bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signaling
Authors: Marechal Elise , Poliard Anne , Henry Kilian , Moreno Mathias , Legrix Mathilde , Macagno Nicolas , Mondielli Grégoire , Fauquier Teddy , Barlier Anne , Etchevers Heather C.
Published: April 2022

Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.
CARP2 regulates the Golgi dynamics upon EGF stimulation
Authors: Sharma Rahul , Srinivasula Srinivasa M.
Published: April 2022

Golgi apparatus regulate diverse cellular functions like protein sorting, vesicular trafficking, secretion, protein modifications like glycosylation etc. In mammalian cells though, Golgi apparatus appear as ribbon architecture, individual stacks laterally linked to each other by tubular structure, it’s architecture changes dynamically to cater to the needs of the cell under physiological and stress conditions. Loss of Golgi integrity is reported to be associated with pathological conditions like cancer and neurodegeneration. Very little is known of molecular regulators of Golgi dynamics. Here, we demonstrate that CARP2 (Caspase −8 and −10 associated RING containing protein 2), an endosomal ubiquitin ligase and a known regulator of cell migration, modulates the Golgi structure. Stimulation with EGF (Epidermal growth factor) modestly increased CARP2 protein levels. CARP2 exogenous expression or EGF treatment resulted in dispersal of the Golgi apparatus. Conversely, CARP2 deletion suppressed EGF induced Golgi dispersal. CARP2 variants that are defective in their endosomal-association or E3 ligase activity were unable to exhibit Golgi dispersal, indicating importance of both the endosomal localization and the E3 activity for this function. Importantly, we provide evidence that in EGF stimulated cells CARP2 mechanistically functions by targeting one of the Golgi structural proteins, Golgin45 for ubiquitination and degradation. Taken together, our findings unravel the existence of crosstalk between endosomal ubiquitin signaling and the Golgi dynamics.

Significance

The Golgi is an organelle that exists in mammals in ribbon form - individual stacks laterally linked with each other - is central to protein and lipid modifications, trafficking and secretion. The Golgi architecture is changed dynamically to cater to the physiological needs of the cells (eg: cell division, migration). Dysfunctional or altered Golgi is reported under pathological conditions like cancer, neurodegenerative diseases etc. This study unravels a complex signaling between endosomal ubiquitin ligase, CARP2 and one of the Golgi structural proteins, Golgin45. Here, we delineate CARP2-Golgin45 signaling as a fundamental mechanism that regulates Golgi dynamics underlying in EGF-stimulated cell migration.
Development of the adult-like larval stomach of Lepidobatrachus laevis
Authors: Austiff Jennifer K. , Hanken James
Published: April 2022

Most frogs’ diets shift between the larval and adult phases, from a filter-feeding herbivore to a bulk-feeding carnivore. This change in diet corresponds to a biphasic mode of gut development that begins during embryogenesis and resumes at metamorphosis, when nearly the entire anatomy of the tadpole is reorganized into its adult morphology. The frog Lepidobatrachus laevis forgoes this metamorphic shift in feeding mode and instead consumes a bulk-feeding, carnivorous diet both as a larvae and as an adult. This unusual larval diet is enabled by the presence of an adult-like stomach in the tadpole. This study investigates the anatomy and embryonic development of the larval stomach of L. laevis and what, if any, further changes occur during metamorphosis. The histology of embryonic and metamorphic stomach development is compared to that of Xenopus tropicalis, a frog with a typical larval stomach. We find that L. laevis directly forms an adult-like stomach during embryonic development without first forming a larval-like configuration. Moreover, no additional major remodeling of the stomach occurs during metamorphosis, although the stomach does gradually and slightly increase in complexity, proliferating more glands and increasing connective tissue and muscle layers, between hatching and the end of metamorphosis. This developmental trajectory of the stomach in L. laevis corresponds with the megalophagous, carnivorous diet these frogs maintain from tadpole to adult, as well as the maintenance of active feeding throughout metamorphosis. These results will facilitate future investigations into the mechanisms underlying the evolution of this unusual larval anuran feeding strategy, as well as the broader study of how development mediates evolutionary change.
SARS-CoV-2 Omicron (BA.1 and BA.2) specific novel CD8+ and CD4+ T cell epitopes targeting spike protein
Authors: Parn Simone , Savsani Kush , Dakshanamurthy Sivanesan
Published: April 2022

The Omicron (BA.1/B.1.1.529) variant of SARS-CoV-2 harbors an alarming 37 mutations on its spike protein, reducing the efficacy of current COVID-19 vaccines. This study identified CD8+ and CD4+ T cell epitopes from SARS-CoV-2 S protein mutants. To identify the highest quality CD8 and CD4 epitopes from the Omicron variant, we selected epitopes with a high binding affinity towards both MHC I and MHC II molecules and applied other clinical checkpoint predictors including immunogenicity, antigenicity, allergenicity, instability, and toxicity. Subsequently, we found eight Omicron (BA.1/B.1.1.529) specific CD8+ and eleven CD4+ T cell epitopes with a world population coverage of 76.16% and 97.46%, respectively. Additionally, we identified common epitopes across Omicron BA.1 and BA.2 lineages that target mutations critical to SARS-CoV-2 virulence. Further, we identified common epitopes across B.1.1.529 and other circulating SARS-CoV-2 variants, such as B.1.617.2 (Delta). We predicted CD8 epitopes’ binding affinity to murine MHC alleles to test the vaccine candidates in preclinical models. The CD8 epitopes were further validated using our previously developed software tool PCOptim. We then modeled the three-dimensional structures of our top CD8 epitopes to investigate the binding interaction between peptide-MHC and peptide-MHC-TCR complexes. Importantly, our identified epitopes are targeting the mutations on the RNA-binding domain and the fusion sites of S protein. This could potentially eliminate viral infections and form long-term immune responses compared to rather short-lived mRNA vaccines and maximize the efficacy of vaccine candidates against the current pandemic and potential future variants.
Propofol disrupts alpha dynamics in distinct thalamocortical networks underlying sensory and cognitive function during loss of consciousness
Authors: Weiner Veronica S. , Zhou David W. , Kahali Pegah , Stephen Emily P. , Peterfreund Robert A. , Aglio Linda S. , Szabo Michele D. , Eskandar Emad N. , Salazar-Gomez Andrés F. , Sampson Aaron L. , Cash Sydney S. , Brown Emery N. , Purdon Patrick L.
Published: April 2022

During propofol-induced general anesthesia, alpha rhythms undergo a striking shift from posterior to anterior, termed anteriorization. We combined human intracranial recordings with diffusion imaging to show that anteriorization occurs with opposing dynamics in two distinct thalamocortical subnetworks. The cortical and thalamic anatomy involved, as well as their known functional roles, suggest multiple means by which propofol dismantles sensory and cognitive processes to achieve loss of consciousness.
Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord
Authors: Wang Yi-wen , Wreden Chris C , Levy Maayan , Marshall Zarion D , MacLean Jason N , Heckscher Ellie S
Published: April 2022

Understanding how circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental biology. Here, we addressed how neurons from different lineages wire with each other to form a specific circuit motif. To do so, we combined developmental genetics—Twin spot MARCM, Multi-color Flip Out, permanent labeling—with circuit analysis—calcium imaging, connectomics, and network science analyses. We find many lineages are organized into temporal cohorts, which are sets of lineage-related neurons born within a tight time window, and that temporal cohort boundaries have sharp transitions in patterns of input connectivity. We identify a feed-forward circuit motif that encodes the onset of vibration stimuli. This feed-forward circuit motif is assembled by preferential connectivity between temporal cohorts from different neuronal stem cell lineages. Further, connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the feed-forward motif is formed by sequential addition of temporal cohorts, with circuit output neurons born before circuit input neurons. Further, we generate multiple new tools for the fly community. Ultimately, our data suggest that sequential addition of neurons (with outputs neurons being oldest and input neurons being youngest) could be a fundamental strategy for assembling feed-forward circuits.
Vascular inflammation exposes perivascular cells to SARS-CoV-2 infection
Authors: Franca Cristiane Miranda , Mansoorifar Amin , Athirasala Avathamsa , Subbiah Ramesh , Tahayeri Anthony , Bertassoni Luiz
Published: April 2022

Pericytes stabilize blood vessels and promote vascular barrier function. However, vessels subjected to pro-inflammatory conditions have impaired barrier function, which has been suggested to potentially expose perivascular cells to SARS-CoV-2. To test this hypothesis, we engineered pericyte-supported vascular capillaries on-a-chip, and determined that the extravasation and binding of spike protein (S1) on perivascular cells of inflamed vessels to be significantly higher that in healthy controls, indicating a potential target to understand COVID-19 vascular complications.
KSHV Topologically Associating Domains in Latent and Reactivated Viral Chromatin
Authors: Campbell Mel , Chantarasrivong Chanikarn , Yanagihashi Yuichi , Inagaki Tomoki , Davis Ryan R. , Nakano Kazushi , Kumar Ashish , Tepper Clifford G. , Izumiya Yoshihiro
Published: April 2022

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 base pair resolution and constructed a 3D KSHV genomic structural model with 2kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by CTCF and SMC1, and the KSHV transactivator was recruited to these sites during reactivation. Triggering KSHV gene expression decreased pre-wired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate-early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible non-coding RNA regions moved toward the inner space of the structure, resembling the configuration of a “bird cage” during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help coordinate viral gene transcription.

