bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged mitosis causes separase deregulation and chromosome nondisjunction
Authors: Shindo Norihisa , Otsuki Makoto , Uchida Kazuhiko S.K. , Hirota Toru
Published: July 2020

Intercellular karyotype diversity, or aneuploidy, is a widespread feature of cancers propagated through continuous mitotic chromosome segregation errors, the condition called chromosomal instability. The protease separase is an essential enzyme that sever cohesin between sister chromatids, and a probe for separase activity has articulated that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed through metaphase; however the relevance of this robust control remains unclear. In this study, we found that separase activates precociously during prolonged metaphase, consistently in multiple types of cancer cell lines. An artificial extension of metaphase alone in chromosomally stable diploid cells was found to induce precocious activation. These kinetic changes resulted in an incomplete removal of cohesin and emergence of chromosomal bridges in anaphase. Conversely, in transformed cells, shortening back of their prolonged metaphase restored the robust activation of separase and ameliorated anaphase bridge formation. These observations suggest that retarded metaphase progression directly affects the separase activation profile, which provides a previously unanticipated etiology for chromosomal instability in cancers and underscores the significance of the swift mitotic transitions for fail-safe chromosome segregation.
The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant specific motif and acts in a partially redundant manner with the tonoplast enzyme
Authors: Lupanga Upendo , Röhrich Rachel , Askani Jana , Hilmer Stefan , Kiefer Christiane , Krebs Melanie , Kanazawa Takehiko , Ueda Takashi , Schumacher Karin
Published: July 2020

Vacuolar-type H+-ATPases (V-ATPases) are versatile proton pumps that control the pH of many intracellular compartments in all eukaryotic cells. The localization of the Arabidopsis V-ATPase was previously shown to be determined by the isoforms of subunit a (VHA-a). The incorporation of VHA-a1 targets the V-ATPase to the trans-Golgi network/early endosome (TGN/EE) whilst the incorporation of VHA-a2 or VHA-a3 targets the V-ATPase to the tonoplast. By employing chimeric proteins and site directed mutagenesis we identified a targeting domain (a1-TD) containing an acidic cluster in the N-terminus of VHA-a1 that serves as both an ER export signal and as a TGN retention motif. The a1-TD is conserved among seed plants and we confirmed experimentally that its presence is predictive of TGN/EE- localization. In contrast to many other non-seed plants, the liverwortMarchantia polymorphaencodes only a single VHA-a subunit (MpVHA-a) and we show here that it is predominantly localized at the tonoplast. In our attempts to determine if MpVHA-a can functionally replace the Arabidopsis VHA-a isoforms, we used CRISPR/Cas9 to generate null-alleles lacking VHA-a1 and discovered that its function is essential for male gametophyte development but can be replaced by VHA-a2 and VHA-a3 during vegetative growth.
Population genomics on the origin of lactase persistence in Europe and South Asia
Authors: Satta Yoko , Takahata Naoyuki
Published: July 2020

The C to T mutation at rs4988235 located upstream of the lactase (LCT) gene is the primary determinant for lactase persistence (LP) that is prevalent among Europeans and South Asians. Here, we review evolutionary studies of this mutation based on ancient and present-day human genomes with the following concluding remarks: the mutation arose in the Pontic Steppe somewhere between 23,000 and 5960 years ago, emigrated into Europe and South Asia in the Bronze Age via the expansion of the Steppe ancestry, and experienced local hard sweeps with their delayed onsets occurring between 5000 and 3280 years ago. We also argue that the G to A mutation at rs182549 arose earlier than 23,000 years ago, the intermediate CA haplotype ancestral to the LP-related TA haplotype is still represented by samples from Tuscans, admixed Americans and South Asians, and the great majority of G to A mutated descendants have hitchhiked since the C to T mutation was favored by local selection.
Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target
Authors: Sobala Łukasz F. , Fernandes Pearl Z , Hakki Zalihe , Thompson Andrew J , Howe Jonathon D , Hill Michelle , Zitzmann Nicole , Davies Scott , Stamataki Zania , Butters Terry D. , Alonzi Dominic S. , Williams Spencer J , Davies Gideon J
Published: July 2020

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the soleendo-acting glycoside hydrolase involved in N-glycan trimming and unusually is located within the Golgi, where it allows ER escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N-glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation.

