Integrated approach to functional analysis of an ERBB2 variant of unknown significance detected by a cancer gene panel test

Purpose Dealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G. Methods ERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo. Results We found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo. Conclusion Our results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

Different Fruit-Specific Promoters Drive AtMYB12 Expression to Improve Phenylpropanoid Accumulation in Tomato

Tomato is an economically crucial vegetable/fruit crop globally. Tomato is rich in nutrition and plays an essential role in a healthy human diet. Phenylpropanoid, a critical compound in tomatoes, reduces common degenerative and chronic diseases risk caused by oxidative stress. As an MYB transcription factor, ATMYB12 can increase phenylpropanoid content by activating phenylpropanoid synthesis related genes, such as PAL, C4H, 4CL, CHS. However, the heterologous expression of AtMYB12 in tomatoes can be altered through transgenic technologies, such as unstable expression vectors and promoters with different efficiency. In the current study, the efficiency of other fruit-specific promoters, namely E8S, 2A12, E4, and PG, were compared and screened, and we determined that the expression efficiency of AtMYB12 was driven by the E8S promoter was the highest. As a result, the expression of phenylpropanoid synthesis related genes was regulated by AtMYB12, and the phenylpropanoid accumulation in transgenic tomato fruits increased 16 times. Additionally, the total antioxidant capacity of fruits was measured through Trolox equivalent antioxidant capacity (TEAC) assay, which was increased by 2.4 times in E8S transgenic lines. TEAC was positively correlated with phenylpropanoid content. Since phenylpropanoid plays a crucial role in the human diet, expressing AtMYB12 with stable and effective fruit-specific promoter E8S could improve tomato’s phenylpropanoid and nutrition content and quality. Our results can provide genetic resources for the subsequent improvement of tomato varieties and quality, which is significant for human health.

The role of the Siglec-G ITIM domain during bacterial infection

Siglecs, membrane-bound lectins of the sialic acid-binding immunoglobulin superfamily, inhibit immune responses by recruiting tyrosine phosphatases (e.g., SHP-1 and SHP-2) through their cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) domain. The role of Siglecs in infection has been extensively studied, but downstream signaling through the ITIM domain remains unclear. Here, we used a GST pull-down assay to identify additional proteins associated with the ITIM domain during bacterial infection. Gdi2 bound to ITIM under normal homeostasis, but Rab1a was recruited to ITIM during bacterial infection. Western blot analysis confirmed the presence of SHP-1 and SHP-2 in eluted ITIM-associated proteins under normal homeostasis. We confirmed the association of ITIM with Gdi2 or Rab1a by transfection of corresponding expression vectors in 293T cells followed by immunoprecipitation-western blot assay. Thus, ITIM’s role in the inhibition of the immune response during bacterial infection may be regulated by interaction with Gdi2 and Rab1a in addition to SHP-1 and SHP-2.

Development of an Oral Salmonella-Based Vaccine Platform against SARS-CoV-2

Effective vaccine development for global outbreaks, such as the coronavirus disease 2019 (COVID-19), has been successful in the short run. However, the currently available vaccines have been associated with a higher frequency of adverse effects compared with other general vaccines. In this study, the possibility of an oral bacteria-based vaccine that can be safely used as a platform for large-scale, long-term immunization was evaluated. A well-known Salmonella strain that was previously considered as a vaccine delivery candidate was used. Recombinant Salmonella cells expressing engineered viral proteins related with COVID-19 pathogenesis were engineered, and the formulation of the oral vaccine candidate strain was evaluated by in vitro and in vivo experiments. First, engineered S proteins were synthesized and cloned into expression vectors, which were than transformed into Salmonella cells. In addition, when orally administrated to mice, the vaccine promoted antigen-specific antibody production and cellular immunity was induced with no significant toxicity effects. These results suggest that Salmonella strains may represent a valuable platform for the development of an oral vaccine for COVID-19 as an alternative to tackle the outbreak of various mutated coronavirus strains and new infectious diseases in the future.

Identification of mercury‐resistant Streptomyces isolated from Cyperus rotundus L. rhizosphere and molecular cloning of mercury (II) reductase gene

Streptomyces is one of mercury‐resistant bacteria which can convert Hg2+ into nontoxic Hg0 . This study aimed to identify mercury‐resistant Streptomyces present in the Cyperus rotundus rhizosphere from artisanal small‐scale gold mining (ASGM) area and clone merA gene to the cloning and expression vectors. Molecular identification was conducted using 16s rRNA gene with the maximum likelihood algorithms. Results revealed that the AS1 and AS2 strains were a group of Streptomyces ardesiacus and the BR28 strain was closed to Brevibacillus agri. The AS2 merA gene was cloned to pMD20 cloning vectors, pGEX‐5x‐1 and pET‐28c expression vectors. The transformation was successfully performed in BL21 and DH5α competent cells. The full length of the merA gene was confirmed to be 1,425 bp. This study is the first research on identifying mercury‐resistant Streptomyces and cloning the full‐length merA gene in Indonesia.

The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa (P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

HPV16 E6 Gene Polymorphisms and the Functions of the Mutation Site in Cervical Cancer Among Uygur and Han Women in Xinjiang

Background: To investigate the genotype distribution of human papillomavirus (HPV) in infected Uygur and Han women in Xinjiang; analyze the HPV16 E6 gene polymorphism site and relationship with the development of cervical cancer.Methods: The HPV16 E6 sequence was analyzed using the European standard prototype to perform an evolutionary tree. HPV16 E6-295T/350T, 295G/350G, and 295T/350G GV230 vectors were stably transfected into cervical cancer C33A cells to analyze the cell proliferation, migration and invasion, apoptosis by CCK8 and clonogenic assays, transwell and cell scratch assays, FACS experiments. Results: The total HPV infection rate was 26.390% (760/2879), whereas the Uygur 22.87% (196/857) and the Han was 27.89% (564/2022) (P < 0.05). Among 110 mutations, 65 cases of E6 genes were mutated at nucleotide 350 (T350G) with the leucine changing to valine (L83V). Moreover, there were 7 cases of E6 gene mutated at nucleotide 295 (T295G) with aspartic changing to glutamic (D64E). When E6 vector(s) of mutations sites were transfected into C33A cells, they were found to promote cellular proliferation, migration, invasion, and inhibit apoptosis. The 295T/350G had the strongest effect on C33A cells and 295G/350G was significantly stronger than 295T/350T (P < 0.05).Conclusions: The positive HPV infection rates differed between the Uygur and Han in Xinjiang, and the genotype distribution of infection was different. After transfecting C33A cells with different eukaryotic expression vectors, the 295T/350G mutation site promoted the proliferation，migration, and invasion of C33A cells to a greater extent than 295G/350G; however, 295G/350G had a stronger effect than 295T/350T.