Importance

The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi’s sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 base pair resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral lifecycle.
Longitudinal lung function assessment of patients hospitalised with COVID-19 using1H and129Xe lung MRI
Authors: Saunders Laura C , Collier Guilhem J , Chan Ho-Fung , Hughes Paul J C , Smith Laurie J , Watson James , Meiring James , Gabriel Zoë , Newman Thomas , Plowright Megan , Wade Phillip , Eaden James A , Bray Jody , Marshall Helen , Capener David J , Armstrong Leanne , Rodgers Jennifer , Brook Martin , Biancardi Alberto M , Rao Madhwesha R , Norquay Graham , Rodgers Oliver , Munro Ryan , Ball James E , Stewart Neil J , Lawrie Allan , Jenkins Gisli , Grist James , Gleeson Fergus , Schulte Rolf F. , Johnson Kevin M , Wilson Frederick , Cahn Anthony , Swift Andrew J , Rajaram Smitha , Mills Gary H , Watson Lisa , Collini Paul J , Lawson Rod , Thompson A A Roger , Wild Jim M
Published: April 2022

Introduction

Microvascular abnormalities and impaired gas transfer have been observed in patients with COVID-19. The progression of pathophysiological pulmonary changes during the post-acute period in these patients remains unclear.

Methods

Patients who were hospitalised due to COVID-19 pneumonia underwent a pulmonary1H and129Xe MRI protocol at 6, 12, 25 and 51 weeks after hospital admission. The imaging protocol included: ultra-short echo time, dynamic contrast enhanced lung perfusion,129Xe lung ventilation,129Xe diffusion weighted and129Xe 3D spectroscopic imaging of gas exchange.

Results

9 patients were recruited and underwent MRI at 6 (n=9), 12 (n=9), 25 (n=6) and 51 (n=8) weeks after hospital admission. Patients with signs of interstitial lung damage at 3 months were excluded from this study. At 6 weeks after hospital admission, patients demonstrated impaired129Xe gas transfer (RBC:M) but normal lung microstructure (ADC, LmD). Minor ventilation abnormalities present in four patients were largely resolved in the 6–25 week period. At 12 week follow up, all patients with lung perfusion data available (n=6) showed an increase in both pulmonary blood volume and flow when compared to 6 weeks, though this was not statistically significant. At 12 week follow up, significant improvements in129Xe gas transfer were observed compared to 6-week examinations, however129Xe gas transfer remained abnormally low at weeks 12, 25 and 51. Changes in129Xe gas transfer correlated significantly with changes in pulmonary blood volume and TLCOZ-score.

Conclusions

This study demonstrates that multinuclear MRI is sensitive to functional pulmonary changes in the follow up of patients who were hospitalised with COVID-19. Impairment of xenon transfer may indicate damage to the pulmonary microcirculation.
Combined Infection Control Interventions Protect the Essential Workforce from Occupationally-Acquired SARS-CoV-2 during Produce Production, Harvesting and Processing Activities
Authors: Cooper D. Kane , Sobolik Julia S. , Kovacevic Jovana , Rock Channah M. , Sajewski Elizabeth T. , Guest Jodie L. , Lopman Ben A. , Jaykus Lee-Ann , Leon Juan S.
Published: April 2022

Essential food workers experience an elevated risk of SARS-CoV-2 infection due to prolonged occupational exposures (e.g., frequent close contact, enclosed spaces) in food production and processing areas, shared transportation (car or bus), and employer-provided shared housing. The purpose of this study was to evaluate the impact of combined food industry interventions and vaccination on reducing the daily cumulative risk of SARS-CoV-2 infection for produce workers. Six linked quantitative microbial risk assessment models were developed in R to simulate daily scenarios experienced by a worker. Standard industry interventions (2 m physical distancing, handwashing, surface disinfection, universal masking, increased ventilation) and two-dose mRNA vaccinations (86–99% efficacy) were modeled individually and jointly to assess risk reductions. The infection risk for an indoor (0.802, 95% Uncertainty Interval [UI]: 0.472–0.984) and outdoor (0.483, 95% UI: 0.255–0.821) worker was reduced to 0.018 (93% reduction) and 0.060 (87.5% reduction) after implementation of combined industry interventions. Upon integration of these interventions with vaccination, the infection risk for indoor (0.001, 95% UI: 0.0001–0.005) and outdoor (0.004, 95% UI: 0.001–0.016) workers was reduced by ≥99.1%. Food workers face considerable risk of occupationally-acquired SARS-CoV-2 infection without interventions; however, consistent implementation of key infection control measures paired with vaccination effectively mitigates these risks.

