Exposure to Multiple Forms of Bias Victimization on Youth and Young Adults: Relationships with Trauma Symptomatology and Social Support.

Research has documented that a significant portion of youth are exposed to bias victimization. However, less is known about whether experiencing certain types of bias victimization (e.g., sexual orientation bias) is more or less likely to be related to a more extensive bias victimization history (i.e., experiencing multiple types of bias victimization) and whether exposure to multiple types of bias victimization explains any relationships between specific types of bias victimization and negative outcomes. To address these gaps, the current study explores relationships between exposure to multiple types of bias-motivated victimization, trauma symptomatology and perceived social support. Participants were 854 youth and young adults (60.9% female) from three higher risk communities who completed a survey on personal experiences with bias-related victimization. The average age of participants was 16.6 years; 28.5% of the sample described themselves as Black or African American; 13.4% as Hispanic or Latino (any race); 45.3% as White, and 12.8% as another race. Sixty-nine percent of the sample described their sexual orientation as heterosexual; 8.9% as gay, lesbian, or homosexual; 12.5% as bisexual; and 9.5% as another sexual orientation. Sixty-three percent of participants reported at least one type of bias victimization in their lifetime, and more than one in three youth (38.7%) experienced two or more types of bias victimization in their lifetimes (18.1% two types, 12.1% three types, and 8.5% four or more types). Experiencing multiple types of bias victimization was related to higher trauma symptomatology and less perceived social support. Experiencing multiple types of bias victimization attenuated or eliminated the association between individual types of bias victimization and well-being. The findings contribute to a growing body of research demonstrating the damaging mental health effects of occupying multiple marginalized statuses, and points to the cumulation of bias victimization experiences as an important factor contributing to significant differences in well-being and support among youth and young adults.

Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry.

Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using cation exchange chromatography is routinely used as a fingerprint of the distribution of posttranslational modifications present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identification of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high-resolution native mass spectrometry.

Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells and conditioned media during apoptosis and necrosis.

Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D-LC-MSE discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control.

Ethical and Practical Considerations for Collecting Research-Related Data from Commercially Sexually Exploited Children.

This article presents seven challenges of collecting primary (i.e., firsthand) data from commercially sexually exploited children (CSEC). We drew on our research team's experience collecting longitudinal data from 28 CSEC survivors with a 12-month follow-up period. We used both face-to-face and electronic group brainstorming methods to nominate a list of research-related challenges. The two main themes that were identified were challenges that can limit data quality and concerns about the impact of research on participants, researchers, and others. The three challenges related to data quality are (1) the age of the research participants; (2) questions about obtaining informed consent from parents or guardians; and (3) the over-interrogation of CSEC youth. The four challenges related to concerns about the impact of research were (4) concerns that research participation may further exploit youth; (5) staying in the role of researcher and refraining from providing advocacy; (6) secondary trauma and burnout experienced by research staff; and (7) the additional burden that research and data collection may place on the advocates and direct service providers. Because the process of collecting data from CSEC youth can be complicated, and rife with ethical and practical challenges, we have relayed our experiences with seven specific research-related challenges in order to stimulate discourse and further progress in the field.

Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer.

The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50-60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.

Religious Beliefs Against Smoking Among Black and White Urban Youth.

This study explores the relation of religiosity to cigarette smoking in a sample of 4776 Black versus White adolescents. Findings show that Black adolescents have significantly stronger religious beliefs against smoking than do White students. Further, teens with strong or very strong religious beliefs are less likely to have smoked. The protective effect of religious beliefs against smoking was stronger for Whites than for Blacks. These findings suggest that efforts in the Black religious community to prevent cigarette smoking have been somewhat successful. Similar efforts in the White community might help stem the tide of tobacco use among White teens.

Quantitative host cell protein analysis using two dimensional data independent LC-MS(E).

Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed. Mass spectrometry methods provide rationale for targeted removal strategies through the provision of both qualitative and quantitative HCP information. A high pH, low pH, reversed-phase data independent 2D-LC-MS(E) proteomic platform was applied to determine HCP repertoires in the Protein A purified monoclonal antibody (mAb) samples as a function of culture harvest time, elution buffer used for DSP and also following inclusion of additional DSP steps. Critical quality attributes (CQAs) were examined for mAbs purified with different Protein A elution buffers to ensure that the selected buffers not only minimized the HCP profile but also exhibited no adverse effect on product quality. Results indicated that an arginine based Protein A elution buffer minimized the levels of HCPs identified and quantified in a purified mAb sample and also demonstrated no impact on product CQAs. It was also observed that mAbs harvested at later stages of cell culture contained higher concentrations of HCPs but that these were successfully removed by the addition of DSP steps complementary to Protein A purification. Taken together, our results showed how mass spectrometry based methods for HCP determination in conjunction with careful consideration of processing parameters such as harvest time, Protein A elution buffers, and subsequent DSP steps can reduce the HCP repertoire of therapeutic mAbs.

