bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous GFP tagging in the diatomThalassiosira pseudonana
Authors: Nam Onyou , Grouneva Irina , Mackinder Luke C. M.
Published: October 2022

The regulated abundance and spatial distribution of proteins determines cellular structure and function. The discovery of green fluorescent protein (GFP) and fusing it to a target protein to determine subcellular localization revolutionized cell biology. Most localization studies involve introducing additional copies of a target gene genetically fused to GFP and under the control of a constitutive promoter, resulting in the expression of the GFP-fusion protein at non-native levels. Here we have developed a single vector CRISPR/Cas9 guided GFP knock-in strategy in the diatomThalassiosira pseudonana. This enables precise and scarless knock-in of GFP at the endogenous genomic location to create GFP fusion proteins under their nativecisandtransregulatory elements with knock-in efficiencies of over 50%. We show that a previously uncharacterized bestrophin-like protein localizes to the CO2-fixing pyrenoid and demonstrate that by measuring GFP fluorescence we can track relative protein abundance in response to environmental change. To enable endogenous tagging, we developed a Golden Gate Molecular Cloning system for the rapid assembly of episomes for transformation intoThalassiosira pseudonanavia bacterial conjugation. In addition, this versatile toolbox enables CRISPR/Cas9 gene editing, provides a broad range of validated fluorophores and enables future large-scale functional studies in diatoms.

Significance statement

Fluorescent protein (FP) tagging is a widely utilized technique for understanding the spatial distribution of proteins. However, introducing extra gene copies under constitutive promoters that randomly integrate into the genome can result in non-biologically relevant expression levels, unwanted genomic mutations and localization artefacts. To overcome this, we developed a novel single vector system capable of CRISPR/Cas9-guided endogenous GFP tagging in a globally important model diatom. This allows scarless GFP knock-in at precise genomic locations resulting in GFP fusions regulated by native promoters/terminators, which facilitates accurate localization and determination of relative protein abundance. Moreover, the developed modular cloning framework is user-friendly and opens the door for high throughput large-scale studies, including FP tagging, knock-out, and knock-in.
Integrated signaling and transcriptome analysis reveals Src-family kinase individualities and novel pathways controlled by their constitutive activity
Authors: Koutras Nikolaos , Morfos Vasileios , Konnaris Kyriakos , Kouvela Adamantia , Shaukat Athanasios-Nasir , Stathopoulos Constantinos , Stamatopoulou Vassiliki , Nika Konstantina
Published: October 2022

The Src family kinases (SFKs) Lck and Lyn are crucial for lymphocyte development and function. Albeit tissue-restricted expression patterns, the two kinases share common functions, the most pronounced one, being the phosphorylation of ITAM motifs in the cytoplasmic tails of antigenic receptors. Lck is predominantly expressed in T-lymphocytes; however, it can be ectopically found in B-1 cell subsets and numerous pathologies including acute and chronic B-cell leukemias. The exact impact of Lck on the B-cell signaling apparatus remains enigmatic and is followed by the long-lasting open question of mechanisms granting selectivity amongst SFK members. In this work we sought to investigate the mechanistic basis of ectopic Lck function in B-cells and compare it to events elicited by the predominant B-cell SFK, Lyn. Our results reveal substrate promiscuity displayed by the two SFKs, which however, is buffered by their differential susceptibility towards regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, we show that Lck- and Lyn-generated signals suffice to induce transcriptome alterations, reminiscent of B-cell activation, in the absence of receptor/co-receptor engagement. Finally, our analyses revealed a yet unrecognized role of SFKs in tipping the balance of cellular stress responses, by promoting the onset of ER-phagy, an as yet completely uncharacterized process in B-lymphocytes.

Significance

The Src-family-kinases Lck and Lyn are mandatory for lymphocyte function. However, several aspects of their regulation and critical pathways they control remain elusive. Using signaling and transcriptome analysis we show that the two kinases share substrate preferences; yet they display differential susceptibility towards regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, overexpression of both kinases suffices to induce receptor-ligation independent signaling responses. Finally, our analyses reveal a novel role of SFKs in tipping the balance of cellular stress responses, by promoting ER-phagy, in the expense of proteasomal degradation and the Unfolded Protein Response. These data advance our understanding of molecular individualities amongst SFK members, and identifies novel networks significant for lymphocyte activation and effector function.
HY5 regulates GLK transcription factors to orchestrate photomorphogenesis inArabidopsis thaliana
Authors: Zhang Ting , Zhang Rui , Zeng Xi-Yu , Lee Sanghwa , Ye Lu-Huan , Tian Shi-Long , Zhang Yi-Jing , Busch Wolfgang , Zhou Wen-Bin , Zhu Xin-Guang , Wang Peng
Published: October 2022

