bluuebunny/crossref_metadata_2025 huggingface dataset:
YAP condensates are highly organized hubs for YAP/TEAD transcription
Authors: Hao Siyuan , Fuehrer Hannah , Flores Eduardo , Demmerle Justin , Lippincott-Schwartz Jennifer , Liu Zhe , Sukenik Shahar , Cai Danfeng
Published: October 2022

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. The transcription factor TEAD1 actively stabilizes YAP condensates, which also recruit BRD4, a coactivator that is enriched at active enhancers. Using single molecule tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.
A HUMANIZED FCRN TRANSGENIC MOUSE FOR PRECLINICAL PHARMACOKINETICS STUDIES
Authors: Conner Christopher M. , van Fossan Don , Read Kristen , Cowley Dale O. , Alvarez Oscar , Xu Shannon (Xiang-Ru) , Webb David R. , Jarnagin Kurt
Published: October 2022

Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation.

Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRNhave been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRNtransgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrnand insertion of a hFCRNmini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira®) demonstrate hFCRN-mediated protection. These newly generated SYNB-hFCRNmice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development.

Materials availability

Cryopreserved germline cells from the strains are available from the MMRRC. Dr. Jarnagin will provide sequencing and other evidence supporting strain identity and the plasmids used for constructions.
Dissecting the genetic architecture of sunflower head diameter using genome-wide association study
Authors: Delen Yavuz , Mural Ravi V. , Xu Gen , Delen Semra Palali , Schnable James C. , Yang Jinliang , Dweikat Ismail
Published: October 2022

Sunflower (Helianthus annuusL.) plays an essential role in meeting the edible oil demand worldwide. Sunflower seed yield can be decomposed into several yield component traits, one of which is the head diameter. In 2019, 2020, and 2022, we evaluated the head diameter phenotypic variation on a set of diverse sunflower accessions (N=342) in replicated field trials. By combining three years of field data, the broad sense heritability (H2) of the head diameter trait was estimated to be 0.88. Then, a subset of N=274 accessions was genotyped by using the tunable genotyping-by-sequencing (tGBS) method, resulting in 226,779 high-quality SNPs. With these SNPs and the head diameter phenotype, the genome-wide association study (GWAS) was conducted using two statistical approaches: 1) the mixed linear model (MLM) and 2) the fixed and random model circulating probability unification (farmCPU). The MLM and farmCPU GWAS approaches identified 106 and 8 significant SNPs that were placed close to 53 and 21 genes, respectively. Two significant peaks were identified in MLM, with a strong signal on chromosome 10 and a less strong signal on chromosome 16. The farmCPU method detected the same signals on chromosomes 10 and 16 and several additional significant signals on other chromosomes. The head diameter associated genetic loci and the underlying candidate genes can be leveraged for further functional validation and serve as a basis for sunflower oil yield improvement.
Spatiotemporal gene expression atlas of the extremophyteSchrenkiella parvula
Authors: Wijesinghege Chathura , Wang Guannan , Pantha Pramod , Tran Kieu-Nga , Dassanayake Maheshi
Published: October 2022

Extremophytes are naturally selected to survive environmental stresses, but scarcity of genetic resources for them developed with spatiotemporal resolution limit their use in stress biology.Schrenkiella parvulais one of the leading extremophyte models with initial molecular genomic resources developed to study its tolerance mechanisms to high salinity. Here we present a transcriptome atlas forS. parvulawith subsequent analyses to highlight its diverse gene expression networks associated with salt responses. We included spatiotemporal expression profiles, expression specificity of each gene, and co-expression and functional gene networks representing 115 transcriptomes sequenced from 35 tissue and developmental stages examining their responses before and after 27 salt treatments in our current study. The highest number of tissue-preferentially expressed genes were found in seeds and siliques while genes in seedlings showed the broadest expression profiles among developmental stages. Seedlings had the highest magnitude of overall transcriptomic responses to salinity compared to mature tissues and developmental stages. Differentially expressed genes in response to salt were largely mutually exclusive but shared common stress response pathways spanning across tissues and developmental stages. Our foundational dataset created forS. parvularepresenting a stress-adapted wild plant lays the groundwork for future functional, comparative, and evolutionary studies using extremophytes aiming to uncover novel stress tolerant mechanisms.

