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Journal articles on the topic 'Sentinel genes'

Author: Grafiati
Published: 10 March 2023

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1

Oh, Chang-Kyu, and Yuseok Moon. "Dietary and Sentinel Factors Leading to Hemochromatosis." Nutrients 11, no. 5 (May 10, 2019): 1047. http://dx.doi.org/10.3390/nu11051047.

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Although hereditary hemochromatosis is associated with the mutation of genes involved in iron transport and metabolism, secondary hemochromatosis is due to external factors, such as intended or unintended iron overload, hemolysis-linked iron exposure or other stress-impaired iron metabolism. The present review addresses diet-linked etiologies of hemochromatosis and their pathogenesis in the network of genes and nutrients. Although the mechanistic association to diet-linked etiologies can be complicated, the stress sentinels are pivotally involved in the pathological processes of secondary hemochromatosis in response to iron excess and other external stresses. Moreover, the mutations in these sentineling pathway-linked genes increase susceptibility to secondary hemochromatosis. Thus, the crosstalk between nutrients and genes would verify the complex procedures in the clinical outcomes of secondary hemochromatosis and chronic complications, such as malignancy. All of this evidence provides crucial insights into comprehensive clinical or nutritional interventions for hemochromatosis.
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2

Lee, J. H., H. Torisu-Itakura, Y. Huynh, and R. Essner. "Quantitative analysis of immunosuppressive genes expressed in melanoma sentinel nodes." Annals of Surgical Oncology 11, S2 (February 2004): S86. http://dx.doi.org/10.1007/bf02524075.

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Hao, Hongying, Deyi Xiao, Jianmin Pan, Jifu Qu, Michael Egger, Sabine Waigel, Mary Ann G. Sanders, Wolfgang Zacharias, Shesh N. Rai, and Kelly M. McMasters. "Sentinel Lymph Node Genes to Predict Prognosis in Node-Positive Melanoma Patients." Annals of Surgical Oncology 24, no. 1 (September 23, 2016): 108–16. http://dx.doi.org/10.1245/s10434-016-5575-7.

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4

Torisu-Itakura, Hitoe, Jonathan H. Lee, Randall P. Scheri, Young Huynh, Xing Ye, Richard Essner, and Donald L. Morton. "Molecular Characterization of Inflammatory Genes in Sentinel and Nonsentinel Nodes in Melanoma." Clinical Cancer Research 13, no. 11 (June 1, 2007): 3125–32. http://dx.doi.org/10.1158/1078-0432.ccr-06-2645.

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5

Krajaejun, T., G. M. Gauthier, C. A. Rappleye, T. D. Sullivan, and B. S. Klein. "Development and Application of a Green Fluorescent Protein Sentinel System for Identification of RNA Interference in Blastomyces dermatitidis Illuminates the Role of Septin in Morphogenesis and Sporulation." Eukaryotic Cell 6, no. 8 (May 11, 2007): 1299–309. http://dx.doi.org/10.1128/ec.00401-06.

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ABSTRACT A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, “X,” next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.
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Torisu-Itakura, Hitoe, Randall Scheri, Young Huynh, Xing Ye, and Richard Essner. "Molecular profiling of inflammatory genes predicts tumor positivity of sentinel nodes in melanoma." Journal of the American College of Surgeons 203, no. 3 (September 2006): S47. http://dx.doi.org/10.1016/j.jamcollsurg.2006.05.113.

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7

Young, Barry P., Kathryn L. Post, Jesse T. Chao, Fabian Meili, Kurt Haas, and Christopher Loewen. "Sentinel interaction mapping – a generic approach for the functional analysis of human disease gene variants using yeast." Disease Models & Mechanisms 13, no. 7 (May 29, 2020): dmm044560. http://dx.doi.org/10.1242/dmm.044560.

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ABSTRACTAdvances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using synthetic dosage lethality screening, ‘sentinel’ yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in a high-throughput fashion through simple growth assays using solid or liquid media. Sentinel interaction mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.
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8

Karapetyan, Lilit, William Gooding, Aofei Li, Xi Yang, Andrew Knight, Hassan M. Abushukair, Danielle Vargas De Stefano, et al. "Sentinel Lymph Node Gene Expression Signature Predicts Recurrence-Free Survival in Cutaneous Melanoma." Cancers 14, no. 20 (October 11, 2022): 4973. http://dx.doi.org/10.3390/cancers14204973.

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We sought to develop a sentinel lymph node gene expression signature score predictive of disease recurrence in patients with cutaneous melanoma. Gene expression profiling was performed on SLN biopsies using U133A 2.0 Affymetrix gene chips. The top 25 genes associated with recurrence-free survival (RFS) were selected and a penalized regression function was used to select 12 genes with a non-zero coefficient. A proportional hazards regression model was used to evaluate the association between clinical covariates, gene signature score, and RFS. Among the 45 patients evaluated, 23 (51%) had a positive SLN. Twenty-one (46.7%) patients developed disease recurrence. For the top 25 differentially expressed genes (DEG), 12 non-zero penalized coefficients were estimated (CLGN, C1QTNF3, ADORA3, ARHGAP8, DCTN1, ASPSCR1, CHRFAM7A, ZNF223, PDE6G, CXCL3, HEXIM1, HLA-DRB). This 12-gene signature score was significantly associated with RFS (p < 0.0001) and produced a bootstrap C index of 0.888. In univariate analysis, Breslow thickness, presence of primary tumor ulceration, SLN positivity were each significantly associated with RFS. After simultaneously adjusting for these prognostic factors in relation to the gene signature, the 12-gene score remained a significant independent predictor for RFS (p < 0.0001). This SLN 12-gene signature risk score is associated with melanoma recurrence regardless of SLN status and may be used as a prognostic factor for RFS.
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Ouellette, Rodney J., Dominique Richard, and Emmanuel Maïcas. "RT-PCR for Mammaglobin Genes,MGB1andMGB2, Identifies Breast Cancer Micrometastases in Sentinel Lymph Nodes." American Journal of Clinical Pathology 121, no. 5 (May 2004): 637–43. http://dx.doi.org/10.1309/mmactxt55l8qtkc1.

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10

Kondragunta, Bhargavi, Bharat H. Joshi, Jing Han, Kurt A. Brorson, Raj K. Puri, Antonio R. Moreira, and Govind Rao. "Bioreactor environment-sensitive sentinel genes as novel metrics for cell culture scale-down comparability." Biotechnology Progress 28, no. 5 (September 2012): 1138–51. http://dx.doi.org/10.1002/btpr.1606.

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11

Liao, Ning, Cheukfai Li, Li Cao, Yanhua Chen, Chongyang Ren, Xiaoqing Chen, Hsiaopei Mok, et al. "Single cell profile of tumor and immune cells in primary triple-negative breast cancer and different sites in the axillary lymph nodes." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e12570-e12570. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e12570.

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e12570 Background: Little is known about the host-tumor interaction in the lymph node basin at a single cell level. This study examines single cell sequences in breast cancer nodal metastasis of a patient with triple negative breast cancer. Methods: The primary breast tumor, sentinel lymph node, an adjacent lymph node with metastatic involvement and a clinically normal-appearing lymph node were collected during operation. Single-cell sequencing was performed on all specimens. Results: 14,016 cells were clustered as 6 cell populations. Cancer cells demonstrated the molecular characteristics of TNBC basal B subtype and highly expressed genes in the MAPK signaling cascade. Tumor associated macrophages regulated antigen processing and presentation and other immune-related pathways to promote tumor invasion. CD8+ and CD4+ T lymphocytes concentrated more in sentinel lymph node and mainly stratified as two transcriptional states. Conclusions: The first single cell report investigates the host-tumor interaction in the lymph node basin of triple-negative breast cancer. Single-cell sequencing analysis suggested that the sentinel lymph node was the initial meeting site of tumor infiltration and immune response, where partial T lymphocytes perform anti-tumor activity while other T cells exhibit an exhaustion state. We proposed a molecular explanation to the well-established clinical principle that the 5-year and 10-year survival outcomes were noninferior between sentinel lymph node dissection (SLND) and axillary lymph node dissection (ALND).
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Liao, Ning, Cheukfai Li, Li Cao, Yanhua Chen, Chongyang Ren, Xiaoqing Chen, Hsiaopei Mok, et al. "Single cell profile of tumor and immune cells in primary triple-negative breast cancer and different sites in the axillary lymph nodes." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e12570-e12570. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e12570.

