Racial disparities in cancer genetic counseling encounters: study protocol for investigating patient-genetic counselor communication in the naturalistic clinical setting using a convergent mixed methods design.

BACKGROUND: Despite decades of effort to reduce racial cancer disparities, Black people continue to die at higher rates from cancer than any other U.S. racial group. Because prevention is a key to the cost-effective and long-term control of cancer, the potential for cancer genetic counseling to play a central role in reducing racial cancer disparities is high. However, the benefits of genetic counseling are not equitable across race. Only 2% of genetic counselors self-identify as Black/African American, so most genetic counseling encounters with Black patients are racially discordant. Patients in racially discordant medical interactions tend to have poorer quality patient-provider communication and receive suboptimal clinical recommendations. One major factor that contributes to these healthcare disparities is racial bias. Drawing on findings from prior research, we hypothesize that genetic counselor providers' implicit racial prejudice will be associated negatively with the quality of patient-provider communication, while providers' explicit negative racial stereotypes will be associated negatively with the comprehensiveness of clinical discussions of cancer risk and genetic testing for Black (vs. White) patients. METHODS: Using a convergent mixed methods research design, we will collect data from at least 15 genetic counseling providers, from two different institutions, and their 220 patients (approximately equal number of Black and White patients per provider) whose appointments are for a hereditary cancer condition. The data sources will include two provider surveys, two patient surveys, video- and/or audio-recordings of genetic counseling encounters, and medical chart reviews. The recorded cancer genetic counseling in-person and telehealth encounters will be analyzed both qualitatively and quantitatively to assess the quality of patient-provider communication and the comprehensiveness of clinical discussion. Those data will be linked to pre- and post-encounter survey data and data from medical chart reviews to test our hypotheses. DISCUSSION: Findings from this multi-site study will highlight specific aspects of cancer genetic counseling encounters (patient-provider communication and clinical recommendations) that are directly associated with patient-centered outcomes (e.g., satisfaction, trust, genetic testing completion). Patient-provider communication and clinical recommendations are modifiable factors that can be integrated into current genetic counseling training curricula and thus can have immediate impact on genetic counseling training and practice.

When resilience is not enough: Imagining novel approaches to supporting Black youth navigating racism.

The narrative surrounding the impact racism has had on the well-being of Black youth has shifted across sociocultural and historical context. Early discourse around these topics were problem-saturated, focusing on deficits "within" Black youth. Over time, an important narrative shift occurred: greater attention was paid to the inherent assets of Black youth, their families, and communities, including how racial-ethnic protective factors such as racial socialization afforded them resilience. What resulted was decades of research seeking to understand the mechanisms that allow Black youth to bounce back in spite of racism-related adversity. Notwithstanding the viable practice and policy implications that have emerged from such inquiry, at what point does our focus on the resilience of Black youth - whether individual or multisystemic - fall short? It is with this question in mind that this paper challenges those committed to the optimal development of Black youth to consider yet another narrative shift: one that stands upon the legacy of cultural ecological frameworks and the seminal models underlying resilience research, and calls us toward not supporting Black youth's adaptation to racism, but toward collective efforts to transform our approach, pushing back against the perniciousness of racism.

Influence of Foreign Versus US Nativity on the Trajectory of Functional Independence Over the 10 Years After Traumatic Brain Injury: A Model Systems Study.

OBJECTIVE: Although racial/ethnic disparities have been documented in numerous outcomes after traumatic brain injury (TBI), research has yet to explore differences in rehabilitation outcomes based upon nativity (foreign-born vs US-born individuals). The purpose of this study was to compare the functional outcomes over the first 10 years after TBI for individuals born in the United States with those who were foreign-born. SETTING/PARTICIPANTS: A sample of 10 695 individuals in the TBI Model Systems database with a coding for country of birth the United States ( n = 9435) versus other than the United States ( n = 1260) was used. DESIGN/MAIN MEASURES: Outcome measures at 1, 2, 5, and 10 years after TBI included the Motor and Cognitive subscales of the Functional Independence Measure and the Supervision Rating Scale. RESULTS: Foreign-born individuals exhibited lower motor functional independence trajectories than those born in the United States, even after controlling for demographic and injury-related covariates. However, foreign-born individuals generally showed a stronger upward quadratic trajectory in motor functional independence with the greatest gains between the 5- and 10-year time points, whereas those born in the United States improved more quickly but then plateaued between the 5- and 10-year time points; these differential effects over time dissipated with the addition of demographic and injury-related covariates. Foreign-born individuals also exhibited lower cognitive functional independence trajectories, as well as greater supervision needs trajectories, than those born in the United States, even after controlling for demographic and injury-related covariates. CONCLUSION: These findings dovetail with literature suggesting that racially/ethnically diverse groups exhibit reduced functional independence after TBI. The findings may also suggest potential systematic barriers such as healthcare access and language barriers that may influence the frequency, rate, and quality of care received. The results highlight the importance of uncovering cultural distinctions and can aid in facilitating research examining foreign nativity-based disparities following TBI.