SIGNIFICANCE STATEMENT

The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
Proteus software for physics-based protein design
Authors: Mignon David , Druart Karen , Opuu Vaitea , Polydorides Savvas , Villa Francesco , Gaillard Thomas , Michael Eleni , Archontis Georgios , Simonson Thomas
Published: July 2020

We describe methods and software for physics-based protein design. The folded state energy combines molecular mechanics with Generalized Born solvent. Sequence and conformation space are sampled with Replica Exchange Monte Carlo, assuming one or a few fixed protein backbone structures and discrete side chain rotamers. Whole protein design and enzyme design are presented as illustrations. Full redesign of three PDZ domains was done using a simple, empirical, unfolded state model. Designed sequences were very similar to natural ones. Enzyme redesign exploited a powerful, adaptive, importance sampling approach that allows the design to directly target substrate binding, reaction rate, catalytic efficiency, or the specificity of these properties. Redesign of tyrosyl-tRNA synthetase stereospecificity is reported as an example.
Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex
Authors: Vishnubhotla Ravikanth , Vankadari Naveen , Ketavarapu Vijayasarathy , Amanchy Ramars , Avanthi Steffie , Bale Govardhan , Reddy Duvvur Nageshwar , Sasikala Mitnala
Published: July 2020

SARS-CoV-2, a highly transmittable pathogen has infected over 3.8 million people around the globe. The spike glycoprotein of SARS-CoV-2 engages host ACE2 for adhesion, TMPRSS2 for activation and entry. With the aid of whole-exome sequencing, we report a variant rs12329760 inTMPRSS2gene and its mutant V160M, which might impede viral entry. Furthermore, we identified TMPRSS2 cleavage sites in S2 domain of spike glycoprotein and report the structure of TMPRSS2 in complex with spike glycoprotein. We also report the structures of protease inhibitors in complex with TMPRSS2, which could hamper the interaction with spike protein. These findings advance our understanding on the role of TMPRSS2 and in the development of potential therapeutics.
Capturing limbal epithelial stem cell population dynamics, signature, and their niche
Authors: Altshuler Anna , Amitai-Lange Aya , Tarazi Noam , Dey Sunanda , Strinkovsky Lior , Bhattacharya Swarnabh , Hadad-Porat Shira , Nasser Waseem , Imeri Jusuf , Ben-David Gil , Tiosano Beatrice , Berkowitz Eran , Karin Nathan , Savir Yonatan , Shalom-Feuerstein Ruby
Published: July 2020

Stem cells (SCs) are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. It was several decades ago, when limbal epithelial SCs (LSCs) that regenerate the corneal epithelium were one of the first sporadic, quiescent SCs ever discovered. However, LSC dynamics, heterogeneity and genetic signature are largely unknown. Moreover, recent accumulating evidence strongly suggested that epithelial SCs are actually abundant, frequently dividing cells that display stochastic behavior.

In this work, we performed an in-depth analysis of the murine limbal epithelium by single-cell RNA sequencing and quantitative lineage tracing. The generated data provided an atlas of cell states of the corneal epithelial lineage, and particularly, revealed the co-existence of two novel LSC populations that reside in separate and well-defined sub-compartments. In the “outer” limbus, we identified a primitive widespread population of quiescent LSCs (qLSCs) that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins, while the “inner” limbus host prevalent active LSCs (aLSCs) co-expressing Krt15-GFP/Atf3/Mt1-2/Socs3. Analysis of LSC population dynamics suggests that while qLSCs and aLSCs possess different proliferation rates, they both follow similar stochastic rules that dictate their self-renewal and differentiation. Finally, T cells were distributed in close proximity to qLSCs. Indeed, their absence or inhibition resulted in the loss of quiescence and delayed wound healing. Taken together, we propose that divergent regenerative strategies are tailored to properly support tissue-specific physiological constraints. The present study suggests that in the case of the cornea, quiescent epithelial SCs are abundant, follow stochastic rules and neutral drift dynamics.
Structure of Full Length Plasmodium Myosin A and its light chain PfELC, dual targets against malaria parasite pathogenesis
Authors: Moussaoui Dihia , Robblee James P. , Auguin Daniel , Krementsova Elena B. , Haase Silvia , Blake Thomas C. A. , Baum Jake , Robert-Paganin Julien , Trybus Kathleen M. , Houdusse Anne
Published: July 2020

Parasites from the genusPlasmodiumare the causative agents of malaria. The mobility, infectivity and ultimately pathogenesis of this parasite relies on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future antimalarials targeting both the glideosome motor and its regulatory elements.

Highlights

The first structures of the full length PfMyoA motor in two states of its motor cycle.

A unique priming of the PfMyoA lever arm results from specific lever arm/motor domain interactions, which allows for a larger powerstroke to enhance speed.

Sequence adaptations within the motor domain and degenerate IQ motifs in the lever arm dictate PfMyoA motor properties.

PfELC is essential for blood cell invasion and is a weak link in the assembly of a fully functional motor, providing a second novel target for antimalarial drug design.
Local Field Potentials in a Pre-motor Region Predict Learned Vocal Sequences
Authors: Brown Daril E. , Chavez Jairo I. , Nguyen Derek H. , Kadwory Adam , Voytek Bradley , Arneodo Ezequiel , Gentner Timothy Q. , Gilja Vikash
Published: July 2020

Neuronal activity within the premotor region HVC is tightly synchronized to, and crucial for, the articulate production of learned song in birds. Characterizations of this neural activity typically focuses on patterns of sequential bursting in small carefully identified subsets of single neurons in the HVC population. Much less is known about population dynamics beyond the scale of individual neurons. There is a rich history of using local field potentials (LFP), to extract information about behavior that extends beyond the contribution of individual cells. These signals have the advantage of being stable over longer periods of time and have been used to study and decode complex motor behaviors, such as human speech. Here we characterize LFP signals in the putative HVC of freely behaving male zebra finches during song production, to determine if population activity may yield similar insights into the mechanisms underlying complex motor-vocal behavior. Following an initial observation that structured changes in the LFP were distinct to all vocalizations during song, we show that it is possible to extract time varying features from multiple frequency bands to decode the identity of specific vocalization elements (syllables) and to predict their temporal onsets within the motif. This demonstrates that LFP is a useful signal for studying motor control in songbirds. Surprisingly, the time frequency structure of putative HVC LFP is qualitatively similar to well established oscillations found in both human and non-human mammalian motor areas. This physiological similarity, despite distinct anatomical structures, may give insight to common computational principles for learning and/or generating complex motor-vocal behaviors.

Author Summary

Vocalizations, such as speech and song, are a motor process that requires the coordination of several muscle groups receiving instructions from specific brain regions. In songbirds, HVC is a premotor brain region required for singing and it is populated by a set of neurons that fire sparsely during song. How HVC enables song generation is not well understood. Here we describe network activity in putative HVC that precedes the initiation of each vocal element during singing. This network activity can be used to predict both the identity of each vocal element (syllable) and when it will occur during song. In addition, this network activity is similar to activity that has been documented in human, non-human primate, and mammalian premotor regions tied to muscle movements. These similarities add to a growing body of literature that finds parallels between songbirds and humans in respect to the motor control of vocal organs. Given the similarities of the songbird and human motor-vocal systems these results suggest that the songbird model could be leveraged to accelerate the development of clinically translatable speech prosthesis.
Comprehensive Live-cell Imaging Analysis of Cryptotanshinone and Synergistic Drug-Screening Effects in Various Human and Canine Cancer Cell Lines
Authors: Bittner Michael L. , Lopes Rosana , Hua Jianping , Sima Chao , Datta Aniruddha , Wilson-Robles Heather M.
Published: July 2020

Background

Several studies have highlighted both the extreme anticancer effects of Cryptotanshinone (CT), a Stat3 crippling component fromSalvia miltiorrhiza, as well as other STAT3 inhibitors to fight cancer.

Methods

Data presented in this experiment incorporates 2 years ofin vitrostudies applying a comprehensive live-cell drug-screening analysis of human and canine cancer cells exposed to CT at 20 μM concentration, as well as to other drug combinations. As previously observed in other studies, dogs are natural cancer models, given to their similarity in cancer genetics, epidemiology and disease progression compared to humans.

Results

Results obtained from several types of human and canine cancer cells exposed to CT and varied drug combinations, verified CT efficacy at combating cancer by achieving an extremely high percentage of apoptosis within 24 hours of drug exposure.

Conclusions

CT anticancer efficacy in various human and canine cancer cell lines denotes its ability to interact across different biological processes and cancer regulatory cell networks, driving inhibition of cancer cell survival.
Efficacy and gut dysbiosis of gentamicin-intercalated smectite as a new therapeutic agent againstHelicobacter pyloriin a mouse model
Authors: Jeong Su Jin , Lee Kyoung Hwa , Kim Jie-Hyun , Park Soon Young , Song Young Goo
Published: July 2020

Background

Helicobacter pylorieradication rate with conventional standard therapy is decreasing owing to antibiotic resistance, necessitating novel antibacterial strategies againstH. pylori. We evaluated the efficacy of a gentamicin-intercalated smectite hybrid (S-GM)-based treatment, and analyzed fecal microbiome composition inH. pylori-infected mice.

Methodology

To evaluate anti-H. pyloriefficacy, mice were divided into eight groups, andH. pylorieradication was assessed byCampylobacter-like organism (CLO) test and PCR assay ofH. pyloriin gastric mucosa. One week afterH. pylorieradication, proinflammatory cytokine levels and atrophic changes in gastric mucosa were examined. Stool specimens were collected and analyzed for microbiome changes. The S-GM-based triple regimen decreased bacterial burdenin vivo, compared with that in untreated mice or mice treated with other regimens. The therapeutic reactions in the CLO test from gastric mucosa were both 90% in standard triple therapy and S-GM therapy group, respectively. Those ofH. pyloriPCR in mouse gastric mucosa were significantly lower in standard triple therapy and S-GM therapy groups than in non-treatment group. Toxicity test results showed that S-GM therapy reduced IL-8 level and atrophic changes in gastric mucosa. Stool microbiome analysis revealed that compared with mice treated with the standard triple therapy, mice treated with the S-GM therapy showed microbiome diversity and abundant microorganisms at the phylum level.

Conclusion

Our results suggested that S-GM is a promising and effective therapeutic agent againstH. pyloriinfection.

Author summary

The eradication rate onHelicobacter pylori(H. pylori) showed decreasing trend due to antibiotic resistance, especially clarithromycin. Therefore, we made a smectite hybrid as a drug delivery system using aminoglycosides antibiotic-gentamicin, and applied it to the mouse stomach wall to confirm the localized therapeutic effect, and set the different treatment duration to verify the effect. As a result, it was confirmed that the therapeutic efficacy of gentamicin (GM)-intercalated smectite hybrid (S-GM) was not inferior to the existing standard triple therapy, based on amoxicillin and clarithromycin, and preserved the diversity of gut microbiome composition. Therefore, a S-GM treatment is expected to be a new alternative regimen toH. pyloriinfection.
The glycosyltransferase UGT76B1 is critical for plant immunity as it governs the homeostasis ofN-hydroxy-pipecolic acid
Authors: Mohnike Lennart , Rekhter Dmitrij , Huang Weijie , Feussner Kirstin , Tian Hainan , Herrfurth Cornelia , Zhang Yuelin , Feussner Ivo
Published: July 2020

The trade-off between growth and defense is a critical aspect of plant immunity. Therefore, plant immune response needs to be tightly regulated. The hormone regulating plant defense against biotrophic pathogens is salicylic acid (SA). Recently,N-hydroxy-pipecolic acid (NHP) was identified as second regulator for plant innate immunity and systemic acquired resistance. Although the biosynthetic pathway leading to NHP formation has already been identified, the route how NHP is further metabolized was unclear. Here, we present UGT76B1 as a UDP-dependent glycosyltransferase that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid (NHP-OGlc). Analysis of T-DNA and CRISPR knock-out mutant lines ofUGT76B1by targeted and non-targeted UHPLC-HRMS underlined NHP and SA as endogenous substrates of this enzyme in response toPseudomonasinfection and UV treatment. UGT76B1 shows similar KMfor NHP and SA.ugt76b1mutant plants have a dwarf phenotype and a constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme FMO1. This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance inugt76b1. Externally applied NHP can move to distal tissue inugt76b1mutant plants. Although glycosylation is not required for the long distance movement of NHP during systemic acquired resistance, it is crucial to balance growth and defense.
Phosphosite T674A mutation in kinesin family member 3A fails to reproduce tissue and ciliary defects characteristic of CILK1 loss of function
Authors: Gailey Casey D. , Wang Eric J. , Jin Li , Ahmadi Sean , Brautigan David L. , Li Xudong , Xu Wenhao , Fu Zheng
Published: July 2020

Background

Kinesin family member 3A (KIF3A) is a molecular motor protein in the heterotrimeric kinesin-2 complex that drives anterograde intraflagellar transport. This process plays a pivotal role in both biogenesis and maintenance of the primary cilium that supports tissue development. Ciliogenesis associated kinase 1 (CILK1) phosphorylates human KIF3A at Thr672. CILK1 loss of function causes ciliopathies that manifest profound and multiplex developmental defects, including polydactyly, shortened and hypoplastic bones and alveoli airspace deficiency, leading to perinatal lethality. Prior studies have raised the hypothesis that CILK1 phosphorylation of KIF3A is critical for its regulation of organ development.

Results

We produced a mouse model with phosphorylation site Thr674 in mouseKif3amutated to Ala.Kif3aT674A homozygotes are viable and exhibit no skeletal abnormalities, and only mildly reduced airspace in alveoli. Mouse embryonic fibroblasts carryingKif3aT674A mutation show a normal rate of ciliation and a moderate increase in cilia length.

Conclusion

These results indicate that eliminating Kif3a Thr674 phosphorylation by CILK1 is insufficient to reproduce the severe developmental defects in ciliopathies caused by Cilk1 loss of function. This suggests KIF3A phosphorylation by CILK1 is not essential for tissue development and other substrates are involved in Cilk1 ciliopathies.
Mapping cellular nanoscale viscoelasticity and relaxation times relevant to growth of living Arabidopsis thaliana plants using multifrequency AFM
Authors: Seifert Jacob , Kirchhelle Charlotte , Moore Ian , Contera Sonia
Published: July 2020

The shapes of living organisms are formed and maintained by precise control in time and space of growth, which is achieved by dynamically fine-tuning the mechanical (viscous and elastic) properties of their hierarchically built structures from the nanometer up. Most organisms on Earth including plants grow by yield (under pressure) of cell walls (bio-polymeric matrices equivalent to extracellular matrix in animal tissues) whose underlying nanoscale viscoelastic properties remain unknown. Multifrequency atomic force microscopy (AFM) techniques exist that are able to map properties to a small subgroup of linear viscoelastic materials (those obeying the Kelvin-Voigt model), but are not applicable to growing materials, and hence are of limited interest to most biological situations. Here, we extend existing dynamic AFM methods to image linear viscoelastic behavior in general, and relaxation times of cells of multicellular organisms in vivo with nanoscale resolution, featuring a simple method to test the validity of the mechanical model used to interpret the data. We use this technique to image cells at the surface of livingArabidopsis thalianahypocotyls to obtain topographical maps of storage E’ = 120 − 200 MPa and loss E’’= 46 − 111 MPa moduli as well as relaxation times τ = 2.2 − 2.7 µs of their cell walls. Our results demonstrate that cell walls, despite their complex molecular composition, display a striking continuity of simple, linear, viscoelastic behavior across scales–following almost perfectly the standard linear solid model–with characteristic nanometer scale patterns of relaxation times, elasticity and viscosity, whose values correlate linearly with the speed of macroscopic growth. We show that the time-scales probed by dynamic AFM experiments (milliseconds) are key to understand macroscopic scale dynamics (e.g. growth) as predicted by physics of polymer dynamics.
USP11 deubiquitinates monoubiquitinated SPRTN to repair DNA-protein crosslinks
Authors: Perry Megan , Kollala Sai Sundeep , Biegert Meghan , Su Grace , Kodavati Manohar , Mallard Halle , Kreiling Natasha , Holbrook Alexander , Ghosal Gargi
Published: July 2020

DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription and recombination. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins crosslinked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN chromatin accessibility during DPC repair. The deubiquitinase regulating SPRTN function in DPC repair is unknown. Here, we identify USP11 as a SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells andin vitro.USP11 depletion impairs SPRTN deubiquitination in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings elucidate the function of USP11 in the regulation of SPRTN monoubiquitination and SPRTN-mediated DPC repair.
Leptin suppresses development of GLP-1 Inputs to the Paraventricular Nucleus of the Hypothalamus
Authors: Biddinger Jessica E. , Lazarenko Roman M. , Scott Michael M. , Simerly Richard B.
Published: July 2020

The nucleus of the solitary tract is critical for the central integration of signals from visceral organs and contains preproglucagon (PPG) neurons, which express leptin receptors and send direct projections to the paraventricular nucleus of the hypothalamus (PVH). Here, we visualized neuronal projections of PPG neurons in leptin-deficient Lepob/obmice and found that projections from PPG neurons are elevated compared with controls, and PPG projections were normalized by targeted rescue of leptin receptors in LepRbTB/TBmice, which lack functional neuronal leptin receptors. Moreover, Lepob/oband LepRbTB/TBmice displayed increased levels of neuronal activation in the PVH following vagal stimulation, and whole-cell patch recordings of GLP-1 receptor-expressing PVH neurons revealed enhanced excitatory neurotransmission, suggesting that leptin acts cell autonomously to suppress representation of excitatory afferents from PPG neurons, thereby diminishing the impact of visceral sensory information on GLP-1 receptor neurons in the PVH.
Structural basis of TRPC4 regulation by calmodulin and pharmacological agents
Authors: Vinayagam Deivanayagabarathy , Quentin Dennis , Sitsel Oleg , Merino Felipe , Stabrin Markus , Hofnagel Oliver , Yu Maolin , Ledeboer Mark W. , Malojcic Goran , Raunser Stefan
Published: July 2020

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin. Molecular details of both calmodulin and drug binding have remained elusive so far. Here we report structures of TRPC4 in complex with a pyridazinone-based inhibitor and a pyridazinone-based activator and calmodulin. The structures reveal that both activator and inhibitor bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand binding site can be transmitted to the central ion-conducting pore of TRPC4. Calmodulin binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel calmodulin-induced regulatory mechanism of canonical TRP channels.
Galectin-8 senses phagosomal damage and recruits selective autophagy adapter TAX1BP1 to controlMycobacterium tuberculosisinfection in macrophages
Authors: Bell Samantha L. , Lopez Kayla L. , Cox Jeffery S. , Patrick Kristin L. , Watson Robert O.
Published: July 2020

