Invariant BECN1 CXXC motifs bind Zn2+ and regulate structure and function of the BECN1 intrinsically disordered region.

ABBREVIATIONS: AFM: aromatic finger mutant; BH3D: BCL2 homology 3 domain; CCD: coiled-coil domain; CD: circular dichroism spectroscopy; [CysDM1]: C18S and C21S double mutant; [CysDM2]: C137S, and C140S double mutant; [CysTM], C18S, C21S, C137S, and C140S tetrad mutant; Dmax: maximum particle diameter; dRI, differential refractive index; EFA: evolving factor analysis; FHD: flexible helical domain; FL: full length; GFP: green fluorescent protein; HDX-MS: hydrogen/deuterium exchange mass spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; IDR: intrinsically disordered region; ITC, isothermal titration calorimetry; MALS, multi angle light scattering; MBP: maltose-binding protein; MoRFs: molecular recognition features; P(r): pairwise-distance distribution; PtdIns3K: class III phosphatidylinositol 3-kinase; Rg: radius of gyration; SASBDB: small angle scattering biological data bank; SEC: size-exclusion chromatography; SEC-SAXS: size-exclusion chromatography in tandem with small angle X-ray scattering; TEV: tobacco-etch virus; TFE: 2,2,2-trifluoroethanol; TPEN: N,N,N,N-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine; Vc: volume of correlation; WT: wild-type.

Phage-derived anti-idiotype and anti-YTE antibodies in development of MK-1654 pharmacokinetic and immune response assays.

Background: MK-1654 is a fully human monoclonal antibody with YTE mutations currently in phase III clinical trials for prophylactic use in protecting infants from human respiratory syncytial virus infection. Materials & methods: We generated anti-idiotype (anti-ID) and anti-YTE antibodies against MK-1654 by panning with MorphoSys HuCal phage libraries, and used the antibodies in the development of MK-1654 pharmacokinetic (PK) and immune response (IR) assays. Results: Detection of MK-1654 in nonhuman primate and human nasal wash samples showed combined use of anti-ID and anti-YTE antibodies can deliver desired sensitivity and accuracy in PK studies. IR studies showed anti-ID can serve as suitable positive control in neutralizing antibody assays. Conclusion: Phage-derived anti-IDs and anti-YTEs are suitable for PK and IR assays.

Successful Criminal Prosecutions of Sex Trafficking and Sexual Abuse of Minors: A Comparative Analysis.

Despite increased awareness of sex trafficking of minors in the U.S., prosecution of traffickers remains difficult, in part because of victim uncooperativeness. There are questions about how that uncooperativeness is expressed, whether it is evident in successfully prosecuted cases, and whether it is unique to trafficked minors or it emerges in similar age victims of sexual abuse. To provide insight relevant to these questions, we compared appellate opinions in two types of successfully prosecuted criminal cases: sex trafficking and sexual abuse of adolescent victims. In the trafficking opinions, victims were rarely described as disclosing on their own or as knowing their trafficker before the victimization. The opinions also often alluded to the trafficking victims' uncooperativeness and delinquency history, and frequently mentioned electronic evidence and prosecution experts. The sexual abuse opinions, in contrast, tended to suggest that victims' own disclosures initiated the case, perpetrators were known and trusted adults, and caregiver support during the case was common. Finally, the sexual abuse opinions never explicitly mentioned victim uncooperativeness or electronic evidence and rarely mentioned expert testimony or delinquency. The different characterizations of the two case types highlight the need for improved education concerning effective prosecution of sex crimes against minors.

Foster youth's placement preferences: The roles of kin, siblings, and age.

BACKGROUND: Debates exist regarding whether foster youth should be asked about their placement preferences following removal, with only youth aged 12 years and older at times assumed legally competent to provide input. OBJECTIVES: The present study evaluated whether placement-related factors known to predict youth's well-being also shape their placement preferences and whether preferences differ between youth below and above the age at which they are considered legally competent to provide input. METHOD: Data (N = 1033, ages 6-17 years, 54 % female) were obtained from NSCAW-I. Youth were asked open- and closed-ended questions about their placement preferences. RESULTS: Among youth removed for shorter periods, placement with kin was related to a greater preference for their current placement (RRR = 0.31, p < .001) and desire for permanency in that placement (OR = 1.95, p = .005) relative to youth placed with non-kin. However, youth removed for longer periods (e.g., a year) were similar in their desires for their current placement to be permanent regardless of whether they were living with kin or non-kin caregivers. Among younger youth, placement with siblings (RRR = 0.42, p = .015) was linked to a preference for their current placement. Racial match between youth and their non-kin caregiver was unrelated to their placement preferences. CONCLUSIONS: Findings revealed that both younger and older youth's placement preferences were shaped by factors objectively linked to youth's well-being and thus align with best practices in placement decisions. The paper discusses the importance of asking youth as young as 6 years about their placement preferences and offers suggestions for social service and legal professionals regarding questioning strategies.

Stress and working memory in children and adolescents: Insights from a multisystem approach.

Despite considerable research with adults suggesting that acute stress negatively affects working memory (WM), a core cognitive function, few studies have assessed these effects in youths. Studies that have been conducted have produced null findings, although these studies did not measure stress via multiple systems (e.g., hypothalamic-pituitary-adrenal [HPA] axis and sympathetic nervous system [SNS]) or include wide developmental age ranges. In the current study, we examined the links between acute stress and WM in 8- to 15-year-olds. Youths completed the Trier Social Stress Test-Modified, during which repeated saliva samples were collected to measure responses of the HPA axis (cortisol) and SNS (salivary alpha-amylase). Immediately afterward, youths completed the n-back task, an established measure of WM. Accuracy and false alarm (FA) scores were computed to explore whether associations between arousal and WM differed when WM versus only the inhibitory control facet of WM processes were considered. Relations varied as a function of age, physiological system, and type of WM process. Accuracy improved and FA scores deceased as age and SNS reactivity increased, particularly in combination. Moreover, when arousal was higher according to only one physiological system (HPA axis or SNS), FA scores were lower, but when arousal was driven by both systems or low in both systems, FA scores were higher. Together, results highlight the need for more complex investigations of stress and WM across development that take into account system-specific responses and multiple facets of WM.

Structural transitions in conserved, ordered Beclin 1 domains essential to regulating autophagy.

Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositol-3-kinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiled-coil domain (CCD). However, the last 16 CCD residues, composing an "overlap helix" (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the C-terminal ß-α repeated, autophagy-specific domain (BARAD). Here, using CD spectroscopy, isothermal titration calorimetry, and small-angle X-ray scattering, we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the two-domain CCD-BARAD. Last, we used cellular assays to demonstrate that mutation of these OH-interface residues abrogates starvation-induced up-regulation of autophagy but does not affect basal autophagy. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains (i.e. the CCD or the BARAD). Our findings have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes.

Interdependence of oxysterols with cholesterol profiles in multiple sclerosis.

PURPOSE: To investigate levels of oxysterols in healthy control (HC) and multiple sclerosis (MS) patients and their interdependence with demographic, clinical characteristics, and cholesterol biomarkers. METHODS: This study included 550 subjects (203 HC, 221 relapsing-remitting MS (RR-MS), 126 progressive MS (P-MS)). A complete lipid profile including total cholesterol (TC); high-density lipoprotein-cholesterol (HDL-C); low-density lipoprotein-cholesterol (LDL-C); apolipoproteins (Apo) A1, A2, B, and E; C-reactive protein (CRP); 24-hydroxycholesterol (HC); 25-HC; 27-HC; 7α-HC; and 7-ketocholesterol (KC) was obtained. Lipoprotein particle sizing by proton nuclear magnetic resonance (H1 NMR) was available for 432 subjects. RESULTS: The levels of 24-HC, 27-HC, and 7α-HC (all p < 0.015) were lower in MS compared to HC, and 7-KC was higher in P-MS compared to RR-MS ( p < 0.001). TC, LDL-C, and ApoB were associated with higher levels of all oxysterols (all p < 0.05) in HC. In MS, LDL-C was associated with higher levels of 24-HC, 25-HC, 7-KC, and 7α-HC (all p < 0.05), while TC and ApoB were associated with increased levels of all oxysterols (all p < 0.005). CONCLUSION: The findings of lower 24-HC, 27-HC, and 7α-HC in MS compared to HC and higher 7-KC in P-MS compared to RR-MS indicate that the oxysterol network is disrupted in MS.

Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome.

Arginine methylation is a posttranslational modification that impacts wide-ranging cellular functions, including transcription, mRNA splicing and translation. RNA binding proteins (RBPs) represent one of the largest classes of arginine methylated proteins in both mammals and the early diverging parasitic protozoan, Trypanosoma brucei. Here, we report the effects of arginine methylation on the functions of the essential and previously uncharacterized T. brucei RBP, DRBD18. RNAseq analysis shows that DRBD18 depletion causes extensive rearrangement of the T. brucei transcriptome, with increases and decreases in hundreds of mRNAs. DRBD18 contains three methylated arginines, and we used complementation of DRBD18 knockdown cells with methylmimic or hypomethylated DRBD18 to assess the functions of these methylmarks. Methylmimic and hypomethylated DRBD18 associate with different ribonucleoprotein complexes. These altered macromolecular interactions translate into differential impacts on the T. brucei transcriptome. Methylmimic DRBD18 preferentially stabilizes target RNAs, while hypomethylated DRBD18 is more efficient at destabilizing RNA. The protein arginine methyltransferase, TbPRMT1, interacts with DRBD18 and knockdown of TbPRMT1 recapitulates the effects of hypomethylated DRBD18 on mRNA levels. Together, these data support a model in which arginine methylation acts as a switch that regulates T. brucei gene expression.