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Background: There is still a dearth of information about grammatical aspects of language production in aphasia. Aims: Making novel use of methods of elicited production aimed at testing the limits of competence, we studied three cases of chronic aphasia, stemming from major stroke. We asked: (1) Whether the elicited production method reveals sparing of language abilities not readily evidenced in spontaneous utterances or on conventional aphasia tests. (2) Which language production abilities survive damage to both Broca's region and Wernicke's region? Materials & Procedures: Targeted words, morphological and syntactic structures were elicited by sentence completion with supporting linguistic and visual context. Targets were never modelled during the procedure. For verbs, visual and auditory contexts emphasise completed actions, targeting past tense forms. Lesion description was based on structural MRI scans. Outcomes & Results: The three participants showed partially spared ability to produce nouns, adjectives, and verb stems in context. The elicitation method proved more productive in some cases than picture prompts or sentence prompts. Past tense inflections were usually omitted. Hence stems and inflections were dissociable. Two participants showed partial success with the passive, and no participant produced a full relative clause, including the relative pronoun, but two produced reduced forms of subject relatives. Partial sparing of production capability in these cases points to the likely importance of portions of the left hemisphere remote from Broca and Wernicke regions. Conclusions: This application of elicited production methodology demonstrates possibilities of lexical, morphological, and syntactic production not evident in spontaneous utterances or by conventional aphasia tests. Some lexical and grammatical capabilities survived massive damage to both anterior and posterior portions of the left hemisphere. [ABSTRACT FROM AUTHOR]

This study addresses the meanings of alcohol/other drug use for those who, during adolescence, moved beyond experimentation to dysfunctional levels of use. Interviews were conducted with 12 students in recovery from alcohol/other drug dependency, all currently participating in a university-based recovery program. Interpretive phenomenological analyses of the interviews revealed that meanings ascribed to use were extensive, complex, and intertwining. Students viewed their alcohol/other drug use in negative ways--as an escape, as a mask to feelings, and as leading to a double life. Yet they also saw it as normative, developmentally significant, and as an integral part of their social interactions. [ABSTRACT FROM AUTHOR]

The development of a safe, efficacious, and widely effective differentiation therapy for AML would dramatically improve the outlook for many patients worldwide. To this aim, our laboratory has discovered a class of differentiation agents that demonstrate tumour regression in murine models in vivo . Herein, we report a lead optimisation process around compound OXS007417, which led to improved potency, solubility, metabolic stability, and off-target toxicity of this compound class. A hERG liability was investigated and successfully alleviated through addition of nitrogen atoms into key positions of the compound. OXS008255 and OXS008474 demonstrated an improved murine PK profile in respect to OXS007417 and a delay in tumour growth in a subcutaneous in vivo model using HL-60 cells., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A. J. R., T. A. M., S. G. D. and P. V. are founders and minor shareholders of OxStem Oncology, a subsidiary company of OxStem Ltd. G. M. W. is a minor shareholder of OxStem Ltd. At the time the work was conducted, A. J. R. and G. M. W. were paid consultant for OxStem Ltd. T. A. M. is currently a paid consultant for and minor shareholder of Dark Blue Therapeutics Ltd., (This journal is © The Royal Society of Chemistry.)

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum .

The role of negative affect in precipitating drug craving and relapse among young adults in recovery from substance use disorder (SUD) is well documented. However, most studies focus on negative affect as a trait-level congregate of multiple negative emotion states. The present study examined the associations between specific facets of negative affect, college stressors, and craving among young adult college students in SUD recovery. Data were drawn from a three-week daily diary study of 50 students participating in a collegiate recovery community at a U.S. university ( M age = 21.42, 76% males). At the within-person level, craving was higher on days when young adults experienced higher than usual anger, fear, and sadness, but not guilt. At the between-person level, individuals higher in agitation reported greater levels of craving on average. Moderation analyses further showed that college stressors heightened the within-person association between anger and craving. Findings demonstrate that negative affect is not monolithic and that its different aspects are uniquely associated with craving at both between- and within-person levels. Findings from this study could guide collegiate SUD recovery programs that wish to provide greater support to their members by helping them identify both individual- and time-specific relapse risks, such as generally high levels of agitation or days when anger, fear, or sadness are higher than usual for a particular individual. Our findings also suggest that future research should consider distinct features and implications of affective structures at between- and within-person levels, and how these may be uniquely associated with craving., Competing Interests: Disclosure statement. The authors report there are no competing interests to declare.

Introduction: Drug development for neurodegenerative diseases such as Friedreich's ataxia (FRDA) is limited by a lack of validated, sensitive biomarkers of pharmacodynamic response in affected tissue and disease progression. Studies employing neuroimaging measures to track FRDA have thus far been limited by their small sample sizes and limited follow up. TRACK-FA, a longitudinal, multi-site, and multi-modal neuroimaging natural history study, aims to address these shortcomings by enabling better understanding of underlying pathology and identifying sensitive, clinical trial ready, neuroimaging biomarkers for FRDA., Methods: 200 individuals with FRDA and 104 control participants will be recruited across seven international study sites. Inclusion criteria for participants with genetically confirmed FRDA involves, age of disease onset ≤ 25 years, Friedreich's Ataxia Rating Scale (FARS) functional staging score of ≤ 5, and a total modified FARS (mFARS) score of ≤ 65 upon enrolment. The control cohort is matched to the FRDA cohort for age, sex, handedness, and years of education. Participants will be evaluated at three study visits over two years. Each visit comprises of a harmonized multimodal Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) scan of the brain and spinal cord; clinical, cognitive, mood and speech assessments and collection of a blood sample. Primary outcome measures, informed by previous neuroimaging studies, include measures of: spinal cord and brain morphometry, spinal cord and brain microstructure (measured using diffusion MRI), brain iron accumulation (using Quantitative Susceptibility Mapping) and spinal cord biochemistry (using MRS). Secondary and exploratory outcome measures include clinical, cognitive assessments and blood biomarkers., Discussion: Prioritising immediate areas of need, TRACK-FA aims to deliver a set of sensitive, clinical trial-ready neuroimaging biomarkers to accelerate drug discovery efforts and better understand disease trajectory. Once validated, these potential pharmacodynamic biomarkers can be used to measure the efficacy of new therapeutics in forestalling disease progression., Clinical Trial Registration: ClinicalTrails.gov Identifier: NCT04349514., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: R.E. and S.Z. are employed by Takeda Pharmaceutical Company Ltd with both authors receiving salary and S.Z. holds stocks in the company. A.S. was employed by Takeda Pharmaceutical Company Ltd at the time of his contribution to the TRACK-FA project. Takeda Pharmaceutical Company Ltd remains committed to FRDA research and will help develop translational tools to monitor patient disease and share with the FRDA community. T.S. and B.Y. are both employees of PTC Therapeutics. D.L. is a grant recipient from the National Institute of Health (NIH), Muscular Dystrophy Association (MDA), Friedreich’s Ataxia Research Alliance (FARA), Reata Pharmaceuticals Inc, Retrotope Inc, Voyager Therapeutics, Novartis Gene Therapies, Audentes Therapeutics (Astellas Gene Therapies) and Minoryx Therapeutics S.L. T.P.L.R. has equity interest in PRISM Clinical Imaging and Proteus Neurodynamics and consulting/advisory board engagement with CTF MEG International Services LP, Ricoh Company Ltd, Spago Nanomedical AB, Avexis (Novartis Gene Therapies) and Acadia Pharmaceuticals Inc. P.G.H. is a grant recipient from the Friedreich’s Ataxia Research Alliance (FARA), GoFAR, Ataxia UK, the Bob Allison Ataxia Research Centre, and the National Institute of Health (NIH). CMRR is supported by NIH grants P41EB027061 and P30NS076408. P.G.H. reports grants from Minoryx Therapeutics for activities outside this study. M.P. and R.J. and M.L. are employed by IXICO plc, ML is a shareholder for IXICO plc. M.C.F. is a grant recipient from PTC Therapeutics and has taken part in advisory board for PTC Therapeutics and Avexis (Novartis Gene Therapies). T.R. is a grant recipient from the Friedreich’s Ataxia Research Alliance (FARA). C.L. is a research grant recipient from the Friedreich’s Ataxia Research Alliance (FARA), GoFAR, Ataxia UK, the Bob Allison Ataxia Research Center, and National Institute of Health (NIH) grants P41. EB027061 and P30 NS076408. C.L. reports research grants from Minoryx Therapeutics and Biogen Inc. for activities outside this study. S.S. is a broad member of the Research Advisory Board for National Ataxia Foundation (USA), a research grant recipient from the Friedreich’s Ataxia Research Alliance (FARA), Wyck Foundation, National Ataxia Foundation, Muscular Dystrophy Association (MDA), National Institute of Health (NIH), FDA and receives industry support from Reata Pharmaceutical Inc, Retrotope Inc, PTC Therapeutics, Biohaven Pharmaceuticals, Avidity Biosciences Inc, and Strides Pharma Science Limited. M.L.K. holds shares in Novartis Gene Therapies. K.R. has received grants from the German Federal Ministry of Education and Research (BMBF 01GQ1402, 01DN18022), the German Research Foundation (IRTG 2150, ZUK32/1), Alzheimer Forschung Initiative e.V. (AFI 13812, NL-18002CB) and honoraria for presentations or advisory boards from Biogen and Roche. J.F. is employed by the Friedreich’s Ataxia Research Alliance (FARA) and receives a salary from this institution. L.C. is a research grant recipient from the Friedreich Ataxia Research Alliance (FARA), Ataxia UK, Medical Research Future Fund and is funded by a Medical Research Futures Fund Next Generation Career Development Fellowship., (Copyright: © 2022 Georgiou-Karistianis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Skeletal metastasis is the leading cause of morbidity and mortality in prostate cancer, with 80% of advanced prostate cancer patients developing bone metastases. Before metastasis, bone remodeling occurs, stimulating pre-metastatic niche formation and bone turnover, and platelets govern this process. Stem cell factor (SCF, Kit Ligand) is increased in advanced prostate cancer patient platelet releasates. Further, SCF and its receptor, CD117/c-kit, correlate with metastatic prostate cancer severity. We hypothesized that bone-derived SCF plays an important role in prostate cancer tumor communication with the bone inducing pre-metastatic niche formation. We generated two cell-specific SCF knockout mouse models deleting SCF in either mature osteoblasts or megakaryocytes and platelets. Using two syngeneic androgen-insensitive murine prostate cancer cell lines, RM1 ( Ras and Myc co-activation) and mPC3 ( Pten and Trp53 deletion), we examined the role of bone marrow-derived SCF in primary tumor growth and bone microenvironment alterations. Platelet-derived SCF was required for mPC3, but not RM1, tumor growth, while osteoblast-derived SCF played no role in tumor size in either cell line. While exogenous SCF induced proangiogenic protein secretion by RM1 and mPC3 prostate cancer cells, no significant changes in tumor angiogenesis were measured by immunohistochemistry. Like our previous studies, tumor-induced bone formation occurred in mice bearing RM1 or mPC3 neoplasms, demonstrated by bone histomorphometry. RM1 tumor-bearing osteoblast SCF knockout mice did not display tumor-induced bone formation. Bone stromal cell composition analysis by flow cytometry showed significant shifts in hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and osteoblast cell percentages in mice bearing RM1 or mPC3 tumors. There were no significant changes in the percentage of macrophages, osteoclasts, or osteocytes. Our study demonstrates that megakaryocyte/platelet-derived SCF regulates primary mPC3 tumor growth, while SCF originating from osteoblasts plays a role in bone marrow-derived progenitor cell composition and pre-metastatic niche formation. Further, we show that both the source of SCF and the genetic profile of prostate cancer determine the effects of SCF. Thus, targeting the SCF/CD117 signaling axis with tyrosine kinase inhibitors could affect primary prostate carcinomas or play a role in reducing bone metastasis dependent on the gene deletions or mutations driving the patients' prostate cancer., Competing Interests: The 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 © 2022 Foster, Shi, Harris, Patel, Surratt, Langsten and Kerr.)

Collegiate Recovery Communities (CRCs) are important sources of support for college students building and maintaining recovery from substance use disorders. The current study used daily diary data from members of a CRC to examine with which sources of social support students engaged daily, and whether students connected with these sources more on days when they indicated higher-than-usual recovery difficulty, negative affect, and/or school stress. Results indicate that on days when students reported having greater difficulty with recovery maintenance than usual, they had higher odds of being in contact with family members and were expected to talk or spend time with family for longer than usual. Students also had higher odds of having recovery-focused conversations with both 12-step sponsors and CRC peers on days of greater-than-usual recovery maintenance difficulty. Recovery maintenance difficulty was uniquely associated with longer duration of family contact, above and beyond negative affect and school stress. Thus, the occurrence, amount, and nature of CRC members' interactions with important social network members varied in relation to perceived recovery challenges that same day. Findings highlight the importance of providing college students with multiple sources of support that they can use to maintain their recoveries despite daily challenges., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Identifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation, and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation, similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers, including SOX9, under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated β 3 integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The development of distant metastasis is the leading cause of prostate cancer (CaP)-related death, with the skeleton being the primary site of metastasis. While the progression of primary tumors and the growth of bone metastatic tumors are well described, the mechanisms controlling pre-metastatic niche formation and homing of CaP to bone remain unclear. Through prior studies, we demonstrated that platelet secretion was required for ongoing tumor growth and pre-metastatic tumor-induced bone formation. Platelets stimulated bone marrow-derived cell (BMDC) mobilization to tumors supporting angiogenesis. We hypothesized that proteins released by the platelet α granules were responsible for inducing changes in the pre-metastatic bone niche. We found that the classically anti-angiogenic protein thrombospondin (TSP)-1 was significantly increased in the platelets of mice with RM1 murine CaP tumors. To determine the role of increased TSP-1, we implanted tumors in TSP-1 null animals and assessed changes in tumor growth and pre-metastatic niche. TSP-1 loss resulted in increased tumor size and enhanced angiogenesis by immunohistochemistry. Conversely, TSP-1 deletion reduced BMDC mobilization and enhanced osteoclast formation resulting in decreased tumor-induced bone formation as measured by microcomputed tomography. We hypothesized that changes in the pre-metastatic niche were due to the retention of TGF-β1 in the platelets of mice after TSP-1 deletion. To assess the importance of platelet-derived TGF-β1, we implanted RM1 CaP tumors in mice with platelet factor 4-driven deletion of TGF-β1 in platelets and megakaryocytes. Like TSP-1 deletion, loss of platelet TGF-β1 resulted in increased angiogenesis with a milder effect on tumor size and BMDC release. Within the bone microenvironment, platelet TGF-β1 deletion prevented tumor-induced bone formation due to increased osteoclastogenesis. Thus, we demonstrate that the TSP-1/TGF-β1 axis regulates pre-metastatic niche formation and tumor-induced bone turnover. Targeting the platelet release of TSP-1 or TGF-β1 represents a potential method to interfere with the process of CaP metastasis to bone., Competing Interests: None., (AJCEU Copyright © 2021.)

Cancer stem-like cells (CSCs) are associated with cancer progression, metastasis, and recurrence, and may also represent a subset of circulating tumor cells (CTCs). In our prior study, CTCs in advanced prostate cancer patients were found to express CD117/c-kit in a liquid biopsy. Whether CD117 expression played an active or passive role in the aggressiveness and migration of these CTCs remained an open question. In this study, we show that CD117 expression in prostate cancer patients is associated with decreased overall and progression-free survival and that activation and phosphorylation of CD117 increases in prostate cancer patients with higher Gleason grades. To determine how CD117 expression and activation by its ligand stem cell factor (SCF, kit ligand, steel factor) alter prostate cancer aggressiveness, we used C4-2 and PC3-mm human prostate cancer cells, which contain a CD117+ subpopulation. We demonstrate that CD117+ cells display increased proliferation and migration. In prostaspheres, CD117 expression enhances sphere formation. In both 2D and 3D cultures, stemness marker gene expression is higher in CD117+ cells. Using xenograft limiting dilution assays and serial tumor initiation assays, we show that CD117+ cells represent a CSC population. Combined, these data indicate that CD117 expression potentially promotes tumor initiation and metastasis. Further, in cell lines, CD117 activation by SCF promotes faster proliferation and invasiveness, while blocking CD117 activation with tyrosine kinase inhibitors (TKIs) decreased progression in a context-dependent manner. We demonstrate that CD117 expression and activation drives prostate cancer aggressiveness through the CSC phenotype and TKI resistance.

This study reveals a new microfluidic biosensor consisting of a multi-constriction microfluidic device with embedded electrodes for measuring the biophysical attributes of single cells. The biosensing platform called the iterative mechano-electrical properties (iMEP) analyzer captures electronic records of biomechanical and bioelectrical properties of cells. The iMEP assay is used in conjunction with standard migration assays, such as chemotaxis-based Boyden chamber and scratch wound healing assays, to evaluate the migratory behavior and biophysical properties of prostate cancer cells. The three cell lines evaluated in the study each represent a stage in the standard progression of prostate cancer, while the fourth cell line serves as a normal/healthy counterpart. Neither the scratch assay nor the chemotaxis assay could fully differentiate the four cell lines. Furthermore, there was not a direct correlation between wound healing rate or the migratory rate with the cells' metastatic potential. However, the iMEP assay, through its multiparametric dataset, could distinguish between all four cell line populations with p-value < 0.05. Further studies are needed to determine if iMEP signatures can be used for a wider range of human cells to assess the tumorigenicity of a cell population or the metastatic potential of cancer cells., Competing Interests: Conflicts of interest There are no conflicts to declare. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis , and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1 b . These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

Cyclization of the peptide backbone by connecting the N- and C-terminus can endow target peptides with favorable properties, such as increased stability or potential oral bioavailability. However, there are few tools available for carrying out this modification. Asparaginyl endopeptidases (AEPs) are a class of enzymes that typically work as proteases, but a subset is highly efficient at cyclization of the peptide backbone. In this chapter we describe how to utilize a cyclizing AEP (OaAEP1 b ) to produce backbone-cyclized peptides both in planta and in vitro. Using the in planta method, OaAEP1 b and the target precursor peptide are coexpressed in the leaves of the model plant Nicotiana benthamiana, and cyclization of the target peptide occurs in planta. Using the in vitro method, purified recombinant OaAEP1 b produced in bacteria is used to cyclize the target precursor peptide in vitro.

Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of diverse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the "marker of ligase activity", together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Affiliating with 12-step groups appears to reduce relapse risk. By relying on between-person designs, extant research has been unable to examine daily mechanisms through which 12-step group affiliation contributes to recovery., Objectives: To examine the daily use and factor structure of the 12 steps and intrapersonal predictors and moderators of 12-step use. To determine whether the 12 steps were used in response to daily craving and, if so, which steps and in what contexts., Methods: Data comprised 1304 end-of-day diary data entries from 55 young adults collected in 2008 from members of a college recovery community, combined with person-level baseline measures. Exploratory factor analysis examined the factor structure, and multi-level models examined both day-level and person-level predictors and moderators of step use, including meeting attendance, drug and alcohol dependence, social support, and coping strategies., Results: Analyses produced two factors: Everyday steps, comprising surrender and maintenance steps, and action steps. Moderation analyses revealed that only action steps were significantly associated with craving, suggesting that craving can spur their use, but only among individuals pursuing certain general strategies for coping with stress: Separate median-split models produced significant associations between craving and action steps only among individuals high in avoidance, high in support-seeking, and/or low in problem-solving. Conclusions/Importance: This is the first study to empirically discern a 2-factor structure underlying the 12 steps, and to show that the two sets of steps are used in different contexts. The study also illustrates the value of person-centered approaches to recovery research and practice.

Metastatic or recurrent tumors are the primary cause of cancer-related death. For prostate cancer, patients diagnosed with local disease have a 99% 5-year survival rate; however, this 5-year survival rate drops to 28% in patients with metastatic disease. This dramatic decline in survival has driven interest in discovering new markers able to identify tumors likely to recur and in developing new methods to prevent metastases from occurring. Biomarker discovery for aggressive tumor cells includes attempts to identify cancer stem cells (CSCs). CSCs are defined as tumor cells capable of self-renewal and regenerating the entire tumor heterogeneity. Thus, it is hypothesized that CSCs may drive primary tumor aggressiveness, metastatic colonization, and therapeutic relapse. The ability to identify these cells in the primary tumor or circulation would provide prognostic information capable of driving prostate cancer treatment decisions. Further, the ability to target these CSCs could prevent tumor metastasis and relapse after therapy allowing for prostate cancer to finally be cured. Here, we will review potential CSC markers and highlight evidence that describes how cells expressing each marker may drive prostate cancer progression, metastatic colonization and growth, tumor recurrence, and resistance to treatment.

Listening to the needs of the community is an important step toward reducing health disparities. Researchers may need to adjust their methods to maximize participation and benefit to the community. This report describes how the project team adjusted its approach to a weight loss intervention to support a community of African American women seeking to improve their health.