SIGNIFICANCE STATEMENT

Concerted transcriptomic responses coordinated across developmental stages and tissues are required to complete a plant lifecycle under salt stress. Transcriptomic resources created with spatiotemporal resolution for plants are rare and for stress-adapted plants rarer. We present a transcriptome atlas enabling discovery of genes and networks evolved as adaptations to salt stress in a model extremophyte,Schrenkiella parvula. The spatiotemporally resolved gene expression networks are largely non-overlapping but functionally connected through synergistic stress responsive pathways.
Hsp47 Promotes Biogenesis of Multi-subunit Neuroreceptors in the Endoplasmic Reticulum
Authors: Wang Ya-Juan , Di Xiao-Jing , Han Dong-Yun , Nashmi Raad , Henderson Brandon J. , Moss Fraser J. , Mu Ting-Wei
Published: October 2022

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurodegenerative, neurological, and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is not well understood. Previous proteomics studies identified Hsp47 (Gene:SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric type A (GABAA) receptors. Here, we show that Hsp47 enhances neuronal GABAAreceptor functional surface expression, acting after Binding immunoglobulin Protein (BiP) to preferentially bind the folded conformation of GABAAreceptors. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAAreceptors. These Hsp47 properties are also extended to other Cys-loop receptors, including nicotinic acetylcholine receptors. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 also plays a critical and general role in the maturation of multi-subunit neuroreceptors.

Highlights

Hsp47 positively regulates the functional surface expression of endogenous GABAAreceptors.

Hsp47 acts after BiP and preferentially binds the folded conformation of GABAAreceptors.

Hsp47 promotes the subunit-subunit assembly of GABAAreceptors.

Hsp47 plays a critical and general role in the maturation of multi-subunit neuroreceptors.
Identification of aPrunusMAX1 Homolog as a Unique Strigol Synthase from Carlactone Bypassing 5-Deoxystrigol
Authors: Wu Sheng , Zhou Anqi , Hiugano Kozue , Yoda Akiyoshi , Xie Xiaonan , Yamane Kenji , Miura Kenji , Nomura Takahito , Li Yanran
Published: October 2022

Strigol was the first strigolactone (SL) to be discovered, but the biosynthetic pathway remains elusive. Here, through rapid gene screening using a microbial SL-producing platform, we functionally identified a strigol synthase (PpMAX1c, a cytochrome P450 711A enzyme) inPrunusthat synthesizes strigol directly from the SL precursor carlactone through catalyzing multi-step oxidations and C-ring cyclization, bypassing the synthesis of 5-deoxystrigol. The function of PpMAX1c was validated through reconstructing the biosynthesis of strigol inNicotiana benthamiana. Additional genomic analysis and functional verification confirm that peach also encodes an orobanchol synthase (PpCYP722C, a cytochrome P450 722C enzyme), which hints at the presence of both strigol-type and orobanchol-type SLs in peach and was confirmed through metabolic analysis of peach seedlings. This work highlights the catalytic diversity of the largely unexplored family of CYP711A homologs and sets the foundation to characterize the roles of different types of SLs in the economically importantPrunus.
Mitoxantrone Targets Both Host and Bacteria to Overcome Vancomycin Resistance inEnterococcus faecalis
Authors: da Silva Ronni A. G. , Wong Jun Jie , Antypas Haris , Choo Pei Yi , Goh Karlyn , Jolly Shreya , Liang Cui , Sing Leona Tay Kwan , Veleba Mark , Hu Guangan , Chen Jianzhu , Kline Kimberly A.
Published: October 2022

Among Enterococci, intrinsic and acquired resistance to antibiotics such as β-lactams and vancomycin critically limit treatment options for infection with these opportunistic pathogens. Antimicrobials that enhance the host immune response are emerging as alternative approaches, with the potential to overcome bacterial resistance. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX)in vitroandin vivoagainst vancomycin resistantEnterococcus faecalis(VRE). We show that,in vitro, MTX is a potent antibiotic against Gram-positive bacteria with a minimal inhibitory concentration (MIC) of ~1 μg/ml through induction of reactive oxygen species and DNA damage. MTX synergises with vancomycin and lowers the vancomycin concentration required to kill VRE by over 140-fold. This synergy is specific to vancomycin-resistant, but not susceptible strains because vancomycin rendered the resistant strains more permeable to MTX and thus MTX-mediated DNA damage. In a murine wound infection model, MTX treatment effectively reduced VRE bacterial numbers by 120-fold and with further reduction when combined with vancomycin. Wounds treated with MTX had significantly higher numbers of macrophages and higher pro-inflammatory cytokines compared to untreated wounds. In addition, MTX augmented intracellular bacterial killing by both murine and human macrophages by upregulating the expression of lysosomal hydrolases cathepsins D and H, and β-Hexosaminidase. These results show that MTX is a potent antibiotic against Gram-positive bacteria, synergizes with vancomycin, enhances macrophage recruitment and intracellular bactericidal activity, and represents a promising dual bacterium- and host-targeted therapeutic for overcoming vancomycin resistance.