Gender Differences in Letters of Recommendation for Gynecology Surgical Fellowship Applicants: Analysis of One Program's Recruitment Cycle.

STUDY OBJECTIVE: To examine whether there are gender differences in letters of recommendation (LORs) written for residents applying to gynecology surgical fellowships. DESIGN: Retrospective study. SETTING: Single, academic institution. PATIENTS: LORs for applicants to gynecology oncology, urogynecology, and minimally invasive gynecology fellowships during the 2019-2020 application cycle. INTERVENTIONS: Not applicable. MEASUREMENTS AND MAIN RESULTS: We analyzed the linguistic content of the letters for the presence of 4 summary variables and 21 word categories based on previous studies using validated computerized text analysis software. We used multivariable analysis using linear mixed models to compare linguistic characteristics of letters by applicant gender. We performed qualitative content analysis on letters and compared the frequency of code themes by gender. The mixed-method design was planned to allow for analysis of domains not captured in text analysis. Among 680 letters written for 186 applicants, 124 (18.2%) were written for men, and 556 (81.8%) were written for women. There were no differences in the least square mean (standard error) word counts for LORs written for men and women applicants, 465 (20.0) vs 458(9.4) words, p = .74. In multivariable analysis, LORs written for men were found to have higher authentic tone and more risk words (p = .005 and p = .03, respectively). LORs written for women contained more communal (relationship-oriented) words (p = .006). The qualitative analysis demonstrated that ability, interpersonal traits, surgical skills, and research were the most often mentioned themes. Comments about compassion/empathy, leadership potential, teaching, interpersonal skills, and patient rapport were found more often in letters for men. More doubt raisers (words that raise doubt or concern) were present in letters for men, but letters for both genders had similar levels of negative criticism. In contrast, comments on ability, being "drama-free," and self-awareness were found more often in letters for women. CONCLUSION: There were gender differences in LORs written for obstetrics and gynecology surgical subspecialty fellowship applicants indicating the presence of gender bias.

Diversifying Clinical Child and Adolescent Psychology: A Change Gonna Come.

OBJECTIVES: Workforce diversity is an ongoing challenge in the field of clinical child and adolescent psychology. This article discusses individual, institutional, and nonspecific factors that contribute to a lack of diversity among clinical child and adolescent psychologists and offers suggestions to diversify and advance the field of clinical child and adolescent mental health. METHOD: Seventeen professors, licensed psychologists, faculty, and clinicians in the field of clinical child and adolescent psychology answered questions about workforce diversity and who is permitted access to the field. No formal research was conducted. RESULTS: Individual factors included: racial discrimination and microaggressions, feelings of isolation, otherness, and not belonging. Institutional factors included: racism in academia, racial underrepresentation, ethnocentric and culturally-biased training, biased admissions selection processes, financial barriers, and lack of institutional commitment. Nonspecific factors were: values misalignment, hidden expectations, suboptimal mentoring, and limited research opportunities. CONCLUSIONS: Drawing on recent scholarship and the Contexts, Actions, and Outcomes (CAO) Model, we recommend institutional changes in programs, policies, practices, resources, climate, partnerships, and inquiry to improve diversity in the field of clinical child and adolescent psychology.

Exploring the association between parental ethnic-racial socialization and parental closeness on Black-White biracial adolescents' choice of racial identification toward blackness.

