Production of Human ATG Proteins for Lipidation Assays.

Humans express several orthologs of yeast Atg8, in the LC3 and GABARAP families, which play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of LC3 and GABARAP regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. LC3 and GABARAP are families of related ubiquitin-like proteins (UBLs) (referred to here collectively as LC3/GABARAP), and their lipidation involves a divergent UBL conjugation cascade including ATG7, ATG3, and ATG12~ATG5-ATG16L1 acting as E1, E2, and E3 enzymes, respectively. ATG7 initiates LC3/GABARAP conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of ATG3. Ultimately, the ATG12~ATG5-ATG16L1 complex catalyzes LC3/GABARAP ligation to a primary amino group on PE or other acceptor lipids. This chapter describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12~ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation.

The contribution of familial internalizing and externalizing liability factors to borderline personality disorder.

BACKGROUND: Individuals with borderline personality disorder (BPD) frequently display co-morbid mental disorders. These disorders include 'internalizing' disorders (such as major depressive disorder and anxiety disorders) and 'externalizing' disorders (such as substance use disorders and antisocial personality disorder). It is hypothesized that these disorders may arise from latent 'internalizing' and 'externalizing' liability factors. Factor analytic studies suggest that internalizing and externalizing factors both contribute to BPD, but the extent to which such contributions are familial is unknown. METHOD: Participants were 368 probands (132 with BPD; 134 without BPD; and 102 with major depressive disorder) and 885 siblings and parents of probands. Participants were administered the Diagnostic Interview for DSM-IV Personality Disorders, the Revised Diagnostic Interview for Borderlines, and the Structured Clinical Interview for DSM-IV. RESULTS: On confirmatory factor analysis of within-person associations of disorders, BPD loaded moderately on internalizing (factor loading 0.53, S.E. = 0.10, p < 0.001) and externalizing latent variables (0.48, S.E. = 0.10, p < 0.001). Within-family associations were assessed using structural equation models of familial and non-familial factors for BPD, internalizing disorders, and externalizing disorders. In a Cholesky decomposition model, 84% (S.E. = 17%, p < 0.001) of the association of BPD with internalizing and externalizing factors was accounted for by familial contributions. CONCLUSIONS: Familial internalizing and externalizing liability factors are both associated with, and therefore may mutually contribute to, BPD. These familial contributions account largely for the pattern of co-morbidity between BPD and internalizing and externalizing disorders.

Comparison of alternative models for personality disorders, II: 6-, 8- and 10-year follow-up.

BACKGROUND: Several conceptual models have been considered for the assessment of personality pathology in DSM-5. This study sought to extend our previous findings to compare the long-term predictive validity of three such models: the five-factor model (FFM), the schedule for nonadaptive and adaptive personality (SNAP), and DSM-IV personality disorders (PDs). METHOD: An inception cohort from the Collaborative Longitudinal Personality Disorder Study (CLPS) was followed for 10 years. Baseline data were used to predict long-term outcomes, including functioning, Axis I psychopathology, and medication use. RESULTS: Each model was significantly valid, predicting a host of important clinical outcomes. Lower-order elements of the FFM system were not more valid than higher-order factors, and DSM-IV diagnostic categories were less valid than dimensional symptom counts. Approaches that integrate normative traits and personality pathology proved to be most predictive, as the SNAP, a system that integrates normal and pathological traits, generally showed the largest validity coefficients overall, and the DSM-IV PD syndromes and FFM traits tended to provide substantial incremental information relative to one another. CONCLUSIONS: DSM-5 PD assessment should involve an integration of personality traits with characteristic features of PDs.

The association of personality disorders with the prospective 7-year course of anxiety disorders.