Comparative analysis of monoclonal antibody N-glycosylation using stable isotope labelling and UPLC-fluorescence-MS.

A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.

Mechanistic insight into Myc stabilization in breast cancer involving aberrant Axin1 expression.

High expression of the oncoprotein Myc has been linked to poor outcome in human tumors. Although MYC gene amplification and translocations have been observed, this can explain Myc overexpression in only a subset of human tumors. Myc expression is in part controlled by its protein stability, which can be regulated by phosphorylation at threonine 58 (T58) and serine 62 (S62). We now report that Myc protein stability is increased in a number of breast cancer cell lines and this correlates with increased phosphorylation at S62 and decreased phosphorylation at T58. Moreover, we find this same shift in phosphorylation in primary breast cancers. The signaling cascade that controls phosphorylation at T58 and S62 is coordinated by the scaffold protein Axin1. We therefore examined Axin1 in breast cancer and report decreased AXIN1 expression and a shift in the ratio of expression of two naturally occurring AXIN1 splice variants. We demonstrate that this contributes to increased Myc protein stability, altered phosphorylation at S62 and T58, and increased oncogenic activity of Myc in breast cancer. Thus, our results reveal an important mode of Myc activation in human breast cancer and a mechanism contributing to Myc deregulation involving unique insight into inactivation of the Axin1 tumor suppressor in breast cancer.

Application of multi-omics techniques for bioprocess design and optimization in chinese hamster ovary cells.

Significant improvements in the productivity and quality of therapeutic proteins produced in Chinese hamster ovary (CHO) cells have been reported since their establishment as host cells for biopharmaceutical production. Initial advances in the field focused on engineering strategies to manipulate genes associated with proliferation, apoptosis, and various metabolic pathways. Process engineering efforts to optimize culture media, batch-feeding strategies and culture conditions, including temperature and osmolarity, were also reported. More recently, focus has shifted toward enhancing process consistency and product quality using systems biology quality by design-based approaches during process development. Integration of different data generated using omics technologies, such as genomics, transcriptomics, proteomics and metabolomics, has facilitated a greater understanding of CHO cell biology. These techniques have enabled the provision of global information on dynamic changes in cellular components associated with different phenotypes. Using systems biology to understand these important host cells at the cellular level will undoubtedly result in further progression in the development and expression of biopharmaceutical products in CHO cells.

Enhancing detection of labor violations in the agricultural sector: A multilevel generalized linear regression model of H-2A violation counts.

Agricultural workers are essential to the supply chain for our daily food, and yet, many face harmful work conditions, including garnished wages, and other labor violations. Workers on H-2A visas are particularly vulnerable due to the precarity of their immigration status being tied to their employer. Although worksite inspections are one mechanism to detect such violations, many labor violations affecting agricultural workers go undetected due to limited inspection resources. In this study, we identify multiple state and industry level factors that correlate with H-2A violations identified by the U.S. Department of Labor's Wage and Hour Division using a multilevel zero-inflated negative binomial model. We find that three state-level factors (average farm acreage size, the number of agricultural establishments with less than 20 employees, and higher poverty rates) are correlated with H-2A violations. These findings offer valuable insights into where H-2A violations are being detected at the state and industry levels.

The Axin1 scaffold protein promotes formation of a degradation complex for c-Myc.

Expression of the c-Myc proto-oncoprotein is tightly regulated in normal cells. Phosphorylation at two conserved residues, threonine58 (T58) and serine62 (S62), regulates c-Myc protein stability. In cancer cells, c-Myc can become aberrantly stabilized associated with altered T58 and S62 phosphorylation. A complex signalling cascade involving GSK3beta kinase, the Pin1 prolyl isomerase, and the PP2A-B56alpha phosphatase controls phosphorylation at these sites. We report here a novel role for the tumour suppressor scaffold protein Axin1 in facilitating the formation of a degradation complex for c-Myc containing GSK3beta, Pin1, and PP2A-B56alpha. Although knockdown of Axin1 decreases the association of c-Myc with these proteins, reduces T58 and enhances S62 phosphorylation, and increases c-Myc stability, acute expression of Axin1 reduces c-Myc levels and suppresses c-Myc transcriptional activity. Moreover, the regulation of c-Myc by Axin1 is impaired in several tested cancer cell lines with known stabilization of c-Myc or loss of Axin1. This study provides critical insight into the regulation of c-Myc expression, how this can be disrupted in three cancer types, and adds to our knowledge of the tumour suppressor activity of Axin1.

