Mock Control Research Articles

A combined approach of DNA metabarcoding collectively enhances the detection efficiency of medicinal plants in single and polyherbal formulations.

Empirical research has refined traditional herbal medicinal systems. The traditional market is expanding globally, but inadequate regulatory guidelines, taxonomic knowledge, and resources are causing herbal product adulteration. With the widespread adoption of barcoding and next-generation sequencing, metabarcoding is emerging as a potential tool for detecting labeled and unlabeled plant species in herbal products. This study validated newly designed rbcL and ITS2 metabarcode primers for metabarcoding using in-house mock controls of medicinal plant gDNA pools and biomass pools. The applicability of the multi-barcode sequencing approach was evaluated on 17 single drugs and 15 polyherbal formulations procured from the Indian market. The rbcL metabarcode demonstrated 86.7% and 71.7% detection efficiencies in gDNA plant pools and biomass mock controls, respectively, while the ITS2 metabarcode demonstrated 82.2% and 69.4%. In the gDNA plant pool and biomass pool mock controls, the cumulative detection efficiency increased by 100% and 90%, respectively. A 79% cumulative detection efficiency of both metabarcodes was observed in single drugs, while 76.3% was observed in polyherbal formulations. An average fidelity of 83.6% was observed for targeted plant species present within mock controls and in herbal formulations. In the present study, we achieved increasing cumulative detection efficiency by combining the high universality of the rbcL locus with the high-resolution power of the ITS2 locus in medicinal plants, which shows applicability of multilocus strategies in metabarcoding as a potential tool for the Pharmacovigilance of labeled and unlabeled plant species in herbal formulations.

Dissecting Rubella Placental Infection in an In Vitro Trophoblast Model.

Vertical transmission of rubella virus (RuV) occurs at a high rate during the first trimester of pregnancy. The modes of vertical transmission including the response of trophoblasts to RuV are not well understood. Here, RuV-trophoblast interaction was studied in the BeWo trophoblast cell line. Analysis included early and late time-point kinetics of virus infection rate and the antiviral innate immune response at mRNA and protein level. BeWo characteristics were addressed through metabolic activity by extracellular flux analysis and syncytiotrophoblast formation through incubation with forskolin. We found that RuV infection of BeWo led to profuse type III interferon (IFN) production. Transfecting trophoblast cells with dsRNA analog induced an increase in the production of type I IFN-β and type III IFNs; however, this did not occur in RuV-infected BeWo trophoblasts. IFN-β and to a lesser extent type III IFN-λ1 were inhibitory to RuV. While no significant metabolic alteration was detected, RuV infection reduced the cell number in the monolayer culture in comparison to the mock control and resulted in detached and floating cells. Syncytia formation restricted RuV infection. The use of BeWo as a relevant cell culture model for infection of trophoblasts highlights cytopathogenicity in the absence of a type I IFN response as a pathogenic alteration by RuV.

Abstract 3602: The metastasis-suppressive function of intracellular galectin-3 in melanoma

Abstract Melanoma is a relatively rare skin cancer. However, it accounts for most of all skin cancer-related deaths worldwide. Once melanoma spreads to distant sites, it confers a poor prognosis characterized by resistance to therapy and high mortality rate. Hence, a favorable clinical outcome is strongly correlated with early diagnosis. Despite the ongoing research to identify novel diagnostic, prognostic, and therapeutic targets for metastatic melanoma (MM), a reliable serum marker to predict whether melanoma is vulnerable to metastasize or has already metastasized is still lacking. Galectin-3 (Gal-3), a ß-galactoside-binding protein, is widely expressed by many human epithelial and immune cells. High extracellular Gal-3 expression levels have been reported to be positively correlated with late-stage disease in melanoma patients, where it is theorized to bind surface glycosylated proteins to promote melanoma cell invasion and metastasis. Gal-3 is also expressed intracellularly where it participates in the regulation of many biological processes. However, intrinsic Gal-3 levels within the melanoma cell and its relationship to melanoma progression is still poorly understood. Using The Cancer Genome Atlas (TCGA) database, we analyzed the Gal-3 expression profiles of 471 tumor samples obtained from melanoma patients. To our surprise, TCGA data analysis has shown significantly higher expression of Gal-3 in primary melanoma samples compared to metastatic melanoma. We hypothesize that intracellular Gal-3 potentially negatively regulates primary melanoma progression to metastatic disease. Using several human melanoma cell lines silenced for Gal-3 expression, we investigated the corresponding malignancy-associated activities of these cells. Our results revealed that Gal-3 silencing in melanoma cells potentiated melanoma migration, invasion, and colony formation, compared with mock controls, suggesting that intracellular Gal-3 could interfere with melanoma metastatic activity. Moreover, loss of Gal-3 boosted the activation of the PI3K/AKT and MAPK signaling pathways and upregulated nuclear factor of activated T cells (NFAT1) expression and its pro-metastatic downstream target genes. Importantly, this study illuminates the apparent opposing roles of Gal-3 in melanoma progression, with intracellular Gal-3 potentially serving as a metastasis-suppressive molecule. Moreover, these studies reveal the potential of analyzing melanoma cell-intrinsic levels of Gal-3 to better predict metastatic potential and clinical outcome in melanoma patients. Citation Format: Norhan B. B. Mohammed, Charles J. Dimitroff. The metastasis-suppressive function of intracellular galectin-3 in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3602.