One sentence summary

Mitoxantrone synergizes with vancomycin against vancomycin resistant bacterial strains via direct antibiotic activity and by augmenting both host macrophage recruitment to the site of infection and macrophage bactericidal activity.
Blended care in patients with knee and/or hip osteoarthritis in physical therapy: a Delphi study on needs and preconditions
Authors: Weber F. , Kloek C. , Arntz A. , Grüneberg C. , Veenhof C.
Published: October 2022

Introduction

Osteoarthritis is a major public health concern. Despite existing evidence-based treatment options, the health care situation remains unsatisfactory. Digital care options, especially combined with in-person sessions seems to be promising. Therefore, the aim of this study was to investigate the needs, preconditions, barriers and facilitators of blended physical therapy.

Methods

This Delphi study consisted of interviews, an online survey and focus groups. Participants were physical therapists, patients with hip or knee osteoarthritis with or without experience in digital care and stakeholders of the health care system. In the first phase, interviews were conducted with patients and physical therapists. The interview guide was based on the “Consolidated Framework For Implementation Research”. The interviews focused on experiences with digital and blended care. Furthermore, needs, facilitators and barriers were discussed. In the second phase, an online survey and focus groups served the process to confirm the needs and collect preconditions.

Results

Nine physical therapists, seven patients and six stakeholders confirm that an increase of acceptance of digital care by physical therapists and patients is crucial. One of the most frequently mentioned facilitator was conducting regular in-person sessions. Physical therapists and patients conclude that blended physical therapy needs to be tailored to the patients’ needs. The reimbursement of blended physical therapy needs to be clarified.

Discussion

Most importantly, it is necessary to strengthen the acceptance of patients and physical therapists towards digital care. Overall, for development and implementation purposes, it is crucial to take the needs and preconditions into account.
Language and Social Regions Are Affected in Toddlers with Autism and Predict Later Language Outcome
Authors: Duan Kuaikuai , Eyler Lisa , Pierce Karen , Lombardo Michael V. , Datko Michael , Hagler Donald , Taluja Vani , Zahiri Javad , Campbell Kathleen , Barnes Cynthia Carter , Arias Steven , Nalabolu Srinivasa , Troxel Jaden , Dale Anders M. , Courchesne Eric
Published: October 2022

Autism spectrum disorder is a heterogeneous neurodevelopmental disorder. Early brain overgrowth yet reduced cerebellar size is well recognized for autism, but cortical regions involved show inconsistent patterns of alteration. No complete and replicable map of early regional brain size alterations has been charted. It is also not clear whether individual differences in brain size relate to autism symptom severity and cognitive deficits and predict later language outcomes. We leveraged structural MRI data from 166 autistic and 109 typical developing toddlers to comprehensively and systematically investigate regional gray matter volume alterations and cortical surface area and thickness perturbations in autism compared to typical developing toddlers using linear mixed-effect models. We then examined their replicability in an independent cohort of 38 autistic and 37 typical developing toddlers. We further investigated associations between regional brain size and symptom severity, Mullen and Vineland cognitive performance using linear regression models. Lastly, we investigated whether early brain size (at intake mean age of 2.5 years) can improve support vector machine prediction of language outcome at 3-4 years of age when added to a model containing intake clinical and behavioral measures. Compared to typical developing toddlers, autistic toddlers presented larger or thicker lateral temporal regions, smaller or thinner frontal lobe and midline structures, larger callosal subregion volume, and smaller cerebellum. Most of these differences were replicated in an independent toddler cohort. Moreover, the identified gray matter alterations were related to autism symptom severity and cognitive impairments at intake, and, remarkably, they improved the accuracy for predicting later language outcome beyond intake clinical and demographic variables. Gray matter volume, thickness, and surface area in regions involved in language, social, and face processing were altered in autistic toddlers. Alterations in these regions are major early-age developmental attributes of autism. The early-age alterations in these cortical attributes in different regions may be the result of dysregulation in multiple neural processes and stages, consistent with prenatal multi-process, multi-stage models of autism. Here we also show these gray matter alterations are promising prognostic biomarkers for language outcome prediction.
CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies
Authors: Gulden Gamze , Sert Berranur , Teymur Tarik , Ay Yasin , Tiryaki Nulifer Neslihan , Ovalı Ercüment , Tarhan Nevzat , Tastan Cihan
Published: October 2022