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e12570 Background: Little is known about the host-tumor interaction in the lymph node basin at a single cell level. This study examines single cell sequences in breast cancer nodal metastasis of a patient with triple negative breast cancer. Methods: The primary breast tumor, sentinel lymph node, an adjacent lymph node with metastatic involvement and a clinically normal-appearing lymph node were collected during operation. Single-cell sequencing was performed on all specimens. Results: 14,016 cells were clustered as 6 cell populations. Cancer cells demonstrated the molecular characteristics of TNBC basal B subtype and highly expressed genes in the MAPK signaling cascade. Tumor associated macrophages regulated antigen processing and presentation and other immune-related pathways to promote tumor invasion. CD8+ and CD4+ T lymphocytes concentrated more in sentinel lymph node and mainly stratified as two transcriptional states. Conclusions: The first single cell report investigates the host-tumor interaction in the lymph node basin of triple-negative breast cancer. Single-cell sequencing analysis suggested that the sentinel lymph node was the initial meeting site of tumor infiltration and immune response, where partial T lymphocytes perform anti-tumor activity while other T cells exhibit an exhaustion state. We proposed a molecular explanation to the well-established clinical principle that the 5-year and 10-year survival outcomes were noninferior between sentinel lymph node dissection (SLND) and axillary lymph node dissection (ALND).
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13

Beasley, Georgia M., Eda K. Holl, Norma E. Farrow, Margaret G. Leddy, April K. Salama, Brent A. Hanks, and Smita K. Nair. "Abstract B16: The immune profile of sentinel lymph nodes in melanoma." Cancer Research 80, no. 19_Supplement (October 1, 2020): B16. http://dx.doi.org/10.1158/1538-7445.mel2019-b16.

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Abstract Background: Immune cell composition and immune-mediated processes occurring in the sentinel lymph node (SLN) of melanoma patients regulate generation of tumor-specific effector T-cell responses and ultimately tumor control. We hypothesized that comprehensive analysis of these immune-associated processes in the SLN biopsy may identify patients at high risk of melanoma recurrence. Methods: We analyzed 38 formalin-fixed, paraffin-embedded sentinel lymph nodes from melanoma patients undergoing SLN biopsy from 2009-2012 using the NanoString nCounter® PanCancer Immune Profiling panel. The panel included 770 immune related genes. Results: Of 38 patients, 19 were SLN negative and 19 were SLN positive. Among SLN-negative patients (n=19), the median Breslow depth was 1.25 mm, and 21% (n=4) had developed recurrence at median follow-up of 6.2 years. Among SLN-positive patients, the median Breslow depth was 3.35 mm and 37% (n=7) had developed recurrence at median follow-up of 6.5 years. There were 31 genes significantly differentially expressed (P&lt;0.01) in SLN positive compared to SLN negative, including higher relative expression of B-cell signaling markers (CD19, CD22, Pax5, CXCR5, CD79B). Among all 38 patients, there were 13 genes differentially expressed (P&lt;0.05) between patients with and without recurrence, including higher relative expression of IL6 in patients with recurrence compared to no recurrence. Further analysis is ongoing. Conclusion: In this retrospective series, we found the immunologic gene expression profile of melanoma SLNs was significantly different in SLN negative versus SLN positive. There were also significant differences in the profile between patients who developed melanoma recurrence versus no recurrence regardless of SLN status. We aim to build on these pilot data. The goal is to use this profile to predict patients at high risk of recurrence after SLNB who may benefit from adjuvant therapy. Citation Format: Georgia M. Beasley, Eda K. Holl, Norma E. Farrow, Margaret G. Leddy, April K. Salama, Brent A. Hanks, Smita K. Nair. The immune profile of sentinel lymph nodes in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B16.
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Karapetyan, Lilit, William Gooding, Xi Yang, Andrew Knight, Aofei Li, Cindy Sander, Monica Panelli, Walter J. Storkus, Ahmad A. Tarhini, and John M. Kirkwood. "Abstract 5169: Sentinel lymph node gene expression signature predicts survival outcome in cutaneous melanoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5169. http://dx.doi.org/10.1158/1538-7445.am2022-5169.

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Abstract Background: Sentinel lymph node status is an important prognostic factor for melanoma progression. We sought to develop a sentinel lymph node gene expression signature score predictive of disease progression in patients with cutaneous melanoma. Methods: Cases were retrieved from University of Pittsburgh protocol “Sentinel Node and Non-Sentinel Lymph Nodes (SLN and non-SLN) Procurement from Melanoma Subjects for Molecular Profiling.” Gene expression profiling was performed on SLN biopsies using U133A 2.0 Affymetrix gene chips. The top 25 genes associated with progression-free survival (PFS) were selected and a penalized regression function was used to select genes with a non-zero coefficient. A risk score was calculated from a 12 gene signature. A proportional hazards regression model was used to evaluate the association between clinical covariates, gene signature score, and PFS. Results: In total, 45 patients were included [female=18 (40%), median age (range)= 56 (16-81) years]. The median Breslow thickness (IQR) was 4.1 (3.3-6.0) mm, ulceration was present in 29 (67%) cases, and 25 (56%) lesions had nodular histology. Among patients, 23 (51%) had a positive SLN via H&E. Twenty-one (46.7%) patients developed disease recurrence/progression with a median follow-up of 8.3 years. The median time to progression was 9.6 years. Among the top 25 genes, 12 non-zero penalized coefficients were estimated (CLGN, C1QTNF3, ADORA3, ARHGAP8, ZNF223, PDE6G, CXCL3, CHRFAM7A, HLA-DRB-decreased risk, DCTN1, ASPSCR1, HEXIM1-increased risk). A 12-gene signature score was significantly associated with PFS (p&lt;0.0001) and produced a bootstrap cross-validated C index of 0.888. Stratifying by SLN status the gene signature risk score remained highly significant (p&lt;0.0001). In univariate analysis, Breslow thickness (HR=1.41), presence of ulceration (HR=3.61), SLN positivity (HR=2.50), and acral-lentiginous histology compared to nodular (HR=4.07) were each significantly associated with PFS (all p&lt;0.05). After simultaneously adjusting for all of these factors plus the gene signature, the 12-gene score remained a significant predictor for PFS (p&lt;0.0001). Conclusion: A SLN 12-gene signature risk score is associated with melanoma progression regardless of SLN status and can be used as a prognostic factor for PFS of patients. External validation of the gene signature is warranted. Citation Format: Lilit Karapetyan, William Gooding, Xi Yang, Andrew Knight, Aofei Li, Cindy Sander, Monica Panelli, Walter J. Storkus, Ahmad A. Tarhini, John M. Kirkwood. Sentinel lymph node gene expression signature predicts survival outcome in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5169.
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15

Tarhini, Ahmad A., William A. LaFramboise, Uma N. M. Rao, Howard Edington, James F. Pingpank, Matthew Peter Holtzman, Hussein Abdul-Hassan Tawbi, et al. "Differential genomic profiles of tumor-involved and tumor-free sentinel lymph nodes in patients with melanoma." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 9043. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.9043.

