bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic activation of tubulin tyrosination in HCM mice and in human iPSC-engineered heart tissues improves heart function
Authors: Pietsch Niels , Chen Christina Y. , Kupsch Svenja , Bacmeister Lucas , Geertz Birgit , Herrera-Rivero Marisol , Voß Hanna , Krämer Elisabeth , Braren Ingke , Westermann Dirk , Schlüter Hartmut , Mearini Giulia , Schlossarek Saskia , van der Velden Jolanda , Caporizzo Matthew , Lindner Diana , Prosser Benjamin L. , Carrier Lucie
Published: May 2023

Rationale

Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular (LV) hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that α-tubulin detyrosination (dTyr-tub) is markedly elevated in heart failure. Acute reduction of dTyr-tub by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, and thus poses a new perspective for HCM treatment.

Objective

In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model (Mybpc3-knock-in; KI), in human HCM cardiomyocytes and in SVBP-deficient human engineered heart tissues (EHTs).

Methods and Results

AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents and 3-week-old KI mice and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show that i) TTL for 6 weeks dose-dependently reduced dTyr-tub and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes; ii) TTL for 12 weeks improved diastolic filling, cardiac output and stroke volume and reduced stiffness in KI mice; iii) TTL for 10 days normalized cell hypertrophy in HCM hiPSC-cardiomyocytes; iv) TTL induced a marked transcription and translation of several tubulins and modulated mRNA or protein levels of components of mitochondria, Z-disc, ribosome, intercalated disc, lysosome and cytoskeleton in KI mice; v) SVBP-deficient EHTs exhibited reduced dTyr-tub levels, higher force and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs.

Conclusion

This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.
EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION
Authors: Adams William P. , Raisch Tristan B. , Zhao Yajun , Davalos Rafael , Barrett Sarah , King D. Ryan , Bain Chandra B. , Colucci-Chang Katrina , Blair Grace A. , Hanlon Alexandra , Lozano Alicia , Veeraraghavan Rengasayee , Wan Xiaoping , Deschenes Isabelle , Smyth James W. , Hoeker Gregory S. , Gourdie Robert G. , Poelzing Steven
Published: May 2023

Rationale

Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular volume. Ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs.

Objective

Determine the relationship between ventricular CV and structural changes to micro and nano-scale extracellular spaces.

Methods

Conduction and connexin43 (Cx43) gap junction protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with albumin, mannitol, dextran 70kDa, or dextran 2MDa. Peak sodium current was quantified from isolated guinea pig ventricular myocytes. Extracellular resistance (Re) was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs.

Results

CV was significantly reduced by mannitol, and increased by albumin, dextran 70kDa and 2MDa. The combination of albumin and dextran 70kDa decreased CV relative to albumin alone. Rewas reduced by mannitol, not significantly changed by albumin, and increased by both dextran 70kDa and dextran 2MDa. Cx43 gap junction expression and conductance, and peak sodium current were not significantly altered by the osmotic agents. The perinexal width in response to osmotic agents, in order of narrowest to widest, was: albumin with dextran 70kDa, albumin or dextran 2MDa alone, dextran 70kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width.

Conclusions

Cardiac conduction does not correlate with bulk tissue impedance, but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume in ventricular myocardium is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.
A high-content screen identified ingenol-3-angelate as an enhancer of B7-H3-CAR T cell activity by increasing B7-H3 protein expression on the target cell surface via PKCα activation
Authors: Lee Ha Won , O’Reilly Carla , Beckett Alex N. , Currier Duane G. , Chen Taosheng , DeRenzo Christopher
Published: May 2023

CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches may ultimately be required to achieve widespread clinical success. In this regard, we developed a novel high-content and high-throughput screen to evaluate 1114 FDA approved drugs to increase expression of the solid tumor antigen B7-H3 in metastatic osteosarcoma cells. In this proof-of-principle screen, we demonstrate that ingenol-3-angelate increased B7-H3 (CD276) mRNA, total protein, and cell surface expression. Mechanistically, ingenol-3-angelate increased B7-H3 expression via protein kinase C alpha activation. Functionally, ingenol-3-angelate induced B7-H3 expression enhanced B7-H3-CAR T cell function, highlighting utility of the approach, and paving the way for expanding this high-throughput and high-content technique to study other tumor and CAR T cell combinations.
Association of genetic variation inCOL11A1with adolescent idiopathic scoliosis
Authors: Yu Hao , Khanshour Anas M. , Ushiki Aki , Otomo Nao , Koike Yoshinao , Einarsdottir Elisabet , Fan Yanhui , Antunes Lilian , Kidane Yared H. , Cornelia Reuel , Sheng Rory , Zhang Yichi , Pei Jimin , Grishin Nick V. , Evers Bret M. , Cheung Jason Pui Yin , Herring John A. , Terao Chikashi , Song You-Qiang , Gurnett Christina A. , Gerdhem Paul , Ikegawa Shiro , Rios Jonathan J. , Ahituv Nadav , Wise Carol A.
Published: May 2023

