bluuebunny/crossref_metadata_2025 huggingface dataset:
British Policy towards the Incorporation of the Baltic States into the USSR: A Dilemma of de facto and de jure Recognition
Authors: Tikhonravov Evgeny
Published: May 2022

Multiple viewpoints have been expressed regarding the British attitude towards the Soviet annexation of Estonia, Latvia, and Lithuania. The vast majority of writers maintain that the United Kingdom extended only de facto recognition to this incorporation. A small minority of commentators, however, hold otherwise and argue that Britain recognised the annexation de jure. To address this controversy, which has previously received almost no consideration, this article traces the development of the British policy towards the incorporation of the Baltic republics into the USSR, giving wide scope to the statements of the British government and to the views of Members of Parliament. Careful attention is also given to judicial practice. This legal and historical analysis helps to identify the meaning of de facto and de jure recognition used by Britain in the Baltic case, as well as clarifies a number of other relevant questions (for example, whether a territorial annexation may constitute a separate object of recognition, and whether a military occupant may be equated with a government recognised de facto). After determining the correct approach to this academic debate, the article suggests how its findings may contribute to studies contrasting the position of the UK with respect to the Baltic annexation with that of other countries.
Identification of genes differentially expressed between prostrate shoots and erect shoots in the lycophyte Selaginella nipponica using an RNA-seq approach
Authors: Sun Jun , Li Gui-Sheng
Published: May 2022

Lycophytes are the earliest vascular plants and Selaginella is the most studied genus among them. Prostrate shoots are produced during early growth and erect shoots emerge later in S. nipponica, thus providing an opportunity for exploring the evolution of the mechanism underlying the transition between growth phases. Six libraries were sequenced for the prostrate and the erect shoots, and a total of 206 768 genes were identified. Some genes were differentially expressed in prostate and erect shoot, with relatively high expression in the prostate shoots being related to hormone responses and defence reactions, while higher expression in the erect shoots was related to spore formation and shoot development. Some SPL genes possessed a miR156 binding site and were highly expressed in the erect shoots, while AP2-like genes were more highly expressed in the prostrate shoots but simultaneously lacked any miR172 binding site. MiR156 was detected at a higher concentration in the prostrate shoots. Thus, the mechanism for the vegetative to reproductive transition of sporophytes probably originated in the common ancestor of vascular plants and must have experienced stepwise development during evolution.
Fungal association and root morphology shift stepwise during ontogenesis of orchidCremastra appendiculatatowards autotrophic nutrition
Authors: Zahn Franziska E , Lee Yung-I , Gebauer Gerhard
Published: May 2022

The chlorophyllous, terrestrial orchid Cremastra appendiculata from East Asia is unique concerning its fungal mycorrhiza partners. The initially mycoheterotrophic protocorms exploit rather specialized non-rhizoctonia saprotrophic Psathyrellaceae. Adult individuals of this orchid species are either linked to Psathyrellaceae being partially mycoheterotrophic or form mycorrhiza with fungi of the ubiquitous saprotrophic rhizoctonia group. This study provides new insights on nutrition mode, subterranean morphology and fungal partners across different life stages of C. appendiculata. We compared different development stages of C. appendiculata to surrounding autotrophic reference plants based on multi-element natural abundance stable isotope analyses (δ13C, δ15N, δ2H, δ18O) and total N concentrations. Site- and sampling-time-independent enrichment factors of stable isotopes were used to reveal trophic strategies. We determined mycorrhizal fungi of C. appendiculata protocorm, seedling and adult samples using high-throughput DNA sequencing. We identified saprotrophic non-rhizoctonia Psathyrellaceae as dominant mycorrhizal fungi in protocorm and seedling rhizomes. In contrast, the roots of seedlings and mature C. appendiculata were mainly colonized with fungi belonging to the polyphyletic assembly of rhizoctonia (Ceratobasidium, Thanatephorus and Serendipitaceae). Mature C. appendiculata did not differ in isotopic signature from autotrophic reference plants suggesting a fully autotrophic nutrition mode. Characteristic of orchid specimens entirely relying on fungal nutrition, C. appendiculata protocorms were enriched in 15N, 13C and 2H compared to reference plants. Seedlings showed an intermediate isotopic signature, underpinning the differences in the fungal community depending on their subterranean morphology. In contrast to the suggestion that C. appendiculata is a partially mycoheterotrophic orchid species, we provide novel evidence that mature C. appendiculata with rhizoctonia mycobionts can be entirely autotrophic. Besides an environmentally driven variability among populations, we suggest high within-individual flexibility in nutrition and mycobionts of C. appendiculata, which is subject to the ontogenetic development stage.
MD DaVis: interactive data visualization of protein molecular dynamics
Authors: Maity Dibyajyoti , Pal Debnath
Published: May 2022

Molecular dynamics (MD) simulations have become an integral part of biomolecular study. Most MD software suites do not include analysis tools and those which do create very basic visualizations. Molecular Dynamics Data Visualizer (MD DaVis) is a python package developed to facilitate quick comparative analysis of MD trajectories of similar proteins or the same protein under different conditions. MD DaVis can quickly generate interactive visualization from molecular dynamics trajectories with a few simple steps. Interactive plots eliminate the need to make multiple plots for comparison, improving productivity and saving time.