Synopsis

Bundled interventions, particularly if they include vaccination, produce significant reductions (>99%) in SARS-CoV-2 infection risk for essential food workers.

Graphical Abstract
Risk factors for severe COVID-19 in hospitalized children in Canada: A national prospective study from March 2020–May 2021
Authors: Farrar Daniel S. , Drouin Olivier , Hepburn Charlotte Moore , Baerg Krista , Chan Kevin , Cyr Claude , Donner Elizabeth J. , Embree Joanne E. , Farrell Catherine , Forgie Sarah , Giroux Ryan , Kang Kristopher T. , King Melanie , Thibodeau Melanie Laffin , Orkin Julia , Ouldali Naïm , Papenburg Jesse , Pound Catherine M. , Price Victoria E. , Proulx-Gauthier Jean-Philippe , Purewal Rupeena , Ricci Christina , Sadarangani Manish , Salvadori Marina I. , Thibeault Roseline , Top Karina A. , Viel-Thériault Isabelle , Kakkar Fatima , Morris Shaun K.
Published: April 2022

Background

Children living with chronic comorbid conditions are at increased risk for severe COVID-19, though there is limited evidence regarding the risks associated with specific conditions and which children may benefit from targeted COVID-19 therapies. The objective of this study was to identify factors associated with severe disease among hospitalized children with COVID-19 in Canada.

Methods

We conducted a national prospective study on hospitalized children with microbiologically confirmed SARS-CoV-2 infection via the Canadian Paediatric Surveillance Program from April 2020–May 2021. Cases were reported voluntarily by a network of >2800 paediatricians. Hospitalizations were classified as COVID-19-related, incidental infection, or infection control/social admissions. Severe disease (among COVID-19-related hospitalizations only) was defined as disease requiring intensive care, ventilatory or hemodynamic support, select organ system complications, or death. Risk factors for severe disease were identified using multivariable Poisson regression, adjusting for age, sex, concomitant infections, and timing of hospitalization.

Findings

We identified 544 children hospitalized with SARS-CoV-2 infection, including 60·7% with COVID-19-related disease and 39·3% with incidental infection or infection control/social admissions. Among COVID-19-related hospitalizations (n=330), the median age was 1·9 years (IQR 0·1–13·3) and 43·0% had chronic comorbid conditions. Severe disease occurred in 29·7% of COVID-19-related hospitalizations (n=98/330), most frequently among children aged 2-4 years (48·7%) and 12-17 years (41·3%). Comorbid conditions associated with severe disease included technology dependence (adjusted risk ratio [aRR] 2·01, 95% confidence interval [CI] 1·37-2·95), neurologic conditions (e.g. epilepsy and select chromosomal/genetic conditions) (aRR 1·84, 95% CI 1·32-2·57), and pulmonary conditions (e.g. bronchopulmonary dysplasia and uncontrolled asthma) (aRR 1·63, 95% CI 1·12-2·39).

Interpretation

While severe outcomes were detected at all ages and among patients with and without comorbidities, neurologic and pulmonary conditions as well as technology dependence were associated with increased risk of severe COVID-19. These findings may help guide vaccination programs and prioritize targeted COVID-19 therapies for children.

Funding

Financial support for the CPSP was received from the Public Health Agency of Canada.
Impact of the COVID-19 epidemic on mortality in rural coastal Kenya
Authors: Otiende M , Nyaguara A , Bottomley C , Walumbe D , Mochamah G , Amadi D , Nyundo C , Kagucia EW , Etyang AO , Adetifa IMO , Maitha E , Chondo E , Nzomo E , Aman R , Mwangangi M , Amoth P , Kasera K , Ng’ang’a W , Barasa E , Tsofa B , Mwangangi J , Bejon P , Agweyu A , Williams TN , Scott JAG
Published: April 2022

Background

The impact of COVID-19 on all-cause mortality in sub-Saharan Africa remains unknown.

