bluuebunny/crossref_metadata_2025 huggingface dataset:
A TLR7 Agonist Conjugated to a Nanofibrous Peptide Hydrogel as a Potent Vaccine Adjuvant
Authors: Euliano Erin M. , Pogostin Brett H. , Agrawal Anushka , Yu Marina H. , Baryakova Tsvetelina H. , Graf Tyler P. , Hartgerink Jeffrey D. , McHugh Kevin J.
Published: March 2024

Toll-like receptors (TLRs) recognize pathogen- and damage-associated molecular patterns and, in turn, trigger the release of cytokines and other immunostimulatory molecules. As a result, TLR agonists are increasingly being investigated as vaccine adjuvants, though many of these agonists are small molecules that quickly diffuse away from the vaccination site, limiting their co-localization with antigens and, thus, their effect. Here, the small-molecule TLR7 agonist 1V209 is conjugated to a positively-charged multidomain peptide (MDP) hydrogel, K2, which was previously shown to act as an adjuvant promoting humoral immunity. Mixing the 1V209-conjugated K250:50 with the unfunctionalized K2produces hydrogels that retain the shear-thinning and self-healing physical properties of the original MDP, while improving the solubility of 1V209 more than 200-fold compared to the unconjugated molecule. When co-delivered with ovalbumin as a model antigen, 1V209-functionalized K2produces antigen-specific IgG titers that were statistically similar to alum, the gold standard adjuvant, and a significantly lower ratio of Th2-associated IgG1 to Th1-associated IgG2a than alum, suggesting a more balanced Th1 and Th2 response. Together, these results suggest that K2MDP hydrogels functionalized with 1V209 are a promising adjuvant for vaccines against infectious diseases, especially those benefiting from a combined Th1 and Th2 immune response.

Table of Contents

Activation of toll-like receptors (TLRs) stimulates a signaling cascade to induce an immune response. A TLR7 agonist was conjugated to an injectable peptide hydrogel, which was then used to deliver a model vaccine antigen. This platform produced antibody titers similar to the gold standard adjuvant alum and demonstrated an improved balance between Th1- and Th2-mediated immunity over alum.
Conveying tactile object characteristics through customized intracortical microstimulation of the human somatosensory cortex
Authors: Verbaarschot C. , Karapetyan V. , Greenspon C.M. , Boninger M. , Bensmaia S.J. , Sorger B. , Gaunt R. A.
Published: March 2024

Microstimulation of the somatosensory cortex can evoke tactile percepts in people with spinal cord injury, providing a means to restore touch. While location and intensity can be reliably conveyed, two issues that prevent creating more complex naturalistic sensations are a lack of methods to effectively scan the large stimulus parameter space and difficulties with assessing percept quality. Here, we addressed both challenges with an experimental paradigm that enabled three individuals with tetraplegia to control their stimulation parameters in a blinded fashion to create sensations for different virtual objects. Participants felt they could reliably create object-specific sensations and reported vivid object-appropriate characteristics. Despite substantial overlap in stimulus parameter selections across objects, both linear classifiers and participants could match stimulus profiles with their respective objects significantly above chance without any visual cues. We conclude that while visual information contributes to the experience of artificial touch, microstimulation in the somatosensory cortex itself can evoke intuitive percepts with a variety of tactile properties. This novel self-guided stimulation approach may be used to effectively characterize percepts from future stimulation paradigms.
Integrating Step-down Care in Community-based Paediatric Palliative Services: A Realist Evaluation
Authors: Yeo Zhi Zheng , Poh Heng Chong
Published: March 2024

Background

Young persons with advanced life-limiting illness living at home have fluctuating and complex needs. Community paediatric palliative care (PPC) is still predominantly specialist-led. This poses a potential care vacuum as medical conditions stabilise and specialised care ceases. One home-based PPC service introduced a step-down care program (COMET) as bridge to continuous yet adaptable support. Above overall effectiveness, how different outcomes are achieved is also investigated for context.

Methods

We conducted a realist evaluation, using a mixed-methods case series design to study COMET operations and impact within the community care context (Context->Mechanism->Outcomes). Patient medical records and in-depth interviews with family caregivers and PPC professionals generated rich quantitative and qualitative data for analysis.

Results

Of 121 patients under specialist PPC homecare, 18 (14.9%) were enrolled in COMET since November 2020; 12 of these formed individual case studies. Interviews with 15 caregivers and 7 PPC clinicians produced three crucial findings: (i) Ongoing access to specialist care is required for ever evolving complexities; (ii) Continuing support at home is vital for regular management and intermittent emergencies; (iii) COMET harmonizes shifting levels of support within a single unified framework, safeguarding existing rapport and care consistency.

