bluuebunny/crossref_metadata_2025 huggingface dataset:
HDAC1 and PRC2 mediate combinatorial control in SPI1/PU.1-dependent gene repression in murine erythroleukaemia
Authors: Gregoricchio Sebastian , Polit Lélia , Esposito Michela , Berthelet Jérémy , Delestré Laure , Evanno Emilie , Diop M’Boyba , Gallais Isabelle , Aleth Hanna , Poplineau Mathilde , Zwart Wilbert , Rosenbauer Frank , Rodrigues-Lima Fernando , Duprez Estelle , Boeva Valentina , Guillouf Christel
Published: July 2022

Although originally described as transcriptional activator, SPI1/PU.1, a major player in haematopoiesis whose alterations are associated with haematological malignancies, has the ability to repress transcription. Here, we investigated the mechanisms underlying gene repression in the erythroid lineage, in which SPI1 exerts an oncogenic function by blocking differentiation. We show that SPI1 represses genes by binding active enhancers that are located in intergenic or gene body regions. HDAC1 acts as a cooperative mediator of SPI1-induced transcriptional repression by deacetylating SPI1-bound enhancers in a subset of genes, including those involved in erythroid differentiation. Enhancer deacetylation impacts on promoter acetylation, chromatin accessibility and RNA pol II occupancy. In addition to the activities of HDAC1, polycomb repressive complex 2 (PRC2) reinforces gene repression by depositing H3K27me3 at promoter sequences when SPI1 is located at enhancer sequences. Moreover, our study identified a synergistic relationship between PRC2 and HDAC1 complexes in mediating the transcriptional repression activity of SPI1, ultimately inducing synergistic adverse effects on leukaemic cell survival. Our results highlight the importance of the mechanism underlying transcriptional repression in leukemic cells, involving complex functional connections between SPI1 and the epigenetic regulators PRC2 and HDAC1.
Efficient quantitative monitoring of translational initiation by RelE cleavage
Authors: Focht Caroline M , Strobel Scott A
Published: July 2022

The sequences of the 5′ untranslated regions (5′-UTRs) of mRNA alter gene expression across domains of life. Transcriptional modulators can be easily assayed through transcription termination, but translational regulators often require indirect, laborious methods. We have leveraged RelE’s ribosome-dependent endonuclease activity to develop a quantitative assay to monitor translation initiation of cis-regulatory mRNAs. RelE cleavage accurately reports ligand-dependent changes in ribosome association for two translational riboswitches and provides quantitative information about each switch's sensitivity and range of response. RelE accurately reads out sequence-driven changes in riboswitch specificity and function and is quantitatively dependent upon ligand concentration. RelE cleavage similarly captures differences in translation initiation between yeast 5′-UTR isoforms. RelE cleavage can thus reveal a plethora of information about translation initiation in different domains of life.
Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1
Authors: Lussier-Price Mathieu , Wahba Haytham M , Mascle Xavier H , Cappadocia Laurent , Bourdeau Veronique , Gagnon Christina , Igelmann Sebastian , Sakaguchi Kazuyasu , Ferbeyre Gerardo , Omichinski James G
Published: July 2022

SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO–SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.
R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline
Authors: Hicks Tara , Koury Emily , McCabe Caleb , Williams Cameron , Crahan Caroline , Smolikove Sarit
Published: July 2022

Accumulation of DNA–RNA hybrids in the form of R-loops can result in replication–transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis.
Decoding YAP dependent transcription in the liver
Authors: Biagioni Francesca , Croci Ottavio , Sberna Silvia , Donato Elisa , Sabò Arianna , Bisso Andrea , Curti Laura , Chiesa Arianna , Campaner Stefano
Published: July 2022

The transcriptional coactivator YAP is emerging as a master regulator of cell growth. In the liver, YAP activity is linked to hepatomegaly, regeneration, dedifferentiation, and aggressive tumor growth. Here we present genomic studies to address how YAP may elicit such profound biological changes in murine models. YAP bound the genome in a TEAD-dependent manner, either at loci constitutively occupied by TEAD or by pioneering enhancers, which comprised a fraction of HNF4a/FOXA-bound embryonic enhancers active during embryonic development but silent in the adult. YAP triggered transcription on promoters by recruiting BRD4, enhancing H3K122 acetylation, and promoting RNApol2 loading and pause-release. YAP also repressed HNF4a target genes by binding to their promoters and enhancers, thus preventing RNApol2 pause-release. YAP activation led to the induction of hepatocyte proliferation, accompanied by tissue remodeling, characterized by polarized macrophages, exhausted T-lymphocytes and dedifferentiation of endothelial cells into proliferative progenitors. Overall, these analyses suggest that YAP is a master regulator of liver function that reshapes the enhancer landscape to control transcription of genes involved in metabolism, proliferation, and inflammation, subverts lineage specification programs by antagonizing HNF4a and modulating the immune infiltrate and the vascular architecture of the liver.
Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility
Authors: Qiao Cuncun , Debiasi-Anders Gianluca , Mir-Sanchis Ignacio
Published: July 2022

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1–DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD′s presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep′s activities as initiators and as helicases.
Structural basis of Streptomyces transcription activation by zinc uptake regulator
Authors: Yang Xu , Wang Yiqun , Liu Guiyang , Deng Zixin , Lin Shuangjun , Zheng Jianting
Published: July 2022

Streptomyces coelicolor (Sc) is a model organism of actinobacteria to study morphological differentiation and production of bioactive metabolites. Sc zinc uptake regulator (Zur) affects both processes by controlling zinc homeostasis. It activates transcription by binding to palindromic Zur-box sequences upstream of −35 elements. Here we deciphered the molecular mechanism by which ScZur interacts with promoter DNA and Sc RNA polymerase (RNAP) by cryo-EM structures and biochemical assays. The ScZur-DNA structures reveal a sequential and cooperative binding of three ScZur dimers surrounding a Zur-box spaced 8 nt upstream from a −35 element. The ScRNAPσHrdB-Zur-DNA structures define protein-protein and protein-DNA interactions involved in the principal housekeeping σHrdB-dependent transcription initiation from a noncanonical promoter with a −10 element lacking the critical adenine residue at position −11 and a TTGCCC −35 element deviating from the canonical TTGACA motif. ScZur interacts with the C-terminal domain of ScRNAP α subunit (αCTD) in a complex structure trapped in an active conformation. Key ScZur-αCTD interfacial residues accounting for ScZur-dependent transcription activation were confirmed by mutational studies. Together, our structural and biochemical results provide a comprehensive model for transcription activation of Zur family regulators.
The L1-ORF1p coiled coil enables formation of a tightly compacted nucleic acid-bound complex that is associated with retrotransposition
Authors: Cashen Ben A , Naufer M Nabuan , Morse Michael , Jones Charles E , Williams Mark C , Furano Anthony V
Published: July 2022

Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.
Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism
Authors: Satange Roshan , Kao Shih-Hao , Chien Ching-Ming , Chou Shan-Ho , Lin Chi-Chien , Neidle Stephen , Hou Ming-Hon
Published: July 2022

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson–Crick base pair at the two drug-binding interfaces can result in a single-stranded ‘chair-shaped’ DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a ‘curved-shaped’ duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs’ potential synergetic use.
mRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation
Authors: Çetin Burak , O’Leary Seán E
Published: July 2022