Light induced de-etiolation is an important aspect of seedling photomorphogenesis. GOLDEN2 LIKE (GLK) transcriptional regulators are involved in chloroplast development, but to what extent they participate in photomorphogenesis is not clear. Here we show that ELONGATED HYPOCOTYL5 (HY5) binds toGLKpromoters to activate their expression, and also interacts with GLK proteins. Chlorophyll content in the de-etiolating Arabidopsis seedlings of thehy5 glk2double mutants was lower than that inhy5single mutant.GLKsinhibited hypocotyl elongation, and the phenotype could superimpose on thehy5phenotype. Correspondingly, GLK2 regulates the expression of photosynthesis and cell elongation genes partially independent of HY5. Before exposed to light, the accumulation of GLK proteins was regulated by DE-ETIOLATED 1 (DET1), while also affected (especially for GLK1) transcriptionally by HY5. The enhanced etioplast development and photosystem gene expression observed indet1mutant were attenuated indet1 glk2double mutant. Our study reveals that GLKs act down-stream of HY5 and likely cooperate with HY5 and DET1, to orchestrate multiple developmental traits during the light-induced skotomorphogenesis to photomorphogenesis transition in Arabidopsis.

One-sentence summary

GLK and GNC act downstream of HY5, and cooperate with HY5 and DET1, to regulate both chloroplast development and hypocotyl elongation during the transition from skotomorphogenesis to photomorphogenesis.
Genetic basis of a seasonal life-history polyphenism
Authors: Caplins Serena A
Published: October 2022

Seasonal polyphenisms are common across the animal and plant kingdom yet we understand the explicit interactions between genetics and environment for only a few taxa. Are the genomic regions and their variants associated with the trait the same or different across environments? Is the response to selection shared or different across different “background” selection environments? Offspring type in the sacoglossan sea slug Alderia willowi is a seasonally modulated interaction between genotype and phenotype that results in offspring of wildly different developmental trajectories. In a genome-wide association test I found 41 SNPs associated with offspring type. In an evolve and resequence experiment I found thousands of loci changed in frequency following selection. These loci were partially shared (37%) between low and high salinity. Of the 41 candidate SNPs identified in the GWAS only seven also showed significant allele frequency change across replicates in the selection experiments with four in high salinity, two in low and one in both. This reveals a broad pattern of allele frequency change that is largely unique to the environment in which selection for the same phenotype occurs. The results presented in this paper showcase the ability of phenotypic plasticity to move the phenotype independent of the genotype and thus maintain the polyphenism that is so striking in this species.
Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors
Authors: Jauregui-Lozano Juan , Bakhle Kimaya M. , Hagins Arrianna C. , Weake Vikki M.
Published: October 2022

Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. Here, we profiled chromatin marks associated with active transcription in the agingDrosophilaeye to understand how chromatin modulates transcriptional outputs. We found that both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, we found no correlation with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because agingDrosophilaundergo a decrease in visual function, our data suggest that H3K36me3 plays a role in maintaining visual function in the aging eye through regulating alternative splicing.
Actin cytoskeleton dynamics affect replication of Human Metapneumovirus
Authors: Rodríguez Pamela Elizabeth , Gil Pedro Ignacio , Cámara Jorge Augusto , Cámara Alicia , Paglini María Gabriela
Published: October 2022

Human Metapneumovirus (hMPV) is responsible for viral respiratory infection with clinical and epidemiological relevance in pediatric, immunocompromised, and elderly populations. Little is known about hMPVin vitroreplication processes and their relationship with cellular structures such as the cytoskeleton. Our goal was to evaluate the role of the actin cytoskeleton in hMPV replication at different stages of viral growth. hMPV was isolated in Vero cells from a clinical sample and identified as A2genotype. The cytopathic effect was detected by the appearance of cell rounding and refractory cell clusters. The growth curve showed that viral replication maximum level was between 48 and 72 hpi. The highest percentage of infected cells and intracellular hMPV-protein were detected at the early stages of the replication cycle. Disruption of actin microfilaments with Cytochalasin D during the early events provoked an increase in both intracellular and extracellular viruses. We demonstrate that the early phase of the hMPV curve is crucial for viral replication; and the disruption of microfilaments during this time increments both viral protein expression and release of viruses to the extracellular space. This study contributes to elucidate wild-type hMPV growth kinetics, providing new insights on the actin cytoskeleton role in viral replication mechanisms.
Ultra-weak protein-protein interactions can modulate proteome-wide searching and binding
Authors: Hofmann Jennifer L. , Maheshwari Akshay J. , Sunol Alp M. , Endy Drew , Zia Roseanna N.
Published: October 2022

