Miracle-dz/UniFAR-FTUs huggingface dataset:
Expansion, in vivo–ex vivo cycling, and genetic manipulation of primary human hepatocytes

Significance The ability to study human liver disease is limited by available hepatocyte models. Primary human hepatocytes (PHH) and xenograft models suffer from limited availability, donor-to-donor variability, and high cost. Here we report two transformative advances. First, the alkaloid retrorsine improves humanization of the murine liver, which allows routine production of highly humanized mice and high-quality mouse-passaged PHH. Second, the ability to genetically modify PHH cultures and retransplant to create highly humanized mice with genetically altered grafts. When combined, these two advances open new frontiers for creating disease-specific PHH models and for performing genetic and other screens in PHH. Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
Virtual screening, molecular dynamics, density functional theory and quantitative structure activity relationship studies to design peroxisome proliferator-activated receptor-γ agonists as anti-diabetic drugs

Abstract Type 2 diabetes (T2D) is generally characterized by elevated blood glucose levels, insulin resistance, and relative lack of insulin; however, insulin resistance is the predominant risk factor. Hence, the use of insulin sensitizer drugs to increase insulin sensitivity has gained immense interest as an attractive treatment option for T2D and their major target is a nuclear receptor PPAR-γ (peroxisome proliferator-activated receptor-γ). A wide range of synthetic insulin sensitizers such as thiazolidinedione act as PPAR-γ agonists thereby enhancing insulin action and improving hyperglycemia in patients. Nonetheless, they pose severe adverse effects for human, necessitating an emergent need to develop effective insulin sensitizer drugs. Herein, virtual screening of 10,000 ligands is performed and the best five ligands are identified. MET364, ILE341, CYS285, ALA292, PHE282, and LEU330 residues are found to play an important role in ligand binding. It is shown from the molecular dynamics simulations results of the top-ranked ligands that increased numbers of hydrogen bonds are formed with PPAR-γ catalytic residues. Quantum chemical calculations reveal that all the best ligands can demonstrate good thermodynamic stability and pharmacokinetic properties. Partial-least-square (PLS) regression of quantitative structural activity relationship (QSAR) is utilized to model and predict the binding energy for ligands. Principal component analysis is further explored for the best ligands’ QSAR pattern recognition. Importantly, the predicted values of the binding energy of the potential ligands by the PLS regression is favourably compared with the values of binding energy obtained from molecular docking with incredible high accuracy of 98%.
The GATOR2–mTORC2 axis mediates Sestrin2-induced AKT Ser/Thr kinase activation

Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet–induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex (mTORC) 1 and AMP-activated protein kinase, are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol 3-kinase and mTORC2, kinases upstream of AKT, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WDR24 (WD repeat domain 24), the GATOR2 component essential for the Sestrin2–GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2–mTORC2 interaction, completely ablated Sestrin2-induced AKT activation. We also noted that Sestrin2 also directly binds to the pleckstrin homology domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2.
Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins

While the GAS M protein has been the leading vaccine target for decades, the bacteria encode many other virulence factors of interest for vaccine development. In this work, we show that emm-like genes are encoded in a remarkable majority of GAS genomes and expressed at a level similar to that for the emm gene. In collaboration with the U.S. Centers for Disease Control, we developed molecular definitions of the different emm and emm-like gene families. This clarification should abrogate mistyping of strains, especially in the area of whole-genome typing. We have also updated the emm-typing collection by removing emm-like gene sequences and provided in-depth analysis of Mrp and Enn protein sequence structure and diversity. ABSTRACT The core Mga (multiple gene activator) regulon of group A Streptococcus (GAS) contains genes encoding proteins involved in adhesion and immune evasion. While all GAS genomes contain genes for Mga and C5a peptidase, the intervening genes encoding M and M-like proteins vary between strains. The genetic make-up of the Mga regulon of GAS was characterized by utilizing a collection of 1,688 GAS genomes that are representative of the global GAS population. Sequence variations were examined with multiple alignments, and the expression of all core Mga regulon genes was examined by quantitative reverse transcription-PCR in a representative strain collection. In 85.2% of the sampled genomes, the Mga locus contained genes encoding Mga, Mrp, M, Enn, and C5a peptidase proteins. These isolates account for 53% of global infections. Only 9.1% of genomes did not contain either an mrp or an enn gene. The pairwise identity within Enn (68.6%) and Mrp (83.2%) protein sequences was higher than within M proteins (44.7%). Gene expression varied between strains tested, but high expression was recorded for all genes in at least one strain. Previous nomenclature issues were clarified with molecular gene definitions. Our findings support a shift in focus in the GAS research field to further consider the role of Mrp and Enn in virulence and vaccine development. IMPORTANCE While the GAS M protein has been the leading vaccine target for decades, the bacteria encode many other virulence factors of interest for vaccine development. In this work, we show that emm-like genes are encoded in a remarkable majority of GAS genomes and expressed at a level similar to that for the emm gene. In collaboration with the U.S. Centers for Disease Control, we developed molecular definitions of the different emm and emm-like gene families. This clarification should abrogate mistyping of strains, especially in the area of whole-genome typing. We have also updated the emm-typing collection by removing emm-like gene sequences and provided in-depth analysis of Mrp and Enn protein sequence structure and diversity.
Clinical outcomes of meniscus repair and partial meniscectomy: Does tear configuration matter?

