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1.  Metal-Free Reductive Cleavage of C–N and S–N Bonds by Photoactivated Electron Transfer from a Neutral Organic Donor** 
A photoactivated neutral organic super electron donor cleaves challenging arenesulfonamides derived from dialkylamines at room temperature. It also cleaves a) ArC–NR and b) ArN–C bonds. This study also highlights the assistance given to these cleavage reactions by the groups attached to N in (a) and to C in (b), by lowering LUMO energies and by stabilizing the products of fragmentation.
doi:10.1002/anie.201306543
PMCID: PMC4227565  PMID: 24311295
electron transfer; cleavage reactions; photochemistry; radical ions; reduction
2.  The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation 
PLoS Genetics  2014;10(10):e1004703.
The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.
Author Summary
What are the mechanisms used by animals to cope with stressful environments that inflict damage or restrict essential processes such as growth, development, and reproduction? One strategy is changes in physiology that increase stress resistance, and an extreme version of this strategy is diapause, an alternative developmental state that is enduring and stress resistant. In the nematode C. elegans, stress tolerance and entry into a diapause state called dauer larvae are mediated by the conserved insulin/IGF-1 pathway. Specifically, the FOXO transcription factor DAF-16 promotes stress tolerance and dauer larvae development. However, the targets of DAF-16 that mediate these processes remain largely elusive. Using an unbiased forward genetic screen to discover new mediators of stress tolerance, we identified natc-1, a novel target of DAF-16 and the insulin/IGF-1 pathway. natc-1 encodes a conserved subunit of the N-terminal acetyltransferase C (NAT) complex. The NatC complex modifies target proteins by acetylating the N-terminus. We demonstrated that natc-1 mediates diapause entry and stress tolerance. Furthermore, we elucidated regulation of NatC by demonstrating that natc-1 is a direct transcriptional target that is repressed by DAF-16. These findings may be relevant to other animals because both the insulin/IGF-1 signaling pathway and the NAT system are conserved during evolution.
doi:10.1371/journal.pgen.1004703
PMCID: PMC4199503  PMID: 25330323
3.  Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry 
Molecular microbiology  2010;76(4):1010-1019.
The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.
doi:10.1111/j.1365-2958.2010.07159.x
PMCID: PMC4113555  PMID: 20398220
diphtheria toxin; coatomer protein complex I
4.  Expanding the scope and relevance of health interventions: Moving beyond clinical trials and behavior change models 
An overemphasis on clinical trials and behavior change models has narrowed the knowledge base that can be used to design interventions. The overarching point is that the process of overanalyzing variables is impeding the process of gaining insight into the everyday experiences that shape how people define health and seek treatment. This claim is especially important to health decision-making and behavior change because subtle interpretations often influence the decisions that people make. This manuscript provides a critique of traditional approaches to developing health interventions, and theoretically justifies what and why changes are warranted. The limited scope of these models is also discussed, and an argument is made to adopt a strategy that includes the perceptions of people as necessary for understanding health and health-related decision-making. Three practical strategies are suggested to be used with the more standard approaches to assessing the effectiveness and relevance of health interventions.
doi:10.3402/qhw.v9.24743
PMCID: PMC4107301  PMID: 25053530
health interventions; clinical trials; behavior change models; photovoice; ethnography; narratives; theory; public health
5.  Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells 
PLoS ONE  2014;9(7):e102625.
The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.
doi:10.1371/journal.pone.0102625
PMCID: PMC4094554  PMID: 25014217
6.  Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein–Barr virus associated nasopharyngeal carcinoma 
Virology  2013;441(2):107-113.
Epstein–Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, as well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients.
doi:10.1016/j.virol.2013.03.016
PMCID: PMC3782090  PMID: 23601786
Epstein–Barr virus; Nasopharyngeal carcinoma; CD8+ T cells; T regulatory cells; Ontak
7.  Understanding the Etiology of Prescription Opioid Abuse: Implications for Prevention and Treatment 
Qualitative health research  2013;23(7):963-975.
Although studies on the initiation of substance abuse abound, the body of literature on prescription opioid abuse (POA) etiology is small. Little is known about why and how the onset of POA occurs, especially among high-risk populations. In this study we aimed to fill this important knowledge gap by exploring the POA initiation experiences of 90 prescription opioid abusers currently in treatment and their narrative accounts of the circumstances surrounding their POA onset. This research was conducted within a storyline framework, which operates on the premise that the path to drug abuse represents a biography or a process rather than a static condition. Audiotapes of in-depth interviews were transcribed, coded, and thematically analyzed. Analyses revealed the presence of four trajectories leading to POA. This study adds to the limited research on POA etiology by not only illuminating the psychosocial factors that contribute to POA onset, but also by situating initiation experiences within broader life processes. The study findings provide crucial insights to policymakers and interventionists in identifying who is at risk for POA, and more important, when and how to intervene most efficaciously.
doi:10.1177/1049732313488837
PMCID: PMC3680787  PMID: 23656723
addiction/substance use; illness and disease, prevention; interviews, semistructured; mental health and illness; psychosocial issues; social constructionism; sociology
8.  Synthesis of Antioxidants for Prevention of Age-related Macular Degeneration.# 
Journal of natural products  2013;76(3):450-454.
Photooxidation of A2E may be involved in diseases of the macula and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidants quercetin and sesamol. These compounds contain water-solubilizing amine groups and several were more potent inhibitors of A2E photooxidation than quercetin.
doi:10.1021/np300769c
PMCID: PMC4069254  PMID: 23346866
9.  Epidemiology of injuries in hurling: a prospective study 2007–2011 
BMJ Open  2014;4(6):e005059.
Objectives
Hurling is a stick handling game which, although native to Ireland, has international reach and presence. The aim of this study was to report incidence and type of injuries incurred by elite male hurling players over five consecutive playing seasons.
Design
Prospective cohort study.
Setting
Male intercounty elite sports teams participating in the National GAA Injury Database, 2007–2011.
Participants
A total of 856 players in 25 county teams were enrolled.
Primary and secondary outcomes
Incidence, nature and mechanism of injury were recorded by team physicians or physiotherapists to a secure online data collection portal. Time-loss injury rates per 1000 training and match play hours were calculated and injury proportions were expressed.
Results
In total 1030 injuries were registered, giving a rate of 1.2 injuries per player. These were sustained by 71% (n=608) of players. Injury incidence rate was 2.99 (95% CI 2.68 to 3.30) per 1000 training hours and 61.75 (56.75 to 66.75) per 1000 match hours. Direct player-to-player contact was recorded in 38.6% injuries, with sprinting (24.5%) and landing (13.7%) the next most commonly reported injury mechanisms. Median duration of time absent from training or games, where the player was able to return in the same season, was 12 days (range 2–127 days). The majority (68.3%) of injuries occurred in the lower limbs, with 18.6% in the upper limbs. The trunk and head/neck regions accounted for 8.6% and 4.1% injuries, respectively. The distribution of injury type was significantly different (p<0.001) between upper and lower extremities: fractures (upper 36.1%, lower 1.5%), muscle strain (upper 5.2%, lower 45.8%).
Conclusions
These data provide stable, multiannual data on injury patterns in hurling, identifying the most common injury problems. This is the first step in applying a systematic, theory-driven injury prevention model in the sport.
doi:10.1136/bmjopen-2014-005059
PMCID: PMC4067887  PMID: 24948748
Athletic Injuries; Sports Medicine; Epidemiology; Incidence
10.  GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro 
Molecular microbiology  2011;81(5):1390-1401.
Summary
Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immuno-electron microscopy and gold-labeled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock down of GRP78 results in the emergence of resistance to anthrax lethal toxin and edema toxin action.
doi:10.1111/j.1365-2958.2011.07770.x
PMCID: PMC4041151  PMID: 21797942
unfoldase; anthrax toxin; siRNA; anthrax toxin entry
11.  Treerunners, cryptic lizards of the Plica plica group (Squamata, Sauria, Tropiduridae) of northern South America 
ZooKeys  2013;49-77.