Methods

We monitored mortality among 306,000 residents of Kilifi Health and Demographic Surveillance System, Kenya, through four COVID-19 waves from April 2020-September 2021. We calculated expected deaths using negative binomial regression fitted to baseline mortality data (2010-2019) and calculated excess mortality as observed-minus-expected deaths. We excluded deaths in infancy because of under-ascertainment of births during lockdown. In February 2021, after two waves of wild-type COVID-19, adult seroprevalence of anti-SARS-CoV-2 was 25.1%. We predicted COVID-19-attributable deaths as the product of age-specific seroprevalence, population size and global infection fatality ratios (IFR). We examined changes in cause of death by Verbal Autopsy (VA).

Results

Between April 2020 and February 2021, we observed 1,000 deaths against 1,012 expected deaths (excess mortality -1.2%, 95% PI -6.6%, 5.8%). Based on SARS-CoV-2 seroprevalence, we predicted 306 COVID-19-attributable deaths (a predicted excess mortality of 30.6%) within this period. Monthly mortality analyses showed a significant excess among adults aged ≥45 years in only two months, July-August 2021, coinciding with the fourth (Delta) wave of COVID-19. By September 2021, overall excess mortality was 3.2% (95% PI -0.6%, 8.1%) and cumulative excess mortality risk was 18.7/100,000. By VA, there was a transient reduction in deaths attributable to acute respiratory infections in 2020.

Conclusions

Normal mortality rates during extensive transmission of wild-type SARS-CoV-2 through February 2021 suggests that the IFR for this variant is lower in Kenya than elsewhere. We found excess mortality associated with the Delta variant but the cumulative excess mortality risk remains low in coastal Kenya compared to global estimates.
Multiplexed promoter and gene editing in wheat using the virus-based guide RNA delivery system
Authors: Wang Wei , Yu Zitong , He Fei , Bai Guihua , Trick Harold N. , Akhunova Alina , Akhunov Eduard
Published: April 2022

The low efficiency of genetic transformation and gene editing across diverse cultivars hinders the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we applied the barley stripe mosaic virus (BSMV) for delivering guide RNAs (sgRNA) into the Cas9-expressing wheat lines to create targeted deletions in the promoter of a transcription factor and to perform multiplexed editing of agronomic genes. We demonstrate that pooled BSMV-sgRNAs could be used to generate heritable targeted deletions and multiple mutations in the genome. We transferred the high-expressing allele of Cas9 into adapted spring and winter cultivars and successfully performed the BSMV-sgRNA-based editing of two agronomic genes. The strategies presented in our study could be applied to any cultivar for creating new cis-regulatory diversity or targeting multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes and accelerating gene discovery and trait improvement efforts.
Extended genomic analyses of the broad-host-range phages vB_KmiM-2Di and vB_KmiM-4Dii reveal slopekviruses have highly conserved genomes
Authors: Smith-Zaitlik Thomas , Shibu Preetha , McCartney Anne L. , Foster Geoffrey , Hoyles Lesley , Negus David
Published: April 2022

High levels of antimicrobial resistance among members of theKlebsiella oxytocacomplex (KoC) have led to renewed interest in the use of bacteriophage (phage) therapy to tackle infections caused by these bacteria. In this study we characterized two lytic phages, vB_KmiM-2Di and vB_KmiM-4Dii, that were isolated from sewage water against two GES-5-positiveKlebsiella michiganensisstrains (PS_Koxy2 and PS_Koxy4, respectively). ViPTree analysis showed both phages belonged to the genusSlopekvirus. rpoBgene-based sequence analysis of 108 presumptiveK. oxytocaisolates (n=59 clinical,n=49 veterinary) foundK. michiganensisto be more prevalent (46 % clinical and 43 % veterinary, respectively) thanK. oxytoca(40 % clinical and 6 % veterinary, respectively). Host range analysis against these 108 isolates found both vB_KmiM-2Di and vB_KmiM-4Dii showed broad lytic activity against KoC species. Several putative homing endonuclease genes were encoded within the genomes of both phages, which may contribute to their broad host range. Pangenome analysis of 24 slopekviruses found that genomes within this genus are highly conserved, with more than 50 % of all predicted coding sequences representing core genes at ≥95 % identity and ≥70 % coverage. Given their broad host ranges, our results suggest vB_KmiM-2Di and vB_KmiM-4Dii represent attractive potential therapeutics. In addition, current recommendations for phage-based pangenome analyses may require revision.
Cryo-EM structure of the diapause chaperone artemin
Authors: Parvate Amar D. , Powell Samantha M. , Brookreson Jory T. , Moser Trevor H. , Novikova Irina V. , Zhou Mowei , Evans James E.
Published: April 2022

