bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin-Binding Motif
Authors: Huang Minqi , Wang Xiaoliang , Banerjee Moumita , Mukherji Shreya T , Kutz Laura C , Zhao Aijie , Sepanski Michael , Fan Chen-Ming , Zhu Guo-Zhang , Tian Jiang , Wang Da-Zhi , Zhu Hua , Xie Zi-Jian , Pierre Sandrine V , Cai Liquan
Published: January 2022

The N-terminal caveolin-binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human-induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared with the parental wild-type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated induced Skm cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells’ ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of β-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/β-catenin signaling.
Organoid Medicine for Inflammatory Bowel Disease
Authors: Wakisaka Yusuke , Sugimoto Shinya , Sato Toshiro
Published: January 2022

Inflammatory bowel disease (IBD) is a chronic relapsing-remitting inflammatory disease of the gastrointestinal tract with an unknown etiology, and its incidence is increasing worldwide. Recent advances in immunomodulatory therapeutic agents such as biologics and small-molecule inhibitors have improved the prognosis of patients with IBD. However, some patients are refractory and resistant to these immunomodulatory therapies, and new therapies are needed. Given the importance of the intestinal epithelium in IBD pathogenesis, the difficulty of culturing intestinal epithelial cells (IECs) for long periods remains an obstacle in IBD research. Over the past 15 years, intestinal stem cells have been identified, and the in vivo microenvironment, called the niche, required for their maintenance has been elucidated, making the permanent culture of IECs possible. Recapitulating the niche in vitro, the intestinal epithelium forms 3-dimensional structures called organoids that simulate the intestinal epithelium in vivo. The intestinal epithelium plays an important role in the intestinal barrier and immunomodulatory functions and serves as a physical structure that separates the intestinal lumen from the body. Recent studies have revealed that functional disruption of the intestinal epithelium is closely related to the pathogenesis of IBD, and IBD research using organoids has attracted attention. In this review, we discuss the application of adult tissue-derived organoids culture technology to elucidate the pathogenesis of IBD and to develop novel therapies, including regenerative treatments.
Urinary Biomarkers for Kidney Allograft Injury
Authors: Jin Paul H. , Sarwal Reuben D. , Sarwal Minnie M.
Published: January 2022

The current standard of serum creatinine and biopsy to monitor allograft health has many limitations. The most significant drawback of the current standard is the lack of sensitivity and specificity to allograft injuries, which are diagnosed only after significant damage to the allograft. Thus, it is of critical need to identify a biomarker that is sensitive and specific to the early detection of allograft injuries. Urine, as the direct renal ultrafiltrate that can be obtained noninvasively, directly reflects intrarenal processes in the allograft at greater accuracy than analysis of peripheral blood. We review transcriptomic, metabolomic, genomic, and proteomic discovery-based approaches to identifying urinary biomarkers for the noninvasive detection of allograft injuries, as well as the use of urine cell-free DNA in the QSant urine assay as a sensitive surrogate for the renal allograft biopsy for rejection diagnosis.
Performance of a Multisensor Smart Ring to Evaluate Sleep: In-Lab and Home-Based Evaluation Relative to Polysomnography and Actigraphy: Importance of Generalized Versus Personalized Scoring
Authors: Grandner Michael A. , Bromberg Zohar , Hadley Aaron , Morrell Zoe , Graf Arnulf , Hutchison Stephen , Freckleton Dustin
Published: January 2022

Study Objectives

Wearable sleep technology has rapidly expanded across the consumer market due to advances in technology and increased interest in personalized sleep assessment to improve health and mental performance. In this study, we tested the performance of a novel device, the Happy Ring, alongside other commercial wearables, against in-lab polysomnography (PSG) and an at-home EEG-derived sleep monitoring device, the Dreem 2 Headband.

Methods

36 healthy adults with no diagnosed sleep disorders and no recent use of medications or substances known to affect sleep pattern were assessed across 77 nights while wearing the Happy Ring, as well as a set of other consumer wearable devices. Subjects participated in a single night of in-lab PSG and 2 nights of at-home data collection. The Happy Ring includes sensors for skin conductance, movement, heart rate, and skin temperature. The Happy Ring utilized two machine-learning derived scoring algorithms: a “generalized” algorithm that applied broadly to all users, and a “personalized” algorithm that adapted to individual subjects’ data. Epoch-by-epoch analyses compared the wearable devices to both in-lab PSG and to the Dreem 2 EEG Headband (“Dreem 2 Headband”) at home.

