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Julia M Fraile,1,2 Soumya Palliyil,1 Caroline Barelle,2 Andrew J Porter,1,2 Marina Kovaleva2 1Scottish Biologics Facility, University of Aberdeen, Aberdeen, AB25 2ZP, UK; 2Elasmogen Ltd, Aberdeen, AB25 2ZP, UKCorrespondence: Julia M Fraile; Marina KovalevaElasmogen Ltd, Liberty Building, Foresterhill Road, Aberdeen, AB25 2ZP, UKTel +44 1224 438574Email julia.martinezfraile@elasmogen.com; marina.kovaleva@elasmogen.comAbstract: Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD), characterized by chronic inflammation and accumulation of fat in liver tissue. Affecting estimated 35 million people globally, NASH is the most common chronic liver condition in Western populations, and with patient numbers growing rapidly, the market for NASH therapy is projected to rise to $27.2 B in 2029. Despite this clinical need and attractive commercial opportunity, there are no Food and Drug Administration (FDA)-approved therapies specifically for this disease. Many have tried and unfortunately failed to find a drug, or drug combination, capable of unravelling the complexities of this metabolic condition. At the time of writing this review, only Zydus Cadila’s new drug application for Saroglitazar had been approved (2020) for NASH therapy in India. However, it is hoped that this dearth of therapy options will improve as several drug candidates progress through late-stage clinical development. Obeticholic acid (Intercept Pharmaceuticals), Cenicriviroc (Allergan), Aramchol (Galmed Pharmaceuticals), Resmetirom (Madrigal Pharmaceuticals), Dapagliflozin and Semaglutide (Novo Nordisk) are in advanced Phase 3 clinical trials, while Belapectin (Galectin Therapeutics), MSDC-0602K (Cirius Therapeutics), Lanifibranor (Inventiva), Efruxifermin (Akero) and Tesamorelin (Theratechnologies) are expected to start Phase 3 trials soon. Here, we have performed an exhaustive review of the current therapeutic landscape for this disease and compared, in some detail, the fortunes of different drug classes (biologics vs small molecules) and target molecules. Given the complex pathophysiology of NASH, the use of drug combination, different mechanisms of actions and the targeting of each stage of the disease will likely be required. Hence, the development of a single therapy for NASH seems challenging and unlikely, despite the plethora of later stage trials due to report. We therefore predict that clinical, patient and company interest in pipeline and next-generation therapies will remain high for some time to come.Keywords: NASH, NAFLD, liver fibrosis, clinical trial

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy., Competing Interests: Authors LF, MK, AP, and CB were employed by Elasmogen Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Smyth, Ferguson, Burrows, Burden, Tracey, Herron, Kovaleva, Williams, Porter, Longley, Barelle and Scott.)

Intracellular delivery of proteins, peptides and biologics is an emerging field which has the potential to provide novel opportunities to target intracellular proteins, previously deemed 'undruggable'. However, the delivery of proteins intracellularly remains a challenge. Here, we present a cationic nanoparticle delivery system for enhanced cellular delivery of proteins through use of a polyethyleneimine and poly-(lactic- co -glycolic acid) polymer blend. Cationic nanoparticles were shown to provide increased cellular uptake compared to anionic and neutral nanoparticles, successfully delivering Variable New Antigen Receptors (vNARs), entrapped within the nanoparticle core, to the cell interior. vNARs were identified as ideal candidates for nanoparticle entrapment due to their remarkable stability. The optimised 10% PEI-PLGA nanoparticle formulation displayed low toxicity, was uniform in size and possessed appropriate cationic charge to limit cellular toxicity, whilst being capable of escaping the endo/lysosomal system and delivering their cargo to the cytosol. This work demonstrates the ability of cationic nanoparticles to facilitate intracellular delivery of vNARs, novel biologic agents with potential utility towards intracellular targets., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Objectives: With the development of new technologies capable of detecting low concentrations of Alzheimer's disease (AD) relevant biomarkers, the idea of a blood-based diagnosis of AD is nearing reality. This study aims to consider the evidence of total and phosphorylated tau as blood-based biomarkers for mild cognitive impairment (MCI) and AD when compared to healthy controls., Methods: Studies published between 1 January 2012 and 1 May 2021 (Embase and MEDLINE databases) measuring plasma/serum levels of tau in AD, MCI, and control cohorts were screened for eligibility, including quality and bias assessment via a modified QUADAS. The meta-analyses comprised 48 studies assessing total tau (t-tau), tau phosphorylated at threonine 181 (p-tau181), and tau phosphorylated at threonine 217 (p-tau217), comparing the ratio of biomarker concentrations in MCI, AD, and cognitively unimpaired (CU) controls., Results: Plasma/serum p-tau181 (mean effect size, 95% CI, 2.02 (1.76-2.27)) and t-tau (mean effect size, 95% CI, 1.77 (1.49-2.04)) were elevated in AD study participants compared to controls. Plasma/serum p-tau181 (mean effect size, 95% CI, 1.34 (1.20-1.49)) and t-tau (mean effect size, 95% CI, 1.47 (1.26-1.67)) were also elevated with moderate effect size in MCI study participants compared to controls. p-tau217 was also assessed, albeit in a small number of eligible studies, for AD vs. CU (mean effect size, 95% CI, 1.89 (1.86-1.92)) and for MCI vs. CU groups (mean effect size, 95% CI, 4.16 (3.61-4.71))., Conclusions: This paper highlights the growing evidence that blood-based tau biomarkers have early diagnostic utility for Alzheimer's disease., Registration: PROSPERO No. CRD42020209482.

