bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo proximity proteomics uncovers palmdelphin (PALMD) as a Z-line-associated mitigator of isoproterenol-induced cardiac injury
Authors: Guo Congting , Jardin Blake D. , Lin Junsen , Ambroise Rachelle L. , Wang Ze , Yang Luzi , Mazumdar Neil , Lu Fujian , Ma Qing , Cao Yangpo , Liu Canzhao , Liu Xujie , Lan Feng , Zhao Mingming , Xiao Han , Dong Erdan , Pu William T. , Guo Yuxuan
Published: December 2023

Z-lines are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-line-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-line proteome in vivo. We found palmdelphin (PALMD) as a novel Z-line-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specificpalmdknockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed transverse tubules (T-tubules) and their association with sarcoplasmic reticulum, which formed the Z-line-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with disrupted localization of T-tubule markers caveolin-3 (CAV3) and junctophilin-2 (JPH2) and the reduction of nexilin (NEXN) protein, a crucial Z-line-associated protein that is essential for both Z-line and JMC structures and functions. PALMD was found to interact with NEXN and enhance its protein stability while theNexnmRNA level was not affected. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis.

Highlights

In vivo proximity proteomics uncover novel Z-line components that are undetected in in vitro proximity proteomics in cardiomyocytes.

PALMD is a novel Z-line-associated protein that is dispensable for baseline cardiomyocyte function in vivo.

PALMD mitigates cardiac dysfunction and myocardial injury after repeated isoproterenol insults.

PALMD stabilizes NEXN, an essential Z-line-associated regulator of the junctional membrane complex and cardiac systolic function.
The Molecular Logic of Gtr1/2 and Pib2 Dependent TORC1 Regulation in Budding Yeast
Authors: Cecil Jacob H. , Padilla Cristina M. , Lipinski Austin A. , Langlais Paul R. , Luo Xiangxia , Capaldi Andrew P.
Published: December 2023

The Target of Rapamycin kinase Complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that, inSaccharomyces cerevisiae, nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2, and the phosphatidylinositol 3-phosphate binding protein, Pib2. However, it was unclear if/how Gtr1/2 and Pib2 cooperate to control TORC1. Here we report that this dual regulator system pushes TORC1 into three distinct signaling states: (i) a Gtr1/2 on, Pib2 on, rapid growth state in nutrient replete conditions; (ii) a Gtr1/2 off, Pib2 on, adaptive/slow growth state in poor-quality growth medium; and (iii) a Gtr1/2 off, Pib2 off, quiescent state in starvation conditions. We suggest that other signaling pathways work in a similar way, to drive a multi-level response via a single kinase, but the behavior has been overlooked since most studies follow signaling to a single reporter protein.
Garden, greenhouse or climate chamber? Experimental conditions influence whether genetic differences are phenotypically expressed
Authors: Karitter Pascal , March-Salas Martí , Ensslin Andreas , Rauschkolb Robert , Godefroid Sandrine , Poorter Hendrik , Scheepens Johannes F.
Published: December 2023

Common-environment experiments are important to study genetically-based phenotypic variation within and among plant populations. Such experiments can be performed in an experimental garden, greenhouse or climate chamber. However, phenotypic expression may be strongly affected by the environmental conditions and influenced by parental and storage effects. Hence, it is unclear if results from common-environment experiments are reproducible across multiple experimental setups.

In this study, we assessed the effects of three different growth facilities – outdoor garden, greenhouse, and climate chamber –, on phenotypic expression. We compared ancestral and descendant genotypes of the same population ofLeontodon hispidus. We also evaluated differences in phenotypic expression between plants grown after one (F1) vs. two (F2) intermediate generations.

We observed strong differences among plants growing in different growth facilities. Furthermore, we found that descendants had larger rosettes than ancestors only in the greenhouse and they flowered later than ancestors exclusively in the climate chamber. We did not find significant differences between intermediate generations within the growth facilities.

Overall, our study demonstrates that environmental variation among growth facilities can dictate the presence and magnitude of phenotypic differences. This implies that absence of evidence for phenotypic differences is not evidence of absence. Experimental systems should be carefully designed to provide meaningful conditions related to the research question. Finally, growing a second intermediate generation did not impact the genetic differences of ancestors and descendants within the facilities, supporting that only one intermediate generation may be sufficient to reduce detectable parental and storage effects.
Causal inference for multiple risk factors and diseases from genomics data
Authors: Machnik Nick , Mahmoudi Mahdi , Borczyk Malgorzata , Krätschmer Ilse , Bauer Markus J. , Robinson Matthew R.
Published: December 2023

