bluuebunny/crossref_metadata_2025_split huggingface dataset:
On-target activity predictions enable improved CRISPR–dCas9 screens in bacteria
Authors: Calvo-Villamañán Alicia , Ng Jérome Wong , Planel Rémi , Ménager Hervé , Chen Arthur , Cui Lun , Bikard David 
Published: April 2020

Abstract
The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.
DXO/Rai1 enzymes remove 5′-end FAD and dephospho-CoA caps on RNAs
Authors: Doamekpor Selom K , Grudzien-Nogalska Ewa , Mlynarska-Cieslak Agnieszka , Kowalska Joanna , Kiledjian Megerditch , Tong Liang 
Published: April 2020

AbstractIn eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3′-FADP or CoA and fission yeast Rai1 with 3′-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.
Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence
Authors: Samolygo Aleksei , Athukoralage Januka S , Graham Shirley , White Malcolm F 
Published: April 2020

Abstract
Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.
Phosphorothioate modified oligonucleotide–protein interactions
Authors: Crooke Stanley T , Vickers Timothy A , Liang Xue-hai 
Published: April 2020

Abstract
Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.
Measured sodium excretion is associated with CKD progression: results from the KNOW-CKD study
Authors: Kang Minjung , Kang Eunjeong , Ryu Hyunjin , Hong Yeji , Han Seung Seok , Park Sue K , Hyun Young Youl , Sung Su Ah , Kim Soo Wan , Yoo Tae-Hyun , Kim Jayoun , Ahn Curie , Oh Kook-Hwan 
Published: April 2020

Abstract

Background
Diet is a modifiable factor of chronic kidney disease (CKD) progression. However, the effect of dietary salt intake on CKD progression remains unclear. Therefore, we analyzed the effect of dietary salt intake on renal outcome in Korean patients with CKD.

Methods
We measured 24-h urinary sodium (Na) excretion as a marker of dietary salt intake in the prospective, multi-center, longitudinal KoreaN cohort study for Outcome in patients With CKD (KNOW-CKD). Data were analyzed from CKD patients at Stages G3a to G5 (n = 1254). We investigated the association between dietary salt intake and CKD progression. Patients were divided into four quartiles of dietary salt intake, which was assessed using measured 24-h urinary Na excretion. The study endpoint was composite renal outcome, which was defined as either halving the estimated glomerular filtration rate or developing end-stage renal disease.

Results
During a median (interquartile range) follow-up of 4.3 (2.8–5.8) years, 480 (38.7%) patients developed the composite renal event. Compared with the reference group (Q2, urinary Na excretion: 104.2 ≤ Na excretion &lt; 145.1 mEq/day), the highest quartile of measured 24-h urinary Na excretion was associated with risk of composite renal outcome [Q4, urinary Na excretion ≥192.9 mEq/day, hazard ratio 1.8 (95% confidence interval 1.12–2.88); P = 0.015] in a multivariable hazards model. Subgroup analyses showed that high-salt intake was particularly associated with a higher risk of composite renal outcome in women, in patients &lt;60 years of age, in those with uncontrolled hypertension and in those with obesity.

Conclusions
High salt intake was associated with increased risk of progression in CKD.
Clinical and immunological characteristics of 56 patients with systemic lupus erythematosus in Uganda
Authors: Bongomin Felix , Sekimpi Maria , Kaddumukasa Mark 
Published: April 2020

Abstract

Objectives
The prevalence and burden of SLE in Africa are poorly understood. This health-facility-based retrospective study aimed to describe the frequency and the clinical and immunological characteristics of SLE in Uganda.

Methods
We reviewed clinical notes of patients presenting with rheumatological complaints in two large rheumatology outpatient clinics in Uganda between January 2014 and December 2019.

Results
Of the 1019 charts reviewed, 5.5% (56) of the patients had confirmed SLE, with a median age of 29 (range: 14–65) years. The male-to-female ratio was ∼1:10, and 19.6% (11/56) of the patients had SLE and RA overlap syndrome. Patients presented with joint pains or swellings (n = 39, 69.6%), typical photosensitive malar rash (n = 34, 60.7%), oral ulceration (n = 23, 41.1%), anaemia (n = 14, 25.0%), hair loss and polyserositis (n = 12, 21.4% each), constitutional symptoms (n = 10, 17.9%), RP (n = 4, 7.1%) or LN (n = 3, 5.4%). ANA and anti-dsDNA autoantibodies were both positive in 25 (75.8%) of the 33 patients with available results. ANA titres were ≥1:160, with a median titre of 1:160 (range: 1:160 to 1:3200). Six patients had titres ≥1:320. The median dsDNA level was 80 (range: 40–283) IU. Ten patients had results of C3 and C4 complement protein levels and, of these, 4 patients had low C3 levels and 3 had low C4 levels.