Availability and implementation

MD DaVis is an open-source Python 3 package (https://pypi.org/project/md-davis/) distributed under MIT license. The source code is available at https://github.com/djmaity/md-davis or https://doi.org/10.5281/zenodo.6227047.

Supplementary information

Supplementary data are available at Bioinformatics online.
‘The Anxiety of a Lifetime’—Dealing with Debt in Desistance from Crime
Authors: Gålnander Robin
Published: May 2022

Debt problems are ubiquitous among people with convictions, yet debts’ implications for processes of desistance from crime remain underexplored. Existing research has primarily focused on ‘punishment debt’. Building on that, this longitudinal study of women in Sweden takes a holistic approach to debts in desistance. Debts are potentially criminogenic forces restricting agency in desistance. However, becoming debt-free and creditworthy is a potent way of approaching mainstream society from a position as ‘excluded other’. Importantly, while some debts are viewed as ‘normal’ and required to live out normative dreams of material prosperity, desisters perceived their debts as the ‘wrong’ type in relation to their view of normalcy. This emphasizes the long road toward the mainstream that desistance processes involve.
Effect of electrical stimulation used in the pulse trawl fishery for common sole on internal injuries in sandeels
Authors: Schram Edward , Molenaar Pieke , Soetaert Maarten , Burggraaf Dirk , Boute Pim G , Lankheet Martin J , Rijnsdorp Adriaan D
Published: May 2022

Electric stimulation was used in the North Sea beam trawl fishery for common sole to reduce its environmental impact. Because electrical stimulation may cause internal injuries in fish, a laboratory experiment was conducted to study the effect of pulse exposure on lesser sandeel (Ammodytes tobianus) and greater sandeel (Hyperoplus lanceolatus), important mid-trophic species in the North Sea ecosystem. We exposed 244 sandeels between two electrodes to a pulsed bipolar current for 2 s in an experimental cage with 5 cm sediment; 221 control fish were handled similarly but not exposed. The occurrence of spinal injuries and internal haemorrhages were scored using X-radiography and dissection. None of the sandeels exposed to a field strength of up to 600 V m–1 showed spinal injury or haemorrhage. Equal numbers of minor spinal abnormalities were found in exposed and control fish. In the absence of spinal injuries, we estimated by bootstrapping the field strength below which spinal injuries are unlikely to occur, i.e. the lower limit threshold, and the corresponding limit dose–response relationship between field strength and injury probability. We conclude that it is unlikely that pulse trawl fishery will have an ecologically significant adverse effect on the population abundance of sandeels, because of the low probabilities of exposure and injury.
Impact of Age on Clinical Outcomes and Efficacy of Adjuvant Dual Anti-HER2 Targeted Therapy
Authors: Lambertini Matteo , Fielding Shona , Loibl Sibylle , Janni Wolfgang , Clark Emma , Franzoi Maria Alice , Fumagalli Debora , Caballero Carmela , Arecco Luca , Salomoni Sharon , Ponde Noam F , Poggio Francesca , Kim Hee Jeong , Villarreal-Garza Cynthia , Pagani Olivia , Paluch-Shimon Shani , Ballestrero Alberto , Del Mastro Lucia , Piccart Martine , Bines Jose , Partridge Ann H , de Azambuja Evandro
Published: May 2022

Background

Young age at breast cancer (BC) diagnosis has historically been a rationale for overtreatment. Limited data with short follow-up exist on the prognostic value of age at diagnosis in HER2-positive BC and the benefit of anti-HER2 therapy in young patients.

Methods

APHINITY (NCT01358877) is an international, placebo-controlled, double-blind randomized phase III trial in HER2-positive early BC patients investigating the addition of pertuzumab to adjuvant chemotherapy plus trastuzumab. The prognostic and predictive value of age on invasive disease-free survival (IDFS) as continuous and dichotomous variable (aged 40 years or younger and older than 40 years) was assessed. A subpopulation treatment effect pattern plot analysis was conducted to illustrate possible treatment-effect heterogeneity based on age as a continuous factor.

Results

Of 4804 included patients, 768 (16.0%) were aged 40 years or younger at enrollment. Median follow-up was 74 (interquartile range = 62-75) months. Young age was not prognostic either as dichotomous (hazard ratio [HR] = 1.06, 95% confidence interval [CI] = 0.84 to 1.33) or continuous (HR = 1.00, 95% CI = 1.00 to 1.01) variable. Lack of prognostic effect of age was observed irrespective of hormone receptor status and treatment arm. No statistically significant interaction was observed between age and pertuzumab effect (Pinteraction = 0.61). Adding pertuzumab improved IDFS for patients in the young (HR = 0.86, 95% CI = 0.56 to 1.32) and older (HR = 0.75, 95% CI = 0.62 to 0.92) cohorts. Similar results were observed irrespective of hormone receptor status. Subpopulation treatment effect pattern plot analysis confirmed the benefit of pertuzumab in 6-year IDFS across age subpopulations.