Biological communities in freshwater streams are often impaired by multiple stressors (e.g., flow or water quality) originating from anthropogenic activities such as urbanization, agriculture, or energy extraction. Restoration efforts in the Chesapeake Bay watershed, USA seek to improve biological conditions in 10% of freshwater tributaries and to protect the biological integrity of existing healthy watersheds. To achieve these goals, resource managers need to better understand which stressors are most likely driving biological impairment. Our study addressed this knowledge gap through two approaches: 1) reviewing and synthesizing published multi-stressor studies, and 2) examining 303(d) listed impairments linked to biological impairment as identified by jurisdiction regulatory agencies (the states within the watershed and the District of Columbia). Results identified geomorphology (i.e., physical habitat), salinity, and toxic contaminants as important for explaining variability in benthic community metrics in the literature review. Geomorphology (i.e., physical habitat and sediment), salinity, and nutrients were the most reported stressors in the jurisdictional impairment analysis. Salinity is likely a major stressor in urban and mining settings, whereas geomorphology was commonly reported in agricultural settings. Toxic contaminants, such as pesticides, were rarely measured; more research is needed to quantify the extent of their effects in the region. Flow alteration was also highlighted as an important urban stressor in the literature review but was rarely measured in the literature or reported by jurisdictions as a cause of impairment. These results can be used to prioritize stressor monitoring by managers, and to improve stressor identification methods for identifying causes of biological impairment., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Mini-Abstract: In this prospective observational cohort of patients with a history of diverticulitis, we assessed the correlation between the diverticulitis quality of life survey (DVQOL) and other patient-reported expressions of disease measures including work and activity impairment, and contentment with gastrointestinal-related health. Then, we assessed whether the DVQOL is better correlated with these measures than diverticulitis episode count. Our study results showed that the DVQOL has a stronger correlation with other disease measures than diverticulitis episode count, and our findings support the broader use of the DVQOL in assessing the burden of diverticulitis and monitoring response to management., Competing Interests: Disclosure: The authors declare that they have nothing to disclose., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Monoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti- Candida mAb reported to date.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape., (© 2021. The Author(s).)

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the "pairing potential" of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the "inside" or "outside" of the infected cell. Their therapeutic potential will be discussed.

This paper assessed the blood pressure, heart rate, and mouth-pressure responses to indoor rock climbing (bouldering) and associated training exercises. Six well-trained male rock climbers (mean ± SD age, 27.7 ± 4.7 yr; stature, 177.7 ± 7.3 cm; mass, 69.8 ± 12.1 kg) completed two boulder problems (6b and 7a+ on the Fontainebleau Scale) and three typical training exercises [maximum voluntary contraction (MVC) isometric pull-up, 80% MVC pull-ups to fatigue, and campus board to fatigue]. Blood pressure and heart rate were measured via an indwelling femoral arterial catheter, and mouth pressure via a mouthpiece manometer. Bouldering evoked a peak systolic pressure of 200 ± 17 mmHg (44 ± 21% increase from baseline), diastolic pressure of 142 ± 26 mmHg (70 ± 32% increase), mean arterial pressure of 163 ± 18 mmHg (56 ± 25% increase), and heart rate of 176 ± 22 beats/min (76 ± 35% increase). The highest systolic pressure was observed during the campus board exercise (218 ± 33 mmHg), although individual values as high as 273/189 mmHg were recorded. Peak mouth pressure during climbing was 31 ± 46 mmHg, and this increased independently of climb difficulty. We concluded that indoor rock climbing and associated exercises evoke a substantial pressor response resulting in high blood pressures that may exceed those observed during other upper-limb resistance exercises. These findings may inform risk stratification for climbers. NEW & NOTEWORTHY This case study provides original data on the exercise pressor response to indoor rock climbing and associated training exercises through the use of an indwelling femoral arterial catheter. Our subjects exhibited systolic/diastolic blood pressures that exceeded values often reported during upper-limb resistance exercise. Our data extend the understanding of the cardiovascular stress associated with indoor rock climbing.

