Steered molecular dynamics simulations reveal critical residues for (un)binding of substrates, inhibitors and a product to the malarial M1 aminopeptidase.

Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the 'water reservoir', (III) ligand reorientation in the 'water reservoir' and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site.

Challenges during diapause and anhydrobiosis: Mitochondrial bioenergetics and desiccation tolerance.

In preparation for the onset of environmental challenges like overwintering, food limitation, anoxia, or water stress, many invertebrates and certain killifish enter diapause. Diapause is a developmentally-programed dormancy characterized by suppression of development and metabolism. For embryos of Artemia franciscana (brine shrimp), the metabolic arrest is profound. These gastrula-stage embryos depress oxidative metabolism by ~99% during diapause and survive years of severe desiccation in a state termed anhydrobiosis. Trehalose is the sole fuel source for this developmental stage. Mitochondrial function during diapause is downregulated primarily by restricting substrate supply, as a result of inhibiting key enzymes of carbohydrate metabolism. Because proton conductance across the inner membrane is not decreased during diapause, the inference is that membrane potential must be compromised. In the absence of any intervention, the possibility exists that the F1 Fo ATP synthase and the adenine nucleotide translocator may reverse, leading to wholesale hydrolysis of cellular ATP. Studies with anhydrobiotes like A. franciscana are revealing multiple traits useful for improving desiccation tolerance that include the expression and accumulation late embryogenesis abundant (LEA) proteins and trehalose. LEA proteins are intrinsically disordered in aqueous solution but gain secondary structure (predominantly α-helix) as water is removed. These protective agents stabilize biological structures including lipid bilayers and mitochondria during severe water stress. © 2018 IUBMB Life, 70(12):1251-1259, 2018.

Cryopreservation of lipid bilayers by LEA proteins from Artemia franciscana and trehalose.

The capacity of Late Embryogenesis Abundant (LEA) proteins and trehalose to protect liposomes against freezing-induced damage was examined by measuring the leakage of 5(6)-carboxyfluorescein (CF). Liposomes were prepared to simulate the lipid compositions of the inner leaflet of the plasma membrane, outer mitochondrial membrane (OMM), and inner mitochondrial membrane (IMM). Two recombinant LEA proteins belonging to Group 3 (AfrLEA2 and AfrLEA3m) were expressed and purified from embryos of Artemia franciscana. Only OMM-like liposomes were significantly protected by AfrLEA2 and AfrLEA3m against freeze-thaw damage; at the highest protein:lipid mass ratio tested, leakage of CF was 56.3% of control with AfrLEA3m and 29.3% with AfrLEA2. By comparison, trehalose provided protection to all compositional types. The greatest stabilization during freezing occurred when trehalose was present on both sides of the bilayer. When mitochondria isolated from rat liver were freeze-thawed in trehalose solution, the OMM remained intact based on the absence of increased oxygen consumption when cytochrome c was added during oxidative phosphorylation (OXPHOS). Respiratory control ratios (OXPHOS/LEAK) were depressed by only 30% after freeze-thawing in trehalose compared to non-frozen controls, which indicated some retention of OXPHOS capacity by the IMM. Trehalose then was loaded into the matrix (0.24 µmol/mg mitochondrial protein) by transient opening of the permeability transition pore, a procedure optimized for retention of OMM integrity. Surprisingly, respiratory control ratios were not improved after freeze-thawing with external plus matrix trehalose, when compared to external trehalose alone. This result could perhaps be explained by insufficient accumulation of matrix trehalose.

Liposomes with diverse compositions are protected during desiccation by LEA proteins from Artemia franciscana and trehalose.

Intracellular accumulation of Late Embryogenesis Abundant (LEA) proteins and the disaccharide trehalose is associated with cellular desiccation tolerance in a number of animal species. Two LEA proteins from anhydrobiotic embryos of the brine shrimp Artemia franciscana were tested for the ability to protect liposomes of various compositions against desiccation-induced damage in the presence and absence of trehalose. Damage was assessed by carboxyfluorescein leakage after drying and rehydration. Further, using a cytoplasmic-localized (AfrLEA2) and a mitochondrial-targeted (AfrLEA3m) LEA protein allowed us to evaluate whether each may preferentially stabilize membranes of a particular lipid composition based on the protein's subcellular location. Both LEA proteins were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane (with cardiolipin), outer mitochondrial membrane, and the inner leaflet of the plasma membrane. Thus liposome stabilization by AfrLEA3m or AfrLEA2 was not dependent on lipid composition, provided physiological amounts of bilayer and non-bilayer-forming lipids were present (liposomes with a non-biological composition of 100% phosphatidylcholine were not protected by either protein). Additive protection by LEA proteins plus trehalose was dependent on the lipid composition of the target membrane. Minimal additional damage occurred to liposomes stored at room temperature in the dried state for one week compared to liposomes rehydrated after 24h. Consistent with the ability to stabilize lipid bilayers, molecular modeling of the secondary structures for AfrLEA2 and AfrLEA3m revealed bands of charged amino acids similar to other amphipathic proteins that interact directly with membranes.

