Miracle-dz/UniFAR-FTUs huggingface dataset:
Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02–1.52 Å) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9–1.16 Å). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information. Significance X-ray crystallography has allowed biologists to visualize the proteins that carry out complex biological processes and has provided powerful insights into how these molecules function. Our next level of understanding requires information about the ensemble of conformations that is at the heart of protein function and energetics. Prior results have shown that room temperature (RT) X-ray crystallography provides extensive ensemble information, but are subject to extenstive X-ray damage. We found that ensemble information with little or no effects from X-ray damage can be collected at RT. We also found that damage effects may be more prevalent than recognized in structures obtained under current standard cryogenic conditions. RT X-ray crystallography can be routinely implemented to obtain needed information about conformational ensembles.
TLR4 regulation in human fetal membranes as an explicative mechanism of a pathological preterm case

The integrity of human fetal membranes is crucial for harmonious fetal development throughout pregnancy. Their premature rupture is often the consequence of a physiological phenomenon previously exacerbated. Beyond all biological processes implied, inflammation is of primary importance and is qualified as “sterile” at the end of pregnancy. Complementary methylomic and transcriptomic strategies on amnion and choriodecidua explants taken from the altered (cervix zone) and intact fetal membranes at term and before labor were used in this study. By cross-analyzing genome-wide studies strengthened by in vitro experiments, we deciphered how the expression of Toll-like receptor 4 (TLR4), a well-known actor of pathological fetal membrane rupture, is controlled. Indeed, it is differentially regulated in the altered zone and between both layers by a dual mechanism: 1) the methylation of TLR4 and miRNA promoters and 2) targeting by miRNA (let-7a-2 and miR-125b-1) acting on the 3’-UTR of TLR4. Consequently, this study demonstrates that a fine regulation of TLR4 is required for sterile inflammation establishment at the end of pregnancy and that it may be dysregulated in the pathological premature rupture of membranes.
GTL1 is required for a robust root hair growth response to avoid nutrient overloading

Root hair growth is tuned in response to the environment surrounding plants. While most of previous studies focused on the enhancement of root hair growth during nutrient starvation, few studies investigated the root hair response in the presence of excess nutrients. We report that the post-embryonic growth of wild-type Arabidopsis plants is strongly suppressed with increasing nutrient availability, particularly in the case of root hair growth. We further used gene expression profiling to analyze how excess nutrient availability affects root hair growth, and found that RHD6 subfamily genes, which are positive regulators of root hair growth, are down-regulated in this condition. On the other hand, defects in GTL1 and DF1, which are negative regulators of root hair growth, cause frail and swollen root hairs to form when excess nutrients are supplied. Additionally, we observed that the RHD6 subfamily genes are mis-expressed in gtl1-1 df1-1. Furthermore, overexpression of RSL4, an RHD6 subfamily gene, induces swollen root hairs in the face of a nutrient overload, while mutation of RSL4 in gtl1-1 df1-1 restore root hair swelling phenotype. In conclusion, our data suggest that GTL1 and DF1 prevent unnecessary root hair formation by repressing RSL4 under excess nutrient conditions.
Radiolabeling of PSMA-617 with 89Zr: A Novel Use of DMSO for Radiochemical Yield Enhancement and Preliminary Small-Animal PET Results

Introduction Prostate-specific membrane antigen (PSMA)-targeted ligands, including PSMA-617, have been developed for theranostics of prostate cancer. 68Ga-PSMA-617 is the de facto standard of PSMA Positron Emission Tomography (PET) for imaging in prostate cancer patients prior to radioligand therapy (RLT) with 177Lu-PSMA-617. The dose-limiting toxicity for PSMA-RLT is damage to the kidney. PET scans using 68Ga-PSMA-617 have to be performed within a few hours of injection due to its short half-life (68 min). However, the presence of radioactivity in urine at the PET imaging timepoint hampers the dose optimization of 177Lu (half-life 6.6 d)-labeled PSMA-617. Thus, the long-lived positron emitter 89Zr (half-life 3.3 d) is suited for optimizing the doses of 177Lu-PSMA-617 because PET scans can be performed after excretion of radioactive urine. Although 89Zr has great potential for PET imaging, its inadequate incorporation into 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), limits its applications. Here, we report the radiolabeling of PSMA-617 with 89Zr and preliminary PET imaging studies using 89Zr-PSMA-617. Methods DMSO and HEPES buffer were used to label PSMA-617 with 89Zr. The dissociation constant (Kd) of 89Zr-PSMA-617 was determined using a cell-binding assay. Delayed-PET scans using 89Zr-PSMA-617 were performed at 24 h (N = 5). Results 89Zr-PSMA-617 was prepared with a radiochemical yield of 70 ± 9%. The Kd value was 6.8 nM. In PET imaging, standardized uptake value (SUV) was highest in LNCaP tumors (SUVmax = 0.98 ± 0.32), whereas it was low in kidney (SUVmax = 0.18 ± 0.7). Conclusion The preparation of 89Zr-PSMA-617 was achieved by using the DMSO and HEPES buffer. 89Zr-PSMA-617 visualize the PSMA positive LNCaP tumors without accumulation in bladder. Advances in knowledge and implications for patient care The use of 89Zr-PSMA-617 to predict the radiation doses in normal tissues lead to safe and effective RLT with 177Lu-PSMA-617.
A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations

Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, that addition of other nsps modulates expression levels, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis. Importance statement The impact of the COVID-19 pandemic has made it essential to better understand the basic biology of SARS-CoV-2, and to search for compounds that can block the activity of key SARS-CoV-2 replication enzymes. However, studies with live SARS-CoV-2 require biosafety level 3 facilities, while existing replicon systems depend on long positive-sense subgenomes that are often difficult to manipulate or produce a high background signal, limiting drug-screens and a rapid analysis of emerging SARS-CoV-2 mutations during the COVID-19 pandemic. To make it easier to study emerging SARS-CoV-2 mutants and screen for inhibitors, we developed a simple mini-replicon that produces a minimal background signal, that can be used in any tissue culture lab, and that only requires four small plasmids to work.
Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes

Abstract In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3′ end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3′ ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3′ ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3′ end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3′ end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.
USP9X Is Required to Maintain Cell Survival in Response to High-LET Radiation

Ionizing radiation (IR) principally acts through induction of DNA damage that promotes cell death, although the biological effects of IR are more broad ranging. In fact, the impact of IR of higher-linear energy transfer (LET) on cell biology is generally not well understood. Critically, therefore, the cellular enzymes and mechanisms responsible for enhancing cell survival following high-LET IR are unclear. To this effect, we have recently performed siRNA screening to identify deubiquitylating enzymes that control cell survival specifically in response to high-LET α-particles and protons, in comparison to low-LET X-rays and protons. From this screening, we have now thoroughly validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using small interfering RNA (siRNA), leads to significantly decreased survival of cells after high-LET radiation. We consequently investigated the mechanism through which this occurs, and demonstrate that an absence of USP9X has no impact on DNA damage repair post-irradiation nor on apoptosis, autophagy, or senescence. We discovered that USP9X is required to stabilize key proteins (CEP55 and CEP131) involved in centrosome and cilia formation and plays an important role in controlling pericentrin-rich foci, particularly in response to high-LET protons. This was also confirmed directly by demonstrating that depletion of CEP55/CEP131 led to both enhanced radiosensitivity of cells to high-LET protons and amplification of pericentrin-rich foci. Our evidence supports the importance of USP9X in maintaining centrosome function and biogenesis and which is crucial particularly in the cellular response to high-LET radiation.
Discovery and Characterization of a Novel Bipartite Botrexvirus From the Phytopathogenic Fungus Botryosphaeria dothidea

In this study, we describe a novel positive, single-stranded (+ss) RNA mycovirus, named Botryosphaeria dothidea botrexvirus 1 (BdBV1), from a phytopathogenic fungus Botryosphaeria dothidea showing abnormal morphology and attenuated virulence. BdBV1 is phylogenetically related to Botrytis virus X (BotVX) and is the second potential member of the proposed genus Botrexvirus in the family Alphaflexiviridae. However, it differs from the monopartite BotVX in that BdBV1 possesses a bipartite genome comprised of two ssRNA segments (RNA1 and RNA2 with lengths of 5,035 and 1,063 nt, respectively). BdBV1 RNA1 and RNA2 encode putative RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes, which share significant identity with corresponding genes in both fungal and plant viruses. Moreover, open reading frames (ORFs) 2–4 of BdBV1 RNA1 shared no detectable identity with any known viral proteins. Immunosorbent electron microscopy (ISEM) analysis using an antibody against the virus CP generated in vitro revealed that BdBV1 is encapsidated in filamentous particles. A comparison of the biological effects of BdBV1 infection on symptoms and growth in isogenic lines of virus-free and virus-infected B. dothidea revealed that BdBV1 is probably involved in reduced growth and virulence of the host fungus. This study describes and characterizes a novel bipartite botrexvirus, which is closely related to uni- and multi-partite fungal and plant viruses and contributes useful information to a better understanding of virus evolution.
Pharmacological Treatment in the Management of Chronic Subdural Hematoma

