Molecular and Clinical Epidemiology of SARS-CoV-2 Infection among Vaccinated and Unvaccinated Individuals in a Large Healthcare Organization from New Jersey.

New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations, from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOCs) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOCs, including Alpha (n = 714), Delta (n = 1877), and Omicron (n = 1802). Omicron isolates were further sub-typed as BA.1 (n = 899), BA.2 (n = 853), or BA.4/BA.5 (n = 50); the remaining 614 isolates were classified as "Other". Approximately 31.5% (1577/5007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p < 0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p < 0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p < 0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.

Using focus groups to plan culturally acceptable primary cervical cancer screening in Grenada, West Indies.

Objective: To document Grenadian women's knowledge about cervical cancer and human papillomavirus (HPV) infection, as well as their attitudes towards primary cervical cancer screening methods. Methods: In this qualitative study, we used focus groups in Grenada to gather information concerning women's knowledge about, attitudes towards and perceptions of screening for cervical cancer and general knowledge about HPV. Ten focus groups comprising 73 participants representing 5 of the 6 parishes in Grenada were conducted with women aged 19-59. Participants were asked about pelvic exams, Pap smears, HPV, reasons for seeking or avoiding cervical cancer screening and how different modalities of testing might affect their decision-making. Responses were then coded and organized into common themes. Results: While many respondents had heard of HPV, far fewer knew about its causative role in cervical cancer, how to prevent HPV infection or testing for the high-risk HPV types that cause almost all cases of cervical cancer. Many participants were aware that cervical cancer screening was beneficial, but numerous barriers to obtaining that screening were noted, including concerns about privacy and stigma, potential discomfort, and the cost and inconvenience involved. Conclusions: Our findings have implications for future cervical cancer screening efforts in Grenada. Central to these efforts should be a focus on educating Grenadians about the role of HPV infection in cervical cancer and the importance of early detection through screening. In addition, addressing issues of stigma and privacy are key to eliminating cervical cancer in Grenada.

Using focus groups to plan culturally acceptable primary cervical cancer screening in Grenada, West Indies / Uso de grupos focales para planificar exámenes primarios de detección del cáncer cervicouterino culturalmente aceptables en Granada, Caribe / Uso de grupos focais para planejar estratégias culturalmente aceitáveis para rastreamento de câncer do colo do útero primário em Granada, Índias Ocidentais

ABSTRACT Objective. To document Grenadian women's knowledge about cervical cancer and human papillomavirus (HPV) infection, as well as their attitudes towards primary cervical cancer screening methods. Methods. In this qualitative study, we used focus groups in Grenada to gather information concerning women's knowledge about, attitudes towards and perceptions of screening for cervical cancer and general knowledge about HPV. Ten focus groups comprising 73 participants representing 5 of the 6 parishes in Grenada were conducted with women aged 19-59. Participants were asked about pelvic exams, Pap smears, HPV, reasons for seeking or avoiding cervical cancer screening and how different modalities of testing might affect their decision-making. Responses were then coded and organized into common themes. Results. While many respondents had heard of HPV, far fewer knew about its causative role in cervical cancer, how to prevent HPV infection or testing for the high-risk HPV types that cause almost all cases of cervical cancer. Many participants were aware that cervical cancer screening was beneficial, but numerous barriers to obtaining that screening were noted, including concerns about privacy and stigma, potential discomfort, and the cost and inconvenience involved. Conclusions. Our findings have implications for future cervical cancer screening efforts in Grenada. Central to these efforts should be a focus on educating Grenadians about the role of HPV infection in cervical cancer and the importance of early detection through screening. In addition, addressing issues of stigma and privacy are key to eliminating cervical cancer in Grenada.

