Miracle-dz/UniFAR-FTUs huggingface dataset:
Rab35–GEFs, DENND1A and folliculin differentially regulate podocalyxin trafficking in two- and three-dimensional epithelial cell cultures

Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin–Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain–containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.
An Influenza Virus Hemagglutinin Computationally Optimized Broadly Reactive Antigen Elicits Antibodies Endowed with Group 1 Heterosubtypic Breadth against Swine Influenza Viruses

Computationally Optimized Broadly Reactive Antigens (COBRA) designed for different influenza virus subtypes (H1N1, H3N2 and H5N1) elicit a subtype-specific broad antibody (Ab) response in naïve as well as in pre-immune influenza virus animal models (1-9). ….
Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1–2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10–15 years on therapy, there were as many as 3.5–5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.
Staphylococcus aureus facilitates its survival in bovine macrophages by blocking autophagic flux

Staphylococcus aureus is a pathogen that is the causative agent of several human and veterinary infections and plays a critical role in the clinical and subclinical mastitis of cattle. Autophagy is a conserved pathogen defence mechanism in eukaryotes. Studies have reported that S aureus can subvert autophagy and survive in cells. Staphylococcus aureus survival in cells is an important cause of chronic persistent mastitis infection. However, it is unclear whether S aureus can escape autophagy in innate immune cells. In this study, initiation of autophagy due to the presence of S aureus was detected in bovine macrophages. We observed autophagic vacuoles increased after S aureus infection of bovine macrophages by transmission electron microscopy (TEM). It was also found that S aureus‐infected bovine macrophages increased the expression of LC3 at different times(0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours). Data also showed the accumulation of p62 induced by S aureus infection. Application of autophagy regulatory agents showed that the degradation of p62 was blocked in S aureus induced bovine macrophages. In addition, we also found that the accumulation of autophagosomes promotes S aureus to survive in macrophage cells. In conclusion, this study indicates that autophagy occurs in S aureus‐infected bovine macrophages but is blocked at a later stage of autophagy. The accumulation of autophagosomes facilitates the survival of S aureus in bovine macrophages. These findings provide new insights into the interaction of S aureus with autophagy in bovine macrophages.
Indoor Trajectory Reconstruction of Walking, Jogging, and Running Activities Based on a Foot-Mounted Inertial Pedestrian Dead-Reckoning System

The evaluation of trajectory reconstruction of the human body obtained by foot-mounted Inertial Pedestrian Dead-Reckoning (IPDR) methods has usually been carried out in controlled environments, with very few participants and limited to walking. In this study, a pipeline for trajectory reconstruction using a foot-mounted IPDR system is proposed and evaluated in two large datasets containing activities that involve walking, jogging, and running, as well as movements such as side and backward strides, sitting, and standing. First, stride segmentation is addressed using a multi-subsequence Dynamic Time Warping method. Then, detection of Toe-Off and Mid-Stance is performed by using two new algorithms. Finally, stride length and orientation estimation are performed using a Zero Velocity Update algorithm empowered by a complementary Kalman filter. As a result, the Toe-Off detection algorithm reached an F-score between 90% and 100% for activities that do not involve stopping, and between 71% and 78% otherwise. Resulting return position errors were in the range of 0.5% to 8.8% for non-stopping activities and 8.8% to 27.4% otherwise. The proposed pipeline is able to reconstruct indoor trajectories of people performing activities that involve walking, jogging, running, side and backward walking, sitting, and standing.
Childhood maltreatment and clinical severity of treatment‐resistant depression in a French cohort of outpatients (FACE‐DR): One‐year follow‐up

Childhood maltreatment is associated with major depressive disorder (MDD). It not only increases the risk of lifetime MDD, but it also aggravates its course. Among depressed patients, 20–30% of them experience treatment‐resistance depression (TRD). We aimed to assess the association between childhood maltreatment, severity of depression in a unipolar TRD sample, and patient outcomes after one‐year of follow‐up.
Assaying Fear Memory Discrimination and Generalization: Methods and Concepts

