Preventing school-based arrest and recidivism through prearrest diversion: Outcomes of the Philadelphia police school diversion program.

OBJECTIVES: Created to combat the school-to-prison pipeline, the Philadelphia Police School Diversion Program offers voluntary community-based services to eligible youth accused of minor school-based offeses in lieu of arrest. This study evaluated program effectiveness in accomplishing goals related to reductions in school-based arrests, serious behavioral incidents, and recidivism. HYPOTHESES: We expected the annual number of school-based arrests in Philadelphia schools to decrease over the program's first 5 years and predicted that the annual number of serious behavioral incidents would not increase. Further, we expected that diverted youth-compared to youth arrested in schools the year before Diversion Program implementation-would have significantly lower rates of recidivism arrests in the 2 years following their school-based incidents. METHOD: Using a quasi-experimental design, we examined data from 2,302 public school students (67.0% male; 76.1% Black; age range: 10-22 years) who were either diverted from arrest through the Diversion Program or arrested in Philadelphia schools in the year prior to Diversion Program implementation. We compared rate of recidivism arrest, number of arrests, and time to arrest between diverted and arrested youth. We also used district-wide descriptive statistics to examine 5-year trends in school-based arrests and serious behavioral incidents. RESULTS: Since program implementation, the annual number of school-based arrests in Philadelphia has declined by 84% and the number of serious behavioral incidents has declined by 34%. Diverted youth demonstrated less recidivism than arrested youth in the 2 years following their initial incident; however, after propensity score matching, we no longer observed significant differences. CONCLUSIONS: Findings indicate that a prearrest diversion program can safely reduce school-based arrests and suggest a need for future research regarding the role of demographic and incident-related characteristics in recidivism outcomes. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Keratin 17 in premalignant and malignant squamous lesions of the cervix: proteomic discovery and immunohistochemical validation as a diagnostic and prognostic biomarker.

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio=14.76, P=0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality.

Optical Genome Mapping for Applications in Repeat Expansion Disorders.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

Elevated expression of keratin 17 in oropharyngeal squamous cell carcinoma is associated with decreased survival.

BACKGROUND: Overexpression of keratin 17 (K17) is highly associated with poor prognosis in squamous cell carcinoma (SCC) of the cervix. This study was performed to (1) determine whether K17 may be a prognostic biomarker in head and neck squamous cell carcinoma (HNSCC) and (2) to establish if K17 expression is associated with human papillomavirus (HPV) status. METHODS: Immunohistochemical staining was performed for K17 of oral, oropharyngeal, and laryngeal SCCs, and normal oropharyngeal mucosa. The HPV status was determined using polymerase chain reaction (PCR). RESULTS: Elevated K17 expression was significantly associated with an overall decreased patient survival (P = .02) and, more specifically, in patients with oropharyngeal SCC (P = .01). When controlling for HPV status and tumor location K17 was still a significant predictor of survival (P = .01). CONCLUSION: Therefore, K17 is a novel prognostic biomarker of poor survival for patients with HNSCCs, controlling for anatomic site and HPV status.

Improving Exclusive Breastfeeding in an Urban Academic Hospital.

BACKGROUND AND OBJECTIVE: Breastfeeding has many well-established health benefits for infants and mothers. There is greater risk reduction in health outcomes with exclusive breastfeeding (EBF). Our urban academic facility has had long-standing low EBF rates, serving a population with breastfeeding disparities. We sought to improve EBF rates through a Learning Collaborative model by participating in the Best Fed Beginnings project. METHODS: Formal improvement science methods were used, including the development of a key driver diagram and plan-do-study-act cycles. Improvement activities followed the Ten Steps to Successful Breastfeeding. RESULTS: We demonstrated significant improvement in the median adherence to 2 process measures, rooming in and skin-to-skin after delivery. Subsequently, the proportion of infants exclusively breastfed at hospital discharge in our facility increased from 37% to 59%. We demonstrated an increase in sustained breastfeeding in a subset of patients at a postpartum follow-up visit. These improvements led to Baby-Friendly designation at our facility. CONCLUSIONS: This quality improvement initiative resulted in a higher number of infants exclusively breastfed in our patient population at "high risk not to breastfeed." Other hospitals can use these described methods and techniques to improve their EBF rates.