Results

Compared to in-lab PSG, the “generalized” and “personalized” algorithms demonstrated good sensitivity (94% and 93%, respectively) and specificity (70% and 83%, respectively). Accuracy was 91% for “generalized” and 92% for “personalized” algorithms. The generalized algorithm demonstrated an accuracy of 67%, 85%, and 85% for light, deep, and REM sleep, respectively. The personalized algorithm was 81%, 95%, and 92% accurate for light, deep, and REM sleep, respectively.

Conclusions

The Happy Ring performed well at home and in the lab, especially regarding sleep detection. The personalized algorithm demonstrated improved detection accuracy over the generalized approach and other devices, suggesting that adaptable, dynamic algorithms can enhance sleep detection accuracy.
Longitudinal serologic and viral testing post–SARS-CoV-2 infection and post-receipt of mRNA COVID-19 vaccine in a nursing home cohort—Georgia, October 2020‒April 2021
Authors: Tobolowsky Farrell A. , Waltenburg Michelle A. , Moritz Erin D. , Haile Melia , DaSilva Juliana C. , Schuh Amy J. , Thornburg Natalie J. , Westbrook Adrianna , McKay Susannah L. , LaVoie Stephen P. , Folster Jennifer M. , Harcourt Jennifer L. , Tamin Azaibi , Stumpf Megan M. , Mills Lisa , Freeman Brandi , Lester Sandra , Beshearse Elizabeth , Lecy Kristin D. , Brown Laura G. , Fajardo Geroncio , Negley Jeanne , McDonald L. Clifford , Kutty Preeta K. , Brown Allison C. ,   for the CDC Infection Prevention and Control Team
Published: January 2022

Importance

There are limited data describing SARS-CoV-2–specific immune responses and their durability following infection and vaccination in nursing home residents.

Objective

To evaluate the quantitative titers and durability of binding antibodies detected after SARS-CoV-2 infection and subsequent COVID-19 vaccination.

Design

A prospective longitudinal evaluation included nine visits over 150 days; visits included questionnaire administration, blood collection for serology, and paired anterior nasal specimen collection for testing by BinaxNOW™ COVID-19 Ag Card (BinaxNOW), reverse transcription polymerase chain reaction (RT-PCR), and viral culture.

Setting

A nursing home during and after a SARS-CoV-2 outbreak.

Participants

11 consenting SARS-CoV-2–positive nursing home residents.

Main Outcomes and Measures

SARS-CoV-2 testing (BinaxNOW™, RT-PCR, viral culture); quantitative titers of binding SARS-CoV-2 antibodies post-infection and post-vaccination (beginning after the first dose of the primary series).

Results

Of 10 participants with post-infection serology results, 9 (90%) had detectable Pan-Ig, IgG, and IgA antibodies and 8 (80%) had detectable IgM antibodies. At first antibody detection post-infection, two-thirds (6/9, 67%) of participants were RT-PCR–positive but none were culture positive. Ten participants received vaccination; all had detectable Pan-Ig, IgG, and IgA antibodies through their final observation ≤90 days post-first dose. Post-vaccination geometric means of IgG titers were 10–200-fold higher than post-infection.

Conclusions and Relevance

Nursing home residents in this cohort mounted robust immune responses to SARS-CoV-2 post-infection and post-vaccination. The augmented antibody responses post-vaccination are potential indicators of enhanced protection that vaccination may confer on previously infected nursing home residents.
Exome sequencing of 628,388 individuals identifies common and rare variant associations with clonal hematopoiesis phenotypes
Authors: Kessler Michael D. , Damask Amy , O’Keeffe Sean , Van Meter Michael , Banerjee Nilanjana , Semrau Stefan , Li Dadong , Watanabe Kyoko , Horowitz Julie , Houvras Yariv , Gillies Christopher , Mbatchou Joelle , White Ryan R. , Kosmicki Jack A. , LeBlanc Michelle G. , Jones Marcus ,   Regeneron Genetics Center,   GHS-RGC DiscovEHR Collaboration, Glass David J. , Lotta Luca A. , Cantor Michael N. , Atwal Gurinder S. , Locke Adam E. , Ferreira Manuel A. R. , Deering Raquel , Paulding Charles , Shuldiner Alan R. , Thurston Gavin , Salerno Will , Reid Jeffrey G. , Overton John D. , Marchini Jonathan , Kang Hyun M. , Baras Aris , Abecasis Gonçalo R. , Jorgenson Eric
Published: January 2022

