bluuebunny/crossref_metadata_2025 huggingface dataset:
Testing for pollinator recognition in multiple species ofHeliconia
Authors: Gannon Dustin G. , Hadley Adam S. , Kormann Urs G. , Andrew Jones F. , Betts Matthew G.
Published: April 2021

Many plants have evolved floral traits that, in effect, filter pollinator communities and promote pollination by efficient pollinators. Most documented pollinator filter traits act to deter or reduce visitation rates by a subsect of the community of floral visitors. However, a recently described pollinator filter termed ‘pollinator recognition’ (PR) acts at a stage after a pollinator visit. PR was first documented experimentally inHeliconia tortuosawhereby pollen tube germination – a proxy for reproduction – was enhanced following visits from morphologically specialized pollinators, but not generalists. This is thought to promote outcrossing among plants by preferentially investing in reproduction following visits by long-ranging hummingbirds with specialized bill shapes. To date, this plant behavior has only been described inH. tortuosa, but, if widespread, could have important ecological implications; given declines in abundances of specialist pollinators, visits by generalists would not buffer the loss of pollination services to plants with PR. We therefore tested for PR in four taxa spread widely across the Heliconiaceae.

We corroborated previous results that visits by long-billed, but not short-billed hummingbirds increased pollen tubes inH. tortuosawith aviary experiments that standardized pollen quality and minimized variation in pollen quantity. Across species, we found great variation in pollen tube responses to experimental treatments. For one species (H. rostrata), we found increased numbers of pollen tubes in those visited by hummingbirds compared to hand pollination alone, regardless of the visiting bird’s bill morphology, indicating recognition of hummingbirds in general. In other cases, hummingbird visits decreased pollen tube counts compared to hand pollinations alone. Furthermore, our results could not substantiate any specific mechanism for pollinator recognition and highlight the need for further work on the complexities of and variability in reproductive strategies across plant taxa.
Proteomic insight into soybean response to flooding stress reveals changes in basic energy metabolism and cell wall modifications
Authors: Khan Mudassar Nawaz , Ahmad Iftikhar , Israr-ud-Din  , Ali Essa , Noureldeen Ahmed , Darwish Hadeer , Khan Muhammad Ishfaq , Tayyab Muhammad , Khan Majid
Published: April 2021

Soybean is a legume crop enriched with proteins and oil. It is frequently exposed to anthropogenic and natural flooding that limits its growth and yield. Current study applied gel-free proteomic techniques to unravel soybean response mechanism to flooding stress. Two-days-old soybeans were flooded for 4 days continuously and root samples were collected at days 2 to 6 for proteomic and enzymatic analyses. Age-matched untreated soybeans were collected as control. After protein extraction, purification and tryptic digestion, the peptides were analyzed on nano-liquid chromatography-mass spectrometry. A total of 539 and 472 proteins with matched peptides 2 or more were identified in control and flooded seedlings, respectively. Among these 364 proteins were commonly identified in both control and flooded soybeans. Fourty-two protein’s abundances were changed 4-fold after 2-days of flooding stress as compared to starting point. The cluster analysis showed that highly increased proteins included cupin family proteins, enolase, pectin methylesterase inhibitor, glyoxalase II, alcohol dehydrogenase and aldolase. The enzyme assay of enolase and pectin methylesterase inhibitor confirmed protein abundance changes. These findings suggest that soybean adopts the less energy consuming strategies and brings biochemical and structural changes in the cell wall to effectively respond to flooding stress and for the survival.
Microvasculature-on-a-Chip: Bridging the interstitial blood-lymph interface via mechanobiological stimuli
Authors: Bachmann Barbara , Spitz Sarah , Jordan Christian , Schuller Patrick , Wanzenböck Heinz D. , Haddadi Bahram , Harasek Michael , Redl Heinz , Holnthoner Wolfgang , Ertl Peter
Published: April 2021

