The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC.
Atrazine induced In vivo immunotoxicity in Bivalves Atrazine is ubiquitously used broad-spectrum herbicide to control the weeds in agriculture. The present study aimed to evaluate the acute toxicity and immunotoxicity of Atrazine in two ecologically and economically important bivalves. Acute toxicity of atrazine evaluated in triplicates by taking control and six experimental groups each comprising of 30 animals and treated with a range of atrazine from 2 PPM to 12 PPM for 96 hours. Mortalities were recorded for every 24 hours until 96 hours and data analyzed by one-way ANOVA and Dunnett T-test. The results indicated a significant increase in mortalities with increase in dose and time of exposure in both species. The values of LC50 were determined as 6.10 PPM and 4.90 PPM respectively for Perna viridis and Paphia malabarica. Furthermore, the immunotoxic potential of atrazine assessed by treating mussels and clams with the five sub-lethal doses of atrazine for 14 days and quantifying the viability of hemocytes by using simple yet reliable Tryphan blue exclusion assay. The results of the present study suggest atrazine-induced immunotoxicity by decreasing the number of viable hemocytes in bivalves. Hemocytes with phagocytic function are indispensable to confer innate immunity in bivalves, decreased viability of these cells leads to compromised immunity. This study is first of its kind to implicate atrazine with the immunotoxicity in bivalves.
Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2 A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development.

One-Sentence SummaryRemoving glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine. AGO1 regulates pericentromeric regions in mouse embryonic stem cells Argonaute proteins (AGOs), that play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse Embryonic Stem Cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1 in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts.

Summary blurbDepletion of AGO1 in mESCs leads to a redistribution of H3K9me3 and HP1 away from pericentromeric regions and is accompanied by an upregulation of major satellites transcripts. DNA-controlled Spatio-temporal Patterning of a Cytoskeletal Active Gel Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton; an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here, we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel comprised of microtubules: a DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration, the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of life-like materials with self-shaping properties. Enhancing Sampling of Water Rehydration on Ligand Binding: A Comparison of Techniques 1Water often plays a key role in protein structure, molecular recognition, and mediating protein-ligand interactions. Thus, free energy calculations must adequately sample water motions, which often proves challenging in typical MD simulation timescales. Thus, the accuracy of methods relying on MD simulations ends up limited by slow water sampling. Particularly, as a ligand is removed or modified, bulk water may not have time to fill or rearrange in the binding site. In this work, we focus on several molecular dynamics (MD) simulation-based methods attempting to help rehydrate buried water sites: BLUES, using nonequilibrium candidate Monte Carlo (NCMC); grand, using grand canonical Monte Carlo (GCMC); and normal MD. We assess the accuracy and efficiency of these methods in rehydrating target water sites. We selected a range of systems with varying numbers of waters in the binding site, as well as those where water occupancy is coupled to the identity or binding mode of the ligand. We analyzed rehydration of buried water sites in binding pockets using both clustering of trajectories and direct analysis of electron density maps. Our results suggest both BLUES and grand enhance water sampling relative to normal MD and grand is more robust than BLUES, but also that water sampling remains a major challenge for all of the methods tested. The lessons we learned for these methods and systems are discussed. Monitoring fish communities through environmental DNA metabarcoding in the fish pass system of the second largest hydropower plant in the world The Itaipu Hydroelectric Power Plant is the second largest in the world in power generation. The artificial barrier created by its dam imposes an obstacle for fish migration. Thus, in 2002, a fish pass system, named Piracema Channel, was built to allow fish to access areas upstream of the reservoir. We tested the potential of environmental DNA metabarcoding to monitor the impact of both the dam and associated fish pass system in the Parana River fish communities and to compare it with traditional monitoring methods. Using a fragment of the 12S gene, we characterized richness and community composition based on amplicon sequence variants, operational taxonomic units, and zero-radius OTUs. We combined GenBank and in-house data for taxonomic assignment. We found that different bioinformatics