Clonal hematopoiesis (CH) refers to the expansion of certain blood cell lineages and has been associated with aging and adverse health outcomes. Here, we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal hematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 27 loci (24 novel) where germline genetic variation influences CH/CHIP predisposition, including missense variants in the DNA-repair gene PARP1 and the lymphocytic antigen coding gene LY75 that are associated with reduced incidence of CH/CHIP. Analysis of 5,194 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID outcomes, cardiovascular disease, hematologic traits, malignancy, smoking, obesity, infection, and all-cause mortality. Longitudinal analyses revealed that one of the CHIP subtypes, DNMT3A-CHIP, is associated with the subsequent development of myeloid but not lymphoid leukemias, and with solid cancers including prostate and lung. Additionally, contrary to previous findings from the initial 50,000 UKB exomes, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogenous phenotypes with shared and unique germline genetic causes and varied clinical implications.
COVIDFast™: A high-throughput and RNA extraction-free method for SARS-CoV-2 detection in swab (SwabFAST™) or saliva (SalivaFAST™)
Authors: Qiu Yue , Lu Ling , Halven Amanda , Terrio Rachel , Yuldelson Sydney , Dougal Natalie , Galbo Filippo , Lu Andrew , Gao Dexiang , Blomquist Bob , Zevallos Jose P. , Harry Brian L. , Yao Xin , Lu Shi-Long
Published: January 2022

There is an urgent need of having a rapid, high throughput, yet accurate SARS-COV-2 PCR testing to control the COVID19 pandemic. However, the RNA extraction step in conventional PCR creates a major bottle neck in the diagnostic process. In this paper we modified the CDC COVID-19 assay and developed an RNA-extraction free RT-qPCR assay for SARS-CoV-2, i.e. COVIDFast™. Depending on sample types, the assay is further divided into SwabFAST™, which uses anterior nares nasal swab, and SalivaFAST™, which uses saliva. By utilizing the proprietary buffer for either swab or saliva samples, the performance of SwabFAST or SalivaFAST is equivalent to RNA-extraction SARS-CoV-2 RT-qPCR in both contrived and clinical samples. The limit of detection of either assay is 4 copies/μL. We further developed a semi-automatic system, which is easy to adapt by clinical lab for implementation of a high-throughput SARS-CoV-2 test. Working together with the COVIDCheck Colorado, we have tested over 400,000 samples using COVIDFast (83.62% SwabFAST and 16.38% SalivaFAST) in less than a year, resulting in significant clinical contribution in the battle against COVID-19 during the pandemic.
P-TEFb is degraded by Siah1/2 in quiescent cells
Authors: Huang Fang , Feng Yongmei , Peterlin B. Matija , Fujinaga Koh
Published: January 2022

P-TEFb, composed of CycT1 and CDK9, regulates the elongation of transcription by RNA polymerase II. In proliferating cells, it is regulated by 7SK snRNA in the 7SK snRNP complex. In resting cells, P-TEFb is absent, because CycT1 is dephosphorylated, released from CDK9 and rapidly degraded. In this study, we identified the mechanism of this degradation. We mapped the ubiquitination and degradation of free CycT1 to its N-terminal region from positions 1 to 280. This region is ubiquitinated at six lysines, where E3 ligases Siah1 and Siah2 bind and degrade these sequences. Importantly, the inhibition of Siah1/2 rescued the expression of free CycT1 in proliferating as well as resting primary cells. We conclude that Siah1/2 are the E3 ligases that bind and degrade the dissociated CycT1 in resting, terminally differentiated, anergic and/or exhausted cells.
Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels
Authors: Cecon Erika , Fernandois Daniela , Renault Nicolas , Coelho Caio Fernando Ferreira , Wenzel Jan , Bedart Corentin , Izabelle Charlotte , Wimez Sarah Gallet , Poder Sophie Le , Klonjkowski Bernard , Schwaninger Markus , Prevot Vincent , Dam Julie , Jockers Ralf
Published: January 2022

COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post COVID condition. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevent SARS-CoV-2 entry in the brain thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Brain entry of SARS-CoV-2 through endothelial cells is prevented by melatonin through allosteric binding to human angiotensin-converting enzyme 2 (ACE2), which interferes with the cell entry receptor function of ACE2 for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.
THE RHIZOSPHERE BACTERIAL COMMUNITIES DIFFER AMONG DOMESTICATED MAIZE LANDRACES – AN EXPERIMENTAL CONFIRMATION
Authors: Lund Mads , Rasmussen Jacob Agerbo , Ramos-Madrigal Jazmín , Gilbert M. Thomas P. , Barnes Christopher James
Published: January 2022

The plant-associated microbiome has been shown to vary considerably between species and across environmental gradients. The effects of genomic variation on the microbiome within single species are less clearly understood, with results often confounded by the larger effects of climatic and edaphic variation.

In this study, the effect of genomic variation on the rhizosphere bacterial communities of maize was investigated by comparing different genotypes grown within controlled environments. Rhizosphere bacterial communities were profiled by metabarcoding the universal bacterial 16S rRNA v3-v4 region. Initially, plants from the inbred B73 line and the Ancho - More 10 landrace were grown for 12- weeks and compared. The experiment was then repeated with an additional four Mexican landraces (Apachito - Chih 172, Tehua - Chis 204, Serrano - Pueb 180 and Hairnoso de Ocho) that were grown alongside additional B73 and Ancho – More 10 genotypes.

In both experiments there were significant genotypic differences in the rhizosphere bacteria. Additionally, the bacterial communities were significantly correlated with genomic distance between genotypes, with the more closely related landraces being more similar in rhizosphere bacterial communities.

Despite limited sampling numbers, here we confirm that genomic variation in maize landraces is associated with differences in the rhizosphere bacterial communities. Further studies that go beyond correlations to identify the mechanisms that determine the genotypic variation of the rhizosphere microbiome are required.
SARS-CoV-2 entry sites are present in all structural elements of the human glossopharyngeal and vagal nerves: clinical implications
Authors: Vitale-Cross L. , Szalayova I , Scoggins A , Palkovits M. , Mezey E
Published: January 2022

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections result in the temporary loss of smell and taste (anosmia and dysgeusia) in about one third of confirmed cases. Several investigators have reported that the viral spike protein receptor is present in olfactory neurons. However, no study has been published to date showing the presence of viral entry sites angiotensin-converting enzyme 2 (ACE2), neuropilin1 (NRP1), and TMPRSS2, the serine protease necessary for priming the viral proteins, in human nerves that are responsible for taste sensation (cranial nerves: VII, IX and X). We used immunocytochemistry to examine three postmortem donor samples of the IXth (glossopharyngeal) and Xth (vagal) cranial nerves where they leave/join the medulla from three donors to confirm the presence of ACE2, NRP1 and TMPRSS2. Two samples were paraffin embedded; one was a frozen sample. In addition to staining sections from the latter, we isolated RNA from it, made cDNA, and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. All three of the proteins required for SARS-CoV-2 infections appear to be present in the human IXth and Xth nerves near the medulla. Direct infection of these nerves by the COVID-19 virus is likely to cause the loss of taste experienced by many patients. In addition, potential viral spread through these nerves into the adjacent brainstem respiratory centers might also aggravate the respiratory problems patients are experiencing.
V1 receptive field structure contributes to neuronal response latency
Authors: Vafaei Amin , Mohammadi Milad , Khadir Alireza , Zabeh Erfan , YazdaniBanafsheDaragh Faraz , Khorasani Mehran , Lashgari Reza
Published: January 2022

The timing of neuronal responses is considered to be important for information transferring and communication across individual neurons. However, the sources of variabilities in the timing of neuronal responses are not well understood and sometimes over-interpreted. A systematic variability in the response latencies of the primary visual cortex has been reported in presence of drifting grating stimulus. Whereas the response latencies are systematically dependent on stimulus orientation. To understand the underlying mechanism of these systematic latencies, we recorded the neuronal response of the cat visual cortex, area 17, and simulated the response latency of V1 neurons, with two geometric models. We showed that outputs of these two models significantly predict the response latencies of the electrophysiology recording during orientation tasks. The periodic patterns created in the raster plots were dependent on the relative position of the stimulus rotation center and the receptive-field sub-regions. We argue the position of stimulus is contributing to systematic response latencies, dependent on drifting orientation. Therefore, we provide a toolbox based on our geometrical model for determining the exact location of RF sub-regions. Our result indicates that a major source of neuronal variability is the lack of fine-tuning in the task parameters. Considering the simplicity of the orientation selectivity task, we argue fine-tuning of stimulus properties is crucial for deduction of neural variability in higher-order cortical areas and understanding their neural dynamics.
Superficial white matter microstructure affects processing speed in cerebral small vessel disease
Authors: Wang Shuyue , Zhang Fan , Huang Peiyu , Hong Hui , Jiaerken Yeerfan , Yu Xinfeng , Zhang Ruiting , Zeng Qingze , Zhang Yao , Kikinis Ron , Rathi Yogesh , Makris Nikos , Lou Min , Pasternak Ofer , Zhang Minming , O’Donnell Lauren J.
Published: January 2022

White matter hyperintensities (WMH) are a typical feature of cerebral small vessel disease (CSVD). This condition contributes to about 50% of dementias worldwide, a massive health burden in aging. Microstructural alterations in the deep white matter (DWM) have been widely examined in CSVD. However, little is known about abnormalities in the superficial white matter (SWM) and their relevance for processing speed, the main cognitive deficit in CSVD. In this paper, 141 patients with CSVD were studied. Processing speed was assessed by the completion time of the Trail Making Test Part A. White matter abnormalities were assessed by WMH burden (lesion volume on T2-FLAIR) and diffusion MRI, including DTI and free-water (FW) imaging microstructure measures. The results of our study indicate that the superficial white matter may play a particularly important role in cognitive decline in CSVD. SWM imaging measures resulted in a large contribution to processing speed, despite a relatively small WMH burden in the SWM. SWM FW had the strongest association with processing speed among all imaging markers and, unlike the other diffusion MRI measures, significantly increased between two patient subgroups with the lowest WMH burdens (possibly representing early stages of disease). When comparing two patient subgroups with the highest WMH burdens, the involvement of WMH in the SWM was accompanied by significant differences in processing speed and white matter microstructure. Given significant effects of WMH volume and regional FW on processing speed, we performed a mediation analysis. SWM FW was found to fully mediate the association between WMH volume and processing speed, while no mediation effect of DWM FW was observed. Overall, our findings identify SWM abnormalities in CSVD and suggest that the SWM has an important contribution to processing speed. Results indicate that FW in the SWM is a sensitive marker of microstructural changes associated with cognition in CSVD. This study extends the current understanding of CSVD-related dysfunction and suggests that the SWM, as an understudied region, can be a potential target for monitoring pathophysiological processes in future research.
Rsc3K, a SMV resistance gene localized on chromosome 2 in soybean cv. Kefeng No.1, interacts with virulence determinant P3
Authors: Jin Tongtong , Yin Jinlong , Xue Song , Li Bowen , Zong Tingxuan , Yang Yunhua , Liu Hui , Liu Mengzhuo , Xu Kai , Li Kai , Wang Liqun , Zhi Haijian
Published: January 2022

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens in Glycine max (L.) Merr (soybean). Twenty-two SMV strains (SC1-SC22) isolated in China were identified based on their responses to ten soybean cultivars. By using the F2-derived F3 (F2:3) and recombinant inbred line (RIL) populations of resistant Soybean cultivar (cv.) Kefeng No.1 × susceptible cv. Nannong 1138-2, we localized the gene mediating resistant to SMV-SC3 strain to a 90 kb interval on chromosome 2 in Kefeng No.1. Bean pod mottle virus (BPMV)-induced gene silencing (VIGS) were used to study the gene function of candidate genes in the mapping interval and revealed that an recombinant gene, later named as Rsc3K, caused by internal deletion of a genomic DNA frag ement in Kefeng No.1, is the resistant gene to SMV-SC3. By shuffling genes between avirulent isolate SC3 and avirulent SMV isolate 1129, we found that P3 is the virulence determinant causing resistance on Kefeng No.1. We showed the interaction between Rsc3K and P3 by the yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays. In conclusion, this study demonstrated that Rsc3K plays a crucial role in resistance of Kefeng No.1 to SMV-SC3 by direct interaction with viral protein P3.

Highlight

Rsc3K resistant to SMV-SC3 by interacting with P3.
In silico Elucidation of Dihydroquinine Mechanism of Action against Toxoplasma gondii
Authors: Ayariga Joseph A. , Huffman Aarin M. , Napier Audrey , Robertson BK , Abugri Daniel A.
Published: January 2022

Dihydroquinine (DHQ), is a quinine-based compound with anti-malarial properties. However, little is known about its mechanism of action against T. gondii inhibition, which shares similar biology with Plasmodium spp. In order to explore DHQ activity as an inhibitor of T. gondii using in vitro assays, we first used an in silico approach to decipher its mechanisms of action based on previous knowledge about its disruption of nucleic acid and protein synthesis. An in silico study was performed on T. gondii parasite replication, transcriptional and translational machinery to decipher the binding potentials of DHQ to some top selected enzymes. We report for the first time, using an in silico analysis that showed that DHQ binds strongly to DNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase and thus could affect DNA replication, transcriptional and translational activities in T. gondii. Also, we found DHQ to effectively bind to mitochondria detoxifying enzymes (i.e., superoxide dismutase (SOD), peroxidoxin, and Catalase (CAT)). In conclusion, DHQ could be a lead compound for the treatment of toxoplasmosis when successfully evaluated using in vitro and in vivo models to confirm its effectiveness and safety.
Fast parametric curve matching (FPCM) for automatic spike detection
Authors: Kleeva Daria , Soghoyan Gurgen , Komoltsev Ilia , Sinkin Mikhail , Ossadtchi Alexei
Published: January 2022

Epilepsy is a widely spread neurological disease, whose treatment often requires resection of the pathological cortical tissue. Interictal spike analysis observed in the non-invasively collected EEG or MEG data offers an attractive way to localize epileptogenic cortical structures for surgery planning purposes. Interictal spike detection in lengthy multichannel data is a daunting task that is still often performed manually. This frequently limits such an analysis to a small portion of the data which renders the appropriate risks of missing the potentially epileptogenic region. While a plethora of automatic spike detection techniques have been developed each with its own assumptions and limitations, non of them is ideal and the best results are achieved when the output of several automatic spike detectors are combined. This is especially true in the low signal-to-noise ratio conditions. To this end we propose a novel biomimetic approach for automatic spike detection based on a constrained mixed spline machinery that we dub as fast parametric curve matching (FPCM). Using the peak-wave shape parametrization, the constrained parametric morphological model is constructed and convolved with the observed multichannel data to efficiently determine mixed spline parameters corresponding to each time-point in the dataset. Then the logical predicates that directly map to verbalized text-book like descriptions of the expected interictal event morphology allow us to accomplish the spike detection task. The results of simulations mimicking typical low SNR scenario show the robustness and high ROC AUC values of the FPCM method as compared to the spike detection performed using more conventional approaches such as wavelet decomposition, template matching or simple amplitude thresholding. Applied to the real MEG and EEG data from the human patients and to rat ECoG data, the FPCM technique demonstrates reliable detection of the interictal events and localization of epileptogenic zones concordant with independent conclusions made by the epileptologist. Since the FPCM is computationally light, tolerant to high amplitude artifacts and flexible to accommodate verbalized descriptions of the arbitrary target morphology, it may complement the existing arsenal of means for analysis of noisy interictal datasets.
Thylakoid grana stacking revealed by multiplex genome editing of LHCII encoding genes
Authors: Guardini Zeno , Gomez Rodrigo L. , Caferri Roberto , Stuttmann Johannes , Dall’Osto Luca , Bassi Roberto
Published: January 2022

Land plant chloroplasts differ from algal ones for their thylakoid membranes being organized in grana: piles of vesicles paired by their stromal surface, forming domains including Photosystem (PS) II and its antenna while excluding PS I and ATPase to stroma membranes, connecting grana stacks. The molecular basis of grana stacking remain unclear. We obtained genotypes lacking the trimeric antenna complex (Lhcb1-2-3), the monomeric Lhcb4-5-6, or both. Full deletion caused loss of grana, while either monomers or trimers support 50% stacking. The expression of Lhcb5 alone restored stacking at 50%, while Lhcb2 alone produced huge grana which broke down upon light exposure. Cyclic electron transport was maintained in the lack of stacking, while excitation energy balance between photosystems and the repair efficiency of damaged Photosystem II were affected. We conclude that grana evolved for need of regulating energy balance between photosystems under terrestrial canopy involving rapid changes in photon spectral distribution.
The SARS-CoV-2 infection in Thailand: analysis of spike variants complemented by protein structure insights
Authors: Ittisoponpisan Sirawit , Yahangkiakan Shalip , Sternberg Michael J E , David Alessia
Published: January 2022

Thailand was the first country outside China to officially report COVID-19 cases. Despite the strict regulations for international arrivals, up until February 2021, Thailand had been hit by two major outbreaks. With a large number of SARS-CoV-2 sequences collected from patients, the effects of many genetic variations, especially those unique to Thai strains, are yet to be elucidated. In this study, we analysed 439,197 sequences of the SARS-CoV-2 spike protein collected from NCBI and GISAID databases. 595 sequences were from Thailand and contained 52 variants, of which 6 had not been observed outside Thailand (p.T51N, p.P57T, p.I68R, p.S205T, p.K278T, p.G832C). These variants were not predicted to be of concern. We demonstrate that the p.D614G, although already present during the first Thai outbreak, became the prevalent strain during the second outbreak, similarly to what was described in other countries. Moreover, we show that the most common variants detected in Thailand (p.A829T, p.S459F and p.S939F) do not appear to cause any major structural change to the spike trimer or the spike-ACE2 interaction. Among the variants identified in Thailand was p.N501T. This variant, which involves an asparagine critical for spike-ACE2 binding, was not predicted to increase SARS-CoV-2 binding, thus in contrast to the variant of global concern p.N501Y. In conclusion, novel variants identified in Thailand are unlikely to increase the fitness of SARS-CoV-2. The insights obtained from this study could aid SARS-CoV-2 variants prioritisations and help molecular biologists and virologists working on strain surveillance.
PIP2-Dependent Thermoring Basis for Cold-Sensing of the TRPM8 Biothermometer
Authors: Wang Guangyu
Published: January 2022

The menthol sensor TRPM8 can be activated by cold and thus serves as a biothermometer in a primary afferent sensory neuron for innocuous-to-noxious cold detection. However, the precise structural origins of specific temperature thresholds and sensitivity have remained elusive. Here, a grid thermodynamic model was employed to examine if the temperature-dependent noncovalent interactions found in the 3D structures of thermo-gated TRPM8 could assemble into a well-organized fluidic grid-like mesh network, featuring the constrained grids as the thermorings for cold-sensing in response to PIP2, Ca2+and chemical agents. The results showed that the different interactions of TRPM8 with PIP2during the thermal incubation induced the formation of the biggest grids with distinct melting temperature threshold ranges. Further, the overlapped threshold ranges between open and pre-open closed states were required for initial cold activation with the matched thermo-sensitivity and the decrease in the systematic thermal instability. Finally, the intact anchor grid near the lower gate was important for channel opening with the active selectivity filter. Thus, PIP2-dependent thermorings in TRPM8 may play a pivotal role in cold sensing. (176 words)
Nested spatial and temporal modeling of environmental conditions associated with genetic markers of Vibrio parahaemolyticus in Washington state Pacific oysters
Authors: Fries Brendan , Davis Benjamin J. K. , Corrigan Anne E. , DePaola Angelo , Curriero Frank C.
Published: January 2022

The Pacific Northwest (PNW) is one of the largest commercial harvesting areas for Pacific oysters (Crassostrea gigas) in the United States. Vibrio parahaemolyticus, a bacterium naturally present in estuarine waters, accumulates in shellfish and is a major cause of seafood-borne illness. Growers, consumers, and public-health officials have raised concerns about rising vibriosis cases in the region. V. parahaemolyticus genetic markers (tlh, tdh, trh) were estimated using an MPN-PCR technique in Washington State Pacific oysters regularly sampled between May and October from 2005 to 2019 (N=2,836); environmental conditions were also measured at each sampling event. Multilevel mixed-effects regression models were used to assess relationships between environmental measures and genetic markers as well as genetic marker ratios (trh:tlh, tdh:tlh, and tdh:trh), accounting for variation across space and time. Spatial and temporal dependence were also accounted for in the model structure. Model fit improved when including environmental measures from previous weeks (1-week lag for air temperature, 3-week lag for salinity). Positive associations were found between tlh and surface water temp, specifically between 15°C and 26°C, and between trh and surface water temperature up to 26°C. tlh and trh were negatively associated with 3-week lagged salinity in the most saline waters (> 27 ppt). There was also a positive relationship between tissue temperature and tdh, but only above 20°C. The tdh:tlh ratio displayed analogous inverted non-linear relationships as tlh. The non-linear associations found between the genetic targets and environmental measures demonstrate the complex habitat suitability of V. parahaemolyticus. Additional associations with both spatial and temporal variables also suggest there are influential unmeasured environmental conditions that could further explain bacterium variability. Overall, these findings confirm previous ecological risk factors for vibriosis in Washington State, while also identifying new associations between lagged temporal effects and pathogenic markers of V. parahaemolyticus.
Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes
Authors: Pineau Judith , Pinon Léa , Mesdjian Olivier , Fattaccioli Jacques , Duménil Ana-Maria Lennon , Pierobon Paolo
Published: January 2022

Immune synapse formation is a key step for lymphocyte activation. In B lymphocytes, the immune synapse controls the production of high-affinity antibodies, thereby defining the efficiency of humoral immune responses. While the key roles played by both the actin and microtubule cytoskeletons in the formation and function of the immune synapse have become increasingly clear, how the different events involved in synapse formation are coordinated in space and time by actin-microtubule interactions is not understood. Using a microfluidic pairing device, we studied with unprecedented resolution the dynamics of the various events leading to immune synapse formation and maintenance. Our results identify two groups of events, local and global dominated by actin and microtubules dynamics, respectively. They further highlight an unexpected role for microtubules and the GEF-H1-RhoA axis in restricting F-actin polymerization at the immune synapse to define the cell polarity axis, allowing the formation and maintenance of a unique competent immune synapse.
A role for Lin28a in aging-associated decline of adult hippocampal neurogenesis
Authors: Hu Zhechun , Ma Jiao , Yue Huimin , Li Xiaofang , Wang Chao , Wang Liang , Sun Binggui , Chen Zhong , Wang Lang , Gu Yan
Published: January 2022

Hippocampal neurogenesis declines with aging. Wnt ligands and antagonists within the hippocampal neurogenic niche regulate the proliferation of neural progenitor cells and the development of new neurons, and the changes of their levels in the niche mediate aging-associated decline of neurogenesis. We found that RNA-binding protein Lin28a remained existent in neural progenitor cells and granule neurons in the adult hippocampus, and decreased with aging. Loss of Lin28a inhibited the responsiveness of neural progenitor cells to niche Wnt agonist and reduced neurogenesis, thus impairing pattern separation. Overexpression of Lin28a increased the proliferation of neural progenitor cells, promoted the functional integration of newborn neurons, restored neurogenesis in Wnt-deficient dentate gyrus, and rescued the impaired pattern separation in aging mice. Our data suggest that Lin28a regulates adult hippocampal neurogenesis as an intracellular mechanism by responding to niche Wnt signals, and its decrease is involved in aging-associated decline of hippocampal neurogenesis as well as related cognitive functions.
Essential regulatory functions of CaMKII T286 phosphorylation in LTP and two distinct forms of LTD
Authors: Cook Sarah G. , Rumian Nicole L. , Bayer K. Ulrich
Published: January 2022

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates both long-term potentiation and depression (LTP and LTD) of excitatory synapses, two opposing forms of synaptic plasticity induced by strong versus weak stimulation of NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD is prevalent in juvenile hippocampus, but in mature hippocampus, LTD is still readily induced by stimulating metabotropic glutamate receptors (mGluRs). Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation that induces Ca2+-independent autonomous kinase activity. This autophosphorylation (i) accelerated CaMKII movement to excitatory synapses after LTP stimuli and (ii) was required for the movement to inhibitory synapses after NMDAR-LTD stimuli. Similar to NMDAR-LTD, the mGluR-LTD stimuli did not induce any CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Taken together, even though CaMKII T286 autophosphorylation has a longstanding prominent role in LTP, it is also required for both major forms of LTD in hippocampal neurons, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.
A triad of kicknet sampling, eDNA metabarcoding, and predictive modeling to assess aquatic macroinvertebrate biodiversity
Authors: Keck François , Hürlemann Samuel , Locher Nadine , Stamm Christian , Deiner Kristy , Altermatt Florian
Published: January 2022

Monitoring freshwater biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution models (SDM). Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared a triad of approaches in order to understand assessment of aquatic macroinvertebrate biodiversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species distribution model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species distribution model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the two approaches, particularly at local scale. Overall, the more specific set of primers, namely fwhF2/EPTDr2n, was most efficient for the detection of target species and for characterizing the diversity of EPT. Moreover, we found that the species richness measured by eDNA was poorly correlated to the richness measured by kicknet sampling and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the statistical model. Overall, however, neither eDNA nor the traditional approach had strong links to the predictive models, indicating inherent limitations in upscaling species richness estimates. Future challenges include improving the accuracy and sensitivity of each approach individually yet also acknowledge their respective limitations, in order to best meet stakeholder demands addressing the biodiversity crisis we are facing.
Continuous Daily Head and Trunk Acceleration Recording for the Assessment of Bone Stimulation in Astronauts
Authors: Sugita Yoshino , Clément Gilles
Published: January 2022

The acceleration of the head and hip along the x-, y-, and z-axis of 14 healthy subjects was recorded during two sessions of 12 consecutive hours. The magnitude, frequency content, and root mean square of the acceleration signals were used to determine the type of physical activity (sitting, standing, walking, etc.) during normal daily life on Earth. The acceleration signal slope (jerk) was also calculated to assess whether these activities were sufficient to maintain bone mineral density. These measurements indicated that the changes in vertical acceleration experienced by our subjects during normal daily life were presumably sufficient to maintain bone mineral density. However, these changes might not be sufficient for postmenopausal women and astronauts during long-term exposure to weightlessness during spaceflight.
Molecular Dynamics of DNA Translocation by FtsK
Authors: Pajak Joshua , Arya Gaurav
Published: January 2022

The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.
Directed evolution of angiotensin-converting enzyme 2 (ACE2) peptidase activity profiles for therapeutic applications
Authors: Heinzelman Pete , Romero Philip A.
Published: January 2022

Angiotensin Converting-Enzyme 2 (ACE2) is currently being investigated for its ability to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases, but its broad substrate scope and diverse physiological roles limit its potential as a therapeutic agent. In this work we engineer ACE2 variants with enhanced hydrolytic activity and specificity toward Angiotensin-II (Ang-II) that may lead to new therapeutic candidates with improved efficacy and reduced off-target side effects. We established a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants with improved Ang-II activity and specificity relative to the off-target peptide substrates Apelin-13 and Angiotensin-I (Ang-I). We screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371 and Y510) that can be mutated to enhance ACE2’s activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wildtype ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat), a sixfold diminished catalytic efficiency (kcat/Km) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes more Ang-II than wildtype ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts.

Significance Statement

The Angiotensin Converting Enzyme 2 (ACE2) carboxypeptidase is being clinically trialed for treatment of acute respiratory distress syndrome (ARDS) related to traumatic injuries and viral infections. We developed an enzyme engineering platform that enabled discovery of ACE2 variants possessing enhanced peptide hydrolysis activity and specificity profiles relevant to ARDS treatment. Beyond their potential utility as ARDS therapeutics, our improved ACE2 variants could be further developed for new unexplored ACE2 therapeutic contexts such as treating Alzheimer’s Disease.