Mitochondrial depolarization and repolarization in the early stages of acetaminophen hepatotoxicity in mice

Mitochondrial injury and depolarization are primary events in acetaminophen hepatotoxicity. Previous studies have shown that restoration of mitochondrial function in surviving hepatocytes, which is critical to recovery, is at least partially accomplished via biogenesis of new mitochondria. However, other studies indicate that mitochondria also have the potential to spontaneously repolarize. Although repolarization was previously observed only at a sub-hepatotoxic dose of acetaminophen, we postulated that mitochondrial repolarization in hepatocytes outside the centrilobular regions of necrosis might contribute to recovery of mitochondrial function following acetaminophen-induced injury. Our studies utilized longitudinal intravital microscopy of millimeter-scale regions of the mouse liver to characterize the spatio-temporal relationship between mitochondrial polarization and necrosis early in acetaminophen-induced liver injury. Treatment of male C57BL/6J mice with a single intraperitoneal 250 mg/kg dose of acetaminophen resulted in hepatotoxicity that was apparent histologically within 2 h of treatment, leading to 20 and 60-fold increases in serum aspartate aminotransferase and alanine aminotransferase, respectively, within 6 h. Intravital microscopy of the livers of mice injected with rhodamine123, TexasRed-dextran, propidium iodide and Hoechst 33342 detected centrilobular foci of necrosis within extended regions of mitochondrial depolarization within 2 h of acetaminophen treatment. Although regions of necrosis were more apparent 6 h after acetaminophen treatment, the vast majority of hepatocytes with depolarized mitochondria did not progress to necrosis, but rather recovered mitochondrial polarization within 6 h. Recovery of mitochondrial function following acetaminophen hepatotoxicity thus involves not only biogenesis of new mitochondria, but also repolarization of existing mitochondria. These studies also revealed a spatial distribution of necrosis and mitochondrial depolarization whose single-cell granularity is inconsistent with the hypothesis that communication between neighboring cells plays an important role in the propagation of necrosis during the early stages of APAP hepatotoxicity. Small islands of healthy, intact cells were frequently found surrounded by necrotic cells, and small islands of necrotic cells were frequently found surrounded by healthy, intact cells. Time-series studies demonstrated that these "islands", consisting in some cases of single cells, are persistent; over a period of hours, injury does not spread from individual necrotic cells to their neighbors

Decreased Expression Of apM1 in Omental and Subcutaneous Adipose Tissue of Humans With Type 2 Diabetes

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes

Research Priorities for Achieving Healthy Marine Ecosystems and Human Communities in a Changing Climate

ABSTRACT: The health of coastal human communities and marine ecosystems are at risk from a host of anthropogenic stressors, in particular, climate change. Because ecological health and human well-being are inextricably connected, effective and positive responses to current risks require multidisciplinary solutions. Yet, the complexity of coupled social-ecological systems has left many potential solutions unidentified or insufficiently explored. The urgent need to achieve positive social and ecological outcomes across local and global scales necessitates rapid and targeted multidisciplinary research to identify solutions that have the greatest chance of promoting benefits for both people and nature. To address these challenges, we conducted a forecasting exercise with a diverse, multidisciplinary team to identify priority research questions needed to promote sustainable and just marine social-ecological systems now and into the future, within the context of climate change and population growth. In contrast to the traditional reactive cycle of science and management, we aimed to generate questions that focus on what we need to know, before we need to know it. Participants were presented with the question, "If we were managing oceans in 2050 and looking back, what research, primary or synthetic, would wish we had invested in today?" We first identified major social and ecological events over the past 60 years that shaped current human relationships with coasts and oceans. We then used a modified Delphi approach to identify nine priority research areas and 46 questions focused on increasing sustainability and well-being in marine social-ecological systems. The research areas we identified include relationships between ecological and human health, access to resources, equity, governance, economics, resilience, and technology. Most questions require increased collaboration across traditionally distinct disciplines and sectors for successful study and implementation. By identifying these questions, we hope to facilitate the discourse, research, and policies needed to rapidly promote healthy marine ecosystems and the human communities that depend upon them