After decades of simply being referred to as the body’s sewage system, the lymphatic system has recently been recognized as a key player in numerous physiological and pathological processes. As an essential site of immune cell interactions, the lymphatic system is a potential target for next-generation drug delivery approaches in treatments for cancer, infections, and inflammatory diseases. However, the lack of cell-based assays capable of recapitulating the required biological complexity combined with unreliablein vivoanimal models currently hamper scientific progress in lymph-targeted drug delivery. To gain more in-depth insight into the blood-lymph interface, we established an advanced chip-based microvascular model to study mechanical stimulation’s importance on lymphatic sprout formation. Our microvascular model’s key feature is the co-cultivation of spatially separated 3D blood and lymphatic vessels under controlled, unidirectional interstitial fluid flow while allowing signaling molecule exchange similar to thein vivosituation. We demonstrate that our microphysiological model recreates biomimetic interstitial fluid flow, mimicking the route of fluidin vivo, where shear stress within blood vessels pushes fluid into the interstitial space, which is subsequently transported to the nearby lymphatic capillaries. Results of our cell culture optimization study clearly show an increased vessel sprouting number, length, and morphological characteristics under dynamic cultivation conditions and physiological relevant mechanobiological stimulation. For the first time, a microvascular on-chip system incorporating microcapillaries of both blood and lymphatic originin vitrorecapitulates the interstitial blood-lymph interface.
Differential roles for DNAJ isoforms in HTT-polyQ and mutant FUS aggregation modulation revealed by chaperone network screens
Authors: Rozales Kinneret , Younis Amal , Kellerman Lior , Heinrich Ronit , Berlin Shai , Shalgi Reut
Published: April 2021

Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families3. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery4,5. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntington’s disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation6,7, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completely different than those of HTT-polyQ. Interestingly, different naturally-occurring isoforms of DNAJ chaperones had opposing effects on HTT-polyQ vs. mutFUS aggregation. We identified a complex of the full length (FL) DNAJB14 and DNAJB12 isoforms which substantially alleviated mutFUS aggregation, in an HSP70-dependent manner. Their naturally occurring short isoforms were unable to form the complex, nor to interact with HSP70, and lost their ability to reduce mutFUS aggregation. In contrast, the short isoform of DNAJB12 significantly alleviated HTT-polyQ aggregation, while DNAJB12-FL aggravated HTT-polyQ aggregation. Finally, we demonstrated that full-length DNAJB14 ameliorated mutFUS aggregation compared to DNAJB14-short in primary neurons. Together, our data unraveled distinct molecular properties required for aggregation protection in different neurodegenerative diseases, and revealed a new layer of complexity of the chaperone network elicited by naturally occurring J-protein isoforms, highlighting functional diversity among the DNAJ family.
Transfer RNA-derived non-coding RNAs (tncRNAs): Uncovering hidden regulators of transcriptional regulatory circuits in plants
Authors: Zahra Shafaque , Singh Ajeet , Poddar Nikita , Kumar Shailesh
Published: April 2021

The lateral emergence of distinct classes of non-coding RNAs has led to better insights into the eukaryotic regulatory networks. Among them, the existence of transfer RNA (tRNA)-derived non-coding RNAs (tncRNAs) demands their exploration in the plant kingdom. Here, we have designed a methodology to uncover the bigger picture of tncRNAome in plants. Using this, we have identified diverse tncRNAs of length 14-50 nt in ~2500 small RNA sequencing (sRNA-seq) samples of six major angiosperms, and further studied their various features including length, codon-usage, cleavage pattern, and modified tRNA nucleosides. Codon-dependent generation of tncRNAs indicates that the process is highly specific rather than being mere random tRNA degradation. Analysis for nucleotide composition of tncRNA cleavage positions indicates that they are generated through precise endoribonucleolytic machinery. Certain tRNA nucleoside modifications on tncRNAs were found to be conserved across the plants, and hence may influence tRNA cleavage, as well as tncRNA functions. Pathway enrichment analysis revealed that common tncRNA targets were majorly involved in the metabolic and developmental processes of plants. Also, many tRFs were found to be associated with transposable elements. Distinct tissue-specific tncRNA clusters indicate their role in plant development under normal physiological conditions. Furthermore, the identification of a significant number of differentially expressed tncRNAs under several abiotic and biotic stresses highlights their probable role as gene expression modulators during various stress conditions. Thus, this study will be beneficial to investigate the emerging role of tncRNAs as prospective biomarkers in plant development and stress.

Highlights

Computational pipeline for accurate identification of genuine transfer RNA-derived non-coding RNAs (tncRNAs) using small RNA sequencing (sRNA-seq) datasets.