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9043 Background: For clinical stage I and II node negative melanoma the histologic status of the sentinel lymph node (SLN) is the most significant predictor of survival. Molecular characterization of node positive and node negative SLNs through gene expression profiling may improve the understanding of the molecular mechanisms of metastasis and identify specific gene signatures for SLN+/SLN- that correlate with clinical outcome. Methods: We characterized 15 SLN+ and 15 SLN- melanoma patients (T3a/b, T4a/b) who underwent SLN dissection for routine staging using transcriptome profiling analysis on 5μ sections of fresh LN samples. The primary endpoint was mRNA expression profiling using the U133A 2.0 Affymetrix gene chips. Significance Analysis of Microarrays v.4 was used to perform non-parametric analysis and statistical comparison for each transcript corrected for false discovery rate (q value) to control for type 1 errors arising from multiple tests. Pathway analysis was performed using Ingenuity Pathway Analysis software. Results: Tumor size ranged from 1-2mm in size. Twenty-one genes were expressed at significantly (q value <0.056) higher levels in SLN+ vs SLN-. These included CCNA2, CEP55, DCT, FEN1, HMMR, MLANA, PAICS, PBK, POSTN, RAB27A, RRM2, SHCBP1, SILV, TYR, CENPE, CCL20, CHEK1, LINS, TPX2, TTK, TYRP1. Pathway analysis (39 genes >1.4 fold; q < 0.12) identified the top disease categories as “Cancer” (26 genes, p = 5.7x10-9) and “Dermatological” (14 genes, p = 3.9x10-6). The top functional categories included “Cell Cycle” (21 genes p= 2.3x10-12) and “Cell Growth and Proliferation” (26 genes p=9x10-9). The relevant canonical pathways were “Eumelanin Biosynthesis” (p=3x10-5) and “Cell Cycle: G2/M DNA Damage Checkpoint Regulation” (p=1.2x10-4). Conclusions: We identified a 21 gene signature that is consistent with metastatic melanoma and its microenvironment and is differentially expressed in SLNs that are tumor involved. These gene families provide a signature of nodal involvement that may assist in diagnosis, and may be further explored towards prediction of outcome and development of therapy. A validation study is ongoing with clinical outcome association analyses as follow up is mature.
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Li, Xiaoyun, Jingling Tang, Hang Du, Xinjun Wang, Liyun Wu, Pingsheng Hu, Hua Zhang, Ruyi Zhang, and Yuan Yang. "Immune Characters and Plasticity of the Sentinel Lymph Node in Colorectal Cancer Patients." Journal of Immunology Research 2021 (August 18, 2021): 1–11. http://dx.doi.org/10.1155/2021/5516399.

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Purpose. This study is aimed at immunologically characterizing sentinel lymph nodes (SNs) in colorectal cancer (CRC) patients and identifying changes in immunological phenotype and function of SNs isolated from the tumor immunosuppressive microenvironment. Methods. A total of 53 pairs of matched SNs and non-SNs (NSNs) were collected by using a lymph node tracer dye. Flow cytometry was performed to detect the immunophenotype of T cells as well as the expression of activation and inhibitory markers. Differential expression and distribution of characteristic immune cell markers were analyzed by multiplex immunohistochemistry (mIHC). Transcriptomics analysis was conducted to compare the differences in the expression of immune-related genes among lymph nodes. The ex vivo culture of lymph nodes was carried out to examine changes in immunological phenotypes and functions. Results. Compared with NSNs, SNs harbored a significantly higher percentage of regulatory T cells (Tregs) but a lower proportion of MoMDSCs. As indicated in the mIHC assays, Tregs, T follicular helper (Tfh) cells, and M2 macrophages were mainly distributed in cortical areas, germinal centers, and subcapsular sinus areas, respectively, while significantly higher numbers of Tregs and Tfh cells were detected in SNs as compared to NSNs. Moreover, GSEA revealed that T cell activation genes and CD8+ T cell exhaustion-related genes are enriched in SNs and NSNs, respectively. The ex vivo culture led to an increase in the proportion of CD4+ cells, while activating T cells in SNs. In addition, SNs displayed a higher increase in the expression of cytokines IFN-γ, TNF-α, and sFas than NSNs. Conclusion. SNs are shown to be in an immune active state in vivo, while highly expressing inhibitory cytokines and suppressive markers. The ex vivo culture enhanced antitumor immunological function of SN-T cells, providing a starting material for adoptive cell therapy for CRC.
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Ma, Jiangang, Biao Tang, Jiahui Lin, Abdelaziz Ed-Dra, Hui Lin, Jing Wu, Yuzhi Dong, Hua Yang, and Min Yue. "Genome Assessment of Carbapenem- and Colistin-Resistant Escherichia coli from Patients in a Sentinel Hospital in China." Cells 11, no. 21 (November 3, 2022): 3480. http://dx.doi.org/10.3390/cells11213480.

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Antimicrobial-resistant (AMR) pathogens are a significant threat to public health worldwide. However, the primary carrier of AMR genes, particularly against last-resort antibiotics, is still only partially studied in Chinese hospitals. In a sentinel hospital in China, we collected 157 E. coli strains from patients between January and July 2021. One blaNDM-1-, nine blaNDM-5-, and one mcr-1-positive E. coli recovered from inpatients were identified as resistant to meropenem and colistin. There are 37 virulence genes discovered in the 11 strains, including astA in strain EC21Z-147 (O128: H4), which belongs to the enteroaggregative E. coli (EAEC). The blaNDM gene is distributed into distinct ST types, including ST48, ST616, ST410, ST711, and ST2003, while the mcr-1 gene was identified in ST117. The conjugative plasmids IncX3, IncI1-I, and IncI2 mediated the blaNDM-5 and mcr-1 genes detected among inpatients. Notably, the youngest age at which mcr-1-positive E. coli has been reported was at one day old, in a child in which the strain is closely related to strains with animal origins. Hospitals are major environments for the spread and dissemination of critical virulence and AMR genes, which requires active monitoring systems at the genome level to surveil the spread of virulence and AMR.
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Sacristán, Irene, Fernando Esperón, Francisca Acuña, Emilio Aguilar, Sebastián García, María José López, Aitor Cevidanes, et al. "Antibiotic resistance genes as landscape anthropization indicators: Using a wild felid as sentinel in Chile." Science of The Total Environment 703 (February 2020): 134900. http://dx.doi.org/10.1016/j.scitotenv.2019.134900.

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Yang, Yunju, Akiko Suzuki, Junichi Iwata, and Goo Jun. "Secondary Genome-Wide Association Study Using Novel Analytical Strategies Disentangle Genetic Components of Cleft Lip and/or Cleft Palate in 1q32.2." Genes 11, no. 11 (October 29, 2020): 1280. http://dx.doi.org/10.3390/genes11111280.

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Orofacial cleft (OFC) is one of the most prevalent birth defects, leading to substantial and long-term burdens in a newborn’s quality of life. Although studies revealed several genetic variants associated with the birth defect, novel approaches may provide additional clues about its etiology. Using the Center for Craniofacial and Dental Genetics project data (n = 10,542), we performed linear mixed-model analyses to study the genetic compositions of OFC and investigated the dependence among identified loci using conditional analyses. To identify genes associated with OFC, we conducted a transcriptome-wide association study (TWAS) based on predicted expression levels. In addition to confirming the previous findings at four loci, 1q32.2, 8q24, 2p24.2 and 17p13.1, we untwined two independent loci at 1q32.2, TRAF3IP3 and IRF6. The sentinel SNP in TRAF3IP3 (rs2235370, p-value = 5.15 × 10−9) was independent of the sentinel SNP at IRF6 (rs2235373, r2 < 0.3). We found that the IRF6 effect became nonsignificant once the 8q24 effect was conditioned, while the TRAF3IP3 effect remained significant. Furthermore, we identified nine genes associated with OFC in TWAS, implicating a glutathione synthesis and drug detoxification pathway. We identified some meaningful additions to the OFC etiology using novel statistical methods in the existing data.
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Gradilone, Angela, Paola Gazzaniga, Diego Ribuffo, Susanna Scarpa, Emanuele Cigna, Fortunata Vasaturo, Ugo Bottoni, et al. "Survivin, bcl-2, bax, and bcl-X Gene Expression in Sentinel Lymph Nodes From Melanoma Patients." Journal of Clinical Oncology 21, no. 2 (January 15, 2003): 306–12. http://dx.doi.org/10.1200/jco.2003.08.066.

