Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Dysfunction in Eph signaling is causally associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been linked to alterations in receptor clustering abilities. Here, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptors in multiple carcinoma cells. We show that cancer cells retain EphA clustering phenotype over several generations, and the degree of clustering reported for migration potential both at population and single-cell levels. Finally, using patient-derived cancer lines, we probed the evolution of EphA signalling in cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our scalable approach provides a reliable scoring scheme for EphA clustering that is consistent over multiple carcinomas and can assay heterogeneity of cancer cell populations in a cost- and time-effective manner.

Author Correction: Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sputum smear-positive, Xpert® MTB/RIF-negative results: magnitude and treatment outcomes of patients in Myanmar.

Setting: Myanmar's National Tuberculosis Programme (NTP) uses the Xpert® MTB/RIF assay to diagnose rifampicin (RMP) resistance in sputum smear-positive (Sm+) pulmonary tuberculosis (TB) patients. The Xpert test may occasionally yield negative results (Xpert-) for Mycobacterium tuberculosis complex, indicating a false-positive sputum smear result, false-negative Xpert result or infection with non-tuberculous mycobacteria (NTM). Patients with NTM may respond poorly to first-line anti-tuberculosis treatment. Objective: To assess the burden of Sm+, Xpert- results at the national level and treatment outcomes of Sm+, Xpert- patients in Yangon Region. Design: A cohort study involving a retrospective record review of routinely collected NTP data. Result: In 2015 and 2016, 4% of the 25 359 Sm+ patients who underwent Xpert testing nationally were Sm+, Xpert-. Similarly, in the Yangon Region, 5% of the 5301 Sm+ patients were also Xpert- and were treated with first-line anti-tuberculosis regimens. Smear grade (scanty/1+) and age ⩾65 years were associated with Sm+, Xpert- results. The 88% treatment success rate for this group was similar to that of Sm+, Xpert+ patients without RMP resistance. Conclusion: Approximately 4-5% of Sm+ TB patients were Xpert-. There is an urgent need to formulate guidelines on how to reassess and manage these patients.

Contexte : Le programme national tuberculose du Myanmar (PNT) recourt au test Xpert® MTB/RIF pour diagnostiquer la résistance à la rifampicine (RMP) chez les patients atteints de tuberculose (TB) pulmonaire à frottis de crachats positif (Sm+). Les résultats du test Xpert peuvent parfois être négatifs (Xpert­) pour le complexe Mycobacterium tuberculosis indiquant soit un résultat de frottis de crachats faussement positif, soit un résultat de test Xpert faussement négatif ou une infection à mycobactéries non-tuberculeuse (NTM). Les patients atteints de NTM peuvent répondre de façon médiocre au traitement anti-tuberculose de première ligne.Objectif : Evaluer la proportion de patients Sm+/Xpert­ au niveau national et les résultats du traitement dans la région de Yangon.Schéma : Etude de cohorte impliquant une revue rétrospective des dossiers de données recueillies en routine par le PNT.Résultats: Au niveau national, en 2015 et 2016, 4% des 25 359 patients Sm+ qui ont eu un test Xpert ont été Xpert­. De même, dans la région de Yangon, 5% des 5301 patients Sm+ ont été Xpert­ et ils ont été traités avec des protocoles anti-tuberculose de première ligne. Le grade du frottis (rare/1+) et l'âge ⩾ 65 ans ont été associés aux résultats Sm+/Xpert­. Le taux de succès du traitement a été de 88% ce qui a été similaire aux résultats des patients Sm+/Xpert+ sans résistance à la RMP.Conclusion : La proportion de patients TB Sm+/Xpert­ a été d'environ 4­5%. Il y a un besoin urgent de formuler des directives sur la manière de réévaluer et de prendre en charge de façon optimale ces patients.