Conclusion
SLE is uncommon among patients presenting with rheumatological complains in Uganda. SLE overlaps with RA in our setting, and a majority of patients present to care with complications.
The ARTE index: Characterizing free‐living physical activity and indices of cardio‐metabolic health among postmenopausal women
Authors: Altherr Cody A. , Baranauskas Marissa N. , Hunter Gary R. , Martins Catia , Carter Stephen J. 
Published: April 2020

PurposeThe doubly‐labeled water (DLW) technique is the gold‐standard used to objectively measure free‐living activity energy expenditure (AEE), however; it does not account for differences in body size or locomotion economy. Whereas the physical activity level (PAL) index, calculated from the ratio of total energy expenditure (TEE) and resting energy expenditure (REE), is employed to adjust for body size – it also does not account for variance in energy expenditure attributed to locomotion economy. Alternatively, the activity‐related time equivalent (ARTE) circumvents the limitations of AEE and PAL. In the present work, DLW was used to compare AEE, PAL, and variations of the ARTE index among a cohort of postmenopausal women dichotomized above/below the 50th percentile of normative data for body fat%. Correlations of AEE, PAL, and ARTE to measures of cardio‐metabolic health including arterial elasticity, body fat% and cardiorespiratory fitness (V̇O2max) were also performed.MethodsNinety‐two, healthy postmenopausal women (65+/−4 years) were recruited to measure free‐living AEE over a 14‐day period using DLW. TEE was reduced by 10% to account for the thermogenesis of feeding whereas AEE was calculated by subtracting REE from adjusted‐TEE. PAL was calculated from the ratio of TEE/REE. ARTE4act was calculated by dividing AEE by the average energy expenditure of non‐graded walking, graded walking, stair climbing, and a carrying task. AEE divided by the average energy expenditure of non‐graded walking was used to calculate ARTEwalk. Arterial elasticity was performed using pulse contour analysis from the radial artery. Dual‐energy X‐ray absorptiometry was used to estimate body fat% whereas a modified‐Balke protocol was used to measure V̇O2max. Between‐group comparisons were performed using independent t‐tests and Pearson’s correlation were used to evaluate relationships.ResultsBetween‐group differences were not detected for AEE, PAL, or the ARTE4act. However, women with less percent body fat (i.e., < 50th percentile) exhibited greater activity as evidenced by the ARTEwalk (min·day−1) (255+/−154 vs. 193+/−130; p < 0.05). Though AEE and PAL did not correlate with any cardio‐metabolic variables, ARTE4act was related to arterial elasticity (r = 0.24) and V̇O2max (r = 0.27) while ARTEwalk was related to arterial elasticity (r = 0.27), body fat% (r = −0.24), and V̇O2max (r = 0.32).ConclusionsConsistent with the ARTEwalk, adjustments for energetic cost of non‐graded walking may improve the sensitivity of AEE for detecting differences in free‐living physical activity and parameters of cardio‐metabolic health – known to be critical for overall well‐being.Support or Funding InformationThis investigation was supported by the following grants from the National Institutes of Health: R01AG027084‐01, R01AG027084‐S, P30DK056336, P60DK079626, and UL1RR025777.
Mapping of the Contraction‐Regulated Phosphoproteome Identifies TRIM28 as a Key Regulator of Skeletal Muscle Size
Authors: Steinert Nathaniel David , Potts Gregory K. , Wilson Gary M. , Klamen Amelia M. , Lin Kuan-Hung , Hermanson Jake Brenner , McNally Rachel M. , Coons Joshua J. , Hornberger Troy A. 
Published: April 2020