The development of genetic modification techniques has led to the opening of a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. Since not only cancerous cells but also healthy cells are damaged by the drugs, intensive efforts are made to develop cancer-targeted techniques. The most promising approach is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapy’s time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the subpopulation of Tcm and Tscm in CAR-T cells, we investigated to produce a long-term stable and cytotoxic efficient CAR-T cell by modifications in the cell activation-dependent production method using Phytohemagglutinin. Phytohemagglutinin (PHA), a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19 specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared to aCD3/aCD28. Two different types of CAR (aCD19 scFv CD8- (CD28 or 41BB)-CD3z-EGFRt) expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm-Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar cytotoxic (anti-tumor) and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells.
Chinese fir genome and the evolution of gymnosperms
Authors: Lin Si-Zu , Chen Yu , Wu Chao , Sun Wei-Hong , Li Zhen , Chen Hengchi , Wang Jieyu , Ji Changmian , Li Shu-Bin , Wang Zhiwen , Tsai Wen-Chieh , Ma Xiang-Qing , Lan Si-Ren , Zhang Fei-Ping , Xie Ya-Cong , Yao Lei , Zhang Yan , Lü Meng-Meng , Zhang Jia-Jun , Zhang Di-Yang , Ye Yi-Quan , Yu Xia , Xu Shan-Shan , Ma Zhi-Hui , Ding Guo-Chang , Cao Guang-Qiu , He Zong-Ming , Wu Peng-Fei , Lin Kai-Min , Liu Ai-Qin , Lin Yan-Qing , Ruan Shao-Ning , Liu Bao , Cao Shi-Jiang , Zhou Li-Li , Li Ming , Shuai Peng , Hou Xiao-Long , Wu Yi-Han , Li Nuo , Xiong Sheng , Hao Yang , Zhou Zhuang , Liu Xue-Die , Zuo Dan-Dan , Li Jia , Wang Pei , Zhang Jian , Liu Ding-Kun , Chen Gui-Zhen , Huang Jie , Huang Ming-Zhong , Li Yuanyuan , Zheng Qinyao , Zhao Xiang , Zhong Wen-Ying , Wang Feng-Ling , Cheng Xin-Chao , Yu Yin , Liu Zhi-Wei , Zheng Hongkun , Ming Ray , Van de Peer Yves , Liu Zhong-Jian
Published: October 2022

Seed plants comprise angiosperms and gymnosperms. The latter includes gnetophytes, cycads, Ginkgo, and conifers. Conifers are distributed worldwide, with 630 species distributed across eight families and 70 genera. Their distinctiveness has triggered much debate on their origin, evolution, and phylogenetic placement among seed plants. To better understand the evolution of gymnosperms and their relation to other seed plants, we report here a high-quality genome sequence for a tree species, Chinese fir (Cunninghamia lanceolata), which has excellent timber quality and high aluminum adaptability and is a member of Cupressaceae with high levels of heterozygosity. We assembled an 11.24 Gb genome with a contig N50 value of 2.15 Mb and anchored the 10.89 Gb sequence to 11 chromosomes. Phylogenomic analyses showed that cycads sister to Ginkgo, which place to sister in all gymnosperm lineages, and Gnetales within conifers sister to Pinaceae. Whole-genome duplication (WGD) analysis showed that the ancestor of seed plants has differentiated into angiosperms and gymnosperms after having experienced a WGD event. The ancestor of extant gymnosperm has experienced a gymnosperm-specific WGD event and the extant angiosperms do not share a common WGD before their most recent common ancestor diverged into existing angiosperms lineages. Analysis of the MADS-box gene family ofC. lanceolatarevealed the developmental mechanism of the reproductive organs inC. lanceolata, which supported the (A)B(C) model of the development of gymnosperms reproductive organs. In addition, astringent seeds and shedding of whole branches (with withered leaves) might be a strategy ofC. lanceolatathat evolved during long-term adaptation to an aluminum-rich environment. The findings also reveal the molecular regulation mechanism of shade tolerance inC. lanceolataseedlings. Our results improve the resolution of ancestral genomic features within seed plants and the knowledge of genome evolution and diversification of gymnosperms.
Optimal dormancy strategies in fluctuating environments given delays in phenotypic switching
Authors: Măgălie Andreea , Schwartz Daniel A. , Lennon Jay T. , Weitz Joshua S.
Published: October 2022