Research on protein-protein interaction (PPIs) tends to focus on high affinity interactions. Weaker interactions (Kd >1μM) recently understood as contributing to intracellular phase separation suggest that even-weaker PPIs might also matter in as-yet unknown ways. However, ultra-weak PPIs (Kd >1mM) are not readily accessible by in vivo techniques. Here we use protein electrostatics to estimate PPI strengths and spatially-resolved dynamic simulations to investigate the potential impacts of ultra-weak PPIs within dense protein suspensions. We find that ultra-weak PPIs can drive formation of transient clusters that last long enough to enable enzyme-catalyzed reactions and accelerate the sampling of protein associations. We apply our method to Mycoplasma genitalium, finding that ultra-weak PPIs should be ubiquitous among cytoplasmic proteins. We also predict that the proteome-wide interactome can be shifted to favor ‘binding-dominant’ ultra-weak PPIs via the introduction of a few charged protein complexes. We speculate that ultra-weak PPIs could contribute to cellular fitness by facilitating sampling and colloidal-scale transport of proteins involved in biological processes, including protein synthesis.
RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes
Authors: Subramanian Megha , McIninch James , Zlatev Ivan , Schlegel Mark K. , Kaittanis Charalambos , Nguyen Tuyen , Agarwal Saket , Racie Timothy , Alvarado Martha Arbaiza , Wassarman Kelly , Collins Thomas S. , Chickering Tyler , Brown Christopher R. , Schmidt Karyn , Castoreno Adam B. , Shulga-Morskaya Svetlana , Stamenova Elena , Buckowing Kira , Berman Daniel , Barry Joseph D. , Bisbe Anna , Maier Martin A. , Fitzgerald Kevin , Jadhav Vasant
Published: October 2022

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein AAV transgene expression is silenced using the clinically validated modality of chemically modified short interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.
Trafficking dynamics of VEGFR1, VEGFR2, and NRP1 in human endothelial cells
Authors: Sarabipour Sarvenaz , Kinghorn Karina , Quigley Kaitlyn M , Kovacs-Kasa Anita , Annex Brian H , Bautch Victoria L , Mac Gabhann Feilim
Published: October 2022

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to ‘see’ (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells - specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.
Phenotypic and genomic comparison of human outbreak and cattle-associated Shiga toxin-producing Escherichia coli O157:H7
Authors: Peroutka-Bigus Nathan , Nielsen Daniel W. , Trachsel Julian , Mou Kathy T. , Sharma Vijay K. , Kudva Indira T. , Loving Crystal L.
Published: October 2022

Escherichia coli O157:H7 (O157)-adulterated food products, including beef and produce, are associated with disease outbreaks in humans. Although cattle feces are a source for the contamination, it is unclear if diverse O157 human-associated outbreak isolates expressing a specific virulence phenotype can colonize and shed in the feces of cattle at a quantitatively similar levels to non-outbreak isolates. It is also unclear if other phenotypes, such as biofilm, cell attachment, and toxin production, differentiate environmental O157 isolates from O157 isolates associated with human illness. Genomic profiling of O157 isolates acquired through routine surveillance can inform if the isolates encode virulence genes associated with human disease, but many genotype-phenotype relationships remain unclear for O157. Therefore, the objective of this study was to compare a diverse set of O157 isolates, with the intent of identifying potential genotypic differences that could inform phenotypes such as cattle colonization and fecal shedding, in vitro cell attachment, biofilm production, and Shiga toxin production. In addition, the relationship between phenotypes and potential for foodborne illness as it relates to genomic virulence traits was explored. No significant differences in cattle colonization and fecal shedding were detected for the tested isolates, despite broad genomic differences. In addition, the in vitro phenotypic differences noted in biofilm and cell attachment did not associate with one LSPA-6 lineage compared to another. Overall, no differences in cattle shedding were observed, yet variations in genotype and phenotype were identified indicating further work is warranted to better understand the relationship between O157 genome and virulence.

