bluuebunny/crossref_metadata_2025 huggingface dataset:
Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China
Authors: Guo Yinjuan , Cao Xingwei , Yang Jinghui , Wu Xiaocui , Liu Yin , Wan Baoshan , Hu Longhua , Wang Hongxiu , Yu Fangyou
Published: January 2021

Introduction. Resistance to rifampin (RIF) in

Mycobacterium tuberculosis

infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive.

Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance.

Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against

M. tuberculosis

.

Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for

M. tuberculosis

isolates were determined using commercial Sensititre

M. tuberculosis

MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis.

Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01).

Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by

M. tuberculosis

strains harbouring these mutations.
Tethered NOS-3, a nematode Nanos RNA-binding protein, enhances reporter expression and mRNA stability
Authors: Doenier Jonathan , Lynch Tina R , Kimble Judith , Aoki Scott T
Published: January 2021

Robust methods are critical for testing thein vivoregulatory mechanism of RNA binding proteins. Here we report improvement of a protein-mRNA tethering assay to probe the function of an RNA binding protein in its natural context within theC. elegansadult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. Thegfpreporter 3’UTR harbors functional binding elements for λN22 peptide, while themCherryreporter 3’UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed aC. elegansNanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its N-terminal intrinsically disordered region, not its C-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: classically Nanos proteins are expected to repress RNA, butnos-3had been found to promotegld-1expression, an effect that could be direct. Regardless, the new dual reporter dramatically improvesin situquantitation of reporter expression after RBP tethering to determine its molecular mechanism in a multicellular tissue.
Discernment of Mediator and Outcome Measurement in the PACE trial
Authors: Carr Ewan , Vitoratou Silia , Chalder Trudie , Goldsmith Kimberley
Published: January 2021

Background

When measuring latent traits, such as those used in psychology and psychiatry, it can be unclear whether the instruments used are measuring different concepts. This issue is particularly important in context of mediation analysis, since for a sound mediation hypothesis the mediator and outcome should be distinct. We sought to assess the extent of measurement overlap between mediators and outcomes in the PACE trial (n=640).

Methods

Potential measurement overlap was assessed using generalised linear latent variable models where confirmatory factor models quantified the extent to which the addition of cross-loading items resulted in significant improvements in model fit.

Results

Out of 26 mediator-outcome pairs considered, only six showed evidence of cross-loading items, supporting the suggestion that mediator and outcome constructs in the PACE trial were conceptually distinct.

Conclusions

This study highlights the importance of assessing measurement overlap in mediation analyses with latent traits to ensure mediator and outcome instruments are distinct.
Evaluation of the rapidBACpro®II kit for the rapid identification of microorganisms directly from blood cultures using MALDI-TOF MS
Authors: Oviaño Marina , Ingebretsen André , Steffensen Anne K , Croxatto Antony , Prod’hom Guy , Quiroga Lidia , Bou Germán , Greub Gilbert , Rodríguez-Sánchez Belén
Published: January 2021

Objectives

Identification of microorganisms directly from blood cultures (BCs) using MALDI-TOF MS has shown to be the application with most impact in this methodology. In this study, a novel commercial method, the rapidBACpro®II, was evaluated in four clinical microbiology laboratories.

Methods

Positive blood culture samples (n=801) were processed using the rapidBACpro®II kit and then compared with routine gold standard. A subset of monomicrobial BCs (n=560) were analyzed in parallel with the Sepsityper®kit (Bruker Daltonics, Bremen, Germany) and compared with the rapidBACpro®II kit. In addition, the rapidBACpro®II kit was also compared with two different in-house methods.

Results

Overall, 80.0% of the monomicrobial isolates (609/761) were correctly identified by the rapidBACpro®II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper®kit yielded higher rates of correct species-level identification provided by the rapidBACpro®II kit for all categories (p>0.0001) except for yeasts identified with score values >1.7. It also proved superior to the ammonium chloride method (p>0.0001) but the differential centrifugation method allowed higher rates of correct identification for Gram negative bacteria (p>0.1).