Organisms have evolved different mechanisms in response to periods of environmental stress, including dormancy – a reversible state of reduced metabolic activity. Transitions to and from dormancy can be random or induced by changes in environmental conditions. Prior theoretical work has shown that stochastic transitioning between active and dormant states at the individual level can maximize fitness at the population level. However, such theories of ‘bet-hedging’ strategies typically neglect certain physiological features of transitions to dormancy, including time lags to gain protective benefits. Here, we construct and analyze a dynamic model that couples stochastic changes in environmental state with the population dynamics of organisms that can initiate dormancy after an explicit time delay. Stochastic environments are simulated using a multi-state Markov chain through which the mean and variance of environmental residence time can be adjusted. In the absence of time lags (or in the limit of very short lags), we find that bet-hedging strategy transition probabilities scale inversely with the mean environmental residence times, consistent with prior theory. We also find that increasing delays in dormancy decreases optimal transitioning probabilities, an effect that can be influenced by the correlations of environmental noise. When environmental residence times - either good or bad - are uncorrelated, the maximum population level fitness is obtained given low levels of transitioning between active and dormant states. However when environmental residence times are correlated, optimal dormancy initiation and termination probabilities increase insofar as the mean environmental persistent time is longer than the delay to reach dormancy. We also find that bet hedging is no longer advantageous when delays to enter dormancy exceed the mean environmental residence times. Altogether, these results show how physiological limits to dormancy and environmental dynamics shape the evolutionary benefits and even viability of bet hedging strategies at population scales.
AdultDrosophilalegs do not regenerate after amputation
Authors: Sustar Anne , Tuthill John C.
Published: October 2022

A recent paper by Abramset al. (2021) claimed that a simple dietary supplement is sufficient to induce appendage regeneration in jellyfish, flies, and mice. This would be remarkable, if true, because it was previously thought that flies and mice lack the capacity for regeneration after injury. We therefore sought to replicate their provocative results. We amputated one tibia of over 1000 fruit flies, fed them control or supplemented diets, and carefully examined their legs three weeks post-injury. We did not, however, observe any instances of leg regeneration. We conducted additional experiments that confirmed the complete absence of neurons, muscles, or other living cells in amputated tibias. Abramset al. also reported the formation of a white blob at the amputation site, which they interpreted as an intermediate regeneration morphology. We tested this hypothesis more rigorously and conclude that the white blob consists of bacteria. Overall, we failed to find any evidence for leg regeneration inDrosophila, even when flies were fed the supplemented diet. Our results therefore contradict the overarching conclusion of Abramset al. that dietary supplements are sufficient to unlock an ancestral mechanism that induces appendage regeneration.
A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogenCryptococcus neoformans
Authors: Probst Corinna , Hallas-Møller Magnus , Ipsen Johan Ø. , Brooks Jacob T. , Andersen Karsten , Haon Mireille , Berrin Jean-Guy , Martens Helle J. , Nichols Connie B. , Johansen Katja S. , Alspaugh J. Andrew
Published: October 2022

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. TheCEL1gene in the human fungal pathogenCryptococcus neoformans(Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. TheCEL1gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of theCEL1gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, acel1Δdeletion mutant was avirulent in two models ofC. neoformansinfection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest thatCnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.

Author summary

Fungi need to adapt quickly to environmental stresses to thrive. The fungal cell wall, which supplies support and integrity to the cell, is an essential compartment to react and interact with the surrounding environment. Rapid changes within the carbohydrate composition and architecture occur in response to environmental stresses. Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper-enzymes, secreted by microbes to assist in the first steps of remodeling and degrading complex and recalcitrant carbohydrates. In this study we explore the role of the putative AA9 family LPMOCnCel1 for growth and virulence of the fungal human pathogenCryptococcus neoformans. TheCEL1gene is highly up-regulated in presence of host stresses and acel1Δmutant strain is avirulent in a murine model of infection. Downstream analysis of virulence-associated phenotypes identified theCEL1gene to be required for thermotolerance as well as cell wall integrity, and efficient cell cycle progression in presence of host-mimicking stresses. Based upon those findings we propose thatCnCel1 likely promotes intrinsic fungal cell wall remodeling events essential for adaptation to the host environment.
The effects of drought and inter-plant competition on the ectomycorrhizal interaction between fungi and Aleppo pine seedlings
Authors: Herol Lior , Shemesh Hagai , Avidar Mor , Yirmiahu Shahar , Zach Yair , Klein Tamir , Livne-Luzon Stav
Published: October 2022

An increase in tree mortality is currently evident in forests around the world. Such mortality could be counterbalanced by the native regeneration of seedlings. Seedling establishment under natural conditions is often limited by inter-plant competition and drought conditions. Many forest ecosystems rely on ectomycorrhizal relationships which could be affected by competition and drought, altering forest resilience.

We carried out an experiment testing the combined effects of drought, herbaceous competition, and ectomycorrhizal fungi (EMF) on the growth and shape of Aleppo pine seedlings and the EMF community composition.