OBJECTIVE: To explore the influences of Black-White biracial adolescents' choice to racially identify with Blackness, parental ethnic-racial socialization (ERS) messages, parental race, and perceived parental closeness were examined. Specifically, this study investigated how messages that promote monoracial Black pride and messages that prepare youth for monoracial Black bias may be related to adolescents' identification with Blackness, and whether parental race or parental closeness moderated these relationships. METHOD: Three hundred thirty Black-White biracial adolescents (Mage = 14.82) were recruited across the United States via social media. Participants completed the Racial Socialization Questionnaire for Biracial Adolescents and a demographic questionnaire to indicate how close they felt to each parent. The analytic sample (n = 280) included respondents who racially identified as exclusively Black, blended Black, or exclusively biracial. RESULTS: Multinomial logistic regression analyses revealed significant differences in the association between ERS messages and adolescent racial identification based on the race of the parent socializer. Further moderation analyses indicated that parental closeness, particularly regarding fathers, amplifies these findings. CONCLUSION: Maternal and paternal ERS messages are differentially associated with biracial adolescents' choice of racial identification toward Blackness. interestingly, ERS messages from White parents seem to significantly influence racial identification compared to Black parents. Parental closeness further elucidates these findings. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Ethnic-racial identity and ethnic-racial socialization competency: How minoritized parents "walk the talk".

OBJECTIVES: Ethnic-racial identity (ERI) has important implications for individual psychosocial functioning as well as familial processes. For example, parents' ERI can shape children's developmental contexts through ethnic-racial socialization (ERS). Yet, existing research has tended to focus on the content or frequency of socialization messages themselves rather than on internal factors like socialization competence. Such competence, as reflected through confidence, skills, and stress, represents critical dimensions that permeate the socialization process and can impact the delivery of messages. The present study examines whether parents' ERI (i.e., private regard, centrality, exploration) is related to perceptions of their socialization competence. METHOD: Data from 203 Black, 194 Asian American, and 188 Latinx parents (N = 585, Mage = 44.46 years, SD = 9.14, 59.70% mothers) of adolescents between the ages of 10-18 were collected via Qualtrics panels. RESULTS: Across all parents, private regard, centrality, and ethnic-racial exploration were positively associated with perceived confidence and skills in engaging in ERS. Regard was additionally associated with lower socialization stress. CONCLUSIONS: The results point to consistent benefits of ERI in helping parents navigate ERS, furthering the understanding of ERI's developmental implications through parents' comfort with and ability to "walk the talk" with their children. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Let's talk: The impact of gendered racial socialization on Black adolescent girls' mental health.

OBJECTIVE: For Black adolescent girls to develop a healthy sense of self, families must prepare them to cope with the realities of experiencing oppression at the nexus of racism and sexism. In this study, we examined the associations of positive and negative gendered racial socialization with mental health outcomes for Black adolescent girls. METHOD: To test the hypothesis that socialization messages of gendered racial pride and empowerment (GRPE) moderate the relationship between messages of internalized gendered racial oppression (IGRO) and indicators of psychological well-being (i.e., depressive, anxiety, and stress symptoms), a series of hierarchical multiple regression analyses were conducted based on the experiences of 282 Black adolescent girls (Mage = 15.40). RESULTS: Findings show that more frequent messages of IGRO were associated with more depression, anxiety, and stress symptoms in Black girls. Findings also suggest that more frequent messages of GRPE reduce the negative associations between messages of IGRO and Black adolescent girls' mental health symptoms to attenuation. CONCLUSIONS: Mental health symptoms in Black girls are able to be moderated by parents' intentional messaging of GRPE. These findings may provide a strategy to protect Black youth from the deleterious consequences of negative messages around race and gender. Future studies may wish to examine the impact of socialization messages of GRPE and IGRO on Black boys' mental health symptoms. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Differentiating competency from content: Parental racial socialization profiles and their associated factors.

For Black parents, the racial socialization (RS) process represents a critical parenting practice. Although the field has historically focused on the content of parents' RS, it is also important to consider caregivers' perception of their competence to effectively teach their children to navigate their racialized world. The present study investigated patterns of RS by exploring 332 Black caregivers' report of both content and competency. Using Latent Profile Analysis (LPA), we identified three profiles of RS: Multifaceted & More Competent (MMC), Unengaged & Moderately Competent (UModC), and Negative, Stressed, & Less Competent (NSLC). Additionally, we explored the role of several previously established correlates of parental RS, including sociodemographic factors (i.e., age, gender, and socioeconomic status), caregiving status (e.g., mother, father, and aunt), and parents' race-related experiences (i.e., history of RS, racial identity, and experiences with racial discrimination). Generally, the NSLC profile consisted of caregivers who were younger than those in the other two profiles, while those in the UModC profile tended to have younger children, relatively. Interestingly, caregivers in the UmodC profile reported receiving significantly less RS in childhood and experienced less racial discrimination than those in the other two profiles. Numerous differences were found across profiles for dimensions of racial identity. The emergence of these varied profiles, as well as the identification of factors that differentiated them, extends our understanding of RS and highlights the importance of considering parents' notions of feeling confident, skillful, and less stressed as they navigate such a vital developmental process for their children.

Para los padres negros, el proceso de socialización racial (SR) representa una práctica fundamental de crianza. Aunque esta área históricamente se ha centrado en el contenido de la SR de los padres, también es importante tener en cuenta la percepción que tienen los cuidadores de su competencia para enseñarles eficazmente a sus hijos a orientarse en su mundo racializado. En el presente estudio se investigaron los patrones de SR analizando el informe de 332 cuidadores negros sobre contenido y competencia. Utilizando el análisis de perfiles latentes (APL), identificamos tres perfiles de SR: polifacéticos y más competentes (PMC), no comprometidos y moderadamente competentes (NCModC) y negativos, menos competentes y estresados (NMCE). Además, analizamos el papel que juegan varias correlaciones de SR de los padres previamente establecidas, incluidos los factores sociodemográficos, (p. ej., la edad, el género y la situación socioeconómica), la situación de cuidado (p. ej.: la madre, el padre, la tía) y las experiencias de los padres relacionadas con la raza (p. ej., los antecedentes de SR, la identidad racial, y las experiencias con la discriminación racial). Generalmente, el perfil NMCE estuvo compuesto por cuidadores que eran más jóvenes que los de los otros dos perfiles, mientras que los del perfil NCModC fueron propensos a tener hijos más pequeños, relativamente. De modo interesante, los cuidadores del perfil NCModC informaron haber recibido una SR considerablemente menor en la niñez y sufrieron menos discriminación racial que los de los otros dos perfiles. Se encontraron numerosas diferencias entre los perfiles con respecto a las dimensiones de identidad racial. El surgimiento de estos perfiles variados, así como la identificación de los factores que los diferenciaron, amplían nuestra comprensión de la SR y destacan la importancia de tener en cuenta las intenciones de los padres de sentirse seguros, habilidosos y menos estresados a medida que transitan este proceso tan fundamental del desarrollo para sus hijos.

Do gender and racial differences exist in letters of recommendation for obstetrics and gynecology residency applicants?

BACKGROUND: Studies in several fields demonstrate gender and racial differences in descriptions of applicants in letters of recommendation. However, gender and racial biases in obstetrics and gynecology residency letters of recommendation are understudied. OBJECTIVE: This study aimed to determine whether there are differences in the letters of recommendation written for medical students applying for obstetrics and gynecology residency based on applicant gender and underrepresented in medicine status. STUDY DESIGN: We analyzed all letters of recommendation submitted to a single obstetrics and gynecology residency program during the 2019-2020 application cycle using a computerized text analysis software to evaluate the presence of 25 word categories. Multivariable regression was used to compare differences in the frequency of word categories by gender and underrepresented in medicine status. Concurrently, we performed a qualitative content analysis of letters from a random sample of applicants balanced for gender and underrepresented in medicine status and explored emerging themes until thematic saturation was reached. RESULTS: We analyzed 3060 letters written for 834 applicants, 721 (87%) of whom were women and 198 (24%) of whom were underrepresented in medicine. Men authored 1605 letters (53%). There was no difference in word categories used in letters written for men and women applicants on multivariable analysis. Similarly, there was no word category difference between letters written for applicants who were underrepresented in medicine compared with White and Asian applicants. However, women letter writers used more communal (relationship-oriented) words compared with men letter writers (P<.001). Moreover, on the qualitative analysis of letters from a random sample of 30 applicants, comments about personality traits were frequent in all letters. Comments on surgical skills, work ethic, and leadership were found more in letters for White and Asian applicants, and phrases that were doubt raisers were found more in letters written for underrepresented medicine applicants. CONCLUSION: On linguistic analysis, letters of recommendation written for obstetrics and gynecology residency applicants were similar overall compared with applicant race and gender.

Interrupting the Pathway From Discrimination to Black Adolescents' Psychosocial Outcomes: The Contribution of Parental Racial Worries and Racial Socialization Competency.