The arboreal, Neotropical lizard Plica plica (Linnaeus, 1758) has been long considered a widespread species with a distribution east of the Andes. A preliminary examination of 101 specimens from about 28 locations mostly north of the Amazon suggests that Plica plica is a cryptic species complex with taxa that can be distinguished on the basis of the number of scale rows at mid-body; the arrangement, shape and ornamentation of scales on the snout; the number of lamellae on the fourth toe; the number of subocular plates; as well as other commonly used external morphological traits. The allopatric species discussed here are concordant with northern South American geography. Plica plica (Linnaeus, 1758) is associated with the Guiana Shield (Suriname, Guyana and Venezuela). A second species, P. caribeana sp. n. is associated with the Caribbean Coastal Range of Venezuela including Trinidad and Tobago. A third, distinctive species, P. rayi sp. n. is associated with the middle Orinoco at the eastern edge of the Guiana Shield. Two other species, P. kathleenae sp. n. and P. medemi sp. n., each based upon a single specimen, one from the Sierra Acarai Mountains of Guyana, and the other from southern Meta, Colombia are described. In addition to morphological analyses, we sequenced 12S and 16S rDNA gene fragments from one Plica plica from Trinidad to assess its relationship and taxonomy to other mainland Plica cf. plica. The results suggest Plica caribeana sp. n. likely diverged prior to the separation of Trinidad from northern Venezuela. Isolation in the Caribbean Coastal Range during its rapid uplift in the late Miocene, combined with a marine incursion into northern Venezuela may have contributed to their genetic divergence from other populations.
doi:10.3897/zookeys.355.5868
PMCID: PMC3867189  PMID: 24363569
Arboreal lizards; Iguania; Neotropics; new species; systematics
12.  Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase 
PLoS ONE  2013;8(9):e75141.
Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.
doi:10.1371/journal.pone.0075141
PMCID: PMC3779220  PMID: 24073241
13.  Overturning Established Chemoselectivities: Selective Reduction of Arenes over Malonates and Cyanoacetates by Photoactivated Organic Electron Donors 
Journal of the American Chemical Society  2013;135(30):10934-10937.
The prevalence of metal-based reducing reagents, including metals, metal complexes, and metal salts, has produced an empirical order of reactivity that governs our approach to chemical synthesis. However, this reactivity may be influenced by stabilization of transition states, intermediates, and products through substrate–metal bonding. This article reports that in the absence of such stabilizing interactions, established chemoselectivities can be overthrown. Thus, photoactivation of the recently developed neutral organic superelectron donor 5 selectively reduces alkyl-substituted benzene rings in the presence of activated esters and nitriles, in direct contrast to metal-based reductions, opening a new perspective on reactivity. The altered outcomes arising from the organic electron donors are attributed to selective interactions between the neutral organic donors and the arene rings of the substrates.
doi:10.1021/ja4050168
PMCID: PMC3755536  PMID: 23859883
14.  Modulation of Quorum Sensing in Acylhomoserine Lactone-Producing or -Degrading Tobacco Plants Leads to Alteration of Induced Systemic Resistance Elicited by the Rhizobacterium Serratia marcescens 90-166 
The Plant Pathology Journal  2013;29(2):182-192.
Numerous root-associated bacteria (rhizobacteria) are known to elicit induced systemic resistance (ISR) in plants. Bacterial cell-density-dependent quorum sensing (QS) is thought to be important for ISR. Here, we investigated the role of QS in the ISR elicited by the rhizobacterium, Serratia marcescens strain 90–166, in tobacco. Since S. marcescens 90–166 produces at least three QS signals, QS-mediated ISR in strain 90–166 has been difficult to understand. Therefore, we investigated the ISR capacity of two transgenic tobacco (Nicotiana tabacum) plants that contained either bacterial acylhomoserine lactone-producing (AHL) or -degrading (AiiA) genes in conjunction with S. marcescens 90–166 to induce resistance against bacterial and viral pathogens. Root application of S. marcescens 90–166 increased ISR to the bacterial pathogens, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. tabaci, in AHL plants and decreased ISR in AiiA plants. In contrast, ISR to Cucumber mosaic virus was reduced in AHL plants treated with S. marcescens 90–166 but enhanced in AiiA plants. Taken together, these data indicate that QS-dependent ISR is elicited by S. marcescens 90–166 in a pathogen-dependent manner. This study provides insight into QS-dependent ISR in tobacco elicited by S. marcescens 90–166.
doi:10.5423/PPJ.SI.11.2012.0173.R2
PMCID: PMC4174776  PMID: 25288945
AiiA; N-Acyl homoserine lactone; plant-growth promoting rhizobacteria; quorum sensing
15.  ZFP36L1 Negatively Regulates Plasmacytoid Differentiation of BCL1 Cells by Targeting BLIMP1 mRNA 
PLoS ONE  2012;7(12):e52187.