Keratin 17 is overexpressed and predicts poor survival in estrogen receptor-negative/human epidermal growth factor receptor-2-negative breast cancer.

Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other keratins (16/14/9) was evaluated in 113 ER-/HER2- ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER-/HER2- (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among keratins 17, 16, 14, and 9 mRNA levels suggesting these keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER-/HER2- breast cancer.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function.

BACKGROUND: Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model. METHODS AND RESULTS: Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3-2×106 cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 µg kg-1 min-1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested If-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration. CONCLUSIONS: iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized.

Her2 and Ki67 Biomarkers Predict Recurrence of Ductal Carcinoma in Situ.

BACKGROUND: A subset of patients with ductal carcinoma in situ (DCIS) experience recurrence or progression to invasive cancer. Current clinical practice is not reliably guided by DCIS recurrence prediction, although recurrence risk for invasive breast cancer can now be assessed. We analyzed a panel of biomarkers (estrogen receptor, Her2, Ki67, p53, cyclin D1, COX-2, caveolin-1, survivin, and PPAR-γ) and DCIS histologic and clinical features to determine associations with DCIS recurrence. MATERIALS AND METHODS: Seventy DCIS cases diagnosed between 1995 and 2010 were divided into 2 groups: 52 had DCIS without known recurrence after excision and 18 had DCIS with subsequent recurrence after excision as DCIS or invasive carcinoma in the ipsilateral or contralateral breast. Tissue microarrays were prepared, immunohistochemistry performed, and expression of the biomarkers scored semiquantitatively. Variables analyzed included age, tumor size, margin status, DCIS grade, necrosis, histologic type, and immunohistochemistry scores. Differences between groups were evaluated using t tests for continuous variables and Fisher exact tests for categorical variables. RESULTS: Intraductal necrosis was associated with increased recurrence risk: 46% of nonrecurrent cases showed necrosis compared with 83% of those who recurred (P=0.007). Her2 (human epidermal growth factor receptor 2) and Ki67 expression distributions were significantly different between nonrecurrent and recurrent cases. Her2 was overexpressed in 14% of nonrecurrent cases compared with 50% in the recurrent cases (P=0.03). A total of 87% of nonrecurrent cases had low Ki67 staining (0% to 10%) compared with 50% among the recurrent cases (P=0.002). CONCLUSION: Our results suggest that Her2 and Ki67 immunohistochemistry and the presence of intraductal necrosis aid in DCIS risk stratification.

Reduction of gastric cancer proliferation and invasion by miR-15a mediated suppression of Bmi-1 translation.

B-cell specific moloney leukemia virus insertion site 1 (Bmi-1) gene plays important roles in gastric cancer, but the epigenetic regulatory mechanism by microRNA (miRNA) and the functional significance of Bmi-1 inhibition in gastric cancer remains elusive. In this study, we systematically investigated the functional roles of miRNA mediated Bmi-1 suppression in gastric cancer. Our results show that the expression of miR-15a is significantly reduced in gastric cancer and the protein expression levels of Bmi-1 are inversely correlated with miR-15a (P = 0.034) in gastric cancer patient samples. Functional studies revealed that ectopic expression of miR-15a decreased Bmi-1 in gastric cancer cell lines with reduced proliferation and tumor invasion. High levels of Bmi-1 in gastric cancer patients are significantly associated with better overall survival (P = 0.024) based on the Kaplan-Meier survival analysis.

Jak/STAT and PI3K signaling pathways have both common and distinct roles in IL-7-mediated activities in human CD8+ T cells.

IL-7 plays an important role in T cell survival, function, and memory cell development, yet the role of cytokine signaling pathways in these processes has not been fully elucidated. Moreover, the underlying mechanisms for the observed impairment of IL-7 activity in diseases, such as HIV infection, breast cancer, and autoimmunity, are not well understood. It was therefore hypothesized that IL-7-induced signaling molecules could be linked with distinct IL-7-associated activities. To address this, the activation and functional associations of IL-7-induced signaling pathways, specifically antigen-independent activities that are relevant to T cell homeostasis, were examined. Low concentrations of IL-7 (100 pg/ml) are capable of activating the Jak-STAT and PI3K signaling pathways, whereas higher concentrations (500-1000 pg/ml) were required to induce Bcl-2 production and glucose uptake. Even higher concentrations of IL-7 (10,000 pg/ml) were needed to induce cell proliferation and intracellular accumulation of perforin. Inhibition of Jak activation reduced IL-7-induced Bcl-2 and perforin production, whereas inhibition of Jak/STAT or PI3K pathways reduced glucose uptake and proliferation. This study suggests a complex control of IL-7-associated activities in the absence of antigen stimulation. These data may provide insights into mechanisms of impaired IL-7 signaling and function in disease and could be relevant for the study of IL-7-based immunotherapeutics. Specifically, this study has linked STAT5 and PI3K activation to shared and distinct IL-7-associated activities in human CD8+ T cells.

Differential expression of miRNAs in colon cancer between African and Caucasian Americans: implications for cancer racial health disparities.

Colorectal cancer (CRC) incidence and mortality are higher in African Americans (AAs) than in Caucasian Americans (CAs) and microRNAs (miRNAs) have been found to be dysregulated in colonic and other neoplasias. The aim of this exploratory study was to identify candidate miRNAs that could contribute to potential biological differences between AA and CA colon cancers. Total RNA was isolated from tumor and paired adjacent normal colon tissue from 30 AA and 31 CA colon cancer patients archived at Stony Brook University (SBU) and Washington University (WU)­St. Louis Medical Center. miRNA profiles were determined by probing human genome-wide miRNA arrays with RNA isolated from each sample. Using repeated measures analysis of variance (RANOVA), miRNAs were selected that exhibited significant (p<0.05) interactions between race and tumor or significant (fold change >1.5, p<0.05) main effects of race and/or tumor. Quantitative polymerase chain reaction (q-PCR) was used to confirm miRNAs identified by microarray analysis. Candidate miRNA targets were analyzed using immunohistochemistry. RANOVA results indicated that miR-182, miR152, miR-204, miR-222 and miR-202 exhibited significant race and tumor main effects. Of these miRNAs, q-PCR analysis confirmed that miR-182 was upregulated in AA vs. CA tumors and exhibited significant race:tumor interaction. Immunohistochemical analysis revealed that the levels of FOXO1 and FOXO3A, two potential miR-182 targets, are reduced in AA tumors. miRNAs may play a role in the differences between AA and CA colon cancer. Specifically, differences in miRNA expression levels of miR-182 may contribute to decreased survival in AA colon cancer patients.

Establishment of highly tumorigenic human colorectal cancer cell line (CR4) with properties of putative cancer stem cells.

BACKGROUND: Colorectal cancer (CRC) has the third highest mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of it is occupied by malignant stem cells (cancer stem cells, CSCs, or cancer-initiating cells, CICs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant cancer models, large banks of characterized patient-derived low-passage cell lines, and especially CIC-enriched cell lines, are urgently needed. PRINCIPAL FINDINGS: Here we report the establishment of a novel CIC-enriched, highly tumorigenic and clonogenic colon cancer cell line, CR4, derived from liver metastasis. This stable cell line was established by combining 3D culturing and 2D culturing in stem cell media, subcloning of cells with particular morphology, co-culture with carcinoma associated fibroblasts (CAFs) and serial transplantation to NOD/SCID mice. Using RNA-Seq complete transcriptome profiling of the tumorigenic fraction of the CR4 cells in comparison to the bulk tumor cells, we have identified about 360 differentially expressed transcripts, many of which represent stemness, pluripotency and resistance to treatment. Majority of the established CR4 cells express common markers of stemness, including CD133, CD44, CD166, EpCAM, CD24 and Lgr5. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express key markers of pluripotency markers, including Sox-2, Oct3/4 and c-Myc. Constitutive overactivation of ABC transporters and NF-kB and absence of tumor suppressors p53 and p21 may partially explain exceptional drug resistance of the CR4 cells. CONCLUSIONS: The highly tumorigenic and clonogenic CIC-enriched CR4 cell line may provide an important new tool to support the discovery of novel diagnostic and/or prognostic biomarkers as well as the development of more effective therapeutic strategies.

Immunocytochemical colocalization of P16(INK4a) and Ki-67 predicts CIN2/3 and AIS/adenocarcinoma.

BACKGROUND: Although previous studies have shown that p16(INK4a) and Ki-67 are sensitive and specific markers for high-grade lesions (≥CIN2) on cervical biopsies, limited information is available regarding the performance of a dual-staining approach as a diagnostic adjunct in cervical cytology. We evaluated a dual p16(INK4a)/Ki-67 immunocytochemistry (ICC) assay to determine its sensitivity and specificity versus that of high-risk HPV (HR-HPV) in a US-based pilot cytology study. METHODS: ThinPrep specimens from 122 cervical cytology specimens encompassing 23 negative (NILM), 20 ASC-US, 22 LSIL, 17 ASCH, 22 HSIL, and 18AGC cases were processed for multiplexed ICC staining using a CINtec Plus Kit. Dual-positive assay results were defined based on the detection of 1 or more epithelial cells that were stained for both p16INK4a and Ki-67 without regard to cellular morphology. HR-HPV testing was performed by multiplex PCR with capillary electrophoresis genotyping. RESULTS: Dual staining for p16(INK4a) and Ki-67 was frequently detected in HSIL and AGC but was rarely detected in NILM cases. The HR-HPV assay showed a sensitivity of 76.2% and a specificity of 55.8% for the detection of clinically significant cervical squamous or endometrial lesions. In contrast, the colocalization of p16(INK4a) plus Ki-67 maintained a high sensitivity of 81.8% and improved specificity to 81.8% for biopsy-confirmed CIN2/3, endocervical adenocarcinoma, or endometrial adenocarcinoma. CONCLUSIONS: Dual staining for p16(INK4a)/Ki-67 immunocytochemistry dramatically increased specificity and maintained high-level sensitivity for the diagnosis of CIN2/3 or glandular lesions compared with PCR-based testing for HR-HPV.

Differential expression of transferrin receptor (TfR) in a spectrum of normal to malignant breast tissues: implications for in situ and invasive carcinoma.

Transferrin receptor (TfR), a type II transmembranous receptor involved in iron uptake, is highly expressed in some cancers. We evaluated the expression of TfR in a spectrum of normal to malignant breast tissues to test the hypothesis that overexpression is associated with malignant transformation. Expression of TfR was studied by immunohistochemistry (CD71-Antibody, Thermo Fisher Scientific, Fremont, CA) for percent positive cells (%) and intensity of staining (0 to 3 score) in normal (n=127), benign (n=172), potentially premalignant and in-situ carcinoma (n=65), and invasive carcinoma (n=38). Normal and benign lesions had significantly lower TfR expression compared with premalignant lesions (atypical hyperplasia and carcinoma in situ) and invasive carcinoma (median %: 0, 10, 50, and 80, respectively; P<0.0001). TfR expression was higher in high-grade ductal carcinoma in situ (DCIS) than in other grades of DCIS (median %: 95 vs 55; P=0.02) and in high-grade invasive carcinoma. Among the latter, medullary carcinoma had the highest expression and there was a trend for invasive lobular carcinoma to have a higher expression than invasive ductal carcinoma. In invasive carcinoma cases, the proportion (%) of cells staining for TfR was inversely correlated with the percentage of estrogen receptor-positive cells, with a decreasing slope on linear regression models. In comparison, the relationship with progesterone receptor was not as well defined and linear regression models revealed close to a flat line. These data show that there is a differential expression of TfR in breast tissues with the highest expression in in-situ and invasive carcinoma and with aggressive phenotypes (higher grade DCIS and lower estrogen receptor positivity in invasive carcinomas). Further studies are indicated to determine whether TfR is an independently significant prognostic marker that may have potential as a therapeutic target in in-situ and invasive breast carcinoma.

Prognostic significance of miR-215 in colon cancer.

BACKGROUND: We have previously shown that miR-215 suppressed the expression of key targets such as thymidylate synthase (TS), dihydrofolate reductase, and denticleless protein homolog (DTL) in colon cancer. miR-215 is a tumor suppressor candidate due to the upregulation of p53 and p21 by targeting DTL. However, high levels of miR-215 conferred chemoresistance due to cell cycle arrest and reduced cell proliferation by suppressing DTL. In this study, the clinical significance of miR-215 was further investigated as a potential prognostic biomarker in colon cancer patients. METHODS: Total RNAs were extracted from 34 paired normal and colon (stage II and III) tumor specimens using the Trizol-based approach. The levels of miR-215 and a closely related miR-192 were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) expression analysis. The expression of DTL mRNA and protein were quantified by real time qRT-PCR and immunohistochemistry. RESULTS: The expression levels of miR-192 (P = .0008) and miR-215 (P < .0001) were significantly decreased in colon tumors compared with normal tissues. DTL was significantly over-expressed and was inversely correlated with miR-215, further suggesting an in vivo physiologic relevance of miR-215 mediated DTL suppression. Kaplan-Meier survival analysis by Cox regression revealed that high levels of miR-215 expression (hazard ratio, 3.516; 95% confidence interval, 1.007-12.28, P = .025) are closely associated with poor patient's overall survival. Furthermore, an elevated expression of a miR-215 target protein DTL was detected in colon cancer tissues whereas no expression was present in normal tissues. CONCLUSION: miR-215 has a unique potential as a prognostic biomarker in stage II and III colon cancer.

p16(INK4a) is superior to high-risk human papillomavirus testing in cervical cytology for the prediction of underlying high-grade dysplasia.

BACKGROUND: The primary goal of this study was to compare the clinical performance of an optimized and rigorously controlled immunocytochemical (ICC) assay for p16(INK4a) to high-risk (HR) human papillomavirus (HPV) detection by polymerase chain reaction (PCR) as diagnostic adjuncts for cytology specimens from colposcopy patients. METHODS: : The study included 403 cervical cytology specimens collected within 3 months of colposcopy. The colposcopic impression and cervical biopsy diagnosis served as the standards for correlation with cytological, p16(INK4a), and HPV data. p16(INK4a) was evaluated using an immunoperoxidase-based assay that was linear over 4 logs for the detection of HeLa-spiked positive control cytology specimens, using a threshold for positive test results that was based on receiver operating characteristic curve analysis. HR-HPV was detected by multiplex PCR using genotype-specific primers. RESULTS: : In all combined diagnostic categories (negative for intraepithelial lesion and malignancy, atypical glandular cells, atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion, low-grade squamous intraepithelial lesion, and high-grade squamous intraepithelial lesion), the p16(INK4a) ICC and HR-HPV assays, respectively, had sensitivity of 81.7% and 83.3% (P = .81) and specificity of 78.1% and 50.9% (P < .001) for the detection of underlying > or =grade 2 cervical intraepithelial neoplasia (CIN) lesions on biopsy. Furthermore, the positive predictive value of p16(INK4a) ICC was greater than that of HR-HPV for patients with biopsies > or =CIN-2 (41.2% and 24.2%, respectively, P = .001). CONCLUSIONS: : This p16(INK4a) immunocytochemical assay has superior specificity but similar sensitivity to HR-HPV testing to predict underlying high-grade dysplastic lesions in patients who are referred for colposcopy. The determination of the overall performance characteristics of p16(INK4a) immunocytochemistry, as an independent test or in combination with HPV testing in low-risk screening populations, however, will require subsequent large-scale prospective clinical trials.

The layered structure of human mitochondrial DNA nucleoids.

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.

Retinal bipolar neurons express the cyclic nucleotide-gated channel of cone photoreceptors.

Cyclic nucleotide-gated (CNG) channels link intracellular cyclic nucleotides to changes in membrane ionic conductance in a variety of physiological contexts. In the retina, in addition to their central role in phototransduction, CNG channels may be involved in nitric oxide signaling in bipolar neurons or in the hyperpolarizing synaptic response to glutamate in ON-type (depolarizing) bipolar cells. Despite their potential physiological significance, however, expression of CNG channels has not yet been demonstrated in bipolar cells. To identify CNG channel subtypes in retinal bipolar neurons, we used single-cell molecular biological techniques in morphologically distinctive ON bipolar cells from goldfish retina. Both single-cell in situ hybridization and single-cell RT-PCR demonstrated in ON bipolar cells the presence of mRNA for the CNG channel subtype that is also found in cone photoreceptors. Other bipolar cells, which likely represent OFF cells, did not express the cone CNG channel. Thus the CNG channel of cone photoreceptors is expressed in ON bipolar cells, where it may be involved in physiological responses to nitric oxide, or in the sign-inverting glutamatergic synapse that gives rise to the ON visual pathway.