Conclusion

Until generalist PPC expertise becomes prevalent outside the hospital setting, novel care models like COMET could plug gaps in community PPC services by offering flexible care options. Continuity of care, efficient resource management, and superior service quality are inherent benefits, if meaningful care tiering through substantive patient assessments are refined further in future iterations.
Generative AI Guidelines in Korean Medical Journals: A Survey Using Human-AI Collaboration
Authors: Ahn Sangzin
Published: March 2024

Background

Generative artificial intelligence (GAI) tools, such as large language models, have the potential to revolutionize medical research and writing, but their use also raises important ethical and practical concerns. This study examines the prevalence and content of GAI guidelines among Korean medical journals to assess the current landscape and inform future policy development.

Methods

Top 100 Korean medical journals by H-index were surveyed. Author guidelines were collected and screened by a human author and AI chatbot to identify GAI-related content. Key components of GAI policies were extracted and compared across journals. Journal characteristics associated with GAI guideline adoption were also analyzed.

Results

Only 18% of the surveyed journals had GAI guidelines, which is much lower than previously reported international journals. However, adoption rates increased over time, reaching 57.1% in the first quarter of 2024. Higher-impact journals were more likely to have GAI guidelines. All journals with GAI guidelines required authors to declare GAI use, and 94.4% prohibited AI authorship. Key policy components included emphasizing human responsibility (72.2%), discouraging AI-generated content (44.4%), and exempting basic AI tools (38.9%).

Conclusion

While GAI guideline adoption among Korean medical journals is lower than global trends, there is a clear increase in implementation over time. The key components of these guidelines align with international standards, but greater standardization and collaboration are needed to ensure responsible and ethical use of GAI in medical research and writing.

Abstract Figure
Blood methylation pattern reflects epigenetic remodelling in adipose tissue after bariatric surgery
Authors: Müller Luise , Hoffmann Anne , Bernhart Stephan H. , Ghosh Adhideb , Zhong Jiawei , Hagemann Tobias , Sun Wenfei , Dong Hua , Noé Falko , Wolfrum Christian , Dietrich Arne , Stumvoll Michael , Massier Lucas , Blüher Matthias , Kovacs Peter , Chakaroun Rima , Keller Maria
Published: March 2024

Background

Studies on DNA methylation following bariatric surgery have primarily focused on blood cells, while it is unclear to which extend it may reflect DNA methylation profiles in specific metabolically relevant organs such as adipose tissue (AT). Here, we investigated whether adipose tissue depots specific methylation changes after bariatric surgery are mirrored in blood.

Methods

Using Illumina 850K EPIC technology, we analysed genome-wide DNA methylation in paired blood, subcutaneous and omental visceral AT (SAT/OVAT) samples from nine individuals with severe obesity pre- and post-surgery.

Findings

The numbers and effect sizes of differentially methylated regions (DMRs) post-bariatric surgery were more pronounced in AT (SAT: 12,865 DMRs from -11.5 to 10.8%; OVAT: 14,632 DMRs from -13.7 to 12.8%) than in blood (9,267 DMRs from -8.8 to 7.7%). Cross-tissue DMRs implicated immune-related genes. Among them, 49 regions could be validated with similar methylation changes in blood from independent individuals. Fourteen DMRs correlated with differentially expressed genes in AT post bariatric surgery, including downregulation ofPIK3AP1in both SAT and OVAT. DNA methylation age acceleration was significantly higher in AT compared to blood, but remained unaffected after surgery.

Interpretation

Concurrent methylation pattern changes in blood and AT, particularly in immune-related genes, suggest blood DNA methylation mirrors inflammatory state of AT post-bariatric surgery.
A dynamic ensemble model for short-term forecasting in pandemic situations
Authors: Botz Jonas , Valderrama Diego , Guski Jannis , Fröhlich Holger
Published: March 2024

During the COVID-19 pandemic, many hospitals reached their capacity limits and could no longer guarantee treatment of all patients. At the same time, governments endeavored to take sensible measures to stop the spread of the virus while at the same time trying to keep the economy afloat. Many models extrapolating confirmed cases and hospitalization rate over short periods of time have been proposed, including several ones coming from the field of machine learning. However, the highly dynamic nature of the pandemic with rapidly introduced interventions and new circulating variants imposed non-trivial challenges for the generalizability of such models.

In the context of this paper, we propose the use of ensemble models, which are allowed to change in their composition or weighting of base models over time and can thus adapt to highly dynamic pandemic or epidemic situations. In that regard, we also explored the use of secondary metadata - Google searches - to inform the ensemble model. We tested our approach using surveillance data from COVID-19, Influenza, and hospital syndromic surveillance of severe acute respiratory infections (SARI). In general, we found ensembles to be more robust than the individual models. Altogether we see our work as a contribution to enhance the preparedness for future pandemic situations.
Enhancing gout management by creating a register using automated queries in electronic health records
Authors: Bürgisser Nils , Mongin Denis , Mehouachi Samia , Buclin Clement P. , Guemara Romain , Darbellay Farhoumand Pauline , Braillard Olivia , Lauper Kim , Courvoisier Delphine S.
Published: March 2024

Objective

To develop an automatic gout register to improve gout management.