Mycobacterium tuberculosis(Mtb) infects a quarter of the world and causes the deadliest infectious disease worldwide. Upon infection, Mtb is phagocytosed by macrophages and uses its virulence-associated ESX-1 secretion system to modulate the host cell and establish a replicative niche. We have previously shown the ESX-1 secretion system permeabilizes the Mtb-containing phagosome and that a population (~30%) of intracellular Mtb are recognized within the cytosol, tagged with ubiquitin, and targeted to the selective autophagy pathway. Despite the importance of selective autophagy in controlling infection, the mechanisms through which macrophages sense and respond to damaged Mtb-containing phagosomes remains unclear. Here, we demonstrate that several cytosolic glycan-binding proteins, known as galectins, recognize Mtb-containing phagosomes. We found that galectins-3, -8, and -9 are all recruited to the same Mtb population that colocalizes with selective autophagy markers like ubiquitin, p62, and LC3, which indicates Mtb damages its phagosomal membrane such that cytosolic host sensors can recognize danger signals in the lumen. To determine which galectins are required for controlling Mtb replication in macrophages, we generated CRISPR/Cas9 knockout macrophages lacking individual or multiple galectins and found that galectin-8-/-and galectin-3/8/9-/-knockout macrophages were similarly defective in targeting Mtb to selective autophagy and controlling replication, suggesting galectin-8 plays a privileged role in anti-Mtb autophagy. In investigating this specificity, we identified a novel and specific interaction between galectin-8 and TAX1BP1, one of several autophagy adaptors that bridges cargo and LC3 during the course of autophagosome formation, and this galectin-8/TAX1BP1 interaction was necessary to efficiently target Mtb to selective autophagy. Remarkably, overexpressing individual galectins increased targeting of Mtb to antibacterial autophagy and limited Mtb replication. Taken together, these data imply that galectins recognize damaged Mtb-containing phagosomes, recruit downstream autophagy machinery, and may represent promising targets for host-directed therapeutics to treat Mtb.
SWR1-independent association of H2A.Z to the LINC complex promotes meiotic chromosome motion
Authors: González-Arranz Sara , Gardner Jennifer M. , Yu Zulin , Patel Neem J. , Heldrich Jonna , Santos Beatriz , Carballo Jesús A. , Jaspersen Sue L. , Hochwagen Andreas , San-Segundo Pedro A.
Published: July 2020

The H2A.Z histone variant is deposited into chromatin by the SWR1 complex affecting multiple aspects of meiosis. Here we describe a SWR1-independent localization of H2A.Z at meiotic telomeres and the centrosome. We demonstrate that H2A.Z colocalizes and interacts with Mps3, the SUN component of the LINC complex that spans the nuclear envelope and links meiotic telomeres to the cytoskeleton promoting meiotic chromosome movement. H2A.Z also interacts with the meiosis-specific Ndj1 protein that anchors telomeres to the nuclear periphery via Mps3. Telomeric localization of H2A.Z depends on Ndj1 and the N-terminal domain of Mps3. Although telomeric attachment to the nuclear envelope is maintained in the absence of H2A.Z, the distribution of Mps3 is altered. The velocity of chromosome movement during meiotic prophase I is reduced in thehtz1Δmutant lacking H2A.Z, but it is unaffected inswr1Δcells. We reveal that H2A.Z is an additional LINC-associated factor that contributes to promote telomere-driven chromosome motion critical for error-free gametogenesis.
Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural divergence among human adenoviruses
Authors: Pérez-Illana Marta , Martínez Marta , Condezo Gabriela N. , Hernando-Pérez Mercedes , Mangroo Casandra , Brown Martha , Marabini Roberto , Martín Carmen San
Published: July 2020

Enteric adenoviruses are one of the main causes of viral gastroenteritis in the world. To carry out a successful infection, the virions must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We have determined the structure of a human enteric adenovirus, HAdV-F41, at 4.0 Å resolution by single particle averaging cryo-electron microscopy, and compared it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base and internal minor coat proteins IIIa and VIII are conserved, we observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, we find an organization of the external minor coat protein IX different from all previously characterized human and non-human mastadenoviruses. Knowledge of the structure of enteric adenoviruses can provide a starting point for the design of vectors suitable for oral delivery or intestinal targeting.
H-Mem: Harnessing synaptic plasticity with Hebbian Memory Networks
Authors: Limbacher Thomas , Legenstein Robert
Published: July 2020

The ability to base current computations on memories from the past is critical for many cognitive tasks such as story understanding. Hebbian-type synaptic plasticity is believed to underlie the retention of memories over medium and long time scales in the brain. However, it is unclear how such plasticity processes are integrated with computations in cortical networks. Here, we propose Hebbian Memory Networks (H-Mems), a simple neural network model that is built around a core hetero-associative network subject to Hebbian plasticity. We show that the network can be optimized to utilize the Hebbian plasticity processes for its computations. H-Mems can one-shot memorize associations between stimulus pairs and use these associations for decisions later on. Furthermore, they can solve demanding question-answering tasks on synthetic stories. Our study shows that neural network models are able to enrich their computations with memories through simple Hebbian plasticity processes.
BDNF Overexpression in the Prelimbic Cortex Does Not Reduce Anxiety- and Depression-like Behavior in Serotonin Knockout Rats
Authors: Diniz Danielle M. , Bosch Kari , Calabrese Francesca , Brivio Paola , Riva Marco A. , Grandjean Joanes , Homberg Judith R.
Published: July 2020

Depressive disorders are one of the leading causes of non-fatal health loss in the last decade. Adding to the burden, the available treatments not always properly work for some individuals. There is, therefore, a constant effort from clinical and preclinical studies to bring forward a better understanding of the disease and look for novel alternative therapies. Two target systems very well explored are the serotonin and the brain-derived neurotrophic factor (BDNF) systems. Selective serotonin reuptake inhibitors (SSRIs), a commonly used class of antidepressants, target the serotonin transporter (SERT) and increase serotonin levels, which in turn also leads to an increase in BDNF. A rat model lacking SERT (SERT knockout) has been a useful tool to study the interplay between serotonin and BDNF. SERT−/−rats present increased extracellular levels of serotonin, yet BDNF levels are decreased, especially in the prefrontal cortex (PFC) and hippocampus. The animals further display anxiety- and depression-like behavior. Therefore, BDNF might mediate the phenotype expressed by the SERT−/−rats. In this study, we sought to investigate whether overexpression of BDNF in the brain of SERT−/−rats would rescue its anxious and depressive-like behavior. Through stereotaxic surgery, SERT−/−and wild-type (WT) rats received BDNF or GFP lentivirus microinfusions into the prelimbic cortex subregion of the mPFC and were submitted to the sucrose consumption, open field test, and forced swim tests. Additionally, we measured hypothalamus-pituitary-adrenal (HPA)-axis reactivity. The results revealed that SERT−/−rats presented decreased sucrose intake, decreased locomotor activity, and increased escape-oriented behavior in the forced swim test compared to WT rats. BDNF upregulation in WT rats caused alterations in the HPA-axis function, resulting in elevated basal plasma corticosterone levels and decreased plasma corticosterone upon stress. In conclusion, BDNF overexpression in the PrL, in general, did not rescue SERT−/−rats from its depression- and anxiety-like behavior, and in WT animals, it caused a malfunction in the HPA-axis.
Functional specialization within the inferior parietal lobes across cognitive domains
Authors: Numssen Ole , Bzdok Danilo , Hartwigsen Gesa
Published: July 2020

The inferior parietal lobe (IPL) is a key neural substrate underlying diverse mental processes, from basic attention to language and social cognition, that define human interactions. Its putative domain-global role appears to tie into poorly understood differences between cognitive domains in both hemispheres. Across attentional, semantic, and social cognitive tasks, our study explored functional specialization within the IPL. The task specificity of IPL subregion activity was substantiated by distinct predictive signatures identified by multivariate pattern-learning algorithms. Moreover, the left and right IPL exerted domain-specific modulation of effective connectivity among their subregions. Task-evoked functional interactions of the anterior and posterior IPL subregions involved recruitment of distributed cortical partners. While anterior IPL subregions were engaged in strongly lateralized coupling links, both posterior subregions showed more symmetric coupling patterns across hemispheres. Our collective results shed light on how under-appreciated functional specialization in the IPL supports some of the most distinctive human mental capacities.
Identification of a long non-coding RNA regulator of liver carcinoma cell survival
Authors: Rybakova Yulia , Gonzalez John T. , Bogorad Roman , Chauhan Vikash P. , Dong Yize L. , Whittaker Charles A. , Koteliansky Victor , Anderson Daniel G.
Published: July 2020

Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, Protein Tyrosine Kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC.
Shear stress-induced pathological changes in endothelial cells occur through Piezo1 activation of TRPV4
Authors: Swain Sandip M. , Liddle Rodger A.
Published: July 2020

