bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional fingerprinting for the developing brain using deep metric
learning: an ABCD study
Authors: Xu Rui , Zhao Shuwan , Jin Qianhui , Wang Zeyao , Zhao Kun , Zhong Suyu , Zhang Jiaying , Liu Yong , Qi Ting , Wei Yongbin
Published: January 2025

Brain fingerprinting is a promising approach for characterizing the
uniqueness of individual brain functioning using functional magnetic resonance
imaging (fMRI) data. Here, we propose a novel deep learning framework, the
metric-BoIT, for brain fingerprinting and demonstrate its effectiveness in
capturing individual variability among early adolescents undergoing dramatic
brain changes. Utilizing resting-state fMRI data from the Adolescent Brain
Cognitive Development (ABCD) dataset, we identified brain functional
fingerprints that achieved remarkable individual identification accuracy,
reaching 97.6% within a single session and maintaining 86.6% accuracy over a
four-year developmental period. Annotation analysis revealed that higher-order
association regions, particularly those within the default-mode network,
contributed most significantly to these distinctive brain fingerprints
(t= 5.618,p<
0.001). Moreover, these brain fingerprints were relevant to cognitive functions,
as evidenced by significant correlations with fluid intelligence
(F= 1.282,p= 0.027)
and crystallized intelligence (F=
1.405,p< 0.001). The extracted brain
fingerprints were additionally associated with genetics, showing that
individuals with strong genomic relationships exhibited more similar brain
fingerprint patterns (t=
−12.330,p< 0.001). Together, our study not
only presents an innovative approach to brain functional fingerprinting but also
provides valuable insights into the individual variability underlying adolescent
neurodevelopment.
MIND-Map; A Comprehensive Toolbox for Estimating Brain Dynamic States
Authors: Khodaei Mohammadreza , Laurienti Paul J. , Simpson Sean L. , Shappell Heather M.
Published: January 2025

Studying dynamic brain states has offered new insights into understanding
functional connectivity. One of the promising approaches for estimating these
brain states is the hidden semi-Markov model (HSMM). However, despite its
potential, its adoption in the neuroscience community has been limited due to
its complexity. We developed the Markov Inference Dynamic Mapping (MIND-Map)
toolbox to overcome this limitation. This interactive user-friendly toolbox
leverages HSMM to identify brain states, analyze their dynamics, and perform
two-sample statistical comparisons of network dynamics. Furthermore, it
introduces a new approach, not yet used in conjunction with these models, for
determining the optimal number of states, addressing a key challenge in the
field. We assessed the performance of the HSMM and our method for identifying
the optimal number of states using two datasets, including a unique dataset
explicitly developed for this purpose.
Spatial drug asymmetry modulates phenotypic diversity-migration relationships
under resistance evolution
Authors: Hu Zhijian , Wu Yuzhen , Wood Kevin
Published: January 2025

At long timescales, resistant phenotypes will emerge and be selected within
the bacterial population as an evolutionary response to drug exposure. This
phenomenon reduces the efficacy of drug therapies and thus compromises patient
health. In spatially heterogeneous drug environments, recent evidence shows that
migration can either promote or decelerate the evolution of antibiotic
resistance, thereby affecting the rate of resistant phenotype emergence.
However, another important quantitative aspect of resistance evolution—bacterial
phenotypic diversity—has often been overlooked and remains challenging to
investigate in spatially extended systems, both experimentally and clinically.
In order to study how diversity is reshaped by migration across space, here we
designed a minimal 2-well experimental system with spatial drug asymmetry. One
well contained a bacteriostatic drug (Linezolid) at the minimum inhibitory
concentration, while the other well served as a sanctuary with just media. We
found that the relationship between diversity and migration follows the
“Intermediate Disturbance Hypothesis” (IDH), with migration as the disturbance
to each well. By varying the selective drug concentrations, we observed that the
diversity-migration relationship changes, and IDH can disappear. This behavior
was explained by an asymmetry parameter derived from a two-phenotype
growth-migration dynamic model.

To further validate how different spatial drug asymmetries modulate the
diversity-migration relationship through this asymmetry parameter, we applied
another bactericidal drug, Ampicillin, and observed similar results. In a more
complex scenario involving both Linezolid and Ampicillin, four distinct
phenotypes, including cross-resistant variants, emerged. Our asymmetry parameter
successfully explained the diversity-migration relationship, with unique
diversity dynamics such as multiple peaks appearing from the model. The minimal
generalist-specialist framework predicted these unique behaviors through global
fitness advantages. Our findings provide experimental support and theoretical
explanations for the emergence of phenotypic diversity in clinical settings,
such as gut-lung translocation. These insights may pave the way for improved
clinical strategies to manage antibiotic resistance evolution.
ConvMut: A Web tool to analyze viral convergent mutations along
phylogenies
Authors: Bernasconi Anna , Fanfoni Emma , Alfonsi Tommaso , Focosi Daniele
Published: January 2025

Convergent evolution in protein antigens is common across pathogens and has
also been documented in SARS-CoV-2; the most likely reason is the need to evade
the selective pressure exerted by previous infection- or vaccine-elicited
immunity. There is a pressing need for tools that allow automated analysis of
convergent mutations.

In response to this need, we developed ConvMut, a tool for identifying
patterns of recurrent mutations in SARS-CoV-2 evolution. To this end, we
exploited the granular phylogenetic tree representation developed by PANGO,
allowing us to observe what we call deltas, i.e., groups of mutations that are
acquired on top of the immediately upstream tree nodes. Deltas comprise both
amino acid substitutions and deletions. ConvMut can perform individual protein
analysis to identify the most common single mutations acquired independently in
a given subtree (starting from a user-selected root). Such mutations are
represented in a barplot that can be sorted by frequency or position and
filtered by region of interest. Lineages are then gathered into clusters
according to their sets of shared mutations. Finally, an interactive graph
orders the clusters, details the acquired amino acid changes for each
sublineage, and allows us to the evolutive path until a selected
lineage.

Other unique tools are paired with the main functionality of ConvMut to
support a complete analysis, such as a frequency analysis for a given nucleotide
or amino acid changes at a given residue across a selected phylogenetic
subtree.

We expect that ConvMut will facilitate the design of antiviral anti-Spike
monoclonal antibodies and Spike-based vaccines with longer-lasting efficacy,
minimizing development and marketing failures.
An Emirati pangenome incorporating a diploid telomere-to-telomere
reference
Authors: Olbrich Michael , Mousa Mira , Wohlers Inken , Aamri Amira Al , Alnaqbi Halima , Alsuwaidi Aisha Hanaya , Manoharan Hima Vadakkeveettil , Marzouka Nour al-dain , Ramachandran Sanjay Erathodi , Thomas Anju Annie , Alameri Mohammed , Tay Guan K , Hamoudi Rifat , Ibrahim Saleh , Ghaithi Noura Al , Alsafar Habiba
Published: January 2025

Reference data on genomic variation forms the basis of genetics research.
Limitations in identifying genetic variation from single reference sequences
have recently been addressed through improvements in sequencing technologies,
allowing the generation of pangenomic references from multiple accurate
chromosome-levelde novoassemblies. Nevertheless,
global pangenomes to date have yet to include genomes from the populations of
the Middle Eastern Region. To address this shortcoming, this study provides an
Emirati genome reference. Its core is a diploid assembly with a Quality Value
(QV) of 60 that includes ten telomere-to-telomere chromosomes. This assembly is
incorporated into a pangenome graph constructed of 52 additional high-quality
assemblies, half of which are trio-based. This Emirati pangenome reveals a
similar level of genomic variation as the one compiled by the Human Pangenome
Reference Consortium, underscoring its utility for the identification of both
global and population-centered genomic variation, even in genome regions that
have been traditionally challenging to assemble but are covered by the Emirati
telomere-to-telomere assembly. As such, the Emirati genome reference
significantly contributes to genomic research globally and is an essential
resource for genomics-based personalized medicine in the United Arab Emirates
and other parts of the Middle East.
From spiking neuronal networks to interpretable dynamics: a
diffusion-approximation framework
Authors: Mohammadi Nosrat , Truccolo Wilson , Cash Sydney S , Giraud Anne-Lise , Proix Timothée
Published: January 2025

Modeling and interpreting the complex recurrent dynamics of neuronal spiking
activity is essential to understanding how networks implement behavior and
cognition. Nonlinear Hawkes process models can capture a large range of spiking
dynamics, but remain difficult to interpret, due to their discontinuous and
stochastic nature. To address this challenge, we introduce a novel framework
based on a piecewise deterministic Markov process representation of the
nonlinear Hawkes process (NH-PDMP) followed by a diffusion approximation. We
analytically derive stability conditions and dynamical properties of the
obtained diffusion processes for single-neuron and network models. We
established the accuracy of the diffusion approximation framework by comparing
it with exact continuous-time simulations of the original neuronal NH-PDMP
models. Our framework offers an analytical and geometric account of the neuronal
dynamics repertoire captured by nonlinear Hawkes process models, both for the
canonical responses of single-neurons and neuronal-network dynamics, such as
winner-take-all and traveling wave phenomena. Applied to human and nonhuman
primate recordings of neuronal spiking activity during speech processing and
motor tasks, respectively, our approach revealed that task features can be
retrieved from the dynamical landscape of the fitted models. The combination of
NH-PDMP representations and diffusion approximations thus provides a novel
dynamical analysis framework to reveal single-neuron and neuronal-population
dynamics directly from models fitted to spiking data.
Premotor cortex hemodynamics reflect internal auditory category, not reported
category
Authors: Boucher Jeff , Shamma Shihab , Boubenec Yves
Published: January 2025