Abstract 657: IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis

Abstract Hematologic malignancy associated with fibroblast growth factor receptor 1 (FGFR1) translocations develops an aggressive stem cell leukemia/lymphoma (SCLL), 80% of which progress to AML and only ~15% show survival of > 15 months. In addition, 23.6% of de novo AML patients overexpress FGFR1, compared with healthy individuals. Therefore, investigating how tumor-intrinsic FGFR1 signaling modulates the tumor microenvironment and their contribution to leukemogenesis is not only important for understanding leukemia initiation and progression but also critical for the development of an efficient cure for AML patients. Interleukin Receptor Associated Kinase 1 (IRAK1) is a protein kinase that initiates a cascade of signaling events eventually leading to activation of proinflammatory genes, including IL-6, IL-10, TNF-α, and IFN-γ, which play critical roles in inflammation and innate immunity. The role of IRAK1 has been reported in several different subtypes of leukemia, including B cell-like diffuse large B-cell lymphoma (ABC DLBCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and melanoma. Most previous IRAK1 studies, however, only focused on its survival and proliferation promoting roles inside the tumor cell itself. Since most of these studies engrafted human cancer cell lines into NSG mice, which lack a functional immune system, it was impossible to investigate the function of IRAK1 in modulating the tumor microenvironment. Using our well-established syngeneic SCLL mouse model, we defined IRAK1 gene as a critical target of FGFR1 in driving leukemogenesis. Our preliminary studies revealed that IRAK1 knockout (KO) in SCLL leukemia cells leads to T cell mediated clearance of the leukemia cells. In contrast, IRAK1 expressing mock control (MC) cells induce the accumulation of myeloid derived suppressor cells (MDSCs), which sequentially suppress T cell function to promote leukemia progression. These observations indicate a critical function for IRAK1 in modulating the tumor microenvironment and promoting leukemogenesis. Most importantly, pilot cytokine profiling of the MC and IRAK1 KO leukemia cells identified IFN-γ as one of the direct targets of IRAK1 in leukemia cells, and scRNA-Seq analysis revealed predominant expression of IFNGR1 and identified activation of the IFN-γ signaling pathway in leukemia induced MDSCs. Genes in this pathway include IFN-γ directly regulated genes, such as CD274 and SOCS3. Further engraftment using either IFNGR1-/- mice or IFNG KO leukemia cells revealed the involvement of this leukemia cell intrinsic, IRAK1 regulated IFN-γ in modulating the tumor microenvironment to promote leukemogenesis. In summary, our data suggests that leukemia cell-derived, IRAK1 regulated inflammatory signals act as critical modulators in establishing a tumor favorable microenvironment during leukemogenesis. Citation Format: Ting Zhang, Xiaocui Lu, Atsuko Matsunaga, Xuexiu Fang, Stephanie Fay Mori, John Cowell, Tianxiang Hu. IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 657.

Abstract 6406: Radiation and STING activation limit tumor development and modulate the immune environment via distinct mechanisms

Abstract Carcinogen-induced lung cancer in A/J mice is an established model of tumor initiation and development. The impact of radiation, immune response, and STING pathway activation on tumor development in this model is not well understood. In this study, we examined the effect of radiation and the STING agonist, DMXAA (Vadimezan), on the development of urethane-induced lung cancer. A/J mice were treated with urethane (i.p.) to induce the development of lung tumors. Three days after urethane treatment, mice received 13 Gy thorax radiation treatment (RT)/mock RT, DMXAA (20 mg/kg i.p.)/vehicle, or a combination treatment (n = 5). Mice were euthanized after 6 hours to 7 days after treatment to assess early events in the lung with RNA-sequencing and flow cytometry, or 5 months after treatment to assess tumor growth and long-term changes to the immune microenvironment with multiplex immunofluorescence. Mice treated with urethane followed by RT had fewer lung surface tumors (p < 0.01) and reduced tumor area (p = 0.0732) compared to their mock RT controls. In parallel, mice that received urethane pre-treatment and RT had high numbers of tertiary lymphoid structures (TLSs; these contained T-cells, B-cells, MHCII+ APCs and CXCL13) compared to the mock RT control mice (p < 0.001). Analysis of immune cell contents of the TLS revealed a notable increase in CD4, CD8 and regulatory T-cells in the RT condition (with urethane pretreatment). Important early events in the lung following RT of urethane pre-treated mice included a transient increase in type I and type II interferons, CD4 and CD8 T-cell infiltration (p < 0.001) and a strong downregulation of cell cycle events. In contrast to these findings, DMXAA treatment after urethane resulted in fewer tumors (p < 0.05), but larger tumors compared to the vehicle-treated group. An increase in immune cells was observed in the lung, however, without clear structures that resembled TLSs. Early events following DMXAA treatment with urethane pre-treatment included dampened levels of type I IFN and TNF-α in circulation and a decrease in T-cell infiltration into the lungs. Interestingly, RT and DMXAA combination treatment (with urethane pre-treatment) drove a synergistic anti-tumor effect, further reducing tumor numbers compared to the RT or DMXAA controls 5 months after treatment. RT and STING agonist treatment of pre-neoplastic lesions have differential impacts on tumor growth in the A/J mouse model, and work synergistically when combined. The different outcomes of RT and DMXAA on tumor growth may be driven by their distinctive mechanism of action on immune responses and capacities to form TLSs. These findings may have future implications for strategies for the early treatment of lung and other cancers, and they suggest that immune responses, including modulation of the STING pathway, may be an important aspect of early tumor development that could be targeted therapeutically. Citation Format: Kay Shigemori, Yanyan Jiang, Bruno Beernaert, Anderson J. Ryan, Eileen E. Parkes. Radiation and STING activation limit tumor development and modulate the immune environment via distinct mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6406.