BACKGROUND: This study prospectively examined the natural clinical course of six anxiety disorders over 7 years of follow-up in individuals with personality disorders (PDs) and/or major depressive disorder. Rates of remission, relapse, new episode onset and chronicity of anxiety disorders were examined for specific associations with PDs. METHOD: Participants were 499 patients with anxiety disorders in the Collaborative Longitudinal Personality Disorders Study, who were assessed with structured interviews for psychiatric disorders at yearly intervals throughout 7 years of follow-up. These data were used to determine probabilities of changes in disorder status for social phobia (SP), generalized anxiety disorder (GAD), obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), panic disorder and panic disorder with agoraphobia. RESULTS: Estimated remission rates for anxiety disorders in this study group ranged from 73% to 94%. For those patients who remitted from an anxiety disorder, relapse rates ranged from 34% to 67%. Rates for new episode onsets of anxiety disorders ranged from 3% to 17%. Specific PDs demonstrated associations with remission, relapse, new episode onsets and chronicity of anxiety disorders. Associations were identified between schizotypal PD with course of SP, PTSD and GAD; avoidant PD with course of SP and OCD; obsessive-compulsive PD with course of GAD, OCD, and agoraphobia; and borderline PD with course of OCD, GAD and panic with agoraphobia. CONCLUSIONS: Findings suggest that specific PD diagnoses have negative prognostic significance for the course of anxiety disorders underscoring the importance of assessing and considering PD diagnoses in patients with anxiety disorders.

Feeding zilpaterol hydrochloride to calf-fed Holsteins has minimal effects on semimembranosus steak color.

To determine the effects of feeding zilpaterol hydrochloride (ZH) for 0, 20, 30, or 40 d (ZH0, ZH20, ZH30, ZH40) on semimembranosus (SM) steak color and color stability in 3 packaging systems, SM subprimals were removed from 60 calf-fed Holstein steers 24 h postmortem. A 7.62-cm-thick portion was removed from each subprimal and stored (2 degrees C) for 21 d; then two 2.54-cm-thick steaks were cut, overwrapped with polyvinyl chloride (PVC) film, and assigned to 0 or 3 d of display. Remaining portions of the subprimals were vacuum packaged for 10 d and then enhanced (10% with a solution containing 0.3% sodium chloride, 0.35% phosphate, and 0.05% rosemary extract), cut into steaks, packaged in high-oxygen (HO-MAP) or carbon monoxide (CO-MAP) modified atmosphere packaging (MAP), and assigned to 0, 3, or 5 d (HO-MAP) or 0 or 9 d (CO-MAP) of display. Panelists evaluated the deep and superficial portions of SM steaks for initial color, display color, discoloration, pH, L*, a*, b*, hue angle, and saturation indices. Feeding duration did not affect (P > 0.05) initial color scores of steaks in PVC. Steaks displayed in PVC from ZH20 or ZH30 diets were slightly brighter and less discolored than the ZH40 treatment. For enhanced steaks in HO-MAP, ZH20 steaks were darker on d 5 (P < 0.05) and more discolored (P < 0.05) on d 3 through 5 than all other diet treatments. For enhanced steaks from steers fed ZH40 and in CO-MAP, the deep and superficial SM tended (P > 0.05) to have improved display color compared with other dietary regimens; however, steaks in CO-MAP from all feeding durations had less than 20% metmyoglobin through d 9 of display. Overall, feeding ZH20 might result in steaks with slightly less color stability when packaged in HO-MAP; however, feeding ZH20 or ZH30 to calf-fed Holstein steers will yield steaks that have equal to or more desirable color traits when packaged in PVC or CO-MAP. Regardless of ZH feeding regimen, HO-MAP and CO-MAP extended the color life of the SM. The CO-MAP system minimized color differences between the superficial and deep portions of the SM muscle and extended total case life compared with traditional and HO-MAP packaging.

Effects of zilpaterol hydrochloride feeding duration on crossbred beef semimembranosus steak color in aerobic or modified atmosphere packaging.