Anti-drug antibodies (ADAs), specific for biotherapeutic drugs, are associated with reduced serum drug levels and compromised therapeutic response. The impact of ADA on the bioavailability and clinical efficacy of blockbuster anti-hTNF- α monoclonal antibodies is well recognised, especially for adalimumab and infliximab treatments, with the large and complex molecular architecture of classical immunoglobulin antibody drugs, in part, responsible for the immunogenicity seen in patients. The initial aim of this study was to develop solid-phase enzyme-linked immunosorbent assays (ELISA) and an in vitro cell-based method to accurately detect ADA and estimate its impact on the preclinical in vivo efficacy outcomes of two novel, nonimmunoglobulin VNAR fusion anti-hTNF- α biologics (Quad-X™ and D1-NDure™-C4) and Humira®, a brand of adalimumab. Serum drug levels and the presence of ADA were determined in a transgenic mouse model of polyarthritis (Tg197) when Quad-X™ and Humira® were dosed at 1 mg/kg and D1-NDure™-C4 was dosed at 30 mg/kg. The serum levels of the Quad-X™ and D1-NDure™-C4 modalities were consistently high and comparable across all mice within the same treatment groups. In 1 mg/kg and 3 mg/kg Quad-X™- and 30 mg/kg D1-NDure™-C4-treated mice, an average trough drug serum concentration of 8  μ g/mL, 50  μ g/mL, and 350  μ g/mL, respectively, were estimated. In stark contrast, Humira® trough serum concentrations in the 1 mg/kg treatment group ranged from <0.008  μ g/mL to 4  μ g/mL with trace levels detected in 7 of the 8 animals treated. Trough serum Humira® and Quad-X™ concentrations in 3 mg/kg treatment samples were comparable; however, the functionality of the detected Humira® serum was significantly compromised due to neutralising ADA. The impact of ADA went beyond the simple and rapid clearance of Humira®, as 7/8 serum samples also showed no detectable capacity to neutralise hTNF- α -mediated cytotoxicity in a murine fibrosarcoma (L929) cell assay. The neutralisation capacity of all the VNAR constructs remained unchanged at the end of the experimental period (10 weeks). The data presented in this manuscript goes some way to explain the exciting outcomes of the previously published preclinical in vivo efficacy data, which showed complete control of disease at Quad-X™ concentrations of 0.5 mg/kg, equivalent to 10x the in vivo potency of Humira®. This independent corroboration also validates the robustness and reliability of the assay techniques reported in this current manuscript, and while it comes with the caveat of a mouse study, it does appear to suggest that these particular VNAR constructs, at least, are of low inherent immunogenicity., Competing Interests: Authors' affiliations are clearly stated including those affiliated with the biotechnology company, Elasmogen Ltd., (Copyright © 2020 Obinna C. Ubah et al.)

Importance: Microinvasive glaucoma surgery (MIGS) combined with phacoemulsification is increasingly utilised in glaucoma management., Objective: To describe the postoperative outcomes of phacoemulsification alone compared to combined phacoemulsification and iStent (Glaukos, San Clemente, CA) or Hydrus (Ivantis, Irvine, CA) for open angle glaucoma., Methods: Retrospective single surgeon comparative case series in a private practice setting. A total of 297 eyes in 190 patients (M:F = 85:105) were included consecutively from March 2011 to June 2017 with the data analysed using linear mixed models. Main outcome measures were intraocular pressure (IOP) and number of medications., Results: By 6 months, all groups showed a reduction in IOP, with the combined iStent group by 4.2 mm Hg and the combined Hydrus group by 4.5 mm Hg, and this trend was sustained with follow-up to two years. All groups also showed a reduction in number of medications, with the phacoemulsification alone cases by 0.3, the combined iStent group by 0.7 and the combined Hydrus group by 1.1 medications. Both the combined surgeries had sustained reduction of number of medications with follow-up to 2 years. Postoperative complications from MIGS device insertion were uncommon and all resolved by one month., Conclusions and Relevance: The combination of phacoemulsification and a MIGS device in open angle glaucoma patients reduced the intraocular pressure and the number of medications by the first postoperative month and had longer-term effects at 2 years follow-up. If the device is inserted without intraoperative complication, the rate of significant short-term risks are low.

Isotope effects depend upon the polarity of the bulk medium in which a chemical process occurs. Implicit solvent calculations with molecule-shaped cavities show that the equilibrium isotope effect (EIE) for heterolysis of the glycosidic bonds in 5'-methylthioadenosine and in 2-( p -nitrophenoxy)tetrahydropyran, both in water, are very sensitive in the range 2 ≤ ε ≤ 10 to the relative permittivity of the continuum surrounding the oxacarbenium ion. However, different implementations of nominally the same PCM method can lead to opposite trends being predicted for the same molecule. Computational modeling of the influence of the inhomogeneous effective dielectric surrounding a substrate within the protein environment of an enzymic reaction requires an explicit treatment. The EIE ( K H / K D ) for transfer of cyclopentyl, cyclohexyl, tetrahydrofuranyl and tetrahydropyranyl cations from water to cyclohexane is predicted by B3LYP/6-31+G(d) calculations with implicit solvation and confirmed by B3LYP/6-31+G(d)/OPLS-AA calculations with averaging over many explicit solvation configurations. Atomic Hessian analysis, whereby the full Hessian is reduced to the elements belonging to a single atom at the site of isotopic substitution, reveals a remarkable result for both implicit and explicit solvation: the influence of the solvent environment on these EIEs is essentially captured completely by only a 3 × 3 block of the Hessian, although these values must correctly reflect the influence of the whole environment. QM/MM simulation with ensemble averaging has an important role to play in assisting the meaningful interpretation of observed isotope effects for chemical reactions both in solution and catalyzed by enzymes.