approaches showed similar results. Also, we found a decrease in fish diversity from 2019 to 2020 probably due to the recent extreme drought experienced in southeastern Brazil. The highest alpha diversity was recorded in the mouth of the fish pass system, located in a protected valley with the highest environmental heterogeneity. Despite the clear indication that the reference databases need to be continuously improved, our results demonstrate the analytical efficiency of the metabarcoding to monitor fish species. Identifying critical transcriptional targets of the MYC oncogene using a novel competitive precision genome editing (CGE) assay MYC is an oncogenic transcription factor that controls major pathways promoting cell growth and proliferation. MYC has been implicated in the regulation of large number of genes, but the exact target genes responsible for its proliferative effects are still not known. Here, we use a novel competitive genome editing (CGE) assay for studying the functional consequence of precise mutations of MYC binding sites on cell proliferation. The CGE method is based on precision genome editing, where a CRISPR/Cas9-induced DNA break is repaired using a template that either reconstitutes the original feature or introduces an altered sequence. Both types of repair templates harbor sequence tags that allow direct comparison between cells that carry original and mutant features and generate a large number of replicate cultures. The CGE method overcomes the limitations of CRISPR/Cas9-technology in analyzing the effect of genotype on phenotype, namely the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation caused by the DNA cuts themselves. Importantly, it provides a powerful method for studying subtle effects elicited by mutation of individual transcription factor binding sites. We show here that E-box mutations at several MYC target gene promoters resulted in reduced cellular fitness, demonstrating a direct correlation between MYC-regulated cellular processes and MYC binding and identifying important transcriptional targets responsible for its functions. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. Naturally occurring combinations of receptors from single cell transcriptomics in endothelial cells VEGF inhibitor drugs have been successful, especially in ophthalmology, but not all patients respond to them. Combinations of drugs are likely to be needed for more effective therapies of angiogenesis-related diseases. In this paper we describe naturally occurring combinations of receptors in endothelial cells that might help to understand how cells communicate and to identify targets for drug combinations. We also develop and share a new software tool called DECNEO to identify them.

Single-cell gene expression data are used to identify a set of co-expressed endothelial cell receptors, conserved among species (mice and humans) and enriched, within a network, of connections to up-regulated genes. This set includes several receptors previously shown to play a role in angiogenesis. Multiple statistical tests from large datasets, including an independent validation set, support the reproducibility, evolutionary conservation and role in angiogenesis of these naturally occurring combinations of receptors. We also show tissue-specific combinations and, in the case of choroid endothelial cells, consistency with both well-established and recent experimental findings, presented in a separate paper.

The results and methods presented here advance the understanding of signaling to endothelial cells. The methods are generally applicable to the decoding of intercellular combinations of signals. Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen While mutation is often deleterious, it can also be adaptive. Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study we investigate two factors that are predicted to influence the trade-off between the costs and benefits of mutation: ecology and genome size. To investigate the relationship between these factors and mutation rate we conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact ecology can have on the adaptive potential of bacterial pathogens. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling. Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Multi-omic meta-analysis of transcriptomes and the bibliome uncovers novel hypoxia-inducible genes Hypoxia is a condition in which cells, tissues, or organisms are deprived of sufficient oxygen supply. Aerobic organisms have a hypoxic response system, represented by hypoxia-inducible factor 1- (HIF1A), to adapt to this condition. Due to publication bias, there has been little focus on genes other than well-known signature hypoxia-inducible genes. Therefore, in this study, we performed a meta-analysis to identify novel hypoxia-inducible genes. We searched publicly available transcriptome databases to obtain hypoxia-related experimental data, retrieved the metadata, and manually curated it. We selected the genes that are differentially expressed by hypoxic stimulation, and evaluated their relevance in hypoxia by performing enrichment analyses. Next, we performed a bibliometric analysis using gene2pubmed data to examine genes that have not been well studied in relation to hypoxia. Gene2pubmed data that provides information about the relationship between gene and publication. We calculated and evaluated the number of reports and similarity coefficients of each gene to HIF1A, which is a representative gene in hypoxia studies. In this data-driven study, we report that several genes that were not known to be associated with hypoxia, including the G protein-coupled receptor 146 gene, are upregulated by hypoxic stimulation. Atg11 promotes Kar9-mediated alignment of the mitotic spindle Asymmetric spindle pole body (SPB) inheritance requires a cascade of events that involve kinases, phosphatases and structural scaffold proteins including molecular motors and microtubule-associated proteins. Higher levels of an SPB component Spc72 and the spindle positioning factor Kar9 at the old SPB, which migrates to the daughter cell, ensure asymmetric SPB inheritance. In this study, by analyzing the protein interaction network of autophagy (Atg)-related proteins with those that constitute the chromosome segregation machinery, and growth inhibition kinetics in the presence of a microtubule poison, we identified Atg11 as a potential regulator of chromosome transmission from a cohort of 35 autophagy mutants in Saccharomyces cerevisiae. Cells lacking Atg11 did not show kinetochore defects but displayed a high rate of chromosome loss and delayed anaphase onset. Atg11 positively interacted with Kar9 and Kip2 and negatively with Dyn1 and Kar3 in mediating proper positioning and alignment of the mitotic spindle that was found to be under the surveillance of the spindle positioning checkpoint. Indeed, atg11{Delta} cells displayed an inverted SPB inheritance. Our experiments further revealed that via the Kar9 pathway, Atg11 promotes asymmetric localization of Spc72 and Kar9 on the old SPB. Atg11 physically interacted with Spc72 and transiently localized close to the old SPB during metaphase-to-anaphase progression. Thus, we uncover an autophagy-independent role of Atg11 in spindle alignment. This study emphasizes the importance of screens to identify factors mediating the complex and intricate crosstalk between biological processes fundamental to genomic integrity. TXN, a Xanthohumol Derivative, Attenuates High-Fat Diet Induced Hepatic Steatosis by Antagonizing PPARγ We previously reported xanthohumol (XN), and its synthetic derivative tetrahydro-XN (TXN) attenuates high-fat diet (HFD) induced obesity and metabolic syndrome in C57BL/6J mice. The objective of the current study was to determine the effect of XN and TXN on lipid accumulation in the liver. Non-supplemented mice were unable to adapt their caloric intake to 60% HFD, resulting in obesity and hepatic steatosis; however, TXN reduced weight gain and decreased hepatic steatosis. Liver transcriptomics indicated TXN might antagonize lipogenic PPAR{gamma} actions in vivo. XN and TXN inhibited rosiglitazone-induced 3T3-L1 cell differentiation concomitant with decreased expression of lipogenesis-related genes. A PPAR{gamma} competitive binding assay showed XN and TXN bind to PPAR{gamma} with an IC50 similar to pioglitazone and 8-10 times stronger than oleate. Molecular docking simulations demonstrated XN and TXN bind in the PPAR{gamma} ligand-binding domain pocket. Our findings are consistent with XN and TXN acting as antagonists of PPAR{gamma}. Linking individual differences in human V1 to perception around the visual field A central question in neuroscience is how the organization of cortical maps relates to perception, for which human primary visual cortex (V1) is an ideal model system. V1 nonuniformly samples the retinal image, with greater cortical magnification (surface area per degree of visual field) at the fovea than periphery and at the horizontal than vertical meridian. Moreover, the size and cortical magnification of V1 varies greatly across individuals. Here, we used fMRI and psychophysics in the same observers to quantify individual differences in V1 cortical magnification and contrast sensitivity at the four polar angle meridians. Across observers, the overall size of V1 and localized cortical magnification positively correlated with contrast sensitivity. Moreover, greater cortical magnification and higher contrast sensitivity at the horizontal than the vertical meridian were strongly correlated. These data reveal a link between cortical anatomy and visual perception at the level of individual observer and stimulus location. Microbiota-instructed regulatory T cell differentiation is mediated by a distinct RORγt+ antigen presenting cell subset The mutualistic relationship of gut-resident microbiota and cells of the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a poised, but largely non-aggressive, immune cell compartment1, 2. Consequences of disturbing this balance, by environmental or genetic factors, include proximal inflammatory conditions, like Crohns disease, and systemic illnesses, both