Conclusions

The rapidBACpro®II kit allowed a high rate of microorganisms correctly identified. The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods. This fact was especially interesting in the case ofStaphylococcussp. andStreptococcussp. in order to elucidate their clinical impact, for example in device-associated bacteremia.
High-fidelity discrimination of ARDS versus other causes of respiratory failure using natural language processing and iterative machine learning
Authors: Afshin-Pour Babak , Qiu Michael , Hosseini Shahrzad , Stewart Molly , Horsky Jan , Aviv Rachel , Zhang Nasen , Narasimhan Mangala , Chelico John , Musso Gabriel , Hajizadeh Negin
Published: January 2021

Despite the high morbidity and mortality associated with Acute Respiratory Distress Syndrome (ARDS), discrimination of ARDS from other causes of acute respiratory failure remains challenging, particularly in the first 24 hours of mechanical ventilation. Delay in ARDS identification prevents lung protective strategies from being initiated and delays clinical trial enrolment and quality improvement interventions. Medical records from 1,263 ICU-admitted, mechanically ventilated patients at Northwell Health were retrospectively examined by a clinical team who assigned each patient a diagnosis of “ARDS” or “non-ARDS” (e.g., pulmonary edema). We then applied an iterative pre-processing and machine learning framework to construct a model that would discriminate ARDS versus non-ARDS, and examined features informative in the patient classification process. Data made available to the model included patient demographics, laboratory test results from before the initiation of mechanical ventilation, and features extracted by natural language processing of radiology reports. The resulting model discriminated well between ARDS and non-ARDS causes of respiratory failure (AUC=0.85, 89% precision at 20% recall), and highlighted features unique among ARDS patients, and among and the subset of ARDS patients who would not recover. Importantly, models built using both clinical notes and laboratory test results out-performed models built using either data source alone, akin to the retrospective clinician-based diagnostic process. This work demonstrates the feasibility of using readily available EHR data to discriminate ARDS patients prospectively in a real-world setting at a critical time in their care and highlights novel patient characteristics indicative of ARDS.
Trajectories of Hypoxemia & Respiratory System Mechanics of COVID-19 ARDS in the NorthCARDS dataset
Authors: Jafari Daniel , Gandomi Amir , Makhnevich Alex , Qiu Michael , Rolston Daniel M , Gottesman Eric P , Tsegaye Adey , Mayo Paul H , Stewart Molly E , Zhang Meng , Hajizadeh Negin
Published: January 2021

Rationale

The preliminary reports of COVID Acute Respiratory Distress Syndrome (COVIDARDS) suggest the existence of a subset of patients with higher lung compliance despite profound hypoxemia. Understanding heterogeneity seen in patients with COVIDARDS and comparing to non-COVIDARDS may inform tailored treatments.

Objectives

To describe the trajectories of hypoxemia and respiratory compliance in COVIDARDS and associations with outcomes.

Methods

A multidisciplinary team of frontline clinicians and data scientists created the Northwell COVIDARDS dataset (NorthCARDS) leveraging over 11,542 COVID-19 hospital admissions. Data was summarized to describe differences based on clinically meaningful categories of lung compliance, and compared to non-COVIDARDS reports. A sophisticated method of extrapolating PaO2 from SpO2, as well estimating FiO2 from non invasive oxygen delivery devices were utilized to create meaningful trends of derived PaO2 to FiO2 (P/F).

Measurements and Main Results

Of the 1595 COVIDARDS patients in the NorthCARDS dataset, there were 538 (34·6%) who had very low lung compliance (<20ml/cmH2O), 982 (63·2%) with low-normal compliance (20-50ml/cmH2O), and 34 (2·2%) with high lung compliance (>50ml/cmH2O). The very low compliance group had double the median time to intubation compared to the low-normal group (107 hours(IQR 26·3, 238·3) vs. 37·9 hours(IQR 4·8, 90·7)). Oxygenation trends have improved in all groups after a nadir immediately post intubation. The P/F ratio improved from a mean of 109 to 155, with the very low compliance group showing a smaller improvement compared to low compliance group. The derived P/F trends closely correlated with blood gas analysis driven P/F trends, except immediately post intubation were the trends diverge as illustrated in the image. Overall, 67·5% (n=1049) of the patients died during the hospitalization. In comparison to non-COVIDARDS reports, there were less patients in the high compliance category (2.2%vs.12%, compliance ≥50mL/cmH20), and more patients with P/F ≤ 150 (57·8% vs. 45.6%). No correlation was apparent between lung compliance and P/F ratio. The Oxygenation Index was similar, (11·12(SD 5·67)vs.12·8(SD 10·8)).

