Zurletrectinib is a next-generation TRK inhibitor with strong intracranial activity against NTRK fusion-positive tumours with on-target resistance to first-generation agents.

BACKGROUND: While NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority. METHODS: In silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations. RESULTS: In vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05). CONCLUSION: Our data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.

Photoelectrochemical sensing of isoniazid and streptomycin based on metal Bi-doped BiOI microspheres grown on book-shape layers of Ti3C2 heterostructures.

Isoniazid and streptomycin are vital drugs for treating tuberculosis, which are utilized as efficient anti-tuberculosis agents. This paper presents a novel visible-light-driven composite photocatalyst Ti3C2/Bi/BiOI, which was built from Ti3C2 nanosheets and Bi/BiOI microspheres. Photoelectrochemical (PEC) sensors based on Ti3C2/Bi/BiOI were synthesized for isoniazid identification, which showed a linear concentration range of 0.1-125 µM with a detection limit of 0.05 µM (S/N = 3). Moreover, we designed a PEC aptasensors based on aptamer/Ti3C2/Bi/BiOI to detect streptomycin in 0.1 M PBS covering the electron donor isoniazid, because the isoniazid consumes photogenerated holes thus increasing the photocurrent effectively and preventing photogenerated electron-hole pairs from being recombined. Furthermore, PEC aptasensors based on aptamer/Ti3C2/Bi/BiOI were synthesized for streptomycin identification, which exhibited a linear concentration range of 0.01-1000 nM with a detection limit of 2.3 × 10-3 nM (S/N = 3), and are well stable in streptomycin sensing.

A comparative study of MassARRAY and GeneXpert assay in detecting rifampicin resistance in tuberculosis patients' clinical specimens.

Objectives: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potent tool for detecting drug resistance in tuberculosis (TB); however, concerns about its reliability have been raised. In this study, we assessed the reliability of MassARRAY (Sequenom, Inc.), which is a MALDI-TOF MS-based method, by comparing it to the well-established GeneXpert assay (Cepheid) as a reference method. Methods: A retrospective study was conducted using laboratory data retrieved from Henan Chest Hospital (Zhengzhou, China). To ensure a rigorous evaluation, we adopted a comprehensive assessment approach by integrating multiple outcomes of the Xpert assay across various specimen types. Results: Among the 170 enrolled TB cases, MassARRAY demonstrated significantly higher sensitivity (85.88%, 146 of 170) compared to the Xpert assay (76.62%, 118 of 154) in TB diagnosis (p < 0.05). The concordance in detecting rifampicin resistance between MassARRAY and the combined outcomes of the Xpert assay was 90%, while it was 97.37% (37 of 38) among smear-positive cases and 89.06% (57 of 64) among culture-positive cases. When compared to the phenotypic susceptibility outcomes of the 12 included drugs, consistency rates of 81.8 to 93.9% were obtained, with 87.9% for multiple drug resistance (MDR) identification. Conclusion: MassARRAY demonstrates high reliability in detecting rifampicin resistance, and these findings may offer a reasonable basis for extrapolation to other drugs included in the test panel.

Extremely high levels of central nervous system involvement in miliary tuberculosis.

BACKGROUND: Miliary tuberculosis (TB) is one of the severest manifestations of TB that can be lethal when concomitant with the central nervous system (CNS) involvement. Bacteriological, biochemical and radiological methods for find CNS comorbidity in miliary TB was evaluated in this study. METHODS: Consecutive miliary TB adults were retrospectively enrolled from two designated TB hospitals in China. The capacities of examinations of cerebrospinal fluid (CSF), cerebral computed tomography (CT) and magnetic resonance imaging (MRI) for diagnosis of CNS involvement were assessed. RESULTS: Assessment of CNS involvement with a lumbar puncture and/or neuroimaging was undertaken in 282 out of 392 of acute miliary TB. Of these 282 patients, 87.59% (247/282) had CNS involvement. Cerebral contrast-enhanced MRI (96.05%, 170/177) and MRI (93.15%, 204/219) yielded significantly higher sensitivities over CSF examination (71.92%, 146/203, P < 0.001) and CT (34.69%, 17/49, P < 0.001). The sensitivity of CSF examination was superior to CT scan (P < 0.001). Although 59.65% (134/225) miliary TB patients acquired bacteriological evidence with sputum examination, the positivity was only 8.82% (21/238) for CSF examination by conventional and molecular tests. CONCLUSION: Almost all miