bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Adjunctive Prefronto-Cerebellar Transcranial Direct Current Stimulation(tDCS) on Craving and Relapse Prevention in Patients with Alcohol Dependence Syndrome (ADS): A Randomized Sham Controlled Study Protocol
Authors: Sarkar Susmita , Singh Lokesh Kumar , Kumar Ajay , Tikka Sai Krishna
Published: August 2022

BACKGROUND

There is growing evidence that tDCS in the Dorsolateral Prefrontal Cortex (DLPFC) technique is effective in controlling craving and relapse in ADS patients. Till now the results are inconclusive. Interestingly, the cerebellum is related to impulsive and compulsive behavior which is supported by both structural and functional neuroimaging findings. There are few clinical trials regarding the effect of cerebellar tDCS in ADS.

OBJECTIVE

To assess the effect of Prefronto-cerebellar tDCS in reducing craving and relapses in ADS patients.

METHODS

The study population will be randomly assigned to either of two groups i.e. Active and Sham tDCS group. The active group will receive 10 sessions over 5 days; 2mA current for 15 minutes. The site of anodal stimulation is 2cm below the Iz (inion) and cathodal stimulation at F3 (left prefrontal) and in the sham group will receive the same except that intensity of the current is 0.2mA. Patients will be assessed for craving at 3-time points.

CONCLUSION

It will be helpful in analyzing the effect of stimulating DLPFC as well as the cerebellum which is a potential novel site in reducing craving and relapse in ADS.

HIGHLIGHTS

tDCS in ADS is being tried for the last decade mainly focusing on the frontal area. With the current understanding of the role of the cerebellum in regulating impulsive and compulsive behavior, we intend to add the cerebellum as a potential site for neuromodulation in reducing craving and relapse in ADS patients.
BlpC-mediated selfish program leads to rapid loss of Streptococcus pneumoniae clonal diversity during infection
Authors: Aggarwal Surya D. , Lees John A. , Jacobs Nathan T. , Bee Gavyn Chern Wei , Abruzzo Annie R. , Weiser Jeffrey N.
Published: August 2022

Chromosomal barcoding and high-throughput sequencing were used to investigate the population dynamics of Streptococcus pneumoniae. During infant mouse colonization, >35-fold reduction in diversity and expansion of a single clonal lineage was observed within 1 day post-inoculation. This loss of diversity was not due to immune factors, host microbiota or exclusively because of genetic drift. Rather, it required the expression of blp bacteriocins induced by the BlpC-quorum sensing pheromone. This points towards the role of intra-strain competition whereby the subpopulation reaching a quorum eliminates others that have yet to activate the blp locus. We show that this loss of diversity also restricts the number of unique clones that could establish colonization during transmission between hosts. Moreover, we show that genetic variation in the blp locus is associated with transmissibility in the human population. We posit this is due to its importance in clonal selection and its role as a selfish element.
Structure of a monomeric photosystem I core associated with iron-stress-induced-A proteins from Anabaena sp. PCC 7120
Authors: Nagao Ryo , Kato Koji , Hamaguchi Tasuku , Ueno Yoshifumi , Tsuboshita Naoki , Shimizu Shota , Furutani Miyu , Ehira Shigeki , Nakajima Yoshiki , Kawakami Keisuke , Suzuki Takehiro , Dohmae Naoshi , Akimoto Seiji , Yonekura Koji , Shen Jian-Ren
Published: August 2022

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions. The cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, their binding property and functional roles in PSI are still missing. We analyzed a cryo-electron microscopy structure of a PSI-IsiA supercomplex isolated from Anabaena grown under an iron-deficient condition. The PSI-IsiA structure contains six IsiA subunits associated with the PsaA side of a PSI core monomer. Three of the six IsiA subunits are identified as IsiA1 and IsiA2. The PSI-IsiA structure lacks a PsaL subunit; instead, a C-terminal domain of IsiA2 is inserted at the position of PsaL, which inhibits the oligomerization of PSI, leading to the formation of a monomer. Furthermore, excitation-energy transfer from IsiAs to PSI appeared with a time constant of 55 ps. These findings provide novel insights into both the molecular assembly of the Anabaena IsiA family and the functional roles of IsiAs.
A fear conditioned cue orchestrates a suite of behaviors
Authors: Chu Amanda , Michel Christa B. , Gordon Nicholas T. , Hanrahan Katherine E. , DuBois Aleah M. , Williams David C. , McDannald Michael A.
Published: August 2022

Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior theoretically linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that - in addition to jumping, and rearing - is theoretically linked to imminent or occurring predation. A criticism of studies observing fear conditioned cue-elicited locomotion is that responding is non-associative. We gave 24 rats (12 female) Pavlovian fear discrimination over a baseline of reward seeking. The within-subjects procedure had full controls for associative learning, consisting of three cues predicting unique foot shock probabilities: danger (p=1), uncertainty (p=0.25), and safety (p=0). TTL-triggered cameras captured 5 behavior frames/s prior to and during cue presentation. We scored 86,400 frames for nine discrete behaviors spanning reward, passive fear, and active fear. Temporal ethograms show that a fear conditioned cue elicits locomotion, jumping, and rearing that is maximal towards cue offset, when foot shock is imminent. A fear conditioned cue further suppresses reward-related behavior, and elicits freezing in a sex-specific manner. The differing temporal profiles and independent expression of these behaviors reveal a fear conditioned cue to orchestrate a rich and intricate suite of behaviors.
Harnessing diversity and antagonism within the pig skin microbiota to identify novel mediators of colonization resistance to methicillin-resistant Staphylococcus aureus
Authors: Flowers Laurice , Wei Monica , Knight Simon A. B. , Zheng Qi , Uberoi Aayushi , Campbell Amy , Pan Ting-Chun Jamie , Walsh Jasmine , Schroeder Erin , Chu Emily W. , Bradley Charles W. , Duran-Struuck Raimon , Grice Elizabeth A.
Published: August 2022

The microbiota mediates multiple aspects of skin barrier function, including colonization resistance to pathogens such as Staphylococcus aureus. The endogenous skin microbiota limits S. aureus colonization via competition and direct inhibition. Novel mechanisms of colonization resistance are promising therapeutic targets for drug-resistant infections, such as those caused by methicillin-resistant S. aureus (MRSA). Here, we developed and characterized a swine model of topical microbiome perturbation and MRSA colonization. As in other model systems, topical antimicrobial treatment had little discernable effect on community diversity though the overall microbial load was sensitive to any type of intervention, including swabbing. In parallel, we established a porcine skin culture collection and screened 7,700 isolates for MRSA inhibition. Inhibitory isolates were represented across all major phyla of the pig skin microbiota and did not have a strong preference for inhibiting closely related species, suggesting that relatedness is not a condition of antagonism. Using genomic and phenotypic criteria, we curated 3 isolates to investigate whether prophylactic colonization would inhibit MRSA colonization in vivo. The 3-member consortium together, but not individually, provided protection against MRSA colonization, suggesting cooperation and/or synergy among the strains. These findings reveal the porcine skin as an underexplored reservoir of skin commensal species with the potential to prevent MRSA colonization and infection.
Sequence features do not drive karyotypic evolution: what are the missing correlates of genome evolution?
Authors: Brekke Thomas D. , Papadopulos Alexander S. T. , Swain Martin T. , Mulley John F.
Published: August 2022

Genome rearrangements are prevalent across the tree of life and even within species. After two decades of research, various suggestions have been proposed to explain which features of the genome are associated with rearrangements and the breakpoints between rearranged regions. These include: recombination rate, GC content, repetitive DNA content, gene density, and markers of chromatin conformation. Here, we use a set of six aligned rodent genomes to identify regions that have not been rearranged and characterize the breakpoint regions where rearrangements have occurred. We found no strong support for any of the expected correlations between breakpoint regions and a variety of genomic features previously identified. These results call into question the utility and repeatability of identifying chromatin characteristics associated with rearranged regions of the genome and suggest that perhaps a different explanation is in order. We analyzed rates of karyotypic evolution in each of the six lineages and found that the Mongolian gerbil genome has had the most rearrangements. That gerbils exhibit very rapid sequence evolution at a number of key DNA repair genes suggests an alternative hypothesis for patterns of genome rearrangement: karyotypic evolution may be driven by variation at a few genes that control the repair pathway used to fix double-stranded DNA breaks. Such variation may explain the heterogeneity in the rates of karyotypic evolution across species. While currently only supported by circumstantial evidence, a systematic survey of this hypothesis is now warranted.
Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5
Authors: Tamura Tomokazu , Yamasoba Daichi , Oda Yoshitaka , Ito Jumpei , Kamasaki Tomoko , Nao Naganori , Hashimoto Rina , Fujioka Yoichiro , Suzuki Rigel , Wang Lei , Ito Hayato , Kimura Izumi , Yokota Isao , Kishimoto Mai , Tsuda Masumi , Sawa Hirofumi , Yoshimatsu Kumiko , Ohba Yusuke , Yamamoto Yuki , Nagamoto Tetsuharu , Kanamune Jun ,   The Genotype to Phenotype Japan (G2P-Japan) Consortium, Matsuno Keita , Takayama Kazuo , Tanaka Shinya , Sato Kei , Fukuhara Takasuke
Published: August 2022

Unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants imposes us to continuous control measurement. Given the rapid spread, new Omicron subvariant named BA.5 is urgently required for characterization. Here we analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1 comprehensively. Although in vitro growth kinetics of BA.5 is comparable among the Omicron subvariants, BA.5 become much more fusogenic than BA.1 and BA.2. The airway-on-a-chip analysis showed that the ability of BA.5 to disrupt the respiratory epithelial and endothelial barriers is enhanced among Omicron subvariants. Furthermore, in our hamster model, in vivo replication of BA.5 is comparable with that of the other Omicrons and less than that of the ancestral B.1.1. Importantly, inflammatory response against BA.5 is strong compared with BA.1 and BA.2. Our data suggest that BA.5 is still low pathogenic compared to ancestral strain but evolved to induce enhanced inflammation when compared to prior Omicron subvariants.
Mixed Heavy Metals Stress Induces Global Iron Starvation as Revealed by System Level Multi-Omic Analyses
Authors: Goff Jennifer L. , Chen Yan , Thorgersen Michael P. , Hoang Linh T. , Poole Farris L. , Szink Elizabeth G. , Siuzdak Gary , Petzold Christopher J. , Adams Michael W.W.
Published: August 2022

Globally, multiple heavy metal contamination is an increasingly common problem. As heavy metals have the potential to disrupt microbially-mediated biogeochemical cycling, it is critical to understand their impact on microbial physiology. However, systems-level studies on the effects of a combination of heavy metals on bacteria are lacking. Here, we use a native Bacillus cereus isolate from the subsurface of the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) — representing a highly abundant species at the site— to assess the combined impact of eight metal contaminants. Using this metal mixture and individual metals, all at concentrations based on the ORR site geochemistry, we performed growth experiments and proteomic analyses of the B. cereus strain, in combination with targeted MS-based metabolomics and gene expression profiling. The combination of eight metals impacts cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Specifically, exposure to the metal mixture elicited global iron starvation responses not observed in any of the individual metal treatments. As nitrate is also a significant contaminant at the ORR site and nitrate and nitrite reductases are iron-containing enzymes, we also examined the effects of the metal mixture on reduction of nitrogen oxides. We found that the metal mixture inhibits the activity of these enzymes through a combination of direct enzymatic damage and post-transcriptional and post-translational regulation. Altogether, these data suggest that metal mixture studies are critical for understanding how multiple rather than individual metals influence microbial processes in the environment.
Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon
Authors: Tenjo-Castaño Francisco , Sofos Nicholas , Lopéz-Méndez Blanca , Stutzke Luisa S. , Fuglsang Anders , Stella Stefano , Montoya Guillermo
Published: August 2022

CRISPR-associated transposons (CASTs) represent unique mobile genetic elements that co-opted CRISPR-Cas immune systems for RNA-guided DNA transposition. Type V-K CAST is composed by Cas12k, TniQ, TnsC and TnsB. Here, we present the 2.46 Å cryoelectron microscopy structure of the Scytonema hofmannii CAST TnsB transposase in complex with the strand transfer DNA in a post-catalytic state. The shTnsB strand transfer complex maintains the intertwined architecture of the MuA phage transpososome. However, the building of the assembly depends on different local interactions. The protein-DNA complex forms a pseudo-symmetrical assembly in which the 4 protomers of shTnsB adopt two different conformations. The recognition of the transposon ends is accomplished by two small helical domains. The two protomers involved in the strand transfer reaction display a catalytically competent active site composed by three acidic residues (DDE), while the other two, which play a key role in the complex architecture, show catalytic pockets where the DDE residues are not properly positioned for cleavage. Quantification of in vivo transposition assays of mutants in key DNA binding residues, reveals that the lack of specificity generally decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of the DDE DNA transposases and offers new insights into RNA-guided transposition in CAST systems.
BackWards – Unveiling the Brain’s Topographic Organization of Paraspinal Sensory Input
Authors: Guekos Alexandros , Cole David M , Dörig Monika , Stämpfli Philipp , Schibli Louis , Schuetz Philipp , Schweinhardt Petra , Meier Michael L
Published: August 2022

Cortical reorganization and its potential pathological significance are being increasingly studied in musculoskeletal disorders such as chronic low back pain (CLBP) patients. However, detailed sensory-topographic maps of the human back are lacking, and a baseline characterization of such representations, reflecting the somatosensory organization of the healthy back, is needed before exploring potential sensory map reorganization. To this end, a novel pneumatic vibrotactile stimulation method was used to stimulate paraspinal sensory afferents, while studying their cortical representations in unprecedented detail. In 41 young healthy participants, vibrotactile stimulations at 20 Hz and 80 Hz were applied bilaterally at nine lo cations along the thoracolumbar axis while functional magnetic resonance imaging (fMRI) was performed. Model-based whole-brain searchlight representational similarity analysis (RSA) was used to investigate the organizational structure of brain activity patterns evoked by thoracolumbar sensory inputs. A model based on seg mental distances best explained the similarity structure of brain activity patterns that were located in different areas of sensorimotor cortices, including the primary somatosensory and motor cortices and parts of the superior parietal cortex, suggesting that these brain areas process sensory input from the back in a “dermatomal” manner. The current findings provide a sound basis for testing the “cortical map reorganization theory” and its pathological relevance in CLBP.

Highlights

Fine-grained cortical activation patterns of paraspinal vibrotactile sensory input were obtained using whole-brain representational similarity analysis.

The patterns were well explained using a model reflecting segmental distances along the thoracolumbar axis.