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than five-fold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near thePAX1gene. Here we sought to define the roles ofPAX1and newly-identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant inCOL11A1encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); P=7.07e-11, OR=1.118). Using CRISPR mutagenesis we generatedPax1knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc (IVD)-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected inPax1-/-spines compared to wildtype. By genetic targeting we found that wildtypeCol11a1expression in costal chondrocytes suppresses expression ofPax1and ofMmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, this suppression was abrogated in the presence of the AIS-associatedCOL11A1P1335Lmutant. Further, we found that either knockdown of the estrogen receptor geneEsr2, or tamoxifen treatment, significantly alteredCol11a1andMmp3expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering aPax1-Col11a1-Mmp3signaling axis in spinal chondrocytes.
Wnt4a is indispensable for genital duct elongation but not for gonadal sex differentiation in the medakaOryzias latipes
Authors: Kanamori Akira , Kitani Ryota , Oota Atsuko , Hirano Koudai , Myosho Taijun , Kobayashi Tohru , Kawamura Kouichi , Kato Naoyuki , Ansai Satoshi , Kinoshita Masato
Published: May 2023

In most vertebrates, oviducts are derived from Mullerian ducts and sperm ducts from Wolffian ducts. In teleosts, however, Mullerian ducts are absent. Wolffian ducts function as nephric ducts in both sexes, and genital ducts are formed by posterior extension of either ovaries or testes. Whether genital ducts of teleosts are newly evolved organs or are a variant of the Mullerian ducts is an important question for evolutionary mechanisms of morphogenesis. One of the genes essential for Mullerian duct formation in mice,wnt4, is expressed in the mesenchyme and induces invagination of the coelomic epithelium and its posterior elongation. Here, we approached to the above question by examining genital duct development in mutants of two Wnt4 genes in medaka (wnt4ais orthologous to mousewnt4andwnt4bparalogous). Thewnt4bmutants had short body but were fertile with normal genital ducts. In contrast, both male and femalewnt4amutants had posterior elongation of the gonads stopped within or just outside the coelom, depending on the mutated alleles. Genetic females of thesclmutants (unable to synthesize testosterone or estrogens) have gonads containing both previtellogenic oocytes and spermatogenic cysts. Their gonads do not have ovarian cavities or sperm duct primordia and are lacking genital ducts completely. The results suggest Wnt4a target organs are posterior parts of the ovarian cavities or the sperm duct primordia. Medakawnt4awas expressed in the mesenchyme ventral to the genital ducts in both sexes. Thus, the aborted elongation of genital ducts in thewnt4amutants, the ortholog of mousewnt4, suggests strongly that mouse Mullerian ducts and teleost genital ducts share homologous developmental processes. To further demonstrate this possible homology, mechanisms of genital duct formation and possible roles of Wnt4 should be compared before and after the appearance of Mullerian ducts in vertebrate phylogeny, namely jawless fish and cartilaginous fish. Additionally,wnt4aandwnt4bsingle mutants or double mutants did not show sex-reversal, suggesting both genes are dispensable for gonadal sex differentiation in medaka. This is in contrast to indispensable function of Wnt4 in mammalian ovarian differentiation.
Plant PAXX has an XLF-like function and stimulates DNA end-joining by the Ku-DNA ligase IV-XRCC4 complex
Authors: Khan Hira , Ochi Takashi
Published: May 2023