Conclusions

Heterogeneity in lung compliance is seen in COVIDARDS, without apparent correlation to degree of hypoxemia. Notably, time to intubation was greater in the very low lung compliance category. Understanding ARDS patient heterogeneity must include consideration of treatment patterns in addition to trajectories of change in patient-level data and demographics.
Resting State Functional Connectivity Predicts Future Changes in Sedentary Behavior
Authors: Morris Timothy P. , Kucyi Aaron , Anteraper Sheeba Arnold , Geddes Maiya Rachel , Nieto-Castañon Alfonso , Burzynska Agnieszka , Gothe Neha , Fanning Jason , Salerno Elizabeth , Whitfield-Gabrieli Susan , Hillman Charles H. , McAuley Edward , Kramer Arthur F.
Published: January 2021

Information about a person’s available energy resources is integrated in daily behavioral choices that weigh motor costs against expected rewards. It has been posited that humans have an innate attraction towards effort minimization and that executive control is required to overcome this prepotent disposition. With sedentary behaviors increasing at the cost of millions of dollars spent in health care and productivity losses due to physical inactivity-related deaths, understanding the predictors of sedentary behaviors will improve future intervention development and precision medicine approaches. In 64 healthy older adults participating in a 6-month aerobic exercise intervention, we use neuroimaging (resting state functional connectivity), baseline measures of executive function and accelerometer measures of time spent sedentary to predict future changes in objectively measured time spent sedentary in daily life. Using cross-validation and bootstrap resampling, our results demonstrate that functional connectivity between 1) the anterior cingulate cortex and the supplementary motor area and 2) the right anterior insula and the left temporoparietal/temporooccipital junction, predict changes in time spent sedentary, whereas baseline cognitive, behavioral and demographic measures do not. Previous research has shown activation in and between the anterior cingulate and supplementary motor area as well as in the right anterior insula during effort avoidance and tasks that integrate motor costs and reward benefits in effort-based decision making. Our results add important knowledge toward understanding mechanistic associations underlying complex sedentary behaviors.
Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart
Authors: Hesse Julia , Owenier Christoph , Lautwein Tobias , Zalfen Ria , Weber Jonas F. , Ding Zhaoping , Alter Christina , Lang Alexander , Grandoch Maria , Gerdes Norbert , Fischer Jens W. , Klau Gunnar W. , Dieterich Christoph , Köhrer Karl , Schrader Jürgen
Published: January 2021

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of WT1+cells the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, that can be classified in three groups each containing a cluster of proliferating cells. Two groups expressed cardiac specification makers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of HIF-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.
Influence of visual feedback persistence on visuo-motor skill improvement
Authors: Unell Alyssa , Eisenstat Zachary M. , Braun Ainsley , Gandhi Abhinav , Gilad-Gutnick Sharon , Ben-Ami Shlomit , Sinha Pawan
Published: January 2021

Towards the larger goal of understanding factors relevant for improving visuo-motor control, we investigated the role of visual feedback for modulating the effectiveness of a simple hand-eye training protocol. The regimen comprised a series of curve tracing tasks undertaken over a period of one week by neurologically healthy individuals with their non-dominant hands. Our three subject groups differed in the training they experienced: those who received ‘Persistent’ visual-feedback by seeing their hand and trace evolve in real-time superimposed upon the reference patterns, those who received ‘Non-Persistent’ visual-feedback seeing their hand movement but not the emerging trace, and a ‘Control’ group that underwent no training. Improvements in performance were evaluated along two dimensions – accuracy and steadiness, to assess visuo-motor and motor skills, respectively. We found that persistent feedback leads to a significantly greater improvement in accuracy than non-persistent feedback. Steadiness, on the other hand, benefits from training irrespective of the persistence of feedback. Our results not only demonstrate the feasibility of rapid visuo-motor learning in adulthood, but more specifically, the influence of visual veridicality and a critical role for dynamically emergent visual information.
Sensitivity enhancement and selection are shared mechanisms for spatial and feature-based attention
Authors: Birman Daniel , Gardner Justin L.
Published: January 2021