VNAR domains are the binding regions of new antigen receptor proteins (IgNAR) which are unique to sharks, skates, and rays (Elasmobranchii). Individual VNAR domains can bind antigens independently and are the smallest reported adaptive immune recognition entities in the vertebrate kingdom. Sharing limited sequence homology with human immunoglobulin domains, their development and use as biotherapeutic agents require that they be humanized to minimize their potential immunogenicity. Efforts to humanize a human serum albumin (HSA)-specific VNAR, E06, resulted in protein molecules that initially had undesirable biophysical properties or reduced affinity for cognate antigen. Two lead humanized anti-HSA clones, v1.10 and v2.4, were subjected to a process of random mutagenesis using error-prone PCR. The mutated sequences for each humanized VNAR variant were screened for improvements in affinity for HSA and biophysical properties, achieved without a predicted increase in overall immunogenicity.

The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding topographies, making it an ideal candidate for accessing cryptic epitopes otherwise intractable to conventional antibodies. These attributes, coupled with high physicochemical stability and amenability to phage display, facilitate the selection of VNAR binders to challenging targets. Here, we explored the unique attributes of these single domains for potential application as bioprocessing reagents in the development of the SEED-Fc platform, designed to generate therapeutic bispecific antibodies. A panel of unique VNARs specific to the SEED homodimeric (monospecific) 'by-products' were isolated from a shark semi-synthetic VNAR library via phage display. The lead VNAR candidate exhibited low nanomolar affinity and superior selectivity to SEED homodimer, with functionality being retained upon exposure to extreme physicochemical conditions that mimic their applicability as purification agents. Ultimately, this work exemplifies the robustness of the semi-synthetic VNAR platform, the predisposition of the VNAR paratope to recognise novel epitopes and the potential for routine generation of tailor-made VNAR-based bioprocessing reagents., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tumor necrosis factor-alpha (TNF-α), an established pro-inflammatory cytokine plays a central role in the induction and progression of several chronic inflammatory and autoimmune diseases. Targeting TNF-α as a treatment modality has shown tremendous success, however there are several limitations associated with the current anti-TNF-α biologic drugs including: immunogenicity, life-threatening infections, resistance to treatment, complexity of manufacture and cost of treatment. Here, we report the in vivo efficacy of novel anti-TNF-α formats generated from molecular engineering of variable new antigen receptors (VNARs), originally derived from the immune system of an immunized nurse shark. Two anti-TNF-α VNAR formats, a tandem multivalent trimer, D1-BA11-C4 and an Fc-fused quadrivalent D1-Fc-C4 (Quad-X™) construct were tested in a clinically relevant, preclinical mouse efficacy model of polyarthritis (Tg197) and compared to the commercial anti-TNF-α "best in class" therapy, Adalimumab (Humira®). Both VNAR formats bind and neutralize TNF-α through an epitope that appears to be different from those recognized by other anti-TNF biologics used clinically. All doses of Quad-X™, from 0.5 to 30 mg/kg, significantly blocked the development of polyarthritis. At 0.5 mg/kg Quad-X™, the arthritis score was improved by 76% and the histopathology score by 63%. At 3 mg/kg Quad-X™, control of disease was almost complete at 90% (arthritis) and 88% (histopathology). In marked contrast, 1 mg/kg Humira® saw profound disease breakthrough with scores of 39 and 16% respectively, increasing to a respectable 82 and 86% inhibition at 10 mg/kg Humira®. We have previously reported the superior potency of anti-TNF-α VNARs in vitro and in these studies translate this superiority into an in vivo setting and demonstrate the potential of VNAR formats to meet the requirements of next-generation anti-TNF-α therapies.

Background: How shoulder injuries treated before the Major League Baseball (MLB) draft affect the player's performance over their career is unclear. The purpose of this study was to determine whether prior shoulder injuries were associated with a difference in the level of performance and advancement of MLB draftees., Methods: Before entering the draft, 119 professional baseball players from 2004 to 2010 were treated for a shoulder injury (73% treated surgically) as an amateur. A 3:1 matched case-control was performed to players without prior shoulder injuries by age, position, round selected, and signing bonus. Follow-up data were collected in 2016, and professional advancement, disabled list time, and in-game performance statistics for pitchers were analyzed and compared., Results: Players with a prior shoulder injury had a statistically higher chance to be assigned to the disabled list then controls (P = .03), but there was no difference in disabled list time or professional advancement. Pitchers with a prior shoulder injury pitched a statistically lower number of innings per game than controls (P = .04). All other in-game performance statistics were not statistically different. The type of treatment did not have any effect on future performance or advancement., Conclusions: Professional baseball players treated for prior shoulder injuries at the amateur level were more likely to sustain future disability than their matched controls, but it did not affect professional advancement., (Copyright © 2018 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Introduction: The aim of this study was to determine the relationship between the way breast cancer is diagnosed and its prognostic features., Methods: We studied new primary invasive and non-invasive breast cancers (670) diagnosed between 2013 and 2015 where detection included at least clinical examination, mammography and breast ultrasound (BUS). The cancers were classified into six Diagnostic Groups according to the results of each modality., Results: Overall, 79% of cancers were positive on mammography, but another 20% were diagnosed on BUS after a negative mammogram. The largest group (37.6% of cases), had all three modalities positive (Group 1). BUS was the only modality positive in 14.6% (Group 4). Mammography alone was positive in 21.2%, of which 73.9% were ductal carcinoma in situ (DCIS). Invasive lobular carcinoma comprised 9.6% of the groups where mammography was positive, but 16.5% of the groups where mammography was negative and BUS positive. Dense breast tissue was more common in the groups where mammography was negative and BUS positive. Invasive cancers comprised 82.7% of Group 4 and 95.2% of Group 1. For those in Group 4, the average size (10.2 mm) and the percentages with lymphovascular invasion (11.1%), lymph node involvement (17.3%) and poor differentiation (12.3%) were less than half the corresponding figures for Group 1 (27.3 mm, 35%, 44%, 44%)., Conclusion: This study illustrates the complementary benefits of mammography and BUS, especially where breast density is high. Tumours diagnosed by BUS alone had better prognostic features in terms of size, lymphovascular invasion, lymph node status, differentiation and hormone receptors, compared with cancers where all three modalities were positive., (© 2018 The Royal Australian and New Zealand College of Radiologists.)