Quadrivalent Formulation of Intranasal Influenza Vaccine M2SR (M2-Deficient Single Replication) Protects against Drifted Influenza A and B Virus Challenge.

Current influenza vaccines demonstrate low vaccine efficacy, especially when the predominantly circulating strain and vaccine are mismatched. The novel influenza vaccine platform M2- or BM2-deficient single replication (M2SR and BM2SR) has been shown to safely induce strong systemic and mucosal antibody responses and provide protection against significantly drifted influenza strains. In this study, we demonstrate that both monovalent and quadrivalent (Quad) formulations of M2SR are non-pathogenic in mouse and ferret models, eliciting robust neutralizing and non-neutralizing serum antibody responses to all strains within the formulation. Following challenge with wildtype influenza strains, vaccinated mice and ferrets demonstrated reduced weight loss, decreased viral replication in the upper and lower airways, and enhanced survival as compared to mock control groups. Mice vaccinated with H1N1 M2SR were completely protected from heterosubtypic H3N2 challenge, and BM2SR vaccines provided sterilizing immunity to mice challenged with a cross-lineage influenza B virus. Heterosubtypic cross-protection was also seen in the ferret model, with M2SR vaccinated animals exhibiting decreased viral titers in nasal washes and lungs following the challenge. BM2SR-vaccinated ferrets elicited robust neutralizing antibodies toward significantly drifted past and future influenza B strains. Mice and ferrets that received quadrivalent M2SR were able to mount immune responses equivalent to those seen with each of the four monovalent vaccines, demonstrating the absence of strain interference in the commercially relevant quadrivalent formulation.

Abstract 1790: Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma

Abstract Antigenic heterogeneity and limited immune cell infiltration has historically hindered immunotherapy approaches to treat the pediatric brain tumor medulloblastoma (MB). Therefore, modern MB treatment modalities rely on broadly cytotoxic chemotherapy and/or radiation, leaving survivors with side effects that diminish quality of life, stressing an urgent need for novel approaches. Gamma delta (γδ) T cells represent an emerging class of cellular immunotherapy with preclinical potency against the CNS tumor glioblastoma and pediatric solid tumors. The ability of γδ T cells to recognize tumor stress antigens, alongside their MHC independence, supports γδ T cell usage as an off-the-shelf allogeneic cellular immunotherapy for MB and other immunologically cold cancers. Our team recently identified protein tyrosine kinase 7 (PTK7) as a novel immunotherapy target highly expressed in neuroblastoma, with low pediatric healthy tissue expression. Alpha beta T cells engineered with CARs targeting PTK7 demonstrated impressive antitumor efficacy against PTK7+ neuroblastoma xenografts. In that study, we also noted MB and other pediatric solid tumors express elevated levels of PTK7 mRNA. This work therefore serves to test the susceptibility of MB to γδ T cell immunotherapy and validate if targeting PTK7 can further enhance γδ T cell potency. Six MB cell lines representing three out of the four MB molecular subgroups were chosen: DAOY, ONS-76, UW228, D341, D425 and D283. Western blot analysis showed five of six MB cell lines express PTK7 protein, and flow cytometry confirmed PTK7 membrane localization. PTK7high (DAOY) and PTK7low (ONS-76) cells were chosen to test for the antigen-specific killing potential of PTK7-targeted CAR γδ T cells against MB. To generate PTK7-targeted γδ T cells, previously cryopreserved γδ T cells were electroporated with mRNA encoding a PTK7-targeted CAR construct containing the CD28 co-stimulatory domain. After a four hour co-culture, both ONS-76 and DAOY showed moderate rates of apoptosis from exposure to mock electroporated or naïve γδ T cells at 1:1, 2:1 and 5:1 effector:target (E:T) ratios, with approximately 30-40% cell death at 5:1. There was no change in susceptibility of ONS-76 cells to PTK7-CAR γδ T cells when compared to mock electroporated or naïve γδ T cell controls. However, co-culture of DAOY cells with PTK7-CAR γδ T cells markedly increased tumor cell death by as much as 40% compared to the mock controls at E:T ratios as low as 1:1. In summary, naïve expanded γδ T cells induce pronounced MB tumor cell death. Cytotoxicity is further increased against PTK7high MB cells following the introduction of an anti-PTK7 CAR. These results provide a strong rationale for additional preclinical studies regarding the feasibility of γδ T cell-based immunotherapy against MB. Citation Format: Benjamin S. Lowry, Hunter C. Jonus, Jasmine Lee, Trent H. Spencer, Anna M. Kenney, Kelly Goldsmith. Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1790.

Penicillium citrinum Provides Transkingdom Growth Benefits in Choy Sum (Brassica rapa var. parachinensis).

Soil-borne beneficial microbes establish symbioses with plant hosts and play key roles during growth and development therein. In this study, two fungal strains, FLP7 and B9, were isolated from the rhizosphere microbiome associated with Choy Sum (Brassica rapa var. parachinensis) and barley (Hordeum vulgare), respectively. Sequence analyses of the internal transcribed spacer and 18S ribosomal RNA genes combined with colony and conidial morphology identified FLP7 and B9 to be Penicillium citrinum strains/isolates. Plant-fungus interaction assays revealed that isolate B9 showed significant growth promotion effects in Choy Sum plants cultivated in normal soil, as well as under phosphate-limiting conditions. In comparison to the mock control, B9-inoculated plants showed a 34% increase in growth in aerial parts and an 85% upsurge in the fresh weight of roots when cultivated in sterilized soil. The dry biomass of such fungus-inoculated Choy Sum increased by 39% and 74% for the shoots and roots, respectively. Root colonization assays showed that P. citrinum associates directly with the root surface but does not enter or invade the root cortex of the inoculated Choy Sum plants. Preliminary results also indicated that P. citrinum can promote growth in Choy Sum via volatile metabolites too. Interestingly, we detected relatively higher amounts of gibberellins and cytokinins in axenic P. citrinum culture filtrates through liquid chromatography-mass spectrometry analyses. This could plausibly explain the overall growth induction in P. citrinum-inoculated Choy Sum plants. Furthermore, the phenotypic growth defects associated with the Arabidopsis ga1 mutant could be chemically complemented by the exogenous application of P. citrinum culture filtrate, which also showed accumulation of fungus-derived active gibberellins. Our study underscores the importance of transkingdom beneficial effects of such mycobiome-assisted nutrient assimilation and beneficial fungus-derived phytohormone-like metabolites in the induction of robust growth in urban farmed crops.

The Infection Dynamics of Experimental Edwardsiella ictaluri and Flavobacterium covae Coinfection in Channel Catfish (Ictalurus punctatus)

Edwardsiella ictaluri and Flavobacterium covae are pervasive bacterial pathogens associated with significant losses in catfish aquaculture. Bacterial coinfections have the potential to increase outbreak severity and can worsen on-farm mortality. A preliminary assessment of in vivo bacterial coinfection with E. ictaluri (S97-773) and F. covae (ALG-00-530) was conducted using juvenile channel catfish (Ictalurus punctatus). Catfish were divided into five treatment groups: (1) mock control; (2) E. ictaluri full dose (immersion; 5.4 × 105 CFU mL-1); (3) F. covae full dose (immersion; 3.6 × 106 CFU mL-1); (4) E. ictaluri half dose (immersion; 2.7 × 105 CFU mL-1) followed by half dose F. covae (immersion; 1.8 × 106 CFU mL-1); and (5) F. covae half dose followed by half dose E. ictaluri. In the coinfection challenges, the second inoculum was delivered 48 h after the initial exposure. At 21 days post-challenge (DPC), the single dose E. ictaluri infection yielded a cumulative percent mortality (CPM) of 90.0 ± 4.1%, compared with 13.3 ± 5.9% in the F. covae group. Mortality patterns in coinfection challenges mimicked the single dose E. ictaluri challenge, with CPM of 93.3 ± 5.4% for fish initially challenged with E. ictaluri followed by F. covae, and 93.3 ± 2.7% for fish exposed to F. covae and subsequently challenged with E. ictaluri. Despite similarities in the final CPM within the coinfection groups, the onset of peak mortality was delayed in fish exposed to F. covae first but was congruent with mortality trends in the E. ictaluri challenge. Catfish exposed to E. ictaluri in both the single and coinfected treatments displayed increased serum lysozyme activity at 4-DPC (p < 0.001). Three pro-inflammatory cytokines (il8, tnfα, il1β) were evaluated for gene expression, revealing an increase in expression at 7-DPC in all E. ictaluri exposed treatments (p < 0.05). These data enhance our understanding of the dynamics of E. ictaluri and F. covae coinfections in US farm-raised catfish.

Therapeutic Effects of Cornuside on Particulate Matter-Induced Lung Injury.

Particulate matter (PM) is a mixture comprising both organic and inorganic particles, both of which are hazardous to health. The inhalation of airborne PM with a diameter of ≤2.5 μm (PM2.5) can cause considerable lung damage. Cornuside (CN), a natural bisiridoid glucoside derived from the fruit of Cornus officinalis Sieb, exerts protective properties against tissue damage via controlling the immunological response and reducing inflammation. However, information regarding the therapeutic potential of CN in patients with PM2.5-induced lung injury is limited. Thus, herein, we examined the protective properties of CN against PM2.5-induced lung damage. Mice were categorized into eight groups (n = 10): a mock control group, a CN control group (0.8 mg/kg mouse body weight), four PM2.5+CN groups (0.2, 0.4, 0.6, and 0.8 mg/kg mouse body weight), and a PM2.5+CN group (0.2, 0.4, 0.6, and 0.8 mg/kg mouse body weight). The mice were administered with CN 30 min following intratracheal tail vein injection of PM2.5. In mice exposed to PM2.5, different parameters including changes in lung tissue wet/dry (W/D) lung weight ratio, total protein/total cell ratio, lymphocyte counts, inflammatory cytokine levels in the bronchoalveolar lavage fluid (BALF), vascular permeability, and histology were examined. Our findings revealed that CN reduced lung damage, the W/D weight ratio, and hyperpermeability caused by PM2.5. Moreover, CN reduced the plasma levels of inflammatory cytokines produced because of PM2.5 exposure, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide, as well as the total protein concentration in the BALF, and successfully attenuated PM2.5-associated lymphocytosis. In addition, CN substantially reduced the expression levels of Toll-like receptors 4 (TLR4), MyD88, and autophagy-related proteins LC3 II and Beclin 1, and increased protein phosphorylation of the mammalian target of rapamycin (mTOR). Thus, the anti-inflammatory property of CN renders it a potential therapeutic agent for treating PM2.5-induced lung injury by controlling the TLR4-MyD88 and mTOR-autophagy pathways.

First report of youcai mosaic virus in firecracker flower (Crossandra infundibuliformis) in Taiwan.

Firecracker flower or crossandra (Crossandra infundibuliformis), an ornamental native to southern Asia, is commonly grown as bedding plants in the garden. In January 2021, crossandra plants showing mosaic, chlorotic ringspot, and leaf deformation (Suppl. Fig. 1) were observed at a recreational farm in Zhuolan Township (Miauli County, Taiwan) (E120°82'62'', N24°33'30'') . Transmission electron microscope (JEM-1400, JOEL, Japan) examination by negative staining of 10 affected plants indicated the presence of particles resembling a tobamovirus in all examined affected samples. However, no tobamovirus-like particles were observed in the crude sap prepared from healthy crossandra leaf tissues. Total RNA was extracted from affected leaves and used for RT-PCR amplification using the tobamovirus group-specific primer pair Tob Uni1 (5'-ATTTAAGTGGAGGGAAAACCACT-3') and Tob Uni2 (5'-GTYGTTGATGAGTTCGTGGA-3') (Letschert et al., 2002). A cDNA fragment of about 650-bp was amplified and Sanger sequenced (ABI PRISM 3730 DNA Sequencer, Biotechnology Center at National Chung Hsing University, Taichung, Taiwan ), revealing 98% sequence identity to that of a Brassica isolate of youcai mosaic virus (YoMV, AY318866). The virus was isolated through mechanical inoculation onto Chenopodium quinoa to yield two pure isolates, designated FC-1 and FC-2. Mechanical inoculation of FC-1 and FC-2 back to virus-free Crossandra infundibuliformis plants (5 for each isolate) resulted in systemic mosaic, chlorotic ringspot, and leaf deformation. All mock and healthy controls were symptomless and failed to obtain any RT-PCR products with YoMV-specific primers CPF1 (5'- ATGGTTTACAACATCACGAG-3') and CPR1 (5'-CTATGTAGCTGGCGCAGTAG-3'). Systemic symptoms of mild mosaic, ringspot, leafroll, and necrosis appeared on some of the tobaccos (Nicotiana benthamiana, N. tabacum, N. rustica), and cruciferous vegetables (Brassica rapa subsp. chinensis cv. Known-You No.2), B. rapa subsp. pekinensis cv. Autumn Sun), B. oleracea var. italica cv. Ching Hua), B. oleracea var. capitata cv. Green Peak), and Raphanus sativus cv. Snow Lady). However, inoculation of FC-1 and FC-2 on pepper (Capsicum annuum cv. Blue Star) resulted in severe necrosis on leaves and necrotic sunken spots on petioles and stems causing acute wilting and quick death. In stark contrast, FC-1 and FC-2 only induced local lesions on inoculated leaves of Chenopodium quinoa, Gomphrena globose, and Carica papaya. The infectivity of FC isolates to all plants used in host range tests were further confirmed by RT-PCR as mentioned. Oligonucleotide primers (Suppl. Table 1) specifically complementary to YoMV sequence were designed and used to amplify full-length genomic sequences of FC-1 and FC-2 isolates by RT-PCR and Sanger sequencing. Sequence analysis revealed that the genome of both FC-1 and FC-2 isolates consists of 6302 nucleotides. The viral genome has four open reading frames encoding a small replicase subunit, a RNA-dependent RNA polymerase , a movement protein, and a coat protein (CP), respectively. Both sequences, which shared 99.6% identity with each other, have been deposited in the NCBI database (Genbank Accession Numbers LC701592 and LC701593). FC-1 and FC-2 and the deduced amino acid sequences of CP shared 91.2% - 98.9% and 93.4 - 99.4% similarities, respectively, to those of published YoMV strains, confirming the identity of FC-1 and FC-2. RT-PCR analyses detected YoMV in all (100%) crossandra samples collected from the field (Suppl. Fig. 2). YoMV, formerly named Chinese rape mosaic virus (CRMV) or oilseed rape mosaic virus (ORMV) (Zhu et al., 2001), has been reported to infect cruciferous, solanaceous, and ornamental crops in Asia and Europe (Ju et al., 2019). The firecracker flower is a common and popular ornamental in Taiwan, even though its economic values are not so important. However, finding YoMV in firecracker flower may have epidemiological impacts as YoMV can infect economically important cruciferous and solanaceous crops. To our knowledge, this is the first report of YoMV infecting firecracker flowers in Taiwan.

First Report of Stem brown spot on 'Thai Gold' Selenicereus megalanthus in Malaysia caused by Nigrospora sphaerica.