RESUMEN Objetivo. Documentar los conocimientos de las mujeres granadinas sobre el cáncer cervicouterino y la infección por el virus de los papilomas humanos (VPH), así como sus actitudes hacia los métodos primarios de detección del cáncer cervicouterino. Métodos. En este estudio cualitativo, se han empleado grupos focales en Granada para recopilar información sobre los conocimientos, las actitudes y las percepciones de las mujeres sobre la detección del cáncer cervicouterino y nociones generales sobre el VPH. Participaron 73 mujeres de 19 a 59 años de edad, representantes de 5 de las 6 parroquias de Granada. Se formaron diez grupos focales, a los que se les preguntó sobre los exámenes pélvicos, las pruebas de Papanicolaou, el VPH, las razones para buscar o evitar la detección del cáncer cervicouterino y cómo las diferentes modalidades de examen podrían afectar sus decisiones. Luego se codificaron las respuestas y se organizaron en temas comunes. Resultados. Si bien muchas participantes habían oído hablar del VPH, un número considerablemente menor conocía su relación causal con el cáncer cervicouterino, cómo prevenir la infección por VPH o los exámenes de detección de los tipos del VPH de alto riesgo que causan casi todos los casos de cáncer cervicouterino. Muchas participantes sabían que los exámenes de detección del cáncer de cuello uterino eran convenientes, pero mencionaron numerosos obstáculos para obtenerlos, como las preocupaciones sobre la privacidad y la estigmatización, posibles molestias, así como el costo y los inconvenientes relacionados. Conclusiones. Nuestros hallazgos tienen implicaciones para la futura labor de detección del cáncer cervicouterino en Granada. En esta labor debería ser esencial adoptar un enfoque dirigido a educar a las granadinas sobre la relación de la infección por VPH con el cáncer cervicouterino y la importancia de la detección temprana mediante exámenes. Además, combatir los problemas de estigmatización y privacidad es clave para eliminar el cáncer cervicouterino en Granada.

RESUMO Objetivo. Documentar o conhecimento das mulheres de Granada sobre o câncer do colo do útero e a infecção por papilomavírus humano (HPV), bem como suas atitudes em relação aos métodos de rastreamento de câncer do colo do útero primário. Métodos. Neste estudo qualitativo, usamos grupos focais em Granada para coletar informações sobre conhecimentos, atitudes e percepções das mulheres sobre o rastreamento de câncer do colo do útero e conhecimentos gerais sobre HPV. Foram conduzidos dez grupos focais, incluindo 73 participantes e representando 5 das 6 paróquias de Granada, com mulheres de 19 a 59 anos de idade. As participantes responderam perguntas sobre exames ginecológicos, Papanicolau, HPV, razões para procurar ou evitar o rastreamento de câncer do colo do útero e como diferentes modalidades de testes podem afetar sua tomada de decisão. As respostas foram codificadas e organizadas por temas comuns. Resultados. Muitas participantes já tinham ouvido falar do HPV, mas um número muito menor conhecia sua relação causal com o câncer do colo do útero, formas de prevenir a infecção por HPV ou os testes para os tipos de HPV de alto risco, que causam quase todos os casos de câncer do colo do útero. Muitas participantes sabiam que o rastreamento de câncer do colo do útero era benéfico, mas várias barreiras para o rastreamento foram indicadas, incluindo preocupações relacionadas à privacidade e ao estigma, o potencial desconforto e o custo e inconveniência envolvidos. Conclusões. Nossos achados têm implicações para as futuras iniciativas de rastreamento de câncer do colo do útero em Granada. Essas iniciativas devem se focar em educar a população de Granada sobre o papel da infecção por HPV no câncer do colo do útero e a importância da detecção precoce por meio do rastreamento. Além disso, é fundamental abordar questões de estigma e privacidade para eliminar o câncer do colo do útero em Granada.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum.

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane ß-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

The significance of the Dewar valence photoisomer as a UV radiation-induced DNA photoproduct in marine microbial communities.

Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by high-performance liquid chromatography-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER.

Viral perturbations of host networks reflect disease etiology.

Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia.

Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3.

Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.

Suppression of hPOT1 in diploid human cells results in an hTERT-dependent alteration of telomere length dynamics.

POT1 is a 3' telomeric single-stranded overhang binding protein that has been implicated in chromosome end protection, the regulation of telomerase function, and defining the 5' chromosome terminus. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Primary human fibroblasts express low levels of catalytically active hTERT in an S-phase-restricted manner that fails to counteract telomere attrition with cell division. Here, we show that diploid human fibroblasts in which hPOT1 expression has been suppressed harbor telomeres that are longer than control cells. This difference in telomere length delays the onset of replicative senescence and is dependent on S-phase-restricted hTERT expression. These findings are consistent with the view that hPOT1 promotes a nonextendable telomere state resistant to extension by S-phase-restricted telomerase. Manipulating this function of hPOT1 may thus hasten the cytotoxic effects of telomerase inhibition.