Six major tncRNA classes of length ranging from 14 to 50 nt were identified in ~2,500 sRNA-seq datasets in six different angiosperms.

tRNA nucleoside modifications may affect tncRNA cleavage in plants.

Conserved tncRNAs target transcripts are involved in plant growth, development, and metabolism.

tncRNAs are expressed in a tissue-dependent, and stress-specific manner in plants.
Motor unit recruitment patterns of the quadriceps differ between continuous high- and low-torque isometric knee extension to momentary failure
Authors: Murphy Jonathan , Hodson-Tole Emma , Vigotsky Andrew D. , Potvin Jim R. , Fisher James P. , Steele James
Published: April 2021

The size principle is a theory of motor unit (MU) recruitment that suggests MUs are recruited in an orderly manner from the smallest (lower threshold) to the largest (higher threshold) MUs. A consequence of this biophysical theory is that, for isometric contractions, recruitment is dependent on the intensity of actual effort required to meet task demands. This concept has been supported by modelling work demonstrating that, in tasks performed to momentary failure, full MU recruitment will have occurred upon reaching failure irrespective of the force requirements of the task. However,in vivostudies examining this are limited. Therefore, the aim of the current study was to examine MU recruitment of the quadriceps under both higher- and lower-torque (70% and 30% of MVC, respectively) isometric knee extension, performed to momentary failure. Specifically, we compared surface electromyography (sEMG) frequency characteristics, determined by wavelet analysis, across the two continuous isometric knee extension tasks to identify potential differences in recruitment patterns. A convenience sample of 10 recreationally active adult males (height: 179.6±6.0 cm; mass: 76.8±7.3 kg; age: 26±7 years) with previous resistance training experience (6±3 years) were recruited. Using a within-session, repeated-measures, randomised crossover design participants performed the knee extension tasks whilst sEMG was collected from the vastus medialis (VM), rectus femoris (RF) and vastus lateralis (VL). Myoelectric signals were decomposed into intensities as a function of time and frequency using an EMG-specific wavelet transformation. Our first analysis compared the mean frequency at momentary failure; second, we investigated the effects of load on relative changes in wavelet intensities; finally, we quantified the degree of wavelet similarity over time. Wavelet-based calculation of the mean signal frequency appeared to show similar mean frequency characteristics occurring when reaching momentary failure. However, individual wavelets revealed that different changes in frequency components occurred between the two tasks, suggesting that patterns of recruitment differed. Low-torque conditions resulted in an increase in intensity of all frequency components across the trials for each muscle whereas high-torque conditions resulted in a wider range of frequency components contained within the myoelectric signals at the beginning of the trials. However, as the low-torque trial neared momentary failure there was an increased agreement between conditions across wavelets. Our results corroborate modelling studies as well as recent biopsy evidence, suggesting overall MU recruitment may largely be similar for isometric tasks performed to momentary failure with the highest threshold MUs likely recruited, despite being achieved with differences in the pattern of recruitment over time utilised.
Universally valid reduction of multiscale stochastic biochemical systems using simple non-elementary propensities
Authors: Song Yun Min , Hong Hyukpyo , Kim Jae Kyoung
Published: April 2021

Biochemical systems consist of numerous elementary reactions governed by the law of mass action. However, experimentally characterizing all the elementary reactions is nearly impossible. Thus, over a century, their deterministic models that typically contain rapid reversible bindings have been simplified with non-elementary reaction functions (e.g., Michaelis-Menten and Morrison equations). Although the non-elementary reaction functions are derived by applying the quasi-steady-state approximation (QSSA) to deterministic systems, they have also been widely used to derive propensities for stochastic simulations due to computational efficiency and simplicity. However, the validity condition for this heuristic approach has not been identified even for the reversible binding between molecules, such as protein-DNA, enzyme-substrate, and receptor-ligand, which is the basis for living cells. Here, we find that the non-elementary propensities based on the deterministic total QSSA can accurately capture the stochastic dynamics of the reversible binding in general. However, serious errors occur when reactant molecules with similar levels tightly bind, unlike deterministic systems. In that case, the non-elementary propensities distort the stochastic dynamics of a bistable switch in the cell cycle and an oscillator in the circadian clock. Accordingly, we derive alternative non-elementary propensities with the stochastic low-state QSSA, developed in this study. This provides a universally valid framework for simplifying multiscale stochastic biochemical systems with rapid reversible bindings, critical for efficient stochastic simulations of cell signaling and gene regulation. To facilitate the framework, we provide a user-friendly open-source computational package, ASSISTER, that automatically performs the present framework.
DIA-based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia
Authors: Liu Wei , Sun Yaoting , Ge Weigang , Zhang Fangfei , Gan Lin , Zhu Yi , Guo Tiannan , Liu Kexin
Published: April 2021