Mechanical signals, such as those which are evoked during maximal intensity contractions (MIC), can induce an increase in skeletal muscle size. It has been widely concluded that this process is driven by the activation of rapamycin‐sensitive / mTORC1‐dependent signaling; however, recent studies have revealed that mTORC1‐independent signaling events might also be involved. Thus, in an effort to identify these events, we generated a comprehensive map of the MIC‐regulated, and rapamycin‐sensitive phosphoproteomes. In total, we identified over 2,400 unique MIC‐regulated phosphorylation events, of which, nearly 2,200 were unaffected by rapamycin. Interestingly, one of the most robust MIC‐regulated and rapamycin‐insensitive phosphorylation events was located on the S473 residue on a protein named TRIM28. This was intriguing because TRIM28 is a transcriptional intermediary factor that functions in a variety of biological processes and many of its regulatory effects are dependent on the phosphorylation of the S473 residue. Moreover, a recent study revealed that TRIM28 interacts with several skeletal muscle regulatory factors (e.g., MyoD and Mef2) and their transcriptional co‐repressors and co‐activators. Thus, to investigate the role of TRIM28 in the regulation of muscle size, we generated skeletal muscle specific and inducible TRIM28 knockout mice and then subjected these mice to various forms of mechanical stimuli. Collectively, our results indicated that: i) TRIM28 is not required for the mechanical activation of mTORC1 signaling, ii) TRIM28 plays a key role in the maintenance of normal muscle size, and iii) TRIM28 significantly contributes to the pathway via which mechanical stimuli induce hypertrophy. Taken together, these outcomes establish TRIM28 as a novel, mTORC1‐independent, component of the pathway via which mechanical signals regulate skeletal muscle size.Support or Funding InformationNIHR01AR057347
Tracking Antigen‐Specific CD8+ T cells Using MHC Class I Pentamers
Authors: Aleksandrovic Emilija , Lazarski Christopher , Bollard Catherine , Stanojevic Maja , Hanley Patrick 
Published: April 2020

The treatment of choice for many patients with Acute Myeloid Leukemia (AML) is hematopoietic stem cell transplantation, where the patient’s immune system is replaced with that of a matched donor. Due to the conditioning required, cancer relapse and viral infections such as Cytomegalovirus (CMV) are common complications. One potential treatment for relapse and/or viral infection is adoptive immunotherapy with ex vivo expanded T cells targeting either viral or tumor antigens. Prior to infusion into patients, these T cells are typically characterized using ELISpot assays which do not directly assess the presence of distinct T cell clones. Therefore, without a reliable assay to test T cell specificity, optimizing the culture conditions for increased antigen specificity has been challenging. We hypothesized that we could directly assess the specificity of T cell receptors (TCRs) from expanded T cell populations towards their target antigens over time using MHC Class I Pentamers. To test this, we generated CMV‐specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) from two consenting CMV+ donors and tumor‐specific T cells from one donor on our IRB approved protocol. After 10 days of culture, the average CMV+ T cells expanded 10‐fold and consisted of 22.2% CD4+ (range 15.9–46.6%) and 68.9% CD8+ (range 32.9–77.9%) T cells. Using flow cytometry, we were able to detect pentamer positive cells by day 7 for three distinct epitopes: RPH (58.1%), TPR (8.45%), and NLV (0.36%) in one of the CMV‐specific T cell lines. In addition, ALY pentamer positive T cells were detected as early as day 6 of culture in the tumor T cell lines stimulated with the entire PRAME antigen and single epitope of the same antigen (0.2 and 0.16% respectively), while Survivin (LML) positive T cells were detected on day 13 for cells stimulated with the PRAME/Survivin/WT‐1 pepmix (0.19%). These results indicate that in two CMV‐specific and one tumor‐specific T cell product, we were successful in tracking T cell populations and their specificity towards their target antigens over time. Additional experiments, however, are required to reach statistical significance and demonstrate reproducibility. Since this method can identify virus‐ and tumor‐specific T cells early in the culturing process, it has the potential to be used for culture optimization and monitoring epitope expansion in real time, as well as sorting T cells according to their specific TCRs.Support or Funding InformationThis project was supported by the Children’s Cancer Foundation grant awarded to Dr. Patrick Hanley.
Total Cellular ATP Production Varies With Primary Substrate in Breast Cancer Cells
Authors: Mookerjee Shona A. , Ton Justin , Brady Maurice L. , Le Diem T. , Mar Jordon N. 
Published: April 2020

Cancer cell growth is predicted to require substantial increases in the rates of substrate catabolism and ATP turnover to drive biosynthesis and cell growth. Strategic limitation of substrate supply is therefore a potentially effective way to slow tumor growth. However, the effects of substrate limitation on the rates of ATP production and consumption, especially over the acute timescales relevant to most experimental design, are not straightforward. First, bioenergetic homeostasis will oppose substrate limitation by increasing catabolism of substrates that remain accessible, in order to meet existing ATP demand. Second, ATP demand itself may be different under different substrate conditions. Empirical evaluation of total cellular ATP production is therefore required. Cultured cells remain an important model system for understanding these mechanisms of bioenergetic control.Using extracellular flux analysis to estimate total cellular ATP production rate, we demonstrate that the MCF7 breast cancer model supports rates of basal ATP production over an approximately 2‐fold range, in the presence of the single substrates glucose, glutamine, and pyruvate, the main substrates commonly present in standard culture media. In comparison, a nontransformed cell culture model shows maintenance of a single ATP production rate across the same single substrates. These novel findings demonstrate that MCF7 cells are highly flexible with respect to total cellular ATP production, over a timeframe that is independent of the slower cellular remodeling caused by altered transcriptional regulation.Support or Funding InformationNIH 1R15ES025917‐01A1; Touro University California College of Pharmacy
Profiling the Spatial Dynamics of Limb Patterning in Regeneration and Development Using Enhanced V3 HCR‐FISH Probe Design
Authors: Mun Evan , Lovely Alexander , Stein David , Monaghan James 
Published: April 2020

