A sensitive and versatile electrochemical sensor based on hybridization chain reaction and CRISPR/Cas12a system for antibiotic detection.

A sensitive electrochemical platform was constructed with NH2-Cu-MOF as electrochemical probe to detect antibiotics using CRISPR/Cas12a system triggered by hybridization chain reaction (HCR). The sensing system consists of two HCR systems. HCR1 occurred on the electrode surface independent of the target, generating long dsDNA to connect signal probes and producing a strong electrochemical signal. HCR2 was triggered by target, and the resulting dsDNA products activated the CRISPR/Cas12a, thereby resulting in effective and rapid cleavage of the trigger of HCR1, hindering the occurrence of HCR1, and reducing the number of NH2-Cu-MOF on the electrode surface. Eventually, significant signal change depended on the target was obtained. On this basis and with the help of the programmability of DNA, kanamycin and ampicillin were sensitively detected with detection limits of 60 fM and 10 fM (S/N = 3), respectively. Furthermore, the sensing platform showed good detection performance in milk and livestock wastewater samples, demonstrating its great application prospects in the detection of antibiotics in food and environmental water samples.

Label-free electrochemical sensing platform for sensitive detection of ampicillin by combining nucleic acid isothermal enzyme-free amplification circuits with CRISPR/Cas12a.

The residue of ampicillin (AMP) in food and ecological environment poses a potential harm to human health. Therefore, a reliable system for detecting AMP is in great demand. Herein, a label-free and sensitive electrochemical sensor utilizing NH2-Co-MOF as an electrocatalytic active material for methylene blue (MB) was developed for rapid and facile AMP detection by combining hybridization chain reaction (HCR), catalytic hairpin assembly (CHA) with CRISPR/Cas12a. The surface of glassy carbon electrode modified with NH2-Co-MOF was able to undergo HCR independent of the AMP, forming long dsDNA complexes to load MB, resulting in strong original electrochemical signal. The presence of AMP could trigger upstream CHA circuit to activate the CRISPR/Cas12a system, thereby achieving rapid non-specific cleavage of the trigger ssDNA of HCR on the electrode surface, hindering the occurrence of HCR and reducing the load of MB. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the AMP with a detection limit as low as 1.60 pM (S/N = 3). The proposed sensor exhibited good stability, selectivity, and stability, and achieved reliable detection of AMP in milk and livestock wastewater samples, demonstrating its promising application prospects in food safety and environmental monitoring.

TaNAM-6A is essential for nitrogen remobilisation and regulates grain protein content in wheat (Triticum aestivum L.).

Grain protein content (GPC) is a crucial quality trait in bread wheat, which is influenced by the key transcription factor TaNAM. However, the regulatory mechanisms of TaNAM have remained largely elusive. In this study, a new role of TaNAM was unveiled in regulating nitrogen remobilisation which impacts GPC. The TaNAM knockout mutants generated by clustered regularly interspaced short palindromic repeats/Cas9 exhibited significantly delayed senescence and lower GPC, while overexpression of TaNAM-6A resulted in premature senility and much higher GPC. Further analysis revealed that TaNAM directly activates the genes TaNRT1.1 and TaNPF5.5s, which are involved in nitrogen remobilisation. This activity aids in the transfer of nitrogen from leaves to grains for protein synthesis. In addition, an elite allele of TaNAM-6A, associated with high GPC, was identified as a candidate gene for breeding high-quality wheat. Overall, our work not only elucidates the potential mechanism of TaNAM-6A affecting bread wheat GPC, but also highlights the significance of nitrogen remobilisation from senescent leaves to grains for protein accumulation. Moreover, our research provides a new target and approach for improving the quality traits of wheat, particularly the GPC.

Prophylactic Vaccination and Intratumoral Boost with HER2-Expressing Oncolytic Herpes Simplex Virus Induces Robust and Persistent Immune Response against HER2-Positive Tumor Cells.