The current results provide a solid basis for revisiting the “cortical map reorganization theory” and its pathological significance in chronic low back pain.
Evaluating material-driven regeneration in a tissue engineered humanin vitrobone defect model
Authors: de Wildt Bregje W.M. , Cramer Esther E.A. , de Silva Leanne S. , Ito Keita , Gawlitta Debby , Hofmann Sandra
Published: August 2022

Advancedin vitrohuman bone defect models can contribute to the evaluation of materials forin situbone regeneration, addressing both translational and ethical concerns regarding animal models. In this study, we attempted to develop such a model to study material-driven regeneration, using a tissue engineering approach. By co-culturing human umbilical vein endothelial cells (HUVECs) with human bone marrow-derived mesenchymal stromal cells (hBMSCs) on silk fibroin scaffolds within vitrocritically sized defects, the growth of vascular-like networks and three-dimensional bone-like tissue was facilitated. After a model build-up phase of 28 days, materials were artificially implanted and HUVEC and hBMSC migration, cell-material interactions, and osteoinduction were evaluated 14 days after implantation. The materials physiologically relevant for bone regeneration included a platelet gel as blood clot mimic, cartilage spheres as soft callus mimics, and a fibrin gel as control. Although thein vitromodel was limited in the evaluation of immune responses, hallmarks of physiological bone regeneration were observedin vitro. These included the endothelial cell chemotaxis induced by the blood clot mimic and the mineralization of the soft callus mimic. Therefore, the presentin vitromodel could contribute to an improved pre-clinical evaluation of biomaterials while reducing the need for animal experiments.

Graphical abstract
Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance
Authors: Copier J.S. , Bootsma M. , Ng C. , Wilde A.A.M. , Bertels R.A. , Bikker H. , Christiaans I. , van der Crabben S.N. , Hol J.A. , Koopmann T.T. , Knijnenburg J. , Lommerse A.A.J. , van der Smagt J.J. , Bezzina C.R. , Vandenberg J.I. , Verkerk A.O. , Barge-Schaapveld D.Q.C.M. , Lodder E.M.
Published: August 2022

Background

Variants in KCNH2, encoding the hERG channel which is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to Long QT Syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C>T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making.

Methods

A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed by manual whole-cell patch-clamp using HEK293a cells expressing: (I) wild type (WT) KCNH2, (II) KCNH2-p.S906L alone (homozygous, Hm) or (III) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz). A calibrated automated patch-clamp assay using Flp-In HEK293 was used to follow up on the functional data.

Results

Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2, or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch-clamp showed a reduced current density by 69.8%, and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared to KCNH2-WT. Assessment of KCNH2-p.S906L-Hz, by calibrated automatic patch-clamp showed a reduction in current density by 35.6%.

Conclusion

The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss of function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.
Amyloid-β targeting immunisation in aged non-human primate (Microcebus murinus)
Authors: G. Trouche Stéphanie , Boutajangout Allal , Asuni Ayodeji , Fontés Pascaline , Sigurdsson Einar M. , Verdier Jean-Michel , Mestre-Francés Nadine
Published: August 2022

Non-human primates have an important translational value given their close phylogenetic relationship to humans. Studies in these animals remain essential for evaluating efficacy and safety of new therapeutic approaches, particularly in aging primates that display Alzheimer’s disease (AD) -like pathology. With the objective to improve amyloid-β (Aβ) targeting immunotherapy, we investigated the safety and efficacy of an active immunisation with an Aβ derivative, K6Aβ1−30-NH2, in old non-human primates. Thirty-two aged (4-10 year-old) mouse lemurs were enrolled in the study, and received up to four subcutaneous injections of the vaccine in alum adjuvant or adjuvant alone.

Even though antibody titres to Aβ were not high, pathological examination of the mouse lemur brains showed significant reduction in intracellular Aβ without inflammatory or haemorrhagic changes. Moreover, a trend for cognitive improvement was observed in the vaccinated primates, which was probably linked to Aβ clearance. This Aβ derivative vaccine appeared to be safe as a prophylactic measure based on the brain analyses and because it did not appear to have detrimental effects on the general health of these old animals.
Discovery of Novel Bilaterian Signaling Peptides Using Cone Snail Toxins
Authors: Koch Thomas Lund , Torres Joshua P. , Baskin Robert P. , Salcedo Paula Flórez , Chase Kevin , Olivera Baldomero M. , Safavi-Hemami Helena
Published: August 2022

Peptide hormones and neuropeptides form a diverse class of signaling molecules that control essential processes in animals. Despite several breakthroughs in peptide discovery, many signaling peptides remain undiscovered. Recently, we demonstrated the use of somatostatin-like toxins from cone snail venom to identify homologous signaling peptides in prey. Here, we demonstrate that this toxin-based approach can be systematically applied to the discovery of other unknown bilaterian signaling peptides. Using large sequencing datasets, we searched for homologies between cone snail toxins and putative peptides from several important model organisms representing the snails’ prey. We identified five toxin families that share strong similarities with previously unknown signaling peptides from mollusks and annelids. One of the peptides was also identified in rotifers, brachiopods, platyhelminths, and arthropods, and another was found to be structurally related to crustacean hyperglycemic hormone, a peptide not previously known to exist in Spiralia. Based on several lines of evidence we propose that these signaling peptides not only exist but serve important physiological functions. Finally, we propose that the discovery pipeline developed here can be more broadly applied to other systems in which one organism has evolved molecules to manipulate the physiology of another.
Inteins in the Loop: A Framework for Engineering Advanced Biomolecular Controllers for Robust Perfect Adaptation
Authors: Anastassov Stanislav , Filo Maurice , Chang Ching-Hsiang , Khammash Mustafa
Published: August 2022

