bluuebunny/crossref_metadata_2025_split huggingface dataset:
MicrobeMod: A computational toolkit for identifying prokaryotic methylation and restriction-modification with nanopore sequencing
Authors: Crits-Christoph Alexander , Kang Shinyoung Clair , Lee Henry H. , Ostrov Nili 
Published: November 2023

AbstractBacteria and archaea use restriction-modification (R-M) systems to distinguish self from foreign DNA by methylating their genomes with DNA methyltransferases with diverse sequence specificities, and these immunity systems often vary at the strain level. Identifying active methylation patterns and R-M systems can reveal barriers to the introduction of recombinant DNA or phage infection. Here, we present the computational MicrobeMod toolkit for identifying 5mC and 6mA methylation sequence motifs and R-M systems in bacterial genomes using nanopore sequencing of native DNA. We benchmark this approach on a set of referenceE. colistrains expressing methyltransferases with known specificities. We then applied these analyses to 31 diverse bacterial and archaeal organisms to reveal the methylation patterns of strains with previously unexplored epigenetics, finding that prokaryotic 5-methylcytosine may be more common than previously reported. In summary, MicrobeMod can rapidly reveal new epigenetics within a prokaryotic genome sequenced with Oxford Nanopore R10.4.1 flow cells at sequencing depths as low as 10x and only requires native DNA. This toolkit can be used to advance fundamental knowledge of bacterial methylation and guide strategies to overcome R-M barriers of genetic tractability in non-model microbes.
Does stomatal patterning in amphistomatous leaves minimize the CO2diffusion path length within leaves?
Authors: Watts Jacob L. , Dow Graham J. , Buckley Thomas N. , Muir Christopher D. 
Published: November 2023

AbstractPhotosynthesis is co-limited by multiple factors depending on the plant and its environment. These include biochemical rate limitations, internal and external water potentials, temperature, irradiance, and carbon dioxide (CO2). Amphistomatous leaves have stomata on both abaxial and adaxial leaf surfaces. This feature is considered an adaptation to alleviate CO2diffusion limitations in productive environments where other factors are not limiting as the diffusion path length from stomate to chloroplast is effectively halved. Plants can also reduce CO2limitations through other aspects of optimal stomatal anatomy: stomatal density, distribution, patterning, and size. A number of studies have demonstrated that stomata are overdispersed on a single leaf surface; however, much less is known about stomatal anatomy in amphistomatous leaves, especially the coordination between leaf surfaces, despite their prevelance in nature and near ubiquity among crop species. Here we use novel spatial statistics based on simulations and photosynthesis modeling to test hypotheses about how amphistomatous plants may optimize CO2limitations in the model angiospermArabidopsis thalianagrown in different light environments. We find that 1) stomata are overdispersed, but not ideally dispersed, on both leaf surfaces across all light treatments; 2) abaxial and adaxial leaf surface patterning are independent; and 3) the theoretical improvements to photosynthesis from abaxial-adaxial stomatal coordination are miniscule (≪ 1%) across the range of feasible parameter space. However, we also find that 4) stomatal size is correlated with the mesophyll volume that it supplies with CO2, suggesting that plants may optimize CO2diffusion limitations through alternative pathways other than ideal, uniform stomatal spacing. We discuss the developmental, physical, and evolutionary constraits which may prohibit plants from reaching the theoretical adaptive peak of uniform stomatal spacing and inter-surface stomatal coordination. These findings contribute to our understanding of variation in the anatomy of amphistomatous leaves.
CRISPR-based editing strategies to rectifyEYA1complex genomic rearrangement linked to haploinsufficiency
Authors: Hwalin Yi , Yun Yejin , Choi Won Hoon , Hwang Hye-Yeon , Cha Ju Hyuen , Seok Heeyoung , Song Jae-Jin , Lee Jun Ho , Oh Seung Ha , Lee Sang-Yeon , Kim Daesik 
Published: November 2023