Therapeutic mAbs have delivered several blockbuster drugs in oncology and autoimmune inflammatory disease. Revenue for mAbs continues to rise, even in the face of competition from a growing portfolio of biosimilars. Despite this success, there are still limitations associated with the use of mAbs as therapeutic molecules. With a molecular mass of 150 kDa, a two-chain structure and complex glycosylation these challenges include a high cost of goods, limited delivery options, and poor solid tumour penetration. There remains an urgency to create alternatives to antibody scaffolds in a bid to circumvent these limitations, while maintaining or improving the therapeutic success of conventional mAb formats. Smaller, less complex binders, with increased domain valency, multi-specific/paratopic targeting, tuneable serum half-life and low inherent immunogenicity are a few of the characteristics being explored by the next generation of biologic molecules. One novel 'antibody-like' binder that has naturally evolved over 450 million years is the variable new antigen receptor (VNAR) identified as a key component of the adaptive immune system of sharks. At only 11 kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery. VNAR attributes include high affinity for target, ease of expression, stability, solubility, multi-specificity, and increased potential for solid tissue penetration. This review article documents the recent drug developmental milestones achieved for therapeutic VNARs and highlights the first reported evidence of the efficacy of these domains in clinically relevant models of disease., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Infectious crystalline keratopathy was first reported by Gorovoy and colleagues in 1983 when they identified bacteria colonizing a cornea after a penetrating keratoplasty. Subsequent cases have elaborated on the organisms responsible and the management outcomes. Patients present with a white or gray branching opacity originating from an epithelial defect, commonly after a penetrating keratoplasty. Local immunosuppression contributes to the quiescent nature and the limited inflammatory response associated with infectious crystalline keratopathy. Diagnosis of the infective pathogens may be difficult, with a corneal scraping often being too superficial to obtain an adequate specimen. A biofilm is present that advantages microorganism survival, reduces antibiotic bioavailability, and inhibits diagnostic microbial detection. Treatment begins with topical antimicrobials, initially broad spectrum and then targeted to microorganism sensitivity. Adjunctive therapies to enhance the efficacy of treatment include disruption of the microorganism biofilm by laser, intrastromal antibiotics, and keratectomy. In recalcitrant cases, or where corneal scarring ensues, corneal transplantation is required., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A 35-year-old man presented after a high-speed motor vehicle accident and the driver's side curtain air bag causing blunt force trauma rupturing his right globe. The tangential force of the air bag resulted in an unusual arcuate horseshoe-shaped rupture through the lateral rectus insertion, causing avulsion of the muscle and tearing the sclera, with the apices of the tear extending anteriorly towards the limbus. Repair of the globe rupture was undertaken, and secondary vitrectomy revealed that the scleral tear had not involved the retina abutting the ora serrata. Silicone oil tamponade was used to fill the globe and the postoperative best corrected vision was 6/9. This is the first reported case of a ruptured globe caused by a side curtain air bag, resulting in a uniquely shaped arcuate scleral wound combined with lateral rectus avulsion, not associated with rhegmatogenous retinal damage, and is the first air bag-related globe rupture with scleral involvement to report a final best-corrected visual acuity better than 6/60., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

