Atoh8 in Development and Disease

Atoh8 belongs to a large superfamily of transcriptional regulators called basic helix-loop-helix (bHLH) proteins. bHLH proteins have been identified in a wide range of organisms from yeast to humans. The members of this special group of transcription factors were found to be involved not only in embryonic development but also in disease initiation and its progression. Given their importance in several fundamental processes, the translation, subcellular location and turnover of bHLH proteins is tightly regulated. Alterations in the expression of bHLH proteins have been associated with multiple diseases also in context with Atoh8 which seems to unfold its functions as both transcriptional activator and repressor. Like many other bHLH transcription factors, so far, Atoh8 has also been observed to be involved in both embryonic development and carcinogenesis where it mainly acts as tumor suppressor. This review summarizes our current understanding of Atoh8 structure, function and regulation and its complex and partially controversial involvement in development and disease.

Abscisic Acid Increases Hydrogen Peroxide in Multiple Subcellular Compartments to Drive Stomatal Closure

Stomatal closure regulates transpiration and gas exchange in response to environmental cues. Drought upregulates ABA signaling, which elevates levels of reactive oxygen species (ROS). However, the subcellular location and identity of these ROS has received limited study. We found that in guard cells, ABA increased fluorescence of the general redox sensor, dichlorofluorescein (DCF), in distinct subcellular locations including chloroplasts, cytosol, nuclei, and cytosolic puncta. These changes were lost in ABA-insensitive quintuple receptor mutant and accentuated in an ABA-hypersensitive mutant. ABA induced ROS accumulation in these subcellular compartments was lost in mutants with defects in genes encoding hydrogen peroxide synthesizing respiratory burst oxidase homolog (RBOH) enzymes and guard cells treated with the RBOH inhibitor VAS2870, while exogenous hydrogen peroxide treatment is sufficient to close guard cells. The hydrogen peroxide-selective probe, peroxy orange1, also showed ABA-dependent increases in chloroplasts and cytosolic puncta. Using the more sensitive genetically-encoded hydrogen peroxide reporter roGFP-Orp1, we also detected significant hydrogen peroxide increases in the cytosol and nucleus. These cytosolic puncta accumulate ROS after ABA treatment show colocalization with Mitotracker and with a mitochondrial targeted mt-roGFP2-Orp1, which also revealed ABA-increased ROS in mitochondria. These results indicate that elevated hydrogen peroxide after ABA treatment in these subcellular compartments is necessary and sufficient to drive stomatal closure.

NRF2 Participates in the Suppressive Tumor Immune Microenvironment of KRAS/KEAP1 Co-Mutant Non-Small Cell Lung Cancer by Inhibiting the STING Pathway

Background: KRAS/KEAP1 (KK) co-mutant lung adenocarcinoma (LUAD) exhibited poor response to immune checkpoint inhibitors (ICI) via shaping a suppressive tumor immune microenvironment, the mechanism remains to be elucidated. Methods: The mRNA and protein expression of target molecules were analyzed by qRT-PCR and Western blot, respectively. The subcellular location of NRF2 was observed by immunofluorescence staining, and nuclear and cytoplasm isolation. After exogenous over-expression and knockdown of NRF2 and the addition of a STING pathway inhibitor in tumor cells, the effects on the CD8+ T cell recruitment was detected using chemotaxis assay, and the secretion of chemokines CCL5 and CXCL10 was analyzed by ELISA. The potential NRF2 target BRCA1 was identified using bioinformatic approaches and verified by a dual luciferase reporter assay. Results: NRF2, the target of KEAP1, was overexpressed and activated in KK type cells. NRF2 effected as a negative regulator of CD8+ T cells recruitment by decreasing CCL5 and CXCL10 chemokines in KK type LUAD. Mechanistically, NRF2 promoted the transcription and expression of BRCA1 to repair DNA damage, resulting in STING pathway inactivation. Conclusion: The combination of NRF2 inhibitor or STING agonist with ICI may be a promising therapeutic approach for patients with KK type LUAD.

MIR-140 TARGETS LNCRNA DNAJC3-AS1 TO SUPPRESS CELL PROLIFERATION IN ACUTE MYELOID LEUKEMIA

Objectives：MiR-140 and DNAJC3-AS1 have been proven to play critical roles in cancer biology, while their participations in acute myeloid leukemia (AML) are unclear. This study aimed to explore the role of miR-140 and DNAJC3-AS1 in AML. Methods：Analysis of the expression of DNAJC3-AS1 and miR-140 was done with RT-qPCR. The role of DNAJC3-AS1 and miR-140 in the expression of each other was explored with overexpression assay. The direct interaction between DNAJC3-AS1 and miR-140 was analyzed with RNA pull-down assay. The subcellular location of DNAJC3-AS1 was explored with cellular subcellular fractionation assay. Cell proliferation analysis was done with BrdU assay. Results：AML patients showed increased expression of DNAJC3-AS1 and decreased expression of miR-140. DNAJC3-AS1 was detected in both nuclear and cytoplasm samples, and a direct interaction between DNAJC3-AS1 and miR-140 was observed. Discussion：Reduced expression of DNAJC3-AS1 was observed after miR-140 overexpression in AML cells. DNAJC3-AS1 increased cell proliferation and inhibited the role of miR-140 in suppressing cell proliferation. Conclusion：In conclusion, miR-140 may target DNAJC3-AS1 to suppress cell proliferation in AML.