Characterization of the DNA binding specificity of Shelterin complexes.

The Shelterin complex associates with telomeres and plays an essential role in telomere protection and telomerase regulation. In its most abundant form, the complex is composed of six core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Of these subunits, three can interact directly with either single-stranded (POT1) or double-stranded (TRF1, TRF2) telomeric DNA. In this report, we have developed assays to measure the DNA binding activity of Shelterin complexes in human cell extracts. With these assays, we have characterized the composition and DNA binding specificity of two Shelterin complexes: a 6-member complex that contains all six core components and a second complex that lacks TRF1. Our results show that both of these complexes bind with high affinity (K(D) = 1.3-1.5 × 10(-9) M) and selectively to ds/ss-DNA junctions that carry both a binding site for POT1 (ss-TTAGGGTTAG) and a binding site for the SANT/Myb domain of TRF1 or TRF2 (ds-TTAGGGTTA). This DNA binding specificity suggests the preferential recruitment of these complexes to areas of the telomere where ss- and ds-DNA are in close proximity, such as the 3'-telomeric overhang, telomeric DNA bubbles and the D-loop at the base of T-loops.

Gender differences in the effects of parental underestimation of youths' secondary exposure to community violence.

Secondary exposure to community violence is particularly detrimental for male youths, who disproportionately report witnessing community violence and suffering associated trauma-related symptoms. Yet, few studies have investigated whether parents perceive and report similar gender disparities among youths. In addition, few studies have examined the potentially negative effects of parent-child discord as to the youth's level of exposure to violence, or whether these effects vary across gender. Therefore, this study investigated whether differences between parents' and youths' reports of youths' exposure to violence, and the consequences of such reporting discord, varied across the gender of the youth informant. Participants were adolescents aged approximately 12 and 15 years at baseline (N = 1,517; 51 % female). Descriptive analyses indicated that male youths reported significantly higher levels of exposure to violence than female youths, but parents similarly under-reported their male and female children's experiences with violence. Hierarchical analyses indicated that parental underestimation of youths' exposure to violence had negative consequences. Moreover, significant interaction effects demonstrated that only females responded to reporting discord with internalizing problems. Conversely, both male and female youths responded to reporting discord with externalizing problems and offending. The results suggest that while parent-child discord is associated with negative outcomes for both male and female youths, discord may be disproportionately associated with negative outcomes among young females. The findings speak to the utility of examining the correlates and consequences of exposure to violence from a "gendered" perspective.

Which Latinos Experience Bias Victimization? An Examination of Acculturation, Immigrant Status, and Socio-economic Status.

The present study examined factors that could be associated with bias victimization according to intergroup threat theory, namely socioeconomic status (SES), acculturation (Anglo orientation and Latino orientation), immigrant status, and their interactions. Self-identified Latino participants (N = 910) from three cities in the United States were queried about experiences with bias victimization, specifically hate crime and noncriminal bias victimization. Findings revealed that levels of bias victimization, hate crime, and noncriminal bias victimization were associated with SES, Anglo orientation, immigrant status, and their interactions, although in some unpredicted ways. Interactions among key variables helped clarify the roles of these factors in concert on bias victimization. The hate crimes against U.S.-born Latinos and the victimization risk associated with increasing Anglo orientations among immigrants contradicts predictions of intergroup threat theory. More nuanced analyses of social locations are needed to examine bias victimization.

Bias Crime and Victimization Among Latinx Adults: Formal and Informal Help Seeking.

Latinx adults have become increasingly vulnerable to bias motivated victimization. The impact of such incidents on Latinx communities is severely understudied, particularly concerning whether or not victims will seek help as a result of such events. Evidence within other victimization contexts demonstrate Latinx populations may be less likely to seek formal help from police, medical providers, and other formal authorities, relying instead on informal support networks such as family and friends. The current study sought to understand formal and informal help-seeking behavior among Latinx adults who experienced bias motivated victimization. The Understanding and Measuring Bias Victimization against Latinos study obtained rates of bias victimization and subsequent help-seeking behavior among Latinx adults who reported experiencing bias victimization (n = 315, 34.6% of full sample of 910). Those who experience bias victimization seek formal help at much lower rates than informal forms of support. Logistic regression analyses controlling the type of victimization demonstrated that participants who experienced a victimization constituting a hate crime were more likely to seek formal help compared to experiencing non-criminal bias events. Implications include addressing barriers to Latinx bias victims seeking forms of help, in addition to understanding the potential polyvictimization histories that predict why Latinx adults may decide to seek help.

