bluuebunny/crossref_metadata_2025 huggingface dataset:
A Wide-Field Micro-Computed Tomography Detector: Micron Resolution at Half-centimeter Scale
Authors: Yakovlev Maksim A. , Vanselow Daniel J. , Ngu Mee Siing , Zaino Carolyn R. , Katz Spencer R. , Ding Yifu , Parkinson Dula , Wang Steve Yuxin , Ang Khai Chung , La Riviere Patrick J. , Cheng Keith C.
Published: August 2021

Ideal 3-dimensional imaging of many complex samples, such as biological tissues made up of micro-scale structures extending over millimeter- to centimeter-scale tissue samples and organisms, requires both a wide field-of-view and high resolution. With existing optics and detectors used for micro-CT imaging, sub-micron pixel resolution can only be achieved for fields-of-view of <2 mm. This manuscript presents a unique detector system with a 6-mm field-of-view image circle and 0.5 μm pixel size that can be used in both synchrotron facilities and tabletop micro-CT units. A resolution-test pattern with linear microstructures and whole adult Daphnia magna were imaged on Beamline 8.3.2 of the Advanced Light Source. Volumes of 10,000 × 10,000 × 7,096 isotropic 0.5 μm voxels were reconstructed over a 5.0 × 3.5 mm field-of-view. Measurements in the projection domain confirmed a 1.182 μm measured spatial resolution that is largely Nyquist-limited. This unprecedented combination of field-of-view and resolution dramatically reduces the need for sectional scans and computational stitching for large samples, ultimately offering the means to elucidate change in tissue and cellular morphology in the context of larger whole, intact model organisms and specimens. This development is also anticipated to benefit micro-CT imaging in materials science, microelectronics, agricultural science, and biomedical engineering.

Synopsis

A custom wide-field lens and a new-generation megapixel camera enabled microCT scanning over a 3.5 × 5 mm field-of-view at a 1 μm resolution / 0.5 μm pixel size at the Berkeley Lawrence Advanced Light Source using a phantom with micron scale features. This novel combination of resolution and field-of-view will be broadly applicable to any setting in which micron-scale structures need to be characterized comprehensively in 3 dimensions over mm to cm scale.
Architecture and evolution of the cis-regulatory system of the echinoderm kirrelL gene
Authors: Khor Jian Ming , Guerrero-Santoro Jennifer , Ettensohn Charles A.
Published: August 2021

The gene regulatory network (GRN) that underlies echinoderm skeletogenesis is a prominent model of GRN architecture and evolution. KirrelL is an essential downstream effector gene in this network and encodes an Ig-superfamily protein required for the fusion of skeletogenic cells and the formation of the skeleton. In this study, we dissected the transcriptional control region of the kirrelL gene of the purple sea urchin, Strongylocentrotus purpuratus. Using plasmid- and BAC-based transgenic reporter assays, we identified key cis-regulatory elements (CREs) and transcription factor inputs that regulate Sp-kirrelL, including direct, positive inputs from two key transcription factors in the skeletogenic GRN, Alx1 and Ets1. We next identified kirrelL cis-regulatory regions from seven other echinoderm species that together represent all classes within the phylum. By introducing these heterologous regulatory regions into developing sea urchin embryos we provide evidence of their remarkable conservation across ~500 million years of evolution. We dissected in detail the kirrelL regulatory region of the sea star, Patiria miniata, and demonstrated that it also receives direct inputs from Alx1 and Ets1. Our findings identify kirrelL as a component of the ancestral echinoderm skeletogenic GRN. They support the view that GRN sub-circuits, including specific transcription factor-CRE interactions, can remain stable over vast periods of evolutionary history. Lastly, our analysis of kirrelL establishes direct linkages between a developmental GRN and an effector gene that controls a key morphogenetic cell behavior, cell-cell fusion, providing a paradigm for extending the explanatory power of GRNs.
ΔGunfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization
Authors: Bharmal Mohammed-Husain M. , Schrader Jared M.
Published: August 2021

Background

Translation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput.

Results

ΔGunfoldleaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfoldleaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5’ UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9).