'Thai Gold' yellow pitahaya (family Cactaceae, Selenicereus megalanthus) is a new crop being planted commercially in Malaysia. In May 2021, reddish-brown necrotic lesions were observed on the stems of approximately 60% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). As the disease progressed, the smaller lesions merged into larger irregularly shaped areas that formed dark brown in color. Stems with reddish-brown spot symptoms from ten plants were collected from the field and brought to the laboratory in sterilized paper bags. The symptom margin was excised into small blocks (5 x 5 x 5 mm). The blocks were surface sterilized based on Khoo et al. (2022), and placed on potato dextrose agar (PDA). The pathogens were isolated (three isolates were obtained) and cultured on potato dextrose agar (PDA) at 25°C for 5 days in the dark. The isolates developed floccose, white colony that darkened with age in PDA. Conidia (n = 30) were single celled, black, smooth, globose to subglobose, 13.9 to 18.7 μm in diameter, and borne singly on a hyaline vesicle at the tip of each conidiophore. Genomic DNA was extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (tef1-a) region and β-tubulin (tub2) genes were performed using ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T10/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) primer sets, respectively. The products were sent to Apical Scientific Sdn. Bhd. for purification and sequencing. BLASTn analysis of the newly generated ITS (OK448496, OM832586, OM832589) were 100% identical to Nigrospora sphaerica isolate 1SS (MN339998) (507/507 bp), tef1-a (OM223859, OM826971, OM826972) were 100% identical to Nigrospora sphaerica isolate F (MT708197) (497/497 bp) and tub2 (OL697400, OM826973, OM826974) were 100% identical to Nigrospora sphaerica isolate SN180517 (MN719407) (434/434 bp). The isolates established a supported clade to the related N. sphaerica type sequences, according to phylogenetic analysis using maximum likelihood based on the concatenated ITS, tef1-a and tub2 sequences. Morphological and molecular characterization matched the description of N. sphaerica (Kee et al. 2019). Koch's postulates were performed by spray inoculation (106 spores/ml) of isolate Keningau on the stem of three 'Thai Gold' yellow pitahaya plants in growth stage 4 (BBCH code: 419) (Kishore, 2016), while water was sprayed on three mock controls. The experiment was repeated using isolate Keningau02 and Keningau03 as inoculants. The inoculated stems on yellow pitahaya plants were covered with plastics for 48 h, and the plants were maintained in a greenhouse at room temperature 25 to 28°C with a relative humidity of 80 to 90%. All the inoculated stems developed symptoms 5 days post-inoculation, whereas no symptoms occurred on mock controls, thus fulfilling the Koch's postulates. No pathogen was isolated from the mock controls. The experiments were repeated two more times for each isolate. The reisolated fungi were identical to N. sphaerica morphologically and molecularly. Previously, N. sphaerica has been reported to cause stem brown spot disease on S. megalanthus in the Philippines (Taguiam et al. 2020). To our knowledge, this is the first report of N. sphaerica causing stem brown spot on 'Thai Gold' S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for the Malaysian yellow pitahaya production.

Abstract C042: Low miR-9 impacts tumorigenesis and chemoresistance in Black compared with White laryngeal squamous cell carcinoma

Abstract Background: Advanced stage laryngeal squamous cell carcinoma (LSCC) has sustained one of the lowest five-year survival rates across all other head and neck squamous cancers. For several decades, Black patients have maintained a greater likelihood of presenting with advanced LSCC and a higher overall mortality rate compared with White patients. Even when accounting for sociodemographics, a disparity remains, underscoring the importance of investigating the biologic basis of this disparity. Differential microRNA (miRNA) expression has been linked to racially disparate clinical outcomes in other cancers including breast and prostate, but remains underdetermined in LSCC. We present our findings where we investigated miR-9 levels and their influence on LSCC tumorigenesis and chemoresistance using in vitro cell lines derived from Black and White patients. Methods: We previously identified miR-9-5p as significantly lower (5X) in Black compared with White advanced stage LSCC in vivo samples. For functional studies, we obtained two LSCC cell lines derived from a Black patient (UM-SCC-12) and from a White patient (UM-SCC-10A) (provided by Tom Carey, University of Michigan). After transfection with either miR-9 mimic or inhibitor, we used the scratch wound assay to assess cell migration and the cell titer blue assay to assess cell proliferation and chemoresistance. Results: Prior to transfection, we found that UM-SCC-12 had baseline increases in cellular migration, proliferation and chemoresistance compared to UM-SCC-10A. By Northern blot analysis, we further determined that mature miR-9-5p was inherently 5X lower in UM-SCC-12 compared with UM-SCC-10A. After transient transfection, we noted that overexpressing miR-9 in UM-SCC-12 resulted in decreased cellular migration, decreased proliferation and increased chemosensitivity compared to a mock oligo control, whereas inhibiting miR-9 in UM-SCC-10A resulted in increased cellular migration, increased proliferation and decreased chemosensitivity compared to a mock oligo control. Conclusion: Collectively, these studies support that low miR-9 may play a role in LSCC tumorigenesis and chemoresistance. In this manner, it may contribute to cancer health disparate outcomes observed in advanced stage LSCC. Future studies will involve characterizing significant downstream mediators of low miR-9-5p gene regulation. Citation Format: Christina Gobin, Samuel Inkabi, Chayil Lattimore, James Menefee, Tengfei Bian, Christopher Fields, Mingyi Xie, Chengguo Xing, Kristianna Fredenburg. Low miR-9 impacts tumorigenesis and chemoresistance in Black compared with White laryngeal squamous cell carcinoma [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C042.