The protein artemin constitutes over 10% of all protein in Artemia cysts during diapause and acts as both an RNA and protein chaperone. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 Å resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.
Hypoxia-mediated suppression of pyruvate carboxylase drives tumor microenvironment immunosuppression
Authors: Coleman Michael F. , Cotul Eylem Kulkoyluoglu , Pfeil Alexander J. , Devericks Emily N. , Chen Hao , Kiesel Violet A. , Safdar Muhammad H. , Teegarden Dorothy , Hursting Stephen D. , Wendt Michael K.
Published: April 2022

Metabolic reprogramming and immune evasion are established hallmarks of the tumor microenvironment (TME). Growing evidence supports tumor metabolic dysregulation as an important mediator of tumor immune evasion. High TME levels of lactate potently suppress antitumor immunity. Pyruvate carboxylase (PC), the enzyme responsible for the anaplerotic conversion of pyruvate to oxaloacetate, is essential for lung metastasis in breast cancer. Moreover, PC may be dispensable in some cells within the TME, and loss of PC expression is associated with immunosuppression. Here we test whether PC suppression alters tumor metabolism and immunosuppression. Using multiple animal models of breast cancer, we identify a dimorphic role for PC expression in mammary cancer cells. Specifically, PC supports metastatic colonization of the lungs, while suppression of PC promotes primary tumor growth and suppresses histological and transcriptomic markers of antitumor immunity. We demonstrate that PC is potently suppressed by hypoxia, and that PC is frequently suppressed in solid tumors, particularly those with higher levels of hypoxia. Using metabolomics, high-resolution respirometry, and extracellular flux analysis, we show that PC-suppressed cells produce more lactate and undergo less oxidative phosphorylation than controls. Finally, we identify lactate metabolism as a targetable dependency of PC-suppressed cells, which is sufficient to restore T cell populations to the TME of PC-suppressed tumors. Taken together, these data demonstrate that elevated lactate following PC suppression by hypoxia may be a key mechanism through which primary tumors limit antitumor immunity. Thus, these data highlight that PC-directed tumor metabolism is a nexus of tumor progression and antitumor immunity.
ViReMaShiny: An Interactive Application for Analysis of Viral Recombination Data
Authors: Yeung Jason , Routh Andrew L
Published: April 2022

Recombination is an essential driver of virus evolution and adaption, giving rise to new chimeric viruses, structural variants, sub-genomic RNAs, and Defective-RNAs. Next-Generation Sequencing of virus samples, either from experimental or clinical settings, has revealed a complex distribution of recombination events that contributes to the intrahost diversity. We and others have previously developed alignment tools to discover and map these diverse recombination events in NGS data. However, there is no standard for data visualization to contextualize events of interest and downstream analysis often requires bespoke coding. To address this, we present ViReMaShiny, a web-based application built using the R Shiny framework to allow interactive exploration and point-and-click visualization of viral recombination data provided in BED format generated by computational pipelines such as ViReMa (Viral-Recombination-Mapper).
Modelling the spatial dynamics of oncolytic virotherapy in the presence of virus-resistant tumor cells
Authors: Bhatt Darshak K. , Janzen Thijs , Daemen Toos , Weissing Franz J.
Published: April 2022

Oncolytic virotherapy is a promising form of cancer treatment that uses native or genetically engineered viruses to target, infect and kill cancer cells. Unfortunately, this form of therapy is not effective in a substantial proportion of cancer patients, partly due to the occurrence of infection-resistant tumor cells. To shed new light on the mechanisms underlying therapeutic failure and to discover strategies that improve therapeutic efficacy we designed a cell-based model of viral infection. The model allows to investigate the dynamics of infection-sensitive and infection-resistant cells in tumor tissue in presence of the virus. To reflect the importance of the spatial configuration of the tumor on the efficacy of virotherapy, we compare three variants of the model: two 2D models of a monolayer of tumor cells and a 3D model. In all model variants, we systematically investigate how the therapeutic outcome is affected by properties of the virus (e.g. the rate of viral spread), the tumor (e.g. production rate of resistant cells, cost of resistance), the healthy stromal cells (e.g. degree of resistance to virus) and the timing of treatment. We find that various therapeutic outcomes are possible when resistant cancer cells arise at low frequency in the tumor. These outcomes depend in an intricate but predictable way on the death rate of infected cells, where faster death leads to rapid virus clearance and cancer persistence. Our simulations reveal three different causes of therapy failure: rapid clearance of the virus, rapid selection of resistant cancer cells, and a low rate of viral spread due to the presence of infection-resistant healthy cells. Our models suggest that improved therapeutic efficacy can be achieved by sensitizing healthy stromal cells to infection, although this remedy has to be weighed against the toxicity induced in the healthy tissue.
Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies
Authors: Bohr Freja , Bohr Søren S.-R. , Mishra Narendra Kumar , Foutel Nicolás Sebastian González , Pinholt Henrik Dahl , Wu Shunliang , Nielsen Emilie Milan , Zhang Min , Kjaergaard Magnus , Jensen Knud J. , Hatzakis Nikos S.
Published: April 2022