mRNA 5′ cap recognition by eIF4F is a key element of eukaryotic translational control. Kinetic differences in eIF4F–mRNA interactions have long been proposed to mediate translation-efficiency differences between mRNAs, and recent transcriptome-wide studies have revealed significant heterogeneity in eIF4F engagement with differentially-translated mRNAs. However, detailed kinetic information exists only for eIF4F interactions with short model RNAs. We developed and applied single-molecule fluorescence approaches to directly observe real-time Saccharomyces cerevisiae eIF4F subunit interactions with full-length polyadenylated mRNAs. We found that eIF4E–mRNA association rates linearly anticorrelate with mRNA length. eIF4G–mRNA interaction accelerates eIF4E–mRNA association in proportion to mRNA length, as does an eIF4F-independent activity of eIF4A, though cap-proximal secondary structure still plays an important role in defining the final association rates. eIF4F–mRNA interactions remained dominated by effects of eIF4G, but were modulated to different extents for different mRNAs by the presence of eIF4A and ATP. We also found that eIF4A-catalyzed ATP hydrolysis ejects eIF4E, and likely eIF4E•eIF4G from the mRNA after initial eIF4F•mRNA complex formation, suggesting a mechanism to prepare the mRNA 5′ end for ribosome recruitment. Our results support a role for mRNA-specific, factor-driven eIF4F association rates in kinetically controlling translation.
Human RNase 4 improves mRNA sequence characterization by LC–MS/MS
Authors: Wolf Eric J , Grünberg Sebastian , Dai Nan , Chen Tien-Hao , Roy Bijoyita , Yigit Erbay , Corrêa Ivan R
Published: July 2022

With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography–mass spectrometry (LC–MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine–two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5′ cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC–MS/MS.
Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly
Authors: Saha Kaushik , Ghosh Gourisankar
Published: July 2022

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.
SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells
Authors: Fischer Veronique , Hisler Vincent , Scheer Elisabeth , Lata Elisabeth , Morlet Bastien , Plassard Damien , Helmlinger Dominique , Devys Didier , Tora László , Vincent Stéphane D
Published: July 2022

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone-fold domain (HFD)-containing proteins forming three histone-fold (HF) pairs, to which the double HFD-containing SUPT3H adds one HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that (i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H, (ii) SUPT3H is not essential for mESC survival, but required for their growth and self-renewal, and (iii) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.
CMDB: the comprehensive population genome variation database of China
Authors: Li Zhichao , Jiang Xiaosen , Fang Mingyan , Bai Yong , Liu Siyang , Huang Shujia , Jin Xin
Published: July 2022

A high-quality genome variation database derived from a large-scale population is one of the most important infrastructures for genomics, clinical and translational medicine research. Here, we developed the Chinese Millionome Database (CMDB), a database that contains 9.04 million single nucleotide variants (SNV) with allele frequency information derived from low-coverage (0.06×–0.1×) whole-genome sequencing (WGS) data of 141 431 unrelated healthy Chinese individuals. These individuals were recruited from 31 out of the 34 administrative divisions in China, covering Han and 36 other ethnic minorities. CMDB, housing the WGS data of a multi-ethnic Chinese population featuring wide geographical distribution, has become the most representative and comprehensive Chinese population genome database to date. Researchers can quickly search for variant, gene or genomic regions to obtain the variant information, including mutation basic information, allele frequency, genic annotation and overview of frequencies in global populations. Furthermore, the CMDB also provides information on the association of the variants with a range of phenotypes, including height, BMI, maternal age and twin pregnancy. Based on these data, researchers can conduct meta-analysis of related phenotypes. CMDB is freely available at https://db.cngb.org/cmdb/.
The Relationship Between Professionalism and Leadership: Parent–Child or Sibling?
Authors: Clark Diane , Wainwright Susan , Tschoepe Barbara A , Green-Wilson Jennifer , Sebelski Chris , Zeigler Stacey , McGinnis Patricia
Published: July 2022

Professionalism has been the foundation of physical therapy’s contract with society, with the American Physical Therapy Association’s (APTA) Core Values and Code of Ethics serving as its building blocks. Professional formation has focused on professionalism and has been taught in a manner that is more implicit than explicit in doctor of physical therapy (DPT) curricula. As a domain of competence, professionalism alone has not been broad enough to meet societal needs. In reaching our centennial year, many have reflected on what competencies are needed to move forward. The need for leadership competencies is not new and has been espoused by our leaders over the past 100 years. Some advocate for the adoption of leadership as a unique domain of competence, separate from the domain of professionalism, whereas others propose that either professionalism or leadership is one domain of competence that subsumes the other. The purpose of this Perspective is twofold: to compare and contrast the concepts of professionalism and leadership, and to make recommendations regarding what constitutes domains of competence within the professional formation of physical therapists. This Perspective offers recommendations addressing professional formation and the adoption of leadership and professionalism as 2 distinct domains of competence and discusses educational and clinical implications of the recommendations. This Perspective asserts that these recommendations must be adopted to move the profession forward into the next century so that physical therapists are recognized as adding value to the health care system and the evolving needs of society.
Disparities Exist in Physical Therapy Utilization and Time to Utilization Between Black and White Patients With Musculoskeletal Pain
Authors: Richter Randy R , Chrusciel Timothy , Salsich Gretchen , Austin Tricia , Scherrer Jeffrey F
Published: July 2022

Objective

Black patients are less likely than White patients to receive physical therapy for musculoskeletal pain conditions. Current evidence, however, is limited to self-reported conditions and health services use. The purpose of this study was to use a large electronic health record database to determine whether a race disparity existed in use of physical therapy within 90 days of a new musculoskeletal diagnosis.

Methods

Eligible patients (n = 52,384) were sampled from an Optum deidentified electronic health record database of 5 million adults distributed throughout the United States. In this database, patients were designated as “Black” and “White.” Patients were eligible if they had a new diagnosis for musculoskeletal neck, shoulder, back, or knee pain between January 1, 2012, and December 31, 2017. Logistic regression and Cox proportional hazard models were computed before and after adjusting for covariates to estimate the association between race and receipt of physical therapy services within 90 days of musculoskeletal pain diagnoses.

Results

Patients were on average 47.5 (SD = 14.9) years of age, 12.8% were Black, 87.2% were White, and 52.7% were female. Ten percent of Black patients and 15.5% of White patients received physical therapy services within 90 days of musculoskeletal pain diagnoses. After adjusting for covariates, White patients were 57% more likely (odds ratio = 1.57; 95% CI = 1.44–1.71) to receive physical therapy compared with Black patients and had significantly shorter time to physical therapy than Black patients (hazard ratio = 1.53; 95% CI = 1.42–1.66).

Conclusions

In a nationally distributed cohort, Black patients were less likely than White patients to utilize physical therapy and had a longer time to utilization of physical therapy for musculoskeletal pain.

Impact

These findings highlight the need to determine the mechanisms underlying the observed disparities and how these disparities influence health outcomes.
Sex-dimorphic and age-dependent organization of 24 hour gene expression rhythms in human
Authors: Talamanca Lorenzo , Gobet Cédric , Naef Felix
Published: July 2022

The circadian clock modulates most of human physiology. However, the organization of tissue-specific gene expression rhythms is still poorly known, as well as age and sex dependencies. We leveraged the Genotype-Tissue Expression project (GTEx) using a novel algorithm to assign a unique circadian phase to 914 individuals and transfer time information from stronger to weaker clocks. These donor internal phases allowed us to identify and compare programs of rhythmic gene expression in 46 tissues. Clock transcripts showed highly conserved phase and amplitude relationships across tissues, and were tightly synchronized across the body. Tissue rhythmic gene expression programs differed in breadth, covering global and tissue-specific functions, including metabolic pathways and systemic responses such as heat shock. The circadian clock structure and amplitude was indistinguishable across sexes and age groups. However, overall gene expression rhythms were highly sex-dimorphic and more sustained in females. Moreover, rhythmic programs dampened with age across the body. Together, our stratified analysis unveiled a rich organization of sex- and age-specific circadian gene expression rhythms in humans.