Marco de referencia: En el Programa Nacional contra la Tuberculosis (PNT) de Birmania se utiliza la prueba Xpert® MTB/RIF con el fin de diagnosticar la resistencia a rifampicina (RMP) en los pacientes con diagnóstico de tuberculosis (TB) pulmonar y baciloscopia positiva (Sm+). El resultado de la prueba Xpert en ocasiones puede ser negativa para el complejo Mycobacterium tuberculosis, lo cual puede corresponder ya sea a un resultado positivo falso de la baciloscopia del esputo, un resultado negativo falso de la prueba Xpert o a la infección por una micobacteria atípica. Los pacientes infectados por micobacterias atípicas pueden tener una respuesta deficiente al tratamiento antituberculoso de primera línea.Objetivo: Evaluar la proporción de casos SM+, Xpert­ a escala nacional y los desenlaces terapéuticos de estos pacientes en la región de Yangon.Método: Se realizó un estudio de cohortes retrospectivo a partir del análisis de los datos corrientes recogidos en las historias clínicas en el PNT.Resultados: A escala nacional, en el 2015 y el 2016, el 4% de los 25 359 pacientes Sm+ que realizaron la prueba Xpert obtuvo un resultado negativo. Asimismo, en la región de Yangon, el 5% de los 5301 pacientes con Sm+ tuvo un resultado negativo de la prueba Xpert y recibió tratamiento con esquemas antituberculosos de primera línea. Los factores que se asociaron con Sm+ y una prueba Xpert­ fueron el grado de la baciloscopia (bacilos escasos o 1+) y la edad a ⩾ 65 años. La tasa de éxito terapéutico en estos casos fue 88%, una proporción equivalente a los desenlaces de los pacientes Sm+ y Xpert+, sin resistencia a RMP.Conclusión: La proporción de pacientes con Sm+ y Xpert­ fue del 4% al 5%. Existe una necesidad urgente de formular directrices sobre la forma reevaluar estos pacientes y tratarlos de manera óptima.

Rifampicin-resistant tuberculosis patients in Myanmar in 2016: how many are lost on the path to treatment?

SETTING: Regional tuberculosis (TB) centres of the Yangon and Mandalay Regions of Myanmar, which account for 65% of all notified rifampicin-resistant tuberculosis (RR-TB) cases countrywide. OBJECTIVE: To determine 1) initial loss to follow-up (LTFU), 2) treatment delay, and 3) factors associated with initial LTFU and treatment delay among RR-TB patients residing in the Yangon and Mandalay regions diagnosed using Xpert® during January-August 2016. DESIGN: This was a retrospective cohort study. Each diagnosed patient was tracked in the drug-resistant TB treatment registers of the Yangon and Mandalay regional treatment centres for January-December 2016 using patient name, age, sex, township and date of diagnosis. If the diagnosed patient was not found in the treatment register by 31 December 2016, he/she was considered 'initial LTFU'. RESULTS: Of the 1037 RR-TB patients diagnosed, 310 (30%) experienced initial LTFU, which was significantly higher among patients aged 55 years and among those diagnosed in the Mandalay Region. A treatment delay of >1 month was observed in 440 (70%) patients (median delay 41 days). Delay was uniformly high across patient subgroups, and was not associated with any factor. CONCLUSION: Initial LTFU and treatment delays among RR-TB patients were high. Future studies using qualitative research methods are needed to ascertain the reasons for this observation.

Expression of Hex mRNA in early murine postimplantation embryo development.

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.

Sp family members stimulate transcription of the hex gene via interactions with GC boxes.

The 5'-flanking region of the mouse Hex gene was examined in order to identify transcription factors regulating its expression in hepatocytes and haematopoietic cells. We have identified two further GC boxes (GC boxes 3 and 4 at nucleotide positions -149 to -140 and -79 to -70, respectively), i.e. in addition to the two previously determined ones (GC boxes 1 and 2 at nucleotide positions -197 to -188 and -176 to -167, respectively). Luciferase reporter assays revealed that all four GC boxes are transcriptionally active in both MH(1)C(1) rat hepatoma and K562 human chronic myelogenous leukemia cells. Electrophoretic mobility shift assays with specific competitors and antibodies showed that members of the Sp family, namely Sp1 and Sp3, bind to these GC boxes. Overexpression of Sp1 and Sp3 in Drosophila SL2 cells stimulated transcription of the Hex gene through interactions with GC boxes 1 to 4, Sp1 being a more potent activator than Sp3. Thus, we conclude that Sp1 and Sp3 stimulate transcription of the Hex gene in both MH(1)C(1) and K562 cells.

Genomic organization and promoter analysis of a mouse homeobox gene, Hex.

A homeobox gene, Hex, is mainly expressed in haematopoietic cells and hepatocytes. It is assumed to play a role in the early stage of differentiation of these cells. To understand the mechanisms involved in the regulation of the Hex gene expression in hepatocytes, we cloned and characterized the mouse Hex gene. The gene consists of four exons and three introns, and spans about 5.7 kb. All the exon-intron boundaries are consistent with the "GT-AG" rule. A single transcription start site was identified by primer extension and S1 mapping analyses. Although the 5'-flanking region is G/C rich (69%), it contains probable "TATA and CCAAT" boxes. Potential binding sequences for transcriptional regulatory proteins including Sp1 and AP-2 are also present in this region. Functional analysis of the Hex promoter was performed by transfecting MH1C1, HeLa, COS-7, and Caco-2 cells with Hex promoter region-luciferase constructs. We found three possible positive regulatory regions, comprising of nucleotides -199 and -172, -154 and -133, and -105 and -68, respectively, required for Hex gene expression in MH1C1 cells by analyses of a series of 5'-deletion constructs of the fusion genes. The activities of these constructs were extremely low in HeLa, COS-7, and Caco-2 cells suggesting that they possess cell-type specificity. Further analysis revealed two GC boxes, GC box1 and GC box2, at nucleotides -197 to -188 and -176 to -167, respectively, necessary for Hex gene expression. Thus, multiple regulatory elements contribute to the Hex gene expression in hepatocytes.

Primary central nervous system T-cell lymphoma in aids patients: case report and literature review.

According to the published data, most primary central nervous system lymphomas (PCNSLs) are B-cell lymphomas; primary T-cell lymphomas are rare. In a search of the MEDLINE database, we found only 6 cases of primary T-cell PCNSL. Here, we present the case of a 43-year-old man with AIDS, not on highly active antiretroviral therapy, who presented with focal neurologic symptoms and was found on magnetic resonance imaging to have multiple brain lesions. A biopsy showed T-cell lymphoma, and the patient was subsequently treated with whole-brain radiation, to marked clinical response. Reported cases from the literature of primary T-cell PCNSL in AIDS patients are summarized in this review.

cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein.

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of alpha-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

Homeobox gene Hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage.

Disruption of the mouse Hex gene resulted in embryonic lethality around embryonic age (E) 10.5, due to no substantial liver formation. Expression of albumin was detectable in heterozygous (Hex(+/-)) but not in homozygous (Hex(-/-)) [corrected] embryos at E8.5. Instead of liver bud formation at E9.5, a liver-like capsule structure was observed in Hex(-/-) [corrected] embryos. In Hex(-/-) [corrected] mutant liver, we found no hepatocytes but no signs of apoptotic cell death in the area. Expression of transcription factors involved in hepatocyte differentiation, hepatocyte nuclear factor (Hnf)3beta, Hnf6, Hnf4alpha and Hnf1alpha, were restricted to the capsule and internal matrix-like structure in the mutant liver and expression of a subset of these factors were reduced. Hematopoiesis of monocytes was impaired in mutant embryos while erythroid lineage was unaffected. These results indicate that Hex plays an essential role in progenitor cells which commit to the hepatic endoderm and in the hematopoietic differentiation of the monocyte lineage.