The development of effective cancer vaccines remains a significant challenge due to immune tolerance and limited clinical benefits. Oncolytic herpes simplex virus type 1 (oHSV-1) has shown promise as a cancer therapy, but efficacy is often limited in advanced cancers. In this study, we constructed and characterized a novel oHSV-1 virus (VG22401) expressing the human epidermal growth factor receptor 2 (HER2), a transmembrane glycoprotein overexpressed in many carcinomas. VG22401 exhibited efficient replication and HER2 payload expression in both human and mouse colorectal cancer cells. Mice immunized with VG22401 showed significant binding of serum anti-HER2 antibodies to HER2-expressing tumor cells, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, mice primed with VG22401 and intratumorally boosted with the same virus showed enhanced antitumor efficacy in a bilateral syngeneic HER2(+) tumor model, compared to HER2-null backbone virus. This effect was accompanied by the induction of anti-HER2 T cell responses. Our findings suggest that peripheral priming with HER2-expressing oHSV-1 followed by an intratumoral boost with the same virus can significantly enhance antitumor immunity and efficacy, presenting a promising strategy for cancer immunotherapy.

Programmable electrochemical biosensing platform based on catalytic hairpin assembly and entropy-driven catalytic cascade amplification circuit.

Herein, powerful DNA strand displacement reaction and sensitive electrochemical analysis method were ingeniously integrated to develop a programmable biosensing platform. Using DNA as the detection model, a cascade amplification system based on catalytic hairpin assembly and entropy-driven catalytic was constructed, and the reaction rate and signal amplification effect were significantly improved. The product of the cascade amplification circuit could undergo strand displacement reaction with the signal probe on the electrode surface to obtain sensitive electrochemical signal changes and realize highly sensitive detection of the target. In addition, without redesigning the DNA sequences in the cascade amplification circuit, the by-product strand typically wasted in traditional entropy-driven catalytic reactions can be fully utilized to construct a single-signal output biosensing system and even a dual-signal output ratiometric biosensing platform, improving the detection repeatability and reliability of the system, and expanding the application of DNA strand displacement reaction in electrochemical biosensing. Furthermore, benefiting from the design flexibility of the DNA molecules, the constructed biosensing platform realized the sensitive detection of aptamer substrate (kanamycin as an example) and certain metal ion (mercury as an example) by simply recoding the corresponding recognition sequence, demonstrating the good versatility of the biosensing platform.

Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins.

Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast Saccharomyces cerevisiae, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that spo20∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation.

Prevalence and influencing factors of hyperuricemia in middle-aged and older adults in the Yao minority area of China: a cross-sectional study.

Hyperuricemia (HUA) endangers human health, and its prevalence has increased rapidly in recent decades. The current study investigated HUA's prevalence and influencing factors in Gongcheng, southern China. A cross-sectional investigation was conducted; 2128 participants aged 30-93 years were included from 2018 to 2019. Univariate and multivariate logistic regression models were used to screen HUA variables. A Bayesian network model was constructed using the PC algorithm to evaluate the association between influencing factors and HUA. The prevalence of HUA was 15.6% (23.2% in men, 10.7% in women). After screening the variables using a logistic regression analysis model, fatty liver disease (FLD), dyslipidemia, abdominal obesity, creatinine (CREA), somatotype, bone mass, drinking, and physical activity level at work were included in the Bayesian network model. The model results showed that dyslipidemia, somatotype, CREA, and drinking were directly related to HUA. Bone mass and FLD were indirectly associated with HUA by affecting the somatotype. The prevalence of HUA in Gongcheng was high in China. The prevalence of HUA was related to somatotype, drinking, bone mass, physical activity level at work, and other metabolic diseases. A good diet and moderate exercise are recommended to maintain a healthy somatotype and reduce the prevalence rate of HUA.

Targeting carcinoembryonic antigen-expressing tumors using a novel transcriptional and translational dual-regulated oncolytic herpes simplex virus type 1.