1

In sensory decision-making tasks, animals’ decisions are driven by
perception, but also by non-perceptual factors. Because of external and internal
noise, stimuli may be internally misclassified, leading to perceptual errors.
But other, non-sensory factors such as impulsivity or exploratory behavior can
lead to non-perceptual errors. Here we exploited the neural traces of these
errors in frontal cortex to provide insights into their role in sensory decision
making. Using functional ultrasound imaging (fUS), we investigated how the
premotor cortex (PMC) in ferrets represents stimuli in a categorization task,
varying the difficulty in order to manipulate the rates of perceptual errors. We
found that PMC activity reflects the objective (and not the chosen) stimulus
category on incorrect Easy trials, when non-perceptual errors are more likely.
In contrast, PMC responses correlate with the chosen category (and not objective
category) on incorrect Difficult trials, when perceptual errors are more likely.
These results suggest that PMC encodes the ferret’s perceptual decision but not
necessarily the final motor decision. Perceptual errors could be refined further
by assessing licking patterns, but licking patterns alone did not explain the
effect. This study advances our understanding of the functional role of the
frontal cortex in decision making, suggesting that the PMC integrates sensory
inputs to guide behavior based on perceptual, rather than motivational,
information.
Study of the immunogenicity, efficacy and safety of recombinant RBD
SARS-CoV-2 vaccine with CpG adjuvant in rodent, non-rodent and Maccaca
fascicularis using Indonesian Strain Virus
Authors: Limeilati Nur Amalia , Nidom Reviany V. , Sari Trilokita Tunjung , Kusumarahayu Nurvita P. , Fransiska Siti V. , Indrasari Setyarina , Garmana Afrillia N. , Ayuningtyas Dhyan K. , Nugroho Gembong S. , Darsono  , Maharani  , Wijayadikusumah Acep R. , Yulinah Elin , Adyana I Ketut , Nidom Astria N. , Nurainy Neni , Nidom Chairul A.
Published: January 2025

SARS-CoV-2 is the leading cause of the COVID-19 pandemic that causes acute
respiratory syndrome, emerged in late 2019, and was declared a global pandemic
on March 11th, 2020. A safe and effective vaccine that
prevents SARS-CoV-2 infection or minimize SARS-CoV-2 disease burden is needed.
However, in 2021, Low- and middle-income countries (LMICs) face challenges
regarding supply of COVID-19 vaccines. Indonesia, as a public sector vaccine
manufacturing in developing countries was developed COVID-19 vaccine using a
platform based on recombinant subunit proteins, a Receptor Binding Domain (RBD)
of SARS-CoV-2 formulated with combination of Alhydrogel and CpG
Oligodeoxynucleotides 1018 (CpG). In this study, we report the preclinical study
including immunogenicity, toxicity and efficacy of vaccine in animal models. The
vaccine immunogenicity tested in mice and non-human primates, the toxicity was
done in rodents and non-rodents and challenged study and efficacy was done in
non-human primates (NHPs) model. The animal model was vaccinated intramuscularly
(IM). The serology result in mice and non-human primates showed significant
antibody titers and neutralizing antibody responses compared to the RBD
formulations adjuvanted with Alhydrogel only. Safety study in Wistar rats and
New Zealand rabbits for single-dose (acute toxicity) and repeated-dose
(sub-chronic toxicity) showed no abnormalities in the animal’s organs and
behaviors and no deaths were reported in tested animals. Two doses of
vaccination have been shown to protect NHPs against SARS-CoV-2 infection, as
detected by drastic viral reduction from sample swab in nasal, anal, trachea and
nasal wash in 7 days after virus challenged, also viral load measurement from
lung and BAL tissue showed negative result, which gave better result than
negative control and control vaccine group. No evidence of disease enhancement
was observed. These results support clinical development of SARS-CoV-2 vaccine,
and in 2022 this vaccine has been approved for emergency use in
Indonesia.
G-quadruplexes catalyze protein folding by reshaping the energetic
landscape
Authors: Huang Zijue , Ghosh Kingshuk , Stull Frederick , Horowitz Scott
Published: January 2025

proteins

Many proteins have slow folding timesin
vitrothat are physiologically untenable. To combat this
challenge, ATP-dependent chaperonins are thought to possess the unique
ability to catalyze protein folding. Performing quantitative model selection
using protein folding and unfolding data, we here show that short nucleic
acids containing G-quadruplex (G4) structure can also catalyze protein
folding. Performing the experiments as a function of temperature
demonstrates that the G4 reshapes the underlying driving forces of protein
folding. As short nucleic acids can catalyze protein folding without the
input of ATP, the ability of the cell to fold proteins is far higher than
previously anticipated.

Significance Statement

How folding of proteins occurs en masse in the cell is still a daunting
unsolved problem. Many proteins have complicated and difficult folding
trajectories, within vitrofolding times that are
physiologically untenable. The acceleration of protein folding to
physiologically relevant timescales is a biologically essential function
thought to be accomplished by a small set of ATP-dependent chaperonins. In
this work, we surprisingly show that small nucleic acid sequences containing
G-quadruplexes can catalyze protein folding and reshape protein folding
energy landscapes. As a result, the capacity for accelerating protein
folding in the cells is far higher than previously suggested, potentially
explaining the accommodation of large number of proteins with
physiologically unreasonable folding times.
A phage-encoded counter-defense inhibits an NAD-degrading anti-phage defense
system
Authors: Loyo Christian L. , Grossman Alan D.
Published: January 2025

Bacteria

Bacteria contain a diverse array of genes that provide defense against
predation by phages. Anti-phage defense genes are frequently located on
mobile genetic elements and spread through horizontal gene transfer. Despite
the many anti-phage defense systems that have been identified, less is known
about how phages overcome the defenses employed by bacteria. The integrative
and conjugative element
ICEBs1inBacillus
subtiliscontains a gene,spbK,
that confers defense against the temperate phage SPβ through an abortive
infection mechanism. Using genetic and biochemical analyses, we found that
SpbK is an NADase that is activated by binding to the SPβ phage portal
protein YonE. The presence of YonE stimulates NADase activity of the TIR
domain of SpbK and causes cell death. We also found that the SPβ-like phage
Φ3T has a counter-defense gene that prevents SpbK-mediated abortive
infection and enables the phage to produce viable progeny, even in cells
expressingspbK. We made SPβ-Φ3T hybrid phages
that were resistant to SpbK-mediated defense and identified a single gene in
Φ3T (phi3T_120,now
callednipfor NADase inhibitor from phage)
that was both necessary and sufficient to block SpbK-mediated anti-phage
defense. We found that Nip binds to the TIR (NADase) domain of SpbK and
inhibits NADase activity. Our results provide insight into how phages
overcome bacterial immunity by inhibiting enzymatic activity of an
anti-phage defense protein.