Homeostasis is one of the cornerstones of life shaped by billions of years of evolution. A notion that is similar to homeostasis, but yet more stringent, is Robust Perfect Adaptation (RPA). A system is endowed with RPA if it is capable of driving a variable of interest to a prescribed level despite the presence of disturbances and uncertainties in the environment. Designing and building biomolecular controllers capable of achieving RPA have been identified as an important task which has immediate implications for various disciplines. Here, we develop systematic theoretical and experimental frameworks for custom-built proteins that exploit split inteins — short amino acid sequences capable of performing protein-splicing reactions — to design, genetically build and analyze a wide class of RPA-achieving integral feedback controllers. We first lay down a theoretical foundation that facilitates the screening of intein-based controller networks for RPA, and then usher an easy-to-use recipe to simplify their, otherwise complex, underlying mathematical models. Furthermore, we genetically engineer and test various controller circuits based on commonly used transcription factors in mammalian cells. We experimentally and theoretically demonstrate their ability of robustly rejecting external disturbances (that is achieving RPA) over an exquisitely broad dynamic range. Due to their small size, flexibility, modularity, lack of side effects and applicability across various forms of life, inteins serve as promising genetic parts to implement RPA-achieving controllers. To this end, we believe “inteins in the control loop” will leave a significant impact on various disciplines spanning synthetic biology, biofuel production, metabolic engineering and cell therapy among others.
Patterns of numtogenesis in sixteen different mice strains
Authors: Biró Bálint , Gál Zoltán , Brookman Michael , Hoffmann Orsolya Ivett
Published: August 2022

1

Numtogenesis is the phenomenon of mitochondrial sequence localisation and integration into the nuclear genome. This is an ongoing process which contributed to the complexity of eukaryotic genomes. The sequences that are integrated into the nuclear genome are called nuclear mitochondrial sequences (numt). numts have a wide variety of applications in tumor biology, phylogenetic studies, forensic research and so on. Mus musculus musculus is the most popular model organism. Numerous mouse strains are used in medical research to model human diseases. Numts were described in the genome of Mus musculus musculus just like in many other species however the characterisation of numts in different mouse strains is missing. In this study we explored the patterns of numtogenesis in 16 mouse strains by aligning the nuclear genomes with the corresponding mitochondria. Investigation of numts shed light on strain specific differences and resembles the phylogenetic relationships as to our current knowledge in most of the cases.
Time-resolved analysis of Wnt-signaling reveals β-catenin temporal genomic repositioning and cell type-specific plastic or elastic chromatin responses
Authors: Pagella Pierfrancesco , Söderholm Simon , Nordin Anna , Zambanini Gianluca , Jauregi-Miguel Amaia , Cantù Claudio
Published: August 2022

Wnt signaling orchestrates gene expression via its effector β-catenin. Whether β-catenin targets genomic regions simultaneously or in a temporal fashion, and how this impacts the chromatin dynamics to modulate cell behavior, is currently unknown. Here we find that β-catenin binds different loci at each time-point after stimulation, implying that the definition of Wnt-targets is fundamentally temporal. This process is intrinsically cell-type specific. In fact, Wnt/β-catenin progressively shapes the chromatin of human embryonic stem cells consistent with their mesodermal differentiation: we call this genomic response plastic. In embryonic kidney cells, on the other hand, Wnt/β-catenin drives a transient chromatin opening, followed by a re-establishment of the pre-stimulation state: a response that we define elastic. Finally, the Wnt-induced transient chromatin opening requires β-catenin, suggesting a previously unappreciated pioneering role for this molecule. We submit that the plastic-vs-elastic behavior constitutes part of the mechanism explaining how Wnt/β-catenin drives divergent cell-fate decisions during development and homeostasis.
Transcriptomic analyses reveal neuronal specificity of Leigh syndrome associated genes
Authors: Wahedi Azizia , Soondram Chandika , Murphy Alan E. , Skene Nathan , Rahman Shamima
Published: August 2022

Leigh syndrome (subacute necrotising encephalomyelopathy) is a rare inherited, complex, and typically early onset mitochondrial disorder with clinical and genetic heterogeneity. It owes its heterogeneous nature to the complex nature of mitochondrial genetics and the significant interactions that occur between the mitochondrial and nuclear genomes. Stepwise developmental regression is a classical feature of patients with Leigh syndrome, but other neurological features include ataxia, tremor, seizures, and occasionally psychiatric manifestations. To date the involvement of more than 100 genes has been identified in patients with Leigh syndrome. Despite this, the pathophysiology of Leigh syndrome remains unknown, although it is thought to be due to primary mitochondrial dysfunction.