metabolic and autoimmune. One of the means by which this equilibrium is achieved is through induction of both effector and suppressor or regulatory arms of the adaptive immune system. In mice, Helicobacter species induce regulatory (iTreg) and follicular helper (Tfh) T cells in the colon-draining mesenteric lymph nodes under homeostatic conditions, but can instead induce inflammatory Th17 cells when iTreg cells are compromised3, 4. How Helicobacter hepaticus and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here, we investigated which cells and molecular components are required to convey the microbial instruction for the iTreg differentiation program. We found that antigen presentation by cells expressing ROR{gamma}t, rather than by classical dendritic cells, was both required and sufficient for iTreg induction. These ROR{gamma}t+ cells, likely to be type 3 innate lymphoid cells (ILC3) and/or a recently-described population of Aire+ cells termed Janus cells5, require the MHC class II antigen presentation machinery, the chemokine receptor CCR7, and v integrin, which activates TGF-{beta}, for iTreg cell differentiation. In the absence of any of these, instead of iTreg cells there was expansion of microbiota-specific pathogenic Th17 cells, which were induced by other antigen presenting cells (APCs) that did not require CCR7. Thus, intestinal commensal microbes and their products target multiple APCs with pre-determined features suited to directing appropriate T cell differentiation programs, rather than a common APC that they endow with appropriate functions. Our results illustrate the ability of microbiota to exploit specialized functions of distinct innate immune system cells, targeting them to achieve the desired composition of equipoised T cells, thus maintaining tolerance. Greater spear nosed bats in Panama do not use social proximity to improve foraging efficiency Group foraging animals may change their spatial relationships in response to the resource landscape. The strength of social network ties across foraging groups could then reflect the energy balance of individual group members through their effect on foraging efficiency. Greater spear-nosed bats (Phyllostomus hastatus) live in social groups where unrelated females develop group-specific social calls, defend each others offspring, and are hypothesized to coordinate social foraging to feed on balsa flowers. To test if social groups forage together, and if this increases foraging efficiency, we tracked 18 individuals from three social groups of P. hastatus on Isla Colon, Panama using high frequency GPS for 1 - 3 days each. We found that individuals commuted alone but then joined group members in small foraging patches with high densities of flowering balsas, 25 km from their roost on the mainland. Proximity-based social network measures did not predict foraging efficiency or energy expenditure, but individuals with higher social associations spent more time outside of the cave. Individuals did not coordinate movement, but close proximity among individuals increased resting bout duration and synchrony, particularly when in the same patch. These results show that stronger social proximity associations were more closely tied to resting behavior, and indicate that factors other than increased feeding efficiency may structure network relationships of group members while foraging.

Lay SummarySocial foraging can increase foraging efficiency on hard-to-find food sources. Miniaturized high-frequency GPS allowed us to test this on groups of greater spear-nosed bats. We estimated food intake rates, energy expenditure, and the strength of bat social relationships. In contrast to our expectations, we did not find a relationship between bat social relationships and energy expenditure or foraging efficiency. However, bats took longer resting breaks when group members were close. Why these bats commute 25 km individually across the ocean to then forage near each other thus requires further investigation. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Identifying and testing marker-trait associations for growth and phenology in three pine species: implications for genomic prediction In tree species, genomic prediction offers the potential to forecast mature trait values in early growth stages, if robust marker-trait associations can be identified. Here we apply a novel multispecies approach using genotypes from a new genotyping array, based on 20,795 SNPs from three closely related pine species (Pinus sylvestris, Pinus uncinata and Pinus mugo), to test for associations with growth and phenology data from a common garden study. Predictive models constructed using significantly associated SNPs were then tested and applied to an independent multisite field trial of P. sylvestris and the capability to predict trait values was evaluated. One hundred and eighteen SNPs showed significant associations with the traits in the pine species. Common SNPs (MAF > 0.05) associated with bud set were only found in genes putatively involved in growth and development, whereas those