Author Summary

Bacterial viruses (bacteriophages or phages) are widespread and abundant
across the planet. Bacteria have a variety of immune systems, often found on
mobile genetic elements, to combat phage predation. Phages can overcome
these immune systems by mutating to avoid recognition or by producing
molecules that prevent the immune system from working. We determined how an
anti-phage defense system encoded by an integrative and conjugative element
recognizes phage infection to cause cell death prior to the generation of
phage progeny. We also identified a phage gene that prevents this defense
system from functioning. The phage-encoded counter-defense protein inhibits
the enzymatic activity of the anti-phage defense protein, enabling evasion
of immunity and production of infectious phage. There are likely many
different phage-encoded counter-defense genes yet to be discovered.
Conditional fusogenic lipid nanocarriers for cytosolic delivery of
macromolecular therapeutics
Authors: Zhong Qian , Tan Edward K.W. , Shyamal Ananth , Ngambenjawong Chayanon , Parisi Tiziana , Ko Henry , Fleming Heather E. , Jacks Tyler , Bhatia Sangeeta N.
Published: January 2025

Macromolecular therapeutics designed for intracellular targets must overcome
systemic delivery barriers, target cell membrane impermeability, and inefficient
endosomal escape. Here, we engineer a class of conditional fusogenic liposomes
(C-FLIPs) that harness the catalytic activity of extracellular proteases present
in the pathological microenvironment. This context-specific activation enables
on-target membrane fusion with cells in diseased tissue, resulting in cytosolic
delivery of therapeutic payloads. We describe the cytoplasmic delivery of three
prototypic macromolecular therapeutic classes: peptide degraders, cytotoxic
proteins, and ribonucleoprotein particles (RNPs). We further develop C-FLIP to
deliver granzyme B (GzmB) to the cytoplasm of cancer cellsin
vivoand induce pyroptosis in immunologically-inert tumors.
Treatment with C-FLIP/GzmB reprograms the immunosuppressive tumor
microenvironment and synergizes with checkpoint blockade to result in the
regression of established tumors and induce immunological memory. This modular,
non-viral, cytosolic delivery platform represents a promising approach to
leverage pathological protease activity for targeted delivery of
biologics.
T cell-derived IFN-γ Suppresses T Follicular Helper Cell Differentiation and Antibody Responses
Authors: Sala Eleonora , Nelli Maria , Laura Chiara , Di Lucia Pietro , Beccaria Cristian Gabriel , Bono Elisa B. , Mangione Marta , Marotta Davide , Grillo Marta , Sperto Valentina , Giustini Leonardo , Tosi Fabio , Nie Jia , Kim Daehong , Furiato Giuliana , Malpighi Chiara , Consolo Eleonora , Becher Burkhard , David Eyal , Cohen Merav , Giladi Amir , Amit Ido , Bosselut Remy , Guidotti Luca G. , Iannacone Matteo , Kuka Mirela
Published: January 2025

CD4+T cells play a critical role in antiviral humoral and cellular immune responses. We have previously reported that subcutaneous lymphocytic choriomeningitis virus (s.c. LCMV) infection is characterized by a stark compartmentalization of CD4+T cells, leading to strong TH1 polarization but virtually absent T follicular helper (TFH) cells, a key driver of humoral immunity. Here, we investigated the mechanisms responsible for this impaired TFHdifferentiation. We found that T-bet+cells induced by s.c. LCMV infection encompass a TH1 subset expressing Granzyme-B (GzmB) and a Tcf-1+subset that retains the potential for TFHdifferentiation without expressing mature TFHmarkers. Interestingly, IFN-γ blockade enables full differentiation of Tcf-1+cells into TFH, formation of germinal centers and increased antibody production. Of note, the suppression of TFHcells by IFN-γ is not directly mediated through CD4+T cells but rather involves another cell type, likely dendritic cells (DCs). Our study provides novel insights into the mechanisms directing early CD4+T cell polarization and affecting humoral responses to viruses, laying a foundation for the development of effective vaccine strategies.
Rescue of non-healing, degenerative salivary glands by cholinergic-calcium signaling
Authors: Li Jianlong , Tan Li Xuan , Sun Bo , Griffin Nathan , Niknezhad Seyyed Vahid , Yu Chieh , Berthoin Lionel , Cruz Pacheco Noel , Mohabbat Seayar , Sinada Hanan , Gaylord Eliza A , Chen Yu Ting , An Luye , Efraim Yael , Bahney Chelsea Shields , Lombaert Isabelle M.A. , Knox Sarah
Published: January 2025