The ZFP36/Tis11 family of zinc-finger proteins regulate cellular processes by binding to adenine uridine rich elements in the 3′ untranslated regions of various mRNAs and promoting their degradation. We show here that ZFP36L1 expression is largely extinguished during the transition from B cells to plasma cells, in a reciprocal pattern to that of ZFP36 and the plasma cell transcription factor, BLIMP1. Enforced expression of ZFP36L1 in the mouse BCL1 cell line blocked cytokine-induced differentiation while shRNA-mediated knock-down enhanced differentiation. Reconstruction of regulatory networks from microarray gene expression data using the ARACNe algorithm identified candidate mRNA targets for ZFP36L1 including BLIMP1. Genes that displayed down-regulation in plasma cells were significantly over-represented (P = <0.0001) in a set of previously validated ZFP36 targets suggesting that ZFP36L1 and ZFP36 target distinct sets of mRNAs during plasmacytoid differentiation. ShRNA-mediated knock-down of ZFP36L1 in BCL1 cells led to an increase in levels of BLIMP1 mRNA and protein, but not for mRNAs of other transcription factors that regulate plasmacytoid differentiation (xbp1, irf4, bcl6). Finally, ZFP36L1 significantly reduced the activity of a BLIMP1 3′ untranslated region-driven luciferase reporter. Taken together, these findings suggest that ZFP36L1 negatively regulates plasmacytoid differentiation, at least in part, by targeting the expression of BLIMP1.
doi:10.1371/journal.pone.0052187
PMCID: PMC3527407  PMID: 23284928
17.  Adhesive Performance of Biomimetic Adhesive-Coated Biologic Scaffolds 
Biomacromolecules  2010;11(11):2976-2984.
Surgical repair of a discontinuity in traumatized or degenerated soft tissues is traditionally accomplished using sutures. A current trend is to reinforce this primary repair with surgical grafts, meshes, or patches secured with perforating mechanical devices (i.e., sutures, staples, or tacks). These fixation methods frequently lead to chronic pain and mesh detachment. We developed a series of biodegradable adhesive polymers that are synthetic mimics of mussel adhesive proteins (MAPs), composed of 3,4-dihydroxyphenylalanine (DOPA)-derivatives, polyethylene glycol (PEG), and polycaprolactone (PCL). These polymers can be cast into films, and their mechanical properties, extent of swelling, and degradation rate can be tailored through the composition of the polymers as well as blending with additives. When coated onto a biologic mesh used for hernia repair, these adhesive constructs demonstrated adhesive strengths significantly higher than fibrin glue. With further development, a pre-coated bioadhesive mesh may represent a new surgical option for soft tissue repair.
doi:10.1021/bm1007794
PMCID: PMC3027843  PMID: 20919699
biomimetic material; tissue adhesive; surgical mesh; multiblock copolymer; DOPA
18.  Bilateral atypical insufficiency fractures of the proximal tibia and a unilateral distal femoral fracture associated with long-term intravenous bisphosphonate therapy: a case report 
Introduction
Atypical insufficiency fractures of the femur in patients on long-term bisphosphonate therapy have been well described in recent literature. The majority of cases are associated with minimal or no trauma and occur in the subtrochanteric or diaphyseal region.
Case presentation
We describe the case of a 76-year-old British Caucasian woman who presented initially to an emergency department and then to her primary care physician with a long-standing history of bilateral knee pain after minor trauma. Plain radiographs showed subtle linear areas of sclerosis bilaterally in her proximal tibiae. Magnetic resonance imaging confirmed the presence of insufficiency fractures in these areas along with her left distal femur. There are very few reports of atypical insufficiency fractures involving the tibia in patients on long-term bisphosphonate therapy and this appears to be the only documented bilateral case involving the metaphyseal regions of the proximal tibia and distal femur.
Conclusion
In addition to existing literature describing atypical fractures in the proximal femur and femoral shaft, there is a need for increased awareness that these fractures can also occur in other weight-bearing areas of the skeleton. All clinicians involved in the care of patients taking long-term bisphosphonates need to be aware of the growing association between new onset lower limb pain and atypical insufficiency fractures.