associated with growth and budburst were also located in genes putatively involved in response to environment and, to a lesser extent, reproduction. At one of the two independent sites, the model we developed produced highly significant correlations between predicted values and observed height data (YA, height 2020: r = 0.376, p < 0.001). Predicted values estimated with our budburst model were weakly but positively correlated with duration of budburst at one of the sites (GS, 2015: r = 0.204, p = 0.034; 2018: r = 0.205, p = 0.034-0.037) and negatively associated with budburst timing at the other (YA: r = -0.202, p = 0.046). Genomic prediction resulted in the selection of sets of trees whose mean height was taller than the average for each site. Our results provide tentative support for the capability of prediction models to forecast trait values in trees, while highlighting the need for caution in applying them to trees grown in different environments. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. New Segmentation and Feature Extraction Algorithm for Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate. Combining Plasma Extracellular Vesicle Let-7b-5p, miR-184 and Circulating miR-22-3p Levels for NSCLC Diagnosis and Drug Resistance Prediction Low-dose computed tomography (LDCT) Non-Small Cell Lung (NSCLC) screening is associated with high false-positive rates, leading to unnecessary expensive and invasive follow ups. There is a need for minimally invasive approaches to improve the accuracy of NSCLC diagnosis. Plasma extracellular vesicle (EV) and circulating microRNA (miRNA) have been proposed as cancer screening biomarkers. However, the identification of highly sensitive and broadly predictive core miRNA signatures remains a challenge. Also, how these systemic and diverse miRNAs impact cancer drug response is not well understood. Using an integrative approach, we examined plasma EV and circulating miRNA isolated from NSCLC patients versus screening controls with a similar risk profile. We found that combining EV (Hsa-miR-184, Let-7b-5p) and circulating (Hsa-miR-22-3p) miRNAs abundance robustly discriminates between NSCLC patients and high-risk cancer-free controls. Diagnosed NSCLC patients harboring sensitizing mutations in epidermal growth factor receptor EGFR (T790M, L578R) are treated with Osimertinib, a potent tyrosine kinase inhibitor (TKI). Nearly all patients develop TKI resistance via complex mechanisms and progress. We found that Hsa-miR-22-3p, Hsa-miR-184, and Let-7b-5p functionally converge on WNT/{beta}catenin and mTOR/AKT signaling axes, known cancer therapy resistance signals. Targeting Hsa-miR-22-3p and Hsa-miR-184 desensitized EGFR-mutated (T790M, L578R) NSCLC cells to Osimertinib. These findings suggest that the expression levels of circulating hsa-miR-22-3p combined with EV hsa-miR-184 and Let-7b-5p levels potentially define a core biomarker signature for improving the accuracy of NSCLC diagnosis. Importantly, these biomarkers have the potential to enable prospective identification of patients who are at risk of responding poorly to Osimertinib alone but likely to benefit from Osimertinib/AKT blockade combination treatments. Prostaglandin EP3 Receptor signaling is required to prevent insulin hypersecretion and metabolic dysfunction in a non-obese mouse model of insulin resistance When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 weeks of age, frankly diabetic. Previous work has shown Prostaglandin EP3 Receptor (EP3) expression and activity is up-regulated in islets from BTBR-Ob mice as compared to lean controls, actively contributing to their beta-cell dysfunction. In this work, we aimed to test the impact of beta-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the Rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse - an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain - generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice, but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in beta-cell fractional area or markers of beta-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective up-regulation of constitutively-active EP3 splice variants in islets from young, lean BTBR mice as compared to C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females vs. Balb/c males was fully consistent with their sexually-dimorphic metabolic phenotype after loss of EP3-coupled Gz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion.

New and NoteworthyIslet Prostaglandin EP3 receptor (EP3) signaling is well-known as up-regulated in the pathophysiological conditions of type 2 diabetes, contributing to beta-cell dysfunction. Unexpected findings in mouse models of non-obese insulin sensitivity and resistance provide a new dimension to our understanding of EP3 as a key modulator of insulin secretion. A previously-unknown relationship between mouse insulin resistance and the penetrance of Rat insulin promoter-driven germline floxed allele recombination is critical to consider when creating beta-cell-specific knockouts.

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