Here, we sought to determine the cellular specificity of all Leigh syndrome associated genes within the brain and investigate a potential common genetic mechanism between Leigh syndrome and other disorders with overlapping clinical features. We utilized co-expression network analyses constructed using existing UK Brain Expression Consortium (UKBEC) data from 10 brain regions to identify regions of the brain where our genes of interest were enriched. Next, expression weighted cell type enrichment (EWCE) was employed to determine the cellular specificity of Leigh syndrome associated genes. Association with pre-or post-synaptic structures was identified using synaptic gene ontology (SynGO) analysis. Finally, heritability analysis was performed on the co-expression network modules demonstrating enrichment of Leigh syndrome associated genes to investigate potential genetic relationships with Parkinson disease, epilepsy, and schizophrenia.

Co-expression network analyses reveal that genes encoding oxidative phosphorylation subunits and assembly factors exhibit the highest levels of expression in brain regions most affected on MRI and neuropathology in Leigh syndrome. Further, over two thirds of the genes were found to be enriched within the substantia nigra and 19 genes appeared to cluster, with selective enrichment in the putamen, substantia nigra, medulla and thalamus. EWCE analyses of single cell RNA-Seq data from mouse brain revealed significant enrichment of Leigh syndrome associated genes in hippocampal and somatosensory pyramidal neurones and interneurons of the brain. SynGO analysis demonstrated that expression of these genes is preferentially associated with pre-synaptic structures. Heritability studies suggested that some modules in which Leigh syndrome associated genes were enriched were also enriched for Parkinson disease and epilepsy.

In conclusion, our findings suggest a primary mitochondrial dysfunction as the underlying basis of Leigh syndrome with causative genes primarily expressed in neuronal cells, explaining the relative sparing of glial cells observed in neuropathological studies.
Gonadal Hormones Impart Male-Biased Behavioral Vulnerabilities to Immune Activation via Microglial Mitochondrial Function
Authors: Bordt Evan A , Moya Haley A , Jo Young Chan , Ravichandran Caitlin T. , Bankowski Izabella M. , Ceasrine Alexis M. , McDougle Christopher J , Carlezon William A. , Bilbo Staci D
Published: August 2022

There is a strong male bias in the prevalence of many neurodevelopmental disorders such as autism spectrum disorder. However, the mechanisms underlying this sex bias remain elusive. Infection during the perinatal period is associated with an increased risk of neurodevelopmental disorder development. Here, we used a mouse model of early-life immune activation that reliably induces deficits in social behaviors only in males. We demonstrate that male-biased alterations in social behavior are dependent upon microglial immune signaling and are coupled to alterations in mitochondrial morphology, gene expression, and function specifically within microglia, the innate immune cells of the brain. Additionally, we show that this behavioral and microglial mitochondrial vulnerability to early-life immune activation is programmed by the male-typical perinatal gonadal hormone surge. These findings demonstrate that social behavior in males over the lifespan are regulated by microglia-specific mechanisms that are shaped by events that occur in early development.
AlphaPulldown – a Python package for protein-protein interaction screens using AlphaFold-Multimer
Authors: Yu Dingquan , Chojnowski Grzegorz , Rosenthal Maria , Kosinski Jan
Published: August 2022

The Artificial Intelligence-based structure prediction program AlphaFold-Multimer enabled structural modelling of protein complexes with unprecedented accuracy. Increasingly, AlphaFold-Multimer is also used to discover new protein-protein interactions. Here, we present AlphaPulldown, a Python package that streamlines protein-protein interaction screens and high-throughput modelling of higher-order oligomers using AlphaFold-Multimer. It provides a convenient command line interface, a variety of confidence scores, and a graphical analysis tool.

Availability and implementation

AlphaPulldown is freely available at https://www.embl-hamburg.de/AlphaPulldown.
Comparison of thigh muscle characteristics between older and young women using tensiomyography
Authors: Chai Jung Hoon , Kim Chul-Hyun , Bae Sang-Won
Published: August 2022

Tensiomyography is a non-invasive method of evaluating neuromuscular function through skeletal muscle contraction. The objective of this study was to compare the thigh muscle characteristics of older and young women using tensiomyography. Nineteen older and fifteen young women without musculoskeletal diseases were included. For the quadriceps, the bilateral vastus medialis (VM), vastus lateralis (VL), and rectus femoris (RF) and for the hamstrings, the bilateral semitendinosus (ST) and biceps femoris (BF) were measured. Result variables—maximal displacement (Dm), contraction time (Tc), and contraction velocity (Vc)—were compared. Dm values of the hamstrings of both legs and their summed values were significantly smaller in older women than in young women; no difference was found in the Dm values of the quadriceps. Tc and Vc of the hamstrings, VM, and VL were longer and slower, respectively, in older women than in young women. There were no significant differences in the Dm, Tc, or Vc of the RF between older and young women. Decreased Dm of the hamstrings in older women occurred due to changes in muscle function, but not muscle mass. The changes in the Tc of the hamstrings, VM, and VL indicate that type II muscle fibers were converted to type I in older women. There was no difference in RF between older and young women, implying that the RF is not affected by age. Our findings indicate that resistance exercises, which preserve the type II fibers, and flexibility exercises, which reduce stiffness, are appropriate for the lower extremity in older women.
Combined online Bayesian and windowed estimation of background and signal localization facilitates active-feedback particle tracking in complex environments
Authors: Niver Anastasia , Welsher Kevin D.
Published: August 2022