Digital PCR: A Tool to Authenticate Herbal Products and Spices.

Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However, poor DNA quality and quantity are the major demerits of conventional PCR and real-time quantitative PCR (qPCR), as herbal products and spices are highly enriched in secondary metabolites such as polyphenolic compounds. The third-generation digital PCR (dPCR) technology is a highly sensitive, accurate, and reliable method to detect target DNA molecules as it is less affected by PCR inhibiting secondary metabolites due to nanopartitions. Therefore, it can be certainly used for the detection of adulteration in herbal formulations. In dPCR, extracted DNA is subjected to get amplification in nanopartitions using target gene primers, the EvaGreen master mix, or fluorescently labeled targeted gene-specific probes. Here, we describe the detection of Carica papaya (CP) adulteration in Piper nigrum (PN) products using species-specific primers. We observed an increase in fluorescence signal as the concentration of target DNA increased in PN-CP blended formulations (mock controls). Using species-specific primers, we successfully demonstrated the use of dPCR in the authentication of medicinal botanicals.

Impaired immune response and barrier function in GSPD-1-deficient C. elegans infected with Klebsiella pneumoniae

gspd-1-RNAi knockdown Caenorhabditis elegans was used as an immune-compromised model to investigate the role of G6PD in host-pathogen interactions. A shorted lifespan, increased bacterial burden and bacterial translocation were observed in gspd-1-knockdown C. elegans infected with Klebsiella pneumoniae (KP). RNAseq revealed that the innate immune pathway, including clc-1 and tsp-1, was affected by gspd-1 knockdown. qPCR confirmed that tight junction (zoo-1, clc-1) and immune-associated genes (tsp-1) were down-regulated in gspd-1-knockdown C. elegans and following infection with KP. The down-regulation of antimicrobial effector lysozymes, including lys-1, lys-2, lys-7, lys-8, ilys-2 and ilys-3, was found in gspd-1-knockdown C. elegans infected with KP. Deletion of clc-1, tsp-1, lys-7, and daf-2 in gspd-1-knockdown C. elegans infected with KP abolished the shorten lifespan seen in the Mock control. GSPD-1 deficiency in C. elegans resulted in bacterial accumulation and lethality, possibly due to a defective immune response. These findings indicate that GSPD-1 has a protective role in microbial defense in C. elegans by preventing bacterial colonization through bacterial clearance.

First Report of Neoscytalidium dimidiatum Causing stem canker on Selenicereus megalanthus in Malaysia.

Selenicereus megalanthus (family Cactaceae), commonly known as yellow pitahaya is a new crop being planted commercially in Malaysia. In May 2021, stem canker symptoms with sign of black pycnidia formed on the surface of canker (30- to 55-mm in diameter) were observed on the stem of 80% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). The infected stems became rotted when black pycnidia formed. To isolate the pathogen, the symptom margin was excised into four small blocks (5 x 5 x 5 mm), and the blocks were surface sterilized based on Khoo et al. (2022) before plating on potato dextrose agar (PDA). Plates were incubated at 25°C for 7 days in the dark. Two isolates were obtained and were named Keningau and Keningau02. Powdery white mycelia were initially observed in two plates, and then became dark grey with age. Dark pigmentation in plates was observed after a week of incubation at 25°C in the dark. Arthroconidia (n= 30) were hyaline to dark brown, circular or cylindrical with round to truncate ends, with zero to one septum, measuring 8.9 x 5.6 µm in size. Conidia (n= 30) exuded in milky white cirrhus from pycnidia were one-celled, aseptate, oblong, measuring 10.3 × 4.6 µm in size. When reached the maturity stage, conidia were brown and septate. Genomic DNA from Keningau and Keningau02 were extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF1) region and β-tubulin (TUB) genes were performed using ITS1/ITS4, EF1-728F/EF1-986R and T10/Bt2b primer sets, respectively (Carbone and Kohn, 1999; O'Donnell et al. 1997; White et al. 1990). The products were sent to Apical Scientific Sdn. Bhd. for sequencing. BLASTn analysis of the newly generated ITS (GenBank OK458559, OM649909), TEF1 (GenBank OM677768, OM677769) and TUB (GenBank OL697398, OM677766) indicated 99% identity to Neoscytalidium novaehollandiae strain CBS 122071 (GenBank MT592760). Phylogenetic analysis using maximum likelihood and Bayesian inference on the concatenated ITS-TEF1-TUB was constructed using IQ-Tree and MrBayes3.2.7. Neoscytalidium hyalinum, N. novaehollandiae and Neoscytalidium orchidacearum are reduced to synonymy with N. dimidiatum (Philips et al. 2013; Zhang et al. 2021). Although N. novaehollandiae is morphologically and phylogenetically similar to N. dimidiatum, but N. novaehollandiae produce muriform, Dichomera-like conidia that distinguish this species from other known Neoscytalidium species (Crous et al. 2006). No muriform, Dichomera-like conidia were observed in the Malaysia' isolates. The pathogen was identified as N. dimidiatum based on molecular data and morphological characterization (Serrato-Diaz and Goenaga, 2021). Pathogenicity tests were performed based on Mohd et al. (2013) by injection inoculation of 0.2 ml of conidial suspensions (1 x 106 conidia/ml) from isolate Keningau to three 30-month-old yellow pitahaya stems using a disposable needle and syringe. Distilled water was injected into three mock controls. The inoculated yellow pitahaya plants were covered with plastics for 48 h and incubated at 25°C. The pathogenicity test was also performed using isolate Keningau02. All inoculated stems developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiments were repeated two more times. The reisolated fungi were identical to the pathogen morphologically and molecularly. To our knowledge, this is the first report of N. dimidiatum causing stem canker on S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared in the Malaysian yellow pitahaya production.