Importance

Foodborne illness has a major impact on the health and wellbeing of the global population, besides creating substantial financial hardships for industry. While many bacteria and viruses are implicated in foodborne illness, Escherichia coli serotype O157:H7 (O157) is a common food adulterant that can cause human disease and food recalls. Cattle feces are a significant source of food-adulterating O157. A greater understanding of O157 genetics and its relation to phenotype is needed to develop mitigation strategies to limit spread of O157 into the food chain. The goal of the research was to identify O157 genomic and phenotypic attributes of O157 associated with cattle colonization and fecal shedding along with other factors involved in environmental persistence and illness in humans. It was observed that variations in biofilm formation and in vitro cellular adherence did not associate with enhanced cattle colonization or fecal shedding, indicating that the processes involved in cattle colonization are complex and not well understood.
16S rRNA sequencing for profiling microbial communities in idiopathic granulomatous mastitis
Authors: Ong Seeu Si , Xu Jia , Sim Choon Kiat , Khng Alexis Jiaying , Ho Peh Joo , Kwan Philip Kam Weng , Ravikrishnan Aarthi , Tan Kiat-Tee Benita , Tan Qing Ting , Tan Ern Yu , Tan Su-Ming , Putti Thomas Choudary , Lim Swee Ho , Serene Tang Ee Ling , Nagarajan Niranjan , Karnani Neerja , Li Jingmei , Hartman Mikael
Published: October 2022

Background

Idiopathic granulomatous mastitis (IGM) is a rare, chronic, and benign inflammatory breast disease with ambiguous aetiology. Contrastingly, lactational mastitis (LM) is commonly diagnosed in breastfeeding women. To investigate IGM aetiology, we profiled the microbial flora of pus and skin in IGM and LM patients.

Methods

IGM patients were recruited from 2018 to 2020; LM patients were recruited as controls. 16S rRNA sequencing libraries were constructed from 16S rRNA gene amplified from total DNA extracted from pus and skin swabs. Metagenomic analysis was conducted using modified microbiome abundance analysis-suite customised R-resource for paired pus and skin samples. Microbiome multivariable association analyses were performed using linear models.

Results

21 IGM and 3 LM paired pus and skin samples underwent metagenomic analysis. Bray-Curtis ecological dissimilarity distance showed dissimilarity across four sample types (IGM-pus, IGM-skin, LM-pus, LM-skin; PERMANOVA, p<0.001). No characteristic dominant genus was observed across IGM samples. IGM pus samples were more diverse than corresponding IGM skin samples (Shannon and Simpson index; Wilcoxon paired signed-rank tests, p=0.022 and p=0.07). Corynebacterium kroppenstedtii, reportedly associated with IGM in literature, was higher in IGM pus samples than paired skin sample (Wilcoxon, p=0.022). Three other species and nineteen genera were statistically significant in paired IGM pus-skin comparison after antibiotic treatment adjustment, and multiple comparisons correction.

Conclusions

Microbial profiles are unique between IGM and LM patients. Inter-patient variability and polymicrobial IGM pus samples cannot implicate specific genus or species as an infectious cause for IGM.
Radiomic Profiling of Chest CT in a Cohort of Sarcoidosis Cases
Authors: Carlson Nichole E , Lippitt William , Ryan Sarah M , Mroz Margaret , Barkes Briana , Liao Shu-Yi , Maier Lisa A , Fingerlin Tasha E
Published: October 2022

Background

High resolution computed tomography (HRCT) of the chest is increasingly used in clinical practice for sarcoidosis. Visual assessment of chest HRCTs in patients with sarcoidosis has high inter- and intra-rater variation. Radiomics offers a reproducible quantitative assessment of HRCT lung parenchyma and could be useful as an additional summary measure of disease. We develop radiomic profiles on HRCT and map them to radiologic, clinical, and patient reported outcomes.

Research Question

Can radiomic analysis of chest HRCT cluster patients into groups that are related to radiologic, clinical, and patient reported outcomes?

Study Design and Methods

Three-dimensional radiomic features were calculated on chest HRCT for both lungs from sarcoidosis cases enrolled in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study (N=320). Robust and sparse K-means was used to cluster sarcoidosis cases using their radiomic profiles. Differences in patterns on visual assessment (VAS) by cluster were identified using chi-squared tests. Linear regression investigated how pulmonary function tests and patient reported outcomes differed between clusters with and without adjustment for other radiologic quantification.

Results

Radiomic-based clustering identified four clusters associated with both Scadding stage and Oberstein score (P<0.001). One of the clusters had markedly few abnormalities. Another cluster had consistently more abnormalities along with more Scadding stage IV. Average pulmonary function testing (PFT) differed between clusters, even after accounting for Scadding stage and Oberstein score (P<0.001), with one cluster having more obstructive disease. The most discriminative radiomic measures explained 10-15% of the variation in PFT beyond demographic variables. Shortness of breath, fatigue, and physical health differed by cluster (P<0.014).