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Purpose: The expression of apoptosis-related genes, such as survivin, bcl-2, bcl-X, and bax, has been evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry in sentinel lymph nodes (SLNs) from melanoma patients and then correlated to the outcome of patients. Patients and Methods: Thirty-six SLNs were examined. After RNA extraction, an RT-PCR followed by Southern blot hybridization was performed to detect survivin, bcl-2, bcl-X, and bax mRNA. bcl-2, survivin, and bax gene expression was evaluated, whenever possible, also by immunohistochemistry at the protein level. Results: We found a significant correlation (P < .005) between survivin expression and outcome of patients; in fact, 61.5% of patients expressing survivin gene progressed or died because of the disease, whereas 38.5% are currently disease-free. Among patients negative for survivin expression, 100% are disease-free after a median follow-up time of 52.9 months. We did not find a significant correlation between bcl-2, bax, and bcl-X gene expression and outcome of patients. In fact, these genes were found equally expressed in patients with disease progression and in disease-free patients. Conclusion: Our findings show a variable expression of apoptosis-related genes in SLNs of melanoma patients; more interestingly, we found that survivin expression correlates to outcome of patients in a statistically significant way, whereas the expression of other genes, such as bcl-2, bax, and bcl-X, did not seem to correlate to progression of disease. We suggest that the detection of survivin gene expression by RT-PCR in SLNs may be a useful prognostic indicator.
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da Fonseca, Flávio G., Giliane S. Trindade, Ricardo L. A. Silva, Cláudio A. Bonjardim, Paulo C. P. Ferreira, and Erna G. Kroon. "Characterization of a vaccinia-like virus isolated in a Brazilian forest." Journal of General Virology 83, no. 1 (January 1, 2002): 223–28. http://dx.doi.org/10.1099/0022-1317-83-1-223.

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The SPAn232 virus (SPAnv) was isolated from sentinel mice in the forest of Cotia, São Paulo, Brazil. It was grouped originally as a Cotia virus (CV) sample due to serological cross-reaction with the latter. However, SPAnv presented genetic characteristics that differed from CV and indicated that SPAnv is a member of the vaccinia virus (VV) subgroup. SPAnv showed a HindIII-digested DNA pattern similar to those of the WR and Lister strains of VV. Also, SPAnv presented genes homologous to the vaccinia growth factor, thymidine kinase and A-type inclusion (ATI) genes from VV. RFLP analysis of the SPAnv ATI homologous gene indicated that the virus belongs to the VV group. Nucleotide sequences from SPAnv genes showed up to 99% similarity with the same genes from VV. Such a relationship was confirmed visually through the drawing of phylogenetic trees. The results point out the occurrence of a VV strain that is possibly in active circulation in the forests of Southeast Brazil.
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Garcias, Biel, Laia Aguirre, Chiara Seminati, Nerea Reyes, Alberto Allepuz, Elena Obón, Rafael A. Molina-Lopez, and Laila Darwich. "Extended-Spectrum β-Lactam Resistant Klebsiella pneumoniae and Escherichia coli in Wild European Hedgehogs (Erinaceus europeus) Living in Populated Areas." Animals 11, no. 10 (September 28, 2021): 2837. http://dx.doi.org/10.3390/ani11102837.

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Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.
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Egger, Michael E., Deyi Xiao, Hongying Hao, Charles W. Kimbrough, Jianmin Pan, Shesh N. Rai, Alexander C. Cambon, Sabine J. Waigel, Wolfgang Zacharias, and Kelly M. McMasters. "Unique Genes in Tumor-Positive Sentinel Lymph Nodes Associated with Nonsentinel Lymph Node Metastases in Melanoma." Annals of Surgical Oncology 25, no. 5 (March 1, 2018): 1296–303. http://dx.doi.org/10.1245/s10434-018-6377-x.

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Riis, Margit L. H., Torben Lüders, Elke K. Markert, Vilde D. Haakensen, Anne-Jorun Nesbakken, Vessela N. Kristensen, and Ida R. K. Bukholm. "Molecular Profiles of Pre- and Postoperative Breast Cancer Tumours Reveal Differentially Expressed Genes." ISRN Oncology 2012 (November 26, 2012): 1–12. http://dx.doi.org/10.5402/2012/450267.

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Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun families as well as genes of which the expression has previously been associated with cancer. The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology.
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Shapovalova, Valeria, Elvira Shaidullina, Ilya Azizov, Eugene Sheck, Alexey Martinovich, Marina Dyachkova, Alina Matsvay, et al. "Molecular Epidemiology of mcr-1-Positive Escherichia coli and Klebsiella pneumoniae Isolates: Results from Russian Sentinel Surveillance (2013–2018)." Microorganisms 10, no. 10 (October 14, 2022): 2034. http://dx.doi.org/10.3390/microorganisms10102034.

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Background: The dissemination of mobile colistin resistance (mcr) genes is a serious healthcare threat because polymyxins represent “last-line” therapeutics for multi-drug-resistant Gram-negative pathogens. This study aimed to assess the prevalence of colistin resistance and mcr genes and characteristics of clinical Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) isolates and plasmids carrying these genes in Russia. Methods: A total of 4324 Eco and 4530 Kpn collected in the frame of sentinel surveillance in 2013–2018 were tested for susceptibility to colistin and other antibiotics using the broth microdilution method. mcr genes were screened by real-time PCR. Phylogeny, genomic features and plasmids of mcr-positive isolates were assessed using whole-genome sequencing and subsequent bioinformatic analysis. Results: Colistin resistance was detected in 2.24% Eco and 9.3% Kpn. Twenty-two (0.51%) Eco and two (0.04%) Kpn from distant sites carried mcr-1.1. Most mcr-positive isolates co-harbored ESBLs and other resistance determinants to various antibiotic classes. The mcr-positive Eco belonged to 16 MLST types, with ST359 being most common; Kpn belonged to ST307 and ST23. mcr-1.1 was carried mainly in IncI2 (n = 18) and IncX4 (n = 5) plasmids highly similar to those identified previously in human, animal and environmental isolates. Conclusion: This study demonstrated a dissemination of “typical” mcr-bearing plasmids among diverse Eco and Kpn genotypes and across a wide geographic area in Russia. Given the frequent association of mcr with other resistance determinants and potential clinical impact, the continual surveillance of this threat is warranted.
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Tarhini, Ahmad A., Brian Hobbs, Arjun Khunger, Ibrahim Yassine, Iyad Kobeissi, Patrick Hwu, Vernon K. Sondak, William LaFramboise, and John M. Kirkwood. "An 11-gene expression signature related to tumorigenesis and immunosuppression in primary cutaneous melanoma predicts sentinel lymph node metastatic status." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e21579-e21579. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e21579.

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e21579 Background: A biomarker derived from the primary melanoma tumor to predict regional sentinel lymph node (SLN) metastatic status can be valuable in guiding the decision making in planning the SLN surgical procedure for candidate patients. Methods: Gene expression profiling was performed on primary cutaneous melanoma tumor biopsies of 49 (24 known SLN+, 25 SLN-) patients (T3a/b, T4a/b) who underwent SLN for staging using transcriptome profiling analysis. U133A 2.0 Affymetrix gene chips were used. Significance Analysis of Microarrays (SAM) was used to test the association between gene expression level of the primary tumor and SLN status. Genes with fold change > 1.5 and q value < 0.05 were considered differentially expressed. Pathway analysis was performed using Ingenuity Pathway Analysis. Benjamini and Hochberg method was used to adjust for multiple testing in pathway analysis. All statistical analyses were implemented in R. Results: A total of 49 patients with primary cutaneous melanoma were studied, of which, 24 were diagnosed as SLN positive and 25 as SLN negative by routine H&E and immunohistochemistry. Using SLN metastatic status as the outcome, a univariate logistic regression model was fitted with individual probe sets. A total of 251 probe sets were filtered and 11 probe sets were considered as differentially expressed (DE) between SLN-ve and SLN+ve groups, with the selection criterion set at Benjamini- Hochberg method -adjusted p -value below 0.05 and the absolute log fold change above 0.5. As each of the 11 probe sets was matched to a unique known gene, an 11 gene signature was derived and among them, the expression level of 7 genes was significantly reduced in the SLN+ve group compared to the SLN-ve group, while 4 genes were overexpressed in the SLN+ve group. Integrative and interactive heatmaps were produced from hierarchical cluster analysis to show the 11 differentially expressed genes. Selected genes were found to be uniformly related to tumorigenesis, malignant progression, DNA repair, cell cycle regulation, chemoresistance, immunosuppression and/or involvement in multiple cancer-related pathways. Several of the selected genes were previously shown to be prognostic in various malignancies. Conclusions: We present a unique 11-gene expression signature derived from primary melanoma tumor biopsy samples and related to tumorigenesis and immunosuppression that may be useful for the prognostic stratification of melanoma patients. This may allow the optimal selection of patients for SLN biopsy.
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Llorente, Lola, Óscar Herrero, Mónica Aquilino, and Rosario Planelló. "Prodiamesa olivacea: de novo biomarker genes in a potential sentinel organism for ecotoxicity studies in natural scenarios." Aquatic Toxicology 227 (October 2020): 105593. http://dx.doi.org/10.1016/j.aquatox.2020.105593.