VG2025 is a recombinant oncolytic herpes simplex virus type 1 (HSV-1) that uses transcriptional and translational dual regulation (TTDR) of critical viral genes to enhance virus safety and promote tumor-specific virus replication without reducing virulence. The TTDR platform is based on transcriptional control of the essential HSV-1 immediate-early protein ICP27 using a tumor-specific carcinoembryonic antigen (CEA) promoter, coupled with translational control of the neurovirulence factor ICP34.5 using multiple microRNA (miR)-binding sites. VG2025 further incorporates IL-12 and the IL-15/IL-15 receptor alpha subunit complex to enhance the antitumor and immune stimulatory properties of oncolytic HSVs. The TTDR strategy was verified in vitro and shown to be highly selective. Strong in vivo antitumor efficacy was observed following both intratumoral and intravenous administration. Clear abscopal and immune memory effects were also evident, indicating a robust antitumor immune response. Gene expression profiling of treated tumors revealed increased immune cell infiltration and activation of multiple immune-signaling pathways when compared with the backbone virus. Absence of neurotoxicity was verified in mice and in rhesus monkeys. Taken together, the enhanced tumor clearance, excellent safety profile, and positive correlation between CEA levels and viral replication efficiency may provide an opportunity for using biomarker-based precision medicine in oncolytic virotherapy.

TaMADS29 interacts with TaNF-YB1 to synergistically regulate early grain development in bread wheat.

Grain development is a crucial determinant of yield and quality in bread wheat (Triticum aestivum L.). However, the regulatory mechanisms underlying wheat grain development remain elusive. Here we report how TaMADS29 interacts with TaNF-YB1 to synergistically regulate early grain development in bread wheat. The tamads29 mutants generated by CRISPR/Cas9 exhibited severe grain filling deficiency, coupled with excessive accumulation of reactive oxygen species (ROS) and abnormal programmed cell death that occurred in early developing grains, while overexpression of TaMADS29 increased grain width and 1,000-kernel weight. Further analysis revealed that TaMADS29 interacted directly with TaNF-YB1; null mutation in TaNF-YB1 caused grain developmental deficiency similar to tamads29 mutants. The regulatory complex composed of TaMADS29 and TaNF-YB1 exercises its possible function that inhibits the excessive accumulation of ROS by regulating the genes involved in chloroplast development and photosynthesis in early developing wheat grains and prevents nucellar projection degradation and endosperm cell death, facilitating transportation of nutrients into the endosperm and wholly filling of developing grains. Collectively, our work not only discloses the molecular mechanism of MADS-box and NF-Y TFs in facilitating bread wheat grain development, but also indicates that caryopsis chloroplast might be a central regulator of grain development rather than merely a photosynthesis organelle. More importantly, our work offers an innovative way to breed high-yield wheat cultivars by controlling the ROS level in developing grains.

Triosephosphate Isomerase and Its Product Glyceraldehyde-3-Phosphate Are Involved in the Regulatory Mechanism That Suppresses Exit from the Quiescent State in Yeast Cells.