Kinase requirements in human cells: I. Comparing kinase requirements across various cell types.

shRNA loss-of-function screens were used to identify kinases that were rate-limiting for promoting cell proliferation and survival. Here, we study the differences in kinase requirements among various human cells, including freshly prepared primary cells, isogenic cells, immortalized cells, and cancer cell lines. Closely related patterns of kinase requirements among the various cell types were observed in three cases: (i) in repeat experiments using the same cells, (ii) with multiple populations of freshly prepared primary epithelial cells isolated from the same tissue source, and (iii) between nearly isogenic cells that differ from each other by the expression of a single gene. Other commonly used cancer cell lines were distinct from one another, even when they were isolated from similar tumor types. Even primary cells of different lineages isolated from the same tissue source showed many differences. The differences in kinase requirements among cell lines observed in this study suggest that the control of proliferation and survival may be significantly different between cell lines and that simple comparisons from any one cell to another may be misleading. Although the regulation of cell proliferation and survival are heavily studied areas, we did not see a bias in these screens toward the identification of previously known and well studied kinases, suggesting that our knowledge of molecular events in these areas is still meager.

Kinase requirements in human cells: II. Genetic interaction screens identify kinase requirements following HPV16 E7 expression in cancer cells.

Human papillomavirus (HPV) oncoproteins subvert cellular signaling pathways, including kinase pathways, during the carcinogenic process. To identify kinases targeted by the HPV16 E7 oncoprotein, shRNA kinase screens were performed in RKO colorectal carcinoma cell lines that differ only in their expression of HPV16 E7. Our screens identified kinases that were essential for the survival of RKO cells, but not essential for RKO cells expressing HPV16 E7. These kinases include CDK6, ERBB3, FYN, AAK1, and TSSK2. We show that, as predicted, CDK6 knockdown inhibits pRb phosphorylation and induces S-phase depletion, thereby inhibiting cell viability. Knockdown of ERBB3, FYN, AAK1, and TSSK2 induces a similar loss of cell viability through an unknown mechanism. Expression of the HPV16 E7 oncoprotein, known to bind and degrade pRb, relieves the requirement of these kinases. These studies demonstate that expression of a single oncoprotein can dramatically alter kinase sensitivity in human cells. The shRNA screens used here perform analogously to genetic interaction screens commonly used in genetically tractable organisms such as yeast, and thus represent an exciting method for unbiased identification of cellular signaling pathways targeted by cancer mutations.

Endogenous optical biomarkers of normal and human papillomavirus immortalized epithelial cells.

Cellular transformation is associated with a number of phenotypic, cell biological, biochemical and metabolic alterations. The detection and classification of morphological cellular abnormalities represents the foundation of classical histopathology and remains an important mainstay in the clinic. More recently, significant effort is being expended towards the development of noninvasive modalities for the detection of cancer at an early stage, when therapeutic interventions are highly successful. Methods that rely on the detection of optical signatures represent one class of such approaches that have yielded promising results. In our study, we have applied two spectroscopic imaging approaches to systematically identify in a quantitative manner the fluorescence and light scattering signatures of subcellular abnormalities that are associated with cellular transformation. Notably, we find that tryptophan images reveal not only intensity but also localization differences between normal and human papillomavirus immortalized cells, possibly originating from changes in the expression, 3D packing and organization of proteins and protein-rich subcellular organelles. Additionally, we detect alterations in cellular metabolism through quantitative evaluation of the NADH, FAD fluorescence and the corresponding redox ratio. Finally, we use light scattering spectroscopy to identify differences in nuclear morphology and subcellular organization that occur from the nanometer to the micrometer scale. Thus, these optical approaches provide complementary biomarkers based on endogenous fluorescence and scattering cellular changes that occur at the molecular, biochemical and morphological level. Since they obviate the need for staining and tissue removal and can be easily combined, they provide desirable options for further clinical development and assessment.