Although the ion channels Piezo1 and TRPV4 have been implicated in high venous pressure- and fluid shear stress-induced vascular hyperpermeability, they have been described as working independently. Moreover, the mechanism by which Piezo1 and TRPV4 channels in endothelial cells execute the same function is poorly understood. Here we demonstrate that Piezo1 regulates TRPV4 channel activation in endothelial cells and that Piezo1-mediated TRPV4 channel opening is a function of the strength and duration of fluid shear stress. Application of the Piezo1 antagonist, GsMTx4, completely blocked the elevation in intracellular calcium ([Ca2+]i) induced by both fluid shear stress and the Piezo1 agonist, Yoda1. High and prolonged shear stress caused sustained [Ca2+]ielevation which required TRPV4 opening and was responsible for fluid shear stress- and Piezo1-mediated disruption of adherens junctions and actin remodeling. We found that Piezo1’s effects were mediated by phospholipase A2 activation. Blockade of TRPV4 channels with the selective TRPV4 blocker, HC067047, prevented the loss of endothelial cell integrity and actin disruption induced by Yoda1 or shear stress and prevented Piezo1-induced monocyte adhesion to endothelial cell monolayers. These findings demonstrate that Piezo1 activation by fluid shear stress initiates a calcium signal that causes TRPV4 opening which in turn is responsible for the sustained phase calcium elevation that triggers pathological events in endothelial cells.
Generation of an X-ray nanobeam of a free-electron laser using reflective optics with speckle interferometry
Authors: Inoue Takato , Matsuyama Satoshi , Yamada Jumpei , Nakamura Nami , Osaka Taito , Inoue Ichiro , Inubushi Yuichi , Tono Kensuke , Yumoto Hirokatsu , Koyama Takahisa , Ohashi Haruhiko , Yabashi Makina , Ishikawa Tetsuya , Yamauchi Kazuto
Published: July 2020

Ultimate focusing of an X-ray free-electron laser (XFEL) enables the generation of ultrahigh-intensity X-ray pulses. Although sub-10 nm focusing has already been achieved using synchrotron light sources, the sub-10 nm focusing of XFEL beams remains difficult mainly because the insufficient stability of the light source hinders the evaluation of a focused beam profile. This problem is specifically disadvantageous for the Kirkpatrick–Baez (KB) mirror focusing system, in which a slight misalignment of ∼300 nrad can degrade the focused beam. In this work, an X-ray nanobeam of a free-electron laser was generated using reflective KB focusing optics combined with speckle interferometry. The speckle profiles generated by 2 nm platinum particles were systematically investigated on a single-shot basis by changing the alignment of the multilayer KB mirror system installed at the SPring-8 Angstrom Compact Free-Electron Laser, in combination with computer simulations. It was verified that the KB mirror alignments were optimized with the required accuracy, and a focused vertical beam of 5.8 nm (±1.2 nm) was achieved after optimization. The speckle interferometry reported in this study is expected to be an effective tool for optimizing the alignment of nano-focusing systems and for generating an unprecedented intensity of up to 1022 W cm−2 using XFEL sources.
Iterative energy self-calibration of Fe XANES spectra. Erratum
Authors: Jones Michael W. M. , Mallmann Guilherme , Wykes Jeremy L. , Knafelc Joseph , Bryan Scott E. , Howard Daryl L.
Published: July 2020

A correction is made to the paper by Jones et al. (2020). [J. Synchrotron Rad. (2020), 27, 207–211].
Crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase
Authors: Guo Youzhong , Qiu Weihua , Roche Thomas E. , Hackert Marvin L.
Published: July 2020

Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space groupP3221, with unit-cell parametersa=b= 75.3,c= 173.2 Å. The structure was solved by molecular-replacement methods and refined to a finalRfactor of 21.9% (Rfree= 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ions in the active site, an additional Mn2+ion coordinated by His410 and His414, two MnSO4ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.
A proposed carbon-utilization and virulence protein A, CuvA (Rv1422), from Mycobacterium tuberculosis H37Rv: crystallization, X-ray diffraction analysis and ligand binding
Authors: Jeong Yoon Chae , Lee Ki Seog
Published: July 2020

Mycobacterium tuberculosis possesses the ability to undergo physiological adaptations in order to persist during the prolonged course of infection despite the active immune response of the host and in order to overcome multiple environmental changes. Previous studies have proposed that M. tuberculosis CuvA (Rv1422; MtCuvA) might play a critical role in the adaptation of the bacterium to environmental changes, such as nutrient utilization and alteration of the growth rate. However, the detailed function of MtCuvA still remains unclear owing to a lack of structural information. To better understand its role in host adaptation, MtCuvA was purified to homogeneity and was crystallized for the first time using the hanging-drop vapor-diffusion method. The crystal of MtCuvA diffracted to a resolution of 2.1 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.27, b = 170.93, c = 178.10 Å. The calculated Matthews coefficient (V
M) was 2.4 Å3 Da−1, with a solvent content of 48.02%, and thus four molecules appeared to be present in the asymmetric unit. Moreover, it is reported that MtCuvA can bind to the cell-wall precursor components uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine.