University of South Dakota

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.

Physical, chemical and perceived stressors can all evoke non-specific responses in fish, which are considered adaptive to enable the fish to cope with the disturbance and maintain its homeostatic state. If the stressor is overly severe or long-lasting to the point that the fish is not capable of regaining homeostasis, then the responses themselves may become maladaptive and threaten the fish's health and well-being. Physiological responses to stress are grouped as primary, which include endocrine changes such as in measurable levels of circulating catecholamines and corticosteroids, and secondary, which include changes in features related to metabolism, hydromineral balance, and cardiovascular, respiratory and immune functions. In some instances, the endocrine responses are directly responsible for these secondary responses resulting in changes in concentration of blood constituents, including metabolites and major ions, and, at the cellular level, the expression of heat-shock or stress proteins. Tertiary or whole-animal changes in performance, such as in growth, disease resistance and behavior, can result from the primary and secondary responses and possibly affect survivorship.Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol in actinopterygian fishes, that occur following a stressful event. The characteristic elevation in circulating cortisol during the first hour after an acute disturbance can vary by more than two orders of magnitude among species and genetic history appears to account for much of this interspecific variation. An appreciation of the factors that affect the magnitude, duration and recovery of cortisol and other physiological changes caused by stress in fishes is important for proper interpretation of experimental data and design of effective biological monitoring programs.

The antibacterial properties of zinc oxide nanoparticles were investigated using both gram-positive and gram-negative microorganisms. These studies demonstrate that ZnO nanoparticles have a wide range of antibacterial activities toward various microorganisms that are commonly found in environmental settings. The antibacterial activity of the ZnO nanoparticles was inversely proportional to the size of the nanoparticles in S. aureus. Surprisingly, the antibacterial activity did not require specific UV activation using artificial lamps, rather activation was achieved under ambient lighting conditions. Northern analyses of various reactive oxygen species (ROS) specific genes and confocal microscopy suggest that the antibacterial activity of ZnO nanoparticles might involve both the production of reactive oxygen species and the accumulation of nanoparticles in the cytoplasm or on the outer membranes. Overall, the experimental results suggest that ZnO nanoparticles could be developed as antibacterial agents against a wide range of microorganisms to control and prevent the spreading and persistence of bacterial infections.

Nanoparticle metal oxides represent a new class of important materials that are increasingly being developed for use in research and health-related applications. Highly ionic metal oxides are interesting not only for their wide variety of physical and chemical properties but also for their antibacterial activity. Although the in vitro antibacterial activity and efficacy of regular zinc oxides have been investigated, little is known about the antibacterial activity of nanoparticles of ZnO. Preliminary growth analysis data suggest that nanoparticles of ZnO have significantly higher antibacterial effects on Staphylococcus aureus than do five other metal oxide nanoparticles. In addition, studies have clearly demonstrated that ZnO nanoparticles have a wide range of antibacterial effects on a number of other microorganisms. The antibacterial activity of ZnO may be dependent on the size and the presence of normal visible light. The data suggest that ZnO nanoparticles have a potential application as a bacteriostatic agent in visible light and may have future applications in the development of derivative agents to control the spread and infection of a variety of bacterial strains.

The Large Underground Xenon (LUX) experiment is a dual-phase xenon time-projection chamber operating at the Sanford Underground Research Facility (Lead, South Dakota). The LUX cryostat was filled for the first time in the underground laboratory in February 2013. We report results of the first WIMP search data set, taken during the period from April to August 2013, presenting the analysis of 85.3 live days of data with a fiducial volume of 118 kg. A profile-likelihood analysis technique shows our data to be consistent with the background-only hypothesis, allowing 90% confidence limits to be set on spin-independent WIMP-nucleon elastic scattering with a minimum upper limit on the cross section of 7.6 × 10(-46) cm(2) at a WIMP mass of 33 GeV/c(2). We find that the LUX data are in disagreement with low-mass WIMP signal interpretations of the results from several recent direct detection experiments.

We report constraints on spin-independent weakly interacting massive particle (WIMP)-nucleon scattering using a 3.35×10^{4} kg day exposure of the Large Underground Xenon (LUX) experiment. A dual-phase xenon time projection chamber with 250 kg of active mass is operated at the Sanford Underground Research Facility under Lead, South Dakota (USA). With roughly fourfold improvement in sensitivity for high WIMP masses relative to our previous results, this search yields no evidence of WIMP nuclear recoils. At a WIMP mass of 50 GeV c^{-2}, WIMP-nucleon spin-independent cross sections above 2.2×10^{-46} cm^{2} are excluded at the 90% confidence level. When combined with the previously reported LUX exposure, this exclusion strengthens to 1.1×10^{-46} cm^{2} at 50 GeV c^{-2}.

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.

Researching the epidemiology and estimating the prevalence of fetal alcohol syndrome (FAS) and other fetal alcohol spectrum disorders (FASD) for mainstream populations anywhere in the world has presented a challenge to researchers. Three major approaches have been used in the past: surveillance and record review systems, clinic-based studies, and active case ascertainment methods. The literature on each of these methods is reviewed citing the strengths, weaknesses, prevalence results, and other practical considerations for each method. Previous conclusions about the prevalence of FAS and total FASD in the United States (US) population are summarized. Active approaches which provide clinical outreach, recruitment, and diagnostic services in specific populations have been demonstrated to produce the highest prevalence estimates. We then describe and review studies utilizing in-school screening and diagnosis, a special type of active case ascertainment. Selected results from a number of in-school studies in South Africa, Italy, and the US are highlighted. The particular focus of the review is on the nature of the data produced from in-school methods and the specific prevalence rates of FAS and total FASD which have emanated from them. We conclude that FAS and other FASD are more prevalent in school populations, and therefore the general population, than previously estimated. We believe that the prevalence of FAS in typical, mixed-racial, and mixed-socioeconomic populations of the US is at least 2 to 7 per 1,000. Regarding all levels of FASD, we estimate that the current prevalence of FASD in populations of younger school children may be as high as 2-5% in the US and some Western European countries.

The adverse effects of prenatal alcohol exposure constitute a continuum of disabilities (fetal alcohol spectrum disorders [FASD]). In 1996, the Institute of Medicine established diagnostic categories delineating the spectrum but not specifying clinical criteria by which diagnoses could be assigned. In 2005, the authors published practical guidelines operationalizing the Institute of Medicine categories, allowing for standardization of FASD diagnoses in clinical settings. The purpose of the current report is to present updated diagnostic guidelines based on a thorough review of the literature and the authors' combined expertise based on the evaluation of >10 000 children for potential FASD in clinical settings and in epidemiologic studies in conjunction with National Institute on Alcohol Abuse and Alcoholism-funded studies, the Collaborative Initiative on Fetal Alcohol Spectrum Disorders, and the Collaboration on FASD Prevalence. The guidelines were formulated through conference calls and meetings held at National Institute on Alcohol Abuse and Alcoholism offices in Rockville, MD. Specific areas addressed include the following: precise definition of documented prenatal alcohol exposure; neurobehavioral criteria for diagnosis of fetal alcohol syndrome, partial fetal alcohol syndrome, and alcohol-related neurodevelopmental disorder; revised diagnostic criteria for alcohol-related birth defects; an updated comprehensive research dysmorphology scoring system; and a new lip/philtrum guide for the white population, incorporating a 45-degree view. The guidelines reflect consensus among a large and experienced cadre of FASD investigators in the fields of dysmorphology, epidemiology, neurology, psychology, developmental/behavioral pediatrics, and educational diagnostics. Their improved clarity and specificity will guide clinicians in accurate diagnosis of infants and children prenatally exposed to alcohol.