Identification of an iron-responsive protein that is antigenic in patients with Chlamydia trachomatis genital infections.

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

NFI is an essential positive transcription factor for human papillomavirus type 16 early gene expression.

Human papillomavirus type 16 (HPV16) is the primary etiologic agent for greater than 50% of all cervical carcinomas. Expression of the HPV16 E6 and E7 oncoproteins is under control of the upstream regulatory region (URR), which contains a myriad of transcription factor binding sites, including 7 half sites for NFI. These NFI binding sites were used as probes in electrophoretic mobility shift assays (EMSAs), and mutational analysis of individual and multiple NFI binding sites was performed in order to demonstrate the relative importance of particular NFI sites to URR activity. By using 5 NFI half sites as an enhancer, we were able to detect a 4-fold increase in URR activity. Our results define the role and relative contribution of NFI binding sites to the basal activity of the HPV16 promoter, and demonstrate that NFI binding sites can act independently to enhance HPV16 URR activity in immortalized keratinocytes.

Human papillomavirus E7 oncoprotein dysregulates steroid receptor coactivator 1 localization and function.

High-risk human papillomaviruses (HPVs) are present in virtually all cervical carcinomas. However, the majority of women infected with high-risk HPVs do not develop cervical cancer. Therefore, cofactors must contribute to the development and progression of cervical cancer. Although numerous studies have implicated steroid hormones as cofactors in the initiation and progression of cervical neoplasia, the molecular mechanisms by which they contribute to cervical carcinogenesis are currently unknown. These observations led us to investigate a newly discovered association of the high-risk HPV type 16 (HPV16) E7 oncoprotein with steroid receptor coactivator 1 (SRC-1), an essential component of steroid hormone signaling. HPV16 E7 has been previously reported to interact with p300 and p300/CBP-associated factor (PCAF), members of some SRC-1 transcriptional complexes. We demonstrate here that HPV16 E7 associates in vivo and in vitro with SRC-1 independently of p300 and PCAF. Luciferase reporter constructs under the control of either the interleukin-8 promoter or a promoter containing multimerized synthetic estrogen response elements were used to determine the effect of high- and low-risk HPV E7 expression on SRC-1-mediated transcription. In addition, histone acetyltransferase (HAT) assays were performed to determine the effect of HPV E7 on SRC-1-associated HAT activity. These experiments reveal that HPV16 E7 expression down-regulates SRC-1-mediated transcription and SRC-1-associated HAT activity. SRC-1 localization experiments show that SRC-1 is relocalized to the cytoplasm in the presence of high- and low-risk HPV E7 proteins. Our data suggest that HPV E7 proteins dysregulate hormone-dependent gene expression by association with and relocalization of SRC-1. Dysregulation of SRC-1 localization and function by HPV E7 may provide insight into the molecular mechanisms by which steroid hormones act as cofactors in the induction and progression of cervical neoplasia.

The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane.

The protein translocation channel at the outer envelope membrane of chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal transit peptide. Within the transit peptide of the pea Toc75, a major portion of the 10 amino acid long stretch that contains nine glycine residues was shown to be necessary for directing the protein to the chloroplast outer membrane in vitro. In order to get insights into the mechanism by which the polyglycine stretch mediates correct targeting, we divided it into three tri-glycine segments and examined the importance of each domain in targeting specificity in vitro. Replacement of the most C-terminal segment with alanine residues resulted in mistargeting the protein to the stroma, while exchange of either of the other two tri-glycine regions had no effect on correct targeting. Furthermore, simultaneous replacement of the N-terminal and middle tri-glycine segments with alanine repeats did not cause mistargeting of the protein as much as those of the N- and C-terminal, or the middle and C-terminal segments. These results indicate that the most C-terminal tri-glycine segment is important for correct targeting. Exchanging this portion with a repeat of leucine or glutamic acid also caused missorting of Toc75 to the stroma. By contrast, its replacement with repeats of asparagine, aspartic acid, serine, and proline did not largely affect correct targeting. These data suggest that relatively compact and nonhydrophobic side chains in this particular region play a crucial role in correct sorting of Toc75.

NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression.

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-beta treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-beta to inhibit the URR. Based on these studies, we propose that TGF-beta inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.