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HOTTER, G. S., I. H. LI, and N. P. FRENCH. "Binary genomotyping using lipooligosaccharide biosynthesis genes distinguishes betweenCampylobacter jejuniisolates within poultry-associated multilocus sequence types." Epidemiology and Infection 138, no. 7 (November 3, 2009): 992–1003. http://dx.doi.org/10.1017/s0950268809991075.

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SUMMARYCampylobacter jejuniis a leading cause of human bacterial gastroenteritis throughout the industrialized world. We investigated whether or not differences in gene complement at the lipooligosaccharide (LOS) biosynthesis locus can identify epidemiologically useful binary genomotypes in 87C. jejuniisolates from poultry-associated multilocus sequence types (STs) collected during the course of a sentinel surveillance study. Using a PCR-based approach, we correlated assignment of both isolate LOS locus class and binary genomotype with ST. We found that isolates within STs 45, 190, 354 and 474 displayed mosaicism in gene complement at the intra-ST level. For example, based upon their binary genomotypes, we assigned individual ST-45 isolates from human clinical cases as probably originating from either a poultry or wild-bird source. However, intra-ST mosaicism in gene complement was observed alongside broader patterns of congruence in LOS locus class and gene complement that distinguished between isolates from different STs.
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Stürzenbaum, S. R., J. Andre, P. Kille, and A. J. Morgan. "Earthworm genomes, genes and proteins: the (re)discovery of Darwin's worms." Proceedings of the Royal Society B: Biological Sciences 276, no. 1658 (December 16, 2008): 789–97. http://dx.doi.org/10.1098/rspb.2008.1510.

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Small incremental biological change, winnowed by natural selection over geological time scales to produce large consequences, was Darwin's singular insight that revolutionized the life sciences. His publications after 1859, including the ‘earthworm book’, were all written to amplify and support the evolutionary theory presented in the Origin . Darwin was unable to provide a physical basis for the inheritance of favoured traits because of the absence of genetic knowledge that much later led to the ‘modern synthesis’. Mistaken though he was in advocating systemic ‘gemmules’ as agents of inheritance, Darwin was perceptive in seeking to underpin his core vision with concrete factors that both determine the nature of a trait in one generation and convey it to subsequent generations. This brief review evaluates the molecular genetic literature on earthworms published during the last decade, and casts light on the specific aspects of earthworm evolutionary biology that more or less engaged Darwin: (i) biogeography, (ii) species diversity, (iii) local adaptations and (iv) sensitivity. We predict that the current understanding will deepen with the announcement of a draft earthworm genome in Darwin's bicentenary year, 2009. Subsequently, the earthworm may be elevated from the status of a soil sentinel to that elusive entity, an ecologically relevant genetic model organism.
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Schnappinger, Dirk, Sabine Ehrt, Martin I. Voskuil, Yang Liu, Joseph A. Mangan, Irene M. Monahan, Gregory Dolganov, et al. "Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages." Journal of Experimental Medicine 198, no. 5 (September 1, 2003): 693–704. http://dx.doi.org/10.1084/jem.20030846.

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Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.
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Kousik, C. S., and D. F. Ritchie. "Response of Bell Pepper Cultivars to Bacterial Spot Pathogen Races that Individually Overcome Major Resistance Genes." Plant Disease 82, no. 2 (February 1998): 181–86. http://dx.doi.org/10.1094/pdis.1998.82.2.181.

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The effect of major resistance genes (Bs1, Bs2, and Bs3) or gene combinations for resistance to bacterial spot of bell peppers (Xanthomonas campestris pv. vesicatoria) in 15 commercial cultivars on disease reduction and yield were studied during 1995 and 1996. Reaction of cultivars to specific races (races 1, 2, or 3) of the pathogen corresponded with seed company claims for resistance against these races. Races 1 to 4 were used as initial inoculum in 1995, and races 1 to 6 in 1996 field experiments. Cultivars with no known resistance genes to bacterial spot (e.g., Camelot, Jupiter, and Valiant), a single resistance gene (X3R Camelot, King Arthur), or a combination of Bs1 and Bs3 genes (Guardian, Sentinel, and Admiral) were severely diseased. Yields were reduced in all inoculated cultivars compared to non-inoculated cultivars used as controls. Although races 4 and 6 caused significant disease in cultivars with only Bs1 (King Arthur) or Bs2 (X3R Camelot) genes, cultivars with a combination of Bs1 and Bs2 (Boynton Bell, PR9300-8) had much lower levels of bacterial spot. Roger 4178, a hybrid with a combination of Bs1, Bs2, and Bs3 genes, had the lowest disease ratings. Overall, race 3 was predominant during 1995, while races 3 and 6 were recovered most frequently in 1996.
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Liao, Ning, Cheukfai Li, Li Cao, Yanhua Chen, Chongyang Ren, Xiaoqing Chen, Hsiaopei Mok, et al. "Abstract P5-01-06: Single cell profile of tumor and immune cells in primary triple-negative breast cancer and different sites in the axillary lymph nodes." Cancer Research 83, no. 5_Supplement (March 1, 2023): P5–01–06—P5–01–06. http://dx.doi.org/10.1158/1538-7445.sabcs22-p5-01-06.

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Abstract Purpose: Little is known about the host-tumor interaction in the lymph node basin at a single cell level. This study examines single cell sequences in breast cancer nodal metastasis of a patient with triple negative breast cancer. Methods: The primary breast tumor, sentinel lymph node, an adjacent lymph node with metastatic involvement and a clinically normal-appearing lymph node were collected during operation. Single-cell sequencing was performed on all specimens. Results: 14,016 cells were clustered as 6 cell populations. Cancer cells demonstrated the molecular characteristics of TNBC basal B subtype and highly expressed genes in the MAPK signaling cascade. Tumor associated macrophages regulated antigen processing and presentation and other immune-related pathways to promote tumor invasion. CD8+ and CD4+ T lymphocytes concentrated more in sentinel lymph node and mainly stratified as two transcriptional states. The immune cell amount variation among primary tumor, sentinel and normal lymph nodes showed the similar tendency between the scRNA-seq profile of TNBC samples and a previous reported bulk RNA-seq profile of a breast cancer cohort including all four breast cancer subtype samples. Discussion: Single-cell sequencing analysis suggested that the sentinel lymph node was the initial meeting site of tumor infiltration and immune response, where partial T lymphocytes perform anti-tumor activity while other T cells exhibit an exhaustion state. We proposed a molecular explanation to the well-established clinical principle that the 5-year and 10-year survival outcomes were noninferior between SLND and ALND. Citation Format: Ning Liao, Cheukfai Li, Li Cao, Yanhua Chen, Chongyang Ren, Xiaoqing Chen, Hsiaopei Mok, Lingzhu Wen, Kai Li, Yulei Wang, Yuchen Zhang, Yingzi Li, Jiaoyi Lv, Fangrong Cao, Yuting Luo, Hongrui Li, Wendy Wu, Charles M. Balch, Armando E. Giuliano. Single cell profile of tumor and immune cells in primary triple-negative breast cancer and different sites in the axillary lymph nodes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-01-06.
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Ingerslev, Hans-Christian, Carlo Gunnar Ossum, Thomas Lindenstrøm, and Michael Engelbrecht Nielsen. "Fibroblasts Express Immune Relevant Genes and Are Important Sentinel Cells during Tissue Damage in Rainbow Trout (Oncorhynchus mykiss)." PLoS ONE 5, no. 2 (February 18, 2010): e9304. http://dx.doi.org/10.1371/journal.pone.0009304.

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Srour, Marissa K., Bowen Gao, Farnaz Dadmanesh, Ying Qu, Xiaojiang Cui, and Armando E. Giuliano. "Gene expression comparison between primary triple-negative breast cancer and matched axillary lymph node metastasis." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 565. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.565.