Background: Several pharmacological treatments have been used to treat patients with chronic subdural hematoma (CSDH), although little is known about the comparative effectiveness of different classes of medication. We performed a Bayesian network meta-analysis to compare and rank the efficacy and safety of five drug regimens to determine the best treatment for this group of patients. Methods: We systematically searched PubMed, Medline, clinicaltrials.gov, the Cochrane database, and Embase to identify relevant randomized clinical trials (RCTs) comparing drug treatments in adult patients with CSDH. A network meta-analysis was conducted using a Bayesian framework. Random- and fixed-effects models were used to pool the network results, and the preferred model was selected by comparing the deviance information criteria (DIC). Efficacy outcomes included recurrence requiring surgery, changes in hematoma volume, and a good recovery. The safety outcomes were treatment-related adverse events and all-cause mortality. Results: In this Bayesian network meta-analysis, available data were obtained from 12 eligible trials, including 2,098 patients and 5 techniques. Compared to placebo, atorvastatin (RR: 0.45, 95% CrI: 0.24–0.81) and dexamethasone (RR: 0.38, 95% CrI: 0.22–0.63) were similarly effective in reducing recurrence requiring surgery by 55% and 62%, respectively. Dexamethasone (RR: 0.46, 95% CrI: 0.23–0.91) was more effective in reducing recurrence requiring surgery than goreisan. Additionally, atorvastatin reduced the hematoma volume to a greater extent than placebo (MD: −7.44, 95% CrI: −9.49 to −5.43) or goreisan (MD: −14.09, 95% CrI: −23.35 to −4.82). Moreover, tranexamic acid (MD: −12.07, 95% CrI: −21.68 to −2.29) reduced the hematoma volume to a greater extent than goreisan. No significant differences were detected between drugs and placebo with regard to a good recovery. In terms of safety, dexamethasone (RR: 1.96, 95% CrI: 1.20–3.28) increased the risk of mortality compared to placebo. Conclusion: These findings suggest that dexamethasone is the best treatment to reduce recurrence and atorvastatin is the best treatment to reduce hematoma volume in patients with CSDH. However, clinicians should pay close attention to the elevated risk of all-cause mortality and potential adverse events caused by dexamethasone. Future well-designed RCTs with more participants are needed to verify these findings. Clinical Trial Registration: http://osf.io/u9hqp.
SIMON: A Digital Protocol to Monitor and Predict Suicidal Ideation

Each year, more than 800,000 persons die by suicide, making it a leading cause of death worldwide. Recent innovations in information and communication technology may offer new opportunities in suicide prevention in individuals, hereby potentially reducing this number. In our project, we design digital indices based on both self-reports and passive mobile sensing and test their ability to predict suicidal ideation, a major predictor for suicide, and psychiatric hospital readmission in high-risk individuals: psychiatric patients after discharge who were admitted in the context of suicidal ideation or a suicidal attempt, or expressed suicidal ideations during their intake. Specifically, two smartphone applications -one for self-reports (SIMON-SELF) and one for passive mobile sensing (SIMON-SENSE)- are installed on participants' smartphones. SIMON-SELF uses a text-based chatbot, called Simon, to guide participants along the study protocol and to ask participants questions about suicidal ideation and relevant other psychological variables five times a day. These self-report data are collected for four consecutive weeks after study participants are discharged from the hospital. SIMON-SENSE collects behavioral variables -such as physical activity, location, and social connectedness- parallel to the first application. We aim to include 100 patients over 12 months to test whether (1) implementation of the digital protocol in such a high-risk population is feasible, and (2) if suicidal ideation and psychiatric hospital readmission can be predicted using a combination of psychological indices and passive sensor information. To this end, a predictive algorithm for suicidal ideation and psychiatric hospital readmission using various learning algorithms (e.g., random forest and support vector machines) and multilevel models will be constructed. Data collected on the basis of psychological theory and digital phenotyping may, in the future and based on our results, help reach vulnerable individuals early and provide links to just-in-time and cost-effective interventions or establish prompt mental health service contact. The current effort may thus lead to saving lives and significantly reduce economic impact by decreasing inpatient treatment and days lost to inability.
NAFLD or MAFLD: Which Has Closer Association With All-Cause and Cause-Specific Mortality?—Results From NHANES III

