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Trypanosoma brucei is an early divergent parasitic protozoan that causes a fatal disease, African trypanosomiasis. T. brucei possesses a unique and essential translocase of the mitochondrial inner membrane, the TbTIM17 complex. TbTim17 associates with 6 small TbTims, (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction pattern of the small TbTims with each other and TbTim17 are not clear. Here, we demonstrated by yeast two-hybrid (Y2H) analysis that all six small TbTims interact with each other, but stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. Each of the small TbTims also interact directly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial to maintain the steady-state levels of the TbTIM17 complex. Co-immunoprecipitation analyses from T. brucei mitochondrial extracts also showed that TbTim10 has a stronger association with TbTim9 and TbTim8/13, but a weaker association with TbTim13, whereas TbTim13 has a stronger connection with TbTim17. Analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except TbTim13, is present in ~70 kDa complexes, which could be heterohexameric forms of the small TbTims. However, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionated with TbTim17. Altogether, our results demonstrated that TbTim13 is a part of the TbTIM complex and the smaller complexes of the small TbTims likely interact with the larger complex dynamically. Therefore, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific in T. brucei.

SummaryOPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans that are expressed as short or long-form isoforms. These isoforms contribute to OPA1’s ability to control mitochondrial functions. However, isolating OPA1 isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function.Tweetable AbstractWestern blot protocol optimization to visualize OPA1 isoforms.HighlightsProtocol for isolating OPA1 isoforms in skeletal muscle tissueSteps for running isolated skeletal muscle cells from muscle tissue on a gelHow to collect samples in preparation for western blottingDetection of OPA1 isoformsGraphical AbstractKey Resources TableMethod SummarySamples for western blot analysis are isolated from lysed cells, loaded onto a gel, and ran using optimized conditions to better isolate OPA1 isoforms. Samples are transferred to a membrane to for incubation and protein detection using OPA1 antibodies.

Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important. Therefore, in this article, we discuss a comprehensive method to isolate mice skeletal muscle, create satellite cells for tissue culture, and use immunofluorescence to validate our approach.Novel imaging technology is increasing our ability to visualize and analyze cellular organelles and compartments.Tweetable AbstractProper antibody validation of cellular populations within isolated skeletal muscle can lead to better elucidation of skeletal muscle structure and function, and their roles in complex diseases.Graphical AbstractMethod SummaryMyoblasts are isolated from mouse limb muscles. They are plated for immunofluorescence-based validation using confocal microscopy. This method demonstrates the need for reliable antibodies to correctly determine and differentiate between cellular populations within isolated skeletal muscles.

SummaryProximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs).Tweetable AbstractProximity Ligation Assays can be used in skeletal muscle tissue and myoblasts to explore the protein-protein interactions involved in MERC sites.HighlightsSkeletal muscle tissue and cells are plated on glass coverslips for evaluation by proximity ligation assay (PLA).Following fixation, cells are probed and stained for Mfn1, Mfn2, mitochondria, and ER and imaged using fluorescence confocal microscopy.This method shows that PLA can be used in mouse SkM and is adaptable to other models.Protocol for detection of protein-protein interactions using PLA.Graphical AbstractKey Resources Table

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that the morphological changes in BAT mitochondria and cristae observed across aging would reflect alterations to energy dynamics. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT. Cristae examination revealed decreased cristae volume, perimeter, and complexity, with cristate appearing more heterogeneous. Overall, these data define the nature of the mitochondrial structure in BAT, showing loss of cristae definition across aging, suggesting alterations in functionality and bioactivity.Graphical AbstractWorkflow overview of serial block facing-scanning electron microscopy (SBF-SEM), data segmentation, and 3D analysis of mitochondria using Amira software in murine BAT.

One of the biggest obstacles to success is a lack of practical time management skills. Here, we provide suggestions on how to optimize time management.

Background: COVID-19 vaccination rates remain suboptimal among Black Americans who disproportionately experience higher hospitalization and death rates than White Americans. Methods: We conducted a multi-method (interviews and surveys) study among 30 Black Americans (n = 16 vaccinated, n = 14 unvaccinated) to explore factors related to vaccination hesitancy, decision-making processes, and communication related to uptake. Participants were recruited by using community-driven approaches, including partner collaborations. Thematic analysis was used to analyze qualitative data, and descriptive and bivariate analysis was used for quantitative data. Results: Of those unvaccinated, 79% (n = 11) stated they were delaying and 21% (n = 3) were declining vaccination indefinitely. When asked about the likelihood of vaccine initiation in 6 months and 12 months, 29% (n = 4) and 36% (n = 5), respectively, stated that they would receive the vaccine. The following themes emerged: (1) COVID-19 vaccination hesitancy exists on a continuum; (2) varied decision-making processes for COVID-19 vaccination; (3) motivators among vaccinated individuals; (4) barriers among unvaccinated individuals; (5) retrieving and navigating vaccine information within the COVID-19 infodemic; and (6) parent perspectives on child vaccination. Conclusions: Findings suggest that vaccinated and unvaccinated participants had similar and dissimilar perspectives in decision-making processes and vaccine concerns as shown in the Decision-making Processes for the COVID-19 vaccination (DePC) model. Based on these findings, future studies should further explore how factors influencing decision-making can lead to divergent outcomes for COVID-19 vaccination.

Black individuals in the USA continue to be underrepresented in clinical trials with low participation rates in COVID-19 research studies. Identifying participation barriers is necessary as we develop more vaccines and other treatments to address SARS-CoV-2 and associated sequelae. The purpose of this explorative, qualitative study is to apply the theory of planned behavior to understand motivators and barriers to COVID-19 research participation at the early stages of the COVID-19 pandemic. Understanding these factors is important to ultimately lead to increased vaccination rates among Black individuals, especially in strategies that increase preparedness in response to public health emergencies.A phenomenological qualitative study design was conducted between May and September 2020 among 62 Black participants. The participants were purposefully selected from vulnerable subgroups of the Black population: essential workers, young adults, parents, and individuals with underlying medical conditions. An inductive-deductive content analysis approach was used to analyze the interview data.Majority (54.8%) reported willingness to participate in COVID-19 research. The following themes emerged from the interviews: (1) positivity toward research exists yet fear and distrust remain; (2) views toward COVID-19 research vary; (3) motivators to COVID-19 research participation; (4) barriers to COVID-19 research participation; and (5) potential strategies to increase COVID-19 research participation.Based on our findings, majority of the participants reported willingness to participate in research with observational research being the most commonly cited type of research. Providing data on the attitudes and perspectives of Black individuals and their intentions for COVID-19 research participation using TPB informs intervention targets for healthcare providers and policy makers for an equitable emergency response. Our results suggest improved communication on the research process, research opportunities, and participant testimonial through trusted sources could increase the likelihood of participation. This is especially important as we continue through the pandemic and new treatments for COVID-19 vaccines become readily available.

The purpose of this sequential, explanatory mixed methods study is to determine changes in attitudes towards research, trust in medical researchers and the process, and willingness to participate in research among African Americans immediately after receiving past study findings in a community listening session (CLS). We developed and implemented four CLSs with a total of 57 African Americans who were either past research participants or members of the community-at-large. In the quantitative (dominant) phase, 32 participants completed pre-post surveys and 10 of those participants completed the follow-up semi-structured interviews. Paired samples t-tests and McNemar's test determined bivariate differences between pre- and post-surveys. Thematic analyses determined emerging themes to further understand these differences. There was a significant increase in: (1) perceived advantages of clinical trials pretest (M = 26.63, SD = 5.43) and post-test (M = 28.53, SD = 4.24, p < .01); and (2) in trust in medical researchers from pre to post (M = 36.16, SD = 10.40 vs. M = 27.53, SD = 9.37, p < 0.001). There was no significant difference in pre- and post-tests as it relates to perceived disadvantages of clinical trials and willingness to participate. Qualitative analysis yielded the following themes: (1) sharing research results and the impact on attitudes towards research; (2) community listening sessions: a trust building strategy; and (3) satisfaction with the community listening session. Community listening sessions hold promise as a method that researchers can use to simultaneously disseminate research findings and positively impact research perceptions and potentially participation among racial and ethnic minorities.