Non-homologous end joining (NHEJ) plays a major role in repairing DNA double-strand breaks (DSBs) and is key to genome stability and editing. The minimal core NHEJ proteins, namely Ku70, Ku80, DNA ligase IV and XRCC4, are conserved, but other factors vary in different eukaryotes groups. In plants, known NHEJ proteins are the core factors only, and the molecular mechanism of plant NHEJ remains unclear. Here, we report a previously unidentified plant ortholog of PAXX, the crystal structure of which showed a similar fold to human PAXX. However, plant PAXX has similar molecular functions to human XLF, by directly interacting with Ku70/80 and XRCC4. This suggests that plant PAXX combines the roles of mammalian PAXX and XLF and that these functions merged into a single protein during evolution. This is consistent with a redundant function of PAXX and XLF in mammals.
The effect of host admixture on wild house mouse gut microbiota is weak when accounting for spatial autocorrelation
Authors: Čížková Dagmar , Schmiedová Lucie , Kváč Martin , Sak Bohumil , Macholán Miloš , Piálek Jaroslav , Kreisinger Jakub
Published: May 2023

The question of how interactions between the gut microbiome and vertebrate hosts contribute to host adaptation and speciation is one of the major problems in current evolutionary research. Using bacteriome and mycobiome metabarcoding, we examined how these two components of the gut microbiota vary with the degree of host admixture in secondary contact between two house mouse subspecies (Mus musculus musculusandM. m. domesticus). We used a large dataset collected at two replicates of the hybrid zone and model-based statistical analyses to ensure the robustness of our results. Assuming that the microbiota of wild hosts suffers from spatial autocorrelation, we directly compared the results of statistical models that were spatially naive with those that accounted for spatial autocorrelation. We showed that neglecting spatial autocorrelation can drastically affect the results and lead to misleading conclusions. The spatial analyses showed little difference between subspecies, both in microbiome composition and in individual bacterial lineages. Similarly, the degree of admixture had minimal effects on the gut bacteriome and mycobiome and was caused by changes in a few microbial lineages that correspond to the common symbionts of free-living house mice. In contrast to previous studies, these data do not support the hypothesis that the microbiota plays an important role in host reproductive isolation in this particular model system.
Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes
Authors: Zhang Qiang , Ma Sai , Liu Zhengzhi , Zhu Bohan , Zhou Zirui , Li Gaoshan , Meana J. Javier , González-Maeso Javier , Lu Chang
Published: May 2023

Genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are all based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. Drop-BS takes advantage of the ultrahigh throughput offered by droplet microfluidics to prepare bisulfite sequencing libraries of up to 10,000 single cells within 2 d. We applied the technology to profile mixed cell lines, mouse and human brain tissues to reveal cell type heterogeneity. Drop-BS will pave the way for single-cell methylomic studies requiring examination of a large cell population.
The combination of differential expression and differential network connectivity analyses identifies RNA splicing and processing as common pathways altered with age across human tissues
Authors: Batalha Caio M.P.F. , Fujita André , de Souza-Pinto Nadja C.
Published: May 2023

Transcriptomic changes occur with age, but the extent of their similarities across tissues is not clear. Previous studies have identified no similarity in age-modulated genes in different tissues. In this study, we sought to identify transcriptional changes with age across tissues using differential network analysis, with the premise that differential expression analysis alone is not capable of detecting all the changes in the transcriptional landscape that occur in tissues with age. Our results show major transcriptional alterations not detected by differential expression analysis that can be detected by differential connectivity analysis. Combining these two analyses, we detected genes changing across tissues, enriched in “RNA splicing” and “RNA processing”, and highly connected in protein-protein interaction networks. Co-expression module analyses demonstrated that other genes with tissue-specific variations with age are enriched in pathways that combat accumulation of aberrant RNAs and proteins, which are caused by defective splicing. Additionally, tissues displayed a major reorganization of their genes’ connectivities with age, with most demonstrating convergent connectivity patterns. Our analyses identify genes and processes which transcriptional changes are conserved across tissues, demonstrating a central role for RNA splicing and processing genes and highlighting the importance of differential network analysis for understanding the ageing transcriptome.
A potent MAPK13-14 inhibitor prevents airway inflammation and mucus production
Authors: Keeler Shamus P. , Wu Kangyun , Zhang Yong , Mao Dailing , Li Ming , Iberg Courtney A. , Austin Stephen R. , Glaser Samuel A. , Yantis Jennifer , Podgorny Stephanie , Brody Steven L. , Chartock Joshua R. , Han Zhenfu , Byers Derek E. , Romero Arthur G. , Holtzman Michael J.
Published: May 2023

Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness ofMAPK13versusMAPK14gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively down-regulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.

