bluuebunny/crossref_metadata_2025 huggingface dataset:
Host genetics and COVID-19 severity: increasing the accuracy of latest severity scores by Boolean quantum features
Authors: Martelloni Gabriele , Turchi Alessio , Fallerini Chiara , Degl’Innocenti Andrea , Baldassarri Margherita ,   GEN-COVID Multicenter study, Olmi Simona , Furini Simone , Renieri Alessandra
Published: February 2023

The impact of common and rare variants in COVID-19 host genetics is widely studied in [16]. Here, common and rare variants were used to define an interpretable machine learning model for predicting COVID-19 severity. Firstly, variants were converted into sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. After that, the Boolean features, selected by these logistic models, were combined into an Integrated PolyGenic Score, the so called IPGS, which offers a very simple description of the contribution of host genetics in COVID-19 severity. IPGS leads to an accuracy of 55-60% on different cohorts and, after a logistic regression with in input both IPGS and the age, it leads to an accuracy of 75%. The goal of this paper is to improve the previous results, using the information on the host organs involved in the disease. We generalized the IPGS adding a statistical weight for each organ, through the transformation of Boolean features into “Boolean quantum features”, inspired by the Quantum Mechanics. The organs’ coefficients were set via the application of the genetic algorithm Pygad and, after that, we defined two new Integrated PolyGenic Score (and). By applying a logistic regression with both(or indifferently) and age as input, we reach an accuracy of 84-86%, thus improving the results previously shown in [16] by a factor of 10%.
Nuclear genetic background influences the phenotype of theDrosophila tko25tmitochondrial protein-synthesis mutant
Authors: Jacobs Howard T. , Tuomela Tea , Lillsunde Päivi
Published: February 2023

TheDrosophila tko25tpoint mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding, and to vary with mitochondrial genetic background. Here we show similarly profound effects conferred by nuclear genetic background. We backcrossedtko25tinto each of two standard nuclear backgrounds, Oregon R andw1118, the latter used as recipient line in many transgenic applications requiring selection for thewhiteminigene marker. In thew1118background,tko25tflies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygoustko25tfemales in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years,tko25tflies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in thew1118background. Phenotypic severity did not correlate with the expression level of thetkogene. Analysis of tko25t hybrids between the two backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal and parent-of-origin dependent determinants. Although some of these effects may betko25t-specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of non-lethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.
Using pose estimation and 3D rendered models to study leg-mediated self-righting by lanternflies
Authors: Bien Theodore , Alexander Benjamin H. , Li Chengpei , Goeler-Slough Natalie , Tonia Hsieh S. , Kane Suzanne Amador
Published: February 2023

The ability to upright quickly and efficiently when overturned on the ground (terrestrial self-righting) is crucial for living organisms and robots. Previous studies have mapped the diverse behaviors used by various animals to self-right on different substrates, and proposed physical models to explain how body morphology can favor specific self-righting methods. However, to our knowledge no studies have quantified and modeled all of an animal’s limb motions during these complicated behaviors. Here, we studied terrestrial self-righting for nymphs of the invasive spotted lanternfly (Lycorma delicatula), an insect that must frequently recover from being overturned after jumping and falling in its native habitat. These nymphs self-righted successfully in 92-100% of trials on three substrates with different friction and roughness, with no significant difference in the time or number of attempts required. They accomplished this using three stereotypic sequences of movements. To understand these motions, we combined 3D poses tracked on multi-view high-speed video with articulated 3D models created using photogrammetry and Blender rendering software. The results were used to calculate the mechanical properties (e.g., potential and kinetic energy, angular speed, stability margin, torque, force, etc.) of these insects during righting trials. We used an inverted physical pendulum model (a “template”) to estimate the kinetic energy available in comparison to the increase in potential energy required to flip over. While these insects began righting using primarily quasistatic motions, they also used dynamic leg motions to achieve final tip-over. However, this template did not describe important features of the insect’s center of mass trajectory and rotational dynamics, necessitating the use of an “anchor” model comprising the 3D rendered body model and six articulated two-segment legs to model the body’s internal degrees of freedom and capture the role of the legs’ contribution to inertial reorientation. This anchor elucidated the sequence of highly coordinated leg movements these insects used for propulsion, adhesion, and inertial reorientation during righting, and how they frequently pivot about a body contact point on the ground to flip upright. In the most frequently used method, diagonal rotation, these motions allowed nymphs to spin their bodies to upright with lower force with a greater stability margin compared to the other less frequently-used methods. We provide a concise overview of necessary background on 3D orientation and rotational dynamics, and the resources required to apply these low-cost modeling methods to other problems in biomechanics.
Development of minimum reporting sets of patient characteristics in epidemiological research: a methodological systematic review
Authors: Vuong My Luong , Trang Tu Pham Hien , Duong Khanh Linh , Vo Tat-Thang
Published: February 2023

Background

Core patient characteristic sets (CPCS) are increasingly developed to identify variables that should be reported to describe the target population of epidemiological studies in the same medical area, while keeping the additional burden on the data collection acceptable.

