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Yes / Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil. / CNPq (scholarships 207422/2014-1, 500769/2014-1, 311554/2013-0; grants 407624/2012-0, 459100/2014-9).

Lung cancer is the leading cause of cancer-related deaths worldwide. One of the most common genetic aberrations in lung cancer patients is Kirsten rat sarcoma viral oncogene homolog (KRAS). The KRAS protein is a Ras superfamily GTPase that switches between an active GTP-bound form and an inactive GDP-bound form. The consequence of KRAS mutations results in constitutively active downstream pathways involved in uncontrolled cell proliferation and survival. Fortunately, there has been a recent development of KRASG12C inhibitors that directly target mutant KRAS, thereby arresting its proliferative effects. A recently FDA-approved KRASG12C inhibitor for the treatment of non-small cell lung cancer, Sotorasib (AMG 510), has been shown to produce insubstantial clinical response rates and a short duration of response. Similar to other targeted therapies, the limitations of this treatment are primarily due to the emergence of drug resistance. Drug resistance has been studied extensively regarding other anticancer treatments; however, the underlying molecular mechanisms remain poorly characterized. Our investigation begins by establishing and analyzing a subpopulation of cancer cells that evolve and mediate drug resistance, known as drug-tolerant persister cells (DTPCs), in KRASG12C mutant cells using AMG 510. First, we observed the reactivation of a pro-proliferative kinase, ERK, in AMG 510 DTPCs. Additionally, whole transcriptomics analysis, RT-qPCR, and immunofluorescent staining demonstrated significant upregulation of AQP4 in AMG 510 DTPCs compared to drug sensitive cells (DSCs). Aquaporin 4 (AQP4) is a water-selective transmembrane protein that regulates fluid homeostasis in many organ systems, including the lungs, and is involved in intracellular calcium signaling. We aim to explore the connections between AQP4, ERK, and calcium signaling in promoting drug tolerance to AMG 510. The insights gained from this research could lead to improved targeted therapies and clinical outcomes by identifying AQP4 as a resistance-driving biomarker.

Yes / Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country. / Erasmus Mundus Joint Doctorate Program of the European Union through a training grant to WS and the European Research Council-INTERRUPTB starting grant (nr.311725) to BdJ.

No / The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

By using the acridine orange (AO) staining method to examine the release of contents from lysosomes, it was found that PD released AO into the cytosol in both cell lines. However, the releasing pattern of HepG2 and RHepG2 was quite different. Upon PD treatment, the release of AO in HepG2 cells was graduate and slow while that in RHepG2 was sudden and sharp. / Cancer is one of the leading causes of death in the world. During cancer treatment, development of multidrug resistance (MDR) is always the major cause of failures of chemotherapy in human cancers. In our project, hepatocarcinoma HepG2 and its drug-resistant derivatives RHepG2 with MDR towards doxorubicin (Dox), fenretinide and Taxol were used to examine the differences in their response towards various anti-cancer agents. / From the AO staining, most of the lysosomes were found in the cytosol near the nucleus. However, some lysosomes were found inside the nucleus occasionally. When we double stained the HepG2 cells with DiOC6(3), it was found that the lysosomes were actually located inside the nuclear tubules. However, no such lysosome migration was observed after treating the HepG2 cells with PD. Thus, lysosomes inside the nuclear tubules might not be involved in the PD-induced lysosomal pathway. The mechanism that leads to the migration of lysosomes into the nuclear tubules is still unclear. / From the Western blot analysis, cathepsin D (Cat D) and cathepsin L (Cat L) were both released from the lysosomes after treating the two cell lines with PD. Also, it seemed likely that Cat L was released earlier than that of cyt c. This implies that lysosomal permeabilization is an early event in apoptosis. With the use of siRNA technology, it was found that RHepG2 with the knockdown of Cat D and Cat L were more tolerant and vulnerable towards PD, respectively. These suggest that Cat D and Cat L might act oppositely in the apoptotic pathway. Furthermore, the addition of Cat D inhibitor, pepstatin A, blocked the PD-mediated cell death in RHepG2 cells further confirms that Cat D is a pro-apoptotic protein that is involved in the apoptotic pathway. / In conclusion, PD was a potent anti-cancer agent that could reverse the MDR properties of RHepG2 and kill more RHepG2 cells through lysosomal and mitochondrial apoptotic pathway. / Next, we investigated the underlying killing mechanism and found out that PD switched on both the mitochondrial and lysosomal apoptotic pathway in both cell lines. Our results indicate that PD was able to depolarize mitochondrial membrane potential and release apoptosis inducing factor (AIF) and cytochrome c (cyt c) from the mitochondria to cytosol. Also, PD was able to act on isolated mitochondria directly, causing a stronger mitochondrial membrane permeabilization and more AIF release from the RHepG2 than that of the parental cells. / Polyphyllin D (PD) is a saponin found in a tradition Chinese herb, Paris polyphylla, which has been used to treat liver cancers in China for many years. Interestingly, from the MTT assays, we found out that RHepG2 (IC50: 2.0 muM) was more sensitive towards PD when compared to that of its parental cells (IC50: 3.9 muM). To keep the MDR properties, RHepG2 cells were routinely cultured with 1.2 muM of Dox. When we cultured RHepG2 in the absence of Dox but with 1.2 muM of PD for 28 days, the Pgp expression could not be maintained. However, such high expression level of Pgp was maintained when RHepG2 cells were treated with vincristine (1.2 muM) in the absence of Dox. This indicates that vincristine was a substrate of Pgp to keep the Pgp expression in RHepG2 cells while PD was not. / When incubated with different concentrations of Dox, RHepG2 accumulated less Dox than that of its parental HepG2 cells. When probed by the antibody against P-glycoprotein (Pgp), RHepG2 showed a strong Pgp expression. With the addition of Pgp modulator, verapamil, RHepG2 accumulated more Dox. All these findings indicate that Pgp is a mediator giving rise the MDR in RHepG2 cells. However, RHepG2 had a higher resistance to Dox than its parental line even co-cultured with verapamil. RHepG2 remained viable at the intracellular Dox concentration that was toxic to HepG2 cells. These observations suggest that the MDR properties of RHepG2 involved multiple mechanisms in addition to the effect of Pgp. / Lee, Kit Ying Rebecca. / "August 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4735. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 241-253). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

L’émergence et l’augmentation continue de la résistance aux médicaments chez Mycobacterium tuberculosis (MTB) constituent un défi majeur pour la lutte antituberculeuse. Pour résoudre le problème de temps (2 à 8 semaines) posé par les tests classiques de sensibilité aux médicaments (DST), des tests moléculaires ont été développés pour la détection précoce des mutations associées à la résistance. Jusqu'à présent, à l'exception du séquençage complet (incompatible avec un diagnostic de routine dans les pays en développement), aucun test ne détecte simultanément les différents types de résistance majeurs en une seule réaction. Sur la base de la littérature et de travaux antérieurs menés au Vietnam, au Laos et au Cambodge, une puce à ADN capable de détecter 184 mutations liées à la résistance aux médicaments de première et de deuxième lignes a été mise au point. En comparaison avec les données de DST, la puce a montré une sensibilité (84,3%-100%) et une spécificité élevées (89,2%-100%). Par rapport au séquençage, la puce a donné des résultats comparables, avec une sensibilité entre 90% et 100% et une spécificité entre 98,2% et 100%. Elle offre, de plus, une meilleure couverture que les tests moléculaires approuvés par l'OMS, car elle permet la détection des résistances aux médicaments de première et de deuxième lignes dans un seul test. Le temps de traitement des isolats issus de la culture est d’environ 6 à 7 heures, ce qui réduit considérablement le temps de diagnostic par rapport au DST. En conclusion, la puce à ADN a été développée avec succès. Certains aspects techniques doivent maintenant être améliorés pour en faire un outil de diagnostic abordable et facile à utiliser. / The emergence and continuous increase of drug resistance in Mycobacterium tuberculosis (MTB) is a major challenge for tuberculosis (TB) control. To overcome the time-consuming problem of conventional drug susceptibility testing (DST), many molecular-based tests have been recently developed for early detection of drug resistance-associated mutations. Up to now, except whole genome sequencing (not ready for routine diagnostic in low and middle-income countries), no test has the capacity to simultaneously detect the different types of drug resistance to first- and second-line drugs in one reaction. Based on the literature and previous works carried out in Vietnam, Laos and Cambodia, in this study, a DNA chip was developed able to detect 184 main mutations conferring resistance to both first- and second-line drugs. Compared to DST, the DNA chip showed high sensitivity (between 84.3% and 100%) and high specificity (between 89.2% and 100%). Compared to sequencing, the DNA chip showed comparable accuracy, with sensitivity between 90% and 100% and specificity between 98.2% and 100%. The DNA chip showed a better coverage than the WHO endorsed molecular tests since it enables the detection of both first- and second-line drug resistances in one test. The turn-around time is about 6-7h from cultured isolates reducing considerably the diagnostic time compared to cultured-based DST. Finally, the DNA chip has been successfully developed, even if certain technical aspects need to be improved to make an affordable and easy-to-use diagnostic tool.

Choi, Pui Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-122). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression. / Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells. / The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance. / Jiang, Lei. / Adviser: Kwok-Pui, Fang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.