The patterning of limbs in development utilizes spatial and temporal signaling dynamics which guide the formation of a complete limb from undefined tissue. Although they may play different roles, a group of key factors guide the development of most species. This process is recapitulated in the limb regeneration of the Mexican axolotl, and the possibility of regeneration in humans will require a precise understanding of how these factors guide this patterning. Previously, observation of limb patterning was restricted by in situ hybridization imaging techniques which had limited resolution and could not reliably compare the collection of essential genes on a single tissue. Now, we have applied V3 Fluorescent in situ Hybridization by Hybridization Chain Reaction (HCR‐FISH) to the model to generate clear, bright, multiplexed images of the expression of these factors. These multiplexed images are the first of their kind in the field, and allow for the overlapping of key factors in both axolotl salamander and murine models to draw essential conclusions regarding each gene’s role in limb patterning within and between species. Development of an efficient probe‐design pipeline and oligonucleotide amplification parameters has improved signal strength, selectivity, and cost effectiveness. Furthermore, we have designed novel V3 HCR‐FISH probes targeting the introns of actively transcribed sequences allows for the identification of actively transcribed RNA via nuclear‐specific signal. Our techniques have not only been applied to confirm predictions derived from single cell RNA‐seq data regarding the roles of Sonic Hedghog (Shh), Fibroblast Growth Factor 8 (Fgf8), and Gremlin 1 (Grem1), in axolotl limb patterning, but also showcased significant differences between the axolotl and murine limb models. These results indicate that application of V3 HCR‐FISH allows for the robust visualization of multi‐gene transcriptional dynamics with a clarity previously impossible in both regenerative and developmental models.Support or Funding InformationThis project was supported by NSF grants 1558017 and 1656429
Antioxidant activity of the ethyl acetate extract of Justicia spicigera in two periods of the year (spring‐summer and autumn‐winter) in the liver of adult diabetic rats
Authors: Murillo-Villicaña Marina , Peña-Montes Donovan J. , Huerta-Cervantes Maribel , Salgado-Garciglia Rafael , Noriega-Cisneros Ruth , Aguilera-Méndez Asdrúbal , Saavedra-Molina Alfredo 
Published: April 2020

BackgroundDiabetes is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. In studies it was shown that the ethanol extract of Justicia spicigera has a hypoglycemic effect in streptozotocin‐induced rats (STZ), related to the presence of secondary metabolites, such as kaempferitrin. However, the concentration of these compounds presents drastic variations under different environmental conditions.ObjectiveThe objective of this project is to study the activity of secondary metabolites produced by Justicia spicigeraduring two periods of the year (spring‐summer and autumn‐winter) in a chronic diabetes model on the liver of streptozotocin‐induced rats.MethodsMale Wistar rats (450–500 g) with 20 weeks of age were used, divided into 6 groups: normoglycemic control (CN) and diabetic (CD), administered with spring‐summer extract (NEPV and DEPV) and autumn‐winter ( NEOI and DEOI) for 60 days, diabetic rats were induced with an intraperitoneal injection of STZ (45 mg / kg). During treatment the weight and blood glucose were determined every 6 days. Subsequently, the rats were submitting euthanasia to obtain weight, homogenate and mitochondria of liver, which were used for the determination of catalase and superoxide dismutase activities and glutathione levels.ResultsThe ethyl acetate extract of Justicia Spicigera collected during the spring‐summer season significantly lowers blood glucose levels and prevents excessive loss of body weight and liver growth increases catalase activity and reduced glutathione levels. The ethyl acetate extract of Justicia Spicigera collected during the autumn‐winter season prevents excessive loss of body weight and liver growth, but does not exhibit antioxidant activity when compared to the DEPV group.In conclusion, the concentration of Justice spicigera compounds related to hypoglycemic and antioxidant activity is affected by environmental factors.Support or Funding InformationThe authors appreciate the partial economic support from CIC‐UMSNH (2.16, to ASM).