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia (CML). To understand the underlying resistance mechanisms in response to imatinib (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for two months to generate derivative cells with mild, intermediate and severe resistance to the drugs as defined by their increasing resistance index (RI). PulseDIA-based quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7,082 proteotypic proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin Signaling Pathway was found to be significantly enriched in both ADR- and IMA-resistant K562 cells. In particular, IDH2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated IDH2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.
Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients
Authors: Cavallaro Sara , Hååg Petra , Sahu Siddharth S. , Berisha Lorenca , Kaminsky Vitaliy O. , Ekman Simon , Lewensohn Rolf , Linnros Jan , Viktorsson Kristina , Dev Apurba
Published: April 2021

Liquid biopsies based on extracellular vesicle (EV) protein profiles represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we present the development of an electrokinetic sensor for multiplexed surface protein profiling of EVs and analysis of clinical samples. The method detects the difference in the streaming current obtained as a result of EV binding to the inner surface of a functionalized microcapillary, thereby estimating the expression level of a surface marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of three NSCLC adenocarcinoma patients with different genomic alterations (ALK-fusion, KRAS and EGFR) and applied treatments (chemotherapy, EGFR or ALK tyrosine kinase inhibitors). These vesicles were targeted against CD9 tetraspanin, as well as EGFR and PD-L1, two markers of interest in NSCLC. The electrokinetic signals showed detection of these markers on sEVs yet highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-mutant one. The sensors also detected differences in PD-L1 expressions, in line with those measured by complementary methods. The analysis of sEVs from a patient prior and post crizotinib treatment also revealed a significant increase in the expression of some markers, e.g. EGFR and PD-L1. The obtained results hold promise for the application of the method for tumor treatment monitoring based on sEVs from liquid biopsies.
Redundancy-selection trade-off in phenotype-structured populations
Authors: Miele Leonardo , Evans R M L , Azaele Sandro
Published: April 2021

Realistic fitness landscapes generally display a redundancy-fitness trade-off: highly fit trait configurations are inevitably rare, while less fit trait configurations are expected to be more redundant. The resulting sub-optimal patterns in the fitness distribution are typically described by means of effective formulations. However, the extent to which effective formulations are compatible with explicitly redundant landscapes is yet to be understood, as well as the consequences of a potential miss-match. Here we investigate the effects of such trade-off on the evolution of phenotype-structured populations, characterised by continuous quantitative traits. We consider a typical replication-mutation dynamics, and we model redundancy by means of two dimensional landscapes displaying both selective and neutral traits. We show that asymmetries of the landscapes will generate neutral contributions to the marginalised fitness-level description, that cannot be described by effective formulations, nor disentangled by the full trait distribution. Rather, they appear as effective sources, whose magnitude depends on the geometry of the landscape. Our results highlight new important aspects on the nature of sub-optimality. We discuss practical implications for rapidly mutant populations such as pathogens and cancer cells, where the qualitative knowledge of their trait and fitness distributions can drive disease management and intervention policies.
Intranasal HD-Ad Vaccine Protects the Upper and Lower Respiratory Tracts of hACE2 Mice against SARS-CoV-2
Authors: Cao Huibi , Mai Juntao , Zhou Zhichang , Li Zhijie , Duan Rongqi , Watt Jacqueline , Chen Ziyan , Bandara Ranmal Avinash , Li Ming , Ahn Sang Kyun , Boon Betty , Christie Natasha , Gray-Owen Scott , Kozak Rob , Mubareka Samira , Rini James M. , Hu Jim , Liu Jun
Published: April 2021

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.
Rapid adaptation of a complex trait during experimental evolution ofMycobacterium tuberculosis
Authors: Smith Tracy M. , Youngblom Madison A. , Kernien John F. , Mohamed Mohamed A. , Fry Sydney S. , Bohr Lindsey L. , Mortimer Tatum D. , O’Neill Mary B. , Pepperell Caitlin S.
Published: April 2021