Statistical causal learning in genomics relies on the instrumental variable method of Mendelian Randomization (MR). Currently, an overwhelming number of MR studies purport to show causal relationships among a wide range of risk factors and outcomes. Here, we show that selecting instrument variables from genome-wide association study estimates leads to high false discovery rates for many MR approaches, which can be greatly reduced by employing a graphical inference approach which: (i) explicitly tests instrumental variable assumptions; (ii) distinguishes direct from indirect factors in very high-dimensional data; (iii) discriminates pleiotropic from trait-specific markers, controlling for LD genome-wide; (iv) accommodates rare variants and binary outcomes in a principled way; and (v) identifies potential unobserved latent confounding. For 17 traits and 8.4M variants recorded for 458,747 individuals in the UK Biobank, we show that standard MR analysis gives an abundance of findings that disappear under stringent assumption checks, with many relationships reflecting potential unmeasured confounding. This implies that mixtures of temporal precedence and potential for reverse-causality prohibit understanding the underlying nature of phenotypic and genetic correlations in biobank data. We propose that well-curated longitudinal records are likely needed and that our approach provides a first-step toward robust principled screening for potential causal links.
Reduced Survival Expectancy Weakens Reproductive Isolation Between Annual FishesGarcialebias reichertiandG. charrua
Authors: Rivas-Ortiz Noelle , Passos Carlos
Published: December 2023

Hybridization depends on reproductive isolation, which can be impacted by mate choice. Mate choice may vary temporally, as it is modulated by several factors, including survival expectancy and future opportunities for reproduction.Garcialebias reichertiandG. charruaare annual fishes with parapatric distributions that hybridize in the overlapping area of their distributions. They inhabit temporary ponds that flood during the autumn and dry out during the spring, resulting in decreased survival expectancy and future opportunities for reproduction during the breeding season. We predicted that a decrease in survival expectancy would promote reproduction and reduce reproductive isolation betweenG. reichertiandG charrua. By simulating desiccation in the early and late breeding season, we investigated the effects of the desiccation risk and the phase of the breeding season on reproductive isolation and reproductive effort of these species. As expected, our findings reveal that decreased survival expectancy influences both reproductive isolation betweenG. reichertiand G. charrua, and their reproductive effort. Notably, reproductive isolation between these species decreased under a high desiccation risk and in the late breeding season. Additionally, we observed an increase in the frequency of mating and courtship events and aggressive behaviours in the late breeding season. Our study suggests that reproductive isolation betweenG. reichertiandG. charruaand their reproductive effort can change rapidly within a short period of time, emphasizing the influence of survival expectancy on the temporal dynamics of reproductive isolation and hybridization.
Doxycycline Levofloxacin or Moxifloxacin are superior to ciprofloxacin in treating anthrax meningitis in rabbits
Authors: Ben-Shmuel Amir , Glinert Itai , Sittner Assa , Bar-David Elad , Schlomovitz Josef , Levy Haim , Weiss Shay
Published: December 2023

Efficient treatment of anthrax related meningitis in patients poses a significant therapeutic challenge. Previously we demonstrated in our anthrax meningitis rabbit model that ciprofloxacin treatment in ineffective with most of the treated animals succumbing to the infection. Herein we tested the efficacy of Doxycycline in our rabbit model and found it highly effective. To test whether the low efficacy of Ciprofloxacin is an example of low efficacy of all fluoroquinolones or only this substance, we treated rabbits that were inoculated intra cisterna magna (ICM) with Levofloxacin or Moxifloxacin. We found that in contrast to Ciprofloxacin, Levofloxacin and Moxifloxacin were highly efficient in treating lethal anthrax related meningitis in rabbits. We demonstrated (in naïve rabbits) that this deference probably results from variances in blood brain barrier (BBB) penetration of the different fluoroquinolones. The combined treatment of doxycycline and any one of the tested fluoroquinolones was highly effective in the rabbit CNS infection model. The combined treatment of doxycycline and levofloxacin was effective in inhalation rabbit model, as good as the doxycycline monotherapy. These findings imply that while Ciprofloxacin is highly effective as a post exposure prophylactic drug, using this drug to treat symptomatic patients should be reconsidered.
A new efficacious Mcl-1 inhibitor maximizes bortezomib and venetoclax responsiveness in resistant multiple myeloma cells
Authors: Al-Odat Omar S. , Gowda Krishne , Srivastava Sandeep K. , Amin Shantu G , Jonnalagadda Subash C. , Budak-Alpdogan Tulin , Pandey Manoj K.
Published: December 2023

Despite a record number of clinical studies investigating various anti-cancer drugs, the 5-year survival rate for multiple myeloma (MM) patients in the United States is only 55%, and nearly all patients relapse. Poor patient outcomes demonstrate that myeloma cells are “born to survive,” which means they can adapt and evolve following treatment. As a result, new therapeutic approaches to combat this survival mechanism and target treatment-resistant malignant cells are required. Mcl-1, an anti-apoptotic protein, is required for the development of MM and resistance to therapy. This study looks at the possibility of KS18, a Mcl-1 inhibitor derived from pyoluteorin, to treat resistant MM. We show that KS18 inhibits Mcl-1 selectively and promotes post-translational modifications, resulting in UPS-dependent Mcl-1 degradation. Our findings show that KS18-induced Mcl-1 degradation results in caspase-dependent apoptosis. Importantly, KS18 triggered apoptosis in MM patient samples and bortezomib-resistant cells, synergizing with venetoclax to boost apoptosis. Furthermore, KS18 inhibits colony formation in bortezomib-resistant cells. KS18 treated NSG mice displayed significant tumor shrinkage without significant toxicity after four weeks of therapy with a single acceptable dose each week, indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance.