Generalization describes the transfer of conditioned responding to stimuli that perceptually differ from the original conditioned stimulus. One arena in which discriminant and generalized responding is of particular relevance is when stimuli signal the potential for harm. Aversive (fear) conditioning is a leading behavioral model for studying associative learning and memory processes related to threatening stimuli. This article describes a step‐by‐step protocol for studying discrimination and generalization using cued fear conditioning in rodents. Alternate conditioning paradigms, including context generalization, differential generalization, discrimination training, and safety learning, are also described. The protocol contains instructions for constructing a cued fear memory generalization gradient and methods for isolating discrete cued‐from‐context cued conditioned responses (i.e., “the baseline issue”). The preclinical study of generalization is highly pertinent in the context of fear learning and memory because a lack of fear discrimination (overgeneralization) likely contributes to the etiology of anxiety‐related disorders and post‐traumatic stress disorder. © 2020 by John Wiley & Sons, Inc.
Predicting Radial Head Instability in Multiple Hereditary Exostoses (MHE): A Multicenter Analysis of Risk Factors

Background: Forearm deformity occurs in one third of patients with multiple hereditary exostoses (MHE). Conservative and surgical treatment are aimed at preventing radial head subluxation and/or dislocation. Dislocation has been associated with isolated distal ulnar lesions, radial bowing, and ulnar shortening. Risk factors for radial head subluxation have not been clearly elucidated. This study aimed to identify risk factors for all radial head instability in MHE, to optimize early detection and prevent frank dislocation. Methods: This multicenter retrospective case-control investigation included MHE patients with forearm lesions seen between 2000 and 2017 at 2 tertiary care children’s hospitals. Demographic, clinical factors, radiographic measures, and surgical history were quantified. Comparisons were made between forearms that developed radial head instability versus those that remained stable and between those that progressed to radial head subluxation versus those that progressed to dislocation. Results: This study included 171 forearms in 113 patients with MHE, who presented at a mean age of 8.0 years with a median follow-up time of 6.0 years. Nine forearms progressed to radial head subluxation (mean age: 10.2 y), and 24 forearms had radial head dislocation (mean age: 9.9 y). Five subluxations and 3 dislocations occurred despite preventative surgery. Initial radial bowing (7.2% vs. 8.5%, P=0.04), ulnar variance (−5.8% vs. 11.0%, P<0.001), and ulnar shortening (−2.5 vs. 9.1 mm, P=0.04) were predictive of radial head instability. Distal ulnar lesions and more severe ulnar variance (−5.8 vs. −10.6, P<0.001) and shortening (−2.5 vs. 13.2 mm, P=0.02) were associated with an increased risk of radial head subluxation. No significant differences were identified between forearms that progressed to subluxation versus those that progressed to dislocation. Conclusions: Distal ulnar lesions and radiographic measures can be used to determine the risk of radial head instability in MHE. Ulnar variance and shortening are early identifiable risk factors for radial head subluxation that can help guide monitoring and treatment. Radial bowing may be a late predictor of instability. Level of Evidence: Level III—prognostic.
Killer‐cell immunoglobulin‐like receptor assessment algorithms in haemopoietic progenitor cell transplantation: current perspectives and future opportunities

Natural killer cells preferentially target and kill malignant and virally infected cells. Both these properties present compelling clinical utility in the field of haemopoietic progenitor cell transplantation (HPCT), potentially promoting a graft vs leukaemia effect in the absence of graft vs host disease and protecting against cytomegalovirus activation. Killer Ig‐like receptors (KIR) play a central role in the cytotoxic action of natural killer cells, providing opportunity for improving transplantation outcomes by prioritising potential donors with optimal characteristics. Numerous algorithms for assessing KIR gene content as part of HPCT donor selection protocols exist, but no single model has been found to be universally applicable in all transplant centres. This review summarises several of the predominant strategies in KIR assessment algorithms, discussing their basic scientific principles, clinical utility and benefits to post‐transplant outcomes. Finally, the review will consider how future donor selection protocols could develop towards unifying the concepts of KIR proteomics and genetics for optimising patient care.
Influence of Tooth Preparation Design and Scan Angulations on the Accuracy of Two Intraoral Digital Scanners: An In Vitro Study Based on 3-Dimensional Comparisons.