One sentence summary

Circadian phase inference of 914 GTEx donors reveals sex- and age-dependent rhythmic gene expression programs across 46 human tissues.
Transferability of cathodal tDCS effects from the primary motor to the dorsolateral prefrontal cortex: a multimodal TMS-EEG study
Authors: Mosayebi-Samani Mohsen , Agboada Desmond , Mutanen Tuomas P. , Haueisen Jens , Kuo Min-Fang , Nitsche Michael A.
Published: July 2022

Neurophysiological effects of transcranial direct current stimulation (tDCS) have been extensively studied over the primary motor cortex (M1). Much less is however known about its effects over non-motor areas, such as the prefrontal cortex (PFC), which is the neuronal foundation for many high-level cognitive functions and involved in neuropsychiatric disorders. In this study, we, therefore, explored the transferability of cathodal tDCS effects over M1 to the PFC. Eighteen healthy human participants (11 males and 8 females) were involved in eight randomized sessions, in which four cathodal tDCS dosages, low, medium, and high, as well as sham stimulation, were applied over the M1 and PFC. After-effects of tDCS were evaluated via transcranial magnetic stimulation (TMS)-electroencephalography (EEG), and TMS-elicited motor evoked potentials (MEP), for the outcome parameters TMS-evoked potentials (TEP), TMS-evoked oscillations, and MEP amplitude alterations. TEPs were studied both at the regional and global scalp levels. The results indicate a regional dosage-dependent nonlinear neurophysiological effect of M1 tDCS, which is not one-to-one transferable to PFC tDCS. Low and high dosages of M1 tDCS reduced early positive TEP peaks (P30, P60), and MEP amplitudes, while an enhancement was observed for medium dosage M1 tDCS (P30 and MEP amplitudes). In contrast, prefrontal low, medium and high dosage tDCS uniformly reduced the early positive TEP peak amplitudes. Furthermore, for both cortical areas, regional tDCS-induced modulatory effects were not observed for late TEP peaks, nor TMS-evoked oscillations. However, at the global scalp level, widespread effects of tDCS were observed for both, TMS-evoked potentials and oscillations. This study provides the first direct physiological comparison of tDCS effects applied over different brain areas and therefore delivers crucial information for future tDCS applications.

SIGNIFICANCE STATEMENT

Modulatory effects of tDCS over the M1 were largely taken as a template so far for the use of this intervention over non-motor regions. However, the neurophysiological effects of tDCS over non-motor regions, such as the prefrontal cortex (PFC), have been much less explored. In the current study, we, using concurrent transcranial magnetic stimulation- electroencephalography, systematically explored the transferability of cathodal tDCS effects on cortical excitability from M1 to the PFC. The results indicate a dosage-dependent nonlinear neurophysiological effect of motor cortex tDCS, which is not one-to-one transferable to prefrontal tDCS. This study provides the first direct physiological comparison of tDCS effects applied over different brain areas, which will further consolidate the rationale for the extension of tDCS applications at both, basic and clinical levels.
Dose-dependent disruption of hepatic zonation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice: integration of single-nuclei RNA sequencing and spatial transcriptomics
Authors: Nault R. , Saha S. , Bhattacharya S. , Sinha S. , Maiti T. , Zacharewski Tim
Published: July 2022

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently induces the development of hepatic fat accumulation and inflammation with fibrosis in mice initially in the portal region. Conversely, differential gene and protein expression is first detected in the central region. To further investigate cell-specific and spatially resolved dose-dependent changes in gene expression elicited by TCDD, single-nuclei RNA sequencing and spatial transcriptomics were used for livers of male mice gavaged with TCDD every 4 days for 28 days. The proportion of 11 cell (sub)types across 131,613 nuclei dose-dependently changed with 68% of all portal and central hepatocyte nuclei in control mice being overtaken by macrophages following TCDD treatment. We identified 368 (portal fibroblasts) to 1,339 (macrophages) differentially expressed genes. Spatial analyses revealed initial loss of portal identity that eventually spanned the entire liver lobule with increasing dose. Induction of R-spondin 3 (Rspo3) and pericentralApc, suggested dysregulation of the Wnt/β-catenin signaling cascade in zonally resolved steatosis. Collectively, the integrated results suggest disruption of zonation contributes to the pattern of TCDD-elicited NAFLD pathologies.

SYNOPSIS

Single-nuclei RNA sequencing (snRNAseq) and spatial transcriptomics were integrated to investigate cell-specific and spatially resolved dose-dependent changes elicited by TCDD. We show that TCDD causes a loss of zonal characteristics that disrupts spatially defined metabolic functions.

- Dose-dependent analyses show higher responsiveness of central hepatocytes despite hepatotoxicity occurring initially in the portal region.

- Integration of snRNAseq and spatial transcriptomics demonstrates a loss of hepatocytes with portal characteristics.

- TCDD disrupted spatially resolved expression of β-catenin signaling members that are critical in maintaining liver zonation.

- Spatial transcriptomics and snRNAseq shows induction of R-spondin3 from nonparenchymal cells which serve as cue for the β-catenin pathway.
Fancm regulates meiotic double-strand break repair pathway choice in mammals
Authors: Tsui Vanessa , Lyu Ruqian , Novakovic Stevan , Stringer Jessica M. , Dunleavy Jessica E. M. , Granger Elissah , Semple Tim , Leichter Anna , Martelotto Luciano G. , Merriner D. Jo , Liu Ruijie , McNeill Lucy , Zerafa Nadeen , Hoffmann Eva , O’Bryan Moira K. , Hutt Karla , Deans Andrew J. , Heierhorst Jörg , McCarthy Davis J. , Crismani Wayne
Published: July 2022

Meiotic crossovers are required for accurate chromosome segregation and to produce new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we describe the tumour suppressor FANCM as a meiotic anti-crossover factor in mammals. Crossover analyses with single-gamete and pedigree datasets both reveal a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss of function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway. Despite the gametogenesis phenotypes in Fancm mutants both sexes were capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.
Mcm2 promotes stem cell differentiation via its ability to bind H3-H4
Authors: Xu Xiaowei , Hua Xu , Brown Kyle , Ren Xiaojun , Zhang Zhiguo
Published: July 2022

Mcm2, a subunit of the Mcm2-7 helicase best known for its role in DNA replication, contains a histone binding motif that facilitates the transfer of parental histones following DNA replication. Here we show that Mcm2 is important for the differentiation of mouse embryonic stem (ES) cells. The Mcm2-2A mutation defective in histone binding impairs differentiation and disrupts the programmatic changes in gene expression and histone modifications during differentiation. Mcm2 localizes at transcription starting sites and the binding of Mcm2 at gene promoters is disrupted in both Mcm2-2A ES cells and neuro-precursor cells (NPCs). Reduced Mcm2 binding at bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications in Mcm2-2A ES cells correlates with decreased chromatin accessibility at corresponding sites in NPCs. Together, our studies reveal a novel function of Mcm2 in ES cell differentiation, likely through manipulating chromatin landscapes at bivalent chromatin domains.
Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device
Authors: Brown Scott D. , Dreolini Lisa , Wilson Jessica F. , Balasundaram Miruna , Holt Robert A.
Published: July 2022

Background

Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.

Results

Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 % to 0.53 %, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100 % accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.

Conclusions

Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.
Diverse Monogenic Subforms of Human Spermatogenic Failure
Authors: Nagirnaja Liina , Lopes Alexandra M. , Charng Wu-Lin , Miller Brian , Stakaitis Rytis , Golubickaite Ieva , Stendahl Alexandra , Luan Tianpengcheng , Friedrich Corinna , Mahyari Eisa , Fadial Eloise , Kasak Laura , Vigh-Conrad Katinka , Oud Manon S. , Xavier Miguel J. , Cheers Samuel R. , James Emma R. , Guo Jingtao , Jenkins Timothy G , Riera-Escamilla Antoni , Barros Alberto , Carvalho Filipa , Fernandes Susana , Gonçalves João , Gurnett Christina A. , Jørgensen Niels , Jezek Davor , Jungheim Emily S , Kliesch Sabine , McLachlan Robert I. , Omurtag Kenan R , Pilatz Adrian , Sandlow Jay , Smith James , Eisenberg Michael L. , Hotaling James M , Jarvi Keith A. , Punab Margus , Rajpert-De Meyts Ewa , Carrell Douglas T. , Krausz Csilla , Laan Maris , O’Bryan Moira K. , Schlegel Peter N. , Tüttelmann Frank , Veltman Joris A. , Almstrup Kristian , Aston Kenneth I. , Conrad Donald F.
Published: July 2022