Pines that germinated in the presence of the EMF spores were taller, had greater biomass, and more side branches. However, under conditions of either competition or drought, the effect of EMF on seedling biomass and height was greatly reduced, while the effect on shoot branching was maintained. Under a combination of drought and competition, EMF had no influence on plant growth and shape. The EMF community was strongly dominated byGeoporaspecies, and its structure was not affected by the treatments. Plants experiencing competition were nitrogen poor but presented the highest levels of EMF sequence abundance.

Stressful conditions seem to alter the relationship between EMF and seedling growth. Specifically, under drought, both colonization and seedling response to EMF was small. However, under competition, colonization was maintained while no growth enhancement was evident. This discrepancy highlights the complexity of the benefits provided to seedlings by EMF under ecologically relevant conditions.
Design and performance of a long-read sequencing panel for pharmacogenomics
Authors: van der Lee Maaike , Busscher Loes , Menafra Roberta , Zhai Qinglian , van den Berg Redmar R. , Kingan Sarah B , Gonzaludo Nina , Hon Ting , Han Ting , Arbiza Leonardo , Numanagić Ibrahim , Kloet Susan L. , Swen Jesse J.
Published: October 2022

Pharmacogenomics (PGx)-guided drug treatment is one of the cornerstones of personalized medicine. However, the genes involved in drug response are highly complex and known to carry many (rare) variants. Current technologies (short-read sequencing and SNP panels) are limited in their ability to resolve these genes and characterize all variants. Moreover, these technologies cannot always phase variants to their allele of origin. Recent advance in long-read sequencing technologies have shown promise in resolving these problems. Here we present a long-read sequencing panel-based approach for PGx using PacBio HiFi sequencing.

A capture based approach was developed using a custom panel of clinically-relevant pharmacogenes including up- and downstream regions. A total of 27 samples were sequenced and panel accuracy was determined using benchmarking variant calls for 3 Genome in a Bottle samples and GeT-RM star(*)-allele calls for 21 samples..

The coverage was uniform for all samples with an average of 94% of bases covered at >30×. When compared to benchmarking results, accuracy was high with an average F1 score of 0.89 for INDELs and 0.98 for SNPs. Phasing was good with an average of 68% the target region phased (compared to ~20% for short-reads) and an average phased haploblock size of 6.6kbp. Using Aldy 4, we compared our variant calls to GeT-RM data for 8 genes (CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, SLCO1B1, TPMT), and observed highly accurate star(*)-allele calling with 98.2% concordance (165/168 calls), with only one discordance inCYP2C9leading to a different predicted phenotype.

We have shown that our long-read panel-based approach results in high accuracy and target phasing for SNVs as well as for clinical star(*)-alleles.
A SNP affects Wnt4 expression in endometrial stroma, with antagonistic implications for pregnancy, endometriosis and reproductive cancers
Authors: Pavlicev Mihaela , McDonough-Goldstein Caitlin E. , Zupan Andreja Moset , Muglia Lisa , Hu Yueh-Chiang , Kong Fansheng , Monangi Nagendra , Dagdas Gülay , Zupancic Nina , Marziaz Jamie , Sinner Debora , Zhang Ge , Wagner Günter , Muglia Louis
Published: October 2022

The common human single nucleotide polymorphism rs3820282 is associated with multiple phenotypes ranging from gestational length to likelihood of endometriosis and ovarian cancer and can thus serve as a paradigm for a highly pleiotropic genetic variant. Pleiotropy makes it challenging to assign specific causal roles to particular genetic variants. Deleterious mutations in multifunctional genes may cause either the co-occurrence of multiple disorders in the same individuals (i.e., syndromes), or be repeatedly associated with a variety of disorders in a population. Moreover, the adverse effects can occur in combination with advantages in other traits, maintaining high frequencies of deleterious alleles in the population. To reveal the causal role of this specific SNP, we investigated the molecular mechanisms affected by rs3820282 in mice. We have shown previously that rs3820282 introduces a high affinity estrogen receptor 1 binding site at theWnt4locus. Having introduced this nucleotide substitution into the homologous site of the mouse genome by CRISPR/Cas 9 we show that this change causes a specific upregulation ofWnt4transcription in the endometrial stromal cells during the preovulatory estrogen peak in late proestrus. Transcriptomic analysis of the whole uterus reveals broad systemic effects on uterine gene expression, including downregulation of proliferation and induction of many progesterone-regulated pro-implantation genes. The effect on proliferation is limited to the luminal epithelium, whereas other effects involve the uterine stromal compartment. We suggest that in the uterus, these changes could contribute to increased permissiveness to embryo invasion. Yet in other estrogen-responsive tissues, the same changes potentially lead to decreased resistance to invasion by cancer cells and endometriotic foci. A single molecular effect of rs3820282 onWnt4expression may thus underlie the various associated phenotypic effects.
Extreme genome diversity and cryptic speciation in a harmful algal bloom forming eukaryote
Authors: Wisecaver Jennifer H. , Auber Robert P. , Pendleton Amanda L. , Watervoort Nathan F. , Fallon Timothy R. , Riedling Olivia L. , Manning Schonna R. , Moore Bradley S. , Driscoll William W.
Published: October 2022