Interpretation

Radiomic quantification of sarcoidosis identifies new subtypes representative of existing radiologic assessment and more predictive of pulmonary function. These findings provide evidence that radiomics may be useful for identifying new imaging-based disease phenotypes.
Validation of diagnosis of acute myocardial infarction and stroke in electronic medical records: a primary care cross-sectional study in Madrid, Spain (the e-MADVEVA Study)
Authors: de Burgos-Lunar C , del Cura-González I , Cárdenas-Valladolid JC , Gómez-Campelo P , Abánades-Herranz JC , López-de Andrés A , Sotos-Prieto M , Iriarte-Campo V , Salinero-Fort MA
Published: October 2022

Objectives

To validate the diagnoses of AMI and stroke recorded in EMR and to estimate the population prevalence of both diseases in people aged ≥ 18 years

Design

Cross-sectional validation study Setting: 45 primary care centres

Participants

Simple random sampling of diagnoses of AMI and stroke (ICPC-2 codes K75 and K90, respectively) registered by 55 physicians and random age- and sex-matched sampling of the records that included in primary care EMRs in Madrid (Spain).

Primary and Secondary Outcomes Measures

Sensitivity, specificity, positive and negative predictive values, and overall agreement were calculated using the kappa statistic. Applied gold standards were electrocardiograms, brain imaging studies, hospital discharge reports, cardiology reports, and neurology reports. In the case of AMI, the ESC/ACCF/AHA/WHF Expert Consensus Document was also used. Secondary outcomes were the estimated prevalence of both diseases considering the sensitivity and specificity obtained (true prevalence).

Results

The sensitivity of a diagnosis of AMI was 98.11% (95% CI, 96.29-99.03), and the specificity was 97.42% (95% CI, 95.44-98.55). The sensitivity of a diagnosis of stroke was 97.56% (95% CI, 95.56-98.68), and the specificity was 94.51% (95% CI, 91.96-96.28). No differences in the results were found after stratification by age and sex (both diseases). The prevalence of AMI and stroke was 1.38% and 1.27%, respectively.

Conclusion

The validation results show that diagnoses of AMI and stroke in primary care EMRs constitute a helpful tool in epidemiological studies. The prevalence of AMI and stroke was lower than 2% in the population aged over 18 years.

Strengths and limitations of this study

The major strength of the e-MADVEVA study is the individual validation (manual validation) of electronic medical records by comparing each case and non-case, matched by age, with an accepted reference gold standard.

The validation method allows for calculating PPV, NPV, Sensitivity, and Specificity, in contrast with other methods such as questionnaires for healthcare practitioners or patients and comparing rates in a comparable population.

In-hospital mortality due to AMI and stroke may not have been recorded in primary care EMRs, with the result that the prevalence of patients who die in hospital due to a first AMI or stroke may be underestimated.

The ICPC-2 codes studied for AMI (ICPC-2 K75) and stroke (ICPC-2 K90) do not allow differentiation between the different types of AMI and stroke.
AMU: Using mRNA Embedding in Self-Attention Network to Predict Melanoma Immune Checkpoint Inhibitor Response
Authors: Yin Yi , Wu Qing , Wang Ziming , Kang Yu , Xie Xianhe
Published: October 2022

Background

To precisely predict drug response and avoid unnecessary treatment have been urgent needs to be resolved in the age of melanoma immunotherapy. Deep learning model is a powerful instrument to predict drug response. Simultaneously extracting the function and expression data characteristics of mRNA may help to improve the prediction performance of the model.

Methods

We designed a deep learning model named AMU with self-attention structure which were fed with the mRNA expression values for predicting melanoma immune checkpoint inhibitor clinical responses.

Results

Comparing with SVM, Random Forest, AdaBoost, XGBoost and the classic convolutional network, AMU showed the preferred performance with the AUC of 0.941 and mAP of 0.960 in validation dataset and AUC of 0.672, mAP of 0.800 in testing dataset, respectively. In model interpretation work, TNF-TNFRSF1A pathway were indicated as a key pathway to influence melanoma immunotherapy responses. Further, gene features extracted from embedding layer and calculated by t-SNE algorithm, showed a local similarity with Functional Protein Association Network (STRING, https://cn.string-db.org/), AMU could predict gene functions and interactions simultaneously.

Conclusions

Deep learning model built with self-attention structure has strong power to process mRNA expression data and gene vector representation is a promising work in biomedical field.

What is already known on this topic

The types of biomarkers for immunotherapy are very complex and transcriptomics biomarker research is one part of it, but currently it is lack of generally acknowledged results with practical value. Combining deep learning models with transcriptomics biomarker markers can help us to predict drug sensitivity. However, the powerful capabilities of deep learning models have not been fully exploited and utilized.

What this study adds

The expression of 160 genes could well predict the efficacy of immunotherapy, even if the tissue samples were after drug administration, and through model training, we could also extract the interactions and connections between genes. The deep learning model could not only do prediction, but were also promising in performing gene vector representation learning.

How this study might affect research, practice or policy

Our research is not only to provide a model with high predictive value, but also to extract gene interaction relations during model training, which is very enlightening for gene vector representation learning. The research of gene vector representation learning can promote the prediction accuracy of deep learning models in various biomedical fields because it can become the common upstream of many biomedical tasks.
Enteroendocrine cells protect the stem cell niche by regulating crypt metabolism in response to nutrients
Authors: McCauley Heather A. , Riedman Anne Marie , Enriquez Jacob R. , Zhang Xinghao , Watanabe-Chailland Miki , Sanchez J. Guillermo , Kechele Daniel O. , Paul Emily F. , Riley Kayle , Burger Courtney , Lang Richard A. , Wells James M.
Published: October 2022

Background and aims

The intestinal stem cell niche is exquisitely sensitive to changes in diet, with high fat diet, caloric restriction, and fasting resulting in altered crypt metabolism and intestinal stem cell function. Unlike cells on the villus, cells in the crypt are not immediately exposed to the dynamically changing contents of the lumen. We hypothesized that enteroendocrine cells (EECs), which sense environmental cues and in response release hormones and metabolites, are essential for relaying the nutrient status of the animal to cells deep in the crypt.

Methods

We used the tamoxifen-inducible VillinCreERT2 mouse model to deplete EECs (Neurog3fl/fl) from adult intestinal epithelium and we generated human intestinal organoids from wild-type and NEUROG3-null human pluripotent stem cells. We used indirect calorimetry, 1H-NMR metabolomics, mitochondrial live imaging, and the Seahorse bioanalyzer to assess metabolism. Intestinal stem cell activity was measured by proliferation and enteroid-forming capacity. Transcriptional changes were assessed using 10X Genomics single-cell sequencing.

Results

Loss of EECs resulted in increased energy expenditure in mice, an abundance of active mitochondria, and a shift of crypt metabolism to fatty acid oxidation. Crypts from mouse and human intestinal organoids lacking EECs displayed increased intestinal stem cell activity and failed to activate phospho-S6 ribosomal protein, a marker for activity of the master metabolic regulator mammalian target of rapamycin (mTOR). These phenotypes were similar to those observed when wild-type mice were deprived of nutrients.

Conclusions

Deletion of EECs recapitulated a fasting phenotype despite normal levels of ingested nutrients. These data suggest that EECs are required to relay nutritional information to the stem cell niche and are essential regulators of intestinal metabolism.
Effects of generations in captivity and elevated rearing temperature on Ontario hatchery brook trout (Salvelinus fontinalis) fry quality and survival
Authors: Wilder A.S. , Wilson C.C. , Warriner T. R. , Semeniuk C.A.D.
Published: October 2022

With increasing environmental temperatures causing concern for the status of freshwater fishes, captive breeding programs may become increasingly important for conservation efforts as well as to support fisheries. Although captive broodstocks provide reliable gamete sources for production stocking, prolonged generations under hatchery conditions selection for hatchery conditions (domestication) and reduced phenotypic plasticity to novel environmental stressors. We assessed the effects of rearing temperature and number of generations spent in captivity on the survival and quality (indicated by lack of malformations) of long-term (F20+) and newly-captive (F1) strains of Ontario hatchery brook trout (Salvelinus fontinalis) with shared genetic history. We found that elevated temperatures decreased likelihood of survival between the hatched and fry stages. Additionally, we found that elevated temperature reduced fry quality of F1fish whereas F20+fish were less thermally sensitive, suggesting no reduction in plasticity due to captivity. The combined effects of elevated rearing temperatures and number of hatchery generations suggest that selection for captivity can occur rapidly (in one generation) even under benign conditions, and that additive stressor effects of captivity and temperature may impact newly established strains.
Three-dimensional structure of inner ear hair cell mitochondrial networks and ribbon synapses in a zebrafish model of Usher syndrome type 1B
Authors: Riley Kenneth C. , Koleilat Alaa , Dugdale Joseph , Cooper Shawna A. , Christensen Trace A. , Schimmenti Lisa A.
Published: October 2022