Methods

We analysed the electronic health records (EHR) of all patients >18 years old from a tertiary academic hospital (2013-2022) based on six criteria: International Classification of Diseases 10 (ICD-10) gout diagnosis, urate-lowering therapy (ULT) prescription, uric acid crystal in joint aspiration and gout-related terms in problem lists, clinical or imaging reports. We assessed the positive and negative predictive value (PPV and NPV) of the query by chart reviews.

Results

Of 2,110,902 out- and inpatients, 10,289 had at least one criterion for gout. The combination of joint aspiration OR diagnostic in the problem list OR ≥ 2 other criteria created a register of 5,138 patients, with a PPV of 92.4% (95%CI: 88.5 to 95.0), and an NPV of 94.3% (95%CI: 91.9 to 96.0). PPV and NPV were similar amongst outpatients and inpatients. Incidence was 2.9 per 1000 person-year and dropped by 30% from the COVID-19 pandemic onward. Patients with gout were on average 71.2 years old (SD 14.9), mainly male (76.5%), overweight (69.5%) and polymorbid (mean number of comorbidities of 3, IQR 1-5). More than half (57.4%) had received a urate lowering treatment, 6.7% had a gout that led to a hospitalisation or ≥2 flares within a year, and 32.9% received a rheumatology consultation.

Conclusion

An automatic EHR-based gout register is feasible, valid and could be used to evaluate and improve gout management. Interestingly, the register uncovered a marked underdiagnosis or underreporting of gout since the COVID-19 pandemic.

Key messages

What is already known on this topic?

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Gout is the most prevalent inflammatory arthritis, but it remains undertreated despite affordable and effective treatment options.

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Quantifying this undertreatment and detecting its causes and risk factors to pilot quality improvement initiative requires an extensive register of gout patients.

What this study adds?

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This is the first automatic EHR-based gout register, allowing frequent, inexpensive, and sustainable updates.

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The automated queries show high positive and negative predictive values to identify gout patients.

How this study might affect research, practice or policy?

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This register can facilitate the assessment of the adequacy of gout management and the monitoring of quality indicators following improvement projects, or change in policies

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It provides an easy platform for cohort studies or adaptive trials

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Its methodology is reproducible, facilitating the establishment of gout or other disease registers within different EHR systems
Specific pelvic shape in patients with developmental dysplasia of the hip on 3D morphometric homologous model analysis
Authors: Sasaki Yui , Suzuki Daisuke , Tokita Ryo , Takashima Hiroyuki , Matsumura Hirofumi , Nagoya Satoshi
Published: March 2024

Purpose

To clarify the morphological factors of the pelvis in patients with developmental dysplasia of the hip (DDH), three-dimensional (3D) pelvic morphology was analyzed using a template-fitting technique.

Methods

Three-dimensional pelvic data of 50 patients with DDH (DDH group) and 3D pelvic data of 50 patients without obvious pelvic deformity (Normal group) were used. All patients were female. A template model was created by averaging the normal pelvises into a symmetrical and isotropic mesh. Next, 100 homologous models were generated by fitting the pelvic data of each group of patients to the template model. Principal component analysis was performed on the coordinates of each vertex (15,235 vertices) of the pelvic homologous model. In addition, a receiver-operating characteristic (ROC) curve was calculated from the sensitivity of DDH positivity for each principal component, and principal components for which the area under the curve was significantly large were extracted (p<0.05). Finally, which components of the pelvic morphology frequently seen in DDH patients are related to these extracted principal components was evaluated.

Results

The first, third, and sixth principal components showed significantly larger areas under the ROC curves. The morphology indicated by the first principal component was associated with a decrease in coxal inclination in both the coronal and horizontal planes. The third principal component was related to the sacral inclination in the sagittal plane. The sixth principal component was associated with narrowing of the superior part of the pelvis.