The objective of this research was to determine the effects of feeding zilpaterol hydrochloride (ZH) for 0, 20, 30, or 40 d before slaughter (ZH0, ZH20, ZH30, or ZH40, respectively) on semimembranosus (SM) color development and stability. A 7.62-cm-thick portion was removed from 60 beef steer SM subprimals and stored (2 degrees C) for 21 d; then two 2.54-cm-thick steaks were cut, overwrapped with polyvinyl chloride (PVC) film, and assigned to 0 or 3 d of display. Remaining portions of the subprimals were stored in a vacuum for 10 d and then enhanced 10% to a meat concentration of 0.3% sodium chloride, 0.35% phosphate, and 0.05% rosemary extract. Steaks were packaged in a high-oxygen (HO-MAP) or carbon monoxide (CO-MAP) modified atmosphere and assigned to 0, 3, or 5 d (HO-MAP) or 0 or 9 d (CO-MAP) of display. The deep (DSM) and superficial (SSM) portions of steaks were evaluated for initial color, display color, discoloration, pH, L*, a*, b*, hue angle, and saturation indices. For steaks in PVC, no differences (P > 0.05) occurred in initial or discoloration color scores because of ZH feeding duration. The enhanced SSM steaks from ZH20 in PVC were brighter red (P < 0.05) than SSM steaks from ZH40 in PVC. The DSM in PVC had less (P < 0.05) pH and paler (P < 0.05) color than the SSM. Display color scores for the DSM of PVC steaks were brighter red (P < 0.05) than the SSM initially (d 0 and 1), but the DSM discolored faster (P < 0.05) than the SSM on d 1 to 3. The SM steaks from steers fed ZH20 or ZH30 were slightly brighter and less discolored during display in PVC than the ZH40 diet. For enhanced steaks in HO-MAP, the DSM of ZH20 and ZH30 diets displayed 4 d and the DSM of ZH20 displayed 5 d was a brighter (P < 0.05) red than the DSM from ZH40. At display d 1 and 5, the SSM of ZH20 steaks in HO-MAP was a brighter (P < 0.05) red than SSM steaks from ZH40. The SSM of ZH40 HO-MAP steaks was darker (P < 0.05) red on d 3 than the SSM from other diets. For enhanced steaks in CO-MAP, ZH30 steaks were brighter (P < 0.05) red than ZH0 or ZH40 steaks on d 0 and 9 of display. Steaks in CO-MAP from all feeding durations were less than 20% discolored through d 9. The DSM was lighter (P < 0.05) than the SSM on d 0 for steaks packaged in HO-MAP and CO-MAP. Feeding cattle ZH for 20 or 30 d will yield steaks with color characteristics equal to or better than steaks from control cattle, whereas feeding ZH for 40 d will likely produce less desirable meat color traits.

Personality traits as prospective predictors of suicide attempts.

OBJECTIVE: To examine higher order personality factors of negative affectivity (NA) and disinhibition (DIS), as well as lower order facets of impulsivity, as prospective predictors of suicide attempts in a predominantly personality disordered sample. METHOD: Data were analyzed from 701 participants of the Collaborative Longitudinal Personality Disorders Study with available follow-up data for up to 7 years. Cox proportional hazards regression analyses was used to examine NA and DIS, and facets of impulsivity (e.g. urgency, lack of perseverance, lack of premeditation and sensation seeking), as prospective predictors of suicide attempts. RESULTS: NA, DIS and all facets of impulsivity except for sensation seeking were significant in univariate analyses. In multivariate models which included sex, childhood sexual abuse, course of major depressive disorder and substance use disorders, only NA and lack of premeditation remained significant in predicting suicide attempts. DIS and the remaining impulsivity facets were not significant. CONCLUSION: NA emerged as a stronger and more robust predictor of suicide attempts than DIS and impulsivity, and warrants greater attention in suicide risk assessment. Distinguishing between facets of impulsivity is important for clinical risk assessment.

Improvement in borderline personality disorder in relationship to age.