Conclusions

In patients with HER2-positive early BC treated with modern anticancer therapies, young age did not demonstrate either prognostic or predictive value, irrespective of hormone receptor status.
The Sense and Nonsense of Antimicrobial Susceptibility Testing in Cystic Fibrosis
Authors: LiPuma John J
Published: May 2022

Antimicrobial susceptibility testing (AST) has been used to guide therapy of airway infection in persons with cystic fibrosis (CF) for decades. However, evidence that AST adds benefit to treatment outcomes in CF is lacking. In fact, the routine use of AST has potential to exacerbate inappropriate antibiotic use. Several features of airway infection in CF contribute to the limitations of AST in predicting treatment outcomes, providing rationale for abandoning this practice altogether. Other features of CF infection suggest, however, that select use of AST can provide worthwhile guidance to antibiotic selection.
Isolated echinococcal infection of the spleen: a rare case report
Authors: Sotirios Botaitis , Savas Deftereos , Maria-Eleni Zachou , Stauros Vasileiadis , Sempachedin Perente
Published: May 2022

Echinococciasis is a zoonotic infection observed in almost every anatomic location in the body. The liver and lungs are the most frequent sites of infection. However, spleen involvement is rare, and isolated splenic hydatid disease is even less common. Herein, we report a case of isolated splenic hydatid cyst without any tissue involvement that was treated with the classical surgical approach.
Evolutionary history of the grass gynoecium
Authors: Sokoloff Dmitry D , Fomichev Constantin I , Rudall Paula J , Macfarlane Terry D , Remizowa Margarita V
Published: May 2022

The grass family (Poaceae) includes cereal crops that provide a key food source for the human population. The food industry uses the starch deposited in the cereal grain, which develops directly from the gynoecium. Morphological interpretation of the grass gynoecium remains controversial. We re-examine earlier hypotheses and studies of morphology and development in the context of more recent analyses of grass phylogenetics and developmental genetics. Taken in isolation, data on gynoecium development in bistigmatic grasses do not contradict its interpretation as a solitary ascidiate carpel. Nevertheless, in the context of other data, this interpretation is untenable. Broad comparative analysis in a modern phylogenetic context clearly demonstrates that the grass gynoecium is pseudomonomerous. A bistigmatic grass gynoecium has two sterile carpels, each producing a stigma, and a fertile carpel that lacks a stigma. To date, studies of grass developmental genetics and developmental morphology have failed to fully demonstrate the composite nature of the grass gynoecium be­cause its complex evolutionary history is hidden by extreme organ integration. It is problematic to interpret the gynoecium of grasses in terms of normal angiosperm gynoecium typology. Even the concept of a carpel becomes misleading in grasses; instead, we recommend the term pistil for descriptive purposes.
The Gaia-ESO survey: Constraining evolutionary models and ages for young low mass stars with measurements of lithium depletion and rotation
Authors: Binks A S , Jeffries R D , Sacco G G , Jackson R J , Cao L , Bayo A , Bergemann M , Bonito R , Gilmore G , Gonneau A , Jiminéz-Esteban F , Morbidelli L , Randich S , Roccatagliata V , Smiljanic R , Zaggia S
Published: May 2022

A growing disquiet has emerged in recent years that standard stellar models are at odds with observations of the colour-magnitude diagrams (CMDs) and lithium depletion patterns of pre main sequence (PMS) stars in clusters. In this work we select 1,246 high probability K/M-type constituent members of 5 young open clusters (5–125 Myr) in the Gaia-ESO Survey to test a series of models that use standard input physics and others that incorporate surface magnetic fields or cool starspots. We find that: standard models provide systematically under-luminous isochrones for low-mass stars in the CMD and fail to predict Li-depletion of the right strength at the right colour; magnetic models provide better CMD fits with isochrones that are ∼1.5 − 2 times older, and provide better matches to Li depletion patterns. We investigate how rotation periods, most of which are determined here for the first time from Transiting Exoplanet Survey Satellite data, correlate with CMD position and Li. Among the K-stars in the older clusters we find the brightest and least Li-depleted are the fastest rotators, demonstrating the classic ‘Li-rotation connection’ for the first time at ∼35 Myr in NGC 2547, and finding some evidence that it exists in the early M-stars of NGC 2264 at <10 Myr. However, the wide dispersion in Li depletion observed in fully-convective M-dwarfs in the γ Vel cluster at ∼20 Myr appears not to be correlated with rotation and is challenging to explain without a very large (>10 Myr) age spread.
PhyloCloud: an online platform for making sense of phylogenomic data
Authors: Deng Ziqi , Botas Jorge , Cantalapiedra Carlos P , Hernández-Plaza Ana , Burguet-Castell Jordi , Huerta-Cepas Jaime
Published: May 2022

Phylogenomics data have grown exponentially over the last decades. It is currently common for genome-wide projects to generate hundreds or even thousands of phylogenetic trees and multiple sequence alignments, which may also be very large in size. However, the analysis and interpretation of such data still depends on custom bioinformatic and visualisation workflows that are largely unattainable for non-expert users. Here, we present PhyloCloud, an online platform aimed at hosting, indexing and exploring large phylogenetic tree collections, providing also seamless access to common analyses and operations, such as node annotation, searching, topology editing, automatic tree rooting, orthology detection and more. In addition, PhyloCloud provides quick access to tools that allow users to build their own phylogenies using fast predefined workflows, graphically compare tree topologies, or query taxonomic databases such as NBCI or GTDB. Finally, PhyloCloud offers a novel tree visualisation system based on ETE Toolkit v4.0, which can be used to explore very large trees and enhance them with custom annotations and multiple sequence alignments. The platform allows for sharing tree collections and specific tree views via private links, or make them fully public, serving also as a repository of phylogenomic data. PhyloCloud is available at https://phylocloud.cgmlab.org
GEOexplorer: a webserver for gene expression analysis and visualisation
Authors: Hunt Guy P , Grassi Luigi , Henkin Rafael , Smeraldi Fabrizio , Spargo Thomas P , Kabiljo Renata , Koks Sulev , Ibrahim Zina , Dobson Richard J B , Al-Chalabi Ammar , Barnes Michael R , Iacoangeli Alfredo
Published: May 2022