Despite successes in tracking single molecules in vitro, the extension of active-feedback single-particle methods to tracking rapidly diffusing and unconfined proteins in live cells has not been realized. Since existing active-feedback localization methods localize particles in real time assuming zero background, they are ill-suited to track in the inhomogeneous background environment of a live cell. Here, we develop a windowed estimation of signal and background levels which uses recent data to estimate the current particle brightness and background intensity. These estimates facilitate recursive Bayesian position estimation, improving upon current Kalman-based localization methods. Combined, online Bayesian and windowed estimation of background and signal (COBWEBS) surpasses existing localization methods. Simulations demonstrate improved localization accuracy and responsivity in a homogenous background for selected particle and background intensity combinations. Improved or similar performance of COBWEBS tracking extends to the majority of signal and background combinations explored. Furthermore, improved tracking durations are demonstrated in the presence of heterogeneous backgrounds for multiple particle intensities, diffusive speeds, and background patterns. COBWEBS can accurately track particles in the presence of high and non-uniform backgrounds including intensity changes of up to three-fold that of the particle’s intensity, making it a prime candidate for advancing active-feedback single-fluorophore tracking to the cellular interior.
Evaluation of an image-derived input function for kinetic modeling of nicotinic acetylcholine receptor-binding PET ligands
Authors: Zammit Matthew , Kao Chien-Min , Zhang Hannah J. , Holderman Nathanial , Mitchell Samuel , Tanios Eve , Zhang Vincent , Bhuiyan Mohammed , Freifelder Richard , Green William N. , Mukherjee Jogeshwar , Chen Chin-Tu
Published: August 2022

Development of positron emission tomography (PET) radiotracers that bind with high-affinity to α4β2-type nicotinic receptors (α4β2Rs) allows for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. One challenge associated with preclinical PET imaging involves the lack of true tissue reference regions free of specific tracer binding in the rodent brain, impeding accurate quantification of the tracer binding potential. Here, we investigate the use of an image-derived arterial input function for kinetic analysis of radiotracer binding in male and female mice. Two radiotracers were explored in this study: 2-[18F]FA85380 (2-FA), which displays similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, which displays similar pKa and binding affinity to nicotine. For both radiotracers, time-activity curves of the left ventricle of the heart displayed similar standardized uptake values (SUVs) across wild type mice, mice lacking the β2 subunit for tracer binding, and acute nicotine-treated mice, whereas typical reference tissue SUVs displayed high variation between groups. Binding potential values estimated from a two-tissue compartment model (2TCM) fit of the data with the image-derived input function were significantly higher than estimates from reference tissue-based estimations. Rate constants of radiotracer dissociation were very slow for 2-FA and very fast for Nifene, similar to the in vitro dissociation rates reported for varenicline and nicotine, respectively. We conclude that use of an image-derived input function for kinetic modeling of nicotinic PET ligands improves quantification compared to reference tissue-based methods, and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.
Endothelial TIE1 restricts angiogenic sprouting to coordinate vein assembly in synergy with its homologue TIE2
Authors: Cao Xudong , Li Taotao , Xu Beibei , Ding Kai , Li Weimin , Shen Bin , Chu Man , Zhu Dengwen , Rui Li , Shang Zhi , Li Xiao , Wang Yinyin , Zheng Shuyu , Alitalo Kari , Liu Ganqiang , Tang Jing , Kubota Yoshiaki , He Yulong
Published: August 2022

Objective

Vascular growth followed by vessel specification is crucial for the establishment of a hierarchical blood vascular network. We have here investigated mechanisms underlying venogenesis, particularly the molecular control over venous fate acquisition during vascular development.

Approach and Results

We analyzed the function of TIE1 as well as its synergy with TIE2 in the regulation of vein formation by employing genetic mouse models targeting Tie1 and Tek. Cardinal vein growth appeared normal in TIE1 deficient mice, whereas TIE2 deficiency altered the identity of cardinal vein endothelial cells with the aberrant expression of DLL4. Interestingly, the parallel growth of murine cutaneous veins along with arteries, which was initiated at approximately embryonic day 13.5, was retarded in mice lack of TIE1. Tie1 deletion disrupted also venous integrity, displaying increased sprouting angiogenesis and vascular bleeding. Abnormal venous sprouts with defective arteriovenous alignment were also observed in the mesenteries of Tie1 deleted mice. Mechanistically, TIE1 deficiency resulted in the decreased expression of venous regulators including TIE2 while angiogenic regulators were upregulated. The alteration of TIE2 level by TIE1 insufficiency was further confirmed by the siRNA-mediated knockdown of Tie1 in cultured endothelial cells. Additionally, combining the endothelial deletion of Tie1 with one null allele of Tek resulted in a progressive increase of vein-associated angiogenesis leading to the formation of vascular tufts in retinas, whereas the loss of Tie1 alone produced only a relatively mild venous defect.