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565 Background: Sentinel lymph node (SLN) biopsy guides breast cancer treatment and prognostication. To date, there have been few studies examining the genetics of SLN metastasis in triple negative breast cancer (TNBC). The aim of this study is to characterize and compare gene expression patterns of primary breast cancers and autologous matched SLN metastases. Methods: Patients with stage 2-3 ER/PR negative, HER2 negative TNBC with macrometastasis to the SLN who did not have neoadjuvant therapy were selected. The tumor-specific area was isolated from breast and SLN paraffin embedded tissue sections. Gene expression of a panel of 2567 cancer-associated genes was analyzed with the HTG EdgeSeq system coupled with the Illumina Next Generation Sequencing (NGS) platform. Results were validated with RNA-scope assays for RNA in situ detection. Results: 18 pairs of TNBC and matched SLN metastasis were analyzed for 2567 genes. Compared with the primary TNBC, SLN metastasis had 34 statistically significant upregulated genes and 31 downregulated genes. SLN metastasis had at least a 5-fold change (FC) in upregulation in 3 genes and downregulation in 3 genes, compared to primary TNBC [Table]. Notably, there was an upregulation of anti-apoptosis and survival signaling genes (i.e. BIRC3) in the SLN metastasis. There was also an upregulation of chemotaxis genes (CCL13, CXCL19, CXCL21, CXCR5, TNFSF11, p<0.001). The most striking feature is the downregulation of genes known to regulate cell microenvironment interaction (MMP2, MMP14, COL1A1, COL1A2, COL5A2, COL6A6, COL11A1, COL17A1). Conclusions: In TNBC, SLN metastasis has a distinct gene expression profile. Genes associated with anti-apoptosis, survival responses, and chemotaxis are upregulated, and genes associated with regulation of extracellular matrix are downregulated. Upregulated and downregulated genes with at least a 5-fold change in gene expression in lymph node metastasis compared with TNBC.[Table: see text]
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Spicer, Lori, Davina Campbell, J. Kristie Johnson, Cynthia Longo, Thomas Balbuena, Thomas Ewing, Maria Karlsson, et al. "Characterization of carbapenem-resistant gram-negative bacteria collected in the Sentinel Surveillance Program, 2018–2019." Antimicrobial Stewardship & Healthcare Epidemiology 2, S1 (May 16, 2022): s52. http://dx.doi.org/10.1017/ash.2022.156.

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Background: Carbapenem resistance in gram-negative organisms is an important public health problem. The CDC conducted Sentinel surveillance in 2018–2019 to characterize these organisms from 9 facilities in 9 different states. Methods: Carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter spp (CRA) obtained from clinical samples of patients in acute-care or long-term care facilities were submitted to the CDC. Identification was confirmed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and antimicrobial susceptibility testing (AST) was performed via broth microdilution for 27 antibiotics. All confirmed CRE and CRPA were tested for carbapenemase production (CP) using the modified carbapenem inactivation method (mCIM). The isolates that were mCIM-positive were assessed by real-time PCR for presence of blaKPC, blaNDM, blaVIM, and blaIMP. CP-CRE were also assessed for blaOXA-48-like. All confirmed CRA were tested for the same genes as CRPA and blaOXA-23–like, blaOXA-24/40-like, blaOXA-58–like, and blaOXA-235–like genes. Difficult-to-treat resistance (DTR) was defined as resistance to all β-lactams (excluding newer β-lactam combination agents) and quinolones tested. Results: The CDC confirmed 208 CRE, 161 CRPA, and 94 CRA. Table 1 summarizes AST results for a selection of drugs. We identified 112 (53.8%) mCIM-positive CRE and 6 (3.7%) mCIM-positive CRPA. The PCR results are summarized in Table 2. One mCIM-positive and PCR-negative isolate was positive in a metallo-β-lactamase screen. Conclusions: Resistance among CRE and CRPA to newer β-lactam combination agents was detected. Options for treating CRA are limited. Of 112 CP-CRE, 85.7% harbored blaKPC; CP-CRPA were rare (3.7%); and most CRA harbored blaOXA-23-like (55.3%) or blaOXA-24/40-like (30.9%). Whole-genome sequencing is planned to better understand gene variants, sequence types, and additional resistance markers present among the isolates.Funding: NoneDisclosures: None
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Coberley, Carter R., James J. Kohler, Joseph N. Brown, Joseph T. Oshier, Henry V. Baker, Michael P. Popp, John W. Sleasman, and Maureen M. Goodenow. "Impact on Genetic Networks in Human Macrophages by a CCR5 Strain of Human Immunodeficiency Virus Type 1." Journal of Virology 78, no. 21 (November 1, 2004): 11477–86. http://dx.doi.org/10.1128/jvi.78.21.11477-11486.2004.

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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) impacts multiple lineages of hematopoietic cells, including lymphocytes and macrophages, either by direct infection or indirectly by perturbations of cell networks, leading to generalized immune deficiency. We designed a study to discover, in primary human macrophages, sentinel genetic targets that are impacted during replication over the course of 7 days by a CCR5-using virus. Expression of mRNA and proteins in virus- or mock-treated macrophages from multiple donors was evaluated. Hierarchical agglomerative cluster analysis grouped into distinct temporal expression patterns >900 known human genes that were induced or repressed at least fourfold by virus. Expression of more than one-third of the genes was induced rapidly by day 2 of infection, while other genes were induced at intermediate (day 4) or late (day 7) time points. More than 200 genes were expressed exclusively in either virus- or mock-treated macrophage cultures, independent of the donor, providing an unequivocal basis to distinguish an effect by virus. HIV-1 altered levels of mRNA and/or protein for diverse cellular programs in macrophages, including multiple genes that can contribute to a transition in the cell cycle from G1 to G2/M, in contrast to expression in mock-treated macrophages of genes that maintain G0/G1. Virus treatment activated mediators of cell cycling, including PP2A, which is impacted by Vpr, as well as GADD45 and BRCA1, potentially novel targets for HIV-1. The results identify interrelated programs conducive to optimal HIV-1 replication and expression of genes that can contribute to macrophage dysfunction.
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Qiao, Chuanling, Qinfang Liu, Bhupinder Bawa, Huigang Shen, Wenbao Qi, Ying Chen, Chris Ka Pun Mok, Adolfo García-Sastre, Jürgen A. Richt, and Wenjun Ma. "Pathogenicity and transmissibility of reassortant H9 influenza viruses with genes from pandemic H1N1 virus." Journal of General Virology 93, no. 11 (November 1, 2012): 2337–45. http://dx.doi.org/10.1099/vir.0.044040-0.

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Both H9N2 avian influenza and 2009 pandemic H1N1 viruses (pH1N1) are able to infect humans and swine, which has raised concerns that novel reassortant H9 viruses with pH1N1 genes might be generated in these hosts by reassortment. Although previous studies have demonstrated that reassortant H9 viruses with pH1N1 genes show increased virulence in mice and transmissibility in ferrets, the virulence and transmissibility of reassortant H9 viruses in natural hosts such as chickens and swine remain unknown. This study generated two reassortant H9 viruses (H9N2/CA09 and H9N1/CA09) in the background of the pH1N1 A/California/04/2009 (CA09) virus by replacing either both the haemagglutinin (HA) and neuraminidase (NA) genes or only the HA gene with the respective genes from the A/quail/Hong Kong/G1/1997 (H9N2) virus and evaluated their replication, pathogenicity and transmission in chickens and pigs compared with the parental viruses. Chickens that were infected with the parental H9N2 and reassortant H9 viruses seroconverted. The parental H9N2 and reassortant H9N2/CA09 viruses were transmitted to sentinel chickens, but H9N1/CA09 virus was not. The parental H9N2 replicated poorly and was not transmitted in pigs, whereas both H9N2/CA09 and H9N1/CA09 viruses replicated and were transmitted efficiently in pigs, similar to the pH1N1 virus. These results demonstrated that reassortant H9 viruses with pH1N1 genes show enhanced replication and transmissibility in pigs compared with the parental H9N2 virus, indicating that they may pose a threat for humans if such reassortants arise in swine.
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Lacroix, C., V. Coquillé, J. Guyomarch, M. Auffret, and D. Moraga. "A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species." Marine Pollution Bulletin 86, no. 1-2 (September 2014): 304–13. http://dx.doi.org/10.1016/j.marpolbul.2014.06.049.