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

Background: Sustained recovery from substance abuse is a dynamic intraindividual-level process., Objectives: We argue that research on recovery process will benefit from a theoretical approach that captures both the dynamic and the idiographic nature of substance abuse recovery. In addition to setting out why we believe that research on recovery can benefit from such an approach, we provide a demonstration of idiographic within-individual analyses of between- and within-day associations among negative affect, substance use craving, and positive social experiences., Design and Subjects: The data used were drawn from 39 abstinent young adults in 12-step recovery from substance abuse (mean age = 22.9, females = 12). Participants provided an average of 26.7 days of daily diary data by end-of-day collections. Unified first-order structural equation models were fit individually to predict daily levels of craving and negative affect from the previous day's same two variables as well as from both the previous day's and the same day's positive social experiences., Results: Model estimates demonstrated substantial interindividual heterogeneity in their day-to-day associations in both direction and magnitude, highlighting the importance of applying idiographic approach to understanding recovery. Cluster analyses were subsequently applied to individual model estimates to identify homogeneous subgroups that demonstrated similar day-to-day association patterns, revealing two distinct subgroups that appeared to manage daily abstinence through different mechanisms., Conclusions: The idiographic approach presented provides the potential value of framing recovery as an idiosyncratic dynamic process and provides targets for tailored and adaptive treatment and recovery supporting intervention in future design and evaluation., (© The Author(s) 2015.)

Background: Polycystic Ovary Syndrome (PCOS) affects approximately 15% of reproductive-age women and increases risk of insulin resistance, type 2 diabetes mellitus, cardiovascular disease, cancer and infertility. Hyperinsulinemia is believed to contribute to or worsen all of these conditions, and increases androgens in women with PCOS. Carbohydrates are the main stimulators of insulin release, but research shows that dairy products and starches elicit greater postprandial insulin secretion than non-starchy vegetables and fruits. The purpose of this study was to determine whether an 8-week low-starch/low-dairy diet results in weight loss, increased insulin sensitivity, and reduced testosterone in women with PCOS., Methods: Prospective 8-week dietary intervention using an ad libitum low starch/low dairy diet in 24 overweight and obese women (BMI ≥ 25 kg/m 2 and ≤ 45 kg/m 2 ) with PCOS. Diagnosis of PCOS was based on the Rotterdam criteria. Weight, BMI, Waist Circumference (WC), Waist-to-Height Ratio (WHtR), fasting and 2-hour glucose and insulin, homeostasis model assessment of Insulin Resistance (HOMA-IR), HbA1c, total and free testosterone, and Ferriman-Gallwey scores were measured before and after the 8-week intervention., Results: There was a reduction in weight (-8.61 ± 2.34 kg, p<0.001), BMI (-3.25 ± 0.88 kg/m 2 , p<0.001), WC (-8.4 ± 3.1 cm, p<0.001), WHtR (-0.05 ± 0.02 inches, p<0.001), fasting insulin (-17.0 ± 13.6 μg/mL, p<0.001) and 2-hour insulin (-82.8 ± 177.7 μg/mL, p=0.03), and HOMA-IR (-1.9 ± 1.2, p<0.001) after diet intervention. Total testosterone (-10.0 ± 17.0 ng/dL, p=0.008), free testosterone (-1.8 pg/dL, p=0.043) and Ferriman-Gallwey scores (-2.1 ± 2.7 points (p=0.001) were also reduced from pre- to post-intervention., Conclusion: An 8-week low-starch/low-dairy diet resulted in weight loss, improved insulin sensitivity and reduced testosterone in women with PCOS.

Background: Addiction science has primarily utilized self-report, continued substance use, and relapse factors to explore the process of recovery. However, the entry into successful abstinence substantially reduces our assessment abilities. Advances in neuroscience may be the key to objective understanding, treating, and monitoring long-term success in addiction recovery., Objectives: To explore functional near infrared spectroscopy (fNIR) as a viable technique in the assessment of addiction-cue reactivity. Specifically, prefrontal cortex (PFC) activation to alcohol cues was explored among formally alcohol-dependent individuals, across varying levels of successful abstinence. The aim of the investigation was to identify patterns of PFC activation change consistent with duration of abstinence., Methods: A total of 15 formally alcohol-dependent individuals, with abstinence durations ranging from 1 month to 10 years, viewed alcohol images during fNIR PFC assessment. Participants also subjectively rated the same images for affect and arousal level., Results: Subjective ratings of alcohol cues did not significantly correlate with duration of abstinence. As expected, days of abstinence did not significantly correlate with neutral cue fNIR reactivity. However, for alcohol cues, fNIR results showed increased days of abstinence was associated with decreased activation within the dorsolateral and dorsomedial prefrontal cortex regions., Conclusions: The present results suggest that fNIR may be a viable tool in the assessment of addiction-cue reactivity. RESULTS also support previous findings on the importance of dorsolateral and dorsomedial PFC in alcohol-cue activation. The findings build upon these past results suggesting that fNIR-assessed activation may represent a robust biological marker of successful addiction recovery.

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli, refolded in vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Polycystic ovary syndrome (PCOS) affects between 4%-18% of reproductive-aged women and is associated with increased risk of obesity and obesity-related disease. PCOS is associated with hyperinsulinemia, which is known to impair fat oxidation. Research shows that carbohydrates from dairy and starch-based foods cause greater postprandial insulin secretion than carbohydrates from nonstarchy vegetables and fruits. The purpose of this study was to determine whether an ad libitum 8-week low-starch/low-dairy diet would improve fasting and postprandial fat oxidation after a high saturated fat liquid meal (HSFLM) in overweight and obese women with PCOS. Prospective 8-week dietary intervention using a low-starch/low-dairy diet in 10 women (body mass index ≥25 kg/m(2) and ≤45 kg/m(2)) with PCOS. Indirect calorimetry was used at fasting and for 5 h following consumption of the HSFLM to determine respiratory exchange ratio (RER), macronutrient oxidation, and energy expenditure (EE) at week 0 and week 8. Participants had a reduction in body weight (-8.1 ± 1.8 kg, p < 0.05) and fasting insulin (-19.5 ± 8.9 μg/mL, p < 0.05) after dietary intervention; however, these were not significantly correlated with improved fat oxidation. There was a reduction in fasting RER, and fasting and postprandial carbohydrate oxidation, and an increase in fasting and postprandial fat oxidation after adjusting for body weight. There was also significant difference in incremental area under the curve from pre- to post-diet for fat (0.06 ± 0.00 g/kg per 5 h; p < 0.001) and carbohydrate oxidation (-0.29 ± 0.06 g/kg per 5 h; p < 0.001), but not for RER or EE. In conclusion, an 8-week low-starch/low-dairy diet increased fat oxidation in overweight and obese women with PCOS.

Psychological constructs, such as negative affect and substance use cravings that closely predict relapse, show substantial intra-individual day-to-day variability. This intra-individual variability of relevant psychological states combined with the "one day of a time" nature of sustained abstinence warrant a day-to-day investigation of substance use recovery. This study examines day-to-day associations among substance use cravings, negative affect, and tobacco use among 30 college students in 12-step recovery from drug and alcohol addictions. To account for individual variability in day-to-day process, it applies an idiographic approach. The sample of 20 males and 10 females (mean age = 21) was drawn from members of a collegiate recovery community at a large university. Data were collected with end-of-day data collections taking place over an average of 26.7 days. First-order vector autoregression models were fit to each individual predicting daily levels of substance use cravings, negative affect, and tobacco use from the same three variables one day prior. Individual model results demonstrated substantial inter-individual differences in intra-individual recovery process. Based on estimates from individual models, cluster analyses were used to group individuals into two homogeneous subgroups. Group comparisons demonstrate distinct patterns in the day-to-day associations among substance use cravings, negative affect, and tobacco use, suggesting the importance of idiographic approaches to recovery management and that the potential value of focusing on negative affect or tobacco use as prevention targets depends on idiosyncratic processes.

Purpose: Despite aggressive therapies, median survival for malignant gliomas is less than 15 months. Patients with unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) fare worse, presumably because of temozolomide resistance. AdV-tk, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, plus prodrug synergizes with surgery and chemoradiotherapy, kills tumor cells, has not shown MGMT dependency, and elicits an antitumor vaccine effect., Patients and Methods: Patients with newly diagnosed malignant glioma received AdV-tk at 3 × 10(10), 1 × 10(11), or 3 × 10(11) vector particles (vp) via tumor bed injection at time of surgery followed by 14 days of valacyclovir. Radiation was initiated within 9 days after AdV-tk injection to overlap with AdV-tk activity. Temozolomide was administered after completing valacyclovir treatment., Results: Accrual began December 2005 and was completed in 13 months. Thirteen patients were enrolled and 12 completed therapy, three at dose levels 1 and 2 and six at dose level 3. There were no dose-limiting or significant added toxicities. One patient withdrew before completing prodrug because of an unrelated surgical complication. Survival at 2 years was 33% and at 3 years was 25%. Patient-reported quality of life assessed with the Functional Assessment of Cancer Therapy-Brain (FACT-Br) was stable or improved after treatment. A significant CD3(+) T-cell infiltrate was found in four of four tumors analyzed after treatment. Three patients with MGMT unmethylated glioblastoma multiforme survived 6.5, 8.7, and 46.4 months., Conclusion: AdV-tk plus valacyclovir can be safely delivered with surgery and accelerated radiation in newly diagnosed malignant gliomas. Temozolomide did not prevent immune responses. Although not powered for efficacy, the survival and MGMT independence trends are encouraging. A phase II trial is ongoing.

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics., (Copyright © 2011 Wiley Periodicals, Inc.)

A series of trivalent lanthanide hydroxysulfates, Ln(OH)SO(4), (Ln = Pr through Yb, except radioactive Pm) has been synthesized via hydrothermal methods from Ln(2)(SO(4))(3)·8H(2)O by reaction with aqueous NaOH at 170 °C in Teflon lined Parr steel autoclaves, and were characterized by single crystal X-ray diffraction and FT-IR spectroscopy. Two types of arrangements were found in the solid state. The lighter (Ln = Pr-Nd, Sm-Gd) and heavier lanthanide(III) hydroxysulfates (Tb-Yb) are each isostructural. Both structure types exhibit the monoclinic space group P2(1)/n, but the unit cell content is doubled with two crystallographically distinct LnO(8) polyhedra for the heavier lanthanide compounds. The lighter complexes maintain the coordination number 9, forming a three-dimensional extended lattice. The heavier counterparts exhibit the coordination number 8, and arrange as infinite columns of two crystallographically different LnO(8) polyhedra, while extending along the "c" axis. These columns of LnO(8) polyhedra are surrounded and separated by six columns of sulfate ions, also elongating in the "c" direction. The rigid sulfate entities seem to obstruct the closing in of the lighter LnO(9) polyhedra, and show an inclining degree of torsion into the "ac" layers. The crystal lattice of the lighter 4f complexes can sufficiently withstand the tension buildup, caused by the decreasing Ln(3+) radius, up to Gd(OH)SO(4). The energy profile of this structural arrangement then seems to exceed levels at which this structure type is favorable. The lattice arrangement of the heavier Ln-analogues seems to offer a lower energy profile. This appears to be the preferred arrangement for the heavier lanthanide hydroxysulfates, whose crystal lattice exhibits more flexibility, as the coordination sphere of these analogues is less crowded. The IR absorbance frequencies of the hydroxide ligands correlate as a function of the Ln(3+) ionic radius. This corresponds well with the X-ray single crystal analysis data.