Insulin formulations are the hallmark of interventions for treatment of diabetes. Understanding the mechanism that governs insulin self assembly or disassembly —and the role of stabilizing additives—are essential for improving insulin formulations. We report here the real-time direct observation of single insulin self-assembly and disassembly events using single molecule fluorescence microscopy. Our direct observations revealed previously unaccounted monomeric additions to occur to all types of assemblies and allowed us to quantify the existence, abundance and kinetic characterization of diverse assembly pathways involving monomeric dimers or tetrameric insulin species. We proposed and experimentally validated a model where the insulin self-assembly pathway is rerouted favoring monomeric or oligomeric assembly events by solution concentration, additives and formulations. Our rate simulation predicted the abundance of each oligomeric species across a concentration range of 6 orders of magnitude. Besides providing fundamental new insights, the results and toolbox here can be universally applied contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for other proteins.
Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress
Authors: Zhu Zhenglin , Gao Shengqiang , Chen Cheng , Xu Wei , Xiao Pengcheng , Chen Zhiyu , Du Chengcheng , Chen Bowen , Gao Yan , Wang Chunli , Liao Junyi , Huang Wei
Published: April 2022

Objectives

To investigate the effect and mechanisms of salicin (SA) on osteoarthritis (OA) progression.

Methods

Primary rat chondrocytes were stimulated with TNF-α and treated with or without SA. CCK-8 was utilized to determine the cytotoxicity of SA. RT-qPCR, Western Blotting and immunofluorescence staining were used to detect inflammatory factors, cartilage matrix degeneration markers, cell proliferation and apoptosis markers expression at mRNA and protein levels respectively. EdU assay and flow cytometer analysis were utilized for evaluating cell proliferation and apoptosis. RNA-sequencing, molecular docking, drug affinity responsive target stability and WB were applied to clarify mechanisms. Rat OA model was used to evaluate the effect of intra-articular injection of SA on OA progression.

Results

No obvious cytotoxicity was found with the treatment of 10 μM SA. SA rescued TNF-α induced degeneration of cartilage matrix, inhibition of chondrocytes proliferation, and promotion of chondrocytes apoptosis. In mechanism, we clarified SA could directly bind on IRE1α and occupy IRE1α phosphorylation site, followed with inhibiting IRE1α phosphorylation and regulating IRE1α mediated endoplasmic reticulum (ER) stress by IRE1α-IκBα-p65 signaling. Finally, intra-articular injection of SA loaded PLGA could ameliorate OA progression by inhibiting IRE1α mediated ER stress in OA model.

Conclusions

SA alleviates OA by directly binding on ER stress regulator IRE1α and inhibits IRE1α mediated ER stress by IRE1α-IκBα-p65 signaling. Topical use of small molecular drug SA holds the potential of modifying OA progression.
A Lysozyme Murein Hydrolase with Broad-spectrum Antibacterial Activity from Enterobacter Phage myPSH1140
Authors: Ramesh Nachimuthu , Manohar Prasanth , Eniyan Kandasamy , Archana Loganathan , Athira Sudarsanan , Loh Belinda , Ma Long , Leptihn Sebastian
Published: April 2022

Bacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multi-drug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp. and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.
Fixed-Target Serial Crystallography at Structural Biology Center
Authors: Sherrell Darren A. , Lavens Alex , Wilamowski Mateusz , Kim Youngchang , Chard Ryan , Lazarski Krzysztof , Rosenbaum Gerold , Vescovi Rafael , Johnson Jessica L. , Akins Chase , Chang Changsoo , Michalska Karolina , Babnigg Gyorgy , Foster Ian , Joachimiak Andrzej
Published: April 2022

Serial synchrotron crystallography enables studies of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed by using a high-performance computing pipeline. Here, the system was applied to two β-lactamases: a class D serine β-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-β -lactamase from Stenotrophomonas maltophilia K279a.
Transformation of the symbiotic alga Oophila amblystomatis : a new toolbox for animal-algae symbiosis studies
Authors: Genot Baptiste , Burns John A
Published: April 2022