Cells of the budding yeast Saccharomyces cerevisiae form spores or stationary cells upon nutrient starvation. These quiescent cells are known to resume mitotic growth in response to nutrient signals, but the mechanism remains elusive. Here, we report that quiescent yeast cells are equipped with a negative regulatory mechanism which suppresses the commencement of mitotic growth. The regulatory process involves a glycolytic enzyme, triosephosphate isomerase (Tpi1), and its product, glyceraldehyde-3-phosphate (GAP). GAP serves as an inhibitory signaling molecule; indeed, the return to growth of spores or stationary cells is suppressed by the addition of GAP even in nutrient-rich growth media, though mitotic cells are not affected. Reciprocally, dormancy is abolished by heat treatment because of the heat sensitivity of Tpi1. For example, spores commence germination merely upon heat treatment, which indicates that the negative regulatory mechanism is actively required for spores to prevent premature germination. Stationary cells of Candida glabrata are also manipulated by heat and GAP, suggesting that the regulatory process is conserved in the pathogenic yeast. IMPORTANCE Our results suggest that, in quiescent cells, nutrient signals do not merely provoke a positive regulatory process to commence mitotic growth. Exit from the quiescent state in yeast cells is regulated by balancing between the positive and negative signaling pathways. Identifying the negative regulatory pathway would provide new insight into the regulation of the transition from the quiescent to the mitotic state. Clinically, quiescent cells are problematic because they are resistant to environmental stresses and antibiotics. Given that the quiescent state is modulated by manipulation of the negative regulatory mechanism, understanding this process is important not only for its biological interest but also as a potential target for antifungal treatment.

Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes.

In mammals, both professional phagocytes and nonprofessional phagocytes (NPPs) can perform phagocytosis. However, limited targets are phagocytosed by NPPs, and thus, the mechanism remains unclear. We find that spores of the yeast Saccharomyces cerevisiae are internalized efficiently by NPPs. Analyses of this phenomenon reveals that RNA fragments derived from cytosolic RNA species are attached to the spore wall, and these fragments serve as ligands to induce spore internalization. Furthermore, we show that a multiligand receptor, RAGE (receptor for advanced glycation end-products), mediates phagocytosis in NPPs. RAGE-mediated phagocytosis is not uniquely induced by spores but is an intrinsic mechanism by which NPPs internalize macromolecules containing RAGE ligands. In fact, artificial particles labeled with polynucleotides, HMGB1, or histone (but not bovine serum albumin) are internalized in NPPs. Our findings provide insight into the molecular basis of phagocytosis by NPPs, a process by which a variety of macromolecules are targeted for internalization.

Heat Stress Tolerance 2 confers basal heat stress tolerance in allohexaploid wheat (Triticum aestivum L.).

Heat stress substantially reduces the yield potential of wheat (Triticum aestivum L.), one of the most widely cultivated staple crops, and greatly threatens global food security in the context of global warming. However, few studies have explored the heat stress tolerance (HST)-related genetic resources in wheat. Here, we identified and fine-mapped a wheat HST locus, TaHST2, which is indispensable for HST in both the vegetative and reproductive stages of the wheat life cycle. The studied pair of near isogenic lines (NILs) exhibited diverse morphologies under heat stress, based on which we mapped TaHST2 to a 485 kb interval on chromosome arm 4DS. Under heat stress, TaHST2 confers a superior conversion rate from soluble sugars to starch in wheat grains, resulting in faster grain filling and a higher yield potential. A further exploration of genetic resources indicated that TaHST2 underwent strong artificial selection during wheat domestication, suggesting it is an essential locus for basal HST in wheat. Our findings provide deeper insights into the genetic basis of wheat HST and might be useful for global efforts to breed heat-stress-tolerant cultivars.

Cloning and Functional Analysis of TaWRI1Ls, the Key Genes for Grain Fatty Acid Synthesis in Bread Wheat.

WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor (TF), critically regulates the processes related to fatty acid synthesis, storage oil accumulation, and seed development in plants. However, the WRI1 genes remain unknown in allohexaploid bread wheat (Triticum aestivum L.). In this study, based on the sequence of Arabidopsis AtWRI1, two TaWRI1Ls genes of bread wheat, TaWRI1L1 and TaWRI1L2, were cloned. TaWRI1L2 was closely related to monocotyledons and clustered in one subgroup with AtWRI1, while TaWRI1L1 was clustered in another subgroup with AtWRI3 and AtWRI4. Both were expressed highly in the developmental grain, subcellular localized in the nucleus, and showed transcriptional activation activity. TaWRI1L2, rather than TaWRI1L1, promoted oil body accumulation and significantly increased triglyceride (TAG) content in tobacco leaves. Overexpression of TaWRI1L2 compensated for the functional loss of AtWRI1 in an Arabidopsis mutant and restored the wild-type phenotypes of seed shape, generation, and fatty acid synthesis and accumulation. Knockout of TaWRI1L2 reduced grain size, 1000 grain weight, and grain fatty acid synthesis in bread wheat. Conclusively, TaWRI1L2, rather than TaWRI1L1, was the key transcriptional factor in the regulation of grain fatty acid synthesis in bread wheat. This study lays a foundation for gene regulation and genetic manipulation of fatty acid synthesis in wheat genetic breeding programs.