Gene Expression Omnibus (GEO) is a database repository hosting a substantial proportion of publicly available high throughput gene expression data. Gene expression analysis is a powerful tool to gain insight into the mechanisms and processes underlying the biological and phenotypic differences between sample groups. Despite the wide availability of gene expression datasets, their access, analysis, and integration are not trivial and require specific expertise and programming proficiency. We developed the GEOexplorer webserver to allow scientists to access, integrate and analyse gene expression datasets without requiring programming proficiency. Via its user-friendly graphic interface, users can easily apply GEOexplorer to perform interactive and reproducible gene expression analysis of microarray and RNA-seq datasets, while producing a wealth of interactive visualisations to facilitate data exploration and interpretation, and generating a range of publication ready figures. The webserver allows users to search and retrieve datasets from GEO as well as to upload user-generated data and combine and harmonise two datasets to perform joint analyses. GEOexplorer, available at https://geoexplorer.rosalind.kcl.ac.uk, provides a solution for performing interactive and reproducible analyses of microarray and RNA-seq gene expression data, empowering life scientists to perform exploratory data analysis and differential gene expression analysis on-the-fly without informatics proficiency.
Behaviour within a Clinical Trial and Implications for Mammography Guidelines
Authors: Kowalski Amanda E
Published: May 2022

Mammography guidelines have weakened in response to evidence that mammograms diagnose breast cancers that would never eventually cause symptoms, a phenomenon called “overdiagnosis.” Given concerns about overdiagnosis, instead of recommending mammograms, US guidelines encourage women aged 40–49 to get them as they see fit. To assess whether these guidelines target women effectively, I propose an approach that examines mammography behaviour within an influential clinical trial that followed participants long enough to find overdiagnosis. I find that women who are more likely to receive mammograms are healthier and have higher socioeconomic status. More importantly, I find that the 20-year level of overdiagnosis is at least 3.5 times higher among women who are most likely to receive mammograms. At least 36$\%$ of their cancers are overdiagnosed. These findings imply that US guidelines encourage mammograms among healthier women who are more likely to be overdiagnosed by them. Guidelines in other countries do not.
Can transparency undermine peer review? A simulation model of scientist behavior under open peer review
Authors: Bianchi Federico , Squazzoni Flaminio
Published: May 2022

Transparency and accountability are keywords in corporate business, politics, and science. As part of the open science movement, many journals have started to adopt forms of open peer review beyond the closed (single- or double-blind) standard model. However, there is contrasting evidence on the impact of these innovations on the quality of peer review. Furthermore, their long-term consequences on scientists’ cooperation and competition are difficult to assess empirically. This paper aims to fill this gap by presenting an agent-based model that simulates competition and status dynamics between scholars in an artificial academic system. The results would suggest that if referees are sensitive to competition and status, the transparency achieved by open peer review could backfire on the quality of the process. Although only abstract and hypothetical, our findings suggest the importance of multidimensional values of peer review and the anonymity and confidentiality of the process.
Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem
Authors: Sweany Rebecca R. , Breunig Mikaela , Opoku Joseph , Clay Keith , Spatafora Joseph W. , Drott Milton T. , Baldwin Thomas T. , Fountain Jake C.
Published: May 2022

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host–pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).
Distribution and Diversity of Prunus Necrotic Ringspot Virus, Prune Dwarf Virus, and Peach Latent Mosaic Viroid in Wild Prunus spp. in South Carolina and Georgia
Authors: Rodriguez Bonilla Fabian , Cieniewicz Elizabeth
Published: May 2022

Prunus-infecting ilarviruses and viroids are important pathogens in southeastern U.S. peach production. To identify potential sources of inoculum, we conducted a survey for two ilarviruses, Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV), and a viroid, peach latent mosaic viroid (PLMVd), all suggested to be pollen transmissible, in wild Prunus spp . in South Carolina and Georgia. PNRSV was detected in 12% (14/117) of wild Prunus spp., mostly in wild black cherry ( Prunus serotina), with one PNRSV isolate detected in Carolina cherry laurel ( P. caroliniana). PDV and PLMVd were not detected in wild Prunus spp. in this survey. No significant relationship was observed between virus/viroid infection and tree age in P. serotina. Sequence analysis of the gene encoding the PNRSV coat protein from isolates recovered from wild Prunus spp. revealed clustering of South Carolina and Georgia isolates with reference isolate ‘PE5’, an isolate obtained from peach ( P. persica) in the United States. Together,  these results suggest that wild Prunus spp. have the potential to serve as inoculum sources for pollen transmissible viruses near orchards. However, the extent of actual virus ingress into orchards from wild Prunus spp. requires further investigation.