Introduction: Modern full-field digital mammography (FFDM) appears to have increased the detection of breast microcalcification. This retrospective study aimed to assess whether this is associated with over-investigation and possible over-diagnosis of breast pathology of lesser significance., Methods: Three 2-year periods were studied, in which different mammographic technologies were used exclusively viz., classical screen-film mammography (SFM) in 2003-04, computed radiography (CR) in 2009-10 and FFDM in 2012-13. The study was limited to women in whom biopsy was done for indeterminate, suspicious or malignant (ISM) calcification as the only mammographic abnormality., Results: Between the first and the third time periods, the use of core biopsies and vacuum-assisted biopsies (VABs) for 'ISM calcification only' increased from 0. 6% to 1.4% of all mammograms, and biopsies with malignant results increased from 0.18% to 0.33% of all mammograms. VAB became the preferred technique for biopsy over the study period. The proportion of cores and VABs with a malignant result did not change significantly over the three time periods (24-28%), nor did the proportion of invasive cancers, ductal carcinoma in situ (DCIS) (60-63%) or atypia on subsequent excision. The proportion of DCIS which was of high grade was greatest in the FFDM era (69%). Less than 9% of DCIS were of low grade in all three time periods., Conclusions: Despite increased numbers of biopsies being performed, there was no increase in findings of breast pathology of lesser significance. Improved technology (FFDM and VAB) allows immediate biopsy of small clusters of indeterminate microcalcification, rather than using mammographic surveillance, and this study found no evidence that this has been associated with over-investigation or over-diagnosis., (© 2017 The Royal Australian and New Zealand College of Radiologists.)

A 68-year-old woman presented with infectious crystalline keratopathy 4 months after she underwent a combined phacoemulsification and Descemet's stripping endothelial keratoplasty for Fuch's endothelial dystrophy in her left eye. After 5 months of topical moxifloxacin 1%, the infiltrate responded well but had not completely resolved, with the resulting endothelial failure requiring a penetrating keratoplasty 9 months after the initial operation. Microbiology identified Enterococcus faecalis with the histopathology demonstrating bacterial colonies within the graft interface. Postoperatively she developed endophthalmitis, needing vitrectomy and intravitreal antibiotics. The infection settled with no recurrence, with topical and oral antibiotics continued for 2 months. A sutured toric piggyback intraocular lens was performed 18 months postvitrectomy for graft astigmatism, achieving a best-corrected vision of 6/15.Infectious crystalline keratopathy can occur following Descemet's stripping endothelial keratoplasty, requiring long-term topical therapy and potentially leading to graft failure with the necessity for further keratoplasty., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The underlying cause(s) of abnormalities expressed by patients with attention deficit hyperactivity disorder (ADHD) have yet to be delineated. One factor that has been associated with increased vulnerability to ADHD is polymorphism(s) of TACR1, which is the human equivalent of the rodent NK1 (substance P-preferring) receptor gene (Nk1r). We have reported previously that genetically altered mice, lacking functional NK1R (NK1R-/-), express locomotor hyperactivity, which was blunted by the first-line treatment for ADHD, methylphenidate. Here, we compared the effects of this psychostimulant (3, 10 and 30 mg/kg, intraperitoneally) on the behaviour of NK1R-/- mice and their wild types in the 5-Choice Continuous Performance Test, which emulates procedures used to study attention and response control in ADHD patients. Methylphenidate increased total trials (a measure of 'productivity') completed by wild types, but not by NK1R-/- mice. Conversely, this drug reduced perseveration by NK1R-/- mice, but not by wild types. Other drug-induced changes in key behaviours were not genotype dependent, especially at the highest dose: for example, % omissions (an index of inattentiveness) was increased, whereas % false alarms and % premature responses (measures of impulsivity) declined in both genotypes, indicating reduced overall response. These findings are discussed in the context of the efficacy of methylphenidate in the treatment of ADHD. Moreover, they lead to several testable proposals. First, methylphenidate does not improve attention in a subgroup of ADHD patients with a functional deficit of TACR1. Second, these patients do not express excessive false alarms when compared with other groups of subjects, but they do express excessive perseveration, which would be ameliorated by methylphenidate., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. (SCS is a named inventor on an EU patent for the NK1R–/– mouse model of ADHD (awarded to UCL Business), but declined the option to receive royalties)., (© The Author(s) 2016.)

Mice lacking functional NK1 (substance P-preferring) receptors typically display excessive inattentiveness (omission errors) and impulsivity (premature responses) when compared with wildtypes in the 5-Choice Serial Reaction-Time Test (5-CSRTT). These abnormal behaviours are analogous to those seen in humans suffering from Attention Deficit Hyperactivity Disorder (ADHD). Here we used the 5-Choice Continuous‑Performance Test (5C-CPT) to ascertain whether NK1R-/- mice also display excessive false alarms (an inappropriate response to a 'no-go' signal), which is another form of impulsive behaviour. NK1R-/- mice completed more trials than wildtypes, confirming their ability to learn and carry out the task. At the start of Stage 1 of training, but not subsequently, they also scored more premature responses than wildtypes. When the mice were tested for the first time, neither false alarms nor premature responses was higher in NK1R-/- mice than wildtypes but, as in the 5-CSRTT, the latter behaviour was strongly dependent on time of day. NK1R-/- mice expressed excessive perseveration during all stages of the 5C-CPT. This behaviour is thought to reflect compulsive checking, which is common in ADHD patients. These findings point to differences in the 5-CSRTT and 5C-CPT protocols that could be important for distinguishing why the cognitive performance and response control of NK1R-/- mice differs from their wildtypes. The results further lead to the prediction that ADHD patients with polymorphism of the TACR1 gene (the human equivalent of Nk1r) would express more perseveration, but not false alarms, in Continuous Performance Tests when compared with other groups of subjects., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Compassion is considered an important quality for a successful physician today, but did ancient physicians display and value this emotion? How did they feel when faced with the pain and suffering of their patients? How did their patients' emotions affect their own? Many ancient physicians are not well-known for expressions of compassion in their writings; however, this seems to change in the second century AD. One medical writer who exemplifies this change is Soranus of Ephesus (c. 98-138 AD). In his Gynecology, there are a number of passages where compassion is addressed or expressed (such as the chapters on the qualities of the best midwife, the symptom of pica, childbirth, and superstition). The same points can be made of Soranus' On Chronic Diseases, preserved to some extent by the Latin version and adaptation by fifth century AD medical writer Caelius Aurelianus (see, for example, the chapters on chronic headache, mania and elephantiasis). Soranus and Caelius display compassion, understanding, and flexibility of approach when dealing with patient issues; they show themselves willing to change their medical technique when they see that it is doing more harm or discomfort than good. In Soranus and Caelius, we have an image of a physician who acknowledges and is aware of their patients' emotions, beliefs and attitudes, and who exhibits compassion for them.

Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Low molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes., Methods: Different hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential., Results: The simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL., Conclusions: The best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.

Mice lacking functional neurokinin-1 receptors (NK1R-/-) display behavioural abnormalities resembling attention deficit hyperactivity disorder (ADHD): locomotor hyperactivity, impulsivity and inattentiveness. The preferred ligand for NK1R, substance P, is metabolised by angiotensin converting enzyme (ACE), which forms part of the brain renin angiotensin system (BRAS). In view of evidence that the BRAS modulates locomotor activity and cognitive performance, we tested the effects of drugs that target the BRAS on these behaviours in NK1R-/- and wildtype mice. We first tested the effects of the ACE inhibitor, captopril, on locomotor activity. Because there are well-established sex differences in both ADHD and ACE activity, we compared the effects of captopril in both male and female mice. Locomotor hyperactivity was evident in male NK1R-/- mice, only, and this was abolished by treatment with captopril. By contrast, male wildtypes and females of both genotypes were unaffected by ACE inhibition. We then investigated the effects of angiotensin AT1 (losartan) and AT2 (PD 123319) receptor antagonists on the locomotor activity of male NK1R-/- and wildtype mice. Both antagonists increased the locomotor activity of NK1R-/- mice, but neither affected the wildtypes. Finally, we tested the effects of captopril on the performance of male NK1R-/- and wildtype mice in the 5-choice serial reaction-time task (5-CSRTT) and found that ACE inhibition prevented the impulsivity of NK1R-/- mice. These results indicate that certain behaviours, disrupted in ADHD, are influenced by an interaction between the BRAS and NK1R, and suggest that ACE inhibitors could provide a novel treatment for this disorder., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: 'Hom') wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents ('Het', derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors., (© 2015 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Background: Mice with functional ablation of the neurokinin-1 receptor gene (NK1R(-/-)) display behavioural abnormalities which resemble the hyperactivity, inattention and impulsivity seen in Attention Deficit Hyperactivity Disorder (ADHD). Here, we investigated whether the established ADHD treatment, atomoxetine, alleviates these abnormalities when tested in the light/dark exploration box (LDEB) and 5-Choice Serial Reaction-Time Task (5-CSRTT)., Methods: Separate cohorts of mice were tested in the 5-CSRTT and LDEB after treatment with no injection, vehicle or atomoxetine (5-CSRTT: 0.3, 3 or 10mg/kg; LDEB: 1, 3 or 10mg/kg)., Results: Atomoxetine reduced the hyperactivity displayed by NK1R(-/-) mice in the LDEB at a dose (3mg/kg) which did not affect the locomotor activity of wildtypes. Atomoxetine (10mg/kg) also reduced impulsivity in NK1R(-/-) mice, but not wildtypes, in the 5-CSRTT. No dose of drug affected attention in either genotype., Conclusions: This evidence that atomoxetine reduces hyperactive/impulsive behaviours in NK1R(-/-) mice consolidates the validity of using NK1R(-/-) mice in research of the aetiology and treatment of ADHD., (Copyright © 2014. Published by Elsevier Inc.)

Background and Purpose: Mice with functional ablation of substance P-preferring neurokinin-1 receptors (NK1R-/- mice) display behavioural abnormalities resembling those in attention deficit hyperactivity disorder (ADHD). Here, we investigated whether the ADHD treatment, guanfacine, alleviated the hyperactivity and impulsivity/inattention displayed by NK1R-/- mice in the light/dark exploration box (LDEB) and 5-choice serial reaction-time task (5-CSRTT), respectively. Following reports of co-morbid anxiety in ADHD, we also investigated effects of guanfacine on anxiety-like behaviour displayed by NK1R-/- and wild-type (WT) mice in the elevated plus maze (EPM)., Experimental Approach: Mice were treated with guanfacine (0.1, 0.3 or 1.0 mg·kg(-1), i.p.), vehicle or no injection and tested in the 5-CSRTT or the LDEB. Only the lowest dose of guanfacine was used in the EPM assays., Key Results: In the 5-CSRTT, a low dose of guanfacine (0.1 mg·kg(-1)) increased attention in NK1R-/- mice, but not in WT mice. This dose did not affect the total number of trials completed, latencies to respond or locomotor activity in the LDEB. Impulsivity was decreased by the high dose (1.0 mg·kg(-1)) of guanfacine, but this was evident in both genotypes and is likely to be secondary to a generalized blunting of behaviour. Although the NK1R-/- mice displayed marked anxiety-like behaviour, guanfacine did not affect the behaviour of either genotype in the EPM., Conclusions and Implications: This evidence that guanfacine improves attention at a dose that did not affect arousal or emotionality supports our proposal that NK1R-/- mice express an attention deficit resembling that of ADHD patients., (© 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