Methods

We conduct a systematic review of primary studies/ protocols published aiming to develop CPCS, using the PubMed database. We particularly focus on the study design and the characteristics of the proposed CPCS. Quality of Delphi studies was assessed by a tool prosposed in the literatue. All results are reported descriptively.

Results

Among 23 eligible studies, Delphi survey is the most frequently used technique to obtain consensus in CPCS development (69.6%, n=16). Most studies do not include patients as stakeholders. The final CPCS rarely include socioeconomic factors. 60.9% (n=14) and 31.6% (n=6) of studies provide definition and recommend measurement methods for items, respectively.

Conclusion

This study identified a considerable variation and suboptimality in many methodological aspects of CPCS studies. To enhance the credibility and adoption of CPCS, a standard for conducting and reporting CPCS studies is warranted.

Funding

No funds, grants, or other support were received during the preparation of this manuscript.

Registration

This review was not pre-registered.
Non-Communicable Disease Service Readiness in Nepal: A Further Analysis of Nepal Health Facility Survey- 2021
Authors: Adhikari Bikram , Pandey Achyut Raj , Lamichhane Bipul , Saugat Pratap KC , Joshi Deepak , Regmi Shophika , Giri Santosh , Baral Sushil Chandra
Published: February 2023

Objective

To assess the readiness of public and private health facilities(HFs) in delivering Cardiovascular Diseases(CVDs), Diabetes Mellitus(DM), Chronic Respiratory Diseases(CRDs), and Mental Health(MH) services in Nepal.

Methods

We analyzed data on service readiness for CVDs, DM, CRDs, and MH from Nepal Health Facility Survey 2021 using Service Availability and Readiness Assessment manual of the World Health Organization. Readiness score was measured as the average availability of tracer items in percent, and facilities were considered “ready” for Non-Communicable Diseases (NCDs) management if scored ≥70 (out of 100). We performed weighted descriptive analysis, univariate and multivariable logistic regression to determine association of readiness of HFs with province, type of HFs, ecological region, quality assurance activities, external supervision, client’s opinion review, and frequency of HF meetings. The result of regression analysis are presented as odds ratio with 95% confidence interval(CI) and p-value.

Results

Of 1581 facilities offering any NCDs related services, 93.1%(95% CI: 90.9 to 94.7), 75.8%(95%CI: 72.4 to 78.8), 99.3%(95%CI: 98.3 to 99.7) and 26.0%(95%CI: 23.0 to 29.2) provide CVDs, DM, CRDs and MH-related services respectively. The overall readiness score for CVDs, DM, CRDs, and MH-related services were 38.1±15.4, 38.5±16.7, 32.6±14.7 and 24.0±23.1 respectively with readiness score lowest for the guidelines and staff training domain and highest for essential equipment and supplies domain. Peripheral public HFs were more likely to be ready to provide all NCDs-related services as compared to federal/provincial facilities. The HFs with external supervision in past 4 months were less likely to be ready to provide CRDs and DM related services and HFs reviewing client’s opinions were more likely to be ready to provide CRDs, CVDs and DM related services.

Conclusion

Readiness of HFs to provide CVDs, DM, CRDs, and MH-related services was sub-optimal in Nepal. It is recommended to reform policy to improve service readiness for NCDs.

Strengths and limitations of this study

Nationally representative sample of health facilities in Nepal, with coverage of all seven provinces and 77 districts

Survey has adopted a highly standardized survey with the globally accepted research protocol. Variables for readiness analysis are based on standardized WHO’s SARA guideline and thus, findings are comparable to findings from other countries.

Weighted analysis has been performed, which takes into account the complex sampling procedures and adjusts for non-response and disproportionate sampling.