Human observers use cues to guide visual attention to the most behaviorally relevant parts of the visual world. Cues are often separated into two forms: those that rely on spatial location and those that use features, such as motion or color. These forms of cueing are known to rely on different populations of neurons. Despite these differences in neural implementation, attention may rely on shared computational principles, enhancing and selecting sensory representations in a similar manner for all types of cues. Here we examine whether evidence for shared computational mechanisms can be obtained from how attentional cues enhance performance in estimation tasks. In our tasks, observers were cued either by spatial location or feature to two of four dot patches. They then estimated the color or motion direction of one of the cued patches, or averaged them. In all cases we found that cueing improved performance. We decomposed the effects of the cues on behavior into model parameters that separated sensitivity enhancement from sensory selection and found that both were important to explain improved performance. We found that a model which shared parameters across forms of cueing was favored by our analysis, suggesting that observers have equal sensitivity and likelihood of making selection errors whether cued by location or feature. Our perceptual data support theories in which a shared computational mechanism is re-used by all forms of attention.

Significance Statement

Cues about important features or locations in visual space are similar from the perspective of visual cortex, both allow relevant sensory representations to be enhanced while irrelevant ones can be ignored. Here we studied these attentional cues in an estimation task designed to separate different computational mechanisms of attention. Despite cueing observers in three different ways, to spatial locations, colors, or motion directions, we found that all cues led to similar perceptual improvements. Our results provide behavioral evidence supporting the idea that all forms of attention can be reconciled as a single repeated computational motif, re-implemented by the brain in different neural architectures for many different visual features.
Response time modelling reveals evidence for multiple, distinct sources of moral decision caution
Authors: Andrejević Milan , White Joshua P. , Feuerriegel Daniel , Laham Simon , Bode Stefan
Published: January 2021

People are often cautious in delivering moral judgments of others’ behaviours, as falsely accusing others of wrongdoing can be costly for social relationships. Caution might further be present when making judgements in information-dynamic environments, as contextual updates can change our minds. This study investigated the processes with which moral valence and context expectancy drive caution in moral judgements. Across two experiments, participants (N= 122) made moral judgements of others’ sharing actions. Prior to judging, participants were informed whether contextual information regarding the deservingness of the recipient would follow. We found that participants slowed their moral judgements when judging negatively valenced actions and when expecting contextual updates. Using a diffusion decision model framework, these changes were explained by shifts in drift rate and decision bias (valence) and boundary setting (context), respectively. These findings demonstrate how moral decision caution can be decomposed into distinct aspects of the unfolding decision process.
The post-translational modification landscape of commercial beers
Authors: Kerr Edward D. , Caboche Christopher H. , Pegg Cassandra L. , Phung Toan K. , Viejo Claudia Gonzalez , Fuentes Sigfredo , Howes Mark T. , Howell Kate , Schulz Benjamin L.
Published: January 2021

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing.O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.
Ecology drives the evolution of diverse siderophore-production strategies in the opportunistic human pathogenPseudomonas aeruginosa
Authors: Figueiredo Alexandre R.T. , Wagner Andreas , Kümmerli Rolf
Published: January 2021

Bacteria often cooperate by secreting molecules that can be shared as public goods between cells. Because the production of public goods is subject to cheating by mutants that exploit the good without contributing to it, there has been great interest in elucidating the evolutionary forces that maintain cooperation. However, little is known on how bacterial cooperation evolves under conditions where cheating is unlikely of importance. Here we use experimental evolution to follow changes in the production of a model public good, the iron-scavenging siderophore pyoverdine, of the bacteriumPseudomonas aeruginosa. After 1200 generations of evolution in nine different environments, we observed that cheaters only reached high frequency in liquid medium with low iron availability. Conversely, when adding iron to reduce the cost of producing pyoverdine, we observed selection for pyoverdine hyper-producers. Similarly, hyper-producers also spread in populations evolved in highly viscous media, where relatedness between interacting individuals is higher. Whole-genome sequencing of evolved clones revealed that hyper-production is associated with mutations/deletions in genes encoding quorum-sensing communication systems, while cheater clones had mutations in the iron-starvation sigma factor or in pyoverdine biosynthesis genes. Our findings demonstrate that bacterial social traits can evolve rapidly in divergent directions, with particularly strong selection for increased levels of cooperation occurring in environments where individual dispersal is reduced, as predicted by social evolution theory. Moreover, we establish a regulatory link between pyoverdine production and quorum-sensing, showing that increased cooperation at one trait (pyoverdine) can be associated with the loss (quorum-sensing) of another social trait.
AMPK-dependent phosphorylation is required for transcriptional activation of TFEB/TFE3
Authors: Paquette Mathieu , El-Houjeiri Leeanna , Zirden Linda C. , Puustinen Pietri , Blanchette Paola , Jeong Hyeonju , Dejgaard Kurt , Siegel Peter M. , Pause Arnim
Published: January 2021