Conclusions

The ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.
Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method
Authors: Mills Caitlin E. , Subramanian Kartik , Hafner Marc , Niepel Mario , Gerosa Luca , Chung Mirra , Victor Chiara , Gaudio Ben , Yapp Clarence , Nirmal Ajit J. , Clark Nicholas , Sorger Peter K.
Published: August 2021

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo®). Here we describe a public domain “Dye Drop” method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.
PySeq2500: An open source toolkit for repurposing HiSeq 2500 sequencing systems as versatile fluidics and imaging platforms
Authors: Pandit Kunal , Petrescu Joana , Cuevas Miguel , Stephenson William , Smibert Peter , Phatnani Hemali , Maniatis Silas
Published: August 2021

Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument into an open platform for programmable applications. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. As demonstrated here, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.
Air-nanobubbles ineffective to reduce pathogenic bacteria in fresh and brackish waters
Authors: Domingos Jose A. , Huang Qianjun , Liu Hong , Dong Ha Thanh , Khongcharoen Nareerat , Van Phan Thi , Nghia Nguyen Huu , Giang Pham Thai , The Viet Pham , St-Hilaire Sophie
Published: August 2021

Nanobubble (NB) technology has been hailed as a novel way to disinfect water. Previous studies suggested that when NBs collapse, they create shock waves that result in OH- free radicals, which can damage cells, including bacteria. In this study, we investigated, through a series of 11 experiments, the potential use of air nanobubbles (128 ± 44 nm, mean ± SD) to reduce the concentration of various pathogenic bacteria including Aeromonas hydrophila, A. veronii, Vibrio parahaemolyticus, and Streptococcus agalactiae under controlled, tank-based laboratory conditions. Despite the high number of nanobubbles continuously added to a relatively small volume of water in experimental tanks (50-100 L), we did not observe a consistent or significant decrease in bacteria that would control disease outbreaks. Although most of the experiments were conducted in fresh water on A. hydrophila, results were consistent across fresh and brackish water experiments, Gram-negative and Gram-positive bacteria, and a range of nanobubble concentrations. This study suggests air nanobubbles on their own are inadequate to significantly reduce high levels of pathogenic bacteria in water. We propose to explore other gases for improving the disinfection properties of this technology.

SIGNIFICANCE STATEMENT

Air nanobubbles did not sufficiently reduce the level of bacteria in laboratory experiments.
Optical Control of Invertebrate nAChR and Behaviors with Dithienylethene-Imidacloprid
Authors: Zhang Chao , Xu Qi , Xu Zhiping , Wang Long , Liu Zewen , Li Zhong , Shao Xusheng
Published: August 2021

Photopharmacology has changed established methods of studying receptor functions, allowing for increasing spatiotemporal resolution. However, no photopharmacological tools are available for the invertebrate nicotinic acetylcholine receptor (nAChR). Here, we report a photochromic ligand, dithienylethene-imidacloprid (DitIMI), targeting invertebrate nAChR. We demonstrated that DitIMI has low spontaneous in vivo and in vitro activity but can be photoisomerized to a highly active closed-form. This photoisomerization can further be translated to photomodulation of neuron membrane potential and behavioral responses of living mosquito larvae and American cockroaches. Furthermore, we discovered that DitIMI is a specific reporter for fluorescence polarization based high-throughput screening of nAChR ligands.
Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria
Authors: Wang Yingying , Chen Xi , Spengler Katharina , Terberger Karoline , Boehm Marko , Appel Jens , Barske Thomas , Timm Stefan , Battchikova Natalia , Hagemann Martin , Gutekunst Kirstin
Published: August 2021

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. Both the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex catalyze this reaction. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygenin vitroand is required for optimal photomixotrophic growth under aerobic conditions while the PDH complex is inactivated under the same conditions. We found that cells rely on a general shift from utilizing NAD(H)-dependent to ferredoxin-dependent enzymes under these conditions.

The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.
Abundant small RNAs in the reproductive tissues of the honey bee, Apis mellifera, are a plausible mechanism for epigenetic inheritance and parental manipulation of gene expression
Authors: Watson Owen T. , Buchmann Gabriele , Young Paul , Lo Kitty , Remnant Emily J. , Yagound Boris , Shambrook Mitch , Hill Andrew F. , Oldroyd Benjamin P. , Ashe Alyson
Published: August 2021

Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in semen in other animals. Intriguingly we find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. We conclude that small RNAs play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of origin-effects on gene expression and reproductive physiology.
The genome sequence of Sorbus pohuashanensis provides insights into population evolution and leaf sunburn response
Authors: Zhao Dongxue , Zhang Yan , Lu Yizeng , Fan Liqiang , Zhang Zhibin , Chai Mao , Zheng Jian
Published: August 2021

Sorbus pohuashanensis is a potential horticulture and medicinal plant, but its genomic and genetic background remains unknown. Here, we de novo sequenced and assembled the S. pohuashanensis (Hance) Hedl. reference genome using PacBio long reads. Based on the new reference genome, we resequenced a core collection of 22 Sorbus spp. samples, which were divided into two groups (G1 and G2) based on phylogenetic and PCA analysis. These phylogenetic clusters were highly in accordance with the classification based on leaf shape. Natural hybridization between the G1 and G2 groups was evidenced by a sample (R21) with a highly heterozygous genotype. Nucleotide diversity (π) analysis showed that G1 has a higher diversity than G2, and that G2 originated from G1. During the evolution process, the gene families involved in photosynthesis pathways expanded and gene families involved in energy consumption contracted. Comparative genome analysis showed that S. pohuashanensis has a high level of chromosomal synteny with Malus domestica and Pyrus communis. RNA-seq data suggested that flavonol biosynthesis and heat-shock protein (HSP)-heat-shock factor (HSF) pathways play important roles in protection against sunburn. This research provides new insight into the evolution of Sorbus spp. genomes. In addition, the genomic resources and the identified genetic variations, especially those genes related to stress resistance, will help future efforts to introduce and breed Sorbus spp.
Stereo-specific Lasofoxifene Derivatives Reveal the Interplay between Estrogen Receptor Alpha Stability and Antagonistic Activity in ESR1 Mutant Breast Cancer Cells
Authors: Hosfield David J. , Weber Sandra , Li Nan-Sheng , Sauvage Madline , Sullivan Emily , Nduwke Estelle , Han Ross , Cush Sydney , Lainé Muriel , Mader Sylvie , Greene Geoffrey L. , Fanning Sean W.
Published: August 2021

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend ERα cellular lifetime, accumulation, and are antagonists in the breast and agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we synthesized a series of methylpyrollidine lasofoxifene derivatives that maintained the drug’s antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated lasofoxifene derivatives, SERMs, and SERDs show that molecules that favor a highly buried helix 12 conformation achieve the greatest transcriptional suppression activities. Together these results show that chemical reduction of ERα cellular lifetime does not necessarily correlate with transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our approach shows how minor chemical additions modulate receptor cellular lifetime while maintaining other activities to achieve desired SERM or SERD profiles.

SIGNIFICANCE

This study shows that antiestrogens that enforce a wild-type-like antagonist conformation demonstrate improved therapeutic activities in hormone-resistant breast cancer cells harboring Y537S and D538G ESR1.
Sexual dimorphism in epilepsy and comorbidities in Dravet syndrome mice carrying a targeted deletion of exon 1 of the Scn1a gene
Authors: Gerbatin Rogério R. , Augusto Joana , Boutouil Halima , Reschke Cristina R. , Henshall David C.
Published: August 2021

Objective

Dravet Syndrome (DS) is a catastrophic form of paediatric epilepsy associated with multiple comorbidities mainly caused by mutations in the SCN1A gene. DS progresses in three different phases termed febrile, worsening and stabilization stage. Mice that are haploinsufficient for Scn1a faithfully model each stage of DS, although various aspects have not been fully described, including the temporal appearance and sex differences of the epilepsy and comorbidities. The aim of the present study was to investigate the epilepsy landscape according to the progression of DS and the long-term co-morbidities in the Scn1a(+/-)tm1Kea DS mouse line that are not fully understood yet.

Methods

Male and female F1.Scn1a(+/+) and F1.Scn1a(+/-)tm1Kea mice were assessed in the hyperthermia model or monitored by video electroencephalogram (vEEG) and wireless video-EEG according to the respective stage of DS. Long-term comorbidities were investigated through a battery of behaviour assessments in ∼6 month-old mice.

Results

At P18, F1.Scn1a(+/-)tm1Kea mice showed the expected sensitivity to hyperthermia-induced seizures. Between P21 and P28, EEG recordings in F1.Scn1a(+/-)tm1Kea mice combined with video monitoring revealed a high frequency of SRS and SUDEP. Power spectral analyses of background EEG activity also revealed that low EEG power in multiple frequency bands was associated with SUDEP risk in F1.Scn1a(+/-)tm1Kea mice during the worsening stage of DS. Later, SRS and SUDEP rates stabilized and then declined in F1.Scn1a(+/-)tm1kea mice. SRS and SUDEP in F1.Scn1a(+/-)tm1kea mice displayed variations with the time of day and sex, with female mice displaying higher numbers of seizures and greater SUDEP risk. F1.Scn1a(+/-)tm1kea mice ∼6 month- old displayed fewer behavioural impairments than expected including hyperactivity, impaired exploratory behaviour and poor nest building performance.

Significance

These results reveal new features of this model that will optimize use and selection of phenotype assays for future studies on the mechanisms, diagnosis, and treatment of DS.

Key point box

Scn1a(+/-)tm1kea DS mouse model faithfully reproduces the three stages of DS

Sex of F1.Scn1a(+/-)tm1kea mice influences the epilepsy phenotype

F1.Scn1a(+/-)tm1kea develop some of the long-term comorbidities of DS
Tightly-coupled inhibitory and excitatory functional networks in the early visual cortex
Authors: Mulholland Haleigh N. , Hein Bettina , Kaschube Matthias , Smith Gordon B.
Published: August 2021

Intracortical inhibition plays a critical role in shaping activity patterns in the mature cortex. However, little is known about the structure of inhibition in early development prior to the onset of sensory experience, a time when spontaneous activity exhibits long-range correlations predictive of mature functional networks. Here, using calcium imaging of GABAergic neurons in the early ferret visual cortex, we show that spontaneous activity in inhibitory neurons is already highly organized into distributed modular networks before visual experience. Inhibitory neurons exhibit spatially modular activity with long-range correlations and precise local organization that is in quantitative agreement with excitatory networks. Furthermore, excitatory and inhibitory networks are strongly co-aligned at both millimeter and cellular scales. These results demonstrate a remarkable degree of organization in inhibitory networks early in the developing cortex, providing support for computational models of self-organizing networks and suggesting a mechanism for the emergence of distributed functional networks during development.
Molecular Basis for CPC-Sgo1 Interaction: Implications for Centromere Localisation and Function of the CPC
Authors: Abad Maria Alba , Gupta Tanmay , Hadders Michael A , Meppelink Amanda , Wopken J Pepijn , Blackburn Elizabeth , Zou Juan , Buzuk Lana , Kelly David A , McHugh Toni , Rappsilber Juri , Lens Susanne M A , Jeyaprakash A Arockia
Published: August 2021

The Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and Aurora B kinase) and Shugoshin 1 (Sgo1) are key regulators of chromosome bi-orientation, a process essential for error-free chromosome segregation. Their functions rely on their ability to associate with centromeres. Two histone phosphorylations, histone H3 Thr3 (H3T3ph; directly recognised by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognised via Sgo1), together with CPC’s intrinsic ability to bind nucleosome, facilitate CPC centromere recruitment. The molecular basis for CPC-Sgo1 binding and how their direct interaction influences CPC centromere localisation and function are lacking. Here, using an integrative structure-function approach, we show that the histone H3-like Sgo1 N-terminal tail interacts with Survivin acting as a hot-spot for CPC-Sgo1 assembly, while downstream Sgo1 residues, mainly with Borealin contributes for high affinity interaction. Disruption of the Sgo1 N-terminal tail-Survivin interaction abolished CPC-Sgo1 assembly in vitro and perturbed centromere localisation and function of CPC. Our findings provide evidence that CPC binding to Sgo1 and histone H3 N-terminal tail are mutually exclusive, suggesting that these interactions will likely take place in a spatially/temporally restricted manner and provide a rationale for the Sgo1-mediated ‘kinetochore proximal centromere’ pool of CPC.
Combining genetic resources and elite material populations to improve the accuracy of genomic prediction in apple
Authors: Cazenave Xabi , Petit Bernard , Laurens François , Durel Charles-Eric , Muranty Hélène
Published: August 2021

Genomic selection is an attractive strategy for apple breeding that could reduce the length of breeding cycles. A possible limitation to the practical implementation of this approach lies in the creation of a training set large and diverse enough to ensure accurate predictions. In this study, we investigated the potential of combining two available populations, i.e. genetic resources and elite material, in order to obtain a large training set with a high genetic diversity. We compared the predictive ability of genomic predictions within-population, across-population or when combining both populations, and tested a model accounting for population-specific marker effects in this last case. The obtained predictive abilities were moderate to high according to the studied trait and were always highest when the two populations were combined into a unique training set. We also investigated the potential of such a training set to predict hybrids resulting from crosses between the two populations, with a focus on the method to design the training set and the best proportion of each population to optimize predictions. The measured predictive abilities were very similar for all the proportions, except for the extreme cases where only one of the two populations was used in the training set, in which case predictive abilities could be lower than when using both populations. Using an optimization algorithm to choose the genotypes in the training set also led to higher predictive abilities than when the genotypes were chosen at random. Our results provide guidelines to initiate breeding programs that use genomic selection when the implementation of the training set is a limitation.
The connecting cilium inner scaffold provides a structural foundation to maintain photoreceptor integrity
Authors: Mercey Olivier , Kostic Corinne , Bertiaux Eloïse , Giroud Alexia , Sadian Yashar , Chang Ning , Arsenijevic Yvan , Guichard Paul , Hamel Virginie
Published: August 2021

Retinal degeneration is a leading cause of human blindness due to progressive loss of ciliated photoreceptors cells. While this degradation can be associated with cohesion defects of the microtubule-based connecting cilium (CC) structure, the underlying mechanism is not understood. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC-inner scaffold (CC-IS) containing POC5, CENTRIN and FAM161A. Monitoring CC-IS assembly during photoreceptor development in mouse reveals that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Consistently, Fam161a mutations lead to a specific CC-IS loss and trigger microtubule doublets spreading, prior to outer segment collapse and photoreceptor degeneration, providing a molecular mechanism for retinitis pigmentosa disease.

One Sentence Summary

The connecting cilium inner scaffold acts as a structural zipper granting photoreceptor integrity.
Multiplex immunohistochemistry differences between Q fever and atherosclerotic abdominal aortic aneurysms indicate immune suppression
Authors: Cortenbach Kimberley R.G. , Staal Alexander H.J. , Schoffelen Teske , Gorris Mark A.J. , van der Woude Lieke L. , Jansen Anne F.M. , Poyck Paul , van Suylen Robert Jan , Wever Peter C. , Bleeker-Rovers Chantal P. , Srinivas Mangala , Hebeda Konnie M. , van Deuren Marcel , van der Meer Jos W.M. , De Vries I. Jolanda M. , van Kimmenade Roland R.J.
Published: August 2021

Background

Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue.

Methods

Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune system.

Results

Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+FoxP3+ regulatory T cells. Lastly, Q fever AAAs did not contain any well-defined granulomas.

Conclusions

These findings demonstrate that despite the presence of pro-inflammatory effector cells, there is an immune suppressive micro environment in Q fever AAA resulting in persistent local infection with C. burnetii.
A functional network target for tic reduction during thalamic stimulation for Tourette Syndrome
Authors: Baldermann Juan Carlos , Hennen Christina , Schüller Thomas , Andrade Pablo , Visser-Vandewalle Veerle , Horn Andreas , Dembek Till A. , Petry-Schmelzer Jan Niklas , Strelow Joshua Niklas , Jergas Hannah , Kuhn Jens , Barbe Michael T. , Huys Daniel
Published: August 2021

Background

Deep brain stimulation (DBS) of the medial thalamus is an evolving therapy for severe, treatment-refractory Tourette syndrome (TS). It remains unanswered which functionally connected networks need to be modulated to obtain optimal treatment results.

Methods

We assessed treatment response of 15 patients with TS untergoing thalamic DBS six and twelve months postoperatively using the Yale Global Tic Severity Scale (YGTSS) tic score. For each time point, functional connectivity maps seeding from stimulation sites were calculated based on a normative functional connectome derived from 1000 healthy subjects. Resulting maps were analyzed in a voxel-wise mixed model for repeated measurements to identify patterns of connectivity associated with tic reduction.

Results

Connectivity of stimulation to the medial frontal cortex, bilateral insulae and sensorimotor cortex was associated with tic reduction. Connectivity with the temporal lobe, cerebellum, ventral striatum and orbitofrontal cortex was negatively associated. The overall connectivity pattern was robust to leave-one-out cross-validation, explaining 25 % of outcome variance (R = 0.500; p = 0.005).

Conclusions

We delineated a functional connectivity profile seeding from stimulation sites associated with TS-DBS outcome. This pattern comprised areas linked to the processing of premonitory urges and tic execution, thereby extending our current understanding of effective neuromodulation for TS.
Neurosecretory protein GL-induced fat accumulation is accompanied by repressing the immune-inflammatory response in the adipose tissue of mice
Authors: Fukumura Keisuke , Narimatsu Yuki , Iwakoshi-Ukena Eiko , Furumitsu Megumi , Bono Hidemasa , Ukena Kazuyoshi
Published: August 2021

We have recently identified neurosecretory protein GL (NPGL), a small secretory protein expressed in the vertebrate hypothalamus, as an orexigenic factor with remarkable fat accumulation by overexpression of the NPGL precursor gene (Npgl) for two months. In the present study, we analyzed the effects of short-term Npgl overexpression for 18 days as the early stage of obesity to address the mechanisms underlying obese-like phenotype. Similar to previous studies, short-term Npgl overexpression stimulated food intake and fat accumulation in the white adipose tissues (WAT), whereas the masses of the brown adipose tissue, testis, liver, heart, and muscle remained unchanged. In addition, we observed increased blood insulin and leptin levels due to Npgl overexpression, while little changes were induced in blood glucose, free fatty acids, triglyceride, and cholesterol levels. Furthermore, transcriptome analysis of the inguinal WAT using RNA-sequencing technique revealed that overexpression of Npgl upregulated the genes involved in cytoskeleton regulation, whereas it decreased those involved in immune-inflammatory responses. These results suggest that NPGL plays a crucial role in enlarging adipocytes and suppressing inflammation to avoid metabolic abnormalities, eventually contributing to accelerating energy storage.
Multimodal measurements of phototoxicity in nonlinear optical microscopy
Authors: Zhang Xinyi , Dorlhiac Gabriel , Landry Markita P. , Streets Aaron
Published: August 2021

Nonlinear optical imaging modalities, such as two-photon microscopy and stimulated Raman scattering (SRS) microscopy, make use of pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we investigated the short and long-term effects of pulsed laser induced phototoxicity. We used bulk RNA sequencing, quantitative measurement of cell proliferation, and measurement of the generation of reactive oxygen species (ROS) to assess phototoxic effects, at different time scales, for a range of laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant ROS generation, differential gene expression, or change in proliferation rates of mouse Neuro2A cells. Changes in proliferation rate and ROS generation were observed under imaging conditions with an excitation intensity of over 600 mW/μm2. Repeated imaging of the same field of view at this excitation intensity of over 600 mW/μm2 resulted in visual damage to N2A cells. Laser induced perturbations in live cells may impact downstream measurements of cell state including subsequent imaging or molecular measurements. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.
HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage
Authors: Smith Rebecca , Zentout Siham , Chapuis Catherine , Timinszky Gyula , Huet Sébastien
Published: August 2021

PARP1 activity is regulated by its cofactor HPF1. The binding of HPF1 on PARP1 controls the grafting of ADP-ribose moieties on serine residues of proteins nearby the DNA lesions, mainly PARP1 and histones. However, the impact of HPF1 on DNA repair regulated by PARP1 remains unclear. Here, we show that HPF1 controls both the number and the length of the ADP-ribose chains generated by PARP1 at DNA lesions. We demonstrate that HPF1-dependent histone ADP-ribosylation, rather than auto-modification of PARP1, triggers the rapid unfolding of the chromatin structure at the DNA damage sites and promotes the recruitment of the repair factors CHD4 and CHD7. Together with the observation that HPF1 contributes to efficient repair both by homologous recombination and non-homologous end joining, our findings highlight the key roles played by this PARP1 cofactor at early stages of the DNA damage response.
Six1 Promotes Skeletal Muscle Thyroid Hormone Response through Regulation of the MCT10 Transporter
Authors: Girgis John , Yang Dabo , Chakroun Imane , Liu Yubing , Blais Alexandre
Published: August 2021

The Six1 transcription factor is implicated in controlling the development of several tissue types, notably skeletal muscle. Six1 also contributes to muscle metabolism and its activity is associated with the fast-twitch, glycolytic phenotype. Six1 regulates the expression of certain genes of the fast muscle program by directly stimulating their transcription or indirectly acting through a long non-coding RNA. Under the hypothesis that additional mechanisms of action might be at play, a combined analysis of gene expression profiling and genome-wide location analysis data was performed. TheSlc16a10gene, encoding the thyroid hormone transmembrane transporter MCT10, was identified as a gene with a transcriptional enhancer directly bound by Six1 and requiring Six1 activity for full expression in adult mouse tibialis anterior, a predominantly fast-twitch muscle. Of the various thyroid hormone transporters, MCT10 mRNA was found to be the most abundant in skeletal muscle, and to have a stronger expression in fast-twitch compared to slow-twitch muscle groups. Loss-of-function of MCT10 in the tibialis anterior recapitulated the effect of Six1 on the expression of fast-twitch muscle genes and led to lower activity of a thyroid hormone receptor-dependent reporter gene. These results shed light on the molecular mechanisms controlling the tissue expression profile of MCT10 and identify modulation of the thyroid hormone signaling pathway as an additional mechanism by which Six1 influences skeletal muscle metabolism.
Interorganelle lipid flux revealed by enzymatic mass tagging in vivo
Authors: John Peter Arun T. , Peter Matthias , Kornmann Benoît
Published: August 2021

The distinct activities of cellular organelles are dependent on the proper function of their membranes. Coordinated membrane biogenesis of the different organelles necessitates interorganelle transport of lipids from their site of synthesis to their destination membranes. Several proteins and trafficking pathways have been proposed to participate in lipid distribution, but despite the basic importance of this process, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. An obvious reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside living cells. In this strategy, two enzymes, one directed to a “donor” and the other to an “acceptor” organelle, add two distinct mass tags to lipids. Mass spectrometry-based detection of lipids bearing the two mass tags is then used as a proxy for lipid exchange between the two organelles. By applying this approach to ER and mitochondria, we show that the ERMES and Vps13-Mcp1 complexes have lipid transport activity in vivo, and unravel their relative contributions to ER-mitochondria lipid exchange.
Renal Interstitial Cells Promote Nephron Regeneration by Secreting Prostaglandin E2
Authors: Liu Xiaoliang , Yu Ting , Tan Xiaoqin , Jin Daqing , Zhang Jiangping , Zhang Yunfeng , Liao Shuyi , Yang Wenmin , Zhao Jinghong , Zhong Tao P , Liu Chi
Published: August 2021

In organ regeneration, progrnitor and stem cells reside in their native microenvironment, which provides dynamic physical and chemical cues essential to their survival, proliferation and differentiation. However, what kind of cells provide a native microenvironment for renal progenitor cells has not been clarified. Here, single-cell sequencing of zebrafish kidney revealed that fabp10 was a marker of renal interstitial cells (RICs), and the Tg(fabp10a:GFP) transgenic line can specifically label RICs in zebrafish kidney. The formation of RICs and nephrons are closely accompanied during nephron regeneration. RICs form a network to wrap the renal progenitor cell aggregates. RICs in close contact with cell aggregates express cyclooxygenase 2 and secrete prostaglandin 2 (PGE2). Inhibiting PGE2 production prevented nephrogenesis by reducing the proliferation and differentiation of progenitor cell aggregates. PGE2 promoted maturation of the nephron by activating the WNT signaling pathway in progenitor cell aggregates in cooperation with Wnt4a. These findings suggest that RICs provide a necessary microenvironment for rapid nephrogenesis during nephron regeneration.
A highly active phosphate-insensitive phosphatase is widely distributed in nature
Authors: Lidbury Ian D.E.A. , Scanlan David J. , Murphy Andrew R. J. , Christie-Oleza Joseph A. , Aguilo-Ferretjans Maria M. , Hitchcock Andrew , Daniell Tim
Published: August 2021

The regeneration of bioavailable phosphate from immobilised organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three major phosphatases, known as PhoA, PhoX and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these three phosphatase families is negatively regulated by phosphate availability and thus these enzymes play a major role in scavenging phosphorus only during times of phosphate scarcity. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent inBacteroidetes, which is highly abundant in nature and represents a major route for the remineralisation of phosphate in the environment. UsingFlavobacterium johnsoniaeas the model, we reveal PafA is highly active towards phosphomonoesters. Unlike other major phosphatases, PafA is fully functional in the presence of its metabolic product, phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. PafA, which is constitutively produced under all growth conditions tested, rapidly remineralises phosphomonoesters producing significant quantities of bioavailable phosphate that can cross feed into neighbouring cells.pafAis both abundant and highly expressed in the global ocean and abundant in plant rhizospheres, highlighting a new and important enzyme in the global phosphorus cycle with applied implications for agriculture as well as biogeochemical cycling. We speculate PafA expands the metabolic niche ofBacteroidetesby enabling utilisation of abundant organophosphorus substrates in the presence of excess phosphate, when other microbes are rendered incapable.

