Areas of strength and opportunities for growth in translational science education and training: Results of a scoping review from the NCATS Education Branch.

Translational science education and training (E&T) aims to prepare the translational workforce to accelerate progress along the translational pipeline toward solutions that improve human health. In 2020-2021, the National Center for Advancing Translational Sciences (NCATS) Education Branch conducted a scoping review of the E&T literature with this focus. The review used the methodological framework proposed by Arksey and O'Malley. PubMed, Education Resources Information Center (ERIC), and Embase were searched, and forward citations conducted. Screening of titles, abstracts, and full text identified 44 included articles. Data extraction facilitated analysis of E&T content, audiences, modalities, evaluations, and recommendations. The NCATS Translational Science Principles were used to identity described or recommended E&T content. Twenty-nine articles described a translational science E&T opportunity or its evaluation, and another 15 articles offered recommendations for translational science E&T. The most prevalent NCATS Translational Science Principles were boundary-crossing partnerships (77%) and cross-disciplinary team science (75%). Among publications describing E&T opportunities, the most reported modalities were experiential learning (64%) and courses (61%) and the most reported participants were graduate students (68%) and postdoctoral fellows (54%). About half of these articles (n = 15) reported an evaluation, covering a range of proximal to distal outcomes. Recommendations emphasized the value of translational science E&T across training and career stages and the use of varied modalities to reach diverse audiences. This review highlights strengths and opportunities for growth in translational science E&T. Enhancements to content, expansion of participants and modalities, and rigorous evaluations will contribute to building a highly qualified, diverse translational science workforce.

DEIA is essential to advance the goals of translational science: Perspectives from NCATS.

The National Center for Advancing Translational Science (NCATS) seeks to improve upon the translational process to advance research and treatment across all diseases and conditions and bring these interventions to all who need them. Addressing the racial/ethnic health disparities and health inequities that persist in screening, diagnosis, treatment, and health outcomes (e.g., morbidity, mortality) is central to NCATS' mission to deliver more interventions to all people more quickly. Working toward this goal will require enhancing diversity, equity, inclusion, and accessibility (DEIA) in the translational workforce and in research conducted across the translational continuum, to support health equity. This paper discusses how aspects of DEIA are integral to the mission of translational science (TS). It describes recent NIH and NCATS efforts to advance DEIA in the TS workforce and in the research we support. Additionally, NCATS is developing approaches to apply a lens of DEIA in its activities and research - with relevance to the activities of the TS community - and will elucidate these approaches through related examples of NCATS-led, partnered, and supported activities, working toward the Center's goal of bringing more treatments to all people more quickly.

RAD6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by aggravating mitotic spindle damage.

BACKGROUND: Paclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation. METHODS: TNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin. RESULTS: Our data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity. CONCLUSIONS: These data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.

The National Center for Advancing Translational Sciences' Intramural Training Program and Fellow Career Outcomes.

The National Center for Advancing Translational Sciences (NCATS) defines translational science as "the field of investigation focused on understanding the scientific and operational principles underlying each step of the translational process." A major goal of translational science is to determine commonalities across projects to identify principles for addressing persistent bottlenecks in this process. To meet this goal, translational scientists must be conversant in multiple disciplines, work in teams, and understand the larger translational science ecosystem. The development of these skills through translational science training opportunities, such as the translational science training offered by the NCATS intramural research program, prepares fellows for a variety of career options. The unique structure of the NCATS intramural program and the career outcomes of its alumni are described herein to demonstrate the distinct features of this training environment, the productivity of fellows during their time in training, and how this prepares fellows to be competitive for a variety of science careers. To date, the NCATS intramural research program has trained 213 people, ranging from high school to postdoctoral levels. These alumni have transitioned into a wide array of career functions, types, and sectors.

Resistance to the CHK1 inhibitor prexasertib involves functionally distinct CHK1 activities in BRCA wild-type ovarian cancer.