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable with current medicine. Due to the biological complexity of sperm production, defining the genetic basis of NOA has proven challenging, and to date, the most advanced classification of NOA subforms is based on simple description of testis histology. In this study, we exome-sequenced over 1,000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. Population-based testing against fertile controls identified 27 genes as significantly associated with azoospermia. The disrupted genes are primarily on the autosomes, enriched for undescribed human “knockouts”, and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing of adult testes shows that, rather than affecting a single cell type or pathway, azoospermia genes can be grouped into molecular subforms with highly synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed specifically in mitotic divisions of type B spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may serve as a basis for disease classification more advanced than histology.
A Randomised -Controlled Phase 2 trial of Molnupiravir in Unvaccinated and Vaccinated Individuals with Early SARS-CoV-2
Authors: Khoo Saye H , FitzGerald Richard , Saunders Geoffrey , Middleton Calley , Ahmad Shazaad , Edwards Christopher J , Hadjiyiannakis Dennis , Walker Lauren , Lyon Rebecca , Shaw Victoria , Mozgunov Pavel , Periselneris Jimstan , Woods Christie , Bullock Katie , Hale Colin , Reynolds Helen , Downs Nichola , Ewings Sean , Buadi Amanda , Cameron David , Edwards Thomas , Knox Emma , Donovan-Banfield I’ah , Greenhalf William , Chiong Justin , Lavelle-Langham Lara , Jacobs Michael , Painter Wendy , Holman Wayne , Lalloo David G , Tetlow Michelle , Hiscox Julian A , Jaki Thomas , Fletcher Thomas , Griffiths Gareth
Published: July 2022

Background

Molnupiravir was licensed for treating high-risk patients with COVID-19 based on data from unvaccinated adults. AGILE CST-2 (NCT04746183) Phase II reports safety and virological efficacy of molnupiravir in vaccinated and unvaccinated individuals.

Methods

Adult out-patients with PCR-confirmed SARS-CoV-2 infection within five days of symptom onset were randomly assigned 1:1 to receive molnupiravir (800mg twice daily for five days) or placebo. The primary outcome was time to swab PCR-negativity, compared using a Bayesian model for estimating the probability of a superior virological response (Hazard Ratio>1) for molnupiravir over placebo. Secondary outcomes included change in viral titre at day 5, safety and tolerability, clinical progression and patient reported outcome measures. We analysed outcomes after the last participant reached day 29.

Findings

Of 180 participants randomised (90 molnupiravir, 90 placebo), 50% were vaccinated. Infections with SARS-CoV-2 variants Delta (40%), Alpha (21%), Omicron (21%) and EU1 (16%) were represented. The median time to negative-PCR was 8 versus 11 days for molnupiravir and placebo (HR=1·30, 95% CrI 0·92-1·71, p=0·07 by Logrank and p=0·03 by Breslow-Gehan tests). Although small numbers precluded subgroup analysis, no obvious differences were observed between vaccinated and unvaccinated participants. Using a two-point prior the probability of molnupiravir being superior to placebo (HR>1) was 75·4%, which was just below our defined threshold of 80% for establishing superiority. Using an uninformative continuous prior, the probability of HR>1 was 94·7%. As an exploratory analysis, the change in viral titre on day 5 (end of treatment) was significantly greater with molnupiravir compared with placebo. A total of 4 participants reported severe adverse events (grade 3+), 3 of whom were in the placebo arm.

Interpretation

We found molnupiravir to be well-tolerated, with evidence for high probability of antiviral efficacy in a population of vaccinated and unvaccinated individuals infected with a broad range of viral variants.