Significance statement

Phosphorus is an essential element for all life on Earth. Global primary production, and thus the ability for oceans and soils to drawdown atmospheric carbon dioxide, is in part controlled by the availability of inorganic phosphate. Likewise, global food production is also reliant on adequate supplies of phosphorus to both plants and animals. A major fraction of the total phosphorus pool exists as organic phosphorus, which requires mineralisation to phosphate prior to incorporation into cellular biomolecules. This important process is performed by enzymes known as phosphatases. Here, we reveal that the unique bacterial phosphatase, PafA, is a key player in the global phosphorus cycle and presents a major route for the regeneration of bioavailable phosphate required for both primary and secondary production.
Evolutionary analyses of visual opsin genes in anurans reveals diversity and positive selection suggestive of functional adaptation to distinct light environments
Authors: Schott Ryan K , Perez Leah , Kwiatkowski Matthew A , Imhoff Vance , Gumm Jennifer M
Published: August 2021

Among major vertebrate groups, anurans (frogs and toads) are understudied with regards to their visual systems and little is known about variation among species that differ in ecology. We sampled North American anurans representing diverse evolutionary and life histories that likely possess visual systems adapted to meet different ecological needs. Using standard molecular techniques, visual opsin genes, which encode the protein component of visual pigments, were obtained from anuran retinas. Additionally, we extracted the visual opsins from publicly available genome and transcriptome assemblies, further increasing the phylogenetic and ecological diversity of our dataset. We found that anurans consistently express four visual opsin genes (RH1, LWS, SWS1, and SWS2, but not RH2) even though reported photoreceptor complements vary widely among species. We found the first evidence of visual opsin duplication in an amphibian with the duplication of the LWS gene in the African bullfrog, which had distinct LWS copies on the sex chromosomes. The proteins encoded by these genes showed considerable sequence variation among species, including at sites known to shift the spectral sensitivity of visual pigments in other vertebrates and thus mediate dim-light and color vision. Using molecular evolutionary analyses of selection (dN/dS) we found significant evidence for positive selection at a subset of sites in the dim-light rod opsin gene RH1 and the long wavelength sensitive cone opsin gene LWS. The function of sites inferred to be under positive selection are largely unknown, but a few are likely to affect spectral sensitivity and other visual pigment functions based on proximity to previously identified sites in other vertebrates. The observed variation cannot fully be explained by evolutionary relationships among species alone. Taken together, our results suggest that other ecological factors, such as habitat and life history, as well as behaviour, may be driving changes to anuran visual systems.
Dietary methionine restriction impairs anti-tumor immunity through gut microbiota
Authors: Ji Ming , Xu Xiaojiang , Xu Qing , Xu Xin , Azcarate-Peril M. Andrea , Wu Xiaoyue , Liu Juan , Locasale Jason W. , Li Jian-Liang , Shats Igor , Li Xiaoling
Published: August 2021

Dietary methionine restriction has been reported to repress cancer growth and improve therapeutic responses in several pre-clinical settings. However, how this dietary intervention impacts cancer progression in the context of the intact immune system is unknown. Here we report that methionine restriction exacerbates cancer growth and influences the outcomes of anti-tumor immunotherapy through gut microbiota and immune suppression in immunocompetent settings. Methionine restriction reduces T cell activation, increases tumor growth, and impairs response to anti-tumor immunotherapy. Mechanistically, methionine restriction alters composition of gut microbiota and reduces microbial production of hydrogen sulfide. Fecal transplantation from methionine-restricted tumor-free animals is sufficient to repress T cell activation and enhance tumor growth in tumor-bearing recipient mice. Conversely, dietary supplementation of a hydrogen sulfide donor or methionine stimulates anti-tumor immunity and suppresses tumor progression. Our findings reveal a vital role of gut microbiota in mediating methionine restriction-induced suppression of anti-tumor immunity and suggest that any possible anti-cancer benefits of methionine restriction require careful considerations of both the microbiota and the immune system.