Many studies in mice have demonstrated that cardiac-specific innate immune signaling pathways can be reprogrammed to modulate inflammation in response to myocardial injury and improve outcomes. While the echocardiography standard parameters of left ventricular (LV) ejection fraction, fractional shortening, and end-diastolic diameter, and others, are used to assess cardiac function, their dependency on loading conditions somewhat limit their utility in completely reflecting the contractile function and global cardiovascular efficiency of the heart. A true measure of global cardiovascular efficiency should include of the interaction between the ventricle and the aorta (ventriculo-vascular coupling, VVC) as well as measures of aortic impedance and pulse wave velocity. We measured cardiac Doppler velocities, blood pressures, along with VVC, aortic impedance, and pulse wave velocity to evaluate global cardiac function in mouse model of cardiac-restricted low levels TRAF2 overexpression that conferred cytoprotection in the heart. While previous studies reported that response to myocardial infraction and reperfusion was improved in the TRAF2 overexpressed mice, we found that TRAF2 mice had significantly lower cardiac systolic velocities and accelerations, diastolic atrial velocity, lower aortic pressures and rate-pressure product, lower LV contractility and relaxation, and lower stroke work when compared to littermate control mice. Also, we found significantly longer aortic ejection time, isovolumic contraction and relaxation times, and significantly higher mitral early/atrial ratio, myocardial performance index, and ventricular vascular coupling in the TRAF2 overexpression mice compared to their littermate controls. We found no significant differences in the aortic impedance and pulse wave velocity. While the reported tolerance to ischemic insults in TRAF2 overexpression mice may suggest enhanced cardiac reserve, our results indicate a diminished cardiac function in these mice.

Scientists at all career stages can benefit from building diverse mentoring networks that transcend boundaries and promote inclusion. In this piece, we define mentoring networks, describe examples of how mentoring networks can reinforce scientific identity, and help minority scientists overcome unique challenges to achieve their goals in cancer research.

Background: Appendiceal cancer incidence among individuals age < 50 years (early-onset appendiceal cancer) is rising with unknown etiologies. Distinct clinicopathologic/demographic features of early-onset appendiceal cancer remain unexplored. We compared patterns of appendiceal cancer among individuals by age of disease-onset. Methods: Using the NIH/NCI's Surveillance, Epidemiology, and End Results program data, we identified individuals age 20+ years diagnosed with appendiceal cancer from 2007 to 2016. Cochran–Armitage trend tests and multinomial logistic regression models were used to examine age-related differences in clinicopathologic/demographic features of appendiceal cancer. Results: We identified 8,851 patients with appendiceal cancer during the 10-year study period. Histologic subtype, tumor grade, stage, sex and race/ethnicity all significantly differed by age of appendiceal cancer diagnosis. After adjustment for race/ethnicity, sex, stage, insurance status, and tumor grade, young patients were 82% more likely to be Hispanic [OR, 1.82; 95% confidence interval (CI), 1.48–2.25; P < 0.001] and 4-fold more likely to be American Indian or Alaska Native (OR, 4.02; 95% CI, 1.77–9.16; P = 0.0009) compared with late-onset cases. Patients with early-onset appendiceal cancer were also 2- to 3.5-fold more likely to be diagnosed with neuroendocrine tumors of the appendix (goblet cell carcinoid: OR, 1.96; 95% CI, 1.59–2.41; P < 0.0001; carcinoid: OR, 3.52; 95% CI, 2.80–4.42; P < 0.0001) compared with patients with late-onset appendiceal cancer. Among patients with neuroendocrine tumors, early-onset cases were also 45% to 61% less likely to present with high-grade (III–IV) tumors. Conclusions: Approximately one in every three patients with appendiceal cancer is diagnosed before age 50 years in the United States. Appendiceal cancer in young patients is classified by distinct histologic and demographic features. Impact: Early-onset appendiceal cancer determinants can inform discovery of risk factors and molecular biomarkers of appendiceal cancer in young patients, with implications for appendiceal cancer prevention, detection, and treatment.

Alzheimer’s disease (AD), the most common cause of dementia in older adults, disproportionally affects African Americans (AA) with an incidence rate as much as three times higher, compared to other racial/ethnic groups. Multiple factors contribute to this racial disparity however, an in-depth understanding of the biological or genetic contributions does not exist. Compelling evidence indicate that genetic variants of the lipid transport protein, ABCA7, is more strongly associated with AD in African Americans. To understand how ABCA7 contributes to AD on the molecular level, we used a combination of structural and cell biology techniques. Our results suggest that the ABCA7 variant (T319A) reported to confer risk in AA, may contribute to AD by reducing the levels of phosphoinositol bisphosphate (PIP2), a phospholipid reported to be decreased in the AD brain. These results provide a framework for targeting mechanisms that increase PIP2 levels as an effective strategy mitigating AD disparities.

Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation. Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity and altered mitochondrial metabolism. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction.Graphical Abstract

Physical distancing and face masks remain frontline prevention strategies due to suboptimal vaccine uptake and the highly infectious COVID-19 variants. Communities of color are disproportionately impacted by a chronic disease burden that places them at higher risk of severe COVID-19 disease. Therefore, they can greatly benefit from face mask use and physical distancing, especially if the individual(s) have not received the vaccine. We applied the Health Belief Model to explore barriers and motivators influencing physical distancing and face mask use among high-risk, Black American subgroups during the early COVID-19 pandemic stages. We conducted 62 semi-structured interviews among four Black American subgroups: young adults, individuals with underlying medical conditions, essential workers, and parents. Thematic analysis, guided by the Health Belief Model, yielded six themes: (1) Knowledge on Face Mask Use and Physical Distancing, (2) Perceived Susceptibility and Severity Varies by Subgroup, (3) Experience with and Perceived Self-Efficacy to Engage in Preventive Behavior, (4) Perceived Benefits to engaging in preventive behaviors, (5) Perceived Barriers to engage in preventive behaviors, and (6) Cues to action to increase participation. Each subgroup’s unique experience informed multilevel, tailored approaches that can be used by health promotion practitioners to improve face mask use and physical distancing among uniquely vulnerable Black American subgroups in the current and future pandemic.

Mentoring success is derived from active and respectful listening and the willingness to learn and accept opportunities for personal growth. Mentoring shapes every trainee and their career path in science, technology, engineering, and mathematics (STEM). Productive mentoring relationships cultivate rapport, stimulate moments of introspection, and provide constructive feedback. Effective mentoring in STEM allows trainees, especially underrepresented minorities (URMs), to flourish in welcoming and supportive environments. However, URM trainees often experience inadequate mentoring due to their mentor’s inexperience with URM groups, poor mentorship training, or a lack of understanding of their mentee’s journey. To promote diversity, equity, and inclusion in STEM education and research, it is essential for mentors and mentees to work together with creativity, authenticity, and networking. In this workshop, we will focus on mentees’ perspective on how mentors can enhance their training, professional and career development, and improve their focus. We analyzed data on feedback obtained from students interested in pursuing graduate education who attended a recent workshop. Our results show that despite low initial expectations for the workshop, many students were satisfied with the knowledge they learned. The future of increasing the URM representation in STEM lies in providing adequate community support and mentorship throughout the careers of URM professionals.

Increasing COVID-testing and contact tracing is necessary to control the COVID-19 pandemic considering suboptimal vaccine rates. We conducted semi-structured interviews to explore views towards contact tracing and testing among 62 African Americans. Based on our findings, participants identified COVID-19 testing and contact tracing as beneficial, yet medical and governmental mistrust, stigma associated with SARS-CoV-2, lack of access, poor communication, and costs as major barriers. This study also highlights intervention targets to improve COVID-testing and contact tracing.

Despite efforts to increase diversity, a glaring underrepresentation of minorities (URM) persists in the fields of science, technology, engineering, and mathematics (STEM). Graduate school can be a stressful step in the STEM pipeline, especially for students previously unaware of the structure and challenges of post-graduate education. To promote successful minority participation in STEM and prepare prospective students for the impending challenges of graduate school, we developed a workshop based on the mentoring and fostering of a champion-oriented mindset entitled, “The Trials and Tribulations of Graduate School: How Do You Make an Impact?”. We administered the workshop to a cohort of university undergraduates and conducted pre- and post-workshop surveys to measure students’ perceived need for instruction on specific workshop topics. The results suggest that the workshop was well received by the students and provided information that they considered helpful to help navigate the graduate school process.

Objectives The disproportionate impact of coronavirus (COVID-19) on African Americans along with associated inequities in social determinants of health (SDOH) and racism increase their vulnerability to the psychosocial impact of COVID-19. This qualitative study applied the socio-ecological model (SEM) to explore psychosocial stressors, coping styles, and needs to improve psychosocial health among unique subgroups of African Americans in early pandemic stages. Methods Sixty-two African Americans (16 parents, 15 young adults, 16 essential workers, and 15 individuals with underlying medical conditions) participated in qualitative, semi-structured interviews between May and September 2020. Interview data were analyzed based on the SEM using thematic analysis. Results The majority (84%) reported being stressed with parents having the highest level. Four themes emerged : (1) our COVID-19 pandemic state of mind, (2) top stressors in the early stages of the COVID-19 pandemic, (3) coping strategies during COVID-19, and (4) needs during the COVID-19 pandemic to reduce stress. While there were similarities, different stressors were experienced among subgroups, which yielded different coping styles and needs from stakeholders across multi-levels to improve their psychosocial health. Conclusions Findings suggest current and future pandemic response plans need targeted strategies across multiple levels of influence to address the psychosocial impact of the COVID-19 pandemic on African Americans.

The COVID-19 pandemic has disproportionately impacted Black Americans. Inequities in systems and social determinants of health along with racial health disparities impact degree of pandemic preparedness.In early pandemic stages, we aimed to explore: 1) state of pandemic preparedness; 2) effects of socio-ecological factors on preparedness; and 3) multi-level strategies to increase preparedness among uniquely, vulnerable Black American subgroups.We conducted 62 in-depth interviews with Black American community members representing parents, individuals with underlying medical conditions, essential workers, and young adults. Based on the McLeroy's Model Ecological for Health Promotion, an inductive-deductive content analysis approach was used to analyze the interview data around the factors influencing preparedness on individual, interpersonal processes and primary groups, community/institutional, and public policy.Majority (56.5%) of the participants stated they were somewhat or very prepared. We identified four themes: 1) Lived Experiences during the COVID-19 Pandemic; 2) Challenges experienced during the COVID-19 Pandemic; 3) I would do this differently they say; 4) Changes Needed to Survive the Pandemic relate to Public Policy, Community/institutional factors, and Interpersonal processes and primary group(s). All participants described their adjustments to live in the new norm. Participants identified perceived challenges and solutions on multi-levels, driven by subgroup.Pandemic response plans should use targeted strategies across multi-levels to enhance the preparedness of Black Americans, especially those in vulnerable groups. This could reduce the disproportionate COVID-19 disease burden exhibited by Black Americans and better prepare for future pandemics.

Tim50 is a receptor subunit of the preprotein-translocase of the mitochondrial inner membrane, TIM23.Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of Tim50 (TbTim50) with a pair of characteristic DXDX(T/V) phosphatase signature motifs. Here, we demonstrated that besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner.In silicostructural homology models identify the putative binding interfaces that may accommodate different phospho-substrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) ofT. bruceireduced cardiolipin (CL) levels and decreased mitochondrial membrane potential (ΔΨ). TbTim50 knockdown (KD) also reduced the population of G2 phase and increased G1 phase; thus, BF cell growth was reduced. Confocal and electron microscopy revealed a defect in regulation of kinetoplast (kDNA) replication due to TbTim50 KD. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed to stumpy-like with upregulation of few stumpy marker gene expressions. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice and rats. Proteomics analysis showed reductions of the translation factors, flagellar transport proteins, and many proteasomal subunits, including the mitochondrial HslVU that is known to play a role in kDNA replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Altogether, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis.ImportanceAfrican trypanosomiasis, a neglected tropical disease caused by parasitic protozoanTrypanosoma brucei, is transmitted by the tsetse fly prevalent in sub-Saharan Africa. During its digenetic life cycle,T. bruceiundergoes multiple developmental changes to adapt in different environments.T. bruceiBF, dwelling in mammalian blood, generates ATP from glycolysis and hydrolyzes ATP in mitochondria for inner membrane potential. We found that TbTim50, a HAD-family phosphatase, is critical forT. bruceiBF survivalin vitroandin vivo. Depletion of TbTim50 in BF reduced CL levels and mitochondrial ΔΨ and caused a detrimental effect on many cellular functions. Cells accumulated in G1-S phase, and kinetoplast was over-replicated due to depletion of mitochondrial proteasomes, HslVU, a master-regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of stumpy-specific gene expression. TbTim50 is essential forT. bruceisurvival and an importantT. bruceitherapeutic target.

Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723–1818 of p300 Taz2 and residues 37–61 of C/EBP∊. The segment corresponding to the C/EBP∊ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBP∊ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP–p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPβ N-terminus, which associates with the C/EBPβ–p300 complex.

VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave alpha-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 A resolution. The shape of the molecule resembles the letter "V," with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

Pax transactivation domain-interacting protein (PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP consists of three pairs of tandem BRCA1 C-terminal (tBRCT) domains required for assembly into multiple protein complexes. Mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines. Surprisingly, we’ve discovered a novel truncated isoform of PTIP, containing the last four C-terminal BRCT domains, (PTIP-delta). RNAi gene depletion studies reveal a post-transcriptional origin of PTIP-delta. Kinetic profiling of PTIP synthesis indicates that a significant portion of PTIP is processed to PTIP-delta within hours of translation. Recombinant forms of this PTIP-delta retain the ability to form nuclear repair foci. Biophysical characterization uncovers new oligomerization in...

Background Breast cancer is the second most lethal cancer in women. Further, death rates for African American women are the highest for any racial/ethnic group. Hormone receptor status is one of the major prognostic factors and a determinant of treatment options for breast cancer, thus suggesting the importance of molecular level characterization for precision treatments. In this study, we have identified transcriptome level differences correlating to receptor specific molecular subtypes of breast cancer in African American women.