Increased autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK activation and mTORC1 inhibition to maintain energy homeostasis and cell survival. TFEB and TFE3 are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by mTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB/TFE3 transcriptional activity upon nutrient starvation, FLCN depletion and pharmacological manipulation of mTORC1 or AMPK. AMPK loss does not affect TFEB/TFE3 nuclear localization nor protein levels but reduces their transcriptional activity. Collectively, we show that mTORC1 specifically controls TFEB/TFE3 cytosolic retention whereas AMPK is essential for TFEB/TFE3 transcriptional activity. This dual and opposing regulation of TFEB/TFE3 by mTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1. Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466/467/469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB/TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance.
Baseline Lateral Habenula Firing Rate Negatively Correlates with Increase in Ethanol Intake over Days in Adult Long Evans Rats
Authors: Tandon Shashank
Published: January 2021

While many adults consume alcohol, yet only some individuals are at a risk to develop alcohol use disorder (AUD). Variability in the risk for alcohol abuse is multifactorial and includes differences in behavioral and neuronal traits. The lateral habenula (LHb) has been shown to mediate aversive state-related behavioral responses. Interestingly, in both humans and rodents, depression-like symptoms are associated with high LHb activity. Additionally, there is a high co-morbidity between major depressive disorder and AUD. However, LHb lesions in rodents increase ethanol intake over time. Thus, we wanted to determine how baseline LHb activity correlates with ethanol intake over days. Specifically, we wanted to test whether individual variation in baseline LHb activity in ethanol-naive rats is related to home-cage ethanol drinking patterns. Hence, in this study, we determined the correlation between individual variability in baseline LHb neural activity, the negative-affective state-related ultrasonic vocalizations (USVs; 22-28 kHz), and the extent of ethanol intake over days. We first surgically implanted a unilateral 16-wire electrode array in the LHb of adult male Long Evans rats (n=11). Following recovery from surgery, rats were placed in sound-insulated chambers for two hours, where they were free to explore while we simultaneously recorded neuronal signals from their LHb and the USVs emitted by them. Next, these rats underwent an intermittent access to ethanol (IAE) paradigm, where they received 20% ethanol for 24 hours on alternate days (Monday-Wednesday-Friday) and ad-libitum water in their home cages for four weeks. The change in ethanol intake over days differed between rats, with some rats escalating their ethanol intake in the four weeks, while other rats showing no meaningful change in ethanol intake over days as compared to the first session. We found a significant negative correlation between average baseline LHb firing rates in rats and the changes in ethanol intake in the first week of IAE. Specifically, rats with higher baseline LHb firing rats, unlike rats with lower baseline firing rates, did not escalate their ethanol intake from the first session to the second and fourth ethanol sessions of the IAE paradigm. We also found a moderate positive correlation between the number of 22-28 kHz USVs and the average LHb firing rates of these rats. These results indicate that higher baseline LHb neuronal activity in normal adult ethanol naive rats is associated with decreased motivation to seek and consume ethanol during the early stages of ethanol consumption.
A noncanonical FLT3 gatekeeper mutation disrupts gilteritinib binding and confers resistance
Authors: Joshi Sunil K. , Sharzehi Setareh , Pittsenbarger Janét , Bottomly Daniel , Tognon Cristina E. , McWeeney Shannon K. , Druker Brian J. , Traer Elie
Published: January 2021