Quantification of cellular protein expression and molecular features of group 3 LEA proteins from embryos of Artemia franciscana.

Late embryogenesis abundant (LEA) proteins are highly hydrophilic, low complexity proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Here, we report the identification of three new mitochondrial LEA proteins in anhydrobiotic embryos of Artemia franciscana, AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29. These new isoforms are recognized by antibody raised against recombinant AfrLEA3m, the original mitochondrial-targeted LEA protein previously reported from these embryos; mass spectrometry confirms all four proteins share sequence similarity. The corresponding messenger RNA (mRNA) species for the four proteins are readily amplified from total complementary DNA (cDNA) prepared from embryos. cDNA sequences of the four mRNAs are quite similar, but each has a stretch of sequence that is absent in at least one of the others, plus multiple single base pair differences. We conclude that all four mitochondrial LEA proteins are products of independent genes. Each possesses a mitochondrial targeting sequence, and indeed Western blots performed on extracts of isolated mitochondria clearly detect all four isoforms. Based on mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis migration, the cytoplasmic-localized AfrLEA2 exists primarily as a homodimer in A. franciscana. Quantification of protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 as a function of development shows that cellular concentrations are highest in diapause embryos and decrease during development to low levels in desiccation-intolerant nauplius larvae. When adjustment is made for mitochondria matrix volume, the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide guidance for the design of in vitro functional studies with these proteins.

Use of inherent anteversion of an intramedullary nail to avoid malrotation in femur fractures.

Rotational malalignment after intramedullary (IM) nailing of femoral fractures remains a significant problem. A technique using intraoperative fluoroscopy and the anteversion inherent to the IM nail for obtaining appropriate femoral rotational alignment is presented. The technique is advocated as a simple alternative to more complex methods for estimation of femoral anteversion during placement of femoral IM nails. This method is simple and requires intraoperative fluoroscopy on the injured extremity alone. It reliably sets the femoral anteversion within a normal physiologic range with minimal additional intraoperative steps and without preoperative measurements.

Trend in the utilization of CT for adolescents admitted to an adult level I trauma center.

PURPOSE: The aims of this study were to evaluate the trend in the utilization of CT for adolescents admitted to an adult level I trauma center and to compare the utilization pattern between adolescents and adults during the past 11 years. METHODS: Trauma registry data (1996-2006) from an adult level I trauma center were used. Patients aged ≥13 years were eligible to be admitted to this hospital. From this trauma registry, the following variables were extracted: age; sex; ethnicity; insurance status; mechanism of injury; injury severity score; length of hospital and intensive care unit stay; International Classification of Diseases, Ninth Revision (ICD-9), codes; and patient disposition. Patients were categorized on the basis of their age in the following groups: 13 to 18, 19 to 55, and ≥56 years. ICD-9 procedure codes were used to create new variables that were reflective of the frequency of use of head CT (ICD-9 code 87.03), abdominal CT (ICD-9 code 88.01), thoracic CT (ICD-9 code 87.41), and other CT studies, including CT of the extremities and spine (ICD-9 code 88.38). RESULTS: All age groups experienced substantial increases in the utilization of CT, and there were minimal differences in crude utilization rates among different age categories. After adjustment for potential confounders, adolescents had a slightly higher chance of being evaluated by head CT (incident risk ratio [IRR], 1.16; 95% confidence interval [CI], 1.11-1.22) and a significantly lower chance of undergoing thoracic CT (IRR, 0.54; 95% CI, 0.48-0.61) in comparison with adults aged 19 to 55 years. Among adolescents, the chance of undergoing head CT was significantly higher in 2006 relative to 1996 (IRR, 1.50; 95% CI, 1.20-1.86). However, there was no linear increase in utilization pattern from 1996 to 2006. Abdominal CT demonstrated a similar pattern. Thoracic CT and other CT studies demonstrated the most drastic increases in utilization pattern among adolescents. The IRR for the use of thoracic CT increased from 1.15 (95% CI, 0.26-5.20) in 1997 (relative to 1996) to 10.53 (95% CI, 3.24-34.26) in 2006. The IRRs for other CT studies in 2005 and 2006, relative to 1996, were 7.24 and 6.91, respectively. CONCLUSIONS: Treatment of adolescents in adult level I trauma centers is challenging. Trauma centers should adopt strategies that could potentially decrease unnecessary utilization, especially among adolescents. To do this, these facilities should be familiar with patient-related and system-related characteristics that might influence overutilization. Furthermore, physicians in adult trauma centers should be reeducated with regard to potential hazardous consequences of CT for adolescents.