AbstractPathogenic structure variations (SVs) and genomic rearrangements are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements. Studies on Cas9-mediated translocations and inversions have mainly focused on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, through whole- genome sequencing, we identified a novel complex genomic rearrangement (CGR), specifically anEYA1inversion with a deletion, implicated in branchio-oto- renal/branchio-oto (BOR/BO) syndrome. The CGR results in a loss-of-function allele, leading to haploinsufficiency. To address this, two CRISPR-based editing approaches were tested. First, we engineered Cas9 nuclease and paired gRNAs tailored to the patient’s genome. The dual CRISPR/Cas9 system induced efficient editing at sites with paracentric inversion in patient-derived fibroblasts (up to 1.6%), and effectively restored the expression levels of theEYA1gene and its downstream targets, restoring overall transcriptional functionality. Additionally, we engineered gene-activating CRISPR-Cas modules (CRISPRa), which increasedEYA1mRNA and protein expression to wild-type levels in humanEYA1monoallelic knockout cells that mimic the haploinsufficiency. Moreover, CRISPRa significantly improved transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies may offer substantial translational potential for approximately 70% of disease-causingEYA1variants responsible for haploinsufficiency. In parallel to deciphering the complexities of the genomic landscape related to human genetic disorders, our findings demonstrate the potential of CIRSPR-guided genome editing for correcting SVs, including those withEYA1CGR linked to haploinsufficiency.
Wastewater surveillance pilot at US military installations: a cost model analysis
Authors: Sanjak Jaleal , McAuley Erin M , Pinkham Richard , Tarnowski Jacob , Miko Nicole , Rasmussen Bridgette , Manalo Christian , Goodson Michael , Stamps Blake , Necciai Bryan D , Sozhamannan Shanmuga , Maier Ezekiel J 
Published: November 2023

AbstractBackgroundThe coronavirus disease 2019 (COVID-19) pandemic highlighted the need for pathogen surveillance systems to augment both early warning and outbreak monitoring/control efforts. Wastewater samples provide a rapid and accurate source of environmental surveillance data to complement direct patient sampling. Due to its global presence and critical missions, the US military is a leader in global pandemic preparedness efforts. Clinical testing for COVID-19 on US Air Force (USAF) bases (AFBs) was effective, but costly with respect to direct costs and indirect costs of lost time. To remain operating at peak capacity such bases sought a more passive surveillance option and piloted wastewater surveillance (WWS) at 17 AFBs to demonstrate feasibility, safety, and utility from May 2021 to January 2022.ObjectiveHere we model the costs of a wastewater program for pathogens of pandemic potential within the specific context of US military installations using assumptions based on the results of the USAF and Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense (JPEO-CBRND) pilot program. The objective was to determine the cost of deploying WWS to all AFBs, relative to clinical swab testing surveillance regimes.MethodsA simple WWS cost projection model was built based on subject matter expert input and actual costs incurred during a WWS pilot program at USAF AFBs. Several SARS-CoV-2 circulation scenarios were considered and costs of both WWS and clinical swab testing were projected. Break even analysis was conducted to determine how reduction in swab testing could open up space to enable WWS to occur in complement.ResultsOur model confirms that wastewater surveillance is complementary and highly cost-effective when compared to existing alternative forms of biosurveillance. We find that the cost of WWS was between $10.5 - $18.5 million less expensive annually in direct costs as compared to clinical swab testing surveillance. When indirect cost of lost work is incorporated, including assumed lost work required to go obtain a clinical swab test, we estimate that over two thirds of clinical swab testing could be maintained with no additional costs upon implementation of WWS.ConclusionsOur results support adoption of wastewater surveillance across US military installations as part of a more comprehensive and early warning system that will enable adaptive monitoring during disease outbreaks.
Scalable Approach to Medical Wearable Post-Market Surveillance
Authors: Yoo Richard M. , Viggiano Ben T. , Pundi Krishna N. , Fries Jason A. , Zahedivash Aydin , Podchiyska Tanya , Din Natasha , Shah Nigam H. 
Published: November 2023

ABSTRACTObjectiveWe sought to develop a weak supervision-based approach to demonstrate feasibility of post-market surveillance of wearable devices that render AF pre-diagnosis.Materials and MethodsTwo approaches were evaluated to reduce clinical note labeling overhead for creating a training set for a classifier: one using programmatic codes, and the other using prompts to large language models (LLMs). Probabilistically labeled notes were then used to fine-tune a classifier, which identified patients with AF pre-diagnosis mentions in a note. A retrospective cohort study was conducted, where the baseline characteristics and subsequent care patterns of patients identified by the classifier were compared against those who did not receive pre-diagnosis.ResultsLabel model derived from prompt-based labeling heuristics using LLMs (precision = 0.67, recall = 0.83, F1 = 0.74) nearly achieved the performance of code-based heuristics (precision = 0.84, recall = 0.72, F1 = 0.77), while cutting down the cost to create a labeled training set. The classifier learned on the labeled notes accurately identified patients with AF pre-diagnosis (precision = 0.85, recall = 0.81, F1 = 0.83). Those patients who received pre-diagnosis exhibited different demographic and comorbidity characteristics, and were enriched for anticoagulation and eventual diagnosis of AF. At the index diagnosis, existence of pre-diagnosis did not stratify patients on clinical characteristics, but did correlate with anticoagulant prescription.Discussion and ConclusionOur work establishes the feasibility of an EHR-based surveillance system for wearable devices that render AF pre-diagnosis. Further work is necessary to generalize these findings for patient populations at other sites.
Barriers and Facilitators to Equitable Implementation of Long-Acting ART for Adolescents and Youth with HIV in Low- and Middle-Income Settings
Authors: Sam-Agudu Nadia A. , Adirieje Chibueze , Agwu Allison L. , Rakhmanina Natella 
Published: November 2023