Key points

Given that KS18 is a robust Mcl-1 inhibitor that targets Mcl-1 efficiently, it has the potential to be a novel treatment for multiple myeloma.

KS18 has shown promise in re-sensitizing myeloma cells to chemotherapy as well as in overcoming resistance to bortezomib, venetoclax, and ABT-737.
Dietary restriction of individual amino acids stimulates unique molecular responses in mouse liver
Authors: Haws Spencer A. , Liu Yang , Green Cara L. , Babygirija Reji , Armstrong Eric A. , Mehendale Anusha T. , Lamming Dudley W. , Denu John M.
Published: December 2023

Dietary protein and essential amino acid (EAA) restriction promotes favorable metabolic reprogramming, ultimately resulting in improvements to both health and lifespan. However, as individual EAAs have distinct catabolites and engage diverse downstream signaling pathways, it remains unclear to what extent shared or AA-specific molecular mechanisms promote diet-associated phenotypes. Here, we investigated the physiological and molecular effects of restricting either dietary methionine, leucine, or isoleucine (Met-R, Leu-R, and Ile-R) for 3 weeks in C57BL/6J male mice. While all 3 AA-depleted diets promoted fat and lean mass loss and slightly improved glucose tolerance, the molecular responses were more diverse; while hepatic metabolites altered by Met-R and Leu-R were highly similar, Ile-R led to dramatic changes in metabolites, including a 3-fold reduction in the oncometabolite 2-hydroxyglutarate. Pathways regulated in an EAA-specific manner included glycolysis, the pentose phosphate pathway (PPP), nucleotide metabolism, the TCA cycle and amino acid metabolism. Transcriptiome analysis and global profiling of histone post-translational modifications (PTMs) revealed different patterns of responses to each diet, although Met-R and Leu-R again shared similar transcriptional responses. While the pattern of global histone PTMs were largely unique for each dietary intervention, Met-R and Ile-R had similar changes in histone-3 methylation/acetylation PTMs at lysine-9. Few similarities were observed between the physiological or molecular responses to EAA restriction and treatment with rapamycin, an inhibitor of the mTORC1 AA-responsive protein kinase, indicating the response to EAA restriction may be largely independent of mTORC1. Together, these results demonstrate that dietary restriction of individual EAAs has unique, EAA-specific effects on the hepatic metabolome, epigenome, and transcriptome, and suggests that the specific EAAs present in dietary protein may play a key role at regulating health at the molecular level.
The evolutionary dynamics of locally adaptive chromosome inversions inMimulus guttatus
Authors: Kollar Leslie M. , Stanley Lauren E. , Kenchanmane Raju Sunil K. , Lowry David B. , Niederhuth Chad E.
Published: December 2023

Chromosomal inversion polymorphisms are ubiquitous across the diversity of diploid organisms and play a significant role in the evolution of adaptations in those species. Inversions are thought to operate as supergenes by trapping adaptive alleles at multiple linked loci through the suppression of recombination. While there is now considerable support for the supergene mechanism of inversion evolution, the extent to which inversions trap pre-existing adaptive genetic variation versus accumulate new adaptive variants over time remains unclear. In this study, we report new insights into the evolutionary dynamics of a locally adaptive chromosomal inversion polymorphism (inv_chr8A), which contributes to the evolutionary divergence between coastal perennial and inland annual ecotypes of the yellow monkeyflower,Mimulus guttatus. This research was enabled by the sequencing, assembly, and annotation of new annual and perennial genomes ofM. guttatususing Oxford Nanopore long-read sequencing technology. In addition to the adaptive inv_chr8A inversion, we identified three other large inversion polymorphisms, including a previously unknown large inversion (inv_chr8B) nested within the inv_chr8A. Through population genomic analyses and comparative genomics, we determined that the nested inv_chr8B inversion is significantly older than the larger chromosomal inversion in which it resides. We also evaluated key candidate genes involved in gibberellin biosynthesis and anthocyanin regulation, which we hypothesize to underlie the adaptive phenotypic effects of the inv_chr8A inversion. Although little evidence was found to suggest that inversion breakpoint mutations drive adaptive phenotypic effects, our findings support the supergene mechanism of adaptive evolution as a dynamic and continuous process.
Hyper-specialized primates possess a reduced suite of xenobiotic-metabolizing cytochrome P450 genes
Authors: Chaney Morgan E. , Tosi Anthony J. , Bergey Christina M.
Published: December 2023

