bluuebunny/crossref_metadata_2025 huggingface dataset:
Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogenCryptococcus neoformans: a target for novel antifungals
Authors: Washington Erica J. , Zhou Ye , Hsu Allen L. , Petrovich Matthew , Tenor Jennifer L. , Toffaletti Dena L. , Guan Ziqiang , Perfect John R. , Borgnia Mario J. , Bartesaghi Alberto , Brennan Richard G.
Published: March 2023

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-lengthCryptococcus neoformansTps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies residues required for substrate-binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the “solvent” but is not visible in density maps. We determined that the IDD is not required forC. neoformansTps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1.In toto, these studies expand our knowledge of trehalose biosynthesis inCryptococcusand highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

Significance Statement

Fungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to transition from the environment to the human host. Enzymes in the trehalose biosynthesis pathway are absent in humans and, therefore, are potentially significant targets for novel antifungal therapeutics. One enzyme in the trehalose biosynthesis is trehalose-6-phosphate synthase (Tps1). Here, we describe the cryo-electron microscopy structures of the CnTps1 homo-tetramer in the unliganded form and in complex with a substrate and a product. These structures and subsequent biochemical analysis reveal key details of substrate-binding residues and substrate specificity. These structures should facilitate structure-guided design of inhibitors against CnTps1.
Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast
Authors: Guesdon Gabrielle , Gourgues Géraldine , Rideau Fabien , Ipoutcha Thomas , Manso-Silván Lucía , Jules Matthieu , Sirand-Pugnet Pascal , Blanchard Alain , Lartigue Carole
Published: March 2023

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeastSaccharomyces cerevisiaeis considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of in-yeast cloning strategies, a new approach combining two already described methods, the Fusion cloning and the CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from severalMycoplasmaspecies belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves cloning efficacy.
Genomic surveillance reveals circulation of multiple variants and lineages of SARS-CoV-2 during COVID-19 pandemic in Indian city of Bengaluru
Authors: Sreenivas Darshan , Kumar Vinay , Kathirvel Kandasamy , Vadnala Rakesh Netha , Mishra Sachin , Shelar Bhagyashree , Marate Srilatha , CP Lakshminarayanan , K Sai Disha , Bhardwaj Manisha , Pandit Awadhesh , Mayor Satyajit , Ramakrishnan Uma , Notani Dimple
Published: March 2023

Genomic surveillance in response to coronavirus disease (COVID-19) pandemic is crucial for tracking spread, identify variants of concern (VoCs) and understand the evolution of its etiological agent, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). India has experienced three waves of COVID-19 cases, which includes a deadly wave of COVID-19 that was driven by the Delta lineages (second/Delta wave) followed by another wave driven by the Omicron lineages (third/Omicron wave). These waves were particularly dramatic in the metropolitan cities due to high population density. We evaluated the prevalence, and mutational spectrum of SARS-CoV-2 variants/lineages in one such megapolis, Bengaluru city, across these three waves between October 2020 and June 2022. 15,134 SARS-CoV-2 samples were subjected to whole genome sequencing (WGS). Phylogenetic analysis revealed, SARS-CoV-2 variants in Bengaluru city belonged to 18 clades and 196 distinct lineages. As expected, the Delta lineages were the most dominant lineages during the second wave of COVID-19. The Omicron lineage BA.2 and its sublineages accounted for most of the COVID-19 cases in the third wave. Most number of amino acid changes were observed in spike protein. Among the 18 clades, majority of the mutations and least similarity at nucleotide sequence level with the reference genome were observed in Omicron clades.
Beta bursts correlate with synchronization of movements to rhythmic sounds
Authors: Chen Qiaoyu , McAllister Craig J. , Elliott Mark T. , Shapiro Kimron L. , Hanslmayr Simon
Published: March 2023

Accumulating evidence indicates transient beta bursts play an important role in the representation of temporal information and prediction. However, the role of beta bursts in sensorimotor synchronization (SMS) involving active interactions between motor and sensory systems to synchronize predictive movements to periodic events remains unclear. To answer this question, 15 participants were invited to complete a finger-tapping task whilst high-density EEG (128 channels) was recorded. Participants tapped with their right index finger in synchrony with 1 Hz and 0.5 Hz tone trains. In line with previous findings, we found a negative mean asynchrony between tone and tap time, i.e., taps preceded tones for both tone frequencies (1 and 0.5 Hz). In the EEG data, beta bursts were detected and their timing in relationship with tapping and auditory tracking was examined. Results revealed that beta bursts tracked tapping and were modulated by the low frequency phase of the tone frequency (i.e., 1 Hz or 0.5 Hz). Importantly, the locking of beta bursts to the phase of auditory tracking correlated with the behavioural variance on a single trial level that occurred while tapping to the tones. These results demonstrate a critical role for an interplay between beta bursts and low frequency phase in coordinating rhythmic behaviour.
Cis-regulatory control of transcriptional timing and noise in response to estrogen
Authors: Ginley-Hidinger Matthew , Abewe Hosiana , Osborne Kyle , Richey Alexandra , Kitchen Noel , Mortenson Katelyn L. , Wissink Erin M. , Lis John , Zhang Xiaoyang , Gertz Jason
Published: March 2023

Cis-regulatory elements control transcription levels, temporal dynamics, and cell-cell variation or transcriptional noise. However, the combination of regulatory features that control these different attributes is not fully understood. Here, we used single cell RNA-seq during an estrogen treatment time course and machine learning to identify predictors of expression timing and noise. We find that genes with multiple active enhancers exhibit faster temporal responses. We verified this finding by showing that manipulation of enhancer activity changes the temporal response of estrogen target genes. Analysis of transcriptional noise uncovered a relationship between promoter and enhancer activity, with active promoters associated with low noise and active enhancers linked to high noise. Finally, we observed that co-expression across single cells is an emergent property associated with chromatin looping, timing, and noise. Overall, our results indicate a fundamental tradeoff between a gene’s ability to quickly respond to incoming signals and maintain low variation across cells.
Development of prefrontal corticostriatal connectivity in mice
Authors: Mesías Roxana E. , Zaki Yosif , Guevara Christopher A. , Friedman Lauren G. , Hussein Ayan , Therrien Karen , Magee Alexandra R. , Tzavaras Nikolaos , Valle Pamela Del , Baxter Mark G. , Huntley George W. , Benson Deanna L.
Published: March 2023

Rational decision making is grounded in learning to associate actions with outcomes, a process that depends on projections from prefrontal cortex to dorsomedial striatum. Symptoms associated with a variety of human pathological conditions ranging from schizophrenia and autism to Huntington’s and Parkinson’s disease point toward functional deficits in this projection, but its development is not well understood, making it difficult to investigate how perturbations in development of this circuitry could contribute to pathophysiology. We applied a novel strategy based on Hotspot Analysis to assess the developmental progression of anatomical positioning of prefrontal cortex to striatal projections. Corticostriatal axonal territories established at P7 expand in concert with striatal growth but remain largely unchanged in positioning through adulthood, indicating they are generated by directed, targeted growth and not modified extensively by postnatal experience. Consistent with these findings, corticostriatal synaptogenesis increased steadily from P7 to P56, with no evidence for widescale pruning. As corticostriatal synapse density increased over late postnatal ages, the strength of evoked PFC input onto dorsomedial striatal projection neurons also increased, but spontaneous glutamatergic synaptic activity was stable. Based on its pattern of expression, we asked whether the adhesion protein, Cdh8, influenced this progression. In mice lacking Cdh8 in PFC corticostriatal projection neurons, axon terminal fields in dorsal striatum shifted ventrally. Corticostriatal synaptogenesis was unimpeded, but spontaneous EPSC frequency declined and mice failed to learn to associate an action with an outcome. Collectively these findings show that corticostriatal axons grow to their target zone and are restrained from an early age, do not undergo postnatal synapse pruning as the most dominant models predict, and that a relatively modest shift in terminal arbor positioning and synapse function has an outsized, negative impact on corticostriatal-dependent behavior.
Deciphering the gut microbiome of grass carp through multi-omics approach
Authors: Li Ming , Liang Hui , Yang Hongwei , Ding Qianwen , Xia Rui , Chen Jie , Zhou Wenhao , Yang Yalin , Zhang Zhen , Yao Yuanyuan , Ran Chao , Zhou Zhigang
Published: March 2023

Background

Aquaculture plays an important role in global protein supplies and food security. The ban on antibiotics as feed additive proposes urgent need to develop alternatives. Gut microbiota plays important roles in the metabolism and immunity of fish, and has the potential to give rise to novel green inputs for fish culture. However, our understanding of fish gut microbiome is still lacking.

Results

We identified 575,856 non-redundant genes by metagenomic sequencing of the intestinal content samples of grass carp. Taxonomic and functional annotation of the gene catalogue revealed specificity of the gut microbiome of grass carp compared with mammals. Co-occurrence analysis indicated exclusive relations between the genera belonging to Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes, suggesting two independent ecological groups of the microbiota. The association pattern of Proteobacteria with the gene expression modules of fish gut and liver was consistently opposite to that of Fusobacteria, Firmicutes and Bacteroidetes, implying differential functionality of Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes. Therefore, the two ecological groups were divided into two functional groups, i.e., Functional Group 1: Proteobacteria; Functional Group 2: Fusobacteria/Firmicutes/Bacteroidetes. Further analysis revealed that the two functional groups differ in genetic capacity for carbohydrate utilization, virulence factors and antibiotic resistance. Finally, we proposed that the ratio of “Functional Group 2/Functional Group 1” can be used as a biomarker that efficiently reflects the structural and functional characteristics of the microbiota of grass carp.

Conclusions

The gene catalogue is an important resource for investigating the gut microbiome of grass carp. Multi-omics analysis provides insights into functional implications of the main phyla that comprise the fish microbiota, and shed lights on targets for microbiota regulation.
How much storage precision can be lost: Guidance for near-lossless compression of untargeted metabolomics mass spectrometry data
Authors: Tong Junjie , Lu Miaoshan , Peng Bichen , An Shaowei , Wang Jinyin , Yu Changbin
Published: March 2023

The size of high-resolution mass spectrometry (HRMS) data has been increasing significantly. Several lossy compressors have been developed for higher compression rates. Currently, a comprehensive evaluation of what and how MS data (m/zand intensities) with precision losses would affect data processing (i.e., feature detection and compound identification) is absent.

Here, we set an error threshold at 1% to assess the significance of the difference between two files in feature and compound detection results obtained from MZmine3. First, we examined that mzML files with bothm/zand intensity encoded in 32-bit precision appear to be a preferred combination via msConvert, which has smaller file size and minor variation with other combinations of storage precision (<0.13%). We then identified that the absolute error of 10−4form/zhad a feature detection error of 0.57% and compound detection error of 1.1%. For intensities, the relative error group of 2×10−2had an error of 4.65% for features and 0.98% for compounds, compared with precision-lossless files. Taken together, we provided a reasonable scene-accuracy proposal, with a maximum absolute error of 10−4form/zand a maximum relative error of 2×10−2for intensity. This guidance aimed to help researchers in improving lossy compression algorithms and minimizing the negative effects of precision losses on downstream data processing.
A rapid genome-wide analysis of isolated giant viruses only using MinION sequencing
Authors: Hikida Hiroyuki , Okazaki Yusuke , Zhang Ruixuan , Nguyen Thi Tuyen , Ogata Hiroyuki
Published: March 2023

Following the discovery of Acanthamoeba polyphaga mimivirus, diverse giant viruses have been isolated. However, only a small fraction of these isolates has been completely sequenced, limiting our understanding of the genomic diversity of giant viruses. MinION is a portable and low-cost long-read sequencer that can be readily used in a laboratory. Although MinION provides highly error-prone reads that require correction through additional short-read sequencing, recent studies assembled high-quality microbial genomes only using MinION sequencing. Here, we evaluated the accuracy of MinION-only genome assemblies for giant viruses by re-sequencing a prototype marseillevirus. Assembled genomes presented over 99.98% identity to the reference genome with a few gaps, demonstrating a high accuracy of the MinION-only assembly. As a proof of concept, wede novoassembled five newly isolated viruses. Average nucleotide identities to their closest known relatives suggest that the isolates represent new species of marseillevirus, pithovirus, and mimivirus. Assembly of subsampled reads demonstrated that their taxonomy and genomic composition could be analyzed at the 50× sequencing coverage. We also identified a pithovirus gene whose homologues were detected only in metagenome-derived relatives. Collectively, we propose that MinION-only assembly is an effective approach to rapidly perform a genome-wide analysis of isolated giant viruses.
WWOX promotes osteosarcoma development via upregulation of Myc
Authors: Akkawi Rania , Hidmi Osama , Yehya Ameen Haji , Monin Jonathon , Diment Judith , Drier Yotam , Stein Gary S. , Aqeilan Rami I.
Published: March 2023

Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion ofWwoxandTrp53usingOsterix1-Cretransgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.
SpatialScope: A unified approach for integrating spatial and single-cell transcriptomics data using deep generative models
Authors: Wan Xiaomeng , Xiao Jiashun , Tam Sindy Sing Ting , Cai Mingxuan , Sugimura Ryohichi , Wang Yang , Wan Xiang , Lin Zhixiang , Wu Angela Ruohao , Yang Can
Published: March 2023

The rapid emergence of spatial transcriptomics (ST) technologies are revolutionizing our under-standing of tissue spatial architecture and their biology. Current ST technologies based on either next generation sequencing (seq-based approaches) or fluorescence in situ hybridization (image-based approaches), while providing hugely informative insights, remain unable to provide spatial characterization at transcriptome-wide single-cell resolution, limiting their usage in resolving detailed tissue structure and detecting cellular communications. To overcome these limitations, we developed SpatialScope, a unified approach to integrating scRNA-seq reference data and ST data that leverages deep generative models. With innovation in model and algorithm designs, SpatialScope not only enhances seq-based ST data to achieve single-cell resolution, but also accurately infers transcriptome-wide expression levels for image-based ST data. We demonstrate the utility of SpatialScope through comprehensive simulation studies and then apply it to real data from both seq-based and image-based ST approaches. SpatialScope provides a spatial characterization of tissue structures at transcriptome-wide single-cell resolution, greatly facilitating the downstream analysis of ST data, such as detection of cellular communication by identifying ligand-receptor interactions from seq-based ST data, localization of cellular subtypes, and detection of spatially differently expressed genes.
Lifespan Neurodegeneration Of The Human Brain In Multiple Sclerosis
Authors: Coupé Pierrick , Planche Vincent , Mansencal Boris , Kamroui Reda A. , Koubiyr Ismail , Manjon José V. , Tourdias Thomas
Published: March 2023

Background

Atrophy related to Multiple Sclerosis (MS) has been found at the early stages of the disease. However, the archetype dynamic trajectories of the neurodegenerative process, even prior to clinical diagnosis, remain unknown.

Methods

We modeled the volumetric trajectories of brain structures across the entire lifespan using 40944 subjects (38295 healthy controls and 2649 MS patients). Then, we estimated the chronological progression of MS by assessing the divergence of lifespan trajectories between normal brain charts and MS brain charts.

Results

Chronologically, the first affected structure was the thalamus, then the putamen and the pallidum (3 years later), followed by the ventral diencephalon (7 years after thalamus) and finally the brainstem (9 years after thalamus). To a lesser extent, the anterior cingulate gyrus, insular cortex, occipital pole, caudate and hippocampus were impacted. Finally, the precuneus and accumbens nuclei exhibited a limited atrophy pattern.

Conclusion

Subcortical atrophy was more pronounced than cortical atrophy. The thalamus was the most impacted structure with a very early divergence in life. It paves the way toward utilization of these lifespan models for future preclinical/prodromal prognosis and monitoring of MS.
Identification of potential selective autophagy receptors from protein-content profiling of autophagosomes
Authors: Cristiani Alberto , Dutta Arghya , Poveda-Cuevas Sergio Alejandro , Kern Andreas , Bhaskara Ramachandra M.
Published: March 2023

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts e.g., under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)–LIR-docking site (LDS) and Ubiquitin-interacting motif (UIM)–UIM-docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy.
Perception of near-threshold visual stimuli is influenced by pre-stimulus alpha-band amplitude but not by alpha phase
Authors: Melcón María , Stern Enrique , Kessel Dominique , Arana Lydia , Poch Claudia , Campo Pablo , Capilla Almudena
Published: March 2023

Ongoing brain activity preceding visual stimulation has been suggested to shape conscious perception. The underlying mechanisms are still under debate, although alpha oscillations have been pointed out as the main explanatory candidate. According to the pulsed-inhibition framework, bouts of functional inhibition arise in each alpha cycle, allowing information to be processed in a pulsatile manner. Consequently, it has been hypothesized that perceptual outcome can be influenced by the specific phase of alpha oscillations prior to the stimulus onset, although empirical findings are controversial. In this study, we aimed to shed light on the role of pre-stimulus alpha oscillations in visual perception. To this end, we recorded electroencephalographic (EEG) activity while participants performed three near-threshold visual detection tasks with different attentional involvement: a no-cue task, a non-informative cue task (50% cue validity), and an informative cue task (100% cue validity). Cluster-based permutation statistics were complemented with Bayesian analyses to test the effect of pre-stimulus oscillatory amplitude and phase on visual awareness. We additionally examined whether these effects differed on trials with low and high oscillatory amplitude, as expected from the pulsed-inhibition theory. Our results show a clear effect of pre-stimulus alpha amplitude on conscious perception, but only when alpha fluctuated spontaneously and was not modulated by attention, supporting the notion that alpha-band power indexes neural excitability. In contrast, we did not find any evidence that pre-stimulus alpha phase influences the perceptual outcome, not even when differentiating between low and high amplitude trials. Furthermore, Bayesian analysis provided moderate evidence in favor of the absence of phase effects. Taken together, our results challenge the central theoretical predictions of the pulsed-inhibition framework, at least for the particular experimental conditions used here.
Engineering the biological conversion of formate into crotonate inCupriavidus necator
Authors: Collas Florent , Dronsella Beau B. , Kubis Armin , Schann Karin , Binder Sebastian , Arto Nils , Claassens Nico J. , Kensy Frank , Orsi Enrico
Published: March 2023

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO2and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacteriumC. necatoras cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growingC. necatorin minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacteriumviaa modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse β-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.
Topography of inputs into the hippocampal formation of a food-caching bird
Authors: Applegate Marissa C. , Gutnichenko Konstantin S. , Aronov Dmitriy
Published: March 2023

The mammalian hippocampal formation (HF) is organized into domains associated with different functions. These differences are driven in part by the pattern of input along the hippocampal long axis, such as visual input to the septal hippocampus and amygdalar input to temporal hippocampus. HF is also organized along the transverse axis, with different patterns of neural activity in the hippocampus and the entorhinal cortex. In some birds, a similar organization has been observed along both of these axes. However, it is not known what role inputs play in this organization. We used retrograde tracing to map inputs into HF of a food-caching bird, the black-capped chickadee. We first compared two locations along the transverse axis: the hippocampus and the dorsolateral hippocampal area (DL), which is analogous to the entorhinal cortex. We found that pallial regions predominantly targeted DL, while some subcortical regions like the lateral hypothalamus (LHy) preferentially targeted the hippocampus. We then examined the hippocampal long axis and found that almost all inputs were topographic along this direction. For example, the anterior hippocampus was preferentially innervated by thalamic regions, while posterior hippocampus received more amygdalar input. Some of the topographies we found bear resemblance to those described in the mammalian brain, revealing a remarkable anatomical similarity of phylogenetically distant animals. More generally, our work establishes the pattern of inputs to HF in chickadees. Some of these patterns may be unique to chickadees, laying the groundwork for studying the anatomical basis of these birds’exceptional hippocampal memory.
Hem25p is a mitochondrial IPP transporter
Authors: Tai Jonathan , Guerra Rachel M. , Rogers Sean W. , Fang Zixiang , Muehlbauer Laura K. , Shishkova Evgenia , Overmyer Katherine A. , Coon Joshua J. , Pagliarini David J.
Published: March 2023

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor comprised of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing, and targeted uptake assays, we reveal that Hem25p—a mitochondrial glycine transporter required for heme biosynthesis—doubles as an isopentenyl pyrophosphate (IPP) transporter inSaccharomyces cerevisiae. Mitochondria lacking Hem25p fail to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p inEscherichia colienables robust IPP uptake demonstrating that Hem25p is sufficient for IPP transport. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in yeast.
Determine transposable genes when the orders of genes are different
Authors: Wang Yue
Published: March 2023

Certain nucleotide sequences in DNA can change their positions. Such nucleotide sequences might be shorter than a general gene. When we restrict to nucleotide sequences that form complete genes, we can still find genes that change their relative locations in a genome. Thus for different individuals of the same species, the orders of genes might be different. Such spatial difference of gene orders might be affected by temporal difference of gene (mutation) orders, and can be used to explain the order of mutation problem in myeloproliferative neoplasm. A practical problem is to determine such transposable genes in given gene sequences. Through an intuitive rule, we transform the biological problem of determining transposable genes into a rigorous mathematical problem of determining the longest common subsequence. Given several number sequences, determining the longest common subsequence is a classical problem in computer science. Depending on whether the gene sequence is linear or circular, and whether genes have multiple copies, we classify the problem of determining transposable genes into different scenarios and design corresponding algorithms. Specifically, we study the situation where the longest common subsequence is not unique.
Titering of Chimeric Antigen Receptors on CAR T Cells enabled by a Microfluidic-based Dosage-Controlled Intracellular mRNA Delivery Platform
Authors: Chen Yu-Hsi , Jiang Ruoyu , Lee Abraham P.
Published: March 2023

Chimeric antigen receptor (CAR) T-cell therapy shows unprecedented efficacy for cancer treatment, particularly in treating patients with various blood cancers, most notably B-cell acute lymphoblastic leukemia (B-ALL). In recent years, CAR T-cell therapies are being investigated for treating other hematologic malignancies and solid tumors. Despite the remarkable success of CAR T-cell therapy, it has unexpected side effects that are potentially life threatening. Here, we demonstrate the delivery of approximately the same amount of CAR gene coding mRNA into each T cell propose an acoustic-electric microfluidic platform to manipulate cell membranes and achieve dosage control via uniform mixing, which delivers approximately the same amount of CAR genes into each T cell. We also show that CAR expression density can be titered on the surface of primary T cells under various input power conditions using the microfluidic platform.
A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State
Authors: Romero-Tejeda Marisol , Fonoudi Hananeh , Weddle Carly J. , DeKeyser Jean-Marc , Lenny Brian , Fetterman K. Ashley , Magdy Tarek , Sapkota Yadav , Epting Conrad , Burridge Paul W.
Published: March 2023

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination ofMYOCD,SMAD6, andTBX20(MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

GRAPHICAL ABSTRACT

HIGHLIGHTS

Using network-based algorithm Mogrify, acoustic liquid handling, and high-content kinetic imaging cytometry we screened the effect of 4,960 unique transcription factor combinations.

Using 24 patient-specific human fibroblast samples we identified the combination ofMYOCD,SMAD6, andTBX20(MST) as the most successful direct reprogramming combination.

MST cocktail results in reprogrammed cells with spontaneous contraction, cardiomyocyte-like calcium transients, and expression of cardiomyocyte associated genes.
Phosphorylation barcodes direct biased chemokine signaling at CXCR3
Authors: Eiger Dylan S. , Smith Jeffrey S. , Shi Tujin , Stepniewski Tomasz Maciej , Tsai Chia-Feng , Honeycutt Christopher , Boldizsar Noelia , Gardner Julia , Nicora Carrie D. , Moghieb Ahmed M. , Kawakami Kouki , Choi Issac , Zheng Kevin , Warman Anmol , Alagesan Priya , Knape Nicole M. , Huang Ouwen , Silverman Justin D. , Smith Richard D. , Inoue Asuka , Selent Jana , Jacobs Jon M. , Rajagopal Sudarshan
Published: March 2023

G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or “phosphorylation barcodes.” At chemokine receptors, ligands act as “biased agonists” with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered β-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes.
Speed-dependent optimization of gravity effects for motor control
Authors: Poirier Gabriel , Mourey France , Sirandre Cyril , Papaxanthis Charalambos , Gaveau Jeremie
Published: March 2023

Several sensorimotor control studies have provided evidence supporting that the central nervous system optimizes gravity’s effects to minimize muscle effort. Recently, this hypothesis has been supported by the consistent observation of direction-specific negative epochs in the phasic electromyographic signal of antigravity muscles during vertical arm movements. This suggests that gravity torque is harvested to produce some of the arm’s motion. However, further investigation is needed to more finely understand how the CNS integrates gravity effects into muscle commands. Here, we aimed to analyze the phasic muscular activity across varying movement speeds during horizontal and vertical arm movements. We quantified the amount of negativity during acceleration and deceleration phases for all movement directions during fast, natural, and slow movements. We found that the negativity was more important during the acceleration phase of downward movements and during the deceleration phase of upward movements, resulting in diminished phasic activity compared to horizontal movements. Concomitantly, we found direction-specific effects of movement speed on phasic EMG activity of gravity muscles. This resulted in altered EMG to kinematics relationships in vertical movements compared to horizontal ones. These results support theEffort-minimization hypothesisand confirm that the negativity of phasic EMG is an important aspect of the motor command. Furthermore, the present results reveal that the CNS finely tunes this feature across a range of movement speeds and directions.
In search of a unifying theory of white matter aging: improving the understanding of tract-wise degeneration using multi-parametric signatures of morphometry and microstructure
Authors: Robinson Tyler D. , Sun Yutong L. , Chang Paul T. H. , Chen J. Jean
Published: March 2023

While tract-wise differences in volume and microstructure are common targets of investigation in age-related changes in the white matter (WM), there has been relatively little exploration into other attributes of tract morphometry or its relation to microstructure in vivo, and limited understanding on how they jointly inform the interpretation of the WM aging trajectory. This study examines ten WM tracts for tract-wise differences in morphometry (i.e. volume, length, and volume-to-length ratio) and microstructural integrity (i.e. fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity) using diffusion MRI data from the Human Connectome Project in Aging (HCP-A) with the goal of laying the foundation for a unified model of age-related WM microstructure-morphometry trajectories with a special focus on sex differences. Results indicated widely heterogeneous patterns of decline and morphometry-microstructural associations across tracts. Multi-parametric signatures of decline suggest stages or mechanisms of degeneration that differ between sexes. This work highlights the value of integrating microstructural and morphometric measures of WM health instead of observing them separately, suggesting multiple modes of WM degeneration.
Rational design of immune gene therapy combinations viain vivoCRISPR activation screen of tumor microenvironment modulators
Authors: Wang Guangchuan , Zhang Feifei , Chow Ryan D. , He Emily , Zhu Lvyun , Han Qin , Chen Sidi
Published: March 2023

The hostile tumor microenvironment (TME) is major challenge for cancer immunotherapies. Here, we design and perform TME-targetedin vivoCRISPR activation (CRISPRa) screens to uncover factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Through adeno-associated virus (AAV) delivery, multiplexed activation of pooled immunoregulatory genes encodingantigenpresentation,cytokine, and co-stimulationmolecules (APCM) leads to enhanced anti-tumor immunity. APCM screen in metastatic tumors identifiesCd80, Tnfsf14, Cxcl10, Tnfsf18, Tnfsf9, andIfngas the top immunostimulatory candidates. AAV-mediated delivery of these factors individually or in combination shows anti-tumor efficacy across different cancer models. Further optimization pinpointsIfng+Tnfsf9+Il12b(Il12/Il23)as a potent therapeutic combination, leading to increased IFN-γ+CD8+and tissue-resident memory T cells. APCM therapy synergizes with CAR-T cell therapy against human solid tumorsin vivo. APCM-based CRISPRa screen and gene activation systems can thus be leveraged for the rapid generation of off-the-shelf immune gene therapies against solid tumors.
Multiplexed inhibition of immunosuppressive genes with Cas13d for on-demand combinatorial cancer immunotherapy
Authors: Zhang Feifei , Guangchuan Wang , Chow Ryan , He Emily , Majety Medha , Zhang Yueqi , Chen Sidi
Published: March 2023

Checkpoint blockade immunotherapy is a potent class of cancer treatment, however, the complex immunosuppressive tumor microenvironment (TME) often requires multi-agent combinations to be effective. Current cancer immunotherapy combination approaches are cumbersome, usually involving one-drug-at-a-time scheme. Here, we devise Multiplex Universal Combinatorial Immunotherapy via Gene-silencing (MUCIG), as a versatile approach for combinatorial cancer immunotherapy. We harness CRISPR-Cas13d to efficiently target multiple endogenous immunosuppressive genes on demand, allowing us to silence various combinations of multiple immunosuppressive factors in the TME. Intratumoral AAV-mediated administration of MUCIG (AAV-MUCIG) elicits significant anti-tumor activity with several Cas13d gRNA compositions. TME target expression analysis driven optimization led to a simplified off-the-shelf MUCIG targeting a four gene combination (PGGC:Pdl1, Galectin9, Galectin3andCd47). AAV-PGGC shows significantin vivoefficacy in syngeneic tumor models. Single cell and flow profiling revealed that AAV-PGGC remodeled the TME by increasing CD8+T cell infiltration and reducing myeloid-derived immunosuppressive cells (MDSCs). MUCIG thus serves as a universal method to silence multiple immune genesin vivo,and can be delivered via AAV as a therapeutic approach.
Divergent roles for caspase-8 and MLKL in high-fat diet induced obesity and NAFLD in mice
Authors: Tye Hazel , Conos Stephanie A. , Djajawi Tirta M. , Abdoulkader Nasteho , Kong Isabella Y. , Kammoun Helene L. , Narayana Vinod K. , Speir Mary , Gottschalk Timothy A. , Emery Jack , Simpson Daniel S. , Hall Cathrine , Vince Angelina J. , Russo Sophia , Crawley Rhiannan , Rashidi Maryam , Hildebrand Joanne M. , Murphy James M. , Whitehead Lachlan , De Souza David P. , Masters Seth L. , Hawkins Edwin D. , Murphy Andrew J. , Vince James E. , Lawlor Kate E.
Published: March 2023

Cell death frequently occurs in the pathogenesis of obesity and non-alcoholic fatty liver disease (NAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we showviathe direct comparison of mice genetically deficient in apoptotic caspase-8 in myeloid cells, or the essential necroptotic regulators, Receptor-interacting protein kinase-3 (RIPK3) and Mixed lineage kinase domain-like (MLKL), that RIPK3-caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and NAFLD upon high-fat diet feeding. In contrast, MLKL, divergent to RIPK3, contributes to both obesity and NAFLD in a manner largely independent of inflammation. We also uncover that MLKL regulates the expression of molecules involved in lipid uptake, transport and metabolism and, congruent with this, we discover a shift in the hepatic lipidome upon MLKL deletion. Collectively, these findings highlight MLKL as an attractive therapeutic target to combat the growing obesity pandemic and metabolic disease.