The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. Here we present an extensive analysis of viral sequences from HIV-1 infected volunteers from the first "mechanism validation" phase II clinical trial of a mutagenic base analog in which individuals previously treated with antiviral drugs received 1600 mg of KP1461 twice per day for 124 days. Plasma viral loads were not reduced, and overall levels of viral mutation were not increased during this short-term study, however, the mutation spectrum of HIV was altered. A large number (N = 105 per sample) of sequences were analyzed, each derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated control individuals (>7.1 million base pairs of unique viral templates were sequenced). We found that private mutations, those not found in more than one viral sequence and likely to have occurred in the most recent rounds of replication, increased in treated individuals relative to controls after 56 (p = 0.038) and 124 (p = 0.002) days of drug treatment. The spectrum of mutations observed in the treated group showed an excess of A to G and G to A mutations (p = 0.01), and to a lesser extent T to C and C to T mutations (p = 0.09), as predicted by the mechanism of action of the drug. These results validate the proposed mechanism of action in humans and should spur development of this novel antiretroviral approach.

Background: Acute care physical therapy is a rapidly evolving practice area, but little is known about the skills, knowledge, and behaviors necessary for a clinician to be most effective in this area., Objective: The objective of this study was to perform the first nationwide survey of acute care physical therapists to validate the knowledge, skill, and behavior sets that reflect practice parameters specific for acute care physical therapy., Design: A survey format was used., Methods: The Acute Care Physical Therapy Practice Analysis Survey was created on the basis of current literature and consensus of a 9-member Subject Matter Expert (SME) Group. The survey sections addressed knowledge areas, professional behaviors, and patient/client management approaches reflecting practice parameters specific for acute care physical therapy. Additionally, respondent demographic information was collected. Pilot testing necessitated minor changes in the survey. After revision, the survey questionnaire was sent to a sample of experienced acute care physical therapists throughout the United States., Results: A convenience sample of 522 physical therapists who identified themselves as having extensive experience in acute care practice was used. Of these, 254 completed the survey, for a response rate of 48.7%. Through the use of predetermined decision rules, 34 items were eliminated on the basis of a descriptive analysis of survey results as well as a failure of the items to meet the threshold of specificity for acute care practice, as determined through SME Group consensus., Limitations: The potential for self-selection bias, a sample weighted heavily with American Physical Therapy Association members and Acute Care Section members, and a small proportion of clinicians (<3%) with less than 1 year of acute care experience may limit generalizability of these results., Conclusions: The results of this practice analysis describe distinct knowledge, skills, and behaviors specific for acute care physical therapy. The outcomes of the survey might assist in the development of professional (entry-level) acute care competencies, a petition for the recognition of specialization in acute care physical therapy, or both. In addition, the findings of this practice analysis could serve as the foundation for the development of residencies or fellowships in acute care practice.

To address the lack of research examining within-person processes associated with the maintenance of abstinence from substance use, this study examines both associations between negative affect and social experiences and same-day levels of substance use cravings among college students in 'Twelve-Step' substance abuse recovery and the role of coping strategies in moderating these within-day associations. The sample consisted of 1222 end-of-day reports made by 55 recovering college students, 39 males and 16 females. Findings include that daily cravings were predicted by same-day negative affect and each of four negative social experiences. Moreover, each of these within-day associations was moderated by individuals' levels of avoidance coping. In contrast, problem-solving coping only moderated the associations between negative affect and cravings. These findings document a within-day mechanism through which coping strategies, especially avoidance coping, may influence daily variation in cravings and in turn affect abstinence.

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters.

An understanding of the relationship of speech and language symptoms to lesions in the frontal region of the dominant hemisphere depends on a fuller description of the speech phenomena than can be provided by transcriptional or acoustic investigation alone. This paper provides examples of how articulatory movement tracking can aid in describing apraxic speech deficits.

Persons with cerebellar ataxia exhibit changes in physical coordination and speech and voice production. Previously, these alterations of speech and voice production were described primarily via perceptual coordinates. In this study, the spatial-temporal properties of syllable production were examined in 12 speakers, six of whom were healthy speakers and six with ataxia. The speaking task was designed to elicit six different prosodic conditions and four contrastive prosodic events. Distinct prosodic patterns were elicited by the examiner for cerebellar patients and healthy speakers. These utterances were digitally recorded and analysed acoustically and statistically. The healthy speakers showed statistically significant differences among all four prosodic contrasts. The normal model described by the prosodic contrasts provided a sensitive index of cerebellar pathology with quantitative acoustic analyses. A significant interaction between subject groups and prosodic conditions revealed a compromised prosody in cerebellar patients. Significant differences were found for durational parameters, F0 and formant frequencies. The cerebellar speakers demonstrated different patterns of syllable lengthening and syllable reduction from that of the healthy speakers.

As adolescents have entered substance abuse treatment programs in greater numbers than ever before [Substance Abuse Mental Health Services Administration, 2004. Substance abuse treatment by primary substance of abuse. (http://www.oas.samhsa.gov/dasis.htm#teds2). Retrieved September 2004], increasing numbers of young adults are beginning college in 12-step recovery. Unfortunately, with social contexts organized around excessive alcohol consumption, colleges may be the most difficult place through which to control drinking behaviors, let alone remain abstinent. This study used data collected during 2004 and 2005 to describe members of the largest collegiate recovery community in the United States. Included in this study are the following: a description of the program that supports the community, the methods and measurements used to collect data, and results describing the substance use history and characteristics of the community members (N = 82). Collected at a large southwestern university, the data provide drug and alcohol counselors, treatment providers, school administrators, and researchers with the first description of a large collegiate recovery community.