Purpose: We report our experience with the effect of location and configuration of meniscal tears on clinical outcomes. Methods: A retrospective review of patients who underwent partial meniscectomy or meniscal repair between 2008 and 2016 was conducted. One hundred fourteen knees in 106 patients were included, comprising 43 partial meniscectomies and 71 meniscal repairs. Patients were graded pre- and postoperatively with the International Knee Documentation Committee (IKDC) score and Tegner Activity Level Scale. Meniscal tears were classified according to location (anterior horn, body, posterior horn, and others) and type (radial, horizontal, longitudinal, and complex) and subgroups were analyzed for their effect on outcomes. Results: All tears, whether treated with partial meniscectomy or repair, showed significant improvement in postoperative scores (p < 0.05); 39.47% of tears involved the posterior horn alone, 10.53% involved the body alone, 3.51% involved the anterior horn alone, and 46.49% were complex tears that spanned more than one area. Complex tears treated with repair had significantly better scores (IKDC, p = 0.002; Tegner, p = 0.008) than complex tears treated with meniscectomy. Longitudinal tears showed results suggesting better short-term outcomes with meniscectomy than with repair (IKDC, p = 0.036; Tegner p = 0.018), a potential statistical anomaly. Horizontal and radial tears showed no significant difference in outcomes, regardless of treatment. Tears in different locations (anterior horn, body, posterior horn, and others) showed no significant difference in outcomes, regardless of treatment. Conclusion: Meniscal surgery yields significant improvement in postoperative scores. Complex tears showed significantly better postoperative scores when treated with repair.
Aspirin responsiveness and 6-month clinical outcome observed in a cohort study of patients with unstable angina pectoris

Abstract Background Whether patients with coronary heart disease (CHD) and resistance to aspirin found by in vitro tests are at a greater risk of major adverse cardiovascular events (MACEs) is controversial. Objective To identify any association between resistance to aspirin found by in vitro tests and MACEs in patients with unstable angina pectoris (UAP). Methods Previously we demonstrated that 38 of 104 patients admitted to hospital with UAP showed resistance to aspirin using whole blood aggregometry (WBA). In the present study, the same cohort was observed during a 6-month follow-up. The primary end points were MACEs, including cardiovascular death, nonfatal myocardial infarction, revascularization, stroke/transient ischemic attack, or worsening UAP that required the patient to be readmitted to hospital. Results During the course of 6 months, only 1 patient in the aspirin-sensitive group was lost in follow-up, and MACEs occurred in 24 patients. Patients with resistance to aspirin found by WBA did not apparently have a higher risk of MACEs compared with patients who were aspirin-sensitive (29% vs 20%, P > 0.05). Cox regression analysis showed that resistance to aspirin found by WBA appeared to have no significant correlation with 6-month clinical outcome (HR 1.56, 95% CI 0.70-3.48, P > 0.05). Conclusion Aspirin resistance, as defined by WBA, was not associated with an increased risk of MACEs in patients with UAP in a 6-month follow-up. Clarification of the clinical significance of aspirin responsiveness detected by platelet function tests requires further investigation in larger longitudinal studies.
Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification

The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca2+ sparks and dual-tilt electron tomography) and dSTORM imaging of permeabilized Wistar rat ventricular myocytes. Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca2+ spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca2+ spark formation.
Adherence to Anti-retroviral Therapy among HIV-Positive Individuals in Ghana: The Role of Stress and Post Traumatic Stress Symptoms

ABSTRACT Aims: To identify stress and post traumatic stress symptoms (PTSS) in HIV patients on Antiretroviral therapy (ART) and further assess the possible relationship between stress, PTSS, immune function and ART medication adherence. Methods: After obtaining written informed consent, 150 HIV out-patients attending Pantang and Amasaman Hospitals in Accra, Ghana, were recruited for the study. Participants were interviewed using a questionnaire comprising of sections measuring demographic characteristics, perceived stress, post traumatic stress symptoms, and medication adherence. A measure of the immune function (CD4+) from individual’s health records was noted at the time of interview. Results : Ninety four (62.7%) patients exhibited higher stress levels compared to the remaining 56(37.3%). The majority of participants (74%) showed PTSS at levels that met the optimal criteria indicative of post traumatic stress disorder (PTSD) and may have required clinical attention and management. Participants with high stress levels were 84% less likely to be adherent to their antiretroviral medication compared to those with lower levels of stress [OR=0.16 (0.050–0.512),
Pressure Ulcers: A Patient Safety Issue

The prevention of pressure ulcers represents a marker of quality of care. Pressure ulcers are a major nurse-sensitive outcome. Hence, nursing care has a major effect on pressure ulcer development and prevention. Prevention of pressure ulcers often involves the use of low technology, but vigilant care is required to address the most consistently reported risk factors for development of pressure ulcers. The literature suggested that not all pressure ulcers can be prevented, but the use of comprehensive pressure ulcer programs can prevent the majority of pressure ulcers. When the pressure ulcer develops, the goals of healing or preventing deterioration and infection are paramount. Randomized controlled trials are needed to determine optimal management strategies dependent on stage and comorbidities/severity of illness. Nursing remains at the forefront of protecting and safeguarding the patient from pressure ulcers.
Characteristics of Childhood Family Violence Experiences in Spanish Batterers

Intimate Partner Violence (IPV) constitutes a major public health problem worldwide. The existing links between childhood family violence experiences and perpetration of IPV during adulthood suggest that violence is transmitted across generations. The aims of this study were to provide new insights into characteristics of the history of childhood family violence in a sample of Spanish convicted male batterers and to examine what typology of batterer (specialist or generalist) is more likely to have had different types of childhood experiences of violence in the family context. Participants were 740 men convicted of IPV whose ages ranged from 18 to 71 years old (M = 39.74; SD = 10.01). Of them, 305 men were classified as specialist batterers, and 391 men were classified as generalist batterers. Participants completed a questionnaire made ad hoc regarding family of origin violence and sociodemographic aspects. Results showed that, in comparison with generalist batterers, specialist batterers had higher likelihood of having experienced child abuse or maltreatment during their childhood, having experienced physical punishment and child neglect, having their father as the aggressor of such experienced situations of abuse, or maltreatment. Likewise, specialist batterers had higher likelihood of having been witnesses of family violence, having witnessed physical and psychological violence in the family, having witnessed their mother and siblings being the victims and their father being the aggressor when family violence occurred. This study emphasized the need for identifying children exposed to direct and indirect family violence because they could be a vulnerable group for future IPV perpetration. Moreover, variables related to childhood family violence must be considered when developing intervention treatment programs focused on male specialist batterers.
Tendon response to matrix unloading is determined by the patho-physiological niche