New and noteworthy

This study describes the discovery of a potent MAPK13-14 inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases (e.g., asthma, COPD, Covid-19, post-viral and allergic respiratory disease) and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.
TGF-β1 activates neutrophil signaling and gene expression but not migration
Authors: Hein Lauren E. , SenGupta Shuvasree , Gunasekaran Gaurie , Johnson Craig , Parent Carole A.
Published: May 2023

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time-and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4(LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.
FHY3/FAR1 transposable elements generate adaptive genetic variation in the Bassia scoparia genome
Authors: Hall Nathan , Chen Jinyi , Saski Christopher A , Matzrafi Maor , Westra Phillip , Gaines Todd A. , Patterson Eric
Published: May 2023

A near complete genome assembly consisting of 14 scaffolds, a total length of 969.6 Mb, and N50 scaffold length of 99.88 Mb, was generated to better understand how transposable element activity has led to adaptive evolution in Bassia scoparia (kochia), an agronomically important weed. The 9 largest scaffolds correspond to the 9 chromosomes of the close relative, Beta vulgaris. From this assembly, 54,387 protein-coding gene loci were annotated. We determined that genes containing Far Red Elongated Hypocotyl 3 (FHY3) or Far-Red Impaired Response 1 (FAR1) functional domains have undergone a large, kochia-specific gene family expansion. We discovered that putative Mutator Don-Robertson (MuDR) transposable elements with detectable FHY3/FAR1 domains were tightly associated with segmental duplications of 5-enolpyruvylshikimate-3-phosphate synthase subsequently conferring resistance to the herbicide glyphosate. Further, we characterized a new MuDR subtype, named here as "Muntjac", which contributes to the evolution of herbicide resistance in kochia through the process of transduplication. Collectively, our study provides insights into the role of a FHY3/FAR1 gene as an active transposable element and contributes new perspectives on the interaction between transposons and herbicide resistance evolution.
ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion
Authors: Menolfi Demis , Lee Brian J. , Zhang Hanwen , Jiang Wenxia , Bowen Nicole E. , Wang Yunyue , Zhao Junfei , Holmes Antony , Gershik Steven , Rabadan Raul , Kim Baek , Zha Shan
Published: May 2023

The ATR kinase, which coordinates cellular responses to DNA replication stress, is also essential for the proliferation of normal unstressed cells. Although its role in the replication stress response is well defined, the mechanisms by which ATR supports normal cell proliferation remain elusive. Here, we show that ATR is dispensable for the viability of G0-arrested naïve B cells. However, upon cytokine-induced proliferation, Atr-deficient B cells initiate DNA replication efficiently in early S phase, but by mid-S phase they display dNTP depletion, fork stalling, and replication failure. Nonetheless, productive DNA replication can be restored in Atr-deficient cells by pathways that suppress origin firing, such as downregulation of CDC7 and CDK1 kinase activities. Together, these findings indicate that ATR supports the proliferation of normal unstressed cells by tempering the pace of origin firing during the early S phase to avoid exhaustion of dNTPs and other replication factors.
Replication initiation in bacteria: precision control based on protein counting
Authors: Fu Haochen , Xiao Fangzhou , Jun Suckjoon
Published: May 2023

Balanced biosynthesis is the hallmark of bacterial cell physiology, where the concentrations of stable proteins remain steady. However, this poses a conceptual challenge to modeling the cell-cycle and cell-size controls in bacteria, as prevailing concentration-based eukaryote models are not directly applicable. In this study, we revisit and significantly extend the initiator-titration model, proposed thirty years ago, and explain how bacteria precisely and robustly control replication initiation based on the mechanism of protein copy-number sensing. Using a mean-field approach, we first derive an analytical expression of the cell size at initiation based on three biological mechanistic control parameters for an extended initiator-titration model. We also study the stability of our model analytically and show that initiation can become unstable in multifork replication conditions. Using simulations, we further show that the presence of the conversion between active and inactive initiator protein forms significantly represses initiation instability. Importantly, the two-step Poisson process set by the initiator titration step results in significantly improved initiation synchrony withCV∼ 1/Nscaling rather than the standardscaling in the Poisson process, whereNis the total number of initiators required for initiation. Our results answer two long-standing questions in replication initiation: (1) Why do bacteria produce almost two orders of magnitude more DnaA, the master initiator proteins, than required for initiation? (2) Why does DnaA exist in active (DnaA-ATP) and inactive (DnaA-ADP) forms if only the active form is competent for initiation? The mechanism presented in this work provides a satisfying general solution to how the cell can achieve precision control without sensing protein concentrations, with broad implications from evolution to the design of synthetic cells.
Divergent evolution of sleep functions
Authors: Joyce Michaela , Falconio Federica A. , Blackhurst Laurence , Prieto-Godino Lucia , French Alice S. , Gilestro Giorgio F.
Published: May 2023