Resistin-like molecule ß acts as a mitogenic factor in hypoxic pulmonary hypertension via the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK signaling pathways.

BACKGROUND: Pulmonary arterial smooth muscle cell (PASMC) proliferation plays a crucial role in hypoxia-induced pulmonary hypertension (HPH). Previous studies have found that resistin-like molecule ß (RELM-ß) is upregulated de novo in response to hypoxia in cultured human PASMCs (hPASMCs). RELM-ß has been reported to promote hPASMC proliferation and is involved in pulmonary vascular remodeling in patients with PAH. However, the expression pattern, effects, and mechanisms of action of RELM-ß in HPH remain unclear. METHODS: We assessed the expression pattern, mitogenetic effect, and mechanism of action of RELM-ß in a rat HPH model and in hPASMCs. RESULTS: Overexpression of RELM-ß caused hemodynamic changes in a rat model of HPH similar to those induced by chronic hypoxia, including increased mean right ventricular systolic pressure (mRVSP), right ventricular hypertrophy index (RVHI) and thickening of small pulmonary arterioles. Knockdown of RELM-ß partially blocked the increases in mRVSP, RVHI, and vascular remodeling induced by hypoxia. The phosphorylation levels of the PI3K, Akt, mTOR, PKC, and MAPK proteins were significantly up- or downregulated by RELM-ß gene overexpression or silencing, respectively. Recombinant RELM-ß protein increased the intracellular Ca2+ concentration in primary cultured hPASMCs and promoted hPASMC proliferation. The mitogenic effects of RELM-ß on hPASMCs and the phosphorylation of PI3K, Akt, mTOR, PKC, and MAPK were suppressed by a Ca2+ inhibitor. CONCLUSIONS: Our findings suggest that RELM-ß acts as a cytokine-like growth factor in the development of HPH and that the effects of RELM-ß are likely to be mediated by the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK pathways.

Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes.

Oncolytic virotherapy is a promising new tool for cancer treatment, but direct lytic destruction of tumor cells is not sufficient and must be accompanied by strong immune activation to elicit anti-tumor immunity. We report here the creation of a novel replication-competent recombinant oncolytic herpes simplex virus type 1 (VG161) that carries genes coding for IL-12, IL-15, and IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interactions. The VG161 virus replicates efficiently and exhibits robust cytotoxicity in multiple tumor cell lines. Moreover, the encoded cytokines and the PD-L1 blocking peptide work cooperatively to boost immune cell function. In vivo testing in syngeneic CT26 and A20 tumor models reveals superior efficacy when compared to a backbone virus that does not express exogenous genes. Intratumoral injection of VG161 induces abscopal responses in non-injected distal tumors and grants resistance to tumor re-challenge. The robust anti-tumor effect of VG161 is associated with T cell and NK cell tumor infiltration, expression of Th1 associated genes in the injection site, and increased frequency of splenic tumor-specific T cells. VG161 also displayed a superb safety profile in GLP acute and repeated injection toxicity studies performed using cynomolgus monkeys. Overall, we demonstrate that VG161 can induce robust oncolysis and stimulate a robust anti-tumor immune response without sacrificing safety.

Functional characteristics of Svl3 and Pam1 that are required for proper cell wall formation in yeast cells.

In the budding yeast Saccharomyces cerevisiae, Svl3 and Pam1 proteins work as functional homologues. Loss of their function causes increased levels of chitin deposition in the cell wall and temperature sensitivity, suggesting their involvement in cell wall formation. We found that the N- and C-termini of these proteins have distinctive and critical functions. They contain an N-terminal part that has a probable 2-dehydropantoate 2-reductase domain. In Svl3, this part can be replaced with the yeast 2-dehydropantoate 2-reductase, Pan5, suggesting that Svl3 and its homologues may be able to mediate 2-dehydropantoate 2-reductase function. On the other hand, Svl3 is recruited to the bud tip and bud neck via multiple localization signals in the C-terminal part. One of such signals is the lysine-rich region located in the C-terminal end. The function and localization of Svl3 are significantly disrupted by the loss of this lysine-rich region; however, its localization is not completely abolished by the mutation because another localization signal enables appropriate transport. Svl3 and Pam1 orthologues are found in cells across fungal species. The Svl3 orthologues of Candida glabrata can complement the loss of Svl3 and Pam1 in S. cerevisiae. C. glabrata cells lacking the SVL3 and PAM1 orthologue genes exhibit phenotypes similar to those observed in svl3∆pam1∆ S. cerevisiae cells. Thus, Svl3 homologues may be generally required for the assembly of the cell wall in fungal cells.

C-Reactive Protein Promotes the Activation of Fibroblast-Like Synoviocytes From Patients With Rheumatoid Arthritis.

Objective: To evaluate the biological effect and mechanisms of C-reactive protein (CRP) on the activation of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). Study design: To understand if CRP is involved in RA, expression of CRP and its receptors CD32/64 was examined in synovial tissues from RA patients and normal controls. In vitro, the potential role and mechanisms of CRP in FLS proliferation and invasion, expression of pro-inflammatory cytokines, and activation of signaling pathways were investigated in both RA - FLS and a normal human fibroblast-like synoviocyte line (HFLS). Results: Compared to normal controls, synovial tissues from 21 RA patients exhibited highly activated CRP signaling, particularly by FLSs as identified by 65% of CRP-expressing cells being CRP+vimentin+ and CD32/64+vimentin+ cells. In vitro, FLSs from RA patients, but not HFLS, showed highly reactive to CRP by largely increasing proliferative and invasive activities and expressing pro-inflammatory cytokines and chemokines, including CCL2, CXCL8, IL-6, and MMP2/9. All these changes were blocked largely by a neutralizing antibody to CD32 and, to a less extent by the anti-CD64 antibody, revealing CD32 as a primary mechanism of CRP signaling during synovial inflammation. Further studies revealed that CRP also induced synovial inflammation differentially via CD32/CD64- NF-κB or p38 pathways as blockade of CRP-CD32-NF-κB signaling inhibited CXCL8, CCL2, IL-6, whereas CRP induced RA-FLS invasiveness through CD32-p38 and MMP9 expression via the CD64-p38-dependent mechanism. Conclusions: CRP signaling is highly activated in synovial FLSs from patients with RA. CRP can induce synovial inflammation via mechanisms associated with activation of CD32/64-p38 and NF-κB signaling.

Studies on the Properties of the Sporulation Specific Protein Dit1 and its Product Formyl Tyrosine.

The dityrosine layer is a unique structure present in the spore wall of the budding yeast Saccharomyces cerevisiae. The primary constituent of this layer is bisformyl dityrosine. A sporulation-specific protein, Dit1 is localized in the spore cytosol and produces a precursor of bisformyl dityrosine. Although Dit1 is similar to isocyanide synthases, the loss of Dit1 is not rescued by heterologous expression of the Pseudomonas aeruginosa isocyanide synthase, PvcA, indicating that Dit1 does not mediate isocyanidation. The product of Dit1 is most likely formyl tyrosine. Dit1 can produce its product when it is expressed in vegetative cells; however, formyl tyrosine was not detected in the crude cell lysate. We reasoned that formyl tyrosine is unstable and reacts with some molecule to form formyl tyrosine-containing molecules in the cell lysate. In support of this hypothesis, formyl tyrosine was detected when the lysate was hydrolyzed with a mild acid. The same property was also found for bisformyl dityrosine. Bisformyl dityrosine molecules assemble to form the dityrosine layer by an unknown mechanism. Given that bisformyl dityrosine can be released from the spore wall by mild hydrolysis, the process of formyl tyrosine-containing molecule formation may resemble the assembly of the dityrosine layer.

UV Light-inactivated HSV-1 Stimulates Natural Killer Cell-induced Killing of Prostate Cancer Cells.

Herein we demonstrate that ultraviolet light-inactivated Herpes Simplex Virus-1 (UV-HSV-1) stimulates peripheral blood mononuclear cells (PBMCs) to lyse both androgen-sensitive and androgen-independent prostate cancer (PrCA) cell lines, but not the benign prostatic hyperplastic epithelial cell line, BPH-1, and is 1000-10,000-fold more potent at stimulating this killing than ultraviolet light-inactivated Vesicular Stomatitis Virus, adenovirus, reovirus or cytomegalovirus. Among PBMCs, natural killer (NK) cells appear to be a major cell type involved in this killing and UV-HSV-1 appears to directly and potently stimulate NK cell expression of CD69, degranulation, cytokine production, and migration to IL-8 in PC3 conditioned medium. We also found that UV-HSV-1 stimulates glycolysis in PBMCs and NK cells, and that 2-deoxyglucose and the protein kinase C inhibitor, Go6976, and the NFκB inhibitor, Bay 11-7082, all abrogate UV-HSV-1 activated killing of PC3 cells by PBMCs and NK cells. Using neutralizing anti-Toll-like receptor 2 (TLR2) we found that UV-HSV-1, like HSV-1, activates NK cells via TLR2. Taken together, these results are consistent with Toll-like receptor 2 ligands on UV-HSV-1 stimulating TLR2 on NK cells to activate protein kinase C, leading to enhanced glycolysis and NFκB activation, both of which play a critical role in this anti-PrCA innate immune response. Importantly, UV-HSV-1 synergizes with IL-15 to increase the cytolytic activity of PBMCs against PC3 cells and there was considerable donor-to-donor variation in killing ability. These results support the preclinical development of UV-HSV-1 as an adjuvant, in combination with IL-15, for cell infusions of healthy, preselected NK cells to treat PrCA.

Plant-mediated gene silencing of an essential olfactory-related Gqα gene enhances resistance to grain aphid in common wheat in greenhouse and field.

BACKGROUND: Grain aphid (Sitobion avenae F.) is a dominant pest that limits cereal crop production around the globe. Gq proteins have important roles in signal transduction in insect olfaction. Plant-mediated RNA interference (RNAi) has been widely studied in insect control, but its application for the control wheat aphid in the field requires further study. Here, we used double-stranded (ds)RNA feeding to verify the potential of selected Gqα fragments for host-mediated RNAi, and then evaluated the effect of RNAi on aphid olfaction in transgenic wheat in the greenhouse and field. RESULTS: Gqα gene was expressed in the aphid life cycle, and a 540 bp fragment shared 98.1% similarity with the reported sequence. dsGqα feeding reduced the expression of Gqα, and both reproduction and molting in the grain aphid. Feeding transgenic lines in the greenhouse downregulated expression of aphid Gqα, and significantly reduced reproduction and molting numbers. Furthermore, our field results indicate that transgenic lines have lower aphid numbers and higher 1000-grain weight than an unsprayed wild-type control. CONCLUSION: Plant-mediated silencing of an essential olfactory-related Gqα gene could enhance resistance to grain aphid in common wheat in both the greenhouse and the field. © 2018 Society of Chemical Industry.