Aberrant matrix turnover with elevated matrix proteolysis is a hallmark of tendon pathology. While tendon disease mechanisms remain obscure, mechanical cues are central regulators. Unloading of tendon explants in standard culture conditions provokes rapid cell-mediated tissue breakdown. Here we show that biological response to tissue unloading depends on the mimicked physiological context. Our experiments reveal that explanted tendon tissues remain functionally stable in a simulated avascular niche of low temperature and oxygen, regardless of the presence of serum. This hyperthermic and hyperoxic niche-dependent catabolic switch was shown by whole transcriptome analysis (RNA-seq) to be a strong pathological driver of an immune-modulatory phenotype, with a stress response to reactive oxygen species (ROS) and associated activation of catabolic extracellular matrix proteolysis that involved lysosomal activation and transcription of a range of proteolytic enzymes. Secretomic and degradomic analysis through terminal amine isotopic labeling of substrates (TAILS) confirmed that proteolytic activity in unloaded tissues was strongly niche dependent. Through targeted pharmacological inhibition we isolated ROS mediated oxidative stress as a major checkpoint for matrix proteolysis. We conclude from these data that the tendon stromal compartment responds to traumatic mechanical unloading in a manner that is highly dependent on the extrinsic niche, with oxidative stress response gating the proteolytic breakdown of the functional collagen backbone.
A simple and cost-efficient technique to generate hyperpolarized long-lived 15N-15N nuclear spin order in a diazine by signal amplification by reversible exchange

Signal Amplification by Reversible Exchange (SABRE) is an inexpensive and simple hyperpolarization technique that is capable of boosting nuclear magnetic resonance sensitivity by several orders of magnitude. It utilizes the reversible binding of para-hydrogen, as hydride ligands, and a substrate of interest to a metal catalyst to allow for polarization transfer from para-hydrogen into substrate nuclear spins. While the resulting nuclear spin populations can be dramatically larger than those normally created, their lifetime sets a strict upper limit on the experimental timeframe. Consequently, short nuclear spin lifetimes are a challenge for hyperpolarized metabolic imaging. In this report, we demonstrate how both hyperpolarization and long nuclear spin lifetime can be simultaneously achieved in nitrogen-15 containing derivatives of pyridazine and phthalazine by SABRE. These substrates were chosen to reflect two distinct classes of 15N2-coupled species that differ according to their chemical symmetry and thereby achieve different nuclear spin lifetimes. The pyridazine derivative proves to exhibit a signal lifetime of ∼2.5 min and can be produced with a signal enhancement of ∼2700. In contrast, while the phthalazine derivative yields a superior 15 000-fold 15N signal enhancement at 11.7 T, it has a much shorter signal lifetime.
Hepatic PPARα function and lipid metabolic pathways are dysregulated in polymicrobial sepsis

Despite intensive research and constant medical progress, sepsis remains one of the most urgent unmet medical needs of today. Most studies have been focused on the inflammatory component of the disease; however, recent advances support the notion that sepsis is accompanied by extensive metabolic perturbations. During times of limited caloric intake and high energy needs, the liver acts as the central metabolic hub in which PPARα is crucial to coordinate the breakdown of fatty acids. The role of hepatic PPARα in liver dysfunction during sepsis has hardly been explored. We demonstrate that sepsis leads to a starvation response that is hindered by the rapid decline of hepatic PPARα levels, causing excess free fatty acids, leading to lipotoxicity, and glycerol. In addition, treatment of mice with the PPARα agonist pemafibrate protects against bacterial sepsis by improving hepatic PPARα function, reducing lipotoxicity and tissue damage. Since lipolysis is also increased in sepsis patients and pemafibrate protects after the onset of sepsis, these findings may point toward new therapeutic leads in sepsis.
Replication of Pyridyloxobutyl Phosphotriester Lesions in Cells.

Genome integrity is constantly challenged by endogenous or exogenous genotoxic agents, which can give rise to various DNA adducts. After metabolic activation, tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can lead to pyridyloxobutylphosphotriesters (POB-PTEs) in DNA. Here, we synthesize oligodeoxyribonucleotides containing a site-specifically inserted SP- or RP-POB-PTE flanked by two thymidines, and we examine the impact that these lesions have on DNA replication in Escherichia coli cells. We found that these two lesions are not strong impediments to DNA replication, and their replicative bypass is not modulated by genetic depletion of the three SOS-induced DNA polymerases or Ada protein. In addition, neither SP- nor RP-POB-PTEs is mutagenic in E. coli cells. Together, our study unveils, for the first time, the influence of tobacco-specific nitrosamine-induced POB-PTE lesions on DNA replication in vivo.