Most living organisms have evolved to synchronize their biological activities with the earth’s rotation, a daily regulation of biology and behaviour controlled by an evolutionary conserved molecular machinery known as the circadian clock. For most animals, circadian mechanisms are meant to maximize their exposure to positive activities (e.g.:social interactions, mating, feeding – generally during the day) and minimize their exposure to peril (e.g.:predation, weather, darkness – generally during the night1). On top of circadian regulation, some behaviours also feature a second layer of homeostatic control acting as a fail-safe to ensure important activities are not ignored. Sleep is one of these behaviours: largely controlled by the circadian clock for its baseline appearance, it is at the same time modulated by a – poorly understood – homeostatic regulator ensuring animals obey their species-specific amount of daily sleep2. An evolutionary conserved homeostatic control is often considered the main evidence for a core biological function of sleep beyond the trivial one (that is: keeping us out of trouble by limiting our energy expenditure and exposure to danger3,4) and it is hypothesized that sleep evolved around this mysterious basic biological function. Here we characterize sleep regulation in a group of seven species of theDrosophilagenus at key evolutionary distances and representing a variety of ecological niche adaptations. We show that the spontaneous circadian-driven aspects of sleep are conserved among all species but the homeostatic regulation, unexpectedly, is not. We uncover differences in the behavioural, cell-biological and neuro-pharmacological aspects of sleep and suggest that, in Drosophilids, sleep primarily evolved to satisfy a circadian role, keeping animals immobile during dangerous hours of the day. The homeostatic functions of sleep evolved independently, in a species-specific fashion, and are not conserved.
Does stress make males? An experiment on the role of glucocorticoids in anuran sex reversal
Authors: Bókony Veronika , Kalina Csenge , Ujhegyi Nikolett , Mikó Zsanett , Lefler Kinga Katalin , Vili Nóra , Gál Zoltán , Gabor Caitlin R. , Hoffmann Orsolya Ivett
Published: May 2023

Environmentally sensitive sex determination may help organisms adapt to environmental change but also makes them vulnerable to anthropogenic stressors, with diverse consequences for population dynamics and evolution. The mechanisms translating environmental stimuli to sex are controversial: although several fish experiments supported the mediator role of glucocorticoid hormones, results on some reptiles challenged it. We tested this hypothesis in amphibians by investigating the effect of corticosterone on sex determination in agile frogs (Rana dalmatina). This species is liable to environmental sex reversal whereby genetic females develop into phenotypic males. After exposing tadpoles during sex determination to waterborne corticosterone, the proportion of genetic females with testes or ovotestes increased from 11% to up to 32% at 3 out of 4 concentrations. These differences were not statistically significant except for the group treated with 10 nM corticosterone, and there was no monotonous dose-effect relationship. These findings suggest that corticosterone is unlikely to mediate sex reversal in frogs. Unexpectedly, animals originating from urban habitats had higher sex-reversal and corticosterone-release rates, reduced body mass and development speed, and lower survival compared to individuals collected from woodland habitats. Thus, anthropogenic environments may affect both sex and fitness, and the underlying mechanisms may vary across ectothermic vertebrates.
Elevated sleep need in a stress-resilientDrosophilaspecies
Authors: Yano Jessica , Nave Ceazar , Larratt Katherine , Honey Phia , Jingco Cassandra , Roberts Makayla , Trotter Damion , He Xin , Elezi Gazmend , Whitelegge Julian P. , Wasserman Sara , Donlea Jeffrey M.
Published: May 2023

Sleep is broadly conserved across the animal kingdom, but can vary widely between species. It is currently unclear which types of selective pressures and sleep regulatory mechanisms influence differences in sleep between species. The fruit flyDrosophila melanogasterhas become a successful model system for examining sleep regulation and function, but little is known about the sleep patterns and need for sleep in many related fly species. Here, we find thatDrosophila mojavensis, a fly species that has adapted to extreme desert environments, exhibits strong increases in sleep compared toD. melanogaster.Long-sleepingD. mojavensisshow intact sleep homeostasis, indicating that these flies carry an elevated need for sleep. In addition,D. mojavensisexhibit altered abundance or distribution of several sleep/wake related neuromodulators and neuropeptides that are consistent with their reduced locomotor activity, and increased sleep. Finally, we find that in a nutrient-deprived environment, the sleep responses of individualD. mojavensisare correlated with their survival time. Our results demonstrate thatD. mojavensisis a novel model for studying organisms with high sleep need, and for exploring sleep strategies that provide resilience in extreme environments.
Gene editing withoutex vivoculture evades genotoxicity in human hematopoietic stem cells
Authors: Zeng Jing , Nguyen My Anh , Liu Pengpeng , Ferreira da Silva Lucas , Lin Linda Y. , Justus David G. , Petri Karl , Clement Kendell , Porter Shaina N. , Verma Archana , Neri Nola R. , Rosanwo Tolulope , Ciuculescu Marioara-Felicia , Abriss Daniela , Mintzer Esther , Maitland Stacy A. , Demirci Selami , Tisdale John F. , Williams David A. , Zhu Lihua Julie , Pruett-Miller Shondra M. , Pinello Luca , Joung J. Keith , Pattanayak Vikram , Manis John P. , Armant Myriam , Pellin Danilo , Brendel Christian , Wolfe Scot A. , Bauer Daniel E.
Published: May 2023

Gene editing theBCL11Aerythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of theBCL11A+58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of theBCL11A+58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.
FluoMALDI microscopy: matrix co-crystallization simultaneously enhances fluorescence and MALDI imaging
Authors: Yang Ethan , Shen Xinyi Elaine , West-Foyle Hoku , Brown Dalton R. , Johnson Cole C. , Kim Jeong Hee , Roker LaToya Ann , Tressler Caitlin M. , Barman Ishan , Kuo Scot C. , Glunde Kristine
Published: May 2023

We report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. Our approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm eliminates the notion that MALDI matrices obscure and obstruct optical microscopy approaches, allowing to image the exact same cells in tissues, free of any physical changes between fluorescence and MALDI imaging, which minimizes data registration processes. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. We showcase the capabilities of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.
CD5L is a canonical component of circulatory IgM
Authors: Oskam Nienke , den Boer Maurits A. , Lukassen Marie V. , Heer Pleuni Ooijevaar-de , Veth Tim S. , Mierlo Gerard van , Lai Szu-Hsueh , Derksen Ninotska I.L. , Yin Victor C. , Streutker Marij , Franc Vojtech , Siborova Marta , Damen Mirjam , Kos Dorien , Barendregt Arjan , Bondt Albert , Goudoever Johannes B. van , Haas Carla J.C. , Aerts Piet C. , Muts Remy M. , Rooijakkers Suzan H.M. , Vidarsson Gestur , Rispens Theo , Heck Albert J.R.
Published: May 2023

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small CD5 antigen-like (CD5L, 36 kDa) protein. In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor (pIgR). On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.
Ontogeny and social context regulate the circadian activity patterns of Lake Malawi cichlids
Authors: Lloyd Evan , Rastogi Aakriti , Holtz Niah , Aaronson Ben , Albertson R. Craig , Keene Alex C.
Published: May 2023

Activity patterns tend to be highly stereotyped and critical for executing many different behaviors including foraging, social interactions and predator avoidance. Differences in the circadian timing of locomotor activity and rest periods can facilitate habitat partitioning and the exploitation of novel niches. As a consequence, closely related species often display highly divergent activity patterns, raising the possibility that a shift from diurnal to nocturnal behavior, or vice versa, can occur rapidly. In Africa’s Lake Malawi alone, there are over 500 species of cichlids, which inhabit diverse environments and exhibit extensive phenotypic variation. We have previously identified a substantial range in activity patterns across adult Lake Malawi cichlid species, from strongly diurnal to strongly nocturnal. In many species, including fishes, ecological pressures differ dramatically across life-history stages, raising the possibility that activity patterns may change over ontogeny. To determine if rest-activity patterns change across life stages we compared the locomotor patterns of six Lake Malawi cichlid species. While total rest and activity did not change between early juvenile and adult stages, rest-activity patterns did, with juveniles displaying distinct activity rhythms that are more robust than adults. One distinct difference between juveniles and adults is the emergence of complex social behavior. To determine whether social context is required for activity rhythms, we next measured locomotor behavior in group housed adult fish. We found that when normal social interactions were allowed, locomotor activity patterns were restored, supporting the notion that social interactions promote circadian regulation of activity in adult fish. These findings reveal a previously unidentified link between developmental stage and social interactions in the circadian timing of cichlid activity.
Symbiotic bacteria, immune-like sentinel cells, and the response to pathogens in a social amoeba
Authors: Scott Trey J. , Larsen Tyler J. , Brock Debra A. , Uhm So Yeon Stacey , Queller David C. , Strassmann Joan E.
Published: May 2023