Toxicokinetic Triage for Environmental Chemicals

Toxicokinetic (TK) models link administered doses to plasma, blood, and tissue concentrations. High-throughput TK (HTTK) performs in vitro to in vivo extrapolation to predict TK from rapid in vitro measurements and chemical structure-based properties. A significant toxicological application of HTTK has been “reverse dosimetry,” in which bioactive concentrations from in vitro screening studies are converted into in vivo doses (mg/kg BW/day). These doses are predicted to produce steady-state plasma concentrations that are equivalent to in vitro bioactive concentrations. In this study, we evaluate the impact of the approximations and assumptions necessary for reverse dosimetry and develop methods to determine whether HTTK tools are appropriate or may lead to false conclusions for a particular chemical. Based on literature in vivo data for 87 chemicals, we identified specific properties (eg, in vitro HTTK data, physico-chemical descriptors, and predicted transporter affinities) that correlate with poor HTTK predictive ability. For 271 chemicals we developed a generic HT physiologically based TK (HTPBTK) model that predicts non-steady-state chemical concentration time-courses for a variety of exposure scenarios. We used this HTPBTK model to find that assumptions previously used for reverse dosimetry are usually appropriate, except most notably for highly bioaccumulative compounds. For the thousands of man-made chemicals in the environment that currently have no TK data, we propose a 4-element framework for chemical TK triage that can group chemicals into 7 different categories associated with varying levels of confidence in HTTK predictions. For 349 chemicals with literature HTTK data, we differentiated those chemicals for which HTTK approaches are likely to be sufficient, from those that may require additional data

Knee movement patterns of injured and uninjured adolescent basketball players when landing from a jump: A case-control study

BACKGROUND: A common knee injury mechanism sustained during basketball is landing badly from a jump. Landing is a complex task and requires good coordination, dynamic muscle control and flexibility. For adolescents whose coordination and motor control has not fully matured, landing badly from a jump can present a significant risk for injury. There is currently limited biomechanical information regarding the lower limb kinetics of adolescents when jumping, specifically regarding jump kinematics comparing injured with uninjured adolescents. This study reports on an investigation of biomechanical differences in landing patterns of uninjured and injured adolescent basketball players. METHODS: A matched case-control study design was employed. Twenty-two basketball players aged 14–16 years participated in the study: eleven previously knee-injured and eleven uninjured players matched with cases for age, gender, weight, height and years of play, and playing for the same club. Six high-speed, three-dimensional Vicon 370 cameras (120 Hz), Vicon biomechanical software and SAS Version 8 software were employed to analyse landing patterns when subjects performed a "jump shot". Linear correlations determined functional relationships between the biomechanical performance of lower limb joints, and paired t-tests determined differences between the normalised peak biomechanical parameters. RESULTS: The average peak vertical ground reaction forces between the cases and controls were similar. The average peak ground reaction forces between the cases and controls were moderately correlated (r = -0.47). The control (uninjured) players had significantly greater hip and knee flexion angles and significantly greater eccentric activity on landing than the uninjured cases (p < 0.01). CONCLUSION: The findings of the study indicate that players with a history of knee injuries had biomechanically compromised landing techniques when compared with uninjured players matched for gender, age and club. Descriptions (norms) of expected levels of knee control, proprioceptive acuity and eccentric strength relative to landing from a jump, at different ages and physical developmental stages, would assist clinicians and coaches to identify players with inappropriate knee performance comparable to their age or developmental stage

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

Abstract: Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions

The benefits of strength training on musculoskeletal system health: practical applications for interdisciplinary care

Global health organizations have provided recommendations regarding exercise for the general population. Strength training has been included in several position statements due to its multi-systemic benefits. In this narrative review, we examine the available literature, first explaining how specific mechanical loading is converted into positive cellular responses. Secondly, benefits related to specific musculoskeletal tissues are discussed, with practical applications and training programmes clearly outlined for both common musculoskeletal disorders and primary prevention strategies