Importance: Fetal alcohol spectrum disorders are costly, life-long disabilities. Older data suggested the prevalence of the disorder in the United States was 10 per 1000 children; however, there are few current estimates based on larger, diverse US population samples. Objective: To estimate the prevalence of fetal alcohol spectrum disorders, including fetal alcohol syndrome, partial fetal alcohol syndrome, and alcohol-related neurodevelopmental disorder, in 4 regions of the United States. Design, Setting, and Participants: Active case ascertainment methods using a cross-sectional design were used to assess children for fetal alcohol spectrum disorders between 2010 and 2016. Children were systematically assessed in the 4 domains that contribute to the fetal alcohol spectrum disorder continuum: dysmorphic features, physical growth, neurobehavioral development, and prenatal alcohol exposure. The settings were 4 communities in the Rocky Mountain, Midwestern, Southeastern, and Pacific Southwestern regions of the United States. First-grade children and their parents or guardians were enrolled. Exposures: Alcohol consumption during pregnancy. Main Outcomes and Measures: Prevalence of fetal alcohol spectrum disorders in the 4 communities was the main outcome. Conservative estimates for the prevalence of the disorder and 95% CIs were calculated using the eligible first-grade population as the denominator. Weighted prevalences and 95% CIs were also estimated, accounting for the sampling schemes and using data restricted to children who received a full evaluation. Results: A total of 6639 children were selected for participation from a population of 13 146 first-graders (boys, 51.9%; mean age, 6.7 years [SD, 0.41] and white maternal race, 79.3%). A total of 222 cases of fetal alcohol spectrum disorders were identified. The conservative prevalence estimates for fetal alcohol spectrum disorders ranged from 11.3 (95% CI, 7.8-15.8) to 50.0 (95% CI, 39.9-61.7) per 1000 children. The weighted prevalence estimates for fetal alcohol spectrum disorders ranged from 31.1 (95% CI, 16.1-54.0) to 98.5 (95% CI, 57.5-139.5) per 1000 children. Conclusions and Relevance: Estimated prevalence of fetal alcohol spectrum disorders among first-graders in 4 US communities ranged from 1.1% to 5.0% using a conservative approach. These findings may represent more accurate US prevalence estimates than previous studies but may not be generalizable to all communities.

1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD), N -methyl-TBD (MTBD), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are effective organocatalysts for the ring-opening polymerization (ROP) of cyclic esters such as lactide (LA), δ-valerolactone (VL), and ε-caprolactone (CL). TBD is shown to polymerize LA, VL, and CL in a fast and controlled manner, whereas MTBD and DBU polymerized LA and addition of a thiourea cocatalyst led to the ROP of VL and CL being achieved. Each of the catalysts produced polymers displaying high end group fidelity, good correlation between theoretical and observed molecular weight, and linear relationships between conversion and molecular weight. The enhanced activity of TBD relative to MTBD and DBU is attributed to its bifunctionality, enabling the simultaneous activation of both the cyclic ester monomer and the alcohol group of the initiator/propagating species. Temperature-dependent NMR studies generated individual association constants for MTBD with benzyl alcohol and thiourea with VL. In combination with temperature-dependent ROP of VL in the presence of benzyl alcohol, MTBD, and thiourea, these data have led to the derivation of the activation energy for the ROP (49 ± 3 kJ mol -1 ). The simplicity of the reaction conditions, the ready availability of the catalysts, the variety of polymerizable cyclic ester monomers, and the exquisite control over the polymerization are demonstrated.

The corpus luteum (CL) is one of the few endocrine glands that forms from the remains of another organ and whose function and survival are limited in scope and time. The CL is the site of rapid remodeling, growth, differentiation, and death of cells originating from granulosa, theca, capillaries, and fibroblasts. The apparent raison d'etre of the CL is the production of progesterone, and all the structural and functional features of this gland are geared toward this end. Because of its unique importance for successful pregnancies, the mammals have evolved a complex series of checks and balances that maintains progesterone at appropriate levels throughout gestation. The formation, maintenance, regression, and steroidogenesis of the CL are among the most significant and closely regulated events in mammalian reproduction. During pregnancy, the fate of the CL depends on the interplay of ovarian, pituitary, and placental regulators. At the end of its life span, the CL undergoes a process of regression leading to its disappearance from the ovary and allowing the initiation of a new cycle. The generation of transgenic, knockout and knockin mice and the development of innovative technologies have revealed a novel role of several molecules in the reprogramming of granulosa cells into luteal cells and in the hormonal and molecular control of the function and demise of the CL. The current review highlights our knowledge on these key molecular events in rodents.

On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship.