High grade serous ovarian cancer (HGSOC) is a fatal gynecologic malignancy in the U.S. with limited treatment options. New therapeutic strategies include targeting of the cell cycle checkpoints, e.g., ATR and CHK1. We recently reported a promising clinical activity of the CHK1 inhibitor (CHK1i) prexasertib monotherapy in BRCA wild-type (BRCAwt) HGSOC patients. In this study, biopsies of treated patients and cell line models were used to investigate possible mechanisms of resistance to CHK1i. We report that BRCAwt HGSOC develops resistance to prexasertib monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death. On the other hand, we noted CHK1's regulation on RAD51-mediated homologous recombination (HR) repair was not altered in CHK1i-resistant cells. Therefore, CHK1i sensitizes CHK1i-resistant cells to DNA damaging agents such as gemcitabine or hydroxyurea by inhibition of HR. In summary, our results demonstrate new mechanistic insights of functionally distinct CHK1 activities and highlight a potential combination treatment approach to overcome CHK1i resistance in BRCAwt HGSOC.

COPD and lung cancer incidence in the Women's Health Initiative Observational Study: A brief report.

OBJECTIVES: Lung cancer is the leading cause of cancer mortality in both men and women in the United States. COPD is associated with lung cancer independently of cigarette smoking, but remains understudied in women. Utilizing data from the Women's Health Initiative Observational Study (WHI-OS), this report investigates the association between COPD and development of lung cancer, with a focus on ethnicity and cancer subtype. MATERIALS AND METHODS: The WHI-OS, part of the larger Women's Health Initiative (WHI), is comprised of postmenopausal women between ages 50 and 79 years old at enrollment. Self-administered questionnaires were utilized to gather baseline demographic, socioeconomic, and behavioral information from participants. For this analysis, COPD status was determined at study entry (baseline) and on annual survey (incident). Information on the primary outcome of interest, diagnosis of lung cancer, was also collected annually. RESULTS AND CONCLUSION: Of the 92,789 women examined, 1,536 developed lung cancer. Overall, women with COPD were 1.64 times more likely to develop lung cancer than those without COPD, after adjusting for smoking status and intensity, ethnicity, education, body mass index, and income (HR = 1.64, 95 % CI: 1.43, 1.89). The relationship between COPD and lung cancer was not found to be significantly different between ethnic groups (p-value = 0.697). The associations between COPD and lung cancer was similar across subtypes (HR range 1.31-2.16), after adjusting for smoking status and intensity. COPD increases risk of lung cancer in women, thus they may benefit from more intensive surveillance compared to similar women without COPD.

RAD6B is a major mediator of triple negative breast cancer cisplatin resistance: Regulation of translesion synthesis/Fanconi anemia crosstalk and BRCA1 independence.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with few therapy options besides chemotherapy. Although platinum-based drugs have shown initial activity in BRCA1-mutated TNBCs, chemoresistance remains a challenge. Here we show that RAD6B (UBE2B), a principal mediator of translesion synthesis (TLS), is overexpressed in BRCA1 wild-type and mutant TNBCs, and RAD6B overexpression correlates with poor survival. Pretreatment with a RAD6-selective inhibitor, SMI#9, enhanced cisplatin chemosensitivity of BRCA1 wild-type and mutant TNBCs. SMI#9 attenuated cisplatin-induced PCNA monoubiquitination (TLS marker), FANCD2 (Fanconi anemia (FA) activation marker), and TLS polymerase POL η. SMI#9-induced decreases in γH2AX levels were associated with concomitant inhibition of H2AX monoubiquitination, suggesting a key role for RAD6 in modulating cisplatin-induced γH2AX via H2AX monoubiquitination. Concordantly, SMI#9 inhibited γH2AX, POL η and FANCD2 foci formation. RAD51 foci formation was unaffected by SMI#9, however, its recruitment to double-strand breaks was inhibited. Using the DR-GFP-based assay, we showed that RAD6B silencing or SMI#9 treatment impairs homologous recombination (HR) in HR-proficient cells. DNA fiber assays confirmed that restart of cisplatin-stalled replicating forks is inhibited by SMI#9 in both BRCA1 wild-type and mutant TNBC cells. Consistent with the in vitro data, SMI#9 and cisplatin combination treatment inhibited BRCA1 wild-type and mutant TNBC growth as compared to controls. These RAD6B activities are unaffected by BRCA1 status of TNBCs suggesting that the RAD6B function in TLS/FA crosstalk could occur in HR-dependent and independent modes. Collectively, these data implicate RAD6 as an important therapeutic target for TNBCs irrespective of their BRCA1 status.