Design, Synthesis and Characterization of Sequence Specific DNA Cleaving Agents

Chapter I: Synthetic, Sequence Specific DNA Cleaving Peptides Synthetic peptides based on the sequence specific DNA binding domain of Hin recombinase have been prepared which are equiped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. Covalent attachment of EDTA converts the sequence specific DNA binding pep tides into peptides which are capable of DNA strand scission in the presence of iron II, a reducing agent and molecular oxygen. The EDTA-equiped peptides cleave DNA at Hin binding sites and provide information on the nucleotide postion and groove identity of the modified amino acid residue when bound to DNA. The EDTA-peptides were also competent at DNA double strand cleavage, which is a useful characteristic for identifying binding sites on large DNA fragments (&gt;5000 base pairs). A general procedure for the incorporation of an EDTA equiped lysine residue at any position in a synthetic peptide is also described. Results with 22 synthetic peptides indicate that Hin specifically recognizes a minor groove 5'-AAA sequence in its binding site with the peptide sequence Arg-Pro-Arg. Furthermore, the minor groove recognition event contributes substantially to the overall binding affinity. The possibility that minor groove recognition of tracks of A's by the peptide sequence Arg-Pro- Arg (and similar sequences), may be a common recognition motif in sequence specific DNA binding proteins is discussed. Chapter II: Distamycin-Actinomycin Hybrid Affinity Cleaving Molecules One approach to the design of sequence specific DNA binding molecules that read large sequences of double helical DNA is to couple DNA binding units of similar or diverse base pair specificities. Covalent attachment of his- and tris-N-methylpyrrolecarboxamides (based on the sequence specific DNA binding antibiotics Netropsin and Distamycin) and an aromatic phenoxazone intercalator (based on the sequence specific DNA intercalator Actinomycin) creates hybrid DNA binding minor groove and intercalation compounds. Using the affinity cleaving method we find that a first generation hybrid, bis-(EDTA-distamycin-glycyl)phenoxazone, binds the sequence 5'-TTATGGTTAA-3' which is consistent with simultaneous minor groove binding of the two tripyrrolecarboxamide units and intercalation of the phenoxazone moeity. Along with the targeted trimeric binding, the first generation hybrid also gave substantial monomeric/ dimeric binding, and exihibited only modest sequence specificity on large DNA fragments. In an attempt to reduce monomeric and dimeric binding, a second generation hybrid was prepared. Replacement of one pyrrolecarboxamide with a γ aminobutyric acid moeity, bis(EDTA-netropsin-glycyl)phenoxazone, generated a hybrid molecule which showed a reduced tendency for monomeric and dimeric DNA binding modes. The second generation hybrid binds to sequences of the form 5'-(A/T)₄GT(A/T)₄-3' and 5'-(A/T)₄CT(A/T)₄-3' and showed substantially improved sequence specificity on large DNA fragments relative to the first generation hybrid. Chapter III: Bleomycin-Distamycin Hybrid Affinity Cleaving Molecules Hybrids of the sequence specific DNA binding bithiazole domain of Bleomycin and tris-N-methylpyrrolecarboxamide of Distamycin are described. The hybrids address the issue of whether or not the bithiazole unit of the GC specific Bleomycin is a minor groove binding function. By the affinity cleaving method we find that hybrids in which the bithiazole is attached to either the amino or carboxy terminus of the tris-N-methylpyrrolecarboxamide, do not significantly alter the sequence specificity of the pyrrolecarboxamide domain. The results indicate that either the bithiazole unit is not a minor groove binding function in the same sense as the tripyrrolecarboxamide, or, alternatively, the bithiazole does not include all of the required sequence specific DNA recognition elements of Bleomycin. Chapter IV: Studies in Metalloporphyrin Mediated Affinity Cleaving Metalloporphyrins are known to cause DNA strand scission using a variety of central metals, and various reducing and oxidizing cofactors. To investigate if a metalloporphyrin can replace an EDTA•Fe chelate in an affinity cleaving molecule, a metalloporphyrin-Distamycin (PD) was prepared. PD•Fe showed very inefficient DNA cleavage compared to EDTA•Fe equiped affinity cleaving compounds. Additionally, PD•Fe showed no detectable sequence specific DNA cleaving ability. PD•Fe was also less efficient at DNA strand scission than ferriprotoporphyrin itself, which indicates that the DNA binding domain (tripyrrolecarboxamide) and cleaving domain (metalloporphyrin) are interfering with each other’s function in the affinity cleaving compounds. </p