Since the survey was conducted while the COVID-19 pandemic was ongoing, there could be some impact of the pandemic on study results.
Who Wears the Face Mask? Preventive Measures Against COVID-19 in Latin America Before Vaccination
Authors: Rentería Elisenda , Gómez-Casillas Amalia , Zueras Pilar
Published: February 2023

The COVID-19 pandemic outbreak imposed the use of the sanitary mask as a protective measure to reduce the spread of the pandemic, recommended by the World Health Organization. However, the use of the face mask has been uneven and determined by individual, regional, cultural, and political factors. Based on data from the Latinobarometer, we aim to understand the profile of people who used a mask in the context of the COVID-19 pandemic in 18 countries of Latin America, between October and November 2020, right before the mass vaccination campaigns. Results show that women, older people, those with higher education, being employed and not working in temporarily jobs, retirees, students, people with a centrist political ideology, and Catholics, had a higher chance of using a face mask on a regular basis. People living in Venezuela, Chile, Costa Rica and Brazil were the most likely to use face masks. These results call attention to the need to understand social forces behind the willingness to adopt non-pharmacological preventive measures in order to make them more effective in health crisis emergencies.
Development and optimisation of cationic lipid nanoparticles for mRNA delivery
Authors: Yan Dongnan , Lu Haonan , Kaur Apanpreet , Fu Ruisi , Wang Ning , Teh Jin Hui , Lou Hantao , Aboagye Eric O , Chen Rongjun
Published: February 2023

Messenger RNA (mRNA) has been proposed as a therapeutic agent for various diseases, including cancer. To ensure effective transfection of cancer cells, mRNA needs to be transported with a delivery system that protects its integrity and functionality. In this regard, cationic lipid nanoparticles composed of dioleoylphosphatidylethanolamine (DOPE) and 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) have emerged as common vectors to deliver mRNA. In this project, we aim to use luciferase mRNA as a reporter to synthesise mRNA-loaded cationic lipid nanoparticles, and optimise their mRNA encapsulation and transfection efficiency in ovarian cancer cells. The optimisation process included: 1) adjusting the lipid formulation; 2) adjusting the input mRNA concentration before lipid nanoparticle extrusion; and 3) adjusting the extrusion methods. After optimisation, the encapsulation efficiency was optimised to 62%, thus achieving a relatively high transfection luminescence signal (9.4 times compared to baseline). The lipid nanoparticles also demonstrated stable physical characteristics and high biocompatibility (above 75% cell viability after treatment) within 24 hours. Overall, this project evaluated the synthesis of DOPE/DC-Chol cationic lipid nanoparticles, and optimised their mRNA encapsulation and transfection efficiency in ovarian cancer cell lines. The optimised lipid nanoparticles can be utilised as an ideal system for mRNA delivery, which could be further developed as a potential platform for the immunotherapy in ovarian cancer.
Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A
Authors: Stergachis Andrew B. , Blue Elizabeth E. , Gillentine Madelyn A , Wang Lee-kai , Schwarze Ulrike , Cortés Adriana Sedeño , Ranchalis Jane , Allworth Aimee , Bland Austin E. , Chanprasert Sirisak , Chen Jingheng , Doherty Daniel , Folta Andrew B. , Glass Ian , Horike-Pyne Martha , Huang Alden Y. , Khan Alyna T. , Leppig Kathleen A. , Miller Danny E. , Mirzaa Ghayda , Parhin Azma , Raskind Wendy , Rosenthal Elisabeth A. , Sheppeard Sam , Strohbehn Samuel , Sybert Virginia P. , Tran Thao T. , Wener Mark ,   University of Washington Center for Mendelian Genomics (UW-CMG), Undiagnosed Diseases Network (UDN), Byers Peter H. , Nelson Stanley F. , Bamshad Michael J. , Dipple Katrina M. , Jarvik Gail P. , Hoppins Suzanne , Hisama Fuki M.
Published: February 2023

Objectives

Transcript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored.

Methods

We applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis.

Results

We identified an intronic homozygousMFN2c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-lengthMFN2transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A).

Discussion

This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.
An exploratory data analysis from ovine and bovine RNA-seq identifies pathways and key genes related to cervical dilatation
Authors: Gonçalves Joedson Dantas , Sterman Ferraz José Bento , Meirelles Flávio Vieira , Nociti Ricardo Perecin , Franco Oliveira Maria Emilia
Published: February 2023

The present study developed a review and exploration of data in public and already validated repositories. The main objective is to identify the pathways involved in ruminant’s cervical dilatation, which are conserved between cattle and sheep in the follicular and luteal phases of the reproductive cycle. In cattle, 1961 genes were found to be more expressed in the follicular phase and 1560 in the luteal phase. 24 genes were considered exclusively expressed from these 18 genes were in the follicular phase and 6 genes were in the luteal phase. In sheep, 2126 genes are more expressed in the follicular phase and 2469 genes are more expressed in the luteal phase. Hoxb genes were identified in both species and are correlated with the PI3K/Akt pathway. PI3K/Akt was also found in both cattle and sheep, appearing prominently in the follicular and luteal phases of both species. Our analyzes have pointed out that the PI3K/Akt pathway and the Hoxb genes appear in prominence, in modulating mechanisms that involve estrus alterations in the cervix. PI3K/Akt appears to be an important pathway in the cervical relaxation process.
Engineering bacteriophages through deep mining of metagenomic motifs
Authors: Huss Phil , Kieft Kristopher , Meger Anthony , Nishikawa Kyle , Anantharaman Karthik , Raman Srivatsan
Published: February 2023

Bacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Metagenomic Sequence Informed Functional Scoring (Meta-SIFT), to discover sequence motifs in metagenomic datasets that can be used to engineer phage activity. Meta-SIFT uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested over 17,000 Meta-SIFT derived sequence motifs in the receptor-binding protein of the T7 phage. The screen revealed thousands of T7 variants with novel host specificity with functional motifs sourced from distant families. Position, substitution and location preferences dictated specificity across a panel of 20 hosts and conditions. To demonstrate therapeutic utility, we engineered active T7 variants against foodborne pathogenE. coliO121. Meta-SIFT is a powerful tool to unlock the functional potential encoded in phage metagenomes to engineer bacteriophages.
Colocalization of Cancer Associated Biomarkers on Single Extracellular Vesicles for Early Cancer Detection
Authors: Salem Daniel P. , Bortolin Laura T. , Gusenleitner Dan , Grosha Jonian , Zabroski Ibukunoluwapo O. , Biette Kelly M. , Banerjee Sanchari , Sedlak Christopher R. , Byrne Delaney M. , Hamzeh Bilal F. , King MacKenzie S. , Cuoco Lauren T. , Santos-Heiman Timothy , Duff Peter A. , Winn-Deen Emily S. , Guettouche Toumy , Mattoon Dawn R. , Huang Eric K. , Schekman Randy , Couvillon Anthony D. , Sedlak Joseph C.
Published: February 2023

Detection of cancer early, when it is most treatable, remains a significant challenge due to the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (i.e., protein biomarkers, circulating tumor DNA, etc.) that would enable detection using a non-invasive diagnostic assay. In addition, many benign conditions manifest in a similar manner, thus discriminating an early-stage cancerous lesion from a benign tumor can present additional challenges and result in unnecessary medical procedures. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. Extracellular vesicles from all cell types, including early-stage tumors, are known to be abundant in blood, are remarkably stable, and serve as a biopsy of their cell of origin. The detection of a colocalized combination of cancer associated biomarkers that provide tumor specificity on the surface of extracellular vesicles enables the discrimination of early- and late-stage cancer from non-malignant conditions.
Different approaches to estimate benthic metazoan diversity associated with free-living macroalgae (Fucus vesiculosus) on shallow soft sediments
Authors: Preston Roxana , Majaneva Markus , Halonen Viivi , Rodil Iván F
Published: February 2023

Habitat complexity can boost biodiversity by providing a wide range of niches allowing species co-existence. Baltic Sea benthic communities are characterised by low species diversity. Thus the occurrence of the habitat forming macroalgaFucus vesiculosusmay influence benthic communities and promote diversity. Here we obtain biodiversity estimates through conventional and eDNA approaches for the benthic assemblages associated with free-livingFucusand the adjacent bare-sediment habitats at six sites from the Northern Baltic Proper and the Gulf of Finland. Free-livingF. vesiculosushabitats are heterogeneous with biodiversity estimates varying considerably among sites. The additional habitat complexity provided byF. vesiculosustends to improve taxa richness as a result of additional epifauna assemblages, although infaunal taxa richness and abundance is often reduced. Consequently the complex habitats provided by free-livingF. vesiculosusoften improves biodiversity, yet alters the composition of assemblages in soft sediment habitats and consequential ecosystem functioning. We emphasise the disparity in biodiversity estimates achieved when employing different biodiversity approaches. Biodiversity estimates were more similar within approaches compared to between habitat types, with each approach detecting exclusive taxa. We suggest that biodiversity estimates benefit from a multi-approach design where both conventional and eDNA approaches are employed in complement.
Tracking components of bilingual language control in speech production: an fMRI study using functional localizers
Authors: Wolna Agata , Szewczyk Jakub , Diaz Michele , Domagalik Aleksandra , Szwed Marcin , Wodniecka Zofia
Published: February 2023

When bilingual speakers switch back to speaking in their native language (L1) after having used their second language (L2), they often experience difficulty in retrieving words in their L1: this phenomenon is referred to as theL2 after-effect.We used the L2 after-effect as a lens to explore the neural bases of bilingual language control mechanisms. Our goal was twofold: first, to explore whether bilingual language control draws on domain-general or language-specific mechanisms; second, to investigate the precise mechanism(s) that drive the L2 after-effect. We used a precision fMRI approach based onfunctional localizersto measure the extent to which the brain activity that reflects the L2 after-effect overlaps with the language network (Fedorenko et al., 2010) and the domain-general Multiple Demand network (Duncan et al., 2010), as well as three task-specific networks that tap into interference resolution, lexical retrieval, and articulation. Forty-two Polish-English bilinguals participated in the study. Our results show that the L2 after-effect reflects increased engagement of domain-general but not language-specific resources. Furthermore, contrary to previously proposed interpretations, we did not find evidence that the effect reflects increased difficulty related to lexical access, articulation, and the resolution of lexical interference. We propose that difficulty of speech production in the picture naming paradigm – manifested as the L2 after-effect – reflects interference at a non-linguistic level of task schemas or a general increase of cognitive control engagement during speech production in L1 after L2.
Deconvolving feeding niches and strategies of abyssal holothurians from their stable isotope, amino acid, and fatty acid composition
Authors: Stratmann Tanja , van Breugel Peter , van Oevelen Dick
Published: February 2023

Holothurians are the dominant megabenthic deposit feeders in the Peru Basin (South-East Pacific) and feed to various degrees of selectively on the heterogenous pool of sedimentary detritus, but diet preferences for most holothurian species are unknown. This study reconstructs the diets of 13 holothurian species of the orders Elasipodida, Holothuriida, and Synallactida, from bulk stable isotope analyses (δ13C, δ15N) of holothurian body walls and guts, gut contents, and feces that were combined with compound-specific stable isotope analyses of amino acids, phospholipid-derived fatty acids, and neutral lipid-derived fatty acids in the body wall. Fatty acid concentrations showed high levels of storage lipids, an likely adaption to limited food supply to abyssal plains. Amino acid δ15N isotope values allowed estimating trophic levels of holothurian species and calculating heterotrophic re-synthesis of amino acids. Fatty acids served as trophic markers for feeding on diatom- and dinoflagellate derived phytodetritus, bacteria, Foraminifera, and detritus containing the PUFA C22:1ω9-cis. Several holothurian species seemed to be secondary consumers of detritus, while bacteria in their guts were primary consumers of this detritus. A Sørensen–Dice coefficient based cluster analysis using data of trophic levels, levels of heterotrophic re-synthesis of amino acids, feeding selectivity, and food sources/ diet suggested three trophic groups, characterized by different trophic levels. We show that this multi-biomarker driven approach allows to deconvolve trophic niches and feeding selectivity in one of the most challenging environments on earth and to identify dependence of deep-sea species to organic matter inputs that vary with season and/or climate.
Inter-kingdom microbial interactions revealed by a comparative machine-learning guided multi-omics analysis of industrial-scale biogas plants
Authors: Wirth Roland , Bagi Zoltán , Shetty Prateek , Szuhaj Márk , Cheung Teur Teur Sally , Kovács Kornél L. , Maróti Gergely
Published: February 2023

Multi-omics analysis is a powerful tool for the detection and study of inter-kingdom interactions, such as those between bacterial and archaeal members of complex biogas-producing microbial communities. In the present study, the microbiomes of three industrial-scale biogas digesters, each fed with different substrates, were analysed using a machine-learning guided genome-centric metagenomics framework complemented with metatranscriptome data. This data permitted us to elucidate the relationship between abundant core methanogenic communities and their syntrophic bacterial partners. In total, we detected 297 high-quality, non-redundant metagenome-assembled genomes (nrMAGs). Moreover, the assembled 16S rRNA gene profiles of these nrMAGs showed that the phylum Firmicutes possessed the highest copy number, while the representatives of the Archaeal domain had the lowest. Further investigation of the three anaerobic microbial communities showed characteristic alterations over time but remained specific to each industrial-scale biogas plant. The relative abundance of various microbes as revealed by metagenome data were independent from corresponding metatranscriptome activity data. Interestingly, Archaea showed considerably higher activity than was expected from their abundance. We detected 53 nrMAGs that were present in all three biogas plant microbiomes with different abundances. The core microbiome correlated with the main chemical fermentation parameters and no individual parameter emerged as a predominant shaper of community composition. Various interspecies H2/electron transfer mechanisms were assigned to hydrogenotrophic methanogens in the biogas plants that ran on agricultural biomass and wastewater. Analysis of metatranscriptome data revealed that methanogenesis pathways were the most active of all main metabolic pathways. These findings highlight the importance of a combinatorial omics data framework to identify and characterise the activity of specific microbes in complex environments.
IVT generation of guideRNAs for Cas9-enrichment Nanopore Sequencing
Authors: Gilpatrick Timothy , Wang Josh Zhiyong , Weiss David , Norris Alexis L , Eshleman James , Timp Winston
Published: February 2023

Generating high-coverage sequencing coverage at select genomic loci has extensive applications in both research science and genetic medicine. Long-read sequencing technologies (e.g. nanopore sequencing) have expanded our ability to generate sequencing data in regions (e.g. repetitive elements) that are difficult to interrogate with short-read sequencing methods. In work presented here, we expand on our previous work using CRISPR/Cas9 for targeted nanopore sequencing by usingin vitrotranscribed guideRNAs, with 1100 guideRNAs in a single experiment. This approach decreases the cost per guideRNA, increases the number of guideRNAs that can be multiplexed in a single experiment, and provides a way to rapidly screen numerous guideRNAs for cutting efficiency. We apply this strategy in multiple patient-derived pancreatic cancer cell lines, demonstrating its ability to unveil structural variation in “deletion hotspots” around the tumor suppressor genesp16(CDKN2A), andSMAD4.
GeneDART: Extending gene coverage in image-based spatial transcriptomics by deep learning-based domain adaptation with barcode-based RNA-sequencing data
Authors: Ohn Jungyoon , Lee Daeseung , Choi Hongyoon
Published: February 2023

Spatial transcriptomics (ST) technologies provide comprehensive biological insights regarding cell-cell interactions and peri-cellular microenvironments. ST technologies are divided into two categories: imaging-based (I-B) and barcode-based (B-B). I-B ST technologies provide high resolution and sensitivity but have limited gene coverage. B-B ST technologies can analyze the whole transcriptome but have lower spatial resolution. To address these limitations, we propose a deep learning-based model that integrates I-B and B-B ST technologies to increase gene coverage while preserving high resolution. A model, trained by a neural network with an adversarial loss based on I-B and B-B datasets from human breast cancer tissue, was able to extend gene coverage to whole transcripts-level and accurately predict gene expression patterns in the I-B dataset with a high resolution. This novel methodology, named GeneDART, could enable researchers to utilize B-B and I-B ST datasets in a complementary way.
Vir1p, the Yeast Homolog of Virilizer, is Required for mRNA m6A Methylation and Meiosis
Authors: Park Zachory M. , Belnap Ethan , Remillard Matthew , Rose Mark D.
Published: February 2023

N6-Methyladenosine (m6A) is one of the most abundant modifications found on eukaryotic mRNAs. mRNA methylation regulates a host of biological processes including meiosis, a specialized diploid cell division program that results in the formation of haploid cells (gametes). During budding yeast meiosis, m6A levels peak early, before the initiation of the meiotic divisions. High-throughput studies and work from our lab showed that Ygl036wp, a previously uncharacterized protein interacts with Kar4p, a meiotic protein required for mRNA m6A-methylation. Ygl036wp has no discernable domains except for several intrinsically disordered regions. However, protein folding prediction tools showed that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m6A-methylation in higher eukaryotes. In addition, Ygl036wp has several conserved motifs shared with VIRMA/Virilizer/VIR proteins. Accordingly, we propose to call the geneVIR1forbudding yeast ortholog ofVIRMA/Virilizer/VIR1. In support, Vir1p interacts with all other members of the yeast methyltransferase complex and is required for mRNA m6A methylation and meiosis. Vir1p is required for the stability of proteins comprising the methyltransferase complex, suggesting that Vir1p acts as a scaffold to stabilize the complex. Thevir1Δ/Δ mutant is defective for premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factorIME1;additional overexpression of the translational regulatorRIM4is required for sporulation.

Consistent withIME1suppression,vir1Δ/Δ exhibits a defect in the abundance ofIME1mRNA, as well as transcripts within Ime1p’s regulon. Suppression byIME1revealed a defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by additional overexpression ofRIM4. Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation.

Author Summary

Ygl036wp is a previously uncharacterized protein that we propose to name Vir1p (budding yeast ortholog ofVIRMA/Virilizer/VIR1). Work from our lab and others initially found an interaction between Vir1p and members of the yeast mRNA methyltransferase complex (Kar4p and Mum2p). We found that Vir1p interacts with all known members of the methyltransferase complex and is required for mRNA methylation. Vir1p is required early in meiosis;vir1Δ/Δ mutants arrest due to the reduced expression of Ime1p. Lower levels of Ime1p cause severe disruption to the meiotic transcriptome invir1Δ/Δ. Thevir1Δ/Δ meiotic defect can be partially suppressed by the overexpression ofIME1; full suppression requires overexpression of bothIME1andRIM4. Using recent advances in protein folding predictions, we found that Vir1p is a remote homolog of VIRMA/Virilizer/VIR and shares conserved motifs with the protein from other organisms. Vir1p, like VIRMA/Virilizer/VIR, stabilizes the methyltransferase complex.
Systematic part transfer by extending a modular toolkit to diverse bacteria
Authors: Keating Kevin , Young Eric M.
Published: February 2023

It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection forE. coli. This enabled testing of a library of DNA constructs across the Pseudomonadota –Escherichia coli, Pseudomonas putida, Cupriavidus necator, andKomagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms – meaning that the same parts can be used to programE. coli, P. putida, C. necator, andK. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently inK. nataicolathan in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, making it possible to create a small number of parts sets that can span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.
Selection of ade novogene that can promote survival ofE. coliby modulating protein homeostasis pathways
Authors: Frumkin Idan , Laub Michael T.
Published: February 2023

Cells sometime adapt to challenging environments by turning non-functional loci into functional genes in a process termedde novogene birth. But how proteins with random amino acid sequences integrate into existing cellular pathways to provide a benefit remains poorly understood. Here, we screened ∼108random genes for their ability to rescue growth arrest ofE. colicells producing the ribonuclease toxin MazF. Approximately 2,000 random genes could promote growth by reducing transcription from the promoter drivingmazFexpression. Additionally, one gene, named randomantitoxin ofMazF(ramF), whose protein product was well-tolerated in cells and neutralized MazF by interacting with chaperones, leading to MazF proteolysis. We show that the specificity of RamF for MazF relative to other toxins relies on the degron-like function of MazF’s first 10 amino acids. Finally, we demonstrate that random proteins can improve during evolution by identifying beneficial mutations that turned RamF into a more efficient inhibitor. Our work provides a mechanistic basis for howde novogene birth can produce new, functional proteins that are integrated into complex cellular systems and provide a benefit to cells.
Extending the dynamic range of biomarker quantification through molecular equalization
Authors: Newman Sharon S. , Wilson Brandon D. , Mamerow Daniel , Wollant Benjamin , Nyein Hnin , Rosenberg-Hasson Yael , Maecker Holden T. , Eisenstein Michael , Soh H. Tom
Published: February 2023

Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3–4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved—in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two- pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our ‘EVROS’ strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 µl sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion.
Paracrine enhancement of tumor cell proliferation provides indirect stroma-mediated chemoresistance via acceleration of tumor recovery between chemotherapy cycles
Authors: Miroshnychenko Daria , Miti Tatiana , Kumar Pragya , Miller Anna , Laurie Mark , Giraldo Nathalia , Bui Marilyn M. , Altrock Philipp M. , Basanta David , Marusyk Andriy
Published: February 2023

The ability of tumors to survive therapy reflects both cell-intrinsic and microenvironmental mechanisms. Across many cancers, including triple-negative breast cancer (TNBC), a high stroma/tumor ratio correlates with poor survival. In many contexts, this correlation can be explained by the direct reduction of therapy sensitivity by stroma-produced paracrine factors. We sought to explore whether this direct effect contributes to the link between stroma and poor responses to chemotherapies. Ourin vitrostudies with panels of TNBC cell line models and stromal isolates failed to detect a direct modulation of chemoresistance. At the same time, consistent with prior studies, we observed treatment-independent enhancement of tumor cell proliferation by fibroblast-produced secreted factors. Using spatial statistics analyses, we found that proximity to stroma is often associated with enhanced tumor cell proliferationin vivo. Based on these observations, we hypothesized an indirect link between stroma and chemoresistance, where stroma-augmented proliferation potentiates the recovery of residual tumors between chemotherapy cycles. To evaluate the feasibility of this hypothesis, we developed a spatial agent-based model of stroma impact on proliferation/death dynamics. The model was quantitatively parameterized using inferences from histological analyses and experimental studies. We found that the observed enhancement of tumor cell proliferation within stroma-proximal niches can enable tumors to avoid elimination over multiple chemotherapy cycles. Therefore, our study supports the existence of a novel, indirect mechanism of environment-mediated chemoresistance that might contribute to the negative correlation between stromal content and poor therapy outcomes.
Quantifying yeast microtubules and spindles using the Toolkit for Automated Microtubule Tracking (TAMiT)
Authors: Ansari Saad , Gergely Zachary R. , Flynn Patrick , Li Gabriella , Moore Jeffrey K. , Betterton Meredith D.
Published: February 2023

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite development of automated microtubule analysis tools forin vitrostudies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that is solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes severalS. pombemutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work, and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules inS. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.
Shared evolutionary processes shape landscapes of genomic variation in the great apes
Authors: Rodrigues Murillo F. , Kern Andrew D. , Ralph Peter L.
Published: February 2023

For at least the past five decades population genetics, as a field, has worked to describe the precise balance of forces that shape patterns of variation in genomes. The problem is challenging because modelling the interactions between evolutionary processes is difficult, and different processes can impact genetic variation in similar ways. In this paper, we describe how diversity and divergence between closely related species change with time, using correlations between landscapes of genetic variation as a tool to understand the interplay between evolutionary processes. We find strong correlations between landscapes of diversity and divergence in a well sampled set of great ape genomes, and explore how various processes such as incomplete lineage sorting, mutation rate variation, GC-biased gene conversion and selection contribute to these correlations. Through highly realistic, chromosome-scale, forward-in-time simulations we show that the landscapes of diversity and divergence in the great apes are too well correlated to be explained via strictly neutral processes alone. Our best fitting simulation includes both deleterious and beneficial mutations in functional portions of the genome, in which 9% of fixations within those regions is driven by positive selection. This study provides a framework for modelling genetic variation in closely related species, an approach which can shed light on the complex balance of forces that have shaped genetic variation.
Separation of function mutations in microcephaly protein CPAP/CENPJ, reveal its role in regulating centriole number and length
Authors: Jaiswal Sonal , Sanghi Srishti , Singh Priyanka
Published: February 2023

Centriole are microtubule-based cylindrical structures characterized by their definite size, and stable, slow growing microtubules. The centriole core protein CPAP/CENPJ is known to act as a molecular cap regulating centriole length by interacting with microtubule/tubulinviathe conserved microtubule destabilizing, PN2-3, and microtubule stabilizing, A5N, domains. The C-terminus of CPAP has a conserved glycine-rich G-box/TCP domain (1050-1338 amino acids). This region is involved in centriole cartwheel assembly by interacting with the cartwheel protein STIL. However, previously reported primary microcephaly mutation mapped in the G-box of CPAP, i.e., E1235V (MCPH6) affects centriole lengthviaan unknown mechanism. Recently, another primary microcephaly mutation has been mapped to this region of CPAP, i.e., D1196N. However, the effect of D1196N on CPAP functioning is not known. We simultaneously characterized these two MCPH mutations in the G-box of CPAP. We identified that despite affecting the same domain of CPAP, they affect distinct CPAP functions at the centriole. The E1235V mutation caused an overly long centriole, and the D1196N mutation increased the centriole number. Interestingly, both these mutations affect CPAP direct interaction with the cartwheel protein STIL, which is involved in CPAP recruitment to the centriole. Accordingly, the CPAP E1235V centriole localization is significantly affected at the centriole. However, CPAP D1196N can still localize to centriole at levels comparable to the wild-type CPAP. We show that CPAP utilizes an alternate CEP152-dependent route for centriole recruitment. Importantly, our work highlights the importance of the CPAP region outside direct microtubule/tubulin interacting domains in influencing CPAP activity in cartwheel assembly and centriole length. Perhaps, this is why deleterious naturally occurring missense mutations are frequently occurring in this particular region of CPAP in primary microcephaly.