The advances of the Civil Rights movement in the mid-20th century and the success of the first African-American neurosurgeons trained at the Montreal Neurological Institute have led to a number of African-Americans receiving neurosurgery training within the United States. Unfortunately, the details regarding the first African-American neurosurgeon trained in the United States, E. Latunde Odeku, have largely remained in obscurity. Born on June 29, 1927 in Lagos, Nigeria, Dr. Odeku received his M.D. from the Howard University College of Medicine in 1954. He spent the next year at the University of Michigan under the tutelage of Edgar A. Kahn, chief of neurosurgery, and was impressive enough to be offered a residency position. From 1956 to 1960, he trained under Dr. Kahn at the University of Michigan. In 1961, he returned to Howard as a member of the neurosurgery faculty, during which time he became the second African-American to be certified by the American Board of Neurological Surgery. Although he received multiple job offers in the United States, he chose to return to Nigeria where he worked tirelessly, providing excellent neurosurgical care and discipleship until his death in 1974. The diligence and intelligence of E. Latunde Odeku, M.D., F.A.C.S., enabled him to become the first African-American neurosurgeon trained in the United States. A truly global pioneer, his selfless service in America and Nigeria opened the door for people from each country to enhance the field of neurosurgery.

Largely because of the advances of the Civil Rights movement in the mid-20th century, an increasing number of African-Americans have had the opportunity to become physicians and enter the distinguished field of neurosurgery. Many have made the most of this opportunity, becoming prominent in both academics and private practice. Unfortunately, the details regarding the first African-American neurosurgeon, Clarence Sumner Greene, Sr., have remained in relative obscurity. Born on December 26, 1901 in Washington, D.C., Dr. Greene received his M.D. from the Howard University College of Medicine with distinction in 1936. After 7 years of general surgery residency and 4 years as a professor of surgery at Howard University, he was granted the opportunity by the legendary Wilder G. Penfield to train in neurosurgery at the world-renowned Montreal Neurological Institute from 1947 to 1949. Receiving high praise from Dr. Penfield, Dr. Greene became the first African-American certified by the American Board of Neurological Surgery on October 22, 1953. Subsequently, he was appointed as chair of neurosurgery at Howard University, where he successfully treated intracranial aneurysms, brain tumors, and herniated intervertebral discs until his tragic death in 1957. The diligence and perseverance of Clarence Sumner Greene, Sr., M.D., D.D.S., F.A.C.S. enabled him to overcome incredible odds to become the first African-American neurosurgeon, trained by Dr. Penfield at the Montreal Neurological Institute. A true pioneer, his achievements have opened the door for subsequent African-Americans to enhance the field of neurosurgery.

Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed alpha-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

DICER is a key enzyme that processes microRNA and small interfering RNA precursors into their short mature forms, enabling them to regulate gene expression. Only a single Dicer gene exists in the mouse genome, and it is broadly expressed in developing tissues. Dicer-null mutants die before gastrulation. Therefore, to study Dicer function in the later event of lung formation, we inactivated it in the mouse lung epithelium using a Dicer conditional allele and the Sonic Hedgehogcre (Shhcre) allele. Branching arrests in these mutant lungs, although epithelial growth continues in distal domains that are expanded compared with normal samples. These defects result in a few large epithelial pouches in the mutant lung instead of numerous fine branches present in a normal lung. Significantly, the initial phenotypes are apparent before an increase in epithelial cell death is observed, leading us to propose that Dicer plays a specific role in regulating lung epithelial morphogenesis independent of its requirement in cell survival. In addition, we found that the expression of Fgf10, a key gene involved in lung development, is up-regulated and expanded in the mesenchyme of Dicer mutant lungs. Previous studies support the hypothesis that precise localization of FGF10 in discrete sites of the lung mesenchyme serves as a chemoattractant for the outgrowth of epithelial branches. The aberrant Fgf10 expression may contribute to the Dicer morphological defects. However, the mechanism by which DICER functions in the epithelium to influence Fgf10 expression in the mesenchyme remains unknown.

Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by Plasmodium falciparum parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a "hot spot" on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.

We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis.

Coarticulation studies in speech of deaf individuals have so far focused on intrasyllabic patterning of various consonant-vowel sequences. In this study, both inter- and intrasyllabic patterning were examined in disyllables /symbol see text #CVC/ and the effects of phonetic context, speaking rate, and segment type were explored. Systematic observation of F2 and durational measurements in disyllables minimally contrasting in vocalic ([i], [u,][a]) and in consonant ([b], [d]) context, respectively, was made at selected locations in the disyllable, in order to relate inferences about articulatory adjustments with their temporal coordinates. Results indicated that intervocalic coarticulation across hearing and deaf speakers varied as a function of the phonetic composition of disyllables (b&#95;b or d&#95;d). The deaf speakers showed reduced intervocalic coarticulation for bilabial but not for alveolar disyllables compared to the hearing speakers. Furthermore, they showed less marked consonant influences on the schwa and stressed vowel of disyllables compared to the hearing controls. Rate effects were minimal and did not alter the coarticulatory patterns observed across hearing status. The above findings modify the conclusions drawn from previous studies and suggest that the speech of deaf and hearing speakers is guided by different gestural organization.