doi:10.1186/1752-1947-6-50
PMCID: PMC3292822  PMID: 22309438
19.  Biomechanical properties of Achilles tendon repair augmented with a bioadhesive-coated scaffold 
The Achilles tendon is the most frequently ruptured tendon. Both acute and chronic (neglected) tendon ruptures can dramatically affect a patient’s quality of life, and require a prolonged period of recovery before return to pre-injury activity levels. This paper describes the use of an adhesive-coated biologic scaffold to augment primary suture repair of transected Achilles tendons. The adhesive portion consisted of a synthetic mimic of mussel adhesive proteins that can adhere to various surfaces in a wet environment, including biologic tissues. When combined with biologic scaffolds such as bovine pericardium or porcine dermal tissues, these adhesive constructs demonstrated lap shear adhesive strengths significantly greater than that of fibrin glue, while reaching up to 60% of the strength of a cyanoacrylate-based adhesive. These adhesive constructs were wrapped around transected cadaveric porcine Achilles tendons repaired with a combination of parallel and three-loop suture patterns. Tensile mechanical testing of the augmented repairs exhibited significantly higher stiffness (22–34%), failure load (24–44%), and energy to failure (27–63%) when compared to control tendons with suture repair alone. Potential clinical implications of this novel adhesive biomaterial are discussed.
doi:10.1088/1748-6041/6/1/015014
PMCID: PMC3046464  PMID: 21266745
20.  Reflections on the Knife Edge 
The Oncologist  2011;16(2):257-259.
Introduction
The accompanying article, written by John Murphy, a retired lawyer and lifelong outdoorsman from his beloved Colorado Rockies, draws the striking parallel between his experiences as a mountain climber and as a patient with metastatic melanoma facing the hope and uncertainty of experimental therapy. Both are life-threatening circumstances, demanding courage and hope, and challenging our soul in a way almost unique to human experience. Both involve a conscious choice to move forward into dangerous and uncertain territory, and require a determination to look death (John's “Reaper”) in the eye. Many remarkable books and films have been written about such experiences. I recall in particular the 2003 documentary film Touching the Void, about the incredible survival of a mountaineer who returned from a perilous fall in Peru. I highly recommend it to the reader. Another is Laura Hillenbrand's Unbroken: A World War II Story of Survival, Resilience, and Redemption (Random House, 2010), about the survival of a prisoner of war, the celebrated miler Louis Zamperini. Again, unbridled courage and undeniable hope turned futility into future.
John Murphy's reflections remind us of the daily heroism of our patients who are holding tight to the lifeline offered by clinical research. Good climbing, John. All of us are with you on that Knife Edge, waiting for our turn to ascend... and hoping to be as courageous as you were then on Capitol Peak and are again now on the Knife Edge of a clinical trial. For our turn will come.
doi:10.1634/theoncologist.2011-0021
PMCID: PMC3228094  PMID: 21349953
21.  Nasu-Hakola Syndrome: An Unusual Cause of Pathological Fractures 
Case Reports in Orthopedics  2012;2012:817189.
Nasu-Hakola syndrome is a hereditary cause of pathological fractures. Uniquely, patients also develop neuropsychiatric symptoms and signs. The disease is ultimately fatal. We propose a management strategy for pathological fractures in sufferers based on the stage of the disease.
doi:10.1155/2012/817189
PMCID: PMC3505912  PMID: 23227394
22.  Marine Reptiles 
PLoS ONE  2011;6(11):e27373.
doi:10.1371/journal.pone.0027373
PMCID: PMC3210815  PMID: 22087300
23.  Characterisation and Deposition Studies of Recrystallised Lactose from Binary Mixtures of Ethanol/Butanol for Improved Drug Delivery from Dry Powder Inhalers 
The AAPS Journal  2010;13(1):30-43.
Dry powder inhaler formulations comprising commercial lactose–drug blends can show restricted detachment of drug from lactose during aerosolisation, which can lead to poor fine particle fractions (FPFs) which are suboptimal. The aim of the present study was to investigate whether the crystallisation of lactose from different ethanol/butanol co-solvent mixtures could be employed as a method of altering the FPF of salbutamol sulphate from powder blends. Lactose particles were prepared by an anti-solvent recrystallisation process using various ratios of the two solvents. Crystallised lactose or commercial lactose was mixed with salbutamol sulphate and in vitro deposition studies were performed using a multistage liquid impinger. Solid-state characterisation results showed that commercial lactose was primarily composed of the α-anomer whilst the crystallised lactose samples comprised a α/β mixture containing a lower number of moles of water per mole of lactose compared to the commercial lactose. The crystallised lactose particles were also less elongated and more irregular in shape with rougher surfaces. Formulation blends containing crystallised lactose showed better aerosolisation performance and dose uniformity when compared to commercial lactose. The highest FPF of salbutamol sulphate (38.0 ± 2.5%) was obtained for the lactose samples that were crystallised from a mixture of ethanol/butanol (20:60) compared to a FPF of 19.7 ± 1.9% obtained for commercial lactose. Engineered lactose carriers with modified anomer content and physicochemical properties, when compared to the commercial grade, produced formulations which generated a high FPF.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-010-9241-x) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-010-9241-x
PMCID: PMC3032097  PMID: 21057906
deposition study; dry powder inhaler; lactose; particle engineering; salbutamol sulphate
24.  Phylogeography of the Mekong mud snake (Enhydris subtaeniata): the biogeographic importance of dynamic river drainages and fluctuating sea levels for semiaquatic taxa in Indochina 
Ecology and Evolution  2011;1(3):330-342.