Racial discrimination can lead to psychosocial problems for Black adolescents, including internalization (e.g., depression) and externalization (e.g., conduct problems). Black parents (N = 186; Mage = 42.9) of adolescents (ages 10-18) were assessed to investigate how parental worries and racial socialization competency (i.e., confidence, skills, and stress) contribute to the association between parental discrimination experiences and their adolescents' psychosocial problems. Mediation analyses indicated that the total direct models with discrimination, worries, and problems had good fit, and that the addition of worry mediated the discrimination-problems association. Furthermore, racial socialization competency moderated the association between worry and problems, wherein greater competency was associated with less impact of worry on problems. Findings illuminate potential intervention targets for buffering discrimination's influence on adolescents' psychosocial functioning.

The initial development and validation of the Racial Socialization Competency Scale: Quality and quantity.

OBJECTIVES: The present study investigated the reliability and validity of the Racial Socialization Competency Scale (RaSCS). As posited by the Racial Encounter Coping Appraisal and Socialization Theory (RECAST), the RaSCS consists of 3 factors representing 3 novel dimensions of racial socialization competency present in families (e.g., stress, skills, and confidence). METHOD: Responses to the RaSCS were collected from 361 self-identified Black and African American parents and primary caretakers across the United States. RESULTS: After factor analysis, three scales and 27 items were maintained. Evidence was consistently found for 2 subscales within the stress dimension, namely "Call to Action" and "General" racial socialization stressors. Dimensions were also related to their respectively hypothesized constructs, including overall stress, racial socialization frequency, and self-efficacy. CONCLUSIONS: Future directions and recommendations on the measure's use, particularly in the context of interventions, are also discussed. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Impostor Phenomenon and Psychological Well-Being: The Moderating Roles of John Henryism and School Racial Composition Among Black College Students.

The impostor phenomenon (IP), or erroneous cognitions of intellectual incompetence, is a risk factor for poor psychological adjustment among Black emerging adults. Grounded in Lazarus and Folkman's Stress and Coping Framework, the current study investigated John Henryism's active coping and institutional racial composition as moderators of the association between IP and indicators of psychological well-being among 266 Black students (77% women; M age = 19.87) attending predominately White institutions (PWIs) and historically Black colleges/universities (HBCUs). Hierarchical moderation regression analyses revealed that IP was associated with decreases in well-being indicators among students attending PWIs and HBCUs. Moreover, students who attended PWIs and reported higher levels of John Henryism (+1 SD) were most vulnerable to increases in social anxiety, particularly at higher levels of IP. Results suggest that the interaction between IP, John Henrysim, and institutional racial composition may negatively influence psychological well-being. We discuss how these findings can be used to inform clinical and educational practices to best support Black college students.

Coping, Discrimination, and Physical Health Conditions Among Predominantly Poor, Urban African Americans: Implications for Community-Level Health Services.

African Americans and ethnic minorities experience racial discrimination in a variety of settings. Racial discrimination is a potent stressor that has been linked to psychosocial stress and poor physical health. To cope with discriminatory experiences and daily life event stressors, African Americans frequently use the concept of John Henryism (a high effort coping strategy with prolonged exposure to stress). This cross-sectional analysis explored the relationship between racism/discrimination, John Henryism, and health problems in a predominately African American sample. Data were collected through health care screenings for hypertension, diabetes, and obesity and a self-report survey to assess experiences of discrimination and use of John Henryism. Logistic and linear regression models were used to assess the relationship between the John Henryism score, racism/discrimination score, and health problems among 352 participants. John Henryism was associated with a decrease in systolic blood pressure (b = - 12.50, 95% CI = - 23.05, - 1.95) among men, after adjusting for experiences of racism/discrimination and demographic characteristics. Experiences of racism/discrimination were associated with an increase in systolic blood pressure (b = 11.23, 95% CI = 0.38, 22.09) among men, after adjusting for John Henryism and demographic characteristics. Among women, there was no association found between John Henryism and experiences of racism/discrimination with systolic blood pressure. No association was found between John Henryism and experiences of racism/discrimination with being overweight/obese in women nor men. The study found that John Henryism was positively associated with the health of men, while experiences of racism/discrimination were negatively associated with their health. Limitations of the study are discussed, and recommendations are made to guide future research exploring the concept of John Henryism as a relevant factor between stress, racial discrimination and poor health.

What's Race Got to Do With It? Racial Socialization's Contribution to Black Adolescent Coping.

While youth generally experience stressors from developmental milestones, Black youth also face racialized stressors. Racial socialization has been found to help Black youth cope with racialized stressors, but research has yet to show its contribution to coping beyond general socialization practices. This study examines how racial socialization contributes beyond that of general coping socialization to coping behaviors. Fifty-eight third-eighth-grade (Mage  = 11.3, SD = 1.54) youth reported general coping socialization and racial socialization practices and coping behaviors. Results indicate that for engagement coping, racial socialization messages contributed significantly to parent-provided engaged socialization strategies. Implications are considered for the ways in which Black youth experience stress and require culturally specific practices for successful coping with frequently encountered stressors.

Racial discrimination, John Henryism coping, and behavioral health conditions among predominantly poor, urban African Americans: Implications for community-level opioid problems and mental health services.

The current study examined the relationship between John Henryism Active Coping (JHAC), experiences of racial discrimination, and behavioral health outcomes in a community sample of 319 Black adults. Assessments included primary health care screenings as well as self-reported survey questions to assess JHAC, experiences of discrimination, and self-reported behavioral health. Logistic regression models, adjusted for control variables, found a significant relationship between JHAC and having an opioid problem (odds ratio [OR] = 0.95, p = 0.003) and needing mental health services (OR = 0.95, p < 0.001), such that higher levels of coping were associated with lower odds of reporting an opioid problem and needing mental health services. Notably, racial discrimination was not significantly independently associated with behavioral health. Implications for interventions and community programming are discussed.

Future Directions in Research on Racism-Related Stress and Racial-Ethnic Protective Factors for Black Youth.

Research on racism-related stress and racial-ethnic protective factors represents an important enterprise for optimizing the mental health of African American and other racial and ethnic minority youth. However, there has been a relative dearth of work on these factors in the clinical psychology research literature, and more work is needed in outlets such as these. To this end, the current article adopts a developmental psychopathology framework and uses recent empirical findings to outline our current understanding of racism-related stress and racial-ethnic protective factors (i.e., racial identity, racial socialization, Africentric worldview) for African American youth. We then provide nine recommendations-across basic, applied, and broader/cross-cutting research lines-that we prioritize as essential to advancing the future scientific investigation of this crucial research agenda. Within and across these recommendations, we issue a charge to researchers and clinicians alike, with the ultimate goal of alleviating the negative mental health impact that racism-related stress can have on the well-being and mental health of African American and other racial and ethnic minority youth.

σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation.

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σ(H) (early), σ(F), σ(E), σ(G), and σ(K) (late). Here we show that the Clostridium acetobutylicum σ(K) acts both early, prior to Spo0A expression, and late, past σ(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σ(G) proteins were not detectable by Western analysis, while σ(F) protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σ(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σ(F), σ(E), and σ(G), but not sporulation, which was blocked past the σ(G) stage of development, thus demonstrating that σ(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for (13)C metabolic flux analysis.

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and (13)C-metabolic flux analysis ((13)C-MFA). Here, cells were grown in parallel cultures with [1-(13)C]glucose and [U-(13)C]glucose as tracers and (13)C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of (13)C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for (13)C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased (13)C-flux measurements in C. acetobutylicum.

Workflow for quantitative proteomic analysis of Clostridium acetobutylicum ATCC 824 using iTRAQ tags.

Clostridium acetobutylicum (Cac) is an anaerobic, endospore-forming, Gram-positive bacterium with tremendous promise for use as a biocatalyst for the production of fuels and solvents. Cac proteomic sample preparation for shotgun analysis typically involves a multitude of reagents for harsh lysis conditions and to maintain protein solubility. We describe a protein extraction and preparation method for Cac that is compatible with proteomic shotgun analysis using isobaric labeling approaches. The method is applied to the analysis of Cac grown under butanol stress and labeled using iTRAQ 4-plex reagents. This method relies on the use of calcium carbonate to facilitate lysis by sonication and a commercially available kit to remove detergents prior to labeling. This workflow resulted in the identification and quantitation of 566 unique proteins using ProteinPilot software with a false discovery rate of 0.01% for peptide matches and 0.70% for protein matches. Ninety-five proteins were found to have statistically higher expression levels in butanol-stressed Cac as compared to non-stressed Cac. Sixty-one proteins were found to have statistically lower expression levels in stressed versus non-stressed cells. This method may be applicable to other Gram-positive organisms.