The cyanotoxin, microcystin (MC), is known to accumulate in the tissues of diverse aquatic biota although factors influencing exposure, such as feeding habits and seasonal patterns in toxin production, are poorly known. We analyzed seasonal variation in the MC content of primary and secondary consumers, and used dietary analysis (gut contents and stable isotopes) to improve understanding of cyanotoxin transport in food webs. Periods of elevated toxin concentration were associated with peaks in the abundance of genes specific to Microcystis and MC toxin production (mcyD). Peak toxin levels in consumer tissues coincided with peak MC concentrations in seston. However, toxins in tissues persisted in overwintering populations suggesting that potential health impacts may not be limited to bloom periods. Interspecific differences in tissue MC concentrations were related to feeding habits and organic matter sources as pelagic fishes ingested a greater proportion of algae in their diet, which resulted in greater MC content in liver and muscle tissues. Sediments contained a greater proportion of allochthonous (terrestrial) organic matter and lower concentrations of MC, resulting in lower toxin concentrations among benthic detritivores. Among shellfish, the benthic suspension feeder Rangia cuneata (wedge clam) showed seasonal avoidance of toxin ingestion due to low feeding rates during periods of elevated MC. Among predators, adult Blue Catfish had low MC concentrations, whereas Blue Crabs exhibited high levels of MC in both muscle and viscera.

Background: The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs., Methods: A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser., Results: Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454., Conclusions: In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.

Introduction: Invasive lobular cancer (ILC) is an important contributor to false negative mammography. This study aims to assess the value of digital mammography and to identify imaging features that could assist the radiologist to suggest the diagnosis of ILC prior to biopsy., Methods: Three hundred sixty-one cases of pure ILC diagnosed at the Wesley Breast Clinic during the period 1995-2010 were reviewed by one of the authors (AP). Radiological features were categorized, and clinical features and needle sampling results were recorded., Results: Mammography was negative in 29.9% of ILCs. The commonest positive finding was a localized spiculated mass (41.8%). Thirty-four point nine per cent of lesions were visible in only one view, usually cranio-caudal. Calcification was not a feature of ILC. The use of digital mammography in 30% of cases did not decrease the false negative rate for ILC. Breast ultrasound (BUS) showed an abnormality in 97.8%, most commonly a localized irregular hypoechoic mass with shadowing., Conclusions: Digital mammography does not reduce false negative mammography in ILC. The poor visibility of ILCs may be partly related to their low density (mass/unit volume). ILCs may sometimes be poor attenuators of X-rays but excellent attenuators of ultrasound, causing marked acoustic shadowing. Bilateral whole BUS has a very low false negative rate in experienced hands and is mandatory in symptomatic women. The combination of poor visibility on mammography with high visibility on ultrasound, as well as certain characteristic ultrasound appearances of ILC, may enable the radiologist to suggest ILC as a diagnostic possibility, prior to biopsy., (© 2013 The Royal Australian and New Zealand College of Radiologists.)

A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

Background: Adoption ofa preprocedural pause (PPP) associated with a checklist and a team briefing has been shown to improve teamwork function in operating rooms (ORs) and has resulted in improved outcomes. The format of the World Health Organization Safe Surgery Saves Lives checklist has been used as a template for a PPP. Performing a PPP, described as a "time-out," is one of the three principal components, along with a preprocedure verification process and marking the procedure site, of the Joint Commission's Universal Protocol for Preventing Wrong Site, Wrong Procedure, Wrong Person Surgery. However, if the surgeon alone leads the pause, its effectiveness may be decreased by lack of input from other operating team members., Methods: In this study, the PPP was assessed to measure participation and input from operating team members. On the basis of low participation levels, the pause was modified to include an attestation from each member of the team., Results: Preliminary analysis of our surgeon-led pause revealed only 54% completion of all items, which increased to 97% after the intervention. With the new format, operating team members stopped for the pause in 96% of cases, compared with 78% before the change. Operating team members introduced themselves in 94% of cases, compared with 44% before the change. Follow-up analysis showed sustained performance at 18 months after implementation., Conclusions: A preprocedural checklist format in which each member of the operating team provides a personal attestation can improve pause compliance and may contribute to improvements in the culture of teamwork within an OR. Successful online implementation of a PPP, which includes participation by all operating team members, requires little or no additional expense and only minimal formal coaching outside working situations.