Some endosymbionts living within a host must modulate their hosts’ immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a facultatively multicellular social amoeba host. Aggregates of the amoebaDictyostelium discoideumcontain a subpopulation of sentinel cells that function akin to the immune systems of more conventional multicellular organisms. Sentinel cells sequester and discard toxins fromD. discoideumaggregates and may play a central role in defense against pathogens. We measured the number and functionality of sentinel cells in aggregates ofD. discoideuminfected by bacterial endosymbionts in the genusParaburkholderia.InfectedD. discoideumproduced fewer and less functional sentinel cells, suggesting thatParaburkholderiamay interfere with its host’s immune system. Despite impaired sentinel cells, however, infectedD. discoideumwere less sensitive to ethidium bromide toxicity, suggesting thatParaburkholderiamay also have a protective effect on its host. By contrast,D. discoideuminfected byParaburkholderiadid not show differences in their sensitivity to two non-symbiotic pathogens. Our results expand previous work on yet another aspect of the complicated relationship betweenD. discoideumandParaburkholderia, which has considerable potential as a model for the study of symbiosis.
Expression and purification of the mitochondrial transmembrane protein FAM210A inEscherichia coli
Authors: Hollinger Jared , Wu Jiangbin , Awayda Kamel M. , O’Connell Mitchell R. , Yao Peng
Published: May 2023

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His10fusion inEscherichia coli. The recombinant FAM210A protein was inserted into theE. colicell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed withE.coliderived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.
Pathogenic variants in Crx have distinct cis-regulatory effects on enhancers and silencers in photoreceptors
Authors: Shepherdson James L. , Friedman Ryan Z. , Zheng Yiqiao , Sun Chi , Oh Inez Y. , Granas David M. , Cohen Barak A. , Chen Shiming , White Michael A.
Published: May 2023

Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with human blinding diseases that vary in their severity and age of onset. It is unclear how different variants in this single TF alter its function in ways that lead to a range of phenotypes. We examined the effects of human disease-causing variants on CRXcis-regulatory function by deploying massively parallel reporter assays (MPRAs) in live mouse retinas carrying knock-ins of two variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation caused by the variants corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, while p.E168d2 has stronger effects on silencers.Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are de-repressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci were partially predictive of episomal MPRA activity, and silencers were notably enriched among distal elements whose accessibility increases later in retinal development. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on itscis-regulatory function, leading to misregulation of similar sets of enhancers, while having a qualitatively different impact on silencers.
BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome
Authors: Zhong Hui , Janer Alexandre , Khalimonchuk Oleh , Antonicka Hana , Shoubridge Eric A , Barrientos Antoni
Published: May 2023

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from “multiple mitochondrial dysfunction” syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly.
Toward single cell tattoos: Biotransfer printing of lithographic gold nanopatterns on live cells
Authors: Kwok Kam Sang , Zuo Yi , Choi Soo Jin , Pahapale Gayatri J. , Gu Luo , Gracias David H.
Published: May 2023

Lithographic nanopatterning techniques like photolithography, electron-beam lithography, and nanoimprint lithography (NIL) have revolutionized modern-day electronics and optics. Yet, their application for creating nano-bio interfaces is limited by the cytotoxic and two-dimensional nature of conventional fabrication methods. Here, we present a biocompatible and cost-effective transfer process that leverages (a) NIL to define sub-300 nm gold (Au) nanopattern arrays, (b) amine functionalization of Au to transfer the NIL-arrays from a rigid substrate to a soft transfer layer, (c) alginate hydrogel as a flexible, degradable transfer layer, and (d) gelatin conjugation of the Au NIL-arrays to achieve conformal contact with live cells. We demonstrate biotransfer printing of the Au NIL-arrays on rat brains and live cells with high pattern fidelity and cell viability and observed differences in cell migration on the Au NIL-dot and NIL-wire printed hydrogels. We anticipate that this nanolithography-compatible biotransfer printing method could advance bionics, biosensing, and biohybrid tissue interfaces.

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