bluuebunny/crossref_metadata_2025 huggingface dataset:
Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping
Authors: Nishimura Kazusa , Kokaji Hiroyuki , Motoki Ko , Yamazaki Akira , Nagasaka Kyoka , Takisawa Rihito , Yasui Yasuo , Kawai Takashi , Ushijima Koichiro , Yamasaki Masanori , Saito Hiroki , Nakano Ryohei , Nakazaki Tetsuya
Published: August 2022

Multiplexed inter-simple sequence repeats genotyping by sequencing (MIG-seq) is an next-generation sequencing library construction method developed for the analysis of DNA in ecology. Although MIG-seq can generate libraries from low-quality DNA, few polymorphisms can be obtained in species with small genomes. In this study, we developed degenerate oligonucleotide primer MIG-seq (dpMIG-seq) as an effective polymorphism discovery method that allows for variation in the number of polymorphisms while retaining the advantages of MIG-seq, including independence from DNA quality. In dpMIG-seq, a proportion of the simple sequence repeats in the primer sequence of the first PCR in MIG-seq was changed to degenerate oligonucleotides to enable annealing to a wider range of sequences. In tests of several crop species other than wheat, the number of loci that could be sequenced using dpMIG-seq with a data volume of 0.3 gigabases (Gb) was increased compared with that sequenced using MIG-seq. In wheat, the number of polymorphisms obtained via dpMIG-seq was higher than that obtained via MIG-seq when a data volume of about ≥2 Gb was obtained. In dpMIG-seq, different loci could be sequenced by changing the positions of the degenerate oligonucleotides. By applying dpMIG-seq, we constructed a linkage map consisting of 5,142 markers for the rice inter-subspecies F2population, and we detected quantitative trait loci for heading date in the regions where known heading-related genes were located. Overall, our results show that dpMIG-seq is a useful tool for the genetic analysis of crop species.
Racial and Sociodemographic Disparities in Blindness Associated with Primary Angle Closure Glaucoma in the United States: An IRIS® Registry Analysis
Authors: Shah Sona N. , Zhou Sarah , Sanvicente Carina , Burkemper Bruce , Apolo Galo , Li Charles , Li Siying , Liu Lynn , Lum Flora , Moghimi Sasan , Xu Benjamin
Published: August 2022

Purpose

To assess the prevalence and risk factors of blindness among patients newly diagnosed with primary angle closure glaucoma (PACG) in the United States (US).

Design

Retrospective cross-sectional study of patients from the American Academy of Ophthalmology IRIS® (Intelligent Research in Sight) Registry.

Participants

Patients in the IRIS® Registry between the years 2015 to 2019 with a new diagnosis of PACG and visual acuity (VA) data on or within 90 days prior to the date of diagnosis.

Methods

Eligible patients were aged 18 years and older and: (1) were observable in the database at least 24 months prior to the index date of PACG diagnosis; (2) had no history of intraocular pressure (IOP) lowering drops, laser peripheral iridotomy (LPI), cataract surgery, or a diagnosis of pseudophakia unless preceded by a diagnosis of anatomical narrow angle (ANA); and (3) had no history of glaucoma surgery. Multivariable logistic regression models were developed to assess risk factors of blindness.

Main Outcome Measures

Any (one or both eyes) or bilateral (both eyes) blindness (VA ≤ 20/200) at first diagnosis of PACG.

Results

43,901 patients with PACG in the IRIS® Registry met inclusion criteria. Overall prevalence of any and bilateral blindness were 11.5% and 1.8%, respectively. Black and Hispanic patients were at higher risk of any (OR=1.42 and 1.21, respectively; p<0.001) and bilateral (OR=2.04 and 1.53, respectively; p<0.001) blindness compared to non-Hispanic White patients adjusted for ocular comorbidities, including cataracts. Other factors associated with any blindness included age <50 or >80 years, male sex, Medicaid or Medicare insurance category, and Southern or Western practice region (ORs>1.28; p≤0.01). Diagnosis of ANA prior to diagnosis of PACG was protective against any (OR=0.56; p<0.001) and bilateral (OR=0.61; p<0.001) blindness.

Conclusions

Blindness affects 1 out of 9 patients with newly diagnosed PACG in the IRIS® Registry; Black and Hispanic patients and Medicaid and Medicare recipients are significantly more vulnerable. These findings highlight the severe ocular morbidity associated with PACG and the need for increased disease awareness and improved detection methods.
Tracking neural correlates of prioritizing working memory representations through retrospective attentional strengthening
Authors: Li Dongwei , Hu Yiqing , Qi Mengdi , Zhao Chenguang , Jensen Ole , Huang Jing , Song Yan
Published: August 2022

Previous work has proposed two potentials benefits of retrospective attention on working memory (WM): target strengthening and non-target inhibition. It remains unknown which hypothesis contributes to the improved WM performance, yet the neural mechanisms responsible for this attentional benefit are unclear. Here, we recorded electroencephalography (EEG) signals while 33 participants performed a retrospective-cue WM task. Multivariate pattern classification analysis revealed that only representations of target features were enhanced by valid retrospective attention during the retention, supporting the target strengthening hypothesis. Further univariate analysis found that mid-frontal theta inter-trial phase coherence (ITPC) and ERP components were modulated by valid retrospective attention and correlated with individual differences and moment-to-moment fluctuations on behavioral outcomes, suggesting that both trait- and state-level variability in attentional preparatory processes influence goal-directed behavior. Furthermore, task-irrelevant target spatial location could be decoded from EEG signals, indicating that enhanced spatial binding of target representation promotes high WM precision. Importantly, frontoparietal theta-alpha phase-amplitude-coupling was increased by valid retrospective attention and predicted the reduced randomly guessing rates. This long-range connection supported top-down information flow in engagement of frontoparietal networks, which might organize attentional states to integrate target features. Altogether, these results provide neurophysiological bases that retrospective attention improves WM precision through enhancing representation of target and emphasize the critical role of frontoparietal attentional network in the control of WM representations.
A thousand-genome panel retraces the global spread and climatic adaptation of a major crop pathogen
Authors: Feurtey Alice , Lorrain Cécile , McDonald Megan C. , Milgate Andrew , Solomo Peter , Warren Rachael , Puccetti Guido , Scalliet Gabriel , Torriani Stefano F. F. , Gout Lilian , Marcel Thierry C. , Suffert Frédéric , Alassimone Julien , Lipzen Anna , Yoshinaga Yuko , Daum Christopher , Barry Kerrie , Grigoriev Igor V. , Goodwin Stephen B. , Genissel Anne , Seidl Michael F. , Stukenbrock Eva , Lebrun Marc-Henri , Kema Gert H. J. , McDonald Bruce A. , Croll Daniel
Published: August 2022

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.
Three-dimensional imaging of vascular development in the mouse epididymis: a prerequisite to better understand the post-testicular immune context of spermatozoa
Authors: Damon-Soubeyrand Christelle , Bongiovanni Antonino , Chorfa Areski , Goubely Chantal , Pirot Nelly , Pardanaud Luc , Pibouin-Fragner Laurence , Vachias Caroline , Bravard Stéphanie , Guiton Rachel , Thomas Jean-Léon , Saez Fabrice , Kocer Ayhan , Tardivel Meryem , Drevet Joël R. , Henry-Berger Joelle
Published: August 2022

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide for the first time a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.

Graphical abstract

Summary of the expansion of the conventional and hybrid lymphatic vasculature during postnatal development of the murine epididymis.
Beyond linearity: Quantification of the mean for linear CRNs in a random environment
Authors: Sinzger-D’Angelo Mark , Startceva Sofia , Koeppl Heinz
Published: August 2022

Molecular reactions within a cell are inherently stochastic, and cells often differ in morphological properties or interact with a heterogeneous environment. Consequently, cell populations exhibit heterogeneity both due to these intrinsic and extrinsic causes. Although state-of-the-art studies that focus on dissecting this heterogeneity use single-cell measurements, the bulk data that shows only the mean expression levels is still in routine use. The fingerprint of the heterogeneity is present also in bulk data, despite being hidden from direct measurement. In particular, this heterogeneity can affect the mean expression levels via bimolecular interactions with low-abundant environment species. We make this statement rigorous for the class of linear reaction systems that are embedded in a discrete state Markov environment. The analytic expression that we provide for the stationary mean depends on the reaction rate constants of the linear subsystem, as well as the generator and stationary distribution of the Markov environment. We demonstrate the effect of the environment on the stationary mean. Namely, we show how the heterogeneous case deviates from the quasi-steady state (Q.SS) case when the embedded system is fast compared to the environment.
The Impact of Task Context on Predicting Finger Movements in a Brain-Machine Interface
Authors: Mender Matthew J. , Nason-Tomaszewski Samuel R. , Temmar Hisham , Costello Joseph T. , Wallace Dylan M. , Willsey Matthew S. , Kumar Nishant Ganesh , Kung Theodore A. , Patil Parag G. , Chestek Cynthia A.
Published: August 2022

A key factor in the clinical translation of brain-machine interfaces (BMIs) for restoring hand motor function will be their robustness to changes in a task. With functional electrical stimulation (FES) for example, the patient’s own hand will be used to produce a wide range of forces in otherwise similar movements. To investigate the impact of task changes on BMI performance, we trained two rhesus macaques to control a virtual hand with their physical hand while we added springs to each finger group (index or middle-ring-small) or altered their wrist posture. Using simultaneously recorded intracortical neural activity, finger positions, and electromyography, we found that predicting finger kinematics and finger-related muscle activations across contexts led to significant increases in prediction error, especially for muscle activations. However, with respect to online BMI control of the virtual hand, changing either training task context or the hand’s physical context during online control had little effect on online performance. We explain this dichotomy by showing that the structure of neural population activity remained similar in new contexts, which could allow for fast adjustment online. Additionally, we found that neural activity shifted trajectories proportional to the required muscle activation in new contexts, possibly explaining biased kinematic predictions and suggesting a feature that could help predict different magnitude muscle activations while producing similar kinematics.
Correlated substitutions reveal SARS-like coronaviruses recombine frequently with a diverse set of structured gene pools
Authors: Steinberg Asher Preska , Silander Olin K. , Kussell Edo
Published: August 2022

Quantifying SARS-like coronavirus (SL-CoV) evolution is critical to understanding the origins of SARS-CoV-2 and the molecular processes that could underlie future epidemic viruses. While genomic evidence implicates recombination as a factor in the emergence of SARS-CoV-2, few studies have quantified recombination rates among SL-CoVs. Here, we infer recombination rates of SL-CoVs from correlated substitutions in sequencing data using a coalescent model with recombination. Our computationally-efficient, non-phylogenetic method infers recombination parameters of both sampled sequences and the unsampled gene pools with which they recombine. We apply this approach to infer recombination parameters for a range of positive-sense RNA viruses. We then analyze a set of 191 SL-CoV sequences (including SARS-CoV-2) and find that ORF1ab and S genes frequently undergo recombination. We identify which SL-CoV sequence clusters have recombined with shared gene pools, and show that these pools have distinct structures and high recombination rates, with multiple recombination events occurring per synonymous substitution. We find that individual genes have recombined with different viral reservoirs. By decoupling contributions from mutation and recombination, we recover the phylogeny of non-recombined portions for many of these SL-CoVs, including the position of SARS-CoV-2 in this clonal phylogeny. Lastly, by analyzing 444,145 SARS-CoV-2 whole genome sequences, we show current diversity levels are insufficient to infer the within-population recombination rate of the virus since the pandemic began. Our work offers new methods for inferring recombination rates in RNA viruses with implications for understanding recombination in SARS-CoV-2 evolution and the structure of clonal relationships and gene pools shaping its origins.

Significance Statement

Quantifying the population genetics of SARS-like coronavirus (SL-CoV) evolution is vital to deciphering the origins of SARS-CoV-2 and pinpointing viruses with epidemic potential. While some Bayesian approaches can quantify recombination for these pathogens, the required simulations of recombination networks do not scale well with the massive amounts of sequences available in the genomics era. Our approach circumvents this by measuring correlated substitutions in sequences and fitting these data to a coalescent model with recombination. This allows us to analyze hundreds of thousands of sample sequences, and infer recombination rates for unsampled viral reservoirs. Our results provide insights into both the clonal relationships of sampled SL-CoV sequence clusters and the evolutionary dynamics of the gene pools with which they recombine.
Construction and rescue of a DNA-launched DENV2 16681 infectious clone
Authors: Holliday Madeline , Corliss Lochlain , Lennemann Nicholas J.
Published: August 2022

Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, production of Zika virus has been successful from CMV-promoter driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued virus maintained similar replication kinetics in several cell lines commonly used in flavivirus research. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.
Neural oscillations in the aging brain associated with interference control in word production
Authors: Zheng Xiaochen Y. , Piai Vitória
Published: August 2022

Speaking is not only about retrieving words and structuring them into sentences, but it also requires top-down control to plan and execute speech. In previous electrophysiological research with young-adult speakers, mid-frontal theta oscillations have been observed using a picture-word interference paradigm. With this paradigm, participants name pictures while ignoring superimposed distractor words. In particular, mid-frontal theta power increases for categorically related distractors relative to other types of distractors, reflecting the top-down interference control in resolving the competition between processing streams during word production (Piai, Roelofs, Jensen, Schoffelen, & Bonnefond, 2014). In the present study, we conceptually replicated the magnetoencephalography study by Piai et al. (2014) with an older group of healthy adults (mean age of 60 years). Behaviorally, we replicated distractor semantic interference and Stroop-like interference effects usually observed in young adults. However, we did not find the corresponding theta modulation associated with these interference effects on the neural level. Instead, we found beta power decreases for both effects, mostly pronounced in the left posterior temporal and inferior parietal cortex. The distinct spectro-spatial-temporal profile of the oscillatory effects in the older population suggests different underlying dynamics relative to the midline frontal effect previously found in young-adult speakers. Our results indicate that the neural underpinnings of top-down interference control may be modified by aging, and that the mid-frontal theta cannot be the exclusive mechanism enabling interference control during spoken word production.
The DUSP1 on CaMKII-positive neurons in mPFC mediates adolescent cocaine exposure-induced higher sensitivity to cocaine during adulthood
Authors: Wei Xiaoyan , Chang Jiasong , Cheng Zhen , Chen Wenwen , Guo Hao , Liu Zhaoyu , Mai Yuning , Hu Tao , Zhang Yuanyuan , Cai Qinglong , Ge Feifei , Fan Yu , Guan Xiaowei
Published: August 2022

Adolescent cocaine abuse increases the risk for developing addiction in later life, which remains a big public health concern, but the underlying molecular mechanism is poorly understood. Here, adolescent cocaine-exposed (ACE) male mice models were established by administrating cocaine during adolescent period. When growing to adult age, mice were subjected to conditioned place preference (CPP) to evaluate the sensitivity to cocaine, then potential molecule of dual specificity phosphatase 1 (DUSP1) were screened out by transcriptomic sequencing. Subthreshold dose of cocaine (sdC), that is insufficient to produce CPP, was used to induce CPP in adulthood. The sdC treatment effectively induced CPP in ACE mice during adulthood, accompanied with the more triggered CaMKII-positive neurons, and induced higherDusp1gene, lower DUSP1 protein, lower DUSP1 activity and lower DUSP1 expression on CaMKII-positive neurons (DUSP1CaMKII) in medial prefrontal cortex (mPFC). Overexpressing DUSP1CaMKIIsuppressed CaMKII-positive neuronal activation, and ultimately blocked sdC-induced CPP in ACE mice during adulthood. While, knocking-down DUSP1CaMKIIactivated more CaMKII-positive neurons, and aggravated sdC-preferred behavior in ACE mice during adulthood. ERK1/2 might be potential subsequent signal for DUSP1 in the process. Collectively, our findings reveal a novel molecular mechanism underlying adolescent drug abuse-induced susceptibility to addiction during adulthood, and mPFC DUSP1CaMKIIis a promising pharmacological target to predict or treat addiction, especially caused by adolescent substance use.

Adolescent cocaine exposure causes higher cocaine-preferred behaviors during adulthood, along with evoked mPFC activity in response to cocaine challenge. Locally overexpressing but not knocking-down the dual specificity phosphatase 1 (DUSP1) on CaMKII-positive neurons (DUSP1CaMKII) suppresses mPFC activation, and ultimately rescues the higher sensitivity to cocaine during adulthood.
Anti-tumour efficacy of Musa paradisiaca flower extracts on DMBA induced Mammary carcinogenesis in female Wistar rats
Authors: Lavudi Kousalya , S Hemalatha , Kokkanti Rekha Rani , G.V. S Harika , Patnaik Srinivas , Penchalaneni Josthna
Published: August 2022

Background

Several reports have shown the beneficial advantages of Musa paradisiaca in wound healing activities and other ailments. Previously, our in vitro studies validated the anti-cancer activities of Musa flower extracts and confirmed the potential. This thread has led to our current investigation to explore the anticancer potential in vivo.

Purpose

This study focused on the efficacy of banana florets on DMBA induced breast cancer in female wistar rats.

Methods

Induction of tumour using Dimethylbenzanthracene (DMBA) in female wistar rats. Hormonal, antioxidant and anticarcinogenic studies were performed in vivo.

Results

In our current study, we investigated that tumour induction has an effect in disrupting the estrous cycle in mice which may result by hormonal fluctuation levels. Antioxidant and hormonal analysis in vivo revealed the imbalance in estrogen and progesterone levels in untreated group compared to the treated ones. Floral crude extract treatment in vivo has shrunken the tumour volume in flower extract treatment group as well as in standard drug compared to the control. Histopathological staining confirms the disruption of epithelial tissues in tumour induced groups compared to the treated ones. Moreover, Musa floral treatment has shown to revert the damaged tissue morphology in the treated groups compared to the saline treated one. In-vitro studies in MCF-7 and MDA-MB-231 breast cancer cell lines has shown the potent anti-tumorigenic activity using Musa floral extracts.

Conclusion

Taken together, our finding confirmed that banana flower extracts showcase anti-carcinogenic activity against breast cancer both in vitro and in vivo. Tumour induction in mice has an effect in showing the disruption of estrous cycles.

Highlights

Musa paradisiaca crude ethanolic flower extracts have a potential in reducing the tumour growth.

DMBA induced cancer has a deleterious effect on estrogen cycles in female wistar rats.

Disruption of epithelial morphology was observed in tumour induced wistar rats.

Treatment with crude Musa flower extracts on cancer induced rats shows the reduced level of damage and oxidative stress compared to the no treatment group.
Uman Type NF-L Antibodies Are Effective Reagents for the Imaging of Neurodegeneration
Authors: Shaw Gerry , Madorsky Irina , Li Ying , Wang YongSheng , Rana Sabhya , Fuller David D.
Published: August 2022

Recent work shows that certain immunological assays for the neurofilament light chain NF-L detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to NF-L, UD1 and UD2, also known as 2.1 and 47.3 respectively. These are the essential components of the Uman Diagnostics NF-Light™ ELISA kit, the Quanterix Simoa™ bead based NF-L assay and others. We show here that the antibodies bind to neighboring epitopes in a short, conserved and unusual peptide in the NF-L “rod” Coil 2 region. We also describe a surprising and useful feature of Uman and similar reagents. While other well characterized NF-L antibodies show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of presumably spontaneously degenerating or degenerated neurons and their processes. However following experimental mid-cervical injuries to rat spinal cord both Uman antibodies recognize numerous profiles in tissue sections. The Uman positive material was associated with fiber tracts expected to be damaged by the injury administered and the profiles had the swollen, beaded, discontinuous and sinusoidal morphology expected for degenerating and degenerated processes. We also found that several antibodies to the C terminal “tail” region of NF-L stain undamaged axonal profiles but fail to recognize the Uman positive material. The unmasking of the Uman epitopes and the loss of the NF-L tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunological changes we have discovered are due to neurodegeneration induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibody reagents directed against the region of NF-L including the Uman epitopes which have staining properties identical to the Uman reagents. Using these we show that the NF-L region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the NF-L biomarker, describes novel and useful properties of Uman type and NF-L tail binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.
Rational engineering of a β-glucosidase (H0HC94) from glycosyl family I (GH1) to improve catalytic performance on cellobiose
Authors: Sengupta Sauratej , Chanda Pinaki , Manna Bharat , Datta Supratim
Published: August 2022

The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-glucosidases catalyze the final step in cellulose breakdown, producing glucose, and is often the rate-limiting step in biomass hydrolysis. Rationally engineering previously characterized enzymes may be one strategy to increase catalytic activity and the efficiency of cellulose hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate p-Nitrophenyl β-D-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. Based on our hypothesis of increasing catalytic activity by reducing the interaction of residues present near the active site tunnel entrance with glucose without disturbing any existing interactions with cellobiose, we report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold specific activity increase (366.3 ± 36 µmol/min/mg), an almost 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in Q319A H0HC94 cellobiose specificity (236.65 mM-1 s-1) over HOHC94. Molecular dynamic simulations and protein structure network analysis indicate that Q319A significantly increased the dynamically stable communities and hub residues, leading to a change in enzyme conformation and higher enzymatic activity. This study shows the impact of rational engineering of non-conserved residue to increase β-glucosidase substrate accessibility and enzyme specificity.

TOC

A rationally engineered β-glucosidase with a 1.5-fold increase in kcat, and a 3-fold increase in cellobiose specificity over the wild-type
Intrinsic neural excitability induces time-dependent overlap of memory engrams
Authors: Delamare Geoffroy , Feitosa Tomé Douglas , Clopath Claudia
Published: August 2022

Memories are thought to be stored in neural ensembles known as engrams that are specifically reactivated during memory recall. Recent studies have found that memory engrams of two events that happened close in time tend to overlap in the hippocampus and the amygdala, and these overlaps have been shown to support memory linking. It has been hypothesised that engram overlaps arise from the mechanisms that regulate memory allocation itself, involving neural excitability, but the exact process remains unclear. Indeed, most theoretical studies focus on synaptic plasticity and little is known about the role of intrinsic plasticity, which could be mediated by neural excitability and serve as a complementary mechanism for forming memory engrams. Here, we developed a rate-based recurrent neural network that includes both synaptic plasticity and neural excitability. We obtained structural and functional overlap of memory engrams for contexts that are presented close in time, consistent with experimental studies. Moreover, we showed that enhancing the initial excitability of a subset of neurons just before presenting a context biases the memory allocation to these neurons. We then explored the role of inhibition as a way of controlling competition among neurons from two ensembles. This work suggests mechanisms underlying the role of intrinsic excitability in memory allocation and linking, and yields predictions regarding the dynamics of memory engrams.
CRISPR-Cas-based Microhomology-Mediated End Joining for Precise Gene Replacement in Plant
Authors: Vu Tien Van , Lim Gah-Hyun , Choi Seung Hee , Moon Ju Yeon , Nguyen Ngan Thi , Das Swati , Tran Mil Thi , Sung Yeon Woo , Kim Jihae , Song Young Jong , Kim Suk Weon , Jeong Jae Cheol , Kim Jae-Yean
Published: August 2022

Precise gene or allele replacement is a desirable technology, but implementing it in plants remains challenging. CRISPR-Cas-based approaches, such as gene targeting (GT) and prime editing (PE), have opened up new possibilities for precise gene replacement in plants. However, their editing size and efficiency still need improvement. Recently, strategies using canonical nonhomologous end-joining (cNHEJ) and microhomology-mediated end joining (MMEJ) have been considered promising alternatives for precise gene replacement in yeast and mammals. However, these approaches have not been extensively explored and applied to plants. Here, we proposed and tested a CRISPR-Cas-based tool, termed PREMJ (precision genereplacement viamicrohomology-mediated end joining), for precision gene replacement in plants. The PREMJ strategy employing 20-bp microhomology MMEJ donors (∼100 bp lengths) and a canonical nonhomologous end-joining (cNHEJ) inhibitor, NU7441, produced high targeted gene replacement efficiencies, up to 1.60 ± 0.14, 4.47 ± 1.98, and 8.98 ± 4.73 % in protoplasts of tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on PREMJ-based precision gene editing in plants. Although obtaining edited plants remains challenging due to inefficient protoplast regenerations andAgrobacterium-mediated delivery, PREMJ may significantly contribute to precision gene editing in plants competent to PREMJ complex delivery and plant regeneration.

Key Message

We designed and tested a method, termed PREMJ, for precise gene replacement using the CRISPR-Cas-mediated double-stranded break (DSB) formation and repairing the DSBs via microhomology-mediated end joining (MMEJ) with MMEJ donor template carrying desired base changes and microhomologies flanking the DSB ends. PREMJ showed feasibility in precise gene replacement in protoplasts of tomatoes, lettuce, and cabbage, albeit its efficacy inAgrobacterium-mediated plant transformation requires further optimization.
High sensitivity single cell RNA sequencing with split pool barcoding
Authors: Tran Vuong , Papalexi Efthymia , Schroeder Sarah , Kim Grace , Sapre Ajay , Pangallo Joey , Sova Alex , Matulich Peter , Kenyon Lauren , Sayar Zeynep , Koehler Ryan , Diaz Daniel , Gadkari Archita , Howitz Kamy , Nigos Maria , Roco Charles M. , Rosenberg Alexander B.
Published: August 2022

Single cell RNA sequencing (scRNA-seq) has become a core tool for researchers to understand biology. As scRNA-seq has become more ubiquitous, many applications demand higher scalability and sensitivity. Split-pool combinatorial barcoding makes it possible to scale projects to hundreds of samples and millions of cells, overcoming limitations of previous droplet based technologies. However, there is still a need for increased sensitivity for both droplet and combinatorial barcoding based scRNA-seq technologies. To meet this need, here we introduce an updated combinatorial barcoding method for scRNA-seq with dramatically improved sensitivity. To assess performance, we profile a variety of sample types, including cell lines, human peripheral blood mononuclear cells (PBMCs), mouse brain nuclei, and mouse liver nuclei. When compared to the previously best performing approach, we find up to a 2.6-fold increase in unique transcripts detected per cell and up to a 1.8-fold increase in genes detected per cell. These improvements to transcript and gene detection increase the resolution of the resulting data, making it easier to distinguish cell types and states in heterogeneous samples. Split-pool combinatorial barcoding already enables scaling to millions of cells, the ability to perform scRNA-seq on previously fixed and frozen samples, and access to scRNA-seq without the need to purchase specialized lab equipment. Our hope is that by combining these previous advantages with the dramatic improvements to sensitivity presented here, we will elevate the standards and capabilities of scRNA-seq for the broader community.
Identification of differentially expressed pyroptosis-related genes in LUAD
Authors: Qian Bao , Jiang Jiuzhou
Published: August 2022

Background

Lung cancer has the highest mortality rate among malignant tumors in the world, and adenocarcinoma, the most important pathological type of lung cancer, accounts for 80%-85% of all lung cancers, causing a significant burden of disease. Pyroptosis is a newly recognized form of programmed cell death (PCD).

Methods

288 normal tissue data from The Genotype-Tissue Expression (GTEx) database and 501 tumor tissue data from The Cancer Genome Atlas (TCGA) database were selected. Differentially expressed genes were identified between normal and LUAD patients.

Results and Conclusions

With a consensus cluster analysis, 501 LUAD patients were divided into two clusters, and there was a significant difference in survival time between the two clusters. Ten differentially expressed genes were identified, with four up-regulated and six down-regulated. Pyroptosis is closely related to LUAD.
A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation
Authors: Ghanbarpour Alireza , Cohen Steven E. , Fei Xue , Bell Tristan A. , Baker Tania A. , Davis Joseph H. , Sauer Robert T.
Published: August 2022

Intracellular proteases must be specific to avoid degrading the wrong proteins. Here, we present cryo-EM structures of E. coli ClpXP, a AAA+ protease, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate. An open-channel ClpX mutation stimulates degradation of casein, a non-specific substrate, indicating that channel closure contributes to increased degradation specificity. We demonstrate that ClpX activates ClpP cleavage of a degron-free decapeptide by a channel-independent mechanism, in which the peptide substrate appears to pass through a symmetry mismatched gap in the interface between ClpX and ClpP before entering the degradation chamber via the axial portal of ClpP. The peptide products of ClpXP protein degradation are likely to exit the chamber by the reverse route.
Highly precise genome editing using enhanced CRISPR-Cas12a nickase module
Authors: Kim Chan Hyoung , Kim Hanseop , Lee Youngjeon , Lee Hyomin , Lim Kyung-Seob , Park Sang Je , Huh Jae-Won , Kim Young-Hyun , Lee Dong-Seok , Kim Kyoung Mi , Hur Junho K. , Lee Seung Hwan
Published: August 2022

The authors have withdrawn their manuscript owing to significant changes in content. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
Changes of urinary proteome in high-fat dietApoE-/-mice
Authors: Yuanrui Hua , Wenshu Meng , Jing Wei , Yongtao Liu , Youhe Gao
Published: August 2022

Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine is more conducive in the accumulation and response of changes in the physiological state of the body and is not regulated by homeostasis mechanisms, so it is a good source of biomarkers in the early stage of disease. In this study,ApoE-/-mice were fed with a high-fat diet for 5 months. Urine samples from the experimental group and control group, which were C57BL/6 mice fed a normal diet, were collected at seven time points. Proteomic analysis was used for internalcontrol and intergroup control. Internal control results showed a significant difference in the urinary proteome before and after a 1-week high-fat diet, and several differential proteins have been reported to be associated with atherosclerosis or for use as candidate biomarkers. The results of the intergroup control indicated that the biological process enriched by the GO analysis of the differential proteins corresponded to the progression of atherosclerosis. Differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to monitor changes in the body sensitively and provides the possibility of identifying early biomarkers of atherosclerosis.
Structural analysis of the macrocyclic inhibitor BI-4020 binding to EGFR kinase
Authors: Beyett Tyler S. , Rana Jaimin K. , Schaeffner Ilse K. , Heppner David E. , Eck Michael J.
Published: August 2022

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. Structures show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure-inspired approaches to developing targeted therapies in medicinal oncology.
Genome assembly of the acoel flatworm Symsagittifera roscoffensis, a model for research on photosymbiosis
Authors: Martinez Pedro , Ustyantsev Kirill , Biryukov Mikhail , Mouton Stijn , Glasenburg Liza , Sprecher Simon G. , Bailly Xavier , Berezikov Eugene
Published: August 2022

Symsagittifera roscoffensis is a well-known member of the order Acoela that lives in symbiosis with the algae Tetraselmis convolutae during its adult stage. Its natural habitat is the eastern coast of the Atlantic, where at specific locations thousands of individuals can be found lying in large pools on the surface of sand at low tide and in the sandy interstitial web at high tide. As a member of the Acoela it has been used as a proxy for early bilaterian animals; however, its phylogenetic position remains debated. In order to understand the basic structural characteristics of the acoel genome, we sequenced and assembled the genome of aposymbiotic S. roscoffensis. The size of S. roscoffensis genome was measured to be in range 910 - 940 Mb. Sequencing of the genome was performed using PacBio Hi-Fi technology. Hi-C and RNA-seq data were also generated to scaffold and annotate the genome. The resulting assembly is 1.1 Gb large (covering 118% of the estimated genome size) and highly continuous, with N50 scaffold size of 1.04 Mb. The repetitive fraction of the genome is 61%, of which 85% (half of the genome) are LTR retrotransposons. Genome-guided transcriptome assembly identified 34,493 genes, of which 29,351 are protein coding (BUSCO score 97.6%), and 30.2% of genes are spliced leader (SL) trans-spliced. The completeness of this genome suggests that it can be used extensively to characterize gene families and conduct accurate phylogenomic reconstructions.

Significance

Symsagittifera is a representative of the phylum Acoela, the first offshoot of bilaterian animals. This key phylogenetic position adds an extra value to the knowledge of its genome, since it will inform us on how the genome of a bilaterian ancestor might have looked like. Moreover, Symsagittifera roscoffensis is a model organism used in symbiogenesis research. Host and algae can be cultured independently and, after mixing, the symbiosis can be followed. Symbiogenesis was established early on during the evolution of Metazoa. In spite of its biological relevance, very little is known on the molecular mechanisms that control it. Here the genome of the acoel host should provide us with insights on the first adaptations to symbiogenesis occurring in bilateral animals.
Spectral Interferences Impede the High-Resolution Mass Analysis of Recombinant Adeno-Associated Viruses
Authors: Yin Victor , Devine Paul W.A. , Saunders Janet C. , Hines Alistair , Shepherd Sam , Dembek Marcin , Dobson Claire L. , Snijder Joost , Bond Nicholas J. , Heck Albert J.R.
Published: August 2022

Recombinant adeno-associated viruses (rAAVs) are the leading platform for in vivo delivery of gene therapies, with several already approved for clinical use. However, the heterogeneity and structural complexity of these viral particles render them challenging targets to characterize. Orbitrap-based native mass spectrometry (MS) is a method capable of directly characterizing intact megadalton protein assemblies. Here we used such an approach to characterize four different preparations of rAAV8 (two empty and two filled) differing in both their transgene and relative capsid protein isoform (i.e. VP1, VP2 and VP3) content. Interestingly, in native MS measurements of these samples, we observe complicated, unusual, and dramatically different spectral appearances between the four rAAV preparations that cannot be rationalized or interpreted using conventional approaches (i.e. charge state deconvolution). By combining high-resolution native MS, single particle charge detection MS, and spectral simulations, we reveal that these unexpected features result from a combination of stochastic assembly-induced heterogeneity and divergent gas phase charging behaviour between the four rAAV preparations. Our results stress the often-neglected heterogeneity of rAAVs, but also highlight the pitfalls of standard high-resolution mass analysis for such particles. Finally, we show that charge detection MS and spectral simulations can be used to tackle these challenges.
A Different Kind of Restraint Suitable for Molecular Dynamics Simulations
Authors: Kolossváry István , Sherman Woody
Published: August 2022

Conformational sampling of complex biomolecules is an emerging frontier in drug discovery. Indeed, advances in lab-based structural biology and related computational approaches like AlphaFold have made great strides in obtaining static protein structures. However, biology is in constant motion and many important biological processes rely on conformationally-driven events. Unrestrained molecular dynamics (MD) simulations require that the simulated time be comparable to the real time of the biological processes of interest, rendering pure MD impractical for many drug design projects, where conformationally-driven biological events can take microseconds to milliseconds or longer. An alternative approach is to accelerate the sampling of specific motions by applying restraints, guided by insights about the underlying biological process of interest. A plethora of restraints exist to limit the size of conformational search space, although each has drawbacks when simulating complex biological motions. In this work, we introduce a new kind of restraint for molecular dynamics simulations (MD) that is particularly well suited for complex conformationallydriven biological events, such as protein-ligand binding, allosteric modulations, conformational signalling, and membrane permeability. The new restraint, which relies on a barrier function (the scaled reciprocal function) is particularly beneficial to MD, where hard-wall restraints are needed with zero tolerance to restraint violation. We have implemented this restraint within a hybrid sampling framework that combines metadynamics and extended-Lagrangian adaptive biasing force (meta-eABF). We use two particular examples to demonstrate the value of this approach: (1) quantification of the approach of E3-loaded ubiquitin to a protein of interest as part of the Cullin ring ligase and (2) membrane permeability of heterobi-functional degrader molecules with a large degree of conformational flexibility. Future work will involve extension to additional systems and benchmarking of this approach compared with other methods.