The recent FDA approval of the FLT3 inhibitor, gilteritinib, for AML represents a major breakthrough for treatment of FLT3 mutated AML. However, patients only respond to gilteritinib for 6-7 months due to the emergence of drug resistance. Clinical resistance to gilteritinib is often associated with expansion of NRAS mutations, and less commonly via gatekeeper mutations in FLT3, with F691L being the most common. We developed anin vitromodel that charts the temporal evolution of resistance to gilteritinib from early microenvironmental-mediated resistance to late intrinsic resistance mutations. Our model system accurately recapitulates the expansion of NRAS mutations and the F691L gatekeeper mutations found in AML patients. As part of this study, we also identified a novel FLT3N701Kmutation that also appeared to promote resistance to gilteritinib. Using the Ba/F3 system, we demonstrate that N701K mutations effectively act like a gatekeeper mutation and block gilteritinib from binding to FLT3, thereby promoting resistance. Structural modeling of FLT3 reveals how N701K, and other reported gilteritinib resistance mutations, obstruct the gilteritinib binding pocket on FLT3. Interestingly, FLT3N701Kdoes not block quizartinib binding, suggesting that FLT3N701Kmutations are more specific for type 1 FLT3 inhibitors (gilteritinib, midostaurin, and crenolanib). Thus, our data suggests that for the FLT3N701Kmutation, switching classes of FLT3 inhibitors may restore clinical response. As the use of gilteritinib expands in the clinic, this information will become critical to define clinical strategies to manage gilteritinib resistance.
Production of functional oocytes requires maternally expressedPIWIgenes and piRNAs in golden hamsters
Authors: Hasuwa Hidetoshi , Iwasaki Yuka W. , Wan Kin Au Yeung , Ishino Kyoko , Masuda Harumi , Sasaki Hiroyuki , Siomi Haruhiko
Published: January 2021

Many animals have a conserved adaptive genome defense system known as the Piwi-interacting RNA (piRNA) pathway which is essential for germ cell development and function. Disruption of individual mousePiwigenes results in male but not female sterility, leading to the assumption thatPIWIgenes play little or no role in mammalian oocytes. Here, we report generation ofPIWI-defective golden hamsters, which reveals defects in the production of functional oocytes. The mechanisms involved vary among the hamsterPIWIgenes; lack ofPIWIL1has a major impact on gene expression, including hamster-specific young transposon de-silencing, whereasPIWIL3deficiency has little impact on gene expression in oocytes, although DNA methylation was found to be reduced to some extent inPIWIL3-defecient oocytes. Our findings serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes, including humans, which is not possible with mice.
Selection and Validation of Reference Genes Desirable for Gene Expression Analysis by qRT-PCR on Seed Germination ofCastanea henryi
Authors: Liu Bin , Jiang Yuting , Lin Ruqiang , Xiong Yuanfang , Jiang Shuzhen , Lian Hui , Liu Xuedie , Liu Zhong-Jian , Chen Shipin
Published: January 2021

Seed germination is the beginning of the plant’s life cycle, and seed biology is one of the most extensively researched areas in plant physiology, however,Castanea henryias an important seed plant, the stable internal reference gene during germination is not clear. In this study, seven candidate genes (TUA, TUB, TIF, UBC, RPL21, RPL30, RPL34) were screened out from transcriptome data, we analyzed the expression of seven candidate reference genes inC. henryiat different germination stages with RT–qPCR, and using common algorithms including NormFinder, geNorm and BestKeeper to evaluate the candidate genes stability. The results showed that RPL34 and RPL30 were selected as the most stable genes by NormFinder; TIF was the most stable gene identified by BestKeeper; RPL34 and RPL21 were the most stable genes ranked by geNorm, and TUB was the most unstable gene identified by all of the three software. The RPL34 gene was used as the reference gene, to detected the expression trend of two starch synthetase genes SS1 and SS2 during germination by RT–qPCR, the results of RT–qPCR and transcriptome sequencing were basically consistent, which verified the stability of RPL34 candidate gene. Our result is not only showed functional genes for germination ofC. henryiseeds and provide useful guidelines for the selection of reliable reference genes for the normalization of RT– qPCR data for germination of seed plants.
Gas6 drives Zika virus-induced neurological complications in humans and congenital syndrome in immunocompetent mice
Authors: Silva-Filho Joao Luiz , de Oliveira Lilian Gomes , Monteiro Leticia , Parise Pierina L. , Zanluqui Nagela G. , Polonio Carolina M. , de Freitas Carla Longo , Toledo-Teixeira Daniel A. , Souza William M. , Bittencourt Najara , Amorim Mariene R. , Forato Julia , Muraro Stéfanie Primon , de Souza Gabriela Fabiano , Martini Matheus Cavalheiro , Bispos-dos-Santos Karina , Judice Carla C. , Costa Maria Laura , Angerami Rodrigo N. , Freitas André R. R. , Resende Mariangela R. , Garcia Márcia T. , Moretti Maria Luiza ,   The Zika-Unicamp Network, Renia Laurent , Ng Lisa F. P. , Rothlin Carla V. , Costa Fabio TM , Schatzmann Peron Jean Pierre , Proença-Modena José Luiz
Published: January 2021