Conclusion

The most important factor in the difference between normal and DDH pelvises was the change in the coxal angle in both the coronal and horizontal planes. That is, in the anterior and superior views, the normal pelvis is a triangle, whereas in DDH, it was more like a quadrilateral.
Transcriptomic profile of normal breast tissue post-mifepristone treatment: secondary outcomes of a randomized controlled trial
Authors: Utjés Deborah , Boggavarapu Nageswara Rao , Rasul Mohammed , Koberg Isabelle , Zulliger Alexander , Ponandai-Srinivasan Sakthivignesh , von Grothusen Carolina , Lalitkumar Parameswaran Grace , Papaikonomou Kiriaki , Alkasalias Twana , Gemzell-Danielsson Kristina
Published: March 2024

Progesterone receptor antagonism is gaining attention due to progesterone’s recognized role as a major mitogen in breast tissue. Limited but promising data suggest the potential efficacy of antiprogestins in breast cancer prevention. The present study presents secondary outcomes from a randomized controlled trial and examine changes in breast mRNA expression following mifepristone treatment in healthy women. We analyzed 32 paired breast biopsies from 16 healthy premenopausal women at baseline and after two months of mifepristone treatment. In total, twenty-seven differentially expressed genes were identified, with enriched biological functions related to extracellular matrix remodeling. Notably, the altered gene signature induced by mifepristonein vivowas rather similar to thein vitrosignature. Furthermore, this expression gene signature was associated with breast carcinogenesis and significantly correlated with progesterone receptor expression status in breast cancer, as validated in The Cancer Genome Atlas dataset using the R2 platform. The present study is the first to explore the breast transcriptome following mifepristone treatment in healthy breast tissuein vivo, enhancing the understanding of progesterone receptor antagonism and its potential protective effect against breast cancer by investigating its action in healthy breast tissue.
Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis
Authors: Nguyen Binh An , Singh Virender , Afrin Shumaila , Singh Preeti , Pekala Maja , Ahmed Yasmin , Pedretti Rose , Canepa Jacob , Lemoff Andrew , Kluve-Beckerman Barbara , Wydorski Pawel , Chhapra Farzeen , Saelices Lorena
Published: March 2024

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found an unprecedented structural variability in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically-different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure’s impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.

One-Sentence Summary:Wild-type cardiac ATTR fibrils are structurally homogeneous.
Non-mechanical disruption ofBacillus subtilis sporesallows sensitive and deep characterization of the minimal proteome for resuming a cellular lifestyle
Authors: Huang Yixuan , de Koster Alphonse , Tu Zhiwei , Gao Xiaowei , Roseboom Winfried , Brul Stanley , Setlow Peter , Kramer Gertjan
Published: March 2024

In response to extreme conditions,Bacillus subtilisgenerates highly resilient spores characterized by a unique multilayered structure. This confers resistance against various chemicals and enzymes yet adding complexity to the analysis of the spore proteome. As the first step in bottom-up proteomics, sample preparation poses a significant challenge. We assessed how an optimized protocol for sample preparation by easy extraction and digestion (SPEED) performed compared to previously established methods “One-pot” (OP) and single-pot, solid phase-enhanced sample-preparation (SP3) for the proteomic analysis ofB. subtiliscell and spore samples. We found that SPEED outperformed both OP and SP3 in terms of peptides and proteins identified, moreover SPEED highly reproducibly quantified over 1000 proteins in limited input samples as low as 1 OD600ofB. subtiliscells and spores. SPEED was applied to analyze spore samples of different purity by applying sequential purification following harvesting of spores. Comparison of the differential abundance of proteins revealed clusters likely partially stemming from remaining vegetative cells in less purified spore samples. We show that ranking of absolute protein abundance in cellular and spore samples further enables us to rationally differentiate integral spore proteins from vegetative remnants. This is of importance in applications and organisms where highly homogenous spore samples are difficult to obtain. A deep proteomic analysis of spore and vegetative cell samples with the new approach led to the identification of 2447 proteins, 2273 of which were further quantified and compared betweenB. subtilisspores and cells. Our findings indicate that pathways related to peptidoglycan biosynthesis, glycolysis, carbon metabolism, and biosynthesis of secondary metabolites are shared between cells and spores. This corroborates and extends earlier work stressing that despite marked differences in their physiological states, spores preserve vegetative cell (core) proteins, essential for revival under conditions conducive to growth.

Abstract Figure
GATLncLoc+C&S: Prediction of LncRNA subcellular localization based on corrective graph attention network
Authors: Deng Xi , Tang Lin , Liu Lin
Published: March 2024

Long non-coding RNAs (LncRNAs) have a wide range of regulatory roles in gene expression, and the subcellular localization identification of LncRNAs is of great value in understanding their biological functions. Graph neural networks can not only utilize sequence characteristics, but also learn hidden features from non-Euclidean data structures to obtain features with powerful characterization capabilities. To learn more fully from the limited LncRNA localization samples and efficiently exploit easily ignored label features, we propose a corrective graph attention network prediction model GATLncLoc+C&S in this paper. Compared with previous methods, the similarity of optimal features is first used to construct the graph. Then, a re-weighted graph attention network R-GAT is constructed and the soft labels obtained from it are used to correct the graph. Finally, the predicted localization label is further obtained by label propagation. Based on the combination of R-GAT and label propagation, GATLncLoc+C&S effectively solves the problems of few samples and data imbalance in LncRNA subcellular localization. The accuracy of GATLncLoc+C&S reached 95.8% and 96.8% in the experiments of 5- and 4-localization benchmark datasets, which reflects the great potential of our proposed method in predicting LncRNA subcellular localization. The source code and data of GATLncLoc+C&S are available athttps://github.com/GATLncLoc-C-S/GATLncLoc-C-S.
Proteolytic activity of surface exposed HtrA determines its expression level and is needed to survive acidic conditions inClostridioides difficile
Authors: Corver Jeroen , Claushuis Bart , Shamorkina Tatiana M. , de Ru Arnoud H. , van Leeuwen Merle M. , Hensbergen Paul J. , Smits Wiep Klaas
Published: March 2024

To survive in the host, pathogenic bacteria need to be able to react to the unfavourable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogenClostridioides difficileencodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA inC. difficileis hitherto unknown but previous work has shown that an insertional mutant ofhtrAdisplayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface ofC. difficileand characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.
Uncovering the translatome impact of transcriptome induced diversity in eukaryotes: framework and innovative insights
Authors: Verma Paras , Thakur Deeksha , Pandit Shashi Bhushan
Published: March 2024

Isoform diversity is known to enhance a gene’s functional repertoire. Insights into the extent of such sequence variability generated through alternative splicing (AS), may unveil the layers of gene function/regulation. Despite studies on transcriptome diversifying processes, the impact of AS or related processes on sequence diversity still needs to be explored. Current study presents an innovative framework that centralizes exonic loci while integrating protein sequence per entity with attention to splice site variability assessment. The resulting framework enables exon (features) to be tractable, facilitating a systematic, detailed analysis of isoform diversity. We analyzed isoform diversity in five representative organisms and detailed the role of AS and related processes influencing exon inclusion in imparting sequence variation for human genome. Through analyses of exonic variations in two maximally diverged isoforms of human genes, we unraveled intricate splicing patterns prevalent in coding and non-coding regions. We observed that alternative splice sites, sequence changes, and skipping of exons are prevalent in coding exons, while the alternate first exon events are predominant in non-coding exons. Our findings offer a comprehensive understanding of isoform diversity as a function of exonic entity framework, providing valuable insights into the orchestration of exonic events in shaping the proteogenomic landscape.
Tripartite interaction of a patescibacterial epibiont, a methylotrophic gammaproteobacterial host and a jumbo phage
Authors: Buderka Feriel , Lopez-Garcia Purificacion , Deschamps Philippe , Krupovic Mart , Gutierrez-Preciado Ana , Ciobanu Maria , Bertolino Paola , David Gwendoline , Moreira David , Jardillier Ludwig
Published: March 2024

Patescibacteria form a very diverse and widely distributed phylum of small bacteria inferred to have an episymbiotic lifestyle. However, the prevalence of this lifestyle within the phylum and their host specificity remain poorly known due to the scarcity of cultured representatives. Here, we describe a tripartite interaction of a patescibacterium, its gammaproteobacterial host, and a patescibacterial phage based on metagenomic data and enrichment cultures from two shallow, geographically close, freshwater ecosystems. The patescibacterium, Strigamonas methylophilicida sp. nov., defines a new genus within the family Absconditicoccaceae. It grows as epibiont on cells of methanotrophic species of the gammaproteobacterial family Methylophilaceae. Strigamonas cells are tightly attached to the host, sometimes forming stacks that connect two host cells. Despite a surprisingly large genome (1.9 Mb) compared to many other patescibacteria, S. methylophilicida lacks many essential biosynthetic pathways, including the complete biosynthesis of phospholipids, amino acids, and nucleic acids, implying a dependence on the host to obtain these molecules. From metagenomes of these ecosystems, we identified and assembled the genome of a jumbo phage likely infecting the patescibacterium based on a CRISPR spacer match. Its large genome (230 kb) contained a wide gene repertoire, including genes probably involved in the modification of the Strigamonas metabolism and cell surface. By manipulating the patescibacterial phenotype during infection, the phage likely influences in turn the interaction of the patescibacterial epibiont with its methylotrophic host in a parasitic menage-a-trois. Our results confirm a prevalent episymbiotic lifestyle in Absconditicoccaceae and further suggest a clade-specific adaptation of these patescibacteria for gammaproteobacterial hosts.
Novel Polymycoviruses Are Encapsidated in Filamentous Virions
Authors: Han Zhenhao , Jiang Jingjing , Xu Wenxing
Published: March 2024

Polymycoviridaeis a new viral family that was established nearly five years ago, but their viral morphologies (naked or encapsidated) remain controversy since only one member namely, Colletotrichum camelliae filamentous virus 1 (CcFV1), was identified as being encapsidated in filamentous virions. Here, three novel dsRNA viruses belonging to the familyPolymycoviridaewere identified in three phytopathogenic fungal strains and tentatively named Pseudopestalotiopsis camelliae-sinensis polymycovirus 1 (PscPmV1), and Phyllosticta capitalensis polymycovirus 1 (PhcPmV1) and -2 (PhcPmV2), respectively. PscPmV1 and PhcPmVs have five or six genomic dsRNAs, ranging from 1055 to 2405 bp, encoding five or seven putative open reading frames (ORFs), of which ORF1 encodes an RNA-dependent RNA polymerase, ORF5 encodes a P-A-S-rich protein behaving as coat protein (CP); and dsRNAs 4 and 6 encode putative proteins with unknown functions and share no detectable identities with known viral sequences. Upon examination under transmission electron microscopy after purification from fungal mycelia, PscPmV1 and PhcPmVs were found to be encapsidated in filamentous particles, as was a known polymycovirus, Botryosphaeria dothidea RNA virus 1 (BdRV1), which was previously assumed to likely have no conventional virions. The morphology of PscPmV1 was further supported by the observation that its particles could be decorated by polyclonal antibodies against its CP and bound by immuno-gold particles conjugated to the specific CP antibody. Together with CcFV1 and BdRV1, PcsPmV1 PhcPmVs provide strong evidence to support the notion that polymycoviruses are encapsidated in filamentous virions constituted by P-A-S-rich CPs. Moreover, their biological effects on their fungal hosts were assessed.

Significance statement

Polymycoviridae, a recently established viral family, has raised questions about encapsidation. Here, we identify and characterize three novel polymycoviral dsRNA viruses in phytopathogenic fungal strains, tentatively named Pseudopestalotiopsis camelliae-sinensis polymycovirus 1, and Phyllosticta capitalensis polymycovirus 1 and -2, respectively. These polymycoviruses possess five or six genomic dsRNAs, ranging from 1055 to 2405 bp, with two encoding putative proteins of unknown functions and sharing no detectable identities with known viral sequences. Their morphologies indicate filamentous virions constituted by P-A-S-rich coat proteins, observed using immunosorbent electron microscopy combined with immune-gold labeling techniques. Additionally, Botryosphaeria dothidea RNA virus 1, previously assumed to lack conventional virions, is also shown to be encapsidated in filamentous particles. This study provides new evidence supporting the encapsidation of polymycoviruses into elongated and flexuous virions, significantly contributing to our understanding of the evolutionary particle architecture within the virosphere and expanding our knowledge of viral diversity and evolution.
Cerebral perfusion metrics calculated directly, model-free, from a hypoxia-induced step change in deoxyhemoglobin
Authors: Duffin James , Sayin Ece Su , Sobczyk Olivia , Poublanc Julien , Mikulis David J. , Fisher Joseph A.
Published: March 2024

Dynamic susceptibility contrast (DSC) perfusion MRI measures blood flow metrics via application of a BOLD pulse sequence during transit of a gadolinium-based contrast agent (GBCA), We have previously shown that can also be performed using endogenous deoxyhemoglobin (dOHb) as a replacement for GBCA since dOHb also has the necessary paramagnetic properties similar to GBCA1. Conventional analysis with these contrast agents includes deconvolution of an arterial input function (AIF) assuming a kinetic model. However, the immediate decrease in dOHb that occurs during reoxygenation from a transient hypoxia constitutes a step reduction in susceptibility that permits direct, model-free, calculation of relative resting perfusion metrics. The metrics from this step analysis for seven healthy volunteers were compared to those from a conventional analysis of GBCA and dOHb. Voxel- wise maps of mean transit time, and relative cerebral blood flow and cerebral blood volume, had a high spatial congruence for all three analyses and were similar in appearance to published maps. The time course of the T2*-weighted signal step response identifies both the arrival time, the distribution of the step increase in arterial oxygen saturation, SaO2, and the voxel filling phase. The analysis of a step change in dOHb during rapid reoxygenation is an alternative way of calculating perfusion metrics directly from measurements of the resulting T2*-weighted signal, avoiding the potential errors from the use of a kinetic model and requirement for selection of an AIF.
Transcriptional plasticity of virulence genes provides malaria parasites with greater adaptive capacity for avoiding host immunity
Authors: Florini Francesca , Visone Joseph E. , Hadjimichael Evi , Malpotra Shivali , Nötzel Christopher , Kafsack Björn F.C. , Deitsch Kirk W.
Published: March 2024

Chronic, asymptomatic malaria infections contribute substantially to disease transmission and likely represent the most significant impediment preventing malaria elimination and eradication.Plasmodium falciparumparasites evade antibody recognition through transcriptional switching between members of thevargene family, which encodes the major virulence factor and surface antigen on infected red blood cells. This process can extend infections for up to a year; however, infections have been documented to last for over a decade, constituting an unseen reservoir of parasites that undermine eradication and control efforts. How parasites remain immunologically “invisible” for such lengthy periods is entirely unknown. Here we show that in addition to the accepted paradigm of mono-allelicvargene expression, individual parasites can simultaneously express multiplevargenes or enter a state in which little or novargene expression is detectable. This unappreciated flexibility provides parasites with greater adaptive capacity than previously understood and challenges the dogma of mutually exclusivevargene expression. It also provides an explanation for the antigenically “invisible” parasites observed in chronic asymptomatic infections.
Evidence of microglial involvement in the childhood abuse-associated increase in perineuronal nets in the ventromedial prefrontal cortex
Authors: Belliveau Claudia , Rahimian Reza , Fakhfouri Gohar , Hosdey Clémentine , Simard Sophie , Davoli Maria Antonietta , Mirault Dominique , Giros Bruno , Turecki Gustavo , Mechawar Naguib
Published: March 2024

Microglia, known for their diverse roles in the central nervous system, have recently been recognized for their involvement in degrading the extracellular matrix. Perineuronal nets (PNNs), a specialized form of this matrix, are crucial for stabilizing neuronal connections and constraining plasticity. Our group recently reported increased PNN densities in the ventromedial prefrontal cortex (vmPFC) of depressed individuals that died by suicide in adulthood after experiencing childhood abuse (DS-CA) compared to matched controls. To explore potential underlying mechanisms, we employed a comprehensive approach in similar postmortem vmPFC samples, combining a human matrix metalloproteinase and chemokine array, isolation of CD11b-positive microglia and enzyme-linked immunosorbent assays (ELISA). Our findings indicate a significant downregulation of matrix metalloproteinase (MMP)-9 and tissue inhibitors of metalloproteinases (TIMP)-2 in both whole vmPFC grey matter and isolated microglial cells from DS-CA samples. Furthermore, our experiments reveal that a history of child abuse is associated with diminished levels of microglial CX3CR1 and IL33R in both vmPFC whole lysate and CD11b isolated cells. However, levels of the CX3CR1 ligand, CX3CL1 (Fractalkine), did not differ between groups. While these data suggest potential long-lasting alterations in microglial markers in the vmPFC of individuals exposed to severe childhood adversity, direct functional assessments were not conducted. Nonetheless, these findings offer insight into how childhood abuse may contribute to PNN alterations via microglial-related mechanisms.

Graphical Abstract
Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)
Authors: Salinas-Rebolledo Constanza , Blesa Javier , Valenzuela-Nieto Guillermo , Schwefel David , del Rey Natalia López González , Méndez-Ruette Maxs , Burkhalter Janine , Bátiz Luis Federico , Jara Ronald , Obeso José A. , Chana-Cuevas Pedro , Sapkota Gopal P. , Rojas-Fernandez Alejandro
Published: March 2024

The p97 protein is a member of the AAA + family of ATPases. It is a mechanoenzyme that uses energy from ATP hydrolysis to promote protein unfolding and segregation actively. The unfolded products of p97 are presented to the 26S Proteasome for degradation. p97 substrate recognition is mediated by adaptors, which interact with substrates directly or indirectly through ubiquitin modifications, resulting in substrate funnelling into the central pore of the p97 hexamer and unfolding. We have engineered synthetic adaptors to target specific substrates to p97, using the extraordinary intracellular binding capabilities of camelid nanobodies fused to the UBX domain of the p97 Adapter FAF1. In such a way, we created a p97-directed proteolysis-targeting chimera (PROTAC), representing a unique E3 ubiquitin ligase-independent strategy to promote specific proteolysis.
Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduceABCA4splice variants in Stargardt disease
Authors: Liou Roxanne Hsiang-Chi , Urrutia-Cabrera Daniel , Westin Ida Maria , Golovleva Irina , Liu Guei-Sheung , Kumar Satheesh , McLenachan Samuel , Chen Fred Kuanfu , Hsu Fei-Ting , Edwards Tom , Martin Keith R , Cheng Albert Wu , Wong Raymond C.B.
Published: March 2024

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in theABCA4gene. The splicing variants c.5461-10T>C and c.4773+3A>G inABCA4cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate theseABCA4splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containingABCA4variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-splicedABCA4c.5461-10T>C variants and up to 78% reduction of theABCA4c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-splicedABCA4c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts forABCA4, providing an important step to advance the development of gene therapy to treat STGD1.
Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species and strain level diversity and evolution
Authors: Zhang Zhenrun J. , Cole Cody G. , Coyne Michael J. , Lin Huaiying , Dylla Nicholas , Smith Rita C. , Waligurski Emily , Ramaswamy Ramanujam , Woodson Che , Burgo Victoria , Little Jessica C. , Moran David , Rose Amber , McMillin Mary , McSpadden Emma , Sundararajan Anitha , Sidebottom Ashley M. , Pamer Eric G. , Comstock Laurie E.
Published: March 2024

Species of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome with diverse impacts on human health. While Bacteroidales strains and species are genomically and functionally diverse, order-wide comparative analyses are lacking. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, mobile gene, and metabolomic analyses. Families, genera, and species could be grouped based on many distinctive features. However, we also show extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-specific diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses can serve as a resource to facilitate informed selection of strains for microbiome reconstitution.
Botrytis cinereadetoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways
Authors: Bulasag Abriel Salaria , Ashida Akira , Miura Atsushi , Pring Sreynich , Kuroyanagi Teruhiko , Camagna Maurizio , Tanaka Aiko , Sato Ikuo , Chiba Sotaro , Ojika Makoto , Takemoto Daigo
Published: March 2024

Botrytis cinereais a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability ofB. cinereato tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogensPhytophthora infestans(late blight) andAlternaria solani(early blight) was severely inhibited by rishitin,B. cinereawas tolerant to rishitin. After incubation of rishitin with the mycelia ofB. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity toP. infestansandA. solani, indicating thatB. cinereahas at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenicBotrytisspecies, namelyB. elliptica, B. allii, B. squamosa,andB. tulipaealso had at least a partial ability to metabolize rishitin asB. cinerea, but their metabolic capacity was significantly weaker than that ofB. cinerea. These results suggest that the ability ofB. cinereato rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.
High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains
Authors: Loes Andrea N. , Tarabi Rosario Araceli L. , Huddleston John , Touyon Lisa , Wong Sook San , Cheng Samuel M. S. , Leung Nancy H.L. , Hannon William W. , Bedford Trevor , Cobey Sarah , Cowling Benjamin J. , Bloom Jesse D.
Published: March 2024

The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we have developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes via a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately one month. We applied the sequencing-based assay to quantify the impact of influenza vaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificities of the neutralizing antibodies elicited by vaccination vary among individuals, and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year—although the titers six months after vaccination were similar in individuals with and without the previous-year vaccination. We also identified a subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. This study demonstrates the utility of high-throughput sequencing-based neutralization assays that enable titers to be simultaneously measured against many different viral strains. We provide a detailed experimental protocol (DOI:https://dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and a computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others.
Analyses ofXenorhabdus griffiniaegenomes reveal two distinct sub-species that display intra-species variation due to prophages
Authors: Heppert Jennifer K. , Awori Ryan Musumba , Cao Mengyi , Chen Grischa , McLeish Jemma , Goodrich-Blair Heidi
Published: March 2024

Nematodes of the genusSteinernemaand theirXenorhabdusbacterial symbionts are lethal entomopathogens that are useful in the biocontrol of insect pests, as sources of diverse natural products, and as research models for mutualism and parasitism.Xenorhabdusplay a central role in all aspects of theSteinernemalifecycle, and a deeper understanding of their genomes therefore has the potential to spur advances in each of these applications. Here, we report a comparative genomics analysis ofXenorhabdus griffiniae, including the symbiont ofSteinernema hermaphroditumnematodes, for which genetic and genomic tools are being developed. We sequenced and assembled circularized genomes for threeXenorhabdusstrains: HGB2511, ID10 and TH1. We then determined their relationships to otherXenorhabdusand delineated their species via phylogenomic analyses, concluding that HGB2511 and ID10 areXenorhabdus griffiniaewhile TH1 is a novel species. These additions to the existingX. griffiniaelandscape further allowed for the identification of two subspecies within the clade. Consistent with otherXenorhabdus, the analysedX. griffiniaegenomes each encode a wide array of antimicrobials and virulence-related proteins. Comparative genomic analyses, including the creation of a pangenome, revealed that a large amount of the intraspecies variation inX. griffiniaeis contained within the mobilome and attributable to prophage loci. In addition, CRISPR arrays, secondary metabolite potential and toxin genes all varied among strains within theX. griffiniaespecies. Our findings suggest that phage-related genes drive the genomic diversity in closely relatedXenorhabdussymbionts, and that these may underlie some of the traits most associated with the lifestyle and survival of entomopathogenic nematodes and their bacteria: virulence and competition. This study establishes a broad knowledge base for further exploration of not only the relationships betweenX. griffiniaespecies and their nematode hosts but also the molecular mechanisms that underlie their entomopathogenic lifestyle.