During the Cenozoic, Southeast Asia was profoundly affected by plate tectonic events, dynamic river systems, fluctuating sea levels, shifting coastlines, and climatic variation, which have influenced the ecological and evolutionary trajectories of the Southeast Asian flora and fauna. We examined the role of these paleogeographic factors on shaping phylogeographic patterns focusing on a species of semiaquatic snake, Enhydris subtaeniata (Serpentes: Homalopsidae) using sequence data from three mitochondrial fragments (cytochrome b, ND4, and ATPase—2785 bp). We sampled E. subtaeniata from seven locations in three river drainage basins that encompassed most of this species’ range. Genetic diversities were typically low within locations but high across locations. Moreover, each location had a unique suite of haplotypes not shared among locations, and pairwise φST values (0.713–0.998) were highly significant between all location pairs. Relationships among phylogroups were well resolved and analysis of molecular variance (AMOVA) revealed strong geographical partitioning of genetic variance among the three river drainage basins surveyed. The genetic differences observed among the populations of E. subtaeniata were likely shaped by the Quaternary landscapes of Indochina and the Sunda Shelf. Historically, the middle and lower Mekong consisted of strongly dissected river valleys separated by low mountain ranges and much of the Sunda Shelf consisted of lowland river valleys that served to connect faunas associated with major regional rivers. It is thus likely that the contemporary genetic patterns observed among populations of E. subtaeniata are the result of their histories in a complex terrain that created abundant opportunities for genetic isolation and divergence yet also provided lowland connections across now drowned river valleys.
doi:10.1002/ece3.29
PMCID: PMC3287308  PMID: 22393504
Freshwater snake; Homalopsidae; Khorat basin; Mekong River; Pleistocene; Sea levels; Sundaland
25.  Short course pre-operative ferrous sulphate supplementation – is it worthwhile in patients with colorectal cancer? 
INTRODUCTION
Pre-operative anaemia is well recognised in patients presenting with colorectal cancer (CRC). While the benefits of long-term FeSO4 supplementation on Fe deficiency anaemia are well established, it is not known if short-course supplementation (2–3 weeks) impacts significantly on pre-operative haemoglobin (Hb) levels. This study examines the impact of short-term, oral FeSO4 supplementation on patients undergoing surgery for CRC.
PATIENTS AND METHODS
All patients with CRC presenting to a single surgeon were included. At diagnosis, baseline Hb and blood film were checked on all patients who then received 200 mg tds of FeSO4. Haemoglobin was rechecked pre-operatively and daily postoperatively. Patients requiring pre-operative blood transfusions were excluded from analysis.
RESULTS
Between 1 January 2004 and 31 December 2006, 117 patients were identified, 14 of whom were excluded. Patients received a median of 39 days’ treatment with FeSO4. Fifty-eight (56.3%) patients were anaemic at presentation gaining a mean of 1.73 g/dl (P < 0.001) from short-course FeSO4 supplementation. Right-sided tumours (lower mean Hb at presentation; P = 0.008) responded more to FeSO4 when compared to left-sided tumours (P < 0.017). Increase in Hb was unrelated to pathological stage. The transfusion rate for all curative resections was 0.69 units/patient. For the historical cohort (patients undergoing curative resection between 1 January 2001 and 31 December 2003), the mean transfusion rate fell from 1.69 units/patient.
CONCLUSIONS
Routine short-course supplementation with iron offers improved pre-operative Hb prior to surgery in CRC, especially in right-sided lesions and those with presenting anaemia.
doi:10.1308/003588410X12699663904277
PMCID: PMC3229346  PMID: 20573311
Colorectal cancer; FeSO4 supplementation; Anaemia; Haemoglobin

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