Zika virus (ZIKV) has the ability to cross placental and brain barriers, causing congenital malformations in neonates and neurological disorders in adults. However, the pathogenic mechanisms of ZIKV-induced neurological complications in adults and congenital malformations remain unknown. Gas6 is a soluble TAM receptor ligand able to promote flavivirus internalization and downregulation of immune responses. Here we demonstrate high Gas6 levels in the serum of patients with neurological complications which correlated with downregulation of genes associated with the type I IFN responses as consequence ofSocs1upregulation. Gas6 gamma-carboxylation is essential for ZIKV replication in monocytes, the main source of this protein. Gas6 also facilitates ZIKV replication in adult immunocompetent mice enabled susceptibility to transplacental infection and congenital malformations. Our data thus indicate that ZIKV promotes the upregulation of its ligand Gas6, which contributes to viral infectivity and drives the development of severe adverse outcomes during ZIKV infection.
Enhanced Cas12a multi-gene regulation using a CRISPR array separator
Authors: Magnusson Jens P. , Rios Antonio R. , Wu Lingling , Qi Lei S.
Published: January 2021

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple crRNAs. Here, we report that Cas12a array performance is hypersensitive to the GC content of crRNA spacers, as high-GC spacers can impair activity of the downstream crRNA. We analyzed naturally occurring CRISPR arrays and observed that repeats always contain an AT-rich fragment that separates crRNAs; we term this fragment aCRISPR separator.Inspired by this observation, we designed short, AT-rich synthetic separators (synSeparators) that successfully removed the disruptive effects between crRNAs. We demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously unknown feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.
A robust method to isolateDrosophilafat body nuclei for transcriptomic analysis
Authors: Gupta Vanika , Lazzaro Brian P.
Published: January 2021

Gene expression profiles are typically described at the level of the tissue or, often inDrosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. TheDrosophilaadult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single cell nuclei sequencing and potentially can be implemented for bulk RNA-seq.
Targeted knockdown of the PAF49 component of the PAF53/PAF49 heterodimer causes the degradation of PAF53
Authors: McNamar Rachel , Rothblum Katrina , Rothblum Lawrence I.
Published: January 2021

There are significant differences in the components of the ribosomal DNA transcription apparatuses of yeast and mammals. Moreover, the patterns of regulation between mammals and yeast are also different. To overcome, deficits in our understanding of mammalian rDNA transcription, we have developed a system to introduce an inducible degron into the endogenous genes of mammalian cells. This allows us to combine a knock out the endogenous gene product and replace it with mutant proteins in order to study their function in ribosomal DNA transcription. Using this system, we show that the knockout of PAF49, the mammalian ortholog of yeast A34, results in the relatively rapid degradation of PAF53, the ortholog of yeast A49. Interestingly, the steady-state levels of the core subunits of RNA polymerase I are unaffected.
Evaluation of solubility and histological effect of 11-keto-β-Boswellic acid on the diabetic mice liver using FTIR-PCA analysis and microscopy
Authors: Al-Amri I.S. , Mabood F. , Kadim I.T. , Alkindi A.Y. , Al-Harrasi A. , Al-Hashmi S. , Abbas G. , Ahmed B. , Al-Shuhaimi J. , Khalaf S.K. , Shaikh J.
Published: January 2021

This study was designed to develop a rapid, sensitive, accurate, and inexpensive Fourier Transform Infrared Reflectance (FT-IR) Spectroscopy coupled with Principle Component Analysis (PCA) as a detection technique to evaluate the solubility of 11-Keto-β-Boswellic acid (KBA), from the gum resin extracted from the Omani frankincense, (Boswellia sacra) in the liver of STZ induced diabetic mice. This study also investigated the effect of KBA on the histological changes of hepatocytes of diabetic mice. Liver tissue samples from three groups of mice included normal control group, diabetic control group and diabetic group treated IP with KBA were scanned with FT-IR spectrophotometer in the reflection mode. FT-IR Spectra were collected in the wavenumber range from 400 to 4000cm-1using ATR accessorry. The results of FT-IR Spectra were analyzed by using multivariate method Principle Component Analysis. The PCA score plot is an exploratory multivariate method indicated that there was a complete segregation among the three groups of liver samples based on change in variation of position of wavenumber in FT-IR spectra, which revealed that there is a clear effect of KBA solubility on treatments. The histological features showed an improvement in the liver tissues with normal structures of hepatocytes with exhibiting mild vacuolations in their cytoplasm. In conclusion, reflectance FT-IR spectroscopy coupled with PCA could be deployed as a new detection method for rapid, low cost and non-destructive method for evaluating of treatment effects in diseased liver tissue based on the solubility of KBA. Histological findings demonstrated the protective effective of KBA on improving the morphology of liver tissue in diabetic mice which resulted in complete recovery to the damage observed in diabetic control group.