Inner ear hair cells are the fundamental unit of sound and vibration detection in inner ear and lateral line structures. Our understanding of the structure and ultrastructure of hair cells has heretofore relied upon two-dimensional imaging. The development of serial block-face scanning electron microscopy (SBFSEM) changes this paradigm and allows for ultrastructural evaluation of hair cells in three-dimensions. This is the first report of SBFSEM analysis in the myo7aa-/- mutant where we evaluated several attributes of zebrafish hair cells from the inner ear cristae in both wildtype and myo7aa-/- mutant zebrafish through three-dimensional reconstruction. We describe ribbon synapse number, location, and volume, mitochondrial localization, and innervation for individual hair cells. We determined that myo7aa-/- mutant ribbon synapses have a smaller volume and surface area; however, all other hair cell attributes investigated were not significantly different between the mutant and wildtype zebrafish. These findings are critical for the development of therapies for deafness caused by mutations in myo7aa, as this study supports that the necessary hair cell machinery for hearing is largely intact. In addition, the methodology and measurements developed in this study provide a guide for the evaluation of zebrafish hair cells using SBFSEM.
Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081
Authors: Du Liyang , Luo Zhipu , Lin Zhonghui
Published: October 2022

The cyclic oligoadenylates (cOAs) act as second messengers of type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an ‘off-switch’ regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the CRISPR-associated ring nuclease 1 (Crn1) from Saccharolobus solfataricus (Sso) 2081 in its apo or bound to cA4 in both pre-cleavage and transient intermediate states. Sso2081 harbors a unique helical insert that encloses cA4 in the central cavity. Two free phosphates symmetrically bind the catalytic site of apo Sso2081 and overlap with the two scissile phosphates of cA4, supporting a bilaterally symmetrical cleavage. The structure of transient intermediate state captured by Ser11Ala mutation immediately illustrates a stepwise cleavage of cA4 by Sso2081. Our study establishes atomic mechanisms of cA4 recognition and degradation by the type III CRISPR ring nuclease Crn1/Sso2081.
Mitochondrial DNA Breaks Activate an Integrated Stress Response to Reestablish Homeostasis
Authors: Fu Yi , Sacco Olivia , Kanshin Evgeny , Ueberheide Beatrix , Sfeir Agnel
Published: October 2022

Double-strand breaks in mitochondrial DNA (mtDSBs) lead to the degradation of the circular genomes and a reduction in copy number. However, it is unclear how mtDSBs are sensed and what signaling pathways are activated in response to mtDNA damage. In this study, we used mitochondrial-targeted restriction enzymes to investigate the cellular response to mtDSBs. Our results showed that a subset of cells with mtDSBs exhibited defects in mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy revealed compromised mitochondrial membrane and cristae ultrastructure. We also found that mtDSBs activated the integrated stress response (ISR) through the phosphorylation of eIF2α by DELE1 and HRI. Notably, inhibition of the ISR exacerbated the mitochondrial import defect and delayed the recovery of mtDNA copy number following break formation. These findings suggest that the ISR plays a role in mitigating mitochondrial dysfunction following mtDNA damage and is critical to promoting mtDNA repopulation. Last, we used proteomics to survey the proteins present in the nucleoids shortly after mtDSBs and identified ATAD3A, a membrane-anchored protein interacting with nucleoids, as a potential factor in transmitting the signal from damaged genomes to the inner mitochondrial membrane. In summary, our study reveals the sequence of events linking damaged mitochondrial genomes with the cytoplasm and highlights the unexpected role of the ISR in reestablishing homeostasis in response to mitochondrial genome instability.
Flavonols contrary affect the interconnected glucosinolate and camalexin biosynthesis pathway inArabidopsis thaliana
Authors: Naik Jogindra , Tyagi Shivi , Rajput Ruchika , Kumar Pawan , Pucker Boas , Bisht Naveen C. , Misra Prashant , Stracke Ralf , Pandey Ashutosh
Published: October 2022

Flavonols are structurally and functionally diverse molecules playing roles in plant biotic and abiotic stress tolerance, auxin transport inhibition, pollen development, etc. Despite their ubiquitous occurrence in land plants and multifunctionality, the effect of perturbation of flavonol biosynthesis over global gene expression and pathways other than flavonoid biosynthesis has not been studied in detail. To understand the signaling role of different flavonol metabolites, herein, we used the flavonol deficientArabidopsis thalianaloss-of-function mutantflavonol synthase1 (fls1-3)as object of study. Comparative transcriptome and metabolic profiling were used to study the effects of genetic flavonol deficiency and exogenous supplementation with flavonol derivatives (kaempferol, quercetin and rutin) on different cellular processes in the seedling. Various flavonol biosynthesis-related regulatory and structural genes were found to be up-regulated in thefls1-3mutant which could be reversed by exogenous flavonol feeding. Our manifold comparative studies indicated the modulation of various biological processes and metabolic pathways by flavonols. Camalexin biosynthesis was found to be negatively regulated by flavonols. Interestingly, flavonols appeared to promote the accumulation of aliphatic glucosinolate through transcription factor-mediated up-regulation of biosynthesis genes. Overall, this study provides new insights into molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets and adds new knowledge to the expanding plethora of biological activity of flavonols in plants.