OBJECTIVE: It is commonly believed that some features of borderline personality disorder (BPD) improve as individuals reach their late 30s and 40s. This study examined age-related change in borderline criteria and functional impairment, testing the hypothesis that older age would be associated with relatively more improvement than younger age. METHOD: A total of 216 male and female participants with BPD were followed prospectively with yearly assessments over 6 years. RESULTS: Participants showed similar rates of improvement in borderline features regardless of age. A significant age by study year interaction showed functioning in older subjects to reverse direction and begin to decline in the latter part of the follow-up, in contrast to younger subjects who maintained or continued improvement over the 6 years. Despite the decline, functioning for the older subjects was comparable with or slightly better at year 6 than at year 1. CONCLUSION: Improvement in borderline features is not specific to the late 30s and 40s. There may be a reversal of improvement in functioning in some borderline patients in this older-age range.

The 10-year course of physically self-destructive acts reported by borderline patients and axis II comparison subjects.

OBJECTIVE: The purpose of this paper was to determine the frequency and methods of two forms of physically self-destructive acts (i.e. self-mutilation and suicide attempts) reported by borderline patients and axis II comparison subjects over 10 years of prospective follow-up. METHOD: Two hundred and ninety borderline patients and 72 axis II comparison subjects were interviewed about their physically self-destructive acts during their index admission and at five contiguous 2-year follow-up periods. RESULTS: It was found that a high percentage of borderline patients reported multiple acts and methods of each of these two forms of physically self-destructive behavior prior to their index admission. It was also found that the percentage of borderline patients reporting multiple acts and methods declined significantly over time. However, these acts remained significantly more common among borderline patients than axis II comparison subjects. CONCLUSION: The course of self-mutilation and suicide attempts among borderline patients is initially more serious and ultimately more benign than previously recognized.

Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil.

Infection by ectomycorrhizal (ECM) fungi may benefit hybrid poplar growing in contaminated soils by providing greater access to water and nutrients and possibly protecting the trees from direct contact with toxic contaminants. The objective of this research was to determine the effect of colonization of the ECM fungus Pisolithus tinctorius (Pers.) Coker & Couch on hybrid poplar fine root production, biomass and N and P uptake when grown in diesel-contaminated soil (5000 mg diesel fuel kg soil(-1)). Commercially available Mycogrow Tree Tabs were the source of inoculum. A minirhizotron camera was used to provide the data necessary for estimating fine root production. Colonization of hybrid poplar roots (P. deltoides x [P. laurifolia x P. nigra] cv. Walker) by P. tinctorius increased total fine root production in diesel-contaminated soil to 56.58 g m(-2) compared to 22.59 g m(-2) in the uncolonized, diesel-contaminated treatment. Hybrid poplar leaf N and P concentrations were significantly greater in the diesel-contaminated/ECM-colonized treatment compared to the diesel-contaminated/uncolonized treatment after 12 wk, while significantly less diesel fuel was recovered from the soil of the uncolonized treatment compared to the colonized treatment. Both planted treatments removed more contaminants from the soil than an unplanted control. Significantly greater concentrations of total petroleum hydrocarbons (TPH) were found sequestered in hybrid poplar root/fungal-sheath complexes from the colonized treatment compared to the roots of the uncolonized treatment. The results of this study indicate that over a 12-wk growth period, ECM colonization of hybrid poplar in diesel-contaminated soils increased fine root production and whole-plant biomass, but inhibited removal of TPH from the soil.

Occurrence of infected amoebae in cooling towers compared with natural aquatic environments: implications for emerging pathogens.

Many species of bacteria pathogenic to humans, such as Legionella, are thought to have evolved in association with amoebal hosts. Several novel unculturable bacteria related to Legionella have also been found in amoebae, a few of which have been thought to be causes of nosocomial infections in humans. Because amoebae can be found in cooling towers, we wanted to know whether cooling tower environments might enhance the association between amoebae and bacterial pathogens of amoebae in order to identify potential "hot spots" for emerging human pathogens. To compare occurrence of infected amoebae in natural environments with those in cooling towers, 40 natural aquatic environments and 40 cooling tower samples were examined. Logistic regression analysis determined variables that were significant predictors of the occurrence of infected amoebae, which were found in 22 of 40 cooling tower samples but in only 3 of the 40 natural samples. An odds ratio showed that it is over 16 times more likely to encounter infected amoebae in cooling towers than in natural environments. Environmental data from cooling towers and natural habitats combined revealed dissolved organic carbon (DOC) and pH were predictors of the occurrence of the pathogens, however, when cooling tower data alone were analyzed, no variables accounted for the occurrence. Several bacteria have novel rRNA sequences, and most strains were not culturable outside of amoebae. Such pathogens of amoebae may spread to the environment via aerosols from cooling towers. Studies of emerging infectious diseases should strongly consider cooling towers as a source of amoeba-associated pathogens.

Hierarchical relationships between borderline, schizotypal, avoidant and obsessive-compulsive personality disorders.

OBJECTIVE: Comorbidity among personality disorders is widely considered problematic. The validity of one proposed solution, diagnostic hierarchies, was investigated in the current study with respect to borderline, schizotypal, avoidant, and obsessive-compulsive personality disorders. METHOD: One approach used discriminant functions, derived from multiple psycho-social domains, that were used to classify comorbid individuals from the Collaborative Longitudinal Personality Disorder study (CLPS) to explore the possibility of hierarchical precedence of one personality disorder over another. A second approach examined the incremental increase in R(2)-value in predicting functioning and personality provided by each diagnosis over each other diagnosis. RESULTS: Obsessive-compulsive personality disorder was consistently subordinate to other diagnoses, whereas other indications of hierarchical relationships were domain-specific. CONCLUSION: Results indicate minimal support for an over-arching hierarchical pattern among studied personality disorders, and suggest the inclusion of all relevant diagnoses in clinical practice.

Avoidant personality disorder and social phobia: distinct enough to be separate disorders?

OBJECTIVE: Existing evidence from anxiety disorder research indicates that social phobics (SP) with avoidant personality disorder (AVPD) experience more anxiety and show more impairment than patients with SP alone. The purpose of this study was to examine whether in patients diagnosed with AVPD, the co-occurrence of SP adds to its severity. We hypothesized that the addition of SP will not add to the severity of AVPD alone. METHOD: Two groups of patients (AVPD=224; AVPD/SP=101) were compared at baseline and 2 years later on multiple demographic and clinical variables. RESULTS: Patients with AVPD and an additional diagnosis of SP differed little from patients with AVPD alone. CONCLUSION: These findings suggest that AVPD and SP may be alternative conceptualizations of the same disorder.

Stressful life events as predictors of functioning: findings from the collaborative longitudinal personality disorders study.

OBJECTIVE: Although much attention has been given to the effects of adverse childhood experiences on the development of personality disorders (PDs), we know far less about how recent life events influence the ongoing course of functioning. We examined the extent to which PD subjects differ in rates of life events and the extent to which life events impact psychosocial functioning. METHOD: A total of 633 subjects were drawn from the Collaborative Longitudinal Personality Disorders Study (CLPS), a multi-site study of four personality disorders--schizotypal (STPD), borderline (BPD), avoidant (AVPD), obsessive-compulsive (OCPD)--and a comparison group of major depressive disorders (MDD) without PD. RESULTS: Borderline personality disorder subjects reported significantly more total negative life events than other PDs or subjects with MDD. Negative events, especially interpersonal events, predicted decreased psychosocial functioning over time. CONCLUSION: Our findings indicate higher rates of negative events in subjects with more severe PDs and suggest that negative life events adversely impact multiple areas of psychosocial functioning.

Longitudinal diagnostic efficiency of DSM-IV criteria for obsessive-compulsive personality disorder: a 2-year prospective study.

OBJECTIVE: To examine the longitudinal diagnostic efficiency of the DSM-IV criteria for obsessive-compulsive personality disorder (OCPD). METHOD: At baseline, criteria and diagnoses were determined using diagnostic interviews, and blinded assessments were performed 24 months later with 550 participants. Diagnostic efficiency indices (conditional probabilities, total predictive power, and kappa) were calculated for each criterion determined at baseline, using the independent OCPD diagnosis at follow-up as the standard. RESULTS: Longitudinal diagnostic efficiencies for the OCPD criteria varied; findings suggested the overall predictive utility of 'preoccupied with details', 'rigid and stubborn', and 'reluctant to delegate'. CONCLUSION: These findings suggest the predictive validity of three cognitive-interpersonal OCPD criteria.

Internal consistency, intercriterion overlap and diagnostic efficiency of criteria sets for DSM-IV schizotypal, borderline, avoidant and obsessive-compulsive personality disorders.

OBJECTIVE: To evaluate performance characteristics of DSM-IV Personality Disorders (PDs) criteria. METHOD: Six hundred and sixty-eight adults recruited for the Collaborative Longitudinal Personality Disorders Study (CLPS) were assessed with diagnostic interviews. RESULTS: Within-category inter-relatedness was evaluated by Cronbach's alpha and median intercriterion correlations (MIC). Cronbach's alpha ranged from 0.47 to 0.87 (median=0.71); seven of the 10 PDs had alphas greater than 0.70. Between-category criterion overlap was evaluated by "inter-category" intercriterion correlations between all PD pairs (ICMIC). ICMIC values (median=0.08) were lower than MIC values (median=0.23). Diagnostic efficiency statistics (sensitivity, specificity, positive predictive power and negative predictive power were calculated for schizotypal, borderline, avoidant and obsessive-compulsive PDs. CONCLUSION: DSM-IV PD criteria sets have some convergent validity and discriminant validity: criteria for individual PDs correlate better with each other than with criteria for other PDs. Diagnostic efficiency statistics provide guidance regarding usefulness of criteria for inclusion or exclusion.

Borderline personality disorder. Suicidality and self-mutilation.

Epidemiological and neurobiological perspectives about suicidality in borderline patients are described, highlighting how self-destructive and seemingly suicidal acts are their "behavioral specialty." Principles for management, including the need for a primary clinician to oversee safety, and the pros and cons of both "contracting for safety" and hospitalization are presented. Clinical material is used to illustrate an approach that involves minimal initiative to rescue by therapists, a readiness to comply with patients' wishes for protection (the principle of false submission), and very active interpretation. This approach is contrasted with those advocated by Linehan and Kernberg.

Differentiating narcissistic and antisocial personality disorders.

The conceptual, clinical, and empirical overlap between the constructs of narcissistic personality disorder (NPD) and antisocial personality disorder (ASPD) is reviewed and their descriptive discriminability is investigated. Twenty-four patients with NPD and 16 patients with ASPD were compared on 33 characteristics for pathological narcissism assessed with the semistructured Diagnostic Interview for Narcissism. The results confirm a sufficiently broad array of similarities that the question of whether these categories should be kept separate (as they are in DSM-IV) is underscored. The results also indicate important areas of difference. The NPD sample was best discriminated from the ASPD sample by their grandiosity, that is, the tendency to exaggerate their talents, and to regard themselves as more unique and superior.

An intensive outpatient program for patients with borderline personality disorder.

Intensive outpatient programs are designed to promote patients' functioning in the community by offering a more intensive level of structure and support than was previously available for outpatients. This paper describes the intensive outpatient program at McLean Hospital in Belmont, Massachusetts, which is tailored for patients with borderline personality disorder. These patients are susceptible to control struggles and regressive behaviors in more restrictive treatment settings. Through frequent contact with clinicians and other patients in this group-oriented program, patients with borderline personality disorders appear to feel sufficiently "held" and understood to develop their functional capacities as outpatients.