Harmful algal blooms (HABs) of the toxic haptophytePrymnesium parvumare a recurrent problem in many inland and estuarine waters around the world. Strains ofP. parvumvary in the toxins they produce and in other physiological traits associated with HABs, but the genetic basis for this variation is unknown. To investigate genome diversity in this morphospecies, we generated genome assemblies for fifteen phylogenetically and geographically diverse strains ofP. parvumincluding Hi-C guided, near-chromosome level assemblies for two strains. Comparative analysis revealed considerable DNA content variation between strains, ranging from 115 Mbp to 845 Mbp. Strains included haploids, diploids, and polyploids, but not all differences in DNA content were due to variation in genome copy number. Haploid genome size between strains of different chemotypes differed by as much as 243 Mbp. Syntenic and phylogenetic analyses indicate that UTEX 2797, a common laboratory strain from Texas, is a hybrid that retains two phylogenetically distinct haplotypes. Investigation of gene families variably present across strains identified several functional categories associated with metabolism, including candidates for the biosynthesis of toxic metabolites, as well as genome size variation, including recent proliferations of transposable elements. Together, our results indicate thatP. parvumis comprised of multiple cryptic species. These genomes provide a robust phylogenetic and genomic framework for investigations into the eco-physiological consequences of the intra- and inter-specific genetic variation present inP. parvumand demonstrate the need for similar resources for other HAB-forming morphospecies.

SIGNIFICANCE STATEMENT

Harmful algal blooms (HABs) are a global concern. Efforts to understand the genetic basis of traits associated with the success of HAB-forming species are limited by a dearth of genomic resources. In this paper we present genomes for fifteen strains ofPrymnesium parvum, a toxic alga that causes ecosystem and societally disruptive HABs around the world. We uncover an unprecedented amount of sequence-level, gene family, and genome architecture evolution inP. parvumand provide evidence for both cryptic speciation and hybridization. These results illustrate how both inter- and intraspecific genetic variation can be dramatically underestimated in a protist morphospecies. More work is needed to understand the eco-physiological consequences of hidden genetic diversity inP. parvumand HAB-forming species more generally.
DRAM1 promotes antibacterial autophagy and lysosomal delivery of Mycobacterium marinum in macrophages
Authors: Banducci-Karp Adrianna , Xie Jiajun , Engels Sem A. G. , Sarantaris Christos , Varela Monica , Meijer Annemarie H. , van der Vaart Michiel
Published: October 2022

Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellular processes underlying DRAM1-mediated host defence, here we studied the interaction of DRAM1 with Mycobacterium marinum in murine RAW264.7 macrophages. We found that shortly after phagocytosis, DRAM1 localised in a punctate pattern to mycobacteria, which gradually progressed to full DRAM1 envelopment of the bacteria. Within the same time frame, DRAM1-positive mycobacteria colocalised with the LC3 marker for autophagosomes and LysoTracker and LAMP1 markers for (endo)lysosomes. Knockdown analysis revealed that DRAM1 is required for recruitment of LC3 and for acidification of mycobacteria-containing vesicles. A reduction in the presence of LAMP1 further suggested reduced fusion of lysosomes with mycobacteria-containing vesicles. Finally, we show that DRAM1 knockdown impairs the ability of macrophages to control mycobacterial infection. Together, these results support that DRAM1 promotes the trafficking of mycobacteria through the degradative (auto)phagolysosomal pathway. Considering its prominent effect on host resistance to intracellular infection, DRAM1 is a promising target for therapeutic modulation of the microbicidal capacity of macrophages.
Metabolic robustness to growth temperature of a cold adapted bacterium
Authors: Riccardi Christopher , Calvanese Marzia , Ghini Veronica , Alonso-Vásquez Tania , Perrin Elena , Turano Paola , Giurato Giorgio , Weisz Alessandro , Parrilli Ermenegilda , Tutino Maria Luisa , Fondi Marco
Published: October 2022