PURPOSE To evaluate the accuracy of two intraoral scanners (IOS) in terms of different preparation designs and scan angulation limitation due to the presence of adjacent teeth. MATERIALS AND METHODS Eight different complete coverage (CC) and partial coverage (PC) tooth preparations were scanned by two IOS, the 3Shape Trios (TRI) and the 3M True Definition (TRU). All teeth preparations were scanned in the presence and absence of adjacent teeth. Four groups were established for each IOS; Group 1: PC preparations with adjacent teeth. Group 2: CC preparations with adjacent teeth. Group 3: PC preparations without adjacent teeth. Group 4: CC preparations without adjacent teeth. 3D analysis was performed to examine average absolute discrepancy (AAD) and maximum absolute discrepancy (MAD). A Two-way ANOVA was performed followed by a post-hoc Tukey's test HSD to evaluate the effect of adjacent teeth, preparation design, and the type of IOS used. RESULTS For TRI, AAD for groups 1, 2, 3, and 4 were 20 ±1.8 µm, 19.6 ±2.4 µm, 15.5 ±2.7 µm, and 12.9 ±1.4 µm respectively, whereas MAD for groups 1, 2, 3, and 4 were 109.7 ±13.5 µm, 93.2 ±28.9 µm, 85.6 ±16.1 µm, and 66 ±20.1 µm respectively. For TRU IOS, AAD for groups 1, 2, 3, and 4 were 22.0 ±3.6 µm, 17.9 ±2 µm, 20 ±5.9 µm, and 14.9 ±1.7 µm respectively, whereas the MAD for groups 1, 2, 3, and 4 were 151.4 ±38.4 µm, 92.2 ±17. µm, 92.6 ±23.6 µm, and 71.4 ±11.9 µm respectively. Two-way ANOVA showed statistically significant differences between the AAD and MAD of TRI and TRU (P<0.001). There were also statistically significant differences for presence or absence of adjacent teeth (P<0.001), and preparation design (P<0.001). CONCLUSIONS PC preparation scans revealed lower accuracy than CC. The presence of adjacent teeth decreased the accuracy of both IOS. TRI gave higher accuracy than TRU for PC, but both IOS showed comparable accuracy for CC groups. This article is protected by copyright. All rights reserved.
The biochemistry of headgroup exchange during triacylglycerol synthesis in canola.

Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFA) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed-oil synthesis. Loss of BnROD1.A3 and . C3 expression in a double mutant, or by RNA-interference, reduced PUFA content of the oil to 26.6%, compared with 32.5% in wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFA that accumulate in the seed oil, compared with 40% in Arabidopsis. Our results demonstrate the care needed in translating results from a model species to crop plants.
Tumor Infiltrating Neutrophils Are Enriched in Basal-Type Urothelial Bladder Cancer

Background: Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy. Methods: By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b+ tumor-associated-neutrophils (TAN) and CD3+ T cells. Bioinformatics analysis of UBC datasets and gene expression analysis of UBC cell lines were additionally performed. Results: Basal type BC contained a significantly higher density of CD66b+ TAN compared to the luminal type. This finding was validated on TCGA, GSE32894 and GSE124305 datasets by computing a neutrophil signature. Of note, basal-type MIBC display a significantly higher level of chemokines (CKs) attracting neutrophils. Moreover, pro-inflammatory stimuli significantly up-regulate CXCL1, CXCL2 and CXCL8 in 5637 and RT4 UBC cell lines and induce neutrophil chemotaxis. In term of survival, a high density of T cells and TAN was significantly associated to a better outcome, with TAN density showing a more limited statistical power and following a non-linear predicting model. Conclusions: TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.
A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis

The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling. ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.
Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition

C-terminal PP2A antibodies widely used to measure PP2A activity do not immunoprecipitate major PP2A holoenzyme classes. Know thy antibodies Knowing whether a given antibody is specific for its intended target or is sensitive to posttranslational modifications of that target is critical for interpreting experimental data generated with antibody reagents. Two Research Resources from the Ogris group highlight the importance of knowing the capabilities and limitations of antibody reagents. Frohner et al. found that various antibodies raised against the catalytic (C) subunit of protein phosphatase 2A (PP2A) were sensitive to methylation and phosphorylation of PP2A C, cross-reacted with related phosphatases, and failed to immunoprecipitate major subsets of trimeric holoenzymes. This implies that findings using a common commercial phosphatase assay kit that relies on one of these antibodies should be reevaluated. Schüchner et al. found that the recognition of Myc-tagged proteins by a widely used Myc-specific antibody varied depending on sequences adjacent to the tag. A Focus by Janes highlights the importance of systemic validation of research antibodies. The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies—including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit—are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits. The tested antibodies preferentially recognized the nonmethylated form of the enzyme, and they did not coimmunoprecipitate trimeric holoenzymes containing the regulatory subunits B or B′, an issue that precludes their use to monitor PP2A holoenzyme activity. Furthermore, some of the antibodies also recognized the phosphatase PP4, demonstrating a lack of specificity for PP2A. Together, these findings suggest that reinterpretation of the data generated by using these reagents is required.