Tuberculosis (TB), caused byMycobacterium tuberculosis(M. tb), is a leading cause of death due to infectious disease. TB is not traditionally associated with biofilms, butM. tbbiofilms are linked with drug and immune tolerance and there is increasing recognition of their contribution to the recalcitrance of TB infections. Here we usedM. tbexperimental evolution to investigate this complex phenotype and identify candidate loci controlling biofilm formation. We identified novel candidate loci, adding to our understanding of the genetic architecture underlyingM. tbbiofilm development. Under selective pressure to grow as a biofilm, regulatory mutations rapidly swept to fixation and were associated with changes in multiple traits including extracellular matrix production, cell size, and growth rate. Genetic and phenotypic paths to enhanced biofilm growth varied according to the genetic background of the parent strain, suggesting that epistatic interactions are important inM. tbadaptation to changing environments.
The Circadian Phase Response Curve to Light: Conservation across Seasons and Anchorage to Sunset
Authors: Dollish Hannah K. , Kaladchibachi Sevag , Negelspach David C. , Fernandez Fabian-Xosé
Published: April 2021

Phototherapy outcomes are dependent on the precision by which the magnitude and direction of light’s circadian phase-shifting effects can be predicted. Such predictions are largely based on photic phase-response curves (PRCs) generated from animals housed under seasonally agnostic equatorial photoperiods with alternating 12-hour segments of light and darkness. Most of the human population, however, lives at northerly latitudes (20-50° N of the Equator) where seasonal variations in the light-dark schedule are pronounced. Here, we address this disconnect by constructing the first high-resolution seasonal atlas for light-induced circadian phase-resetting. Testing the light responses of nearly 4,000Drosophilaat 120 timepoints across 5 seasonally relevant photoperiods (i.e., LD 8:16, 10:14, 12:12, 14:10, and 16:8; 24 hourly intervals surveyed in each), we determined that many aspects of the circadian PRC waveform are conserved with increasing daylength. Surprisingly though, irrespective of LD schedule, the start of the PRCs always remained anchored to the timing of subjective sunset, creating a differential overlap of the advance zone with the morning hours after subjective sunrise that was maximized under summer photoperiods and minimized under winter photoperiods. These data suggest that circadian photosensitivity is effectively extinguished by the early winter morning and out of optimal phase alignment with the wake schedules of many individuals (which revolve around the timing of sunrise regardless of season). They raise the possibility that phototherapy protocols for conditions such as seasonal depression might be improved with programmed light exposure during the final hours of sleep.
Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis
Authors: Darp Revati , Vittoria Marc A. , Ganem Neil J. , Ceol Craig J.
Published: April 2021

Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAFV600E, the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failurein vitroand in a zebrafish melanoma model. Mechanistically, BRAFV600Ecauses decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAFV600Eactivity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAFV600Eactivity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.

Statement of Significance

Whole-genome doubling is prevalent in many types of solid tumors and important in shaping tumor genomes, yet the causes of whole-genome doubling are not well understood. Here, we discover that oncogenic BRAFV600Ecan induce whole-genome doubling through suppression of cytokinesis, and BRAFV600E-induced whole-genome doubling can occur in melanocytes and be present in nascent melanoma cells upon tumorigenesis.
Chitinase 3-like-1 Contributes to Acetaminophen-induced Liver Injury by Promoting Hepatic Platelet Recruitment
Authors: Shan Zhao , Li Leike , Atkins Constance Lynn , Wang Meng , Wen Yankai , Jeong Jongmin , Moreno Nicolas F. , Feng Dechun , Gui Xun , Zhang Ningyan , Lee Chun Geun , Elias Jack Angel , Lee William , Gao Bin , Lam Fong Wilson , An Zhiqiang , Ju Cynthia
Published: April 2021

Hepatic platelet accumulation contributes to acetaminophen (APAP)-induced liver injury (AILI). However, little is known about the molecular pathways involved in platelet recruitment to the liver and whether targeting such pathways could attenuate AILI. The present study unveiled a critical role of chitinase 3-like-1 (Chi3l1) in hepatic platelet recruitment during AILI. Increased Chi3l1 and platelets in the liver were observed in patients and mice overdosed with APAP. Compared to wild-type (WT) mice, Chi3l1-/-mice developed attenuated AILI with markedly reduced hepatic platelet accumulation. Mechanistic studies revealed that Chi3l1 signaled through CD44 on macrophages to induce podoplanin expression, which mediated platelet recruitment through C-type lectin-like receptor 2. Moreover, APAP treatment of CD44-/-mice resulted in much lower numbers of hepatic platelets and liver injury than WT mice, a phenotype similar to that in Chi3l1-/-mice. Recombinant Chi3l1 could restore hepatic platelet accumulation and AILI in Chi3l1-/-mice, but not in CD44-/-mice. Importantly, we generated anti-Chi3l1 monoclonal antibodies and demonstrated that they could effectively inhibit hepatic platelet accumulation and AILI. Overall, we uncovered the Chi3l1/CD44 axis as a critical pathway mediating APAP-induced hepatic platelet recruitment and tissue injury. We demonstrated the feasibility and potential of targeting Chi3l1 to treat AILI.
Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity
Authors: LaCroix Rebecca , Lin Benjamin , Levchenko Andre
Published: April 2021

Kinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus complexin vivopatterns of PKA-R localization can drive complex phenotypes, including cell migration.
Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts
Authors: Leonen Calvin J. A. , Shimada Miho , Weller Caroline E. , Nakadai Tomoyoshi , Hsu Peter , Shelton Patrick M. M. , Sadilek Martin , Zheng Ning , Roeder Robert G. , Chatterjee Champak
Published: April 2021

Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, and transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we have uncovered negative biochemical crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.
A neural circuit linking two sugar sensors regulates satiety-dependent fructose drive inDrosophila
Authors: Musso Pierre-Yves , Junca Pierre , Gordon Michael D
Published: April 2021

Ingestion of certain sugars leads to activation of fructose sensors within the brain of flies, which then sustain or terminate feeding behavior depending on internal state. Here, we describe a three-part neural circuit that links satiety with fructose sensing. We show that AB-FBl8 neurons of the Fan-shaped body display oscillatory calcium activity when hemolymph glycemia is high, and that these oscillations require synaptic input from SLP-AB neurons projecting from the protocerebrum to the asymmetric body. Suppression of activity in this circuit, either by starvation or genetic silencing, promotes specific drive for fructose ingestion. Moreover, neuropeptidergic signaling by tachykinin bridges fan-shaped body activity and Gr43a-mediated fructose sensing. Together, our results demonstrate how a three-layer neural circuit links the detection of two sugars to impart precise satiety-dependent control over feeding behavior.
Ubiquitin specific protease 1 expression and function in T cell immunity
Authors: Omilusik Kyla D , Nadjsombati Marija S , Yoshida Tomomi M , Shaw Laura A , Goulding John , Goldrath Ananda W
Published: April 2021

T cells are essential mediators of the immune responses against infectious diseases and provide long-lived protection from reinfection. The differentiation of naive T cells to effector T cells and subsequent differentiation and persistence of memory T cell populations in response to infection is a highly regulated process. E protein transcription factors and their inhibitors, Id proteins, are important regulators of both CD4+and CD8+T cell responses; however, their regulation at the protein level has not been explored. Recently, the deubiquitinase USP1 was shown to stabilize Id2 and modulate cellular differentiation in osteosarcomas. Here, we investigated a role for Usp1 in posttranslational control of Id2 and Id3 in T cells. We show that Usp1 was upregulated in T cells following activationin vitroor following infectionin vivo, and the extent of Usp1 expression correlated with the degree of T cell expansion. Usp1 directly interacted with Id2 and Id3 following T cell activation. However, Usp1-deficiency did not impact Id protein abundance in effector T cells or alter effector CD8+T cell expansion or differentiation following a primary infection. Usp1 deficiency did result in a gradual loss of memory cells over time and impaired accumulation and altered differentiation following a secondary infection. Together, these results identify Usp1 as a player in modulating recall responses at the protein level and highlight differences in regulation of T cell responses between primary and subsequent infection encounters. Finally, our observations reveal that differential regulation of Id2/3 proteins between immune vs non-immune cell types.
Immunogenicity of a public neoantigen derived from mutatedPIK3CA
Authors: Chandran Smita S. , Ma Jiaqi , Klatt Martin G. , Dundar Friederike , Bandlamudi Chaitanya , Razavi Pedram , Wen Hannah Y. , Weigelt Britta , Zumbo Paul , Fu Si Ning , Banks Lauren B. , Bestman Watchain D. , Drilon Alexander , Betel Doron , Scheinberg David A. , Baker Brian M. , Klebanoff Christopher A.
Published: April 2021

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrent mutations in driver genes that are restricted by prevalent HLA alleles. Here, we report on a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant (Mut)PIK3CA, among the most common genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this method, we developed a library of TCRs that recognize an endogenously processed neoepitope containing a commonPIK3CAhotspot mutation that is restricted by HLA-A*03:01. Mechanistically, immunogenicity to this public NeoAg arises primarily from enhanced stability of the neopeptide/HLA complex caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a relatively “featureless” surface dominated by the peptide’s backbone. To overcome the challenge of binding such an epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface aided by an unusually long β-chain complementarity-determining region 3 (CDR3β) loop. In a pan-cancer cohort of patients with diverse malignancies that express thePIK3CApublic NeoAg, we observed spontaneous immunogenicity, NeoAg clonal conservation, and in a limited number of cases, evidence of targeted immune escape. Together, these results establish the immunogenic potential of MutPIK3CA, creating a framework for off-the-shelf immunotherapies targeting this public NeoAg.
Class II LBD genesZmLBD5andZmLBD33regulate gibberellin and abscisic acid biosynthesis
Authors: Xiong Jing , Feng Xuanjun , Zhang Weixiao , Wang Xianqiu , Hu Yue , Zhang Xuemei , Wu Fengkai , Guo Wei , Xie Wubing , Wang Qingjun , Xu Jie , Lu Yanli
Published: April 2021

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors. Class ILBDmembers are widely reported to be pivotal for organ development, however, the role of class II members is unknown in cereal crops. Class II LBD proteins are distinguished from class I by the lack of a Gly-Ala-Ser (GAS) peptide and leucine-zipper-like coiled-coil domain, which is thought to be essential for protein dimerization. In this study, ZmLBD5 and ZmLBD33 form homo- and hetero-dimers, like class I members. At seedling stage,ZmLBD5promoted biomass accumulation (shoot dry weight and root dry weight), root development (root length, root number, and root volume), and organ expansion (leaf area), whileZmLBD33repressed these processes and display a dwarf phenotype. BothZmLBD5andZmLBD33displayed negative roles in drought tolerance mainly by increasing stomatal density and stomatal aperture. RNA sequencing, gene ontology enrichment analysis, and transient luciferase expression assays indicated thatZmLBD5andZmLBD33are mainly involved in the regulation of theTPS-KS-GA2oxgene module, which comprises key enzymatic genes upstream of GA and ABA biosynthesis. GA1content increased inZmLBD5-overexpressing seedlings, while GA3and abscisic acid content decreased in both transgenic seedlings. Consequently, exogenous GA1or GA3undoubtedly rescued the dwarf phenotype ofZmLBD33-overexpressing plants, with GA1performing better. The study ofZmLBD5andZmLBD33sheds light on the function of the class IILBDgene family in maize.
Discovery and characterization of Hv1-type proton channels in reef-building corals
Authors: Rangel-Yescas Gisela E. , Cervantes Cecilia , Cervantes-Rocha Miguel A. , Suarez-Delgado Esteban , Banaszak Anastazia T. , Maldonado Ernesto , Ramsey Ian. S. , Rosenbaum Tamara , Islas León D.
Published: April 2021

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding for Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a distinct manner. The biophysical properties of these novel channels make them interesting model systems. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH regulation processes and possible consequences of ocean acidification related to the function of these channels.
ModernAcinetobacter baumanniiclinical isolates replicate inside spacious vacuoles and egress from macrophages
Authors: Sycz Gabriela , Di Venanzio Gisela , Sartorio Mariana G. , Nguyen-Hung Le , Scott Nichollas E. , Beatty Wandy L. , Feldman Mario F.
Published: April 2021

Multidrug-resistantAcinetobacter baumanniiinfections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat theseA. baumanniiinfections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium’s pathobiology, which remains poorly understood.A. baumanniiis regarded as an extracellular opportunistic pathogen. However, research onAcinetobacterhas largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modernA. baumanniiclinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication ofA. baumannii. We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance ofA. baumannii.
Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host
Authors: Borvinskaya Ekaterina V. , Kochneva Albina A. , Drozdova Polina B. , Balan Olga V. , Zgoda Victor G.
Published: April 2021

