1,359 DEGs at 24 and 48 h post-inoculation (hpi), respectively. Flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids, and proanthocyanidins accumulated and many hormonal- and receptor-kinase related genes were expressed, caused by P. brassicae infection during its early phase. Therefore, the early interaction between A. thaliana and P. brassicae plays an important role in the entire infection process. The results provide a new contribution to a better understanding of the interaction between host plants and P. brassicae, as well as the development of future measures for the prevention of clubroot.

Integrated omics study of lipid droplets from Plasmodiophora brassicae.

Plasmodiophora brassicae causes clubroot disease in cruciferous. In this report, lipid droplets were observed in the resting spores of P. brassicae. 295 lipid droplet-associated proteins were identified and categorized into nine groups. Transcriptome analysis of these proteins during three different zoosporic stages revealed differences in gene expression pattern. GO enrichment analysis revealed that these proteins associated with lipid droplets were mainly linked to biosynthesis and metabolism. GC-MS analysis revealed that lipid droplets contain seven types of free fatty acids: saturated fatty acids C16:0 and C18:0, and unsaturated fatty acids C18:1Δ9, C18:1Δ11, C18:2, C20:4 and C20:5. P. brassicae accumulated a large amount of triacylglycerols (TAGs). We systematically analyzed the putative proteins involved in TAG biosynthesis and its metabolic pathway. KEGG pathway analysis defined 3390 genes, including 167 genes involved in lipid metabolism. Transcriptome analysis revealed that 162 candidate enzymes involved in lipid metabolism were differential expressed. Our omics studies are the first to investigate the lipid droplet organelles in P. brassicae, providing a reference resource to study protist lipid droplets.

Arabidopsis Mutant bik1 Exhibits Strong Resistance to Plasmodiophora brassicae.

Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1, and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2, and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 [non-expresser of PR genes that regulate systemic acquired resistance (SAR)] mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms.