bluuebunny/crossref_metadata_2025 huggingface dataset:
Results From a Pilot Study of an Automated Directly Observed Therapy Intervention Using Artificial Intelligence With Conditional Economic Incentives Among Young Adults With HIV
Authors: Stoner Marie C. D. , Smith Louis , Ming Kristin , Mancuso Noah , Patani Henna , Sukhija-Cohen Adam , Granados Yancy , Wagner Danielle , Johnson Mallory O. , Napierala Sue , Neilands Torsten B. , Saberi Parya
Published: February 2024

Background:

Despite improvements in antiretroviral therapy (ART) availability, suboptimal adherence is common among youth with HIV (YWH) and can increase drug resistance and poor clinical outcomes. Our study examined an innovative mobile app-based intervention that used automated directly observed therapy (aDOT) using artificial intelligence, along with conditional economic incentives (CEIs) to improve ART adherence and enhance viral suppression among YWH.

Setting:

We conducted a pilot study of the aDOT-CEI intervention, informed by the operant framework of Key Principles in Contingency Management Implementation, to improve ART adherence among YWH (18–29) in California and Florida who had an unsuppressed HIV viral load.

Methods:

We recruited 28 virally unsuppressed YWH from AIDS Healthcare Foundation clinics, who used the aDOT platform for 3 months. Study outcomes included feasibility and acceptability, self-reported ART adherence, and HIV viral load.

Results:

Participants reported high satisfaction with the app (91%), and 82% said that it helped them take their medication. Comfort with the security and privacy of the app was moderate (55%), and 59% indicated the incentives helped improve daily adherence.

Conclusions:

Acceptability and feasibility of the aDOT-CEI intervention were high with potential to improve viral suppression, although some a priori metrics were not met. Pilot results suggest refinements which may improve intervention outcomes, including increased incentive amounts, provision of additional information, and reassurance about app privacy and security. Additional research is recommended to test the efficacy of the aDOT-CEI intervention to improve viral suppression in a larger sample.
Advanced Techniques in Upper Blepharoplasty: Cauterization + Plication for Enhanced Surgical Outcomes
Authors: Franco Ana C. , Tavares Marconi G. , Grillo Ricardo
Published: February 2024

Blepharoplasty is a commonly performed cosmetic surgery worldwide to address the excess skin and fat on the upper eyelids. The purpose of this paper is to present a surgical variation on blepharoplasty, achieving more pleasant outcomes. This paper describes the cauterization + plication technique, which involves using a cauterizing tool to remove excess skin and fat from the upper eyelid before using sutures to create a crease. It provides valuable insights into the advantages and limitations of this technique, drawing on relevant literature and clinical experience. Muscle cauterization and plication technique is an upgrade from the traditional technique, producing more esthetically pleasing results. This study highlights the importance of understanding the different techniques available for blepharoplasty to achieve the best possible outcomes for patients.
Bioarchaeological investigation of individuals with suspected multibacillary leprosy from the mediaeval leprosarium of St Mary Magdalen, Winchester, Hampshire, UK
Authors: Taylor G. Michael , White-Iribhogbe Katie , Cole Garrard , Ashby David , Stewart Graham R. , Dawson-Hobbis Heidi
Published: February 2024

Introduction. We have examined four burials from the St Mary Magdalen mediaeval leprosarium cemetery in Winchester, Hampshire, UK. One (Sk.8) was a male child, two (Sk.45 and Sk.52) were adolescent females and the fourth (Sk.512) was an adult male. The cemetery was in use between the 10th and 12th centuries. All showed skeletal lesions of leprosy. Additionally, one of the two females (Sk.45) had lesions suggestive of multi-cystic tuberculosis and the second (Sk.52) of leprogenic odontodysplasia (LO), a rare malformation of the roots of the permanent maxillary incisors.

Gap statement. Relatively little is known of the manifestations of lepromatous leprosy (LL) in younger individuals from the archaeological record.

Aims and Methodology. To address this, we have used ancient DNA testing and osteological examination of the individuals, supplemented with X-ray and microcomputed tomography (micro-CT) scan as necessary to assess the disease status.

Results and Conclusions. The presence of Mycobacterium leprae DNA was confirmed in both females, and genotyping showed SNP type 3I-1 strains but with a clear genotypic variation. We could not confirm Mycobacterium tuberculosis complex DNA in the female individual SK.45. High levels of M. leprae DNA were found within the pulp cavities of four maxillary teeth from the male child (Sk.8) with LO, consistent with the theory that the replication of M. leprae in alveolar bone may interfere with root formation at key stages of development. We report our biomolecular findings in these individuals and review the evidence this site has contributed to our knowledge of mediaeval leprosy.
A missense variant effect map for the human tumour suppressor protein CHK2
Authors: Gebbia Marinella , Zimmerman Daniel , Jiang Rosanna , Nguyen Maria , Weile Jochen , Li Roujia , Gavac Michelle , Kishore Nishka , Sun Song , Boonen Rick A , Dines Jennifer N. , Wahl Alexander , Reuter Jason , Johnson Britt , Fowler Douglas M , van Attikum Haico , Roth Frederick P
Published: February 2024

The tumour suppressorCHEK2encodes the serine/threonine protein kinase CHK2 which, upon DNA damage, is important for pausing the cell cycle, initiating DNA repair and inducing apoptosis. CHK2 phosphorylation of the tumour suppressor BRCA1 is also important for mitotic spindle assembly and chromosomal stability. Consistent with its cell cycle checkpoint role, both germline and somatic variants inCHEK2have been linked to breast and multiple other cancer types. Over 90% of clinical germlineCHEK2missense variants are classified as variants of uncertain significance, complicating diagnosis of CHK2-dependent cancer. We therefore sought to test the functional impact of all possible missense variants in CHK2. Using a scalable multiplexed assay based on the ability of human CHK2 to complement DNA sensitivity of aS. cerevisiaelacking its orthologRAD53, we generated a systematic ‘missense variant effect map’ forCHEK2missense variation. Map scores reflect known biochemical features of CHK2 and exhibit good performance in separating pathogenic from benign clinical missense variants. Thus, the missense variant effect map for CHK2 offers value in understanding both known and yet-to-be-observed CHK2 variants.
Age-associated B cell infiltration in salivary glands represents a hallmark of Sjögren’s-like disease in aging mice
Authors: Bagavant Harini , Durslewicz Justyna , Pyclik Marcelina , Makuch Magdalena , Papinska Joanna A. , Deshmukh Umesh S.
Published: February 2024

Sjögren’s disease (SjD), characterized by circulating autoantibodies and exocrine gland inflammation, is typically diagnosed in women over 50 years of age. However, the contribution of age to SjD pathogenesis is unclear. C57BL/6 female mice at different ages were studied to investigate how aging influences the dynamics of salivary gland inflammation. Salivary glands were characterized for immune cell infiltration, inflammatory gene expression, oxidative stress, and saliva production. At 8 months, gene expression of several chemokines involved in immune cell trafficking was significantly elevated. At this age, Age-associated B cells (ABCs), a unique subset of B cells expressing the myeloid markers CD11b and/or CD11c, were preferentially enriched in the salivary glands compared to other organs like the spleen or liver. The salivary gland ABCs increased with age and positively correlated with increased CD4 T follicular helper cells. By 14 months, lymphocytic foci of well-organized T and B cells spontaneously developed in the salivary glands. In addition, the mice progressively developed high titers of serum autoantibodies. A subset of aged mice developed salivary gland dysfunction mimicking SjD patients. Our data demonstrates that aging is a significant confounding factor for SjD. Thus, aged female C57BL/6 mice are more appropriate and a valuable preclinical model for investigating SjD pathogenesis and novel therapeutic interventions.
Maternal and Neonatal Colonization with Multidrug Resistant and Extended Spectrum ß-Lactamase ProducingEscherichia coliandKlebsiella pneumoniaein a Cameroonian Labour Ward
Authors: Njeuna Axelle , Founou Luria Leslie , Founou Raspail Carrel , Koudoum Patrice Landry , Mbossi Aurelia , Blocker Ariel , Bentley Stephen D. , Sone Lucien Etame
Published: February 2024

Background

Escherichia coliandKlebsiella pneumoniaerank among the primary bacterial culprits in neonatal infections and fatalities in sub-Saharan Africa. This study sought to characterize the phenotypic and genotypic features ofEscherichia coliandKlebsiella pneumoniaein a labour ward in Yaoundé, Cameroon.

Methods

A prospective and cross-sectional study spanning five months, from February 21 to June 30, 2022. Recto-vaginal swabs were obtained from expectant mothers, and nasopharyngeal swabs were collected from their babies. The samples were cultured on eosin methylene blue agar and isolates identified using the Enterosystem 18R kit. Extended-spectrum ß-lactamase (ESBL) production was assessed using CHROMAgar ESBL™ and the double disc synergy test. Antibiotic susceptibility was determined by the Kirby-Bauer disk diffusion method. Polymerase chain reaction (PCR) was employed to detect ß-lactamase genesblaSHV,blaCTX-MandblaTEM. ERIC-PCR was used to assess the clonal relatedness of isolates.

Results

E. coliwas predominantly found in pregnant women (81%) and neonates (55%) whileK. pneumoniaepredominated in healthcare workers. Almost all pregnant women (90%) were colonized by one or more multi-drug resistant (MDR) isolates with 52% being concomitantly ESBL producers. Altogether, 22 neonates were positive forE. coliand/orK. pneumoniaeand 19 (91%) were colonized by a MDR isolate. TheblaCTX-M(75%) was the leading ß-lactamase gene detected.

Conclusion

Our study suggests that MDR- and ESBL-E. coliandK. pneumoniaeare circulating at high prevalence in labour Yaoundé. It emphasizes the necessity for strict infection prevention and control measures in conjunction with effective antimicrobial stewardship in the country.
DNA methylation machinery is involved in development and reproduction in the viviparous pea aphid (Acyrthosiphon pisum)
Authors: Yoon Kane , Williams Stephanie , Duncan Elizabeth J.
Published: February 2024

The pea aphid (Acyrthosiphon pisum) like the majority of extant aphids displays cyclical parthenogenesis - the ability of mothers to switch the reproductive mode of their offspring from reproducing parthenogenetically to sexually in response to environmental cues. The pea aphid genome encodes two paralogs of thede novoDNA methyltransferase gene,dnmt3aanddnmt3x. Here we show, using phylogenetic analysis, that this gene duplication event occurred at least 106 million years ago, likely after the divergence of the lineage leading to the Aphidomorpha (aphids, phylloxera and adelgids) from that leading to the scale insects (Coccoidea) and that the two paralogs are maintained in the genomes of all aphids examined. We also show that the mRNA of bothdnmt3paralogs are maternally expressed in the viviparous aphid ovary. During development both paralogs are expressed in the germ cells of embryos beginning at stage 5 and persisting throughout development. Chemical inhibition of the DNA methylation machinery leads to defects of oocytes and early stage embryos, and causes a proportion of later stage embryos to be born dead or die soon after birth. These phenotypes suggest a role for DNA methyltransferases in reproduction, consistent with that seen in other insects. Taking the vast evolutionary history of thednmt3paralogs, and the localization of their mRNAs in the ovary, we suggest there is a role fordnmt3aand/ordnmt3xin early development, and a role for DNA methylation machinery in reproduction and development of the viviparous pea aphid.
Bending DNA increases its helical repeat
Authors: Chandrasekhar Soumya , Swope Thomas P. , Fadaei Fatemeh , Hollis Daniel R. , Bricker Rachel , Houser Draven , Portman John J. , Schmidt Thorsten L.
Published: February 2024

In all biological systems, DNA is under high mechanical stress from bending and twisting. For example, DNA is tightly bent in nucleosome complexes, virus capsids, bacterial chromosomes, or complexes with transcription factors that regulate gene expression. A structurally and mechanically accurate model of DNA is therefore necessary to understand some of the most fundamental molecular mechanisms in biology including DNA packaging, replication, transcription and gene regulation. An iconic feature of DNA is its double helical nature with an average repeath0of ∼10.45 base pairs per turn, which is commonly believed to be independent of curvature. We developed a ligation assay on nicked DNA circles of variable curvature that reveals a strong unwinding of DNA to over 11 bp/turn for radii around 3-4 nm. Our work constitutes a major modification of the standard mechanical model of DNA and requires reassessing the molecular mechanisms and energetics of all processes involving tightly bent DNA.
Axolotl mandible regeneration occurs through mechanical gap closure and a shared regenerative program with the limb
Authors: Kramer Julia , Aires Rita , Keeley Sean D. , Schröder Tom Alexander , Lauer Günter , Sandoval-Guzmán Tatiana
Published: February 2024

The mandible plays an essential part in human life and, thus, defects in this structure can dramatically impair the quality of life in patients. Axolotls, unlike humans, are capable of regenerating their lower jaws; however, the underlying mechanisms and their similarity to those in limb regeneration are unknown. In this work, we used morphological, histological, and transcriptomic approaches to analyze the regeneration of lateral resection defects in the axolotl mandible. We found that this structure can regenerate all missing tissues in 90 days through gap minimization, blastema formation, and finally tissue growth, differentiation, and integration. Moreover, transcriptomic comparisons of regenerating mandibles and limbs showed that they share molecular phases of regeneration, that these similarities peak during blastema stages, and that mandible regeneration occurs at a slower pacing.

Altogether, our study demonstrates the existence of a shared regenerative program used in two different regenerating body structures with different embryonic origins in the axolotl, and contributes to our understanding of the minimum requirements for a successful regeneration in vertebrates, bringing us closer to understand similar lesions in human mandibles.
Biophysics of microRNA-34a targeting and its influence on down-regulation
Authors: Sweetapple Lara , Kosek David M , Banijamali Elnaz , Becker Walter , Müller Juliane , Karadiakos Christina , Baronti Lorenzo , Guzzetti Ileana , Schritt Dimitri , Chen Alan , Andersson Emma R , Petzold Katja
Published: February 2024

microRNAs (miRNAs) regulate target mRNA expression post-transcriptionally through their association with Argonaute 2 (AGO2) proteins. Predicting the efficiency of mRNA repression by miRNA has been limited by our comprehension of the structure-function relationship within miRNA binding sites. Using a combination of EMSA, luciferase reporter assays, and structural probing, we investigated the interaction between the human tumour suppressormiR-34aand 12 mRNA targets. Comparison of direct RNA:RNA interactions and those within the functional AGO2 protein revealed that the isolated mRNA:miRNA duplex serves as a strong predictor for duplex affinity and structure within AGO2. Our findings reveal that AGO2 has a bidirectional capacity to modulate affinity; weakening tight RNA:RNA binders while strengthening weak ones. We identified three distinct structural groups that form uponmiR-34abinding and reveal a novel structural group that exhibits a guide strand bulge. MD simulations indicate a conceivable fit of this miRNA-bulge structure within AGO2. Our results demonstrate that the structural characteristics of mRNA:miRNA duplexes could serve as contributing determinants of repression efficacy.
LXA4 promotes the browning of white adipose through miR-133a-3p/SIRT1 pathway
Authors: Yu Dan , Ruan Yuan , Wang Yisu , Chen Xiaopei , Wang Dan , Wu Tianfeng
Published: February 2024

Lipoxin A4 (LXA4) promotes the browning of white adipose and energy consumption. The specific mechanism of which involved in white adipose browning is less clear. A high-fat diet (HFD) mouse model was constructed. Different groups of mice were treated with LXA4 accordingly. The body weight of mouse, subcutaneous and visceral fat, and food intake were recorded. The effect of LXA4 was examined by observing changes in pathology, serum insulin and lipid accumulation indices. The effects of LXA4/miR-133a-3p/Sirtuin1 on lipid droplet formation, fat browning-related genes, and the insulin receptor-AKT pathway in cells were examined after induction of adipocyte differentiation in 3T3-L1 precursors. At the cellular level, LXA4 promoted lipid droplet formation, expressions of fat browning genes and activation of the insulin receptor-AKT pathway in differentiated 3T3-L1 cells. MiR-133a-3p agomir partially offset the effects of LXA4. SIRT1 was a downstream target gene of miR-133a-3p, participating in the promotive effects of LXA4 on fat browning. LXA4 promotes white adipose browning and relieves insulin resistance through miR-133a-3p/SIRT1 pathway.
T cell interaction partners of DHHC20
Authors: Haxhiraj Deana , Kuropka Benno , Brencher Eva , Rosenberger David , Brandt Helena , Speck David , Scheerer Patrick , Brügger Britta , Clementi Cecilia , Freund Christian
Published: February 2024

Reversible palmitoylation of proteins at cysteine residues represents a post-translational modification that can alter the cellular localization of proteins, change their distribution within lipid membranes or modulate their conformation and molecular interaction patterns. DHHC enzymes catalyze protein palmitoylation, while thioesterases or hydrolases can rapidly remove the acyl-chain. In human T cells, DHHC proteins have been shown to modify proteins that are involved in major signaling pathways such as Ca2+-signaling or kinase-dependent activation of transcription factors for cytokines. For DHHC20, a role in the palmitoylation of the Orai1 Ca2+-channel has been demonstrated, but otherwise its T cell interaction partners are largely unknown. Here, we show that recombinantly expressed DHHC20 robustly interacts with 28 proteins from Jurkat T cells, as shown by affinity enrichment combined with mass spectrometric analysis. We find a robust interaction between DHHC20 and the β-subunit of the trimeric G protein Gαsβ1γ2, while the typically palmitoylated Ga subunit is not identified. Cross-linking mass spectrometry with purified DHHC20 and Gαsβ1γ2 then confirms a direct interaction between the β1γ2 domains and the enzyme, while rigid docking offers structural poses that are in agreement with the observed intermolecular cross-linking constraints. Thus, we suggest a model where the β1γ2 subunits of a trimeric G protein serve as a stable interaction partner of a DHHC enzyme, presumably acting as a landing platform for the Gα subunit that is subsequently palmitoylated by the enzyme.
Klumpy: A Tool to Evaluate the Integrity of Long-Read Genome Assemblies and Illusive Sequence Motifs
Authors: Madrigal Giovanni , Minhas Bushra Fazal , Catchen Julian
Published: February 2024

The improvement and decreasing costs of third-generation sequencing technologies has widened the scope of biological questions researchers can address with de novo genome assemblies. With the increasing number of reference genomes, validating their integrity with minimal overhead is vital for establishing confident results in their applications. Here, we present Klumpy, a tool for detecting and visualizing both misassembled regions in a genome assembly and genetic elements (e.g., genes, promotors, or transposable elements) of interest in a set of sequences. By leveraging the initial raw reads in combination with their respective genome assembly, we illustrate Klumpy’s utility by investigating antifreeze glycoprotein (afgp) loci across two icefishes, by searching for a reported absent gene in the northern snakehead fish, and by scanning the reference genomes of a mudskipper and bumblebee for misassembled regions. In the two former cases, we were able to provide support for the noncanonical placement of an afgp locus in the icefishes and locate the missing snakehead gene. Furthermore, our genome scans were able to identify an cryptic locus in the mudskipper reference genome, and identify a putative repetitive element shared amongst several species of bees.
Decoding functional proteome information in model organisms using protein language models
Authors: Barrios-Núñez Israel , Martínez-Redondo Gemma I. , Medina-Burgos Patricia , Cases Ildefonso , Fernández Rosa , Rojas Ana M.
Published: February 2024

Protein language models have been tested and proved to be reliable when used on curated datasets but have not yet been applied to full proteomes. Accordingly, we tested how two different machine learning based methods performed when decoding functional information from the proteomes of selected model organisms. We found that protein Language Models are more precise and informative than Deep Learning methods for all the species tested and across the three gene ontologies studied, and that they better recover functional information from transcriptomics experiments. The results obtained indicate that these Language Models are likely to be suitable for large scale annotation and downstream analyses, and we recommend a guide for their use.
TheBacillus subtilis yqgC-sodAoperon protects magnesium-dependent enzymes by supporting manganese efflux
Authors: Sachla Ankita J. , Soni Vijay , Piñeros Miguel , Luo Yuanchan , Im Janice J. , Rhee Kyu Y. , Helmann John D.
Published: February 2024

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of theBacillus subtilis yqgC-sodA(YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficientmneP mneSdouble mutant both accumulate Mn and have similar metabolic perturbations they also display phenotypic differences. Several mutations that suppressed Mn intoxication of themneP mneSefflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not themneP mneSstrain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of themenaquinone,siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in themneP mneSand theyqgC-sodAdeletion mutants results from distinct enzymatic vulnerabilities.

Importance

Bacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. InBacillus subtilis, MnSOD is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual mutations) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes.
The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA
Authors: Hengel Sarah R. , Oppenheimer Katherine , Smith Chelsea , Schaich Matthew A. , Rein Hayley L. , Martino Julieta , Darrah Kristie , Ezekwenna Oluchi , Burton Kyle , Van Houten Bennett , Spies Maria , Bernstein Kara A.
Published: February 2024

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1 and SWS1. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show humansgSWSAP1andsgSWS1knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.
Performance characteristics of highly automated HSV-1 and HSV-2 IgG testing
Authors: Crawford Katharine H. D. , Selke Stacy , Pepper Gregory , Goecker Erin , Sobel Aniela , Wald Anna , Johnston Christine , Greninger Alexander L.
Published: February 2024

Herpes simplex virus (HSV) infections are one of the most common and stigmatized infections of humankind, affecting more than 4 billion people around the world and more than 100 million Americans. Yet most people do not know their infection status and antibody testing is not recommended, partly due to poor test performance. Here, we compared the test performance of the Roche Elecsys HSV-1 IgG and HSV-2 IgG, DiaSorin LIAISON HSV-1/2 IgG, and Bio-Rad BioPlex 2200 HSV-1 & HSV-2 IgG assays with the gold-standard HSV western blot in 1994 persons, including 1017 persons with PCR or culture-confirmed HSV-1 and/or HSV-2 infection. Across all samples, the Bio-Rad and Roche assays had similar performance metrics with low sensitivity (<85%), but high specificity (>97%) for detecting HSV-1 IgG and both high sensitivity (>97%) and high specificity (>98%) for detecting HSV-2 IgG. The DiaSorin assay had a higher sensitivity (92.1%) but much lower specificity (88.7%) for detecting HSV-1 IgG and comparatively poor sensitivity (94.5%) and specificity (94.2%) for detecting HSV-2 IgG. The DiaSorin assay performed poorly at low-positive index values with 60.9% of DiaSorin HSV-1 results and 20.8% of DiaSorin HSV-2 results with positive index values <3.0 yielding false positive results. Based on an estimated HSV-2 seroprevalence of 12% in the United States, positive predictive values for HSV-2 IgG were 96.1% for Roche, 87.4% for Bio-Rad, and 69.0% for DiaSorin, meaning nearly 1 of every 3 positive DiaSorin HSV-2 IgG results would be falsely positive. Further development in HSV antibody diagnostics is needed to provide appropriate patient care.
Adeno-Associated Viral Tools to Trace Neural Development and Connectivity Across Amphibians
Authors: Jaeger Eliza C.B. , Vijatovic David , Deryckere Astrid , Zorin Nikol , Nguyen Akemi L. , Ivanian Georgiy , Woych Jamie , Arnold Rebecca C. , Ortega Gurrola Alonso , Shvartsman Arik , Barbieri Francesca , Toma Florina A. , Gorbsky Gary J. , Horb Marko E. , Cline Hollis T. , Shay Timothy F. , Kelley Darcy B. , Yamaguchi Ayako , Shein-Idelson Mark , Tosches Maria Antonietta , Sweeney Lora B.
Published: February 2024

The development, evolution, and function of the vertebrate central nervous system (CNS) can be best studied using diverse model organisms. Amphibians, with their unique phylogenetic position at the transition between aquatic and terrestrial lifestyles, are valuable for understanding the origin and evolution of the tetrapod brain and spinal cord. Their metamorphic developmental transitions and unique regenerative abilities also facilitate the discovery of mechanisms for neural circuit remodeling and replacement. The genetic toolkit for amphibians, however, remains limited, with only a few species having sequenced genomes and a small number of transgenic lines available. In mammals, recombinant adeno-associated viral vectors (AAVs) have become a powerful alternative to genome modification for visualizing and perturbing the nervous system. AAVs are DNA viruses that enable neuronal transduction in both developing and adult animals with low toxicity and spatial, temporal, and cell-type specificity. However, AAVs have never been shown to transduce amphibian cells efficiently. To bridge this gap, we established a simple, scalable, and robust strategy to screen AAV serotypes in three distantly-related amphibian species: the frogsXenopus laevisandPelophylax bedriagae,and the salamanderPleurodeles waltl,in both developing larval tadpoles and post-metamorphic animals. For each species, we successfully identified at least two AAV serotypes capable of infecting the CNS; however, no pan-amphibian serotype was identified, indicating rapid evolution of AAV tropism. In addition, we developed an AAV-based strategy that targets isochronic cohorts of developing neurons – a critical tool for parsing neural circuit assembly. Finally, to enable visualization and manipulation of neural circuits, we identified AAV variants for retrograde tracing of neuronal projections in adult animals. Our findings expand the toolkit for amphibians to include AAVs, establish a generalizable workflow for AAV screening in non-canonical research organisms, generate testable hypotheses for the evolution of AAV tropism, and lay the foundation for modern cross-species comparisons of vertebrate CNS development, function, and evolution.
Lipid A double bond position determination using ozone and laser-induced dissociation
Authors: Mikhael Abanoub , Pětrošová Helena , Smith Derek , Ernst Robert K. , Goodlett David R.
Published: February 2024

Ozone coupled with tandem MS is a traditional tool to assign double bond position in lipids. However, few studies were reported on the use of ozone for double bond determination of bacterial lipids. Most MS-based double bond localization methods used in bacterial lipidomics focused on low molecular weight phospholipids, but never tested for assigning double bond position in complex glycolipids such as Lipid A. Here, we present a simple approach to identify unsaturated lipid A double bond position using on-MALDI plate ozone treatment of intact Lipid A.
Discovery of Alkenyl Oxindole as a Novel PROTAC Moiety for Targeted Protein Degradation via CRL4DCAF11Recruitment
Authors: Wanga Ying , Wei Tianzi , Zhao Man , Huang Aima , Sun Fan , Chen Lu , Lin Risheng , Xie Yubao , Zhang Ming , Xu Shiyu , Sun Zhihui , Hong Liang , Wang Rui , Tian Ruilin , Li Guofeng
Published: February 2024

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of hetero-bifunctional compounds by conjugating different alkenyl oxindoles with the BRD4 inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11for substrate degradation. Furthermore, we validated the most potent hetero-bifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity bothin vitroandin vivo. Our research provides new employable PROTAC moieties for targeted protein degradation, providing new possibilities for drug discovery.
Structural basis for activation and mechanism of the bacterial hexameric motors RadA and ComM in DNA branch migration during homologous recombination
Authors: Rosa Leonardo Talachia , Vernhes Emeline , Soulet Anne-Lise , Polard Patrice , Fronzes Rémi
Published: February 2024

Some DNA helicases play key roles in genome maintenance and plasticity through their branch migration activity in distinct pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in both DNA repair in all species and in natural transformation in Gram positive firmicutes. In others, ComM is the natural transformation-specific helicase. Both RadA and ComM form hexameric rings and use ATP hydrolysis as a source of energy. In this work we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogues. This is the first structure reported for ComM, and the first structure of RadA in its active state. In particular, this study unveiled a molecular switch for ATP hydrolysis and DNA binding coupling in RadA and the role of the Lon protease-like domain, shared by RadA and ComM. Together our results provide new insights on the branch migration mechanism at the molecular level in the context of homologous recombination.
Innate face detectors in the nidopallium of young domestic chicks
Authors: Kobylkov Dmitry , Rosa-Salva Orsola , Zanon Mirko , Vallortigara Giorgio
Published: February 2024

Soon after birth, naïve animals and newborn babies show spontaneous attraction towards faces and face-like stimuli with three dark features representing eyes and a mouth/beak. While neurons selectively responding to faces have been found in the inferotemporal cortex of adult primates, face-selective domains in the brains of young monkeys seem to develop only later in life after exposure to faces. This has fueled a debate on the role of experience in the development of face-detector mechanisms, since face preferences are well documented in naïve animals, such as domestic chicks reared without exposure to faces. Here we demonstrate that neurons in a cortex-homologue area of one-week-old face-naïve domestic chicks selectively respond to a face-like configuration. Our single-cell recordings show that these face detectors do not respond to alternative configurations or isolated facial features. Moreover, the population activity of face-selective neurons accurately encoded the face-like stimulus as a unique category. Thus, our findings show that face detectors are present in the brains of very young animals without pre-existing experience.
Spatiotemporal Changes in Netrin/Dscam1 Signaling Dictate Axonal Projection Direction inDrosophilaSmall Ventral Lateral Clock Neurons
Authors: Liu Jingjing , Wang Yuedong , Liu Xian , Han Junhai , Tian Yao
Published: February 2024

Axon projection is a spatial and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections inDrosophila’s small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons (DNs) secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compellingin vivomodel to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions.
A Vascular Dissection and Rupture Linked Metabolite Acts Via BLT2 Receptor
Authors: Li Yuyu , Yu Jiaqi , Chen Weiyao , Tan Xin , Xu Xuan , Lin Rui , Wang Xue , Jiang Wenxi , Du Jie , Wang Yuan
Published: February 2024

BACKGROUND

Thoracic aortic dissection (TAD) is a life-threatening vascular disease that requires effective drug treatment to prevent progression and rupture. Because arachidonic acid metabolism is involved in inflammation and vascular homeostasis, we investigated the roles of arachidonic acid metabolites in TAD pathogenesis and their utility as therapeutic targets.

METHODS

Serum metabolomics analysis was performed to characterize arachidonic acid metabolites in TAD patients and a TAD mouse model. 12/15-LOX expression was profiled in the aortic tissues of TAD patients and the TAD mouse model. Four-week-old male Alox15 knockout mice (Alox15−/−), 12-HETE-treated mice, ML351 (12/15-LOX inhibitor)-treated mice, and LY255283 (leukotriene B 4 receptor 2 [BLT2] antagonist)-treated mice received β-aminopropionitrile monofumarate (BAPN, 1 g/kg/day) for 4 weeks to model TAD, then underwent assessment of TAD progression. Interaction of 12-HETE produced by macrophages with BLT2 receptor-expressing cells was detected by molecular docking and immunoblotting.

RESULTS

Serum levels of 12-HETE and the expression of 12/15-LOX in aortic tissue were significantly increased in TAD patients and BAPN-treated TAD mice. BAPN-induced TAD progression was significantly ameliorated in Alox15-deficient or -suppressed mice. 12-HETE directly interacted with BLT2 receptors on macrophages, activating the downstream NOX-1/ROS/NF-κB signaling pathway to induce inflammatory cytokine release. This initiated inflammatory cell recruitment and exacerbated extracellular matrix degradation, leading to phenotype switching in vascular smooth muscle cells (VSMCs). Additionally, treatment with ML351 and LY255283 significantly reduced the rates of dissection rupture and combined treatment could maximize the curative effect.

CONCLUSIONS

12-HETE may amplify the inflammatory cascade and trigger aberrant phenotype switching in VSMCs during TAD development. The reduction of circulating 12-HETE or antagonism of its receptor may be new targets for TAD prevention and treatment.

Clinical Perspective

What Is New?

The expression levels of 12/15-LOX and its metabolite 12-HETE were elevated in TAD patients and TAD mice.

Increased levels of 12-HETE directly bind to BLT2 receptors in macrophages, thereby initiating inflammatory cascades that downregulate VSMC differentiation markers through the suppression of IL-6.

Deletion or pharmacologic inhibition of 12/15-LOX and suppression of BLT2 mitigated TAD development by alleviating inflammation and VSMC phenotype switching.

What Are the Clinical Implications?

The inhibition of 12-HETE-related pathways, through mechanisms such as reducing the plasma 12-HETE content or blocking its receptor, may represent a novel therapeutic strategy for TAD.

Further studies are needed to explore the diagnostic value of serum 12-HETE as a novel biomarker for TAD.
Oxytocin signaling regulates maternally-directed behavior during early life
Authors: Zelmanoff Daniel , Kaufman Menachem , Dine Julien , Wietek Jonas , Litvin Anna , Abraham Shaked , Cohen Savanna , Yizhar Ofer
Published: February 2024

Oxytocin is essential in shaping social behavior across the lifespan. While the role of oxytocin signaling in parental care has been widely investigated, little is known about its function in social behavior during early life. This is partly due to the lack of precise technologies for studying the developing brain. Here, we studied the role of oxytocin in pup social behavior under acute separation from and reunion with the mother. We show that the activity of oxytocin neurons was increased by acute maternal separation and returned to baseline after reunion. Behaviorally, maternally-separated pups emitted more ultrasonic vocalizations upon reunion, which were further modulated by nipple attachment behavior. These effects were attenuated by blocking the oxytocin receptor during maternal separation. To investigate the role of oxytocin neurons with higher precision, we established a method for transcranial optogenetic silencing of neuronal activity in untethered and freely behaving pups. Using this approach, we found that silencing of oxytocin neurons during maternal separation disrupted vocal behavior during separation and reunion in a sex-specific manner. Our findings reveal an important role of oxytocin in context-dependent vocal communication in pups, offering new insights into the mechanisms of social behavior during early life.
Functional correlation between WRN and SAMHD1 in end-resection
Authors: Perdichizzi Benedetta , Pichierri Pietro
Published: February 2024

Double-strand breaks (DSBs) can cause chromosome rearrangements, leading to cancer and some genetic diseases. WRN and SAMHD1 are proteins implicated in DSB processing and form a complex. Our study shows that SAMHD1 influences the nuclear recruitment of WRN in response to CPT-induced DSBs. Silencing SAMHD1 restores single-stranded DNA formation in WRN-deficient cells. However, DSB accumulation from CPT treatment is not recovered in WRN S1133A or WS cells when SAMHD1 is silenced. This suggests SAMHD1 cooperates with WRN in DNA damage repair and may have additional protective roles when WRN function in DSBs processing is impaired.
Single Transcript Level Atlas of Oxytocin and the Oxytocin Receptor in the Mouse Brain
Authors: Ryu Vitaly , Gumerova Anisa , Pevnev Georgii , Korkmaz Funda , Kannangara Hasni , Cullen Liam , Witztum Ronit , Sims Steven , Frolinger Tal , Moldavski Ofer , Barak Orly , Cao Jay J. , Lizneva Daria , Goosens Ki A. , Yuen Tony , Zaidi Mone
Published: February 2024

Oxytocin (OXT), a primitive nonapeptide known to regulate reproduction and social behaviors, is synthesized primarily in the hypothalamus and is secretedviahypophyseal-portal system of the posterior pituitary gland. Given that pituitary hormones, traditionally thought of as regulators of single targets, display an array of central and peripheral actions, OXT also directly affects bone and body composition. Its effects on bone remodeling are physiologically relevant, as elevated OXT levels during pregnancy and lactation could cause calcium mobilization from the maternal skeleton for intergenerational calcium transfer towards fetal bone growth. There is an equally large body of evidence that has established the presence of OXT receptors (OXTRs) in the brain through which central functions, such as social bonding, and peripheral functions, such as the regulation of body composition, can be exerted. To purposefully address the effects of OXT on the brain, we used RNAscope to map OXT and OXTR expression, at the single transcript level, in the whole mouse brain. Identification of brain nuclei with the highest OXT and OXTR transcript density will shed further light on functional OXT nodes that could be further interrogated experimentally to define new physiologic circuitry.
A fungal metabolic regulator underlies infectious synergism duringCandida albicans-Staphylococcusaureus intra-abdominal co-infection
Authors: Paul Saikat , Todd Olivia A , Eichelberger Kara R , Tkaczyk Christine , Sellman Bret R , Noverr Mairi C , Cassat James E , Fidel Paul L , Peters Brian M
Published: February 2024

Candida albicansandStaphylococcus aureusare two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven byCandida-induced upregulation of functionalS. aureus⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen ofC. albicanstranscription factor mutants was undertaken and revealed thatzcf13Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches,ZCF13was shown to regulate genes involved in pentose metabolism, includingRBK1andHGT7that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcalagrquorum sensing system and concomitantly repressed toxicity. Unlike wild-typeC. albicans,zcf13Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation andagrrepression. Forced expression ofRBK1andHGT7in thezcf13Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.
A whole-genome CRISPR screen identifies the spindle accessory checkpoint as a locus of nab-paclitaxel resistance in pancreatic cancer cells
Authors: Mondal Priya , Alyateem George , Mitchell Allison V. , Gottesman Michael M.
Published: February 2024

Pancreatic adenocarcinoma is one of the most aggressive and lethal forms of cancer. Chemotherapy is the primary treatment for pancreatic cancer, but resistance to the drugs used remains a major challenge. A genome-wide CRISPR interference and knockout screen in the PANC-1 cell line with the drug nab-paclitaxel has identified a group of spindle assembly checkpoint (SAC) genes that enhance survival in nab-paclitaxel. Knockdown of these SAC genes (BUB1B, BUB3, and TTK) attenuates paclitaxel-induced cell death. Cells treated with the small molecule inhibitors BAY 1217389 or MPI 0479605, targeting the threonine tyrosine kinase (TTK), also enhance survival in paclitaxel. Overexpression of these SAC genes does not affect sensitivity to paclitaxel. These discoveries have helped to elucidate the mechanisms behind paclitaxel cytotoxicity. The outcomes of this investigation may pave the way for a deeper comprehension of the diverse responses of pancreatic cancer to therapies including paclitaxel. Additionally, they could facilitate the formulation of novel treatment approaches for pancreatic cancer.