Funding

Funded by Ridgeback Biotherapeutics and UK National Institute for Health and Care Research infrastructure funding. The AGILE platform infrastructure is supported by the Medical Research Council (grant number MR/V028391/1) and the Wellcome Trust (grant number 221590/Z/20/Z).
Structural basis of transcriptional activation by the OmpR/PhoB-family response regulator PmrA
Authors: Lou Yuan-Chou , Huang Hsuan-Yu , Yeh Hsin-Hong , Chiang Wei-Hung , Chen Chinpan , Wu Kuen-Phon
Published: July 2022

PmrA, an OmpR/PhoB-family response regulator, activates gene transcription responsible for polymyxin resistance in bacteria by recognizing promoters in which the canonical -35 element is replaced by the pmra-box, representing the PmrA recognition sequence. Here, we report a cryo-electron microscopy-derived structure of a bacterial PmrA-dependent transcription activation complex (TAC) containing a PmrA dimer, an RNA polymerase σ70-holoenzyme (RNAPH), and the pbgP promoter DNA. Our structure reveals that the RNAPH mainly contacts the PmrA C-terminal DNA binding domain (DBD) via electrostatic interactions and reorients the DBD three base pairs upstream of the pmra-box, resulting in a dynamic TAC conformation. In vivo assays show that substitution of PmrA DNA-recognition residues eliminated its transcriptional activity, but variants with altered RNAPH-interacting residues exhibited elevated transcriptional activity. Our study indicates that both PmrA recognition-induced DNA distortion and PmrA promoter escape play important roles in its transcriptional activation.
SP1 affects the migration and invasion of laryngeal cancer cells by regulating methylation of claudin 4 promoter region through the p-JNK pathway
Authors: Ni Jinsong , Shi Hailian , Peng Kaiyue , Liu Ya , Liu Yafang
Published: July 2022

Claudin 4 (CLDN4) is abnormally expressed in various tumors, but the mechanisms controlling CLDN4 expression in laryngeal cancer are poorly understood. Here, we describe hypermethylation of the promoter region of the CLDN4 gene and a positive correlation between the expression of CLDN4 and transcription factor Sp1 in laryngeal carcinoma. Specifically, CLDN4 expression was downregulated when laryngeal cancer cells were treated with Sp1 transcription factor inhibitor MTM. Immunohistochemical staining showed that while the expression of CLDN4 and p-JNK was negatively correlated, p-JNK positively correlated with MMP-2 and MMP-9 expression. The expressions of p-JNK, MMP-2, and MMP-9 were significantly downregulated in HEp-2 cells transfected with CLDN4, decreasing migration and invasiveness of HEp-2 cells. Thus, we show that SP1-mediated hypermethylation of the CLDN4 promoter region may activate the JNK signaling pathway and increase MMP-2/MMP-9 expression, which can then affect the migration and invasiveness of laryngeal cancer cells. These results contribute to our understanding of the regulatory mechanisms of CLDN4 in laryngeal cancer and identify the Sp1-CLDN4-jnk signaling pathway-MMP-2/MMP-9 axis as a potential target for the treatment of laryngeal cancer.
Experimental determination and mathematical modeling of standard shapes of forming autophagosomes
Authors: Sakai Yuji , Takahashi Satoru , Koyama-Honda Ikuko , Saito Chieko , Mizushima Noboru
Published: July 2022

The formation of autophagosomes involves dynamic morphological changes of a phagophore from a disk-shaped membrane cisterna into a cup-shaped intermediate and a spherical autophagosome. However, the physical mechanism behind these morphological changes remains elusive. Here, we determined the average shapes of phagophores by statistically investigating three-dimensional electron micrographs of more than 100 phagophores. The results showed that the cup-shaped structures adopted a characteristic morphology; they were longitudinally elongated, and the rim was catenoidal with an outwardly recurved shape. To understand these characteristic shapes, we established a theoretical model of the shape of entire phagophores. The model quantitatively reproduced the average morphology and revealed that the characteristic shape of phagophores (i.e., an elongated shape with a catenoidal rim) was primarily determined by the relative size of the open rim to the total surface area. These results suggest that autophagosomal membranes are highly flexible and that the morphological changes during autophagosome formation follow a stable path determined by elastic bending energy minimization.

The formation of autophagosomes involves dynamic morphological changes of membrane cisternae. Sakai et al. determined the average shapes of forming autophagosomes by statistically investigating three-dimensional electron micrographs and established a theoretical model that quantitatively reproduces the phagophore shapes.