OBJECTIVE: Systematic review of randomized control trials (RCTs) for the effectiveness of pain neuroscience education (PNE) on pain, function, disability, psychosocial factors, movement, and healthcare utilization in individuals with chronic musculoskeletal (MSK) pain. DATA SOURCES: Systematic searches were conducted on 11 databases. Secondary searching (PEARLing) was undertaken, whereby reference lists of the selected articles were reviewed for additional references not identified in the primary search. STUDY SELECTION: All experimental RCTs evaluating the effect of PNE on chronic MSK pain were considered for inclusion. Additional Limitations: Studies published in English, published within the last 20 years, and patients older than 18 years. No limitations were set on specific outcome measures. DATA EXTRACTION: Data were extracted using the participants, interventions, comparison, and outcomes (PICO) approach. DATA SYNTHESIS: Study quality of the 13 RCTs used in this review was assessed by 2 reviewers using the PEDro scale. Narrative summary of results is provided for each study in relation to outcomes measurements and effectiveness. CONCLUSIONS: Current evidence supports the use of PNE for chronic MSK disorders in reducing pain and improving patient knowledge of pain, improving function and lowering disability, reducing psychosocial factors, enhancing movement, and minimizing healthcare utilization.

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

Prostate cancer has the highest prevalence of any nonskin cancer in the human body, with similar likelihood of neoplastic foci found within the prostates of men around the world regardless of diet, occupation, lifestyle, or other factors. Essentially all men with circulating androgens will develop microscopic prostate cancer if they live long enough. This review is a contemporary and comprehensive, literature-based analysis of the putative risk factors for human prostate cancer, and the results were presented at a multidisciplinary consensus conference held in Crystal City, Virginia, in the fall of 2002. The objectives were to evaluate known environmental factors and mechanisms of prostatic carcinogenesis and to identify existing data gaps and future research needs. The review is divided into four sections, including 1) epidemiology (endogenous factors [family history, hormones, race, aging and oxidative stress] and exogenous factors [diet, environmental agents, occupation and other factors, including lifestyle factors]); 2) animal and cell culture models for prediction of human risk (rodent models, transgenic models, mouse reconstitution models, severe combined immunodeficiency syndrome mouse models, canine models, xenograft models, and cell culture models); 3) biomarkers in prostate cancer, most of which have been tested only as predictive factors for patient outcome after treatment rather than as risk factors; and 4) genotoxic and nongenotoxic mechanisms of carcinogenesis. The authors conclude that most of the data regarding risk relies, of necessity, on epidemiologic studies, but animal and cell culture models offer promise in confirming some important findings. The current understanding of biomarkers of disease and risk factors is limited. An understanding of the risk factors for prostate cancer has practical importance for public health research and policy, genetic and nutritional education and chemoprevention, and prevention strategies.

STUDY DESIGN: Prospective, single-group, repeated-measures design. OBJECTIVE: To identify exercises that could be used for strength development and the exercises that would be more appropriate for endurance or stabilization training. BACKGROUND: The exercises analyzed are often used in rehabilitation programs for the spine, hip, and knee. They are active exercises using body weight for resistance; thus a clinician is unable to determine the amount of resistance being applied to a muscle group. Electromyographic (EMG) analysis can provide a measure of muscle activation so that the clinician can have a better idea about the effect the exercise may have on the muscle for strength, endurance, or stabilization. METHODS AND MEASURES: Surface EMG analysis was carried out in 19 males and 11 females while performing the following 9 exercises: active hip abduction, bridge, unilateral-bridge, side-bridge, prone-bridge on the elbows and toes, quadruped arm/lower extremity lift, lateral step-up, standing lunge, and using the Dynamic Edge. The rectus abdominis, external oblique abdominis, longissimus thoracis, lumbar multifidus, gluteus maximus, gluteus medius, vastus medialis obliquus, and hamstring muscles were studied. RESULTS: In healthy subjects, the lateral step-up and the lunge exercises produced EMG levels greater than 45% maximum voluntary isometric contraction (MVIC) in the vastus medialis obliquus, which suggests that they may be beneficial for strengthening that muscle. The side-bridge exercise could be used for strengthening the gluteus medius and the external oblique abdominis muscles, and the quadruped arm/lower extremity lift exercise may help strengthen the gluteus maximus muscle. All the other exercises produced EMG levels less than 45% MVIC, so they may be more beneficial for training endurance or stabilization in healthy subjects. CONCLUSION: Our results suggest these exercises could be used for a core rehabilitation or performance enhancement program. Depending on the individual needs of a patient or athlete, some of the exercises may be more beneficial than others for achieving strength.

Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.