[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
NMR‐Guided Directed Evolution of a Kemp Eliminase
Authors: Bhattacharya Sagar , Margheritis Eleanor , D'Souza Areetha , van Nuland Nico , Tame Jeremy , Volkov Alexander , Makhlynets Olga , Korendovych Ivan
Published: May 2022

Directed evolution has emerged as a powerful tool for improving protein properties and imparting completely new functionalities onto existing proteins. Several computational methods have been successful in predicting potential hotspots for directed evolution. However, they rely heavily on prior structural and/or functional information. To address these fundamental limitations, we focused on Nuclear Magnetic Resonance (NMR) as a tool to rapidly evolve enzymes without structural characterization. AlleyCat, a computationally designed Kemp eliminase produced by introducing a single mutation on C‐terminal domain of a non‐enzymatic metalloprotein Calmodulin, has recently been evolved into AlleyCat7. This traditional directed evolution process required seven rounds of mutagenesis and screening. A retrospective analysis of the evolved variants of AlleyCat series showed a remarkable correlation between chemical shift perturbation and probability of finding a productive mutation in the vicinity of that residue. Using our novel approach, AlleyCat7 has been rapidly evolved into AlleyCat10 with an improvement of about four‐fold in catalytic efficiency after three successive rounds of evolution. NMR‐guided evolution strategy eliminates excessive mutagenesis and has the potential to unlock exciting avenues in directed evolution of protein catalysts.
Deciphering the Role of Cancer‐Testis Antigen (CTA) MAGEA9 in Regulating DNA Repair Pathway Proteins MSH2 and MSH6
Authors: Chu Josephine , Johnson Jodiene , Ramanathan Saumya
Published: May 2022

Cancer is a disease characterized by uncontrolled cell division and is one of the leading causes of death worldwide. In 2021, 1.8 million new cancer cases and 608,570 cancer‐related deaths are projected in the United States. Current cancer treatments are limiting, as they do not consider the differences in the epigenetic and genetic makeup of every patient, which explains why certain therapies may work for one patient but not another. Cancer‐specific proteins have garnered significant interest for precision medicine applications. Cancer testis antigens (CTAs) are a group of tumor antigens with gene expression restricted to the male germ cells in the testis. The Melanoma Antigen Gene (MAGE) protein family is one of the largest groups of CTAs. The MAGE protein family is highly conserved, and all share a common MAGE homology domain. Interestingly, while MAGEs are typically restricted to reproductive tissues, they are aberrantly expressed in cancer. MAGEs are categorized by expression pattern, as Type I MAGEs are strictly expressed in the testis while Type II MAGEs are ubiquitously expressed throughout the body. MAGEA9 belongs to the Type I family of MAGEs and expression correlates with poor patient outcome. Data from our lab indicates that expression of MAGEA9 results in increased cell proliferation and anchorage‐independent growth. Furthermore, we have identified MAGEA9 as a potential regulator of MSH2 and MSH6, two DNA repair pathway proteins that are involved in mismatch recognition and repair. Our goals are therefore to elucidate the molecular mechanism by which MAGEA9 affects MSH2 and MSH6 protein levels. To confirm interaction of MAGEA9 with MSH2 and MSH6 at the protein level, we will use co‐immunoprecipitation, co‐localization, and in vitro binding studies. In addition, we will perform CHX chase assays to determine the effect of MAGEA9 on MSH2 and MSH6 steady‐state levels. We will then use DNA repair assays to assess the overall effect of MAGEA9 on DNA mismatch repair. Collectively, these findings will provide a clearer understanding for the function of MAGEA9 in tumorigenesis, which may provide further insight for the development of novel therapeutic interventions.
Novel Polyanionic Tracts Improve Expression of a TPP1:K16ApoE Chimera
Authors: Modak Anurag , Banach‐Petrosky Whitney , Sleat David E. , Lobel Peter
Published: May 2022

Background

Low expression of highly‐charged recombinant proteins remains a critical challenge in developing protein‐based therapeutics. It has been shown that inclusion of a polyanionic tract (PAT) may improve expression through inter‐ and intramolecular interactions. We previously developed a chimeric construct consisting of tripeptidyl‐peptidase 1 (TPP1), a glycine‐serine linker ([GS]5), a glycine‐aspartate PAT ([GD3]3), and a basic cell‐penetrating peptide K16ApoE as a potential therapeutic agent for late‐infantile neuronal ceroid lipofuscinosis. While TPP1‐(GS)5‐(GD3)3‐(GS)5‐K16ApoE was expressed efficiently, it was cleaved at the PAT. We therefore sought to design novel PATs that improve expression of the full‐length chimera.

Methods

TPP1:K16ApoE chimeric proteins were synthesized to contain tri‐glutamate sequences instead of tri‐aspartate sequences in the original (GD3)3PAT. All seven possible combinations of substitutions were engineered: (GE3)3, (GE3)‐(GD3)2, (GD3)‐(GE3)‐(GD3), (GD3)2‐(GE3), (GE3)2‐(GD3), (GE3)‐(GD3)‐(GE3), or (GD3)‐(GE3)2. Chimeric proteins were expressed in Chinese hamster ovary (CHO) cells and media samples were assayed for TPP1 activity. Activity was normalized to a wild‐type (unmodified) TPP1 positive control expressed in CHO cells. The identity of each construct was verified using SDS‐PAGE and mass spectrometry. Welch’s ANOVA test was used to determine expressional differences between TPP1‐(GS)5‐K16ApoE and PAT‐modified chimeras and the resulting p‐values were adjusted for multiple comparisons using Dunnett’s T3 multiple comparison test.