The ability to conduct reverse genetic studies in symbiotic systems is enabled by transgene expression and transformation of at least one partner. The symbiotic relationship between the yellow spotted salamander, Ambystoma maculatum, and the green alga, Oophila amblystomatis, is a unique model of vertebrate-algae symbiosis. Despite over 130 years of scientific study, there are still many open questions in this symbiosis. Transgene expression in one partner will accelerate research into the symbiotic relationship. In this paper we describe a tool and method for expression of foreign DNA in, and presumed transformation of, the alga O. amblystomatis. We successfully introduced heritable antibiotic resistance to algal cultures, and observed expression of a green fluorescent reporter protein in all transfected and presumably transformed algal populations. The outcomes of this work enable genetic manipulation of the symbiotic alga Oophila amblystomatis, allowing direct testing of hypotheses derived from gene expression or genomic studies that will usher in a deeper understanding of the A. maculatum-O. amblystomatis symbiotic system.

Summary statement

Genetic tools stimulate new possibilities for research in living systems. This work describes a new tool for transformation of a symbiotic alga that enters vertebrate tissues and cells.
Rapid Rerouting of Myosin Traffic at the T Cell Immunological Synapse
Authors: Mąka Robert , Plewa Natalia , Cichoń Urszula , Krysztofiak Katarzyna , Rokicka Jagoda J. , Rock Ronald S.
Published: April 2022

Cytoskeletal motors travel in patterns set by the architecture of their tracks. Nevertheless, we have a limited understanding of how cells dynamically reorganize their traffic patterns in response to signaling events. To investigate cytoskeletal motor rerouting, we used T cells as a model system. Upon an encounter between a T cell and an antigen presenting cell, the T cell builds a specialized interface with spatially organized immunoreceptors and adhesion molecules called the immunological synapse (IS). The IS also constructs new actin networks within minutes that define the synaptic structure. Here we track the movements of single myosin motors along presynaptic and synaptic actin networks of the T cell. We find that both myosin-5 and myosin-6 reroute after IS construction. For example, most myosin-5 traffic moves inward at the IS, although most of the IS actin filaments have a barbed end out orientation. This anomalous myosin-5 traffic pattern indicates that the IS makes two types of actin networks: a structural network that controls IS shape, and a distinct trafficking network that supports myosin motility. We disrupt these trafficking networks with chemical probes against actin, which inhibits the appearance of cell surface markers of T cell activation. Our results highlight the importance of the sparse actin networks at the center of the IS in T cell function.
A salt bridge-mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a cell-based screen
Authors: Sharma Ajay Kumar , Poddar Sakshi Mahesh , Chakraborty Joyeeta , Nayak Bhagyashri Soumya , Kalathil Srilakshmi , Mitra Nivedita , Gayathri Pananghat , Srinivasan Ramanujam
Published: April 2022

Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affectsHelicobacter pyloriFtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-strands S9 and S10 mediate resistance to PC190723 in FtsZ. The single-stepin vivocell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.
Circadian clock control of MRTF-SRF pathway suppresses beige adipocyte thermogenic recruitment
Authors: Xiong Xuekai , Li Weini , Liu Ruya , Saha Pradip , Yechoor Vijay , Ma Ke
Published: April 2022

The morphological transformation of adipogenic progenitors into mature adipocytes requires dissolution of cytoskeleton driven by progressive loss of MRTF-SRF activity. Circadian clock confers temporal control in adipogenic differentiation, and MRTF-SRF inhibits beige adipocyte development. Here we identify that key components of the actin cytoskeleton-MRTF/SRF signaling cascade are direct transcriptional targets of circadian clock, and a clock-MRTF/SRF regulatory axis suppresses beige adipocyte thermogenic recruitment. Genetic loss- or gain-of-functions of the core clock regulator, Brain and Muscle Arnt-like 1 (Bmal1), altered actin cytoskeleton organization, cell shape and SRF activity. Genes involved in actin dynamic-MRTF-SRF pathway display diurnal expression profiles in beige adipose tissue, with identification of Bmal1 direct transcriptional regulation. Using beige adipocyte-selective genetic models together with pharmacological approaches, we demonstrate that Bmal1 inhibits beige adipogenesis and thermogenic capacity via the MRTF-SRF pathway. Selective ablation of Bmal1 induced beigeing, whereas its targeted overexpression attenuated the beige thermogenic program resulting in obesity. Collectively, our findings define a novel circadian clock control in actin cytoskeleton-MRTF-SRF signaling that suppresses beige adipogenesis to maintain metabolic homeostasis.