Restored replication fork stabilization, a mechanism of PARP inhibitor resistance, can be overcome by cell cycle checkpoint inhibition.

Poly(ADP-ribose) polymerase (PARP) inhibition serves as a potent therapeutic option eliciting synthetic lethality in cancers harboring homologous recombination (HR) repair defects, such as BRCA mutations. However, the development of resistance to PARP inhibitors (PARPis) poses a clinical challenge. Restoration of HR competency is one of the many molecular factors contributing to PARPi resistance. Combination therapy with cell cycle checkpoint (ATR, CHK1, and WEE1) inhibitors is being investigated clinically in many cancers, particularly in ovarian cancer, to enhance the efficacy and circumvent resistance to PARPis. Ideally, inhibition of ATR, CHK1 and WEE1 proteins will abrogate G2 arrest and subsequent DNA repair via restored HR in PARPi-treated cells. Replication fork stabilization has recently been identified as a potential compensatory PARPi resistance mechanism, found in the absence of restored HR. ATR, CHK1, and WEE1 each possess different roles in replication fork stabilization, providing different mechanisms to consider when developing combination therapies to avoid continued development of drug resistance. This review examines the impact of ATR, CHK1, and WEE1 on replication fork stabilization. We also address the therapeutic potential for combining PARPis with cell cycle inhibitors and the possible consequence of combination therapies which do not adequately address both restored HR and replication fork stabilization as PARPi resistance mechanisms.

Nano-delivery of RAD6/Translesion Synthesis Inhibitor SMI#9 for Triple-negative Breast Cancer Therapy.

The triple-negative breast cancer (TNBC) subtype, regardless of their BRCA1 status, has the poorest outcome compared with other breast cancer subtypes, and currently there are no approved targeted therapies for TNBC. We have previously demonstrated the importance of RAD6-mediated translesion synthesis pathway in TNBC development/progression and chemoresistance, and the potential therapeutic benefit of targeting RAD6 with a RAD6-selective small-molecule inhibitor, SMI#9. To overcome SMI#9 solubility limitations, we recently developed a gold nanoparticle (GNP)-based platform for conjugation and intracellular release of SMI#9, and demonstrated its in vitro cytotoxic activity toward TNBC cells. Here, we characterized the in vivo pharmacokinetic and therapeutic properties of PEGylated GNP-conjugated SMI#9 in BRCA1 wild-type and BRCA1-mutant TNBC xenograft models, and investigated the impact of RAD6 inhibition on TNBC metabolism by 1H-NMR spectroscopy. GNP conjugation allowed the released SMI#9 to achieve higher systemic exposure and longer retention as compared with the unconjugated drug. Systemically administered SMI#9-GNP inhibited the TNBC growth as effectively as intratumorally injected unconjugated SMI#9. Inductively coupled mass spectrometry analysis showed highest GNP concentrations in tumors and liver of SMI#9-GNP and blank-GNP-treated mice; however, tumor growth inhibition occurred only in the SMI#9-GNP-treated group. SMI#9-GNP was tolerated without overt signs of toxicity. SMI#9-induced sensitization was associated with perturbation of a common set of glycolytic pathways in BRCA1 wild-type and BRCA1-mutant TNBC cells. These data reveal novel SMI#9 sensitive markers of metabolic vulnerability for TNBC management and suggest that nanotherapy-mediated RAD6 inhibition offers a promising strategy for TNBC treatment.

Breast cancer complexity: implications of intratumoral heterogeneity in clinical management.

Generation of intratumoral phenotypic and genetic heterogeneity has been attributed to clonal evolution and cancer stem cells that together give rise to a tumor with complex ecosystems. Each ecosystem contains various tumor cell subpopulations and stromal entities, which, depending upon their composition, can influence survival, therapy responses, and global growth of the tumor. Despite recent advances in breast cancer management, the disease has not been completely eradicated as tumors recur despite initial response to treatment. In this review, using data from clinically relevant breast cancer models, we show that the fates of tumor stem cells/progenitor cells in the individual tumor ecosystems comprising a tumor are predetermined to follow a limited (unipotent) and/or unlimited (multipotent) path of differentiation which create conditions for active generation and maintenance of heterogeneity. The resultant dynamic systems respond differently to treatments, thus disrupting the delicate stability maintained in the heterogeneous tumor. This raises the question whether it is better then to preserve stability by preventing takeover by otherwise dormant ecosystems in the tumor following therapy. The ultimate strategy for personalized therapy would require serial assessments of the patient's tumor for biomarker validation during the entire course of treatment that is combined with their three-dimensional mapping to the tumor architecture and landscape.

Pharmacological targeting of RAD6 enzyme-mediated translesion synthesis overcomes resistance to platinum-based drugs.

Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.

Gold nanoparticle conjugated Rad6 inhibitor induces cell death in triple negative breast cancer cells by inducing mitochondrial dysfunction and PARP-1 hyperactivation: Synthesis and characterization.

We recently developed a small molecule inhibitor SMI#9 for Rad6, a protein overexpressed in aggressive breast cancers and involved in DNA damage tolerance. SMI#9 induces cytotoxicity in cancerous cells but spares normal breast cells; however, its therapeutic efficacy is limited by poor solubility. Here we chemically modified SMI#9 to enable its conjugation and hydrolysis from gold nanoparticle (GNP). SMI#9-GNP and parent SMI#9 activities were compared in mesenchymal and basal triple negative breast cancer (TNBC) subtype cells. Whereas SMI#9 is cytotoxic to all TNBC cells, SMI#9-GNP is endocytosed and cytotoxic only in mesenchymal TNBC cells. SMI#9-GNP endocytosis in basal TNBCs is compromised by aggregation. However, when combined with cisplatin, SMI#9-GNP is imported and synergistically increases cisplatin sensitivity. Like SMI#9, SMI#9-GNP spares normal breast cells. The released SMI#9 is active and induces cell death via mitochondrial dysfunction and PARP-1 stabilization/hyperactivation. This work signifies the development of a nanotechnology-based Rad6-targeting therapy for TNBCs. FROM THE CLINICAL EDITOR: Protein Rad6 is overexpressed in breast cancer cells and its blockade may provide a new treatment against 3N breast cancer. The authors conjugated a small molecule inhibitor SMI#9 for Rad6 to gold nanoparticles in this study and showed that this new formulation specifically targeted chemo-resistant breast cancer cells and highlighted the importance of nanotechnology in drug carrier development.

Ultrasound-guided thoracic paravertebral block catheter experience in 2 neonates.

The study objective is to describe our experience with placement and management of thoracic paravertebral block catheters in 2 neonates. The design is retrospective chart review of 2 consecutive newborns undergoing repair of tracheoesophageal fistula. Ultrasound-guided oblique intercostal approach for catheter placement and infusions of dilute ropivicaine for 4-5 days. The measurements are postoperative narcotics and sedatives administered. The main results are neither infant required continuous infusions of sedatives or narcotics. Thoracic paravertebral block catheter infusions for neonatal thoracotomy and chest tube placement may eliminate the need for continuous narcotic infusions and may avoid the need for narcotic and benzodiazepine taper or weaning.

Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance.

Bifunctional alkylating and platinum based drugs are chemotherapeutic agents used to treat cancer. These agents induce DNA adducts via formation of intrastrand or interstrand (ICL) DNA crosslinks, and DNA lesions of the ICL type are particularly toxic as they block DNA replication and/or DNA transcription. However, the therapeutic efficacies of these drugs are frequently limited due to the cancer cell's enhanced ability to repair and tolerate these toxic DNA lesions. This ability to tolerate and survive the DNA damage is accomplished by a set of specialized low fidelity DNA polymerases called translesion synthesis (TLS) polymerases since high fidelity DNA polymerases are unable to replicate the damaged DNA template. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the homologous recombination (HR) pathway and Fanconi anemia (FA) network, processes critical for ICL repair. Here we review the molecular features and functional roles of TLS polymerases, discuss the collaborative interactions and cross-regulation of the TLS DNA damage tolerance pathway, the FA network and the BRCA-dependent HRR pathway, and the impact of TLS hyperactivation on development of chemoresistance. Finally, since TLS hyperactivation results from overexpression of Rad6/Rad18 ubiquitinating enzymes (fundamental components of the TLS pathway), increased PCNA ubiquitination, and/or increased recruitment of TLS polymerases, the potential benefits of selectively targeting critical components of the TLS pathway for enhancing anti-cancer therapeutic efficacy and curtailing chemotherapy-induced mutagenesis are also discussed.

Melanoma Development and Progression Are Associated with Rad6 Upregulation and ß -Catenin Relocation to the Cell Membrane.

We have previously demonstrated that Rad6 and ß -catenin enhance each other's expression through a positive feedback loop to promote breast cancer development/progression. While ß -catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and ß -catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti- ß -catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. ß -Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear ß -catenin. ß -Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic ß -catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited ß -catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of ß -catenin and that ß -catenin and Rad6 play independent roles in melanoma development.

Rad6 is a Potential Early Marker of Melanoma Development.

Melanoma is the leading cause of death from skin cancer in industrialized countries. Several melanoma-related biomarkers and signaling pathways have been identified; however, their relevance to melanoma development/progression or to clinical outcome remains to be established. Aberrant activation of Wnt/ß-catenin pathway is implicated in various cancers including melanoma. We have previously demonstrated Rad6, an ubiquitin-conjugating enzyme, as an important mediator of ß-catenin stability in breast cancer cells. Similar to breast cancer, ß-catenin-activating mutations are rare in melanomas, and since ß-catenin signaling is implicated in melanoma, we examined the relationship between ß-catenin levels/activity and expression of ß-catenin transcriptional targets Rad6 and microphthalmia-associated transcription factor-M (Mitf-M) in melanoma cell models, and expression of Rad6, ß-catenin, and Melan-A in nevi and cutaneous melanoma tissue specimens. Our data show that Rad6 is only weakly expressed in normal human melanocytes but is overexpressed in melanoma lines. Unlike Mitf-M, Rad6 overexpression in melanoma lines is positively associated with high molecular weight ß-catenin protein levels and ß-catenin transcriptional activity. Double-immunofluorescence staining of Rad6 and Melan-A in melanoma tissue microarray showed that histological diagnosis of melanoma is significantly associated with Rad6/Melan-A dual positivity in the melanoma group compared to the nevi group (P=.0029). In contrast to strong ß-catenin expression in normal and tumor areas of superficial spreading malignant melanoma (SSMM), Rad6 expression is undetectable in normal areas and Rad6 expression increases coincide with increased Melan-A in the transformed regions of SSMM. These data suggest a role for Rad6 in melanoma pathogenesis and that Rad6 expression status may serve as an early marker for melanoma development.

The Escherichia coli CydX protein is a member of the CydAB cytochrome bd oxidase complex and is required for cytochrome bd oxidase activity.

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.