Results

Seven constructs were synthesized, corresponding to each of the aforementioned tri‐glutamate‐substituted PATs, following the general format of TPP1‐(GS)5‐(PAT)‐(GS)5‐K16ApoE. All chimeras expressed successfully as full‐length constructs (Figure 1). No TPP1 activity was detected in the media of untransfected CHO cells. TPP1‐(GS)5‐K16ApoE was previously determined to express at 1.4±1.0% of unmodified TPP1. Of these chimeras with novel PATs, TPP1‐(GS)5‐(GE3)‐(GD3)2‐(GS)5‐K16ApoE [mean ± standard error: 7.3±1.7%, p=0.0040] and TPP1‐(GS)5‐(GE3)2‐(GD3)‐(GS)5‐K16ApoE [5.8±3.1%, p=0.0397] exhibited significantly increased expression relative to TPP1‐(GS)5‐K16ApoE at the 0.05 level of significance.

Conclusion

Alteration of the a priori (GD3)3 PAT sequence by varying the aspartate and glutamate content was sufficient to prevent cleavage of the resulting TPP1:K16ApoE chimeras. However, only the (GE3)‐(GD3)2 and (GE3)2‐(GD3) PATs yielded a 5‐fold and a 4‐fold, respectively, improvement in expression over the original TPP1‐(GS)5‐K16ApoE construct. Further investigation is required to understand the electrostatic and/or structural basis of these findings.
A Small‐molecule Inhibitor VM17 Targeting a Transcriptional Regulator HlyU Attenuates the Virulence of Vibrio vulnificus
Authors: Choi Sang Ho
Published: May 2022

Vibrio vulnificusis a causative agent of food‐borne diseases including gastroenteritis and life‐threatening septicemia. Exotoxins such as a multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxin, a hemolysin, and a phospholipase A2 encoded by rtxA, vvhA, and plpAgenes, respectively, are essential virulence factors. Interestingly, the expression of rtxA, vvhA, and plpA is activated by a transcriptional regulator HlyU. Therefore, HlyU can be an attractive target to control the virulence of V. vulnificus. We performed a high‐throughput screening of 8,364 molecules at 20 µM using Escherichia coli containing HlyU‐expressing and HlyU‐dependent reporter plasmids. A small molecule VM17 exhibiting the most effective HlyU‐inhibiting activity was selected and verified. VM17 did not affect the cellular HlyU levels, indicating that VM17 could inhibit the HlyU activity. The expression of rtxA, vvhA, and plpAin the wild type was significantly reduced in the presence of 20 µM of VM17. Consistent with this, VM17 enhanced the survival rate of the mice infected with V. vulnificus, indicating that the small molecule inhibits the virulence of V. vulnificus in vitroand in vivo. In conclusion, the combined results suggested that VM17 could be a novel lead compound to attenuate the virulence of V. vulnificuswith a low probability of developing resistance.
What Doesn't Kill You Makes You Stronger—Even Heart Radiation?
Authors: SenthilKumar Gopika , Heisner James S. , Schlaak Rachel , Mishra Jyotsna , Katunaric Boran , Camara Amadou K. , Bergom Carmen
Published: May 2022

Radiation therapy (RT) is a commonly used treatment for thoracic cancers. However, the risk of coronary events increase by 4‐16% per Gy of heart dose. Our prior studies show that localized heart RT leads to radiation‐induced heart disease (RIHD) and increased mortality in Dahl Salt Sensitive (SS) rats starting at 3mo post‐RT. We have also shown that whole thoracic RT increases susceptibility to ischemia‐reperfusion (IR) injury in Wistar rats. Taken together, we hypothesized that 10weeks after targeted cardiac RT (prior to RIHD) SS rat hearts would have worsened cardiac function as measured by echocardiography and be more susceptible to IR injury. Echocardiograms were performed 10weeks after 24Gy dose of CT‐guided localized cardiac RT and in age‐matched controls (C). Next, hearts were isolated and perfused ex vivo using the Langendorf method ‐ without ischemia reperfusion (time controls; TC; n=3 in C and RT) or exposed to 25min of global ischemia followed by 60min reperfusion (IR; n=6 in C and RT). During TC and IR, cardiac function, mitochondrial redox state, and vascular reactivity were assessed; the hearts were then sectioned and stained to assess infarct size. Echocardiography of the RT group when compared to C showed an increase in interventricular septal thickness (1.32±0.1 vs 0.78±0.07 cm, p<0.0001; mean±SD) and left ventricular posterior wall thickness (1.03±0.08 vs 0.75±0.06 cm, p<0.005). Accordingly, a decrease in end diastolic (1.79±0.46 vs 2.38±0.35 ml/kg, p<0.05) and end systolic (0.10±0.04 vs 0.23±0.08 ml/kg, p<0.05) volume was also seen, with no significant differences in ejection fraction (94.5±2.1 vs 90.3±3.9 %) or stroke volume (1.69±0.44 vs 2.15±0.27 ml; p>0.05). Interestingly, in the ex vivo perfused hearts, TC RT had a 2 times higher rate‐pressure product – a measure of cardiac work – compared to C (p<0.05), and following IR, the RT group recovered to 2.5x higher levels to C (p<0.0001). Following IR, the RT group compared to C also showed a greater recovery of coronary flow rate (1.6x, p<0.05) and rates of contractility (2.25x) and relaxation (lusitropy; 2.05x) (p<0.0001). No difference in mitochondrial redox state, infarct size, and endothelium‐dependent and independent vascular reactivity were seen between the groups. In conclusion, our results show for the first time that 10weeks post‐targeted cardiac RT, ex vivo hearts show better recovery and cardiac function compared to C after IR through mechanisms other than differences in redox state and vascular reactivity.
Morphological characterization of Mast cells in the cochlea during postnatal rodent development
Authors: Smorodchenko Alina , Dudnik Tatyana , Karayay Betül , Sergeeva Valentina , Olze Heidi , Szczepek Agnieszka J.
Published: May 2022