Protein Subcellular Localization Based on Evolutionary Information and Segmented Distribution

The prediction of protein subcellular localization not only is important for the study of protein structure and function but also can facilitate the design and development of new drugs. In recent years, feature extraction methods based on protein evolution information have attracted much attention and made good progress. Based on the protein position-specific score matrix (PSSM) obtained by PSI-BLAST, PSSM-GSD method is proposed according to the data distribution characteristics. In order to reflect the protein sequence information as much as possible, AAO method, PSSM-AAO method, and PSSM-GSD method are fused together. Then, conditional entropy-based classifier chain algorithm and support vector machine are used to locate multilabel proteins. Finally, we test Gpos-mPLoc and Gneg-mPLoc datasets, considering the severe imbalance of data, and select SMOTE algorithm to expand a few sample; the experiment shows that the AAO + PSSM ∗ method in the paper achieved 83.1% and 86.8% overall accuracy, respectively. After experimental comparison of different methods, AAO + PSSM ∗ has good performance and can effectively predict protein subcellular location.

Consequences of COVID-19 on my experiment of gene overexpression in beans

<p>My name is Salma Meléndez and I am currently a graduate in Agrogenomic Sciences. In March 2020, when COVID-19 was detected in Mexico, I was in my eighth semester of my undergraduate degree. At that time, he had an experiment of overexpression of a gene in bean roots, in order to explore its function during symbiosis with rhizobial bacteria. Unfortunately, the laboratory and the entire campus canceled their face-to-face activities in order to reduce the risk of contagion. An alternative was to take the experimental plants to my house to give the proper care, however, the situation became difficult as I did not have the space or the required conditions at home. On the other hand, other research centers with which we had collaboration agreements also canceled access, such is the case of the Optical Research Center, where we used the confocal microscope to detect subcellular location of proteins. The closure of institutions allowed me to write theoretical parts of my thesis, however, the experimental phase was definitely affected for at least six months. The experiment with the plants was almost completely lost. In the subsequent months I had the opportunity to re-enter my institution; however, under strict conditions and on staggered days, which made certain measurements that require daily continuity difficult. Currently, the laboratory is not as it used to look, full of colleagues sharing results and difficulties, exchanging advice and even certain materials. I think the pandemic has pushed us to do our work more individually and slowly. Consequently, my degree was delayed and transferred from 2020 to 2021. There are still many challenges to overcome, although activities have not been fully restored, science does not stop and we have found a way to face it, slowly but surely.</p>

Visualizing Phytochemical-Protein Interaction Networks: Momordica charantia and Cancer

The in silico study of medicinal plants is a rapidly growing field. Techniques such as reverse screening and network pharmacology are used to study the complex cellular action of medicinal plants against disease. However, it is difficult to produce a meaningful visualization of phytochemical-protein interactions (PCPIs) in the cell. This study introduces a novel workflow combining various tools to visualize a PCPI network for a medicinal plant against a disease. The five steps are 1) phytochemical compilation, 2) reverse screening, 3) network building, 4) network visualization, and 5) evaluation. The output is a PCPI network that encodes multiple dimensions of information, including subcellular location, phytochemical class, pharmacokinetic data, and prediction probability. As a proof of concept, we built a PCPI network for bitter gourd (Momordica charantia L.) against colorectal cancer. The network and workflow are available at https://yumibriones.github.io/network/. The PCPI network highlights high-confidence interactions for further in vitro or in vivo study. The overall workflow is broadly transferable and can be used to visualize the action of other medicinal plants or small molecules against other diseases.

New Insights Into Invertase Gene Family and Functional Characterization of Critical Cell Wall Invertase TaCWINV40 For Male Fertility in Wheat (Triticum Aestivum L.)

Invertase (INV, ec3.2.1.26) irreversibly hydrolyzes sucrose into fructose and glucose, and it is regulated by the environment to affect pollen fertility in some plant species. However, there has been a lack of systematic identification of INV gene family in wheat. In order to reveal the potential influence on the male fertility, a total of 130 wheat INVs that unevenly distributed on 21 chromosomes were systematically identified and analyzed in this study. According to physical and chemical properties, subcellular location, and phylogenetic tree, they were divided into two acidic INV (AINV) subtypes: cell wall group (TaCWINV1-68), vacuole group (TaVINV1-42), and two neutral/alkaline INV (A/NINV) subtypes: cytoplasmic α group (TaA/NINV1-11) and cytoplasmic β group (TaA/NINV12-20). The amplification of A/NINVs is mainly attributed to the polyploidization of wheat, and the multiple duplication events experienced in AINVs revealed their non-dose sensitivity characteristic. The wheat RNA-seq data revealed the tissue specificity of A/NINVs and AINVs, and six spike-specific CWINVs showed significant differential expression between the fertile and sterile anthers of thermo-sensitive male-sterile wheat KTM3315A. TaCWINV40 localized in cell wall was effectively silenced in the fertile KTM3315A, and the malformed pollen grains and non-germinating pollen tubes shed light on its indispensability in the development of wheat anthers. This study will spur the interest on manipulating the novel genetic characteristics of TaCWINVs for the construction and improvement of wheat male sterile materials.

Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.)

Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis-elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.