Subfamilies of cytochrome P450 proteins have been strongly linked to the metabolism of physiologically disruptive compounds such as alkaloids, terpenoids, and other xenobiotics. Consistent with this function, these genes have adaptively evolved in response to environmental pressures exerted on animals, such as herbivores, that consume elevated amounts of toxic xenobiotics or plant secondary metabolites (PSMs). Theory on evolutionary tradeoffs predicts that highly specialized herbivores should exhibit a relatively narrow toolkit of adaptations to accommodate the concomitantly narrow arrays of PSMs in their diets. The bamboo lemurs of Madagascar (generaProlemurandHapalemur) represent an interesting test case for this theory because of their dietary hyper-specialization, as these lemurs consume bamboo and grasses at rates otherwise unseen in the order Primates. To test whether the hyper-specialized folivory of these primates is reflected in a similarly specialized and narrow P450 gene suite, we assembled a dataset of confidently assembledCYP1-3genes for two species of bamboo lemur as well as additional lemur species. We tested the predictions that bamboo lemurs would exhibit, first, greater rates of gene loss for xenobiotic-metabolizing P450s and, second, relaxed selection on xenobiotic-metabolizing P450 subfamilies relative to lemurs without such dietary hyper-specialization. We found support for the first prediction, related to gene loss, in theCYP2B, CYP2C, CYP2D, CYP2J,andCYP3Asubfamilies, all of which encode xenobiotic metabolizers. We additionally inferred relaxation of selection for theCYP2FandCYP2Jsubfamilies. The evolution of the P450 genes in bamboo lemurs provides support for the evolutionary tradeoff hypothesis, and we further hypothesize that, rather than adapting to a general array of PSMs, bamboo lemurs have instead adapted to the primary toxin in their diet, the highly potent poison cyanide.

Highlights

Some of the most specialized diets among primates are those of bamboo lemurs.

Bamboo lemurs have fewer xenobiotic-metabolizing P450 genes than other lemurs.

Natural selection has relaxed on theCYP2FandCYP2Jsubfamilies in bamboo lemurs.
Single Amplified Genome Catalog Reveals the Dynamics of Mobilome and Resistome in the Human Microbiome
Authors: Kawano-Sugaya Tetsuro , Arikawa Koji , Saeki Tatsuya , Endoh Taruho , Kamata Kazuma , Matsuhashi Ayumi , Hosokawa Masahito
Published: December 2023

The increase in metagenome-assembled genomes (MAGs) has significantly advanced our understanding of the functional characterization and taxonomic assignment within the human microbiome. However, MAGs, as population consensus genomes, often mask heterogeneity among species and strains, thereby obfuscating the precise relationships between microbial hosts and mobile genetic elements (MGEs). In contrast, single amplified genomes (SAGs) derived via single-cell genome sequencing can capture individual genomic content, including MGEs. We present the bbsag20 dataset, which encompasses 17,202 human-associated prokaryotic SAGs and 869 MAGs, spanning 647 gut and 312 oral bacterial species. The SAGs revealed diverse bacterial lineages and MGEs with a broad host range that were absent in the MAGs and traced the translocation of oral bacteria to the gut. Importantly, our SAGs linked individual mobilomes to resistomes and meticulously charted a dynamic network of antibiotic resistance genes (ARGs) on MGEs, pinpointing potential ARG reservoirs in the microbial community.
Infant attachment does not depend on neonatal amygdala and hippocampal structure and connectivity
Authors: Jiménez-Sánchez Lorena , Cabez Manuel Blesa , Vaher Kadi , Corrigan Amy , Thrippleton Michael J. , Bastin Mark E. , Quigley Alan J. , Fletcher-Watson Sue , Boardman James P.
Published: December 2023

Infant attachment is an antecedent of later socioemotional abilities, which can be adversely affected by preterm birth. The structural integrity of amygdalae and hippocampi are associated with attachment in childhood. We aimed to investigate associations between neonatal amygdalae and hippocampi structure and their whole-brain connections and attachment behaviours at nine months of age in a sample of infants enriched for preterm birth. In 133 neonates (median gestational age 32 weeks, range 22.14–42.14), we calculated measures of amygdala and hippocampal structure (volume, fractional anisotropy, mean diffusivity, neurite dispersion index, orientation dispersion index) and structural connectivity, and coded attachment behaviours (distress, fretfulness, attentiveness to caregiver) from responses to the Still-Face Paradigm at nine months. After multiple comparisons correction, there were no significant associations between neonatal amygdala or hippocampal structure and structural connectivity and attachment behaviours: standardised β values -0.23 to 0.21, adjusted p-values > 0.34. Findings indicate that the neural basis of infant attachment in term and preterm infants is not contingent on the structure or connectivity of the amygdalae and hippocampi in the neonatal period, which implies that it is more widely distributed in early life and or that network specialisation takes place in the months after hospital discharge.

Highlights

133 infants had brain MRI and attachment data based on the Still-Face Paradigm.

The structure of amygdalae and hippocampi and their brain networks was examined.

Neonatal amygdalae/hippocampi structure did not associate with infant attachment.

Infant attachment is not contingent on neonatal amygdala/hippocampal connectivity.

Graphical abstract
From Text to Tables: A Local Privacy Preserving Large Language Model for Structured Information Retrieval from Medical Documents
Authors: Wiest Isabella C. , Ferber Dyke , Zhu Jiefu , van Treeck Marko , Meyer Sonja K. , Juglan Radhika , Carrero Zunamys I. , Paech Daniel , Kleesiek Jens , Ebert Matthias P. , Truhn Daniel , Kather Jakob Nikolas
Published: December 2023

Background and Aims

Most clinical information is encoded as text, but extracting quantitative information from text is challenging. Large Language Models (LLMs) have emerged as powerful tools for natural language processing and can parse clinical text. However, many LLMs including ChatGPT reside in remote data centers, which disqualifies them from processing personal healthcare data. We present an open-source pipeline using the local LLM “Llama 2” for extracting quantitative information from clinical text and evaluate its use to detect clinical features of decompensated liver cirrhosis.

Methods

We tasked the LLM to identify five key clinical features of decompensated liver cirrhosis in a zero- and one-shot way without any model training. Our specific objective was to identify abdominal pain, shortness of breath, confusion, liver cirrhosis, and ascites from 500 patient medical histories from the MIMIC IV dataset. We compared LLMs with three different sizes and a variety of pre-specified prompt engineering approaches. Model predictions were compared against the ground truth provided by the consent of three blinded medical experts.

Results

Our open-source pipeline yielded in highly accurate extraction of quantitative features from medical free text. Clinical features which were explicitly mentioned in the source text, such as liver cirrhosis and ascites, were detected with a sensitivity of 100% and 95% and a specificity of 96% and 95%, respectively from the 70 billion parameter model. Other clinical features, which are often paraphrased in a variety of ways, such as the presence of confusion, were detected only with a sensitivity of 76% and a specificity of 94%. Abdominal pain was detected with a sensitivity of 84% and a specificity of 97%. Shortness of breath was detected with a sensitivity of 87% and a specificity of 96%. The larger version of Llama 2 with 70b parameters outperformed the smaller version with 7b parameters in all tasks. Prompt engineering improved zero-shot performance, particularly for smaller model sizes.

Conclusion

Our study successfully demonstrates the capability of using locally deployed LLMs to extract clinical information from free text. The hardware requirements are so low that not only on-premise, but also point-of-care deployment of LLMs are possible.

Lay summary

We leveraged the large language model Llama 2 to extract five key features of decompensated liver cirrhosis from medical history texts, simplifying the analysis of complex text-based healthcare data.
Cell-state transitions and frequency-dependent interactions among subpopulations together explain the dynamics of spontaneous epithelial-mesenchymal heterogeneity in breast cancer
Authors: Jain Paras , Kizhuttil Ramanarayanan , Nair Madhav B , Bhatia Sugandha , Thompson Erik W , George Jason T. , Jolly Mohit Kumar
Published: December 2023

Individual cells in a tumour can be distributed among Epithelial (E) and Mesenchymal (M) cell-states, as characterised by the levels of canonical E and M markers. Even after E and M (E-M) subpopulations are isolated and then cultured independently, E-M heterogeneity can re-equilibrate in each population over time, sometimes regaining the initial distribution of the parental cell population. However, it remains unclear which population-level processes give rise to the dynamical changes in E-M heterogeneity observed experimentally, including 1) differential growth, 2) cell-state switching, and 3) frequency-dependent growth or state-transition rates. Here, we analyse the necessity of these three processes in explaining the dynamics of E-M population distributions as observed in PMC42-LA and HCC38 breast cancer cells. We find that growth differences among E and M subpopulations, with and without any frequency-dependent interactions (cooperation or suppression) among E-M sub-populations, are insufficient to explain the observed population dynamics. This insufficiency is ameliorated by including cell-state transitions, albeit at slow rates, in explaining both PMC42-LA and HCC38 cells data. Further, our models predict that treatment of HCC38 cells with TGFβ signalling and JAK2/3 inhibitors could significantly enhance the transition rates from M state to E state, but does not prevent transitions from E to M. Finally, we devise a selection criterion to identify the next most informative time points for which future experimental data can optimally improve the identifiability of our estimated best fit model parameters. Overall, our study identifies the necessary population-level processes shaping the dynamics of E-M heterogeneity in breast cancer cells.
Human monoclonal antibodies reveal subdominant gonococcal and meningococcal cross-protective antigens
Authors: Troisi Marco , Fabbrini Monica , Stazzoni Samuele , Viviani Viola , Carboni Filippo , Abbiento Valentina , Fontana Lucia Eleonora , Tomei Sara , Audagnotto Martina , Santini Laura , Spagnuolo Angela , Antonelli Giada , Paciello Ida , Vacca Fabiola , Cardamone Dario , Marini Eleonora , Mokhtary Pardis , Finetti Francesca , Giusti Fabiola , Bodini Margherita , Torricelli Giulia , Limongi Chiara , Del Vecchio Mariangela , Favaron Sara , Tavarini Simona , Sammicheli Chiara , Rossi Alessandro , Mandelli Andrea Paola , Fortini Pietro , Caffarelli Carla , Gonnelli Stefano , Nuti Ranuccio , Baldari Cosima T. , Sala Claudia , Tagliabue Aldo , Savino Silvana , Brunelli Brunella , Norais Nathalie , Frigimelica Elisabetta , Bardelli Monia , Pizza Mariagrazia , Margarit Immaculada , Delany Isabel , Finco Oretta , Andreano Emanuele , Rappuoli Rino
Published: December 2023

Gonococcus (Gc), a bacterium resistant to most antibiotics causing more than 80 million cases of gonorrhea annually, is a WHO high priority pathogen. Recently, vaccine development prospects were boosted by reports that licensed meningococcus serogroup B (MenB) vaccines provided partial protection against Gc infection. To determine antigens responsible for cross-protection, memory B cells from 4CMenB vaccinated volunteers were single-cell sorted to identify antibodies that kill Gc in a bactericidal assay. Nine different antibodies, all deriving from the IGHV4-34 germline carrying unusually long HCDR3s, recognized the PorB protein, four recognized the lipooligosaccharide (LOS), and four unknown antigens. One of the PorB antibodies, tested in vivo, provided protection from Gc infection. The identification of PorB and LOS as key antigens of gonococcal and meningococcal immunity provides a mechanistic explanation of the cross-protection observed in the clinic and shows that isolating human monoclonal antibodies from vaccinees can be instrumental for bacterial antigen discovery.
Control of a type III-Dv CRISPR‒Cas system by the transcription factor RpaB and interaction of its leader transcript with the DEAD-box RNA helicase CrhR
Authors: Bilger Raphael , Migur Angela , Wulf Alexander , Steglich Claudia , Urlaub Henning , Hess Wolfgang R.
Published: December 2023

CRISPR‒Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR‒Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacteriumSynechocystissp. PCC 6803 encodes three different CRISPR‒Cas systems. The expression of one of these, a type III-Dv system, responds to changes in environmental conditions, such as nitrogen starvation or varying light intensities. Here, we found that the promoter of the six-genecasoperon for the type III-Dv system is controlled by the light-and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif located 53 to 70 nt upstream of the transcription start site, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, we found that the 125 nt leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled to mass spectrometry analysis revealed six residues involved in the CrhR-RNA interaction. Of these, L103, F104, H225, and C371 were predicted to be on the surface of a dimeric CrhR model, while C184 was not on the surface, and P443 could not be assigned to a structural element. These results showed that the expression of the CRISPR‒Cas system is linked to the redox status of the photosynthetic cyanobacterial cell at two different levels. While RpaB affects transcription, CrhR interacts with the leader transcript posttranscription. These results highlight the complex interplay between a CRISPR‒Cas system and its host cell.
Syndrome-informed phenotyping identifies a polygenic background for achondroplasia-like facial variation in the general population
Authors: Vanneste Michiel , Hoskens Hanne , Goovaerts Seppe , Matthews Harold , Aponte Jose D , Cole Joanne , Shriver Mark , Marazita Mary L. , Weinberg Seth M. , Walsh Susan , Richmond Stephen , Klein Ophir D , Spritz Richard A , Peeters Hilde , Hallgrímsson Benedikt , Claes Peter
Published: December 2023

Human craniofacial shape is highly variable yet highly heritable with genetic variants interacting through multiple layers of development. Here, we hypothesize that Mendelian phenotypes represent the extremes of a phenotypic spectrum and, using achondroplasia as an example, we introduce a syndrome-informed phenotyping approach to identify genomic loci associated with achondroplasia-like facial variation in the normal population. We compared three-dimensional facial scans from 43 individuals with achondroplasia and 8246 controls to calculate achondroplasia-like facial scores. Multivariate GWAS of the control scores revealed a polygenic basis for normal facial variation along an achondroplasia-specific shape axis, identifying genes primarily involved in skeletal development. Jointly modeling these genes in two independent control samples showed craniofacial effects approximating the characteristic achondroplasia phenotype. These findings suggest that both complex and Mendelian genetic variation act on the same developmentally determined axes of facial variation, providing new insights into the genetic intersection of complex traits and Mendelian disorders.
O-glycans Expand Lubricin and Attenuate its Viscosity and Shear Thinning
Authors: Boushehri Saber , Holey Hannes , Brosz Matthias , Gumbsch Peter , Pastewka Lars , Aponte-Santamaría Camilo , Gräter Frauke
Published: December 2023

Lubricin, an intrinsically disordered glycoprotein, plays a pivotal role in facilitating smooth movement and ensuring the enduring functionality of synovial joints. The central domain of this protein serves as a source of this excellent lubrication, and is characterized by its highly glycosylated, negatively charged, and disordered structure. However, the influence of O-glycans on the viscosity of lubricin remains unclear. In this study, we employ molecular dynamics simulations in absence and presence of shear, along with continuum simulations, to elucidate the intricate interplay between O-glycans and lubricin and the impact of O-glycans on lubricin’s conformational properties and viscosity. We find the presence of O-glycans to induce a more extended conformation in fragments of the disordered region of lubricin. These O-glycans contribute to a reduction in solution viscosity but at the same time weaken shear thinning at high shear rates, compared to non-glycosylated systems with the same density. This effect is attributed to the steric and electrostatic repulsion between the fragments, which prevent their conglomeration and structuring. Our computational study yields a mechanistic mechanism underlying previous experimental observations of lubricin and paves the way to more rationally understanding its function in the synovial fluid.
The cryoEM structure of theHendra henipavirusnucleoprotein reveals insights into paramyxoviral nucleocapsid architectures
Authors: Passchier Tim C , White Joshua B R , Maskell Daniel P , Byrne Matthew J , Ranson Neil A , Edwards Thomas A , Barr John N
Published: December 2023

We report the first cryoEM structure of theHendra henipavirusnucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of homotetradecameric RNA-bound N protein rings exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent N and N+1protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional interaction between N+1and N-1protomers. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.
Extracellular vesicles released by keratinocytes regulate melanosome maturation, melanocyte dendricity and pigment transfer
Authors: Prospéri Marie-Thérèse , Giordano Cécile , Gomez-Duro Mireia , Hurbain Ilse , Macé Anne-Sophie , Raposo Graça , D’Angelo Gisela
Published: December 2023

Extracellular vesicles (EVs) facilitate the transfer of proteins, lipids and genetic material molecules between cells, and are recognized as an additional mechanism for sustaining intercellular communication. In the epidermis, the communication between melanocytes and keratinocytes is tightly regulated to warrant skin pigmentation. Melanocytes synthetize the melanin pigment in melanosomes that are transported along the dendrites prior to the transfer of melanin pigment to keratinocytes. EVs secreted by keratinocytes modulate pigmentation in melanocytes (Lo Cicero et al., Nat. Comm. 2015). However, whether EVs secreted by keratinocytes contribute to additional processes essential for melanocyte functions remains elusive. Here we show that keratinocyte EVs enhance the ability of melanocytes to generate dendrites, mature melanosomes and their efficient transfer. Further, keratinocyte EVs carrying Rac1 induce important morphological changes, promote dendrite outgrowth, and potentiate melanin transfer to keratinocytes. Hence, in addition to modulate pigmentation, keratinocytes exploit EVs to control melanocyte plasticity and transfer capacity. These data demonstrate that keratinocyte-derived EVs, by regulating melanocyte functions, are major contributors of cutaneous pigmentation and expand our understanding of the mechanism underlying skin pigmentation via a paracrine EV-mediated communication.

SIGNIFICANCE STATEMENT

Our work uncovers how keratinocyte-derived EVs control melanocyte physiology and functions. By promoting the growth of melanocyte dendrites, maturation, accumulation and peripheral positioning of pigmented melanosomes within the dendrites, and transfer of melanin to keratinocytes, EVs released by keratinocytes control crucial processes in skin photo protection. Importantly, given that dysregulation of these pathways could underlie pigment disorders, melanoma or skin carcinoma, our results open avenues to exploit keratinocyte EVs as tools for the design of new therapies to enhance the ability of melanocytes to provide skin photoprotection, and thus decrease the incidence pigmentary disorders and skin cancers.
Three-dimensional reconstruction of fetal rhesus macaque kidneys at single-cell resolution reveals complex inter-relation of structures
Authors: Dequiedt Lucie , Forjaz André , Lo Jamie O. , McCarty Owen , Wu Pei-Hsun. , Rosenberg Avi , Wirtz Denis , Kiemen Ashley
Published: December 2023

Kidneys are among the most structurally complex organs in the body. Their architecture is critical to ensure proper function and is often impacted by diseases such as diabetes and hypertension. Understanding the spatial interplay between the different structures of the nephron and renal vasculature is crucial. Recent efforts have demonstrated the value of three-dimensional (3D) imaging in revealing new insights into the various components of the kidney; however, these studies used antibodies or autofluorescence to detect structures and so were limited in their ability to compare the many subtle structures of the kidney at once. Here, through 3D reconstruction of fetal rhesus macaque kidneys at cellular resolution, we demonstrate the power of deep learning in exhaustively labelling seventeen microstructures of the kidney. Using these tissue maps, we interrogate the spatial distribution and spatial correlation of the glomeruli, renal arteries, and the nephron. This work demonstrates the power of deep learning applied to 3D tissue images to improve our ability to compare many microanatomical structures at once, paving the way for further works investigating renal pathologies.
Modulation ofSalmonellavirulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex
Authors: Yu Xiu-Jun , Xie Haixia , Li Yan , Liu Mei , Hou Ruhong , Predeus Alexander V. , Perez Sepulveda Blanca M. , Hinton Jay C. D. , Holden David W. , Thurston Teresa L. M.
Published: December 2023

The injectisome encoded bySalmonellapathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterise the secretome of a clinical strain of invasive non-typhoidalSalmonella entericaserovar Enteritidis, that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein.sseMis widely distributed between the five subspecies ofSalmonella enterica,is found in many clinically-relevant serovars, and is co-transcribed withpipB2, aSPI-2 effector gene. Translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely β-2-syntrophin, utrophin/ dystrophin, α-catulin, α-dystrobrevin and β-dystrobrevin. The interaction between SseM and β-2-syntrophin and α-dystrobrevin was verified inS.Typhimurium-infected cells and relied on the PDZ domain of β-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseMmutant strain had a small competitive advantage over the wild-type strain in theS.Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild type SseM fromS.Typhimurium orS.Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within aSalmonellaeffector mediates interactions with the DAPC and modulates systemic growth of bacteria in mice.

Importance

InSalmonella enterica, the injectisome machinery encoded bySalmonellapathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells that are required forSalmonellavirulence. The identification and functional characterisation of SPI-2 injectisome effectors advances our understanding of the interplay betweenSalmonellaand its host(s). Using an optimised method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal (iNTS)Salmonella entericaserovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector - SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a PDZ-binding motif that is essential for interaction with the PDZ-containing host protein β-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique amongSalmonellaeffectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.
SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized COVID-19 patients
Authors: Basting Christopher M. , Langat Robert , Broedlow Courtney A. , Guerrero Candace , Bold Tyler D. , Bailey Melisa , Velez Adrian , Schroeder Ty , Short-Miller Jonah , Cromarty Ross , Mayer Zach , Southern Peter J. , Schacker Timothy W. , Safo Sandra E. , Bramante Carolyn T. , Tignanelli Christopher J. , Schifanella Luca , Klatt Nichole R.
Published: December 2023

Coronavirus disease 2019 (COVID-19) and associated severity has been linked to uncontrolled inflammation and may be associated with changes in the microbiome of mucosal sites including the gastrointestinal tract and oral cavity. These sites play an important role in host-microbe homeostasis and disruption of epithelial barrier integrity during COVID-19 may potentially lead to exacerbated inflammation and immune dysfunction. Outcomes in COVID-19 are highly disparate, ranging from asymptomatic to fatal, and the impact of microbial dysbiosis on disease severity is unclear. Here, we obtained plasma, rectal swabs, oropharyngeal swabs, and nasal swabs from 86 patients hospitalized with COVID-19 and 12 healthy volunteers. We performed 16S rRNA sequencing to characterize the microbial communities in the mucosal swabs and measured circulating cytokines, markers of gut barrier integrity, and fatty acids in the plasma samples. We compared these plasma concentrations and microbiomes between healthy volunteers and the COVID-19 patients who had survived or unfortunately died by the end of study enrollment, and between severe disease and healthy controls, as well as performed a correlation analysis between plasma variables and bacterial abundances. The rectal swabs of COVID-19 patients had reduced abundances of several commensal bacteria includingFaecalibacterium prausnitsii, and an increased abundance of the opportunistic pathogensEggerthella lentaandHungatella hathewayi. Furthermore, the oral pathogenScardovia wiggsiaewas more abundant in the oropharyngeal swabs of COVID-19 patients who died. The abundance of bothH. hathewayiandS. wiggsiaecorrelated with circulating inflammatory markers including IL-6, highlighting the possible role of the microbiome in COVID-19 severity, and providing potential therapeutic targets for managing COVID-19.
Viral gene drive spread during herpes simplex virus 1 infection in mice
Authors: Walter Marius , Haick Anoria K , Riley Rebeccah , Massa Paola A , Strongin Daniel E , Klouser Lindsay M , Loprieno Michelle A , Stensland Laurence , Santo Tracy K , Aubert Martine , Taylor Matthew P , Jerome Keith R , Verdin Eric
Published: December 2023

Gene drives are genetic modifications designed to propagate efficiently through a population. Most applications rely on homologous recombination during sexual reproduction in diploid organisms such as insects, but we recently developed a gene drive in herpesviruses that relies on co-infection of cells by wild-type and engineered viruses. Here, we developed a viral gene drive against human herpes simplex virus 1 (HSV-1) and showed that it propagated efficientlyin vitroand during HSV-1 infection in mice. We observed high levels of co-infection and gene drive-mediated recombination in neuronal tissues during herpes encephalitis as the infection progressed from the site of inoculation to the peripheral and central nervous systems. In addition, we found evidence that a superinfecting gene drive virus could recombine with wild-type viruses during latent infection. These findings indicated that HSV-1 achieves high rates of co-infection and recombination during viral infection, a phenomenon that is currently underappreciated. Overall, this study showed that a viral gene drive could spreadin vivoduring HSV-1 infection, paving the way toward therapeutic applications.