Significance

Comparative transcriptome and metabolomic profiling of genetic flavonol deficiency and exogenous flavonol supplementation inA. thalianaseedlings, for the first-time revealed the inverse regulation of interconnected specialized metabolite pathways by flavonol aglycones, and -glycosides. Flavonols negatively regulate camalexin biosynthesis, while promoting the accumulation of aliphatic glucosinolates. Our study adds new insights into the expanding plethora of biological activity of flavonols in plants and will help to uncover the molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets.
Quantitative fluorescence emission anisotropy microscopy for implementing homo-FRET measurements in living cells
Authors: van Zanten Thomas S , Pradeep S Greeshma , Mayor Satyajit
Published: October 2022

Quantitative fluorescence emission anisotropy microscopy reveals the organization of fluorescently labelled cellular components and allows for their characterization in terms of changes in either rotational diffusion or homo-Förster’s energy transfer characteristics in living cells. These properties provide insights into molecular organization, such as orientation, confinement and oligomerization in situ. Here we elucidate how quantitative measurements of anisotropy using multiple microscope systems may be made, by bringing out the main parameters that influence the quantification of fluorescence emission anisotropy. We focus on a variety of parameters that contribute to errors associated with the measurement of emission anisotropy in a microscope. These include the requirement for adequate photon counts for the necessary discrimination of anisotropy values, the influence of extinction coefficients of the illumination source, the detector system, the role of numerical aperture and excitation wavelength. All these parameters also affect the ability to capture the dynamic range of emission anisotropy necessary for quantifying its reduction due to homo-FRET and other processes. Finally, we provide easily implementable tests to assess whether homo-FRET is a cause for the observed emission depolarization.
Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts
Authors: Square Tyler A. , Mackey Emma J. , Sundaram Shivani , Weksler Naama C. , Chen Zoe Z. , Narayanan Sujanya , Miller Craig T.
Published: October 2022

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in direction predicted by a hair regeneration model:Wnt10aandGrem2aincreased tooth replacement rate, whileBmp6andDkk2strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA-seq on stickleback dental tissue showed thatBmp6overexpression resulted in an upregulation of Wnt inhibitors. Together these data support a model where different epithelial organs like teeth and hair share genetic circuitry driving organ regeneration.
An FGF Timer for Zygotic Genome Activation
Authors: Treen Nicholas , Chavarria Emily , Weaver Claire J. , Brangwynne Clifford P. , Levine Michael
Published: October 2022

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timings of gene induction during the earliest phases of embryogenesis. Here we employ high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate, Ciona intestinalis with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by derepression of the ERF repressor by ERK activity, which works in concert with localized activators such as Foxa.a. Absence of ERF results in derepression of target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the 8- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also employed by other vertebrates.
Single-cell chromatin accessibility of developing murine pancreas identifies cell state-specific gene regulatory programs
Authors: de la O Sean , Liu Zhe , Chang Sean , Sneddon Julie B.
Published: October 2022

Numerous studies have characterized the existence of cell subtypes, along with their corresponding transcriptional profiles, within the developing mouse pancreas. The upstream mechanisms that initiate and maintain gene expression programs across cell states, however, remain largely unknown. Here, we generate single-nucleus ATAC-Sequencing data of developing murine pancreas and perform an integrated, multi-omic analysis of both chromatin accessibility and RNA expression to describe the chromatin landscape of both the developing epithelium and mesenchyme at E14.5 at single-cell resolution. We identify candidate transcription factors regulating cell fate and construct gene regulatory networks of active transcription factor binding to regulatory regions of downstream target genes. This work serves as a valuable resource for the field of pancreatic biology in general and contributes to our understanding of lineage plasticity among endocrine cell types. In addition, these data identify which epigenetic states should be represented in the differentiation of stem cells to the pancreatic beta cell fate in order to best recapitulate in vitro the gene regulatory networks that are critical for progression along the beta cell lineage in vivo.