Conclusions

Findings from this study imply that TIE1 and TIE2 act in a synergistic manner to restrict sprouting angiogenesis during vein formation.
State-dependent evolutionary models reveal modes of solid tumor growth
Authors: Lewinsohn Maya A. , Bedford Trevor , Müller Nicola F. , Feder Alison F.
Published: August 2022

Spatial properties of tumor growth have profound implications for cancer progression, therapeutic resistance and metastasis. Yet, how spatial position governs tumor cell division remains difficult to evaluate in clinical tumors. Here, we demonstrate that elevated cellular growth rates on the tumor periphery leave characteristic patterns in the genomes of cells sampled from different parts of a tumor, which become evident when they are used to construct a tumor phylogenetic tree. Namely, rapidly-dividing peripheral lineages branch more extensively and acquire more mutations than slower-dividing lineages in the tumor center. We develop a Bayesian state-dependent evolutionary phylodynamic model (SDevo) that quantifies these patterns to infer the differential cell division rates between peripheral and central cells jointly from the branching and mutational patterns of single-time point, multi-region sequencing data. We validate this approach on simulated tumors by demonstrating its ability to accurately infer spatially-varying birth rates under a range of growth conditions and sampling strategies. We then show that SDevo outperforms state-of-the-art, non-cancer multi-state phylodynamic methods which ignore differential mutational acquisition. Finally, we apply SDevo to multi-region sequencing data from clinical hepatocellular carcinomas and find evidence that cells on the tumor edge divide 3-6x faster than those in the center. As multi-region and single-cell sequencing increase in resolution and availability, we anticipate that SDevo will be useful in interrogating spatial restrictions on tumor growth and could be extended to model non-spatial factors that influence tumor progression, including hypoxia and immune infiltration.
Characterizing Transient Protein-Protein Interactions by Trp-Cys Quenching and Computer Simulations
Authors: Heo Lim , Gamage Katukurunde , Valdes-Garcia Gilberto , Lapidus Lisa J. , Feig Michael
Published: August 2022

Transient protein-protein interactions occur frequently under the crowded conditions encountered in biological environments, yet they remain poorly understood. Here, tryptophan-cysteine quenching is introduced as an experimental approach that is ideally suited to characterize such interactions between proteins with minimal labeling due to its sensitivity to nano- to microsecond dynamics on sub-nanometer length scales. The experiments are paired with computational modeling at different resolutions including fully atomistic molecular dynamics simulations to provide interpretation of the experimental observables and add further insights at the molecular level. This approach is applied to model systems, villin variants and the drkN SH3 domain, in the presence of protein G crowders. It is demonstrated that Trp-Cys quenching experiments are able to not only distinguish between overall attractive and repulsive interactions between different proteins, but they can also discern variations in interaction preferences at different protein surface locations. The close integration between experiment and simulations also provides an opportunity to evaluate different molecular force fields for the simulation of concentrated protein solutions.

Significance Statement

Biological environments typically involve a variety of different proteins at very high concentrations where non-specific interactions are unavoidable. These interactions may go beyond simple crowding effects and involve transient contacts that may impact structure, dynamics, and ultimately function of proteins in vivo. While computer simulations have partially characterized such interactions, experimental data remain limited because established techniques are generally not well-suited to the characterization of dynamic processes on microsecond time and nanometer length scales. Tryptophan quenching by cysteine is introduced here as a new approach for studying transient protein encounters under concentrated conditions with the support of computational modeling. The study demonstrates that such experiments can resolve not just differences between different proteins but also residue-specific interaction preferences.
MetaTiME: Meta-components of the Tumor Immune Microenvironment
Authors: Zhang Yi , Xiang Guanjue , Jiang Alva Yijia , Lynch Allen , Zeng Zexian , Wang Chenfei , Zhang Wubing , Fan Jingyu , Kang Jiajinlong , Gu Shengqing Stan , Wan Changxin , Zhang Boning , Liu X. Shirley , Brown Myles , Meyer Clifford A
Published: August 2022

Recent advances in single-cell RNA sequencing have revealed heterogeneous cell types and gene expression states in the non-cancerous cells in tumors. The integration of multiple scRNA-seq datasets across tumors can reveal common cell types and states in the tumor microenvironment (TME). We developed a data driven framework, MetaTiME, to overcome the limitations in resolution and consistency that result from manual labelling using known gene markers. Using millions of TME single cells, MetaTiME learns meta-components that encode independent components of gene expression observed across cancer types. The meta-components are biologically interpretable as cell types, cell states, and signaling activities. By projecting onto the MetaTiME space, we provide a tool to annotate cell states and signature continuums for TME scRNA-seq data. Leveraging epigenetics data, MetaTiME reveals critical transcriptional regulators for the cell states. Overall, MetaTiME learns data-driven meta-components that depict cellular states and gene regulators for tumor immunity and cancer immunotherapy.