bluuebunny/crossref_metadata_2025 huggingface dataset:
Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins
Authors: Gerson Julia E. , Linton Hunter , Xing Jiazheng , Sutter Alexandra B. , Kakos Fayth S. , Ryou Jaimie , Liggans Nyjerus , Sharkey Lisa M. , Safren Nathaniel , Paulson Henry L. , Ivanova Magdalena I.
Published: August 2020

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown.

We report that the three ubiquilins differ in aggregation propensity, revealed byin-vitroexperiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrilsin vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by the brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements.

We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and levels of aggregation. This variability among ubiquilins along the continuum from liquid-like to solid likely informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in various neurodegenerative diseases.
An easy and reliable whole blood freezing method for flow cytometry immunophenotyping and functional analyses
Authors: Braudeau Cecile , Guen Nina Salabert-Le , Justine Chevreuil , Marie Rimbert , Martin Jerome C. , Josien Regis
Published: August 2020

Background

Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.

Methods

In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor®CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.

Results

Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).

Conclusions

In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.
Functional Evidence for Biased Inhibition of G protein Signaling by YM-254890 in Human Coronary Artery Endothelial Cells
Authors: Peng Qianman , Alqahtani Saud , Nasrullah Mohammed Zahid A , Shen Jianzhong
Published: August 2020

Small molecular chemicals targeting individual subtype of G proteins including Gs, Gi/o and Gq has been lacking, except forpertussis toxinbeing an established selective peptide inhibitor of the Gi/o protein. Recently, a cyclic depsipeptide compound YM-254890 isolated from culture broth ofChromobacterium sp. was reported as a selective inhibitor for the Gq protein by blocking GDP exchange of GTP on the α subunit of Gq complex. However, functional selectivity of YM-254890 towards various G proteins was not fully characterized, primarily due to its restricted availability before 2017. Here, using human coronary artery endothelial cells as a model, we performed a systemic pharmacological evaluation on the functional selectivity of YM-254890 on multiple G protein-mediated receptor signaling. First, we confirmed that YM-254890, at 30 nM, abolished UTP-activated P2Y2 receptor-mediated Ca2+signaling and ERK1/2 phosphorylation, indicating its potent inhibition on the Gq protein. However, we unexpectedly found that YM-254890 also significantly suppressed cAMP elevation and ERK1/2 phosphorylation induced by multiple Gs-coupled receptors including β2-adrenegic, adenosine A2and PGI2receptors. Surprisingly, although YM-254890 had no impact on CXCR4/Gi/o protein-mediated suppression of cAMP production, it abolished ERK1/2 activation. Further, no cellular toxicity was observed for YM-254890, and it neither affected A23187- or thapsigargin-induced Ca2+signaling, nor forskolin-induced cAMP elevation and growth factor-induced MAPK signaling. We conclude that YM-254890 is not a selective inhibitor for Gq protein; instead, it acts as a broad spectrum inhibitor for Gq and Gs proteins and exhibits a biased inhibition on Gi/o signaling, without affecting non-GPCR-mediated cellular signaling.
Efferent retargeting in above-knee amputees is positively related to phantom limb pain
Authors: Therrien Amanda S. , Howard Cortney , Buxbaum Laurel J.
Published: August 2020

Many individuals who undergo limb amputation experience persistent phantom limb pain (PLP). The underlying mechanism of PLP is unknown, but the phenomenon has been associated with reorganization in sensorimotor cortex following amputation. The traditional view is that cortical reorganization degrades the missing limb’s representation. However, recent work suggests that an amputated limb’s cortical representation remains intact and that reorganization reflects a retargeting of efferent projections to residual muscles proximal to the amputation site. Evidence of retargeting has only been shown in individuals with upper limb amputations, and the relationship of retargeting to PLP is controversial. This study assessed retargeting and its relationship to PLP in 10 individuals with lower limb amputations. We recorded electromyographic (EMG) activity in a residual thigh muscle (vastus lateralis, VL) in patients with above-knee amputations during cyclical movements of the foot. VL activity on the amputated side was compared to that recorded on patients’ intact side while they moved their phantom and intact feet, respectively. VL activity in the patient group was also compared to VL activity from a sample of 9 control participants with no amputation. We show that phantom foot movement is associated with greater VL activity in the amputated leg than that seen in the intact leg as well as that exhibited by controls. The magnitude of residual VL activity was also positively related to ratings of PLP. These results provide the first support for retargeting in lower limb amputees and suggest that retargeting is related to the experience of phantom pain.

New and Noteworthy

Previous work has only examined retargeting in upper limb amputees. This study provides evidence for retargeting in lower limb amputees and suggests that retargeting is related to phantom limb pain.
Matching a snail’s pace: Successful use of environmental DNA techniques to detect early stages of invasion by the destructive New Zealand mud snail
Authors: Woodell James D. , Neiman Maurine , Levri Edward P.
Published: August 2020

Early detection of invasive species allows for a more rapid and effective response. Restoration of the native ecosystem after an invasive population has established is expensive and difficult but more likely to succeed when invasions are detected early in the invasion process. Containment efforts to prevent the spread of known invasions also benefit from earlier knowledge of invaded sites. Environmental DNA (eDNA) techniques have emerged as a tool that can identify invasive species at a distinctly earlier time point than traditional methods of detection. Due to expected range expansion in eastern North America, we focus on the destructive New Zealand Mud SnailPotamopyrgus antipodarum(NZMS) invasion. We collected water samples from eight sites that prior evidence indicated were not yet invaded by the NZMS. After filtering these samples to collect eDNA, we used a species-specific probe with qPCR to identify NZMS eDNA. We found evidence for NZMS invasion at five of the eight sites, with later physical confirmation of mud snails at one of these sites. This study is the first example of successful detection of a previously unidentified invasive population of NZMS, setting the stage for further monitoring of at-risk sites to detect and control new invasions of this destructive snail. This study also shows potential opportunities for invasion monitoring offered by using low-cost efforts and methods that are adaptable for citizen science.
Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate
Authors: Addetia Amin , Crawford Katharine HD , Dingens Adam , Zhu Haiying , Roychoudhury Pavitra , Huang Meei-Li , Jerome Keith R. , Bloom Jesse D. , Greninger Alexander L.
Published: August 2020

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have only been performed in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral RT-PCR testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR positive viral test with Ct <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested the outbreak originated largely from a single viral clade. Only three crewmembers tested seropositive prior to the boat’s departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of these crewmembers with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against re-infection (Fisher’s exact test, p=0.002).
LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons
Authors: Sherrill-Mix Scott , Hwang Young , Roche Aoife M. , Glascock Abigail , Weiss Susan R. , Li Yize , Haddad Leila , Deraska Peter , Monahan Caitlin , Kromer Andrew , Graham-Wooten Jevon , Taylor Louis J. , Abella Benjamin S. , Ganguly Arupa , Collman Ronald G. , Van Duyne Gregory D. , Bushman Frederic D.
Published: August 2020

Background

Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse Transcription and Loop-Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.

Results

Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.

Conclusions

LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
Broiler Chickens and Early Life Programming: Microbiome transplant-induced cecal bacteriome dynamics and phenotypic effects
Authors: Ramírez Gustavo A. , Richardson Ella , Clark Jory , Keshri Jitendra , Drechsler Yvonne , Berrang Mark E. , Meinersmann Richard J. , Cox Nelson A. , Oakley Brian B.
Published: August 2020

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first derive a stable source of inoculum. Subsequently, we compared microbiome development between chicks receiving this passaged cecal material, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa were identified across treatment types that may inform future studies aimed at identifying strains associated with beneficial phenotypes.
Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion
Authors: Papa Guido , Mallery Donna L. , Albecka Anna , Welch Lawrence , Cattin-Ortolá Jérôme , Luptak Jakub , Paul David , McMahon Harvey T. , Goodfellow Ian G. , Carter Andrew , Munro Sean , James Leo C.
Published: August 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.
CRISPR-Decryptr reveals cis-regulatory elements from noncoding perturbation screens
Authors: Rasmussen Anders , Äijö Tarmo , Gabitto Mariano Ignacio , Carriero Nicholas , Sanjana Neville , Skok Jane , Bonneau Richard
Published: August 2020

Clustered Regularly Interspace Short Palindromic Repeats (CRISPR)-Cas9 genome editing methods provide the tools necessary to examine phenotypic impacts of targeted perturbations in high-throughput screens. While these technologies have the potential to reveal functional elements with direct therapeutic applications, statistical techniques to analyze noncoding screen data remain limited. We present CRISPR-Decryptr, a computational tool for the analysis of CRISPR noncoding screens. Our method leverages experimental design: accounting for multiple conditions, controls, and replicates to infer the regulatory landscape of noncoding genomic regions. We validate our method on a variety of mutagenesis, CRISPR activation, and CRISPR interference screens, extracting new insights from previously published data.
Characterisation of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification
Authors: Baddock Hannah T. , Brolih Sanja , Yosaatmadja Yuliana , Ratnaweera Malitha , Bielinski Marcin , Swift Lonnie P. , Cruz-Migoni Abimael , Morris Garrett M. , Schofield Christopher J. , Gileadi Opher , McHugh Peter J.
Published: August 2020

The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.
The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer
Authors: Carrique Loic , Duyvesteyn Helen ME , Malinauskas Tomas , Zhao Yuguang , Ren Jingshan , Zhou Daming , Walter Thomas S , Radecke Julika , Huo Jiandong , Ruza Reinis R , Shah Pranav NM , Fry Elizabeth E , Stuart David I
Published: August 2020

Large trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is ‘locked’ into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the ‘locked’ form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the ‘locked’ form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies.

Highlights

SARS-CoV-2 Spike can adopt a ‘locked’ conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting state

This ‘locked’ conformation is compact and stable, braced by lipid bound within a potentially druggable pocket

Key neutralization epitopes are shielded in the ‘locked’ form

Loss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry
New Genotypes and Diversity ofOrientia tsutsugamushiIsolated from Scrub Typhus Patients in South Korea as Determined by Multilocus Sequence Typing
Authors: Hwang Joo-Hee , Kim Jeongsik , Sun In O , Lee Tae Hee , Chung Kyung Min , Lee Chang-Seop
Published: August 2020

Orientia tsutsugamushi, an obligate intracellular organism, is the causative agent of scrub typhus, which is endemic in the Asia-Pacific region. No comparative studies on the genotypic properties ofO. tsutsugamushihave been performed using multilocus sequence typing (MLST) in South Korea. Here, we characterized 51 clinical isolates from Jeonju, in southwestern Korea, and we compared them to isolates from Thailand, Laos, and Japan. We also identified 10 new alleles and six novel sequence types. Overall, our results suggest that the relative genetic stability and the clonal populations ofO. tsutsugamushistrains in South Korea have remained mostly conserved.

Author summary

Scrub typhus is a life-threatening disease, caused by infection withO. tsutsugamushi, a Gram-negative intracellular bacterium. Approximately one million people are infected globally every year, especially in the Asia-Pacific region. Strains ofO. tsutsugamushiare typically distinguished serologically on the basis of sequences of the highly polymorphic 56-kDa outer membrane protein. Multilocus sequence typing (MLST) is a generic typing method that provides a unified bacterial isolate characterization approach that can be used for evolutionary and population studies of bacteria. In this study, we describe the development and application of a MLST scheme that was applied to 51O. tsutsugamushiisolates. We found 10 new alleles and six new STs, which yielded a total of sevenO. tsutsugamushiSTs in South Korea. Among seven different STs (ST 48, 93-98), ST 48 account for the largest proportion (49.0%) ofO. tsutsugamushiSTs in South Korea. With the exception of the appearance of six novel STs, the clonal populations have remained conserved but further study of population structure and evolutionary trends is warranted.
Ilomastat contributes to the survival of mouse after irradiation via promoting the recovery of hematopoietic system
Authors: Zhao Baoquan , Li Xiaoman , Li Xingzhou , Quan Dongqin , Zhang Fang , Hu Burong
Published: August 2020

Ilomastat, a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), has drawn attentions for its function in alleviating radiation damage. However, the detailed mechanisms of Ilomastat’s protection from animal model remain not fully clear. In this study, the C57BL/6 mice were pre-administrated with Ilomastat or vihicle for 2 h, and then total body of mice were exposed to 6 Gy of γ-rays. The protective effect of Ilomastat on the hematopoietic system in the irradiated mice were investigated. We found that pretreatment with Ilomastat significantly reduced the level of TGF-β1 and TNF-α, and elevated the number of bone marrow (BM) mononuclear cells in the irradiated mice. Ilomastat pretreatment also increased the fraction of BM hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) at day 30 after irradiation, and protected the spleen of mouse from irradiation. These results suggest that Ilomastat promotes the recovery of hematopoietic injury in the irradiated mice, and thus contributes to the survival of mouse after irradiation.
The nitrogen-fixing symbiotic cyanobacterium,Nostoc punctiformecan regulate plant programmed cell death
Authors: Belton Samuel P. , McCabe Paul F. , Ng Carl K. Y.
Published: August 2020

Cyanobacteria such asNostocspp. can form nitrogen-fixing symbioses with a broad range of plant species. Unlike other plant-bacteria symbioses, little is understood about the immunological and developmental signalling events induced byNostoccyanobionts (symbiotic cyanobacteria). Here, we used suspension cell cultures to elucidate the early molecular mechanisms underpinning the association between cyanobionts and plants by studying the effects of conditioned medium (CM) fromNostoc punctiformecultures on plant programmed cell death (PCD), a typical immune response activated during incompatible interactions. We showed thatN. punctiforme-CM could suppress PCD induced by a temperature stress. Interestingly, this was preceded by significant transcriptional reprogramming, as evidenced by the differential regulation of a network of defence-associated genes, as well as genes implicated in regulating cell growth and differentiation. This work is the first to show that cyanobionts can regulate PCD in plants and provides a valuable transcriptome resource for the early immunological and developmental signalling events elicited byNostoccyanobionts.
Evolution of Chi motifs in Proteobacteria
Authors: Buton Angélique , Bobay Louis-Marie
Published: August 2020

Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized inEscherichia coliand analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species:E. coli, Haemophilus influenzae,Bacillus subtilis,Lactococcus lactisandStaphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif ofE. coliin 85 species of Proteobacteria. Most Chi motifs were detected inEnterobacteriaceaeand this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.
Mammalian deubiquitinating enzyme inhibitors displayin vitroandin vivoactivity against malaria parasites and potentiate artemisinin action
Authors: Simwela Nelson V. , Hughes Katie R. , Rennie Michael T. , Barrett Michael P. , Waters Andrew P.
Published: August 2020

Current malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasitesin vitroand possess antiparasitic activityin vivoand can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisininin vitroas well asin vivoto the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed intoin vivoantimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.

Graphical abstract
A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes
Authors: Pinch Benika J. , Buckley Dennis L. , Gleim Scott , Brittain Scott M. , Tandeske Laura , D’Alessandro Pier Luca , Harvey Edward P. , Hauseman Zachary J. , Schirle Markus , Sprague Elizabeth R. , Forrester William C. , Dovala Dustin , McGregor Lynn M. , Thoma Claudio R.
Published: August 2020

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates.
Ultra-Low Colcemid Doses Induce Microtubule Dysfunction as Revealed by Super-Resolution Microscopy
Authors: Rozario Ashley M , Duwé Sam , Elliott Cade , Hargreaves Riley B , Dedecker Peter , Whelan Donna R , Bell Toby D M
Published: August 2020

Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt micro-tubules. However, their side effects require the development of safer drug regimens that still retain clinical efficacy. Currently, many questions remain regarding microtubule-interacting drugs at clinically relevant and ultra-low doses. Here, we use super-resolution microscopies (single molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Short exposure to 30 - 80 nM colcemid results in aberrant microtubule curvature while microtubule fragmentation is detected upon treatment with ≥100 nM colcemid. Remarkably, even ultra-low doses (5 hours at <20 nM) led to subtle but significant microtubule architecture remodeling and suppression of microtubule dynamics. These challenges to microtubule function represent less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs, and therefore offer potential for improved understanding and design of anti-cancer agents.
DREAM represses distinct targets by cooperating with different THAP domain proteins
Authors: Gal Csenge , Carelli Francesco Nicola , Appert Alex , Cerrato Chiara , Huang Ni , Dong Yan , Murphy Jane , Ahringer Julie
Published: August 2020

The DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex controls cellular quiescence by repressing cell cycle and other genes, but its mechanism of action is unclear. Here we demonstrate that two C. elegans THAP domain proteins, LIN-15B and LIN-36, co-localize with DREAM and function by different mechanisms for repression of distinct sets of targets. LIN-36 represses classical cell cycle targets by promoting DREAM binding and gene body enrichment of H2A.Z, and we find that DREAM subunit EFL-1/E2F is specific for LIN-36 targets. In contrast, LIN-15B represses germline specific targets in the soma by facilitating H3K9me2 promoter marking. We further find that LIN-36 and LIN-15B differently regulate DREAM binding. In humans, THAP proteins have been implicated in cell cycle regulation by poorly understood mechanisms. We propose that THAP domain proteins are key mediators of Rb/DREAM function.
Kinetic analysis reveals the rates and mechanisms of protein aggregation in a multicellular organism
Authors: Sinnige Tessa , Meisl Georg , Michaels Thomas C. T. , Vendruscolo Michele , Knowles Tuomas P.J. , Morimoto Richard I.
Published: August 2020

The accumulation of insoluble protein aggregates containing amyloid fibrils has been observed in many different human protein misfolding diseases1,2, and their pathological features have been recapitulated in diverse model systems3.In vitrokinetic studies have provided a quantitative understanding of how the fundamental molecular level processes of nucleation and growth lead to amyloid formation4. However, it is not yet clear to what extent these basic biophysical processes translate to amyloid formationin vivo, given the complexity of the cellular and organismal environment. Here we show that the aggregation of a fluorescently tagged polyglutamine (polyQ) protein into µm-sized inclusions in the muscle tissue of livingC. eleganscan be quantitatively described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h-1per cell at 1 mM intracellular protein concentration, and shows that the intrinsic stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results are consistent with observations for the aggregation of polyQ proteinsin vitro5and in cell culture6, and highlight how nucleation events control the overall progression of aggregation in the organism through the spatial confinement into individual cells. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation ofin vivodata from a wide range of protein aggregation diseases.
PTEN Inhibition Ameliorates Muscle Degeneration and Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy
Authors: Yue Feng , Song Changyou , Huang Di , Narayanan Naagarajan , Qiu Jiamin , Jia Zhihao , Yuan Zhengrong , Oprescu Stephanie N , Roseguini Bruno T , Deng Meng , Kuang Shihuan
Published: August 2020

Duchenne Muscular Dystrophy (DMD) is caused by mutation of the muscle membrane protein dystrophin and characterized by severe degeneration of myofibers, progressive muscle wasting and loss of mobility, ultimately cardiorespiratory failure and premature death. Here we report that skeletal muscle-specific knockout (KO) ofPhosphatase and tensin homolog(Pten) gene in an animal model of DMD (mdxmice) alleviates myofiber degeneration and restores muscle function without increasing tumor incidences. Specifically,PtenKO normalizes myofiber size and prevents muscular atrophy, and improves grip strength and exercise performance ofmdxmice.PtenKO also reduces fibrosis and inflammation; and ameliorates muscle pathology inmdxmice. Moreover, we found thatPtenKO upregulates extracellular matrix and basement membrane components positively correlated to wound healing, but suppresses negative regulators of wound healing and lipid biosynthesis; and restores the integrity of muscle basement membrane inmdxmice. Importantly, pharmacological inhibition of PTEN similarly ameliorates muscle pathology and improves muscle integrity and function inmdxmice. Our finding provides evidence that PTEN inhibition may represent a potential therapeutic strategy to restore muscle function in DMD.
Blocking palmitoylation ofToxoplasma gondiimyosin light chain 1 disrupts glideosome composition but has little impact on parasite motility
Authors: Rompikuntal Pramod K. , Foe Ian T. , Deng Bin , Bogyo Matthew , Ward Gary E.
Published: August 2020

Toxoplasma gondiiis a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans,T. gondiiuses an unusual form of gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin-based motor consisting of a Class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. The binding of TgMLC1 to TgGAP45 is therefore considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation disrupt the association of TgMLC1 with the membrane fraction of the parasite in phase partitioning experiments and completely block the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.

Importance

Gliding motility plays a central role in the life cycle ofT. gondiiand other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention and treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely-held “linear motor” model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of and between cells of the hosts they infect.
Bacterial gene essentiality under modeled microgravity
Authors: Burgos Emanuel , Vroom Madeline M. , Rotman Ella , Murphy-Belcaster Megan , Foster Jamie S. , Mandel Mark J.
Published: August 2020

The health of eukaryotic hosts is tightly connected to relationships with symbiotic microorganisms, yet how these relationships develop and evolve during long-duration spaceflight is not well understood. In this study, we asked what bacterial genes are required for growth under modeled, or simulated, microgravity conditions compared to normal gravity controls. To conduct this study, we focused on the marine bacteriumVibrio fischeri, which forms a monospecific symbiosis with the Hawaiian bobtail squid,Euprymna scolopes. The symbiosis has been studied during spaceflight and in ground-based modeled microgravity conditions. We employed a library of over 40,000V. fischeritransposon mutants and compared the fitness of mutants in modeled microgravity compared to the gravity controls using transposon insertion sequencing (INSeq). We identified dozens of genes that exhibited fitness defects under both conditions, likely due to the controlled anaerobic environment, yet we identified relatively few genes with differential effects under modeled microgravity or gravity specifically: only mutants inrodAwere more depleted under modeled microgravity, and mutants in 12 genes exhibited greater depletion under gravity conditions. We additionally compared RNA-seq and INSeq data and determined that expression under microgravity was not predictive of the essentiality of a given gene. In summary, empirical determination of conditional gene essentiality identifies few microgravity-specific genes for environmental growth ofV. fischeri, suggesting that the condition of microgravity has a minimal impact on symbiont gene requirement.

IMPORTANCE

There is substantial evidence that both the host immune system and microbial physiology are altered during space travel. It is difficult to discern the molecular mechanisms of these processes in a complex microbial consortium and during the short durations of experiments in space. By using a model organism that is amenable to high-throughput genetic approaches, we have determined thatV. fischeridoes not require a separate genetic repertoire for media growth in modeled microgravity versus gravity conditions. Our results argue that future studies on how this organism forms a specific and stable association with its animal host will not be confounded by growth effects in the environment. The identification of similar genetic requirements under modeled microgravity and gravity suggest that fitness pressures on microbiome growth in space may be similar to those on Earth and may not negatively impact their animal hosts during long-duration spaceflight.
Population analysis ofLegionella pneumophilareveals the basis of resistance to complement-mediated killing
Authors: Wee Bryan A. , Alves Joana , Lindsay Diane S. J. , Cameron Ross L. , Pickering Amy , Gorzynski Jamie , Corander Jukka , Marttinen Pekka , Smith Andrew J. , Fitzgerald J. Ross
Published: August 2020

Legionella pneumophilais the most common cause of the severe respiratory infection known as Legionnaires’ disease.L. pneumophilais typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 900L. pneumophilaisolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We found that although some clones are more commonly associated with clinical infections, the capacity for human disease is representative of the breadth of species diversity. To investigate the bacterial genetic basis for human disease potential, we carried out a genome-wide association study that identified a single gene (lag-1), to be most strongly associated with clinical isolates. Molecular evolutionary analysis showed thatlag-1, which encodes anO-acetyltransferase responsible for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades ofL. pneumophilaby frequent recent recombination events. Functional analysis revealed a correlation between the presence of a functionallag-1gene and resistance to killing in human serum and bovine broncho-alveolar lavage. In addition,L. pneumophilastrains that expresslag-1escaped complement-mediated phagocytosis by neutrophils. Importantly, we discovered that the expression oflag-1confers the capacity to evade complement-mediated killing by inhibiting deposition of classical pathway molecules on the bacterial surface. In summary, our combined population and functional analyses identifiedL. pneumophilagenetic traits linked to human disease and revealed the molecular basis for resistance to complement-mediated killing, a previously elusive trait of direct relevance to human disease pathogenicity.

Significance

Legionella pneumophilais an environmental bacterium associated with a severe pneumonia known as Legionnaires’ disease. A small number ofL. pneumophilaclones are responsible for a large proportion of human infections suggesting they have enhanced pathogenicity. Here, we employed a large-scale population analysis to investigate the evolution of human pathogenicity and identified a single gene (lag-1) that was more frequently found in clinical isolates. Functional analysis revealed that thelag-1-encodedO-acetyltransferase, involved in modification of lipopolysaccharide, conferred resistance to the classical pathway of complement in human serum. These findings solve a long-standing mystery in the field regardingL. pneumophilaresistance to serum killing, revealing a novel mechanism by whichL. pneumophilamay avoid immune defences during infection.