ABSTRACTRecent approvals of long-acting (LA) antiretroviral treatment (ART) support an innovative alternative to daily oral pills that can improve adherence and treatment outcomes among adolescents and youth (AY) with HIV. We solicited stakeholder feedback on the implementation of LA ART for AY in low-and middle-income countries (LMICs) through a consensus-building forum at the 2022 International Workshop on HIV and Adolescence.We used the nominal group technique to generate, record, discuss, vote on, and rank perceived barriers and facilitators to implementing LA ART for AY. All in-person attendees were invited to participate and were assigned to six groups, each representing an intentional mix of AY, clinicians, researchers, program implementers and policymakers. We collected self-reported de-identified demographics and group rankings of barriers and facilitators. Responses were coded and categorized using the social-ecological model’s five levels of influence.137 Workshop delegates (67.9% male, 27.7% female; 0.7% non-binary and 46.7% less than 35 years old) participated in the group discussions. A large proportion of participants (51.9%) reported working in public health/program implementation. Most participants (88.4%) were from and/or worked in the African region. We identified 55 barriers and 48 facilitators of LA ART implementation and ranked them in social-ecological model categories of public policy, community, institutional/organizational, interpersonal, and individual levels. The highest number of ranked barriers was at the institutional/organizational level. The themes of “equitable access” and “choices of ART” were cross-cutting across individual and interpersonal levels. Other cross-cutting themes were “cost of LA ART” and “need for funding and sustainability of LA ART programs”. Proposed facilitators addressed identified barriers at each social-ecological level of influence and emphasized peer engagement.Our nominal groups identified key barriers and proposed facilitators at five different social ecological levels, which can inform implementation science-guided design and equitable implementation of youth centered LA ART in LMICs and globally.
Genome-wide CRISPR screen identifies KEAP1 perturbation as a vulnerability of ARID1A-deficient cells
Authors: Fournier LA , Kalantari F , Wells JP. , Lee JS , Trigo-Gonzalez G , Moksa MM , Smith T , White J , Shanks A , Wang L , Su E , Wang Y , Huntsman DG , Hirst M , Stirling PC 
Published: November 2023

ABSTRACTARID1A is the core DNA binding subunit of the BAF chromatin remodeling complex and is mutated in about ∼8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at >50% of cases. Despite a growing understanding of the consequences of ARID1A-loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A-deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of wARID1A-deficient cells.
A double-ring of human RAD52 remodels replication forks restricting fork reversal
Authors: Honda Masayoshi , Razzaghi Mortezaali , Gaur Paras , Malacaria Eva , Marozzi Giorgia , Biagi Ludovica Di , Aiello Francesca Antonella , Paintsil Emeleeta A. , Stanfield Andrew J. , Deppe Bailey J. , Gakhar Lokesh , Schnicker Nicholas J. , Ashley Spies M. , Pichierri Pietro , Spies Maria 
Published: November 2023

SummaryHuman RAD521,2is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation3–7. In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL15. The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.One Sentence SummaryUsing cryo-EM, biochemical and single-molecule approaches we show that the structure of stalled DNA replication fork promotes a unique two-ring organization of human RAD52 protein which remodels the fork via DNA strand exchange.
Proteoform-level deconvolution reveals a broader spectrum of ibrutinib off-targets
Authors: Leo Isabelle , Kunold Elena , Anastasia Audrey , Tampere Marianna , Eirich Jürgen , Jafari Rozbeh 
Published: November 2023

AbstractOver the last decade, proteome-wide mapping of drug interactions has revealed that most targeted drugs bind to not only their intended targets, but additional proteins as well. However, the majority of these studies have focused on analyzing proteins as encoded by their genes, thus neglecting the fact that most proteins exist as dynamic populations of multiple proteoforms. Here, we addressed this problem by combining the use of thermal proteome profiling (TPP), a powerful method for proteome analysis, with proteoform detection to refine the target landscape of an approved drug, ibrutinib. We revealed that, in addition to known targets, ibrutinib exhibits an intricate network of interactions involving multiple different proteoforms. Notably, we discovered affinity for specific proteoforms that link ibrutinib to mechanisms in immunomodulation and cellular processes like Golgi trafficking, endosomal trafficking, and glycosylation. These insights provide a framework for interpreting clinically observed off-target and adverse events. More generally, our findings highlight the importance of proteoform-level deconvolution in understanding drug interactions and their functional impacts, and offer a critical perspective for drug mechanism studies and potential applications in precision medicine.
Melanoma-derived DNA polymerase theta variants exhibit altered DNA polymerase activity
Authors: Thomas Corey , Avalos-Irving Lisbeth , Victorino Jorge , Green Sydney , Andrews Morgan , Rodrigues Naisha , Ebirim Sarah , Mudd Ayden , Towle-Weicksel Jamie B. 
Published: November 2023