Mast cells (MC) are immune cells of hematopoietic origin widely distributed in the mammalian body's connective tissue. Their activity plays a potential immunoregulatory role associated with allergic reactions, autoimmune, cardiovascular, and neuronal diseases. Previously, the inner ear‐related MCs were described in the endolymphatic sac. However, the presence of MCs in the mammalian cochlea was not yet investigated.

Here, we demonstrate for the first time that the MCs are present in the healthy cochlea of C57BL/6 mice and Wistar rats. Using histochemistry and immunohistochemistry on the cryo‐/paraffine sections of the whole cochlea and cochlea explants, we detected MCs in the modiolus, spiral ligament, scala vestibuli, and occasionally in scala tympani. The identified MCs expressed various MC‐specific markers: cKit/CD117, chymase, tryptase, and FcεRIa. Quantitative analysis demonstrated a significant decrease in the number of MCs during the postnatal development, resulting in only a few MCs present in the cochlea of rats and mice. In addition, 24h incubation of the cochlear explant with 40 µM of cisplatin lead to a significant reduction of MC numbers.

The presence of MCs in the cochlea may shed new light on postnatal maturation of the auditory periphery and possible involvement in cisplatin ototoxicity. Currently, our findings expand the knowledge about the physiology and pathology of the auditory periphery.

Considering the pivotal role of MCs in inflammation, future functional studies should explain this new basic knowledge and translate it into clinical application.
Enzymatic removal of cell surface antigens as a route towards universal O type blood and organs
Authors: Withers Stephen G.
Published: May 2022

Mammalian cell surfaces are coated in specific sugar structures, many of which function as antigens and are involved in cellular recognition. Important examples are the oligosaccharide A, B, and H antigens present on red blood cells that differentiate the A, B and O blood types. Enzymatic cleavage of the GalNAc and Gal residues from the cell surface would allow conversion of A and B red blood cells, respectively, to O type. Since Type O blood can be universally donated to patients with the same Rh factor, access to efficient enzymes would greatly broaden and simplify blood supply. We have sought such enzymes in metagenomic libraries derived from the human gut microbiome.

Total DNA was extracted from feces samples, fragmented into pieces containing ~30‐40 genes (40‐50 kB) and transformed into E. coli. After picking colonies into 384 well plates we screened them for enzymes that can be used to remove the Gal or GalNAc residues that function as the antigenic determinants from A and B type red blood cells, thereby generating “universal” O type blood. A set of efficient enzymes of a new class has been identified and characterized and used to convert whole units of A blood to O. These enzymes work approximately 30 times faster than any previously characterized and with high specificity. Further, they function well in whole blood thus can be hopefully integrated into the current blood processing process.1

These enzymes also function well on tissue surfaces and, when perfused through donated human A‐type lungs successfully cleared A‐antigen from surfaces exposed to the blood. Subsequent perfusion of the lungs with O‐type plasma to mimic an ABO‐incompatible transplant showed that antigen removal minimized antibody binding and antibody‐mediated injury relative to the control lung, thus may greatly temper the acute rejection phase.2 This could allow expansion of ABO‐incompatible lung transplantations, leading to significant improvements in logistics and fairness of organ allocation.

Rahfeld, P. et al. “An enzymatic pathway in the human gut microbiome that converts A to universal O‐type blood” (2019) Nature Microbiology 4, 1475‐1485.

Wang et al Conversion of blood type A donor lungs into universal blood type lungs using ex vivo ABO enzymatic treatment” (2022) Science Translational Medicine In Press, Scheduled Feb 2022
Conformational dynamics of nuclear pores
Authors: Beck Martin
Published: May 2022

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remained unknown. We combined cryo‐electron tomography (cryo‐ET) with integrative structural modeling to capture the respective large‐scale conformational changes in cellulo. While NPCs of exponentially growing cells adopt a dilated conformation, they reversibly constrict upon cellular energy depletion or conditions of hypertonic osmotic stress. We propose a model in which nuclear envelope membrane tension regulates the conformation of the NPC.
Tracing copper utilization by kinase signal transduction pathways: Implications for cancer cell processes
Authors: Brady Donita C.
Published: May 2022