Summary Statement

Reflectance FT-IR spectroscopy coupled with PCA has been deployed as a new rapid, inexpensive and non-destructive detection method to examine the solubility of 11-keto-β-Boswellic acid (KBA) in streptozotocin (STZ) induced-diabetes mice liver tissue following intraperitoneal treatment. Moreover, microscopic study of liver tissue histopathology revealed that KBA has a protecting effect against STZ damage.
Aureochromes are necessary for maintaining polyunsaturated fatty acid content inNannochloropsis oceanica
Authors: Poliner Eric , Busch Andrea W. U. , Newton Linsey , Kim Young Uk , Clark Rachel , Gonzalez-Martinez Sofía C. , Jeong Byeong-ryool , Montgomery Beronda L. , Farré Eva M.
Published: January 2021

Nannochloropsis oceanica, as other stramenopile microalgae, is rich in long-chain polyunsaturated fatty acids (LC-PUFA) such as eiconsapentaenoic acid (EPA). We observed that fatty acid desaturases (FAD) involved in LC-PUFA biosynthesis were among the strongest blue light induced genes inN. oceanicaCCMP1779. Blue light was also necessary for maintaining LC-PUFA levels in CCMP1779 cells, and growth under red light led to a reduction in EPA content. Aureochromes are stramenopile specific proteins that contain a light-oxygen-voltage-sensing (LOV) domain that associates with a flavin mononucleotide and is able to sense blue light. These proteins also contain a bZIP DNA binding motif and can act as blue light regulated transcription factors by associating with a E-box like motif, which we found enriched in the promoters of blue light induced genes. We demonstrated that,in vitro, two CCMP1779 aureochromes were able to absorb blue light. Moreover, the loss or reduction of any of the three aureochromes led to a decrease in the blue light specific induction of several FADs in CCMP1779. EPA content was also significantly reduced inNoAureo2 andNoAureo4 mutants. Taken together, our results indicate that aureochromes mediate blue light dependent regulation of LC-PUFA content inN. oceanicaCCMP1779 cells.
Associations of psychiatric disease and aging onFKBP5expression converge on cortical supragranular neurons
Authors: Matosin Natalie , Arloth Janine , Czamara Darina , Edmond Katrina Z. , Maitra Malosree , Fröhlich Anna Sophie , Martinelli Silvia , Kaul Dominic , Bartlett Rachael , Curry Amber R. , Gassen Nils C. , Hafner Kathrin , Müller Nikola S , Worf Karolina , Rehawi Ghalia , Nagy Corina , Halldorsdottir Thorhildur , Cruceanu Cristiana , Gagliardi Miriam , Gerstner Nathalie , Ködel Maik , Murek Vanessa , Ziller Michael J , Scarr Elizabeth , Tao Ran , Jaffe Andrew E. , Arzberger Thomas , Falkai Peter , Kleinmann Joel E. , Weinberger Daniel R. , Mechawar Naguib , Schmitt Andrea , Dean Brian , Turecki Gustavo , Hyde Thomas M. , Binder Elisabeth B.
Published: January 2021

Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry.FKBP5is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported thatFKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell-types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propelFKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n=1024) ofFKBP5/1 examining prefrontal cortex (BA9, BA11, BA24) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease-state, aging and genotype on corticalFKBP5/1 expression including in a cell-type specific manner. We identified consistently heightenedFKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq) and targeted histology, we established that these disease- and aging-effects onFKBP5/1 expression were most pronounced in excitatory supragranular neurons. We then found that this increase inFKBP5levels likely impacts on synaptic plasticity, asFKBP5gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in supragranular neurons. These findings pinpoint a novel cellular and molecular mechanism that has significant potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.