First report of Macrophomina phaseolina causing leaf blight on Pometia pinnata in Malaysia.

Pometia pinnata (family Sapindaceae), locally known as 'Kasai', is a tropical hardwood and fruit tree species grown in Malaysia. The decoction of the bark is used for the treatment of fever, sores and colds, while the fruits are edible (Adema et al. 1996). In May 2021, irregular brown spots and necrotic lesions were observed on 'Kasai' with an incidence and severity of approximately 60% and 10% on 10 plants in a nursery (5°55'30.7"N 116°04'36.2"E) in Penampang, Sabah province. When the disease progressed, the spots coalesced into extended patches, blightening the leaves and, gradually, the entire foliage. Small pieces (5 x 5 mm) of infected leaves were excised from the infected margin, and then surface sterilized according to Khoo et al. (2022b), and plated on potato dextrose agar (PDA), and cultured at 25 °C. for 6 days. Colonies were dark brown in color initially whitish on the PDA. The color of fungal colony was dark as the culture aged. Semi-appressed mycelia were observed on the plates with abundant microsclerotia engrossed in the agar. Aggregation of hyphae formed black and round to oblong or irregular shaped microsclerotia. Thirty sclerotia from a representative isolate measured average 63-171 μM length x 57-128 μM wide. The morphological features matched the description of Macrophomina phaseolina (Abd Rahim et al. 2019). The fungal genomic DNA was extracted based on Khoo et al. (2022a and 2022b). PCR was performed using primer sets ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T1/T22 (O'Donnell and Cigelnik 1997) to amplify the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF-1α) region and partial β-tubulin (TUB) gene. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. in Malaysia for sequencing. According to results (GenBank Accession No. OK465197, OM677767, ON237461), they were 100% identical with the reference sequence of Macrophomina phaseolina containing approximately 537 bp, 438 bp and 659 bp of the presented ITS, TEF-1α and TUB region (GenBank Accession No. MN629245, MN136199 and KF952208, respectively). The pathogen was identified as M. phaseolina based on its morphological and molecular data (Abd Rahim et al. 2019). To confirm the pathogenicity test, three non-wounded and healthy leaves of one-month-old 'Kasai' seedlings were inoculated with mycelium plug (1 x 1 cm) of M. phaseolina. Additional three 'Kasai' seedlings were inoculated with sterile PDA agar plug (1 x 1 cm) to serve as controls. The seedlings were monitored and incubated in a greenhouse at ambient temperature based on Iftikhar et al. (2022). After 6 days of inoculation, all infected leaves exhibited the symptoms as observed in the nursery, while the controls remained asymptomatic. The experiment was repeated twice. Re-isolation was performed from the symptomatic leaves and controls. The reisolated fungal isolates were identical to M. phaseolina morphologically and molecularly. No pathogens were isolated from the mock controls. M. phaseolina has been reported to cause leaf blight on Jasminium multiflorum in India (Mahadevakumar and Janardhana, 2016), and Crinum asiaticum and Hymenocallis littoralis in Malaysia (Abd Rahim et al. 2019). To our knowledge, this is the first report of M. phaseolina causing leaf blight on 'Kasai' in Malaysia and worldwide. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for 'Kasai' in Malaysia.

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

BackgroundSushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor.MethodsIn a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method.ResultsWe discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells.ConclusionsIn this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.

Enlightenments of DITTO framework and its application for setting up mock acupuncture in clinical research

How to set up a placebo control scientifically and effectively is a problem in clinical research on acupuncture therapy. In 2020, the UK National Institute of Health Research established a standardized methodological framework DITTO, that is, "Deconstruct""Identify""Take out""Think" and "Optimize" to standardize the setting of invasive console-control intervention. Acupuncture therapy theoretically belongs to the category of invasive therapeutic intervention, and the percutaneous puncture therapy was also considered when the DITTO framework was developed. Therefore, this framework can be applied to clinical research of acupuncture, and may provide a reference for the appropriate setting of simulated acupuncture. In this article, we introduce the formulation process and main contents of the DITTO framework, and analyze the adjustments needed to be made and possible problems encountered in the application of each part of the framework in acupuncture clinical research through medical cases. The DITTO framework can guide and standardize the design of mock acupuncture control in acupuncture clinical research to a certain extent, which is conducive to finding a more suitable implementation method of mock acupuncture, thus promoting the improvement of the quality of acupuncture clinical research. However, because there is no precedent in the field of acupuncture in traditional Chinese medicine, further research is needed in the future practical application so that it can be better combined with the characteristics of acupuncture and the purpose of clinical research.