AbstractDNA Polymerase θ (Pol θ or POLQ) is primarily involved in repairing double-stranded breaks in DNA through the alternative pathway known as microhomology-mediated end joining (MMEJ) or theta-mediated end joining (TMEJ). Unlike other DNA repair polymerases, Pol θ is thought to be highly error prone, yet critical for cell survival. We have identified several mutations in the POLQ gene from human melanoma tumors. Through biochemical analysis, we have demonstrated that all three cancer-associated variants experienced altered DNA polymerase activity including a propensity for incorrect nucleotide selection and reduced polymerization rates compared to WT Pol θ. Moreover, the variants are 30 fold less efficient at incorporating a nucleotide during repair and up to 70 fold less accurate at selecting the correct nucleotide opposite a templating base. Taken together, this suggests that aberrant Pol θ has reduced DNA repair capabilities and may also contribute to increased mutagenesis. While this may be beneficial to normal cell survival, the variants were identified in established tumors suggesting that cancer cells may use this promiscuous polymerase to its advantage to promote metastasis and drug resistance.
Plant pathogenic fungi hijack phosphate starvation signaling with conserved enzymatic effectors
Authors: McCombe Carl L. , Wegner Alex , Zamora Chenie S. , Casanova Florencia , Aditya Shouvik , Greenwood Julian R. , Wirtz Louisa , de Paula Samuel , England Eleanor , Shang Sascha , Ericsson Daniel J. , Oliveira-Garcia Ely , Williams Simon J. , Schaffrath Ulrich 
Published: November 2023

AbstractPhosphate availability modulates plant immune function and regulates interactions with beneficial, phosphate-providing, microbes. Here, we describe the hijacking of plant phosphate sensing by a family of Nudix hydrolase effectors from pathogenicMagnaporthe oryzaeandColletotrichumfungi. Structural and enzymatic analyses of the Nudix effector family demonstrate that they selectively hydrolyze inositol pyrophosphates, a molecule used by plants to monitor phosphate status and regulate starvation responses. InM. oryzae, gene deletion and complementation experiments reveal that the enzymatic activity of a Nudix effector significantly contributes to pathogen virulence. Further, we show that this conserved effector family induces phosphate starvation signaling in plants. Our study elucidates a molecular mechanism, utilized by multiple phytopathogenic fungi, that manipulates the highly conserved plant phosphate sensing pathway to exacerbate disease.One-Sentence SummaryA family of conserved enzyme effectors from pathogenic fungi manipulate plant phosphate sensing to promote infection.
A tomato ethylene-insensitive mutant displays altered growth and higher β-carotene levels in fruit
Authors: Gupta Suresh Kumar , Santisree Parankusam , Gupta Prateek , Kilambi Himabindu Vasuki , Sreelakshmi Yellamaraju , Sharma Rameshwar 
Published: November 2023

AbstractThe mutants insensitive to ethylene are helpful in deciphering the role of ethylene in plant development. We isolated an ethylene-insensitive tomato (Solanum lycopersicum) mutant by screening for acetylene-resistant (atr-1) seedlings. Theatr-1mutant displayed resistance to kinetin, suggesting attenuation of the ethylene sensing response.atr-1also exhibited resistance to ABA- and glucose-mediated inhibition of seed germination. Unlike theNever- ripe(Nr) mutant,atr-1seedlings were resistant to glucose, indicating ethylene sensing inatr-1is located in a component distinct fromNr. Metabolically,atr-1seedlings had lower levels of amino acids but higher levels of several phytohormones, including ABA.atr-1plants grew faster and produced more flowers, leading to a higher fruit set. However, theatr- 1fruits took a longer duration to reach the red-ripe (RR) stage. The ripenedatr-1fruits had higher β-carotene levels, retained high β-carotene and lycopene levels post-RR stage. The metabolome profiles of post-RR stageatr-1fruits revealed increased levels of sugars. Theatr-1had a P279L mutation in the GAF domain of theETR4, a key ethylene receptor regulating tomato ripening. Our study highlights that novel alleles in ethylene receptors may aid in enhancing the nutritional quality of tomato.