Microbial communities experience continuous environmental changes, among which temperature fluctuations are arguably the most impacting. This is particularly important considering the ongoing global warming but also in the “simpler” context of seasonal variability of sea-surface temperature. Understanding how microorganisms react at the cellular level can improve our understanding of possible adaptations of microbial communities to a changing environment. In this work, we investigated which are the mechanisms through which metabolic homeostasis is maintained in a cold-adapted bacterium during growth at temperatures that differ widely (15 and 0°C). We have quantified its intracellular and extracellular central metabolomes together with changes occurring at the transcriptomic level in the same growth conditions. This information was then used to contextualize a genome-scale metabolic reconstruction and to provide a systemic understanding of cellular adaptation to growth at two different temperatures. Our findings indicate a strong metabolic robustness at the level of the main central metabolites, counteracted by a relatively deep transcriptomic reprogramming that includes changes in gene expression of hundreds of metabolic genes. We interpret this as a transcriptomic buffering of cellular metabolism, able to produce overlapping metabolic phenotypes despite the wide temperature gap. Moreover, we show that metabolic adaptation seems to be mostly played at the level of few key intermediates (e.g. phosphoenolpyruvate) and in the cross-talk between the main central metabolic pathways. Overall, our findings reveal a complex interplay at gene expression level that contributes to the robustness/resilience of core metabolism, also promoting the leveraging of state-of-the-art multi-disciplinary approaches to fully comprehend molecular adaptations to environmental fluctuations.
Ribozyme-phenotype coupling in peptide-based coacervate protocells
Authors: Vay Kristian Le , Salibi Elia , Ghosh Basusree , Dora Tang T-Y , Mutschler Hannes
Published: October 2022

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin of life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into extremely long chains. Our results show that formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype – phenotype linkage.
HSPB1 facilitates chemoresistance through inhibiting ferroptotic cancer cell death and regulating NF-κB signaling pathway in breast cancer
Authors: Liang Yiran , Wang Yajie , Zhang Yan , Ye Fangzhou , Luo Dan , Li Yaming , Jin Yuhan , Han Dianwen , Wang Zekun , Chen Bing , Zhao Wenjing , Wang Lijuan , Chen Xi , Ma Tingting , Kong Xiaoli , Yang Qifeng
Published: October 2022

Chemoresistance is one of the major causes of therapeutic failure and poor prognosis for breast cancer patients, especially for triple-negative breast cancer patients. However, the underlying mechanism remains elusive. Here, we identified novel functional roles of heat shock protein beta-1 (HSPB1), regulating the chemoresistance and ferroptotic cell death in breast cancer. Based on TCGA and GEO databases, HSPB1 expression was upregulated in breast cancer tissues and associated with poor prognosis of breast cancer patients, which was considered as an independent prognostic factor for breast cancer. Functional assays revealed that HSPB1 could promote cancer growth and metastasis in vitro and in vivo. Furthermore, HSPB1 facilitated doxorubicin resistance through protecting breast cancer cells from drug-induced ferroptosis. Mechanistically, HSPB1 could bind with Ikβ-α and promote its ubiquitination-mediated degradation, leading to increased nuclear translocation and activation of NF-κB signaling. In addition, HSPB1 overexpression led to enhanced secretion of IL6, which further facilitated breast cancer progression. These findings revealed that HSPB1 upregulation might be a key driver to progression and chemoresistance through regulating ferroptosis in breast cancer, while targeting HSPB1 could be an effective strategy against breast cancer.
On the genetic consequences of habitat contraction: edge effects and habitat loss
Authors: Sgarlata Gabriele Maria , Maié Tiago , de Zoeten Tiago , Rasteiro Rita , Chikhi Lounès
Published: October 2022

Natural climate change and recent anthropogenic activities have largely contributed to habitat loss and fragmentation across the world, leading to 70% of worldwide remaining forests to be within 1 km of forest’s edges (Haddad et al., 2015). Ecological studies have shown that edge-effect influences ecological communities, species richness and abundance across many taxa, contributing to worldwide decline in biodiversity. Since edge-effect reduces species abundance and connectivity, it is also expected to negatively influence species genetic variation. In fact, previous theoretical studies had showed that populations closer to the edges of a finite stepping-stone model tends to have shorter coalescence times, and therefore, lower genetic diversity, than central populations. However, predicting the impact of edge effect on local genetic diversity remains challenging in realistic and more complex habitat fragments, where the additive effect of multiple edges is expected to take place. In the present study we explore the genetic consequence of habitat loss at the scale of a habitat fragment (patch-scale), looking at the interplay betweenpatch-sizeandedge-effecton spatial genetic diversity. We propose a statistical approach to estimate ‘edge-impacted effective population size’ from habitat cover information and use this measure to predict spatial genetic diversity in both equilibrium and non-equilibrium populations. We address these questions using spatially-explicit simulations and propose a spatially-explicit analytical framework able to model spatio-temporal changes in genetic diversity due to edge-effect and habitat loss.