Pregnancy-related claustrophobia.

A 35-year-old primigravida presented with significant anxiety symptoms at 26 weeks' gestation. Symptoms were preceded by a nightmare about her upcoming labour. She developed repetitive intrusive thoughts of being trapped emotionally and physically in her pregnancy. Her symptoms were suggestive of new-onset claustrophobia associated with pregnancy, which has not been previously reported on. Her symptoms ameliorated with a combination of cognitive-behavioural therapy and pharmacotherapy (sertraline and low dose quetiapine). The later stages of pregnancy were associated with minimal symptoms and the resolution of her subjective 'entrapment'. A subsequent pregnancy resulted in similar although less severe symptomatology. No postpartum anxiety symptoms were demonstrated on both occasions. Anxiety symptoms can adversely impact both the mother and fetus, and thus correct identification and management of pregnancy-related claustrophobia improved symptomatology and functioning and allowed for earlier detection and reduced symptomatology in a subsequent pregnancy.

The tumor suppressor protein HBP1 is a novel c-myc-binding protein that negatively regulates c-myc transcriptional activity.

c-Myc is an important transcription factor that regulates cellular proliferation, cell growth, and differentiation. A number of transcriptional co-factors for c-Myc have been described that have binding sites within highly conserved regions of the c-Myc transactivational domain (TAD). Given the importance of the c-Myc TAD, we set out to identify new proteins that interact with this region using a yeast two-hybrid assay. HBP1 was identified in our screen as a protein that interacts with full-length c-Myc but not a c-Myc mutant lacking the TAD. HBP1 is a transcriptional repressor and has been shown to negatively regulate the cell cycle. A correlation between HBP1 under-expression and breast cancer relapse has been described, suggesting that HBP1 may be an important tumor suppressor protein. We have found that HBP1 binds c-Myc in cells, and expression of HBP1 inhibits c-Myc transactivational activity at least partly by preventing c-Myc binding to target gene promoters. c-Myc binds to the C terminus of HBP1, a region lost in some breast tumors, and some HBP1 mutants found in breast cancer weakly interact with and/or no longer negatively regulate c-Myc. This work adds to our understanding of c-Myc regulation and mechanisms of tumor suppression by HBP1.

Understanding factors associated with re-referral of youth for commercial sexual exploitation.

BACKGROUND: Since the passage of the Victims of Trafficking and Violence Prevention Act of 2000 in the United States, awareness has increased about the problem of commercial sexual exploitation of children (CSEC). Moreover, United States Federal law calls upon child welfare agencies to establish policies and practices that help identify and provide services to minors at risk of CSEC. Although awareness has increased about the problem of CSEC, little attention has been paid to understanding why some youths are re-referred to CSEC programs after initial referral and intervention. OBJECTIVE: The present study examines which factors drive re-referrals of youths for CSEC concerns. PARTICIPANTS AND SETTING: Data about a population of high-risk youths (n = 416) referred to a CSEC-specific program in the Northeast of the United States between 2011 and 2018. METHODS: Using logistic regression analyses, we investigate what factors are associated with youth having multiple referrals. RESULTS: Youths with multiple referrals were more likely to have histories of missing from care (OR = 2.996, p < 0.001), substance misuse (OR = 2.802, p < 0.01), and greater agency involvement (OR = 1.260, p < 0.05). However, youths with multiple referrals were not at heightened risk of CSEC as compared to youths that were not re-referred. CONCLUSION: The results demonstrate that re-referred youths meet the profile of a victim as depicted on screening and assessment instruments, which has important implications for victim services and referral systems dedicated to identifying CSEC and protecting youths who may experience re-victimization for CSEC.

Improved outcomes with delayed admission to post-acute care: results of a natural experiment.

PURPOSE: To determine the effectiveness of a post-acute care scheme by exploiting a natural experiment. METHODS: We used a reduction in funding for an Irish PAC scheme based in private nursing homes as a natural experiment to explore the effectiveness of this scheme in a single large general hospital. RESULTS: Compared with an equivalent 3-month period in 2017 (pre-change, N = 169), those admitted to PAC in 2019 (post-change, N = 179), spent a median 6 days longer in acute care, although total duration spent in healthcare settings was the same. Compared with 2017, readmissions to hospital within 90 days of discharge (43/179 (24.0% v 58/169 (34.3%), p = 0.03) and discharge to long-term care from the PAC facility (3 (1.7%) v 14 (8.3%), p = 0.004) were significantly lower in 2019. CONCLUSION: Our results suggest that the longer stay in acute care and shorter stay in PAC was beneficial for patients and led to improved outcomes.