Akin to organic nutrients, such as oxygen, lipids, amino acids, and carbohydrates, the transition metal copper (Cu) is an essential dietary nutrient for normal physiology and development. Decades of research highlight the physiological and disease associated consequences of disrupting homeostatic mechanisms that ensure proper Cu acquisition, storage, and distribution to Cu‐dependent enzymes. However, phenotypes associated with alterations in Cu availability cannot be fully explained by the limited number of enzymes that traditionally harness the redox potential of Cu as a catalytic cofactor. Recent discoveries in Cu biology have revealed direct Cu binding at non‐catalytic sites within signaling molecules that modulate cell proliferation via the protein kinases MEK1/2, lipid metabolism via the phosphodiesterase PDE3B, and nutrient recycling via the autophagic kinases ULK1/2. The emergence of this new paradigm in nutrient sensing and protein regulation has established that Cu is a critical mediator of intracellular signaling, provided evidence for the existence of molecular mechanisms for sensing changes in Cu abundance, and expanded the contribution of Cu to cellular processes necessary for adaptation to nutrient scarcity. Our presentation will focus on the intersections between Cu homeostasis, nutrient signaling, and metabolism by examining the interplay between mechanisms of Cu‐sensing necessary for cellular energy homeostasis and evaluating the necessity of Cu for metabolic flexibility under nutrient and oxygen stress. We will present novel findings on Cu‐controlled autophagy‐lysosomal biogenesis and function, and interconnectivity between mitochondrial Cu transport and cytosolic nutrient sensing signaling pathways necessary for metabolism. These studies increase our fundamental knowledge of the molecular and cellular features of Cu‐dependent enzymes and cellular processes and enable therapeutic targeting of Cu‐dependent disease vulnerabilities.
Decoding molecular plasticity of the dark proteome
Authors: Lemke Edward A.
Published: May 2022

The mechanisms by which intrinsically disordered proteins (IDPs) engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function. Nuclear transport receptors (NTRs) can move through the central channel of the NPC which is filled with hundreds of phenylalanine‐glycine‐rich nucleoporins (FG‐Nups) reaching millimolar concentrations with elusive conformational plasticity. Since site‐specific labeling of proteins with small but highly photostable fluorescent dyes inside cells remains the major bottleneck for directly studying protein dynamics in the cellular interior, we have now developed a semi‐synthetic strategy based on novel artificial amino acids that are easily and site‐specifically introduced into any protein by the natural machinery of the living cell via a newly developed thin‐film synthetic organelle that equips the living cell with up to three genetic codes. This allowed us to develop an experimental approach combining site‐specific fluorescent labeling of IDPs in non‐fixed cells with fluorescent lifetime imaging microscopy (FLIM) to directly decipher the plasticity of FG‐Nups via FRET. Our study enabled a conformational look on the condensated IDPs in the sub‐resolution (roughly (50 nm)3 small cavity) cavity of the NPC. By measuring the end‐to‐end distances of different segments of the labeled FG‐Nups using FLIM‐FRET, we can extract the scaling exponent, which directly describes the conformations of FG‐Nups at their functional status as well as the solvent quality in the cellular and even inner NPC environment.

Reinkemeier CD, Lemke EA. Dual film‐like organelles enable spatial separation of orthogonal eukaryotic translation. Cell. 2021 Sep 16;184(19):4886‐4903.e21

Celetti G, Paci G, Caria J, VanDelinder V, Bachand G, Lemke EA. The liquid state of FG‐nucleoporins mimics permeability barrier properties of nuclear pore complexes. J Cell Bio. (2020) Jan 6;219(1).

Reinkemeier CD, Estrada Girona G, Lemke EA, 2019 Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science, Mar 29;363(6434)

Nikić I, Estrada Girona G, Kang JH, Paci G, rei S, Koehler C, Shymanska NV, Ventura Santos C, Spitz D, Lemke EA, Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy. Angew Chem Int Ed Engl. (2016) Dec 23;55(52):16172‐16176
Genetic and small molecule strategies to edit the glycocalyx
Authors: Neelamegham Siriram
Published: May 2022

The glycocalyx covers all living cells and is a key regulator of cell communication with the extracellular milieu/matrix, other cells, foreign pathogens and the microbiome. A variety of glycoconjugate types decorate mammalian cells including N‐ and O‐linked glycans attached to proteins, glycolipids, and glycosaminoglycans. A complete understanding of the molecular and cellular features controlling living systems is not possible without delineating the structural and functional contributions of the glycocalyx. My laboratory studies this aspect by developing systems based experimental and computational tools to characterize the role of the glycocalyx in normal physiological and selected pathophysiological contexts. For such investigations, we have developed both novel CRISPR‐Cas9 based tools to perturb cell surface glycans and small molecule inhibitors of the same. Additionally, we have developed a Synthetic Glycan toolkit and liquid‐chromatography mass‐spectrometry (LC‐MS) workflow to longitudinally track glycan changes in living cells in a non‐destructive manner. My talk will present the development and application of these technologies in two contexts. First, I will discuss studies of human inflammatory diseases which will present some new molecular players regulating glycosylation and leukocyte‐endothelial cell adhesion mechanics under fluid shear flow. Second, I will present our studies on the role of glycans in regulating SARS‐CoV‐2 viral entry into human cells expressing ACE2. The data demonstrate a critical role for N‐glycans and O‐linked glycans in viral entry and propagation.