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Bujold, Mattéa, Akila Gopalakrishnan, Emma Nally, and Kirst King-Jones. "Nuclear Receptor DHR96 Acts as a Sentinel for Low Cholesterol Concentrations in Drosophila melanogaster." Molecular and Cellular Biology 30, no. 3 (November 23, 2009): 793–805. http://dx.doi.org/10.1128/mcb.01327-09.

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ABSTRACT All eukaryotic cells have to maintain cholesterol concentrations within defined margins in order to function normally. Perturbing cholesterol homeostasis can result in a wide range of cellular and systemic defects, including cardiovascular diseases, as well as Niemann-Pick and Tangier diseases. Here, we show that DHR96 is indispensable for mediating the transcriptional response to dietary cholesterol and that it acts as a key regulator of the Niemann-Pick type C gene family, as well as of other genes involved in cholesterol uptake, metabolism, and transport. DHR96 mutants are viable and phenotypically normal on a standard medium but fail to survive on diets that are low in cholesterol. DHR96 mutants have aberrant cholesterol levels, demonstrating a defect in maintaining cholesterol homeostasis. Remarkably, we found that a high-cholesterol diet phenocopied the genomic profile of the DHR96 mutation, indicating that DHR96 resides at the top of a genetic hierarchy controlling cholesterol homeostasis in insects. We propose a model whereby DHR96 is activated when cellular cholesterol concentrations drop below a critical threshold in order to protect cells from severe cholesterol deprivation.
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Gante, Inês, Joana Martins Ribeiro, João Mendes, Ana Gomes, Vânia Almeida, Frederico Soares Regateiro, Francisco Caramelo, Henriqueta Coimbra Silva, and Margarida Figueiredo-Dias. "One Step Nucleic Acid Amplification (OSNA) Lysate Samples Are Suitable to Establish a Transcriptional Metastatic Signature in Patients with Early Stage Hormone Receptors-Positive Breast Cancer." Cancers 14, no. 23 (November 28, 2022): 5855. http://dx.doi.org/10.3390/cancers14235855.

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The One Step Nucleic Acid Amplification (OSNA) is being adopted worldwide for sentinel lymph nodes (SLNs) staging in breast cancer (BC). As major disadvantage, OSNA precludes prognostic information based on structural evaluation of SLNs. Our aim is to identify biomarkers related to tumor-microenvironment interplay exploring gene expression data from the OSNA remaining lysate. This study included 32 patients with early stage hormone receptors-positive BC. Remaining OSNA lysates were prepared for targeted RNA-sequencing analysis. Identification of differentially expressed genes (DEGs) was performed by DESeq2 in R and data analysis in STATA. The results show that, in metastatic SLNs, several genes were upregulated: KRT7, VTCN1, CD44, GATA3, ALOX15B, RORC, NECTIN2, LRG1, CD276, FOXM1 and IGF1R. Hierarchical clustering analysis revealed three different clusters. The identified DEGs codify proteins mainly involved in cancer aggressiveness and with impact in immune response. The overexpression of the immune suppressive genes VTCN1 and CD276 may explain that no direct evidence of activation of immune response in metastatic SLNs was found. We show that OSNA results may be improved incorporating microenvironment-related biomarkers that may be useful in the future for prognosis stratification and immunotherapy selection. As OSNA assay is being implemented for SLNs staging in other cancers, this approach could also have a wider utility.
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41

Bellomo, Domenico, Suzette M. Arias-Mejias, Chandru Ramana, Joel B. Heim, Enrica Quattrocchi, Sindhuja Sominidi-Damodaran, Alina G. Bridges, et al. "Model Combining Tumor Molecular and Clinicopathologic Risk Factors Predicts Sentinel Lymph Node Metastasis in Primary Cutaneous Melanoma." JCO Precision Oncology, no. 4 (September 2020): 319–34. http://dx.doi.org/10.1200/po.19.00206.

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PURPOSE More than 80% of patients who undergo sentinel lymph node (SLN) biopsy have no nodal metastasis. Here, we describe a model that combines clinicopathologic and molecular variables to identify patients with thin- and intermediate-thickness melanomas who may forgo the SLN biopsy procedure because of their low risk of nodal metastasis. PATIENTS AND METHODS Genes with functional roles in melanoma metastasis were discovered by analysis of next-generation sequencing data and case-control studies. We then used polymerase chain reaction to quantify gene expression in diagnostic biopsy tissue across a prospectively designed archival cohort of 754 consecutive thin- and intermediate-thickness primary cutaneous melanomas. Outcome of interest was SLN biopsy metastasis within 90 days of melanoma diagnosis. A penalized maximum likelihood estimation algorithm was used to train logistic regression models in a repeated cross-validation scheme to predict the presence of SLN metastasis from molecular, clinical, and histologic variables. RESULTS Expression of genes with roles in epithelial-to-mesenchymal transition (glia-derived nexin, growth differentiation factor 15, integrin-β3, interleukin 8, lysyl oxidase homolog 4, transforming growth factor-β receptor type 1, and tissue-type plasminogen activator) and melanosome function (melanoma antigen recognized by T cells 1) were associated with SLN metastasis. The predictive ability of a model that only considered clinicopathologic or gene expression variables was outperformed by a model that included molecular variables in combination with the clinicopathologic predictors Breslow thickness and patient age (area under the receiver operating characteristic curve, 0.82; 95% CI, 0.78 to 0.86; SLN biopsy reduction rate, 42%; negative predictive value, 96%). CONCLUSION A combined model that included clinicopathologic and gene expression variables improved the identification of patients with melanoma who may forgo the SLN biopsy procedure because of their low risk of nodal metastasis.
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42

Purtov, Yuri A., Olga A. Glazunova, Sergey S. Antipov, Viktoria O. Pokusaeva, Eugeny E. Fesenko, Elena V. Preobrazhenskaya, Konstantin S. Shavkunov, Maria N. Tutukina, Viktor I. Lukyanov, and Olga N. Ozoline. "Promoter islandsas a platform for interaction with nucleoid proteins and transcription factors." Journal of Bioinformatics and Computational Biology 12, no. 02 (April 2014): 1441006. http://dx.doi.org/10.1142/s0219720014410066.

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Seventy-eight promoter islands with an extraordinarily high density of potential promoters have been recently found in the genome of Escherichia coli. It has been shown that RNA polymerase binds internal promoters of these islands and produces short oligonucleotides, while the synthesis of normal mRNAs is suppressed. This quenching may be biologically relevant, as most islands are associated with foreign genes, which expression may deplete cellular resources. However, a molecular mechanism of silencing with the participation of these promoter-rich regions remains obscure. It has been demonstrated that all islands interact with histone-like protein H-NS — a specific sentinel of foreign genes. In this study, we demonstrated the inhibitory effect of H-NS using Δhns mutant of Escherichia coli and showed that deletion of dps, encoding another protein of bacterial nucleoid, tended to decrease rather than increase the amount of island-specific transcripts. This observation precluded consideration of promoter islands as sites for targeted heterochromatization only and a computer search for the binding sites of 53 transcription factors (TFs) revealed six proteins, which may specifically regulate their transcriptional output.
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43

Scoccianti, Chiara, Fulvio Ricceri, Pietro Ferrari, Cyrille Cuenin, Carlotta Sacerdote, Silvia Polidoro, Mazda Jenab, Pierre Hainaut, Paolo Vineis, and Zdenko Herceg. "Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: Influence of cruciferous vegetables in an intervention study." Epigenetics 6, no. 9 (September 2011): 1114–19. http://dx.doi.org/10.4161/epi.6.9.16515.

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44

Gardete, S., S. W. Wu, S. Gill, and A. Tomasz. "Role of VraSR in Antibiotic Resistance and Antibiotic-Induced Stress Response in Staphylococcus aureus." Antimicrobial Agents and Chemotherapy 50, no. 10 (October 2006): 3424–34. http://dx.doi.org/10.1128/aac.00356-06.

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ABSTRACT Exposure of Staphylococcus aureus to cell wall inhibitors induces massive overexpression of a number of genes, provided that the VraSR two-component sensory regulatory system is intact. Inactivation of vraS blocks this transcriptional response and also causes a drastic reduction in the levels of resistance to beta-lactam antibiotics and vancomycin. We used an experimental system in which the essential cell wall synthesis gene of S. aureus, pbpB, was put under the control of an isopropyl-β-d-thiogalactopyranoside-inducible promoter in order to induce reversible perturbations in cell wall synthesis without the use of any cell wall-active inhibitor. Changes in the level of transcription of pbpB were rapidly followed by parallel changes in the vraSR signal, and the abundance of the pbpB transcript was precisely mirrored by the abundance of the transcripts of vraSR and some additional genes that belong to the VraSR regulon. Beta-lactam resistance in S. aureus appears to involve a complex stress response in which VraSR performs the critical role of a sentinel system capable of sensing the perturbation of cell wall synthesis and allowing mobilization of genes that are essential for the generation of a highly resistant phenotype. One of the sites in cell wall synthesis “sensed” by the VraSR system appears to be a step catalyzed by PBP 2.
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45

Veldsman, Werner P., Yaqin Wang, Jiaojiao Niu, J. Antonio Baeza, and Ka Hou Chu. "Characterization of the complete mitochondrial genome of a coconut crab, Birgus latro (Linnaeus, 1767) (Decapoda: Anomura: Coenobitidae), from Okinawa, Japan." Journal of Crustacean Biology 40, no. 4 (June 13, 2020): 390–400. http://dx.doi.org/10.1093/jcbiol/ruaa032.

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Abstract We present a full description and analysis of the complete mitochondrial genome of a Pacific Ocean specimen of the coconut crab Birgus latro (Linnaeus, 1767), the largest extant terrestrial arthropod in the world. Our de novo-assembled mitogenome has a massive 16,161 times organelle read coverage, a length of 16,411 bp, contains 22 tDNAs (20 unique), 13 protein-coding genes, two rDNAs, and a putative control region of length 1,381 bp. The control region contains three microsatellites and two pairs of inverted repeats. Contrary to the mitochondrial sentinel gene concept, two-dimensional nucleotide analysis reveals higher GC-content in cox gene families than in nadh gene families. Moreover, cox gene families are more conserved than nadh gene families among the species of Coenobitidae selected for comparison. Secondary structure prediction of the 22 tDNAs shows major deviations from the cloverleaf pattern, which points to a relatively high rate of mutation in these genes. We also present a repertoire of mitochondrial variation between our male Okinawan coconut crab and an Indian Ocean specimen that consists of one insertion, one deletion, 135 SNPs, three MNPs and nine complex polymorphisms. We provide confirmatory evidence that the superfamily Paguroidea, to which the coconut crab belongs, is polyphyletic, that all the protein-coding genes of B. latro are under purifying selection, and that a Pacific versus Indian Ocean coconut crab population divergence occurred during the Pleistocene.
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46

Ramos, Romela Irene, Misa A. Shaw, Leland Foshag, Stacey L. Stern, Negin Rahimzadeh, David Elashoff, and Dave S. B. Hoon. "Genetic Variants in Immune Related Genes as Predictors of Responsiveness to BCG Immunotherapy in Metastatic Melanoma Patients." Cancers 13, no. 1 (December 30, 2020): 91. http://dx.doi.org/10.3390/cancers13010091.

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Adjuvant immunotherapy in melanoma patients improves clinical outcomes. However, success is unpredictable due to inherited heterogeneity of immune responses. Inherent immune genes associated with single nucleotide polymorphisms (SNPs) may influence anti-tumor immune responses. We assessed the predictive ability of 26 immune-gene SNPs genomic panels for a clinical response to adjuvant BCG (Bacillus Calmette-Guérin) immunotherapy, using melanoma patient cohorts derived from three phase III multicenter clinical trials: AJCC (American Joint Committee on Cancer) stage IV patients given adjuvant BCG (pilot cohort; n = 92), AJCC stage III patients given adjuvant BCG (verification cohort; n = 269), and AJCC stage III patients that are sentinel lymph node (SLN) positive receiving no immunotherapy (control cohort; n = 80). The SNP panel analysis demonstrated that the responder patient group had an improved disease-free survival (DFS) (hazard ratio [HR] 1.84, 95% CI 1.09–3.13, p = 0.021) in the pilot cohort. In the verification cohort, an improved overall survival (OS) (HR 1.67, 95% CI 1.07–2.67, p = 0.025) was observed. No significant differences of SNPs were observed in DFS or OS in the control patient cohort. This study demonstrates that SNP immune genes can be utilized as a predictive tool for identifying melanoma patients that are inherently responsive to BCG and potentially other immunotherapies in the future.
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47

Revillion, F., V. Lhotellier, L. Hornez, A. Leroy, M. C. Baranzelli, S. Giard, J. Bonneterre, and J. P. Peyrat. "Real-time reverse-transcription PCR to quantify a panel of 19 genes in breast cancer: relationships with sentinel lymph node invasion." International Journal of Biological Markers 23, no. 1 (2008): 10–17. http://dx.doi.org/10.5301/jbm.2008.2066.

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48

Révillion, F., V. Lhotellier, L. Hornez, A. Leroy, M. C. Baranzelli, S. Giard, J. Bonneterre, and J. P. Peyrat. "Real-Time Reverse-Transcription PCR to Quantify a Panel of 19 Genes in Breast Cancer: Relationships with Sentinel Lymph Node Invasion." International Journal of Biological Markers 23, no. 1 (January 2008): 10–17. http://dx.doi.org/10.1177/172460080802300102.

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At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0–T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2–4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERcx, ERβ), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35–40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.
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49

Darwich, Laila, Chiara Seminati, Jorge R. López-Olvera, Anna Vidal, Laia Aguirre, Marina Cerdá, Biel Garcias, et al. "Detection of Beta-Lactam-Resistant Escherichia coli and Toxigenic Clostridioides difficile Strains in Wild Boars Foraging in an Anthropization Gradient." Animals 11, no. 6 (May 28, 2021): 1585. http://dx.doi.org/10.3390/ani11061585.

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Disease transmission among wild boars, domestic animals and humans is a public health concern, especially in areas with high wild boar densities. In this study, fecal samples of wild boars (n = 200) from different locations of the Metropolitan Area of Barcelona were analyzed by PCR to explore the frequency of β-lactamases and extended cephalosporin and carbapenem resistance genes (ESBLs) in Escherichia coli strains and the presence of toxigenic Clostridioides difficile. The prevalence of genes conferring resistance to β-lactam antimicrobials was 8.0% (16/200): blaCMY-2 (3.0%), blaTEM-1b (2.5%), blaCTX-M-14 (1.0%), blaSHV-28 (1.0%), blaCTX-M-15 (0.5%) and blaCMY-1 (0.5%). Clostridioides difficile TcdA+ was detected in two wild boars (1.0%), which is the first report of this pathogen in wild boars in Spain. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. In conclusion, the detection of E. coli carrying ESBL/AmpC genes and toxigenic C. difficile in wild boars foraging in urban areas reinforces the value of this game species as a sentinel of environmental AMRB sources. In addition, these wild boars can be a public and environmental health concern by disseminating AMRB and other zoonotic agents. Although this study provides the first hints of the potential anthropogenic sources of AMR, further efforts should be conducted to identify and control them.
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50

Takeuchi, Hiroya, Donald L. Morton, Christine Kuo, Roderick R. Turner, David Elashoff, Robert Elashoff, Bret Taback, Akihide Fujimoto, and Dave S. B. Hoon. "Prognostic Significance of Molecular Upstaging of Paraffin-Embedded Sentinel Lymph Nodes in Melanoma Patients." Journal of Clinical Oncology 22, no. 13 (July 1, 2004): 2671–80. http://dx.doi.org/10.1200/jco.2004.12.009.

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PurposeDetection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome.Patients and MethodsqRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (β1→4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3).ResultsFifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P < .0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of ≥ one marker in histopathology-negative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P = .0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1).ConclusionMolecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.
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