Background: The recent change of terminology from non-alcoholic fatty liver disease (NAFLD) to metabolic dysfunction-associated fatty liver disease (MAFLD) has raised heated discussion. We aim to investigate the association of MAFLD or NAFLD with all-cause and cause-specific mortality to compare the outcomes of the two diagnostic criteria in population-based study. Methods: We recruited 12,480 participants from the Third National Health and Nutrition Examination Survey (NHANES III) with matched mortality data in 2015. Participants were divided into four groups for survival analysis: without NAFLD or MAFLD, with only NAFLD, only MAFLD. Cox proportional hazard regression was used to estimate multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for all-cause and cause-specific mortality. Subgroup analysis were applied in MAFLD patients. Results: The weighted prevalence of MAFLD and NAFLD was relatively 27.4 and 27.9%. Participants with NAFLD or MAFLD were largely overlapped (weighted Cohen's kappa coefficient 0.76). MAFLD increased the overall risk for total mortality in a greater magnitude than NAFLD [HR 2.07 (95% CI 1.86, 2.29) vs. 1.47 (1.20, 1.79)], However, the difference was non-significant after metabolic parameters were adjusted. Risks for cardiovascular, neoplasm, and diabetes-related mortality were similar between MAFLD and NAFLD. Referring to individuals without both NAFLD and MAFLD, individuals with only NAFLD showed reduced total mortality [HR 0.48 (0.34, 0.68)] and neoplasm mortality [HR 0.46 (0.24, 0.89)] in crude. Nevertheless, individuals with only MAFLD independently increased the risk for total mortality [adjusted HR 1.47 (1.22, 1.77)] and neoplasm mortality [aHR 1.58 (1.09, 2.28)]. The risk for overall mortality in MAFLD was consistent between subgroups except for race-ethnicity and whether secondary to viral hepatitis. Conclusions: Participants with MAFLD or NAFLD were highly concordant. MAFLD showed greater risk for all-cause mortality and equal risk for cause-specific mortality referring to NAFLD. The new terminology excluded participants with lower mortality risk and included participants with higher risk. Drug development for MAFLD should consider ethnic differences.
Immediate and Delayed Effects of Cupping Therapy on Reducing Neuromuscular Fatigue

Cupping therapy has been popular in elite athletes in recent years. However, the effect of cupping therapy on reducing muscle fatigue has not been investigated. The purpose of this study was to investigate the immediate and delayed effects of cupping therapy on reducing biceps brachii fatigue during biceps curls. Twelve healthy untrained participants were recruited for this repeated-measures study. Cupping therapy (–300 mmHg pressure for 5 min) and sham control (no negative pressure for 5 min) were applied after biceps fatigue induced by performing repeated biceps curls at 75% of the 10 repetitions of maximum of the non-dominant hand. Surface electromyography (EMG) with spectral analyses [mean frequency (MNF), median frequency (MDF), and spectral moments ratio (SMR)] were used to assess muscle fatigue during the fatigue task. EMG signals during the first 10 repetitions and the last 10 repetitions of biceps curls were used to assess neuromuscular fatigue. There were significant decreases in MNF and MDF and a significant increase in SMR immediately and 24 h after the sham control (no intervention). When comparing the MNF, MDF, and SMR after cupping therapy to the sham control, there was no significant immediate effect on reducing muscle fatigue. However, there was a significant delayed effect on improving recovery following fatigue for the cupping therapy compared to the sham control (MNF changes: sham 0.87 ± 0.02 vs. cupping 0.91 ± 0.02, p < 0.05; MDF changes sham: 0.85 ± 0.03 vs. cupping: 0.91 ± 0.02, p < 0.05; SMR changes: sham 1.89 ± 0.15 vs. cupping 1.58 ± 0.13, p < 0.05). The findings of this study demonstrate that there is a time effect of cupping therapy for reducing muscle fatigue. Cupping therapy is effective on reducing biceps brachii muscle fatigue after 24 h.