Chronic degenerative wounds are often deemed irreparable, directing research efforts to focus predominantly on acute tissue injury regeneration while leaving endogenous repair mechanisms for chronically damaged tissues largely unexplored. In this study, we demonstrate that non-healing, severely degenerated salivary gland tissues can be fundamentally restored through first-line treatment with muscarinic agonists. This approach rescues tissue structure and function, returning it to a homeostatic-like state, and reactivates endogenous regeneration processes to drive new cell expansion that persists for months post-treatment. Furthermore, neuromimetic activation profoundly depletes radiation-induced DNA damage and re-establishes the nerve-acinar relationship, ultimately restoring the tissues physiological capacity to maintain homeostasis, even in the absence of treatment. We show that full recovery of organ function, comparable to uninjured controls, is primarily mediated by the re-differentiation of aberrantly de-differentiated epithelial acinar cells and the restoration of mitochondrial function via a muscarinic-calcium signaling pathway. These findings challenge the prevailing notion that chronic organ degeneration is irreversible and propose a readily testable therapeutic strategy for epithelial restoration with potential applications across a spectrum of chronic injuries.
BORC segregates synaptic vesicle and lysosomal proteins through motors UNC-104/KIF1A and UNC-116/KIF5
Authors: Mathew Amal , Seal Sohan , Dandekar Aditee , Chauhan Badal Singh , Sivadasan Sruthi , Nonet Michael L , Koushika Sandhya P.
Published: January 2025

While synaptic vesicle proteins (SVPs) and lysosomal proteins can be present together in axons, the origin of these compartments is unclear. These SV-lysosomes are however thought to segregate further to SVPs and lysosomal proteins. In this study, we identify genes and characterize a genetic pathway involved in the segregation of SVPs and lysosomal proteins in the neuronal cell body. We identify a novel role for BORC (BLOC-1-related complex) in segregating SVPs and lysosomal proteins in the cell body. BORC subunit SAM-4/Myrlysin acts through ARL-8 and Kinesin motor proteins UNC-116/KIF5 and UNC-104/KIF1A in segregating SVPs and lysosomal proteins. Additionally, we also show that LRK-1/LRRK2 and APB-3/AP-3 (β3), involved in pre-SV biogenesis, regulate the segregation of SVPs and lysosomal proteins in the neuronal cell body. LRK-1 recruits SAM-4 that in turn governs the localisation of APB-3 suggesting a hierarchical pathway of LRK-1-SAM-4-APB-3 for the segregation of SVPs and lysosomal proteins. Additionally, we also observe that the size of lysosomal protein-containing compartments (LPCCs) is smaller in SAM-4 and LRK-1. This size regulation depends on UNC-116. Together, we show that BORC recruited by LRK-1 and in turn via motors and along with AP-3 mediates the segregation of SVPs and lysosomal proteins in the neuronal cell body.
Carbon source, cell density, and the microbial community control inhibition ofV. choleraesurface colonization by environmental nitrate
Authors: James Jamaurie , Santos Renato ERS , Watnick Paula I
Published: January 2025

The intestinal diarrheal pathogenVibrio choleraecolonizes the host terminal ileum, a microaerophilic, glucose-poor, nitrate-rich environment. In this environment,V. choleraerespires nitrate and increases transport and utilization of alternative carbon sources via the cAMP receptor protein (CRP), a transcription factor that is active during glucose scarcity. Here we show thatV. choleraenitrate respiration in aerated cultures is under control of CRP and, therefore, glucose availability.V. choleraenitrate respiration results in extracellular accumulation of nitrite becauseV. choleraedoes not possess the machinery for nitrite reduction. This nitrite inhibitsV. choleraebiofilm formation via an as yet unelucidated mechanism that depends on the high cell density master regulator HapR. The genome ofParacoccus aminovorans, an intestinal microbe shown to enhanceV. choleraebiofilm accumulation in the neonatal mouse gut and predispose household contacts to cholera, encodes enzymes that reduce nitrite to nitrogen gas. We report that, in nitrate-supplemented co-cultures,P. aminovoransmetabolizes the nitrite generated byV. choleraeand, thereby, enhancesV. choleraesurface accumulation. We propose thatV. choleraebiofilm formation in the host intestine is limited by nitrite production but can be rescued by intestinal microbes such asP. aminovoransthat have the capacity to metabolize nitrite. Such microbes increaseV. choleraecolonization of the host ileum and predispose to infection.

Importance

V. choleraecolonizes the terminal ileum where both oxygen and nitrate are available as terminal electron acceptors.V. choleraebiofilm formation is inhibited by nitrate due to its conversion to nitrite duringV. choleraerespiration. When co-cultured with a microbe that can further reduce nitrite,V. choleraesurface accumulation in the presence of nitrate is rescued. The contribution of biofilm formation to ileal colonization depends on the composition of the microbiota. We propose that the intestinal microbiota predisposes mammalian hosts to cholera by consuming the nitrite generated byV. choleraein the terminal ileum. Differences in the intestinal abundance of nitrite-reducing microbes may partially explain the differential susceptibility of humans to cholera and the resistance of non-human mammalian models to intestinal colonization withV. cholerae.
Single-cell mapping of regulatory DNA:Protein interactions
Authors: Chi Wei-Yu , Yoon Sang-Ho , Mekerishvili Levan , Ganesan Saravanan , Potenski Catherine , Izzo Franco , Landau Dan , Raimondi Ivan
Published: January 2025

Gene expression is coordinated by a multitude of transcription factors (TFs), whose binding to the genome is directed through multiple interconnected epigenetic signals, including chromatin accessibility and histone modifications. These complex networks have been shown to be disrupted during aging, disease, and cancer. However, profiling these networks across diverse cell types and states has been limited due to the technical constraints of existing methods for mapping DNA:Protein interactions in single cells. As a result, a critical gap remains in understanding where TFs or other chromatin remodelers bind to DNA and how these interactions are perturbed in pathological contexts. To address this challenge, we developed a transformative single-cell immuno-tethering DNA:Protein mapping technology. By coupling a species-specific antibody-binding nanobody to a cytosine base editing enzyme, this approach enables profiling of even weak or transient factor binding to DNA, a task that was previously unachievable in single cells. Thus, our Docking & Deamination followed by sequencing (D&D-seq) technique induces cytosine-to-uracil edits in genomic regions bound by the target protein, offering a novel means to capture DNA:Protein interactions with unprecedented resolution. Importantly, this technique can be seamlessly incorporated into common single-cell multiomics workflows, enabling multimodal analysis of gene regulation in single cells. We tested the ability of D&D-seq to record TF binding both in bulk and at the single-cell level by profiling CTCF and GATA family members, obtaining high specificity and efficiency, with clear identification of TF footprint and signal retention in the targeted cell subpopulations. Furthermore, the deamination reaction showed minimal off-target activity, with high concordance to bulk ChIP-seq reference data. Applied to primary human peripheral blood mononuclear cells (PBMCs), D&D-seq successfully identified CTCF binding sites and enabled integration with advanced machine-learning algorithms for predicting 3D chromatin structure. Furthermore, we integrated D&D-seq with single-cell genotyping to assess the impact ofIDH2mutations on CTCF binding in a human clonal hematopoiesis sample, uncovering altered binding and chromatin co-accessibility patterns in mutant cells. Altogether, D&D-seq represents an important technological advance enabling the direct mapping of TF or chromatin remodeler binding to the DNA in primary human samples, opening new avenues for understanding chromatin and transcriptional regulation in health and disease.
Methanogens are associated with altered microbial production of short-chain fatty acids and human-host metabolizable energy
Authors: Dirks Blake , Davis Taylor L. , Carnero Elvis A. , Corbin Karen D. , Smith Steven R. , Rittmann Bruce E. , Krajmalnik-Brown Rosa
Published: January 2025

Methanogens are CH4-producing, H2-oxidizing (i.e. hydrogenotrophic) archaea. Numerous studies have associated methanogens with obesity, but these results have been inconsistent. One link to host metabolism may be methanogens’ ability to consume H2, thus reducing H2partial pressure and thermodynamically enhancing fermentation of sugars to short-chain fatty acids (SCFA) that the host can absorb. Because research linking methanogenesis to human metabolism is limited, our goal with this exploratory analysis was to investigate relationships between methanogens and other hydrogenotrophs, and the association of methanogens with human metabolizable energy (ME). Using results from a randomized crossover feeding study with well-characterized human participants and novel continuous methane measurements, we analyzed hydrogenotroph abundance and activity, fecal and serum SCFAs, and host ME between high and low CH4producers. Methanogens were detected in about one-half of participants, and most were high CH4producers. We found no evidence that methanogens’ consumption of H2to produce CH4affected other hydrogenotrophs. High CH4producers had greater serum propionate and host ME irrespective of diet, as well as greater gene and transcript abundance of a key enzyme of the H2-consuming, propionate-producing succinate pathway. Finally, a network analysis revealed positive relationships betweenMethanobrevibacter smithii(the most prevalent methanogen in the human colon) and bacteria capable of degrading fiber and fermenting fiber- degradation products, thus forming a trophic chain to extract additional energy from undigested substrates. Our results show that methanogens in a microbial consortium were linked to host metabolizable energy through enhanced SCFA production and host SCFA absorption.
Structure and Post-Translational Modification of the Prostaglandin Transporter
Authors: Yu Peixuan , Orlando Melanie A. , Orlando Benjamin J.
Published: January 2025

The prostaglandin transporter (PGT) is a member of the Organic Anion Transporting Polypeptide (OATP) family of membrane transporters. PGT mediates the uptake of prostaglandins from the extracellular environment to enable intracellular enzymatic degradation and termination of signaling. In addition to importing prostaglandins, PGT is also an essential core component of the Maxi-Cl channel, which facilitates cellular release of ATP and other small organic anions. Despite progress on understanding the (patho)physiological roles of PGT, and development of small molecules to inhibit this transporter, molecular details of the overall structure and transport mechanism remain elusive. Here we determined the cryo-EM structure of human PGT, which demonstrates an overall topology consistent with other OATPs despite possessing a dual transporter/channel functionality. We additionally investigated the role of eight potential disulfide bonds found in the extracellular loops of PGT and paralogous transporters. Through biochemical and functional characterization we demonstrate that six intra-loop disulfide bonds (C420-C511, C450-C470, C492-C474, C459-C507, C444-C494, C580-C594) are essential for proper N-glycosylation, plasma membrane trafficking, and prostaglandin import activity of PGT. In contrast, two inter-loop disulfides (C155-C587 and C143-C448) were found to restrict maximal prostaglandin uptake, suggesting a possible regulatory role in modulating PGT activity. In total, our studies provide a fresh molecular perspective on the structure, post-translational modification, and overall function of PGT.

Significance Statement

The prostaglandin transporter (PGT) is essential for cellular uptake of prostaglandins and serves as a core component of the Maxi-Cl channel. Using cryo-EM we resolved the structure of human PGT, revealing a similar overall topology as other OATP transporters. Extensive site-directed mutagenesis and functional assays identified eight disulfide bonds in PGT’s extracellular domain as key regulators of glycosylation, trafficking, and transport activity. These findings provide a structural basis for PGT function and lay a foundation to further explore substrate recognition and inhibitor design for this biologically significant transporter.
Biochemical and physiological characterization ofAedes aegyptimidgut chymotrypsin
Authors: Ramirez Abigail G. , Isoe Jun , Serafim Mateus Sá M , Fong Daniel , Le My Anh , Nguyen James T. , Burata Olive E. , Lucero Rachael M. , Rascón Alberto A.
Published: January 2025

TheAedes aegyptimosquito is a vector of dengue, Zika, and chikungunya. The mosquito’s reliance on blood facilitates the transmission of these viral pathogens to humans. Digestion of blood proteins depends on the biphasic expression of serine proteases, with trypsin-like activity contributing to most of the activity in the midgut. Other proteases found (serine collagenase- and chymotrypsin-like) are thought to contribute to digestion, but their roles are largely understudied. Thus, elucidating the activity and specific roles of all midgut proteases will help understand the complexity of the digestion process and help validate them as potential targets for the development of a new vector control strategy. Herein, we focused on characterizing the activity profile and role ofAe. aegyptichymotrypsin (AaCHYMO). Knockdown studies resulted in elimination and significant reduction of chymotrypsin-like activity in blood fed midgut extracts, whilein vitrofluorescent and blood protein digestion assays revealed important substrate specificity differences. Interestingly, knockdown of AaCHYMO did not impact fecundity, indicating the presence of an intricate network of proteases working collectively to degrade blood proteins. Further, knockdown of the ecdysone receptor (EcR) led to a decrease in overall AaCHYMO expression and activity in the mosquito, which may play an important regulatory role.
From Data to Insights: A Tool for Comprehensive Quantification of Continuous Glucose Monitoring (QoCGM)
Authors: Cichosz Simon Lebech , Hangaard Stine , Kronborg Thomas , Vestergaard Peter , Jensen Morten Hasselstrøm
Published: January 2025

Continuous Glucose Monitoring (CGM) has become a important technology in the management and research of both type 1 and type 2 diabetes, providing real-time data on glucose fluctuations that were previously inaccessible with traditional monitoring methods. Numerous analytical tools, such as cgmquantify, iglu, GLU, rGV, and CGManalyzer, have been developed for platforms like R and Python to calculate standard metrics and extract insights from CGM data. However, these tools often fail to address the full spectrum of analytical requirements. Furthermore, there is a significant lack of updated, open-source tools tailored for MATLAB—a platform widely used by the research community. To address this gap, we introduce Quantification of Continuous Glucose Monitoring (QoCGM), a comprehensive, open-source analytical tool for CGM data specifically designed for MATLAB. A case study involving 324 individuals with insulin-treated type 2 diabetes mellitus (T2DM) demonstrates the utility of QoCGM, highlighting the distinct aspects of glucose dynamics captured by different CGM-derived metrics through an analysis of their coefficients of determination (R²).
Evaluation of Candidate “Kill or Cure” Strategies to Treat MFN2-related Lipodystrophy
Authors: Luijten Ineke , Weng Xiong , Kibildyte Ula , Buchan Jana , Onishi Ami , Mann Jake , McKay Eleanor , Savage David , Semple Robert K.
Published: January 2025

The R707W mutation in mitofusin 2, encoded by MFN2, causes a form of Multiple Symmetrical Lipomatosis (MFN2-MSL). This resembles sporadic, alcohol-associated MSL, combining loss of lower body adipose tissue with upper body adipose hyperplasia. Morbidity and sometimes mortality arise both from mechanical complications of head and neck adipose overgrowth, and metabolic complications. We reasoned that interventions that either mitigate the underlying cellular pathology, or that exacerbate it to induce selective death of hyperplastic adipose tissue may be beneficial. We thus assessed the effect of a metabolic or pharmacologic stressors or rapamycin inMfn2R707W/R707Wmice and or derived preadipocytes. 50mmol ethanol had little effect on WT orMfn2R707W/R707Wwhite preadipocytes, but increased mitochondrial content and blunted mitolysosome formation inMfn2R707W/R707Wbrown preadipocytes.Daily consumption of 20% EtOH increased brown adipose tissue mass in femaleMfn2R707W/R707Wmice, and serum lactate in males. 200nM rapamycin – a candidate treatment - increased size and mitolysosome content of WT andMfn2R707W/R707Wwhite and brown preadipocytes, but these effects were blunted inMfn2R707W/R707Wcells. In male but not femaleMfn2R707W/R707Wmice, rapamycin reduced or reversed weight gain, reduced brown adipose mass, and increased serum Fgf21. Finally, a panel of other metabolic and pharmacological mitochondrial stressors solicited no selective death or ISR inMfn2R707W/R707Wpreadipocytes. We conclude that ethanol mildly exacerbates MFN-MSL in mice, while rapamycin is tolerated. Lack of sensitisation to mitochondrial stressors implies that the MSL-inducing effect of MFN2 R707W may not be exerted through compromised oxidative phosphorylation.
Hepatic fructose metabolism is antagonized by growth hormone/insulin-like growth factor signaling via regulation of ketohexokinase expression
Authors: Elsaid Salaheldeen , Meesala Prathibha , Choi Youngshim , Yu Liqing , Xiao Yang , Lazar Mitchell A. , Perez-Matute Patrica , List Edward O. , Faccioli Lanuza AP , Sun Yiyue , Florentino Rodrigo M , Pichel Jose G. , Berryman Darlene E. , Mayer Dirk , Kopchick John J. , Tee Sui Seng
Published: January 2025

Overconsumption of added sugars such as fructose has been associated with a remarkable decline in metabolic health. Fructose is primarily metabolized by the small intestines and the liver, via phosphorylation mediated by ketohexokinase (KHK). KHK activity is traditionally viewed as lacking negative feedback mechanisms, such as those present to limit glucose metabolism, leading to excessive fat accumulation characteristic of metabolic dysfunction-associated liver disease (MASLD). In this study, we observe KHK downregulation in hepatocytes of diet-induced and genetic models of MASLD. Reduced KHK coincides with decreased flux of fructose-derived carbons into glycolytic and amino acid metabolic pathways, suggesting the presence of mechanisms that limit KHK-mediated fructolysis in the liver. We subsequently focused on the growth hormone (GH)/insulin-like growth factor (IGF) signaling pathway as a potential mechanism antagonizing KHK expression. In transgenic mice with enhanced GH signaling, KHK levels are reduced, whereas reduced GH activity leads to increased KHK expression. Additionally, administration of GH and IGF-1 in liver cell cultures induces time-dependent degradation of KHK, facilitated by direct interactions between KHK and the IGF-1 receptor (IGF-1R). Single-nuclei RNA sequencing revealed elevated IGF-1R expression in hepatocytes from diet-induced MASLD mice, supported by human MASLD patient samples, which also show reduced KHK expression. Taken together, these findings describe a novel pathway by which GH/IGF-1 signaling regulates KHK, offering new insights into how the liver adapts to metabolic stress to limit fructose-driven liver dysfunction.
G-Quadruplex-Protein Interactome at Human Gene Promoters
Authors: Duan Rui-fang , Zheng Jin-ping , Hao Yu-hua , Tan Zheng
Published: January 2025

DNA-protein interactions at gene promoters play a critical role in gene expression. The promoters of human cells are highly enriched in guanine-rich sequences, which can form four-stranded G-quadruplex (G4) structures. G4s are emerging as a distinct class of structure-based regulatory elements in gene regulation, and their interaction with proteins is essential for the role G4s play. Currently, our understanding of G4-protein interaction is mainly on a case-by-case basis, without systematic information. In this work, we examined the spatial occupancy of 1,183 human DNA-binding proteins, including transcription factors, histones and their modifying enzymes, around the consensus G4-forming region, G4(+), using data from the ENCODE project. We found that the G4(+), its immediate proximal side, and its distal side serve as three primary protein binding sites. Nearly all proteins are either enriched or depleted at these sites, likely due to competition, or in a spatiotemporal transition between the sites, resulting in different degrees of variation or persistence within or across cell/tissue types. Notably, histones were excluded from the proximal side of G4(+), and their binding to G4(+) was turned on and off by acetylation and methylation, respectively. Furthermore, the distal side is preferentially enriched for H3K23me2 and H3K4me2. Our experiments also revealed corresponding patterns of G4-protein interaction. Taken together, our results suggest a general role for G4s in dynamically defining and coordinating chromatin architecture and DNA-protein interactions at gene promoters for transcriptional regulation, a task that is unlikely to be accomplished by sequence-based DNA recognition.

Abstract Figure

Graphical abstract:

A protein can be either enriched or suppressed at G-quadruplexes (G4s), at the proximal side of G4s, or enriched at the distal side of G4s in a 2 kb neighborhood. Regulatory roles of proteins may result from spatial transitions between the different binding regions.
A tool to dissect heterotypic determinants of homotypic protein phase behavior
Authors: Kimbrough Hannah , Jensen Jacob , Weber Caleb , Miller Tayla , Maddera Lucinda E. , Babu Vignesh , Redwine William B. , Halfmann Randal
Published: January 2025

Proteins commonly self-assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each protein’s sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self-assembly in cells. Here, we extend DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether and thereby recruit candidate modifier proteins to mEos3-fused query proteins. This accessorization allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valency of mEosNb-fused modifiers. We show that our system recapitulates known effects of multivalency on liquid-liquid phase separation and can discriminate between nucleation mechanisms of amyloid and amyloid-like assemblies. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions.

Lay summary

Protein self-assemblies are essential for cellular function, but can also contribute to disease. We develop a new tool to study how their formation is influenced by other cellular factors. This tool allows us to control the location and number of interactions between a protein of interest and other proteins that may influence it. Our results provide new insight into mechanisms of self-assembly and will aid research toward treating diseases associated with aberrant assembly.