The protein composition (proteome) of cestode Schistocephalus solidus was measured in an experiment simulating the transition of the parasite from a cold-blooded to a warm-blooded host. Infective S. solidus plerocercoids obtained from the three-spined stickleback Gasterosteus aculeatus were heated at 40 °C for 1 hour or cultured in vitro at 40 °C and 22 °C for 48 hours. In the short-term experiment, the content of only one tegument protein decreased after heating. After long-term heating, which triggered parasite sexual maturation, an increase in the content of ribosomal proteins, translation initiation factors and enzymes of the amino acid biosynthesis pathway was observed. The synthesis of certain gene products for carbohydrate metabolism, including glycolysis/gluconeogenesis, was found to be regulated in the parasite by temperature.

Summary statement

The study focuses on the processes that determine the survival of parasites in warm-blooded animals, and thus demonstrates the potential for the application of proteomics in veterinary medicine.
TheORGAN SIZE(ORG) locus contributes to isometric gigantism in domesticated tomato
Authors: Vicente Mateus Henrique , MacLeod Kyle , Fernandes Figueiredo Cassia Regina , de Oliveira Figueira Antonio Vargas , Mohareb Fady , Kevei Zoltán , Thompson Andrew J. , Zsögön Agustin , Pereira Peres Lázaro Eustáquio
Published: April 2021

Gigantism is a key component of the domestication syndrome, a suite of traits that differentiates crops from their wild relatives. Allometric gigantism is strongly marked in horticultural crops, causing disproportionate increases in the size of edible parts such as stems, leaves or fruits. Tomato (Solanum lycopersicum) has attracted attention as a model for fruit gigantism, and many genes have been described controlling this trait. However, the genetic basis of a corresponding increase in size of vegetative organs contributing to isometric gigantism, has remained relatively unexplored. Here, we identified a 0.4 Mbp region on chromosome 7 in introgression lines (ILs) from the wild speciesSolanum pennelliiin two different tomato genetic backgrounds (cv. M82 and cv. Micro-Tom) that controls vegetative and reproductive organ size in tomato. The locus, namedORGAN SIZE(ORG), was fine-mapped using genotype-by-sequencing. A survey of literature revealed thatORGoverlaps with previously mapped QTLs controlling tomato fruit weight during domestication. Alleles from the wild species led to reduced cell number in different organs, which was partially compensated by greater cell expansion in leaves but not in fruits. The result was a proportional reduction in leaf, flower and fruit size in the ILs harbouring the wild alleles. These findings suggest that selection for large fruit during domestication also tends to select for increases in leaf size by influencing cell division. Since leaf size is relevant for both source-sink balance and crop adaptation to different environments, the discovery ofORGcould allow fine-tuning of these parameters.

One sentence summary

A locus that controls isometric size increase in vegetative and reproductive organs of tomato through changes in cell division
FRAME-tags: genetically encoded fluorescent markers for multiplexed barcoding and time-resolved tracking of live cells
Authors: Anzalone Andrew V. , Jimenez Miguel , Cornish Virginia W.
Published: April 2021

Cellular barcodes offer critical tools for tracking cellular identity in biological systems. Although genetically encoded fluorescent barcodes are ideal for real-time tracking, their scalability is constrained by the broad, overlapping emission spectra characteristic of fluorescent proteins (FPs). Here, we describe a palette of genetically encoded fluorescent barcodes called FRAME-tags, which break this scalability barrier by encoding barcode identity as unique FP expression ratios. FRAME-tags use −1 programmed ribosomal frameshifting RNA motifs to precisely control the translational output of multiple FPs from a single mRNA, leading to extremely narrow and resolvable ratios of the corresponding cellular fluorescence distributions. With this platform, we constructed 20 resolvable FRAME-tags in yeast using just two FPs, and further demonstrated that 100 or more distinguishable FRAME-tags could be made by the addition of a third FP. We used FRAME-tags to map the dynamic fitness landscape of yeast co-cultures, and to characterize the expression pattern of 20 yeast promoters in multiplex across diverse conditions. FRAME-tags offer a valuable new tool for cellular barcoding that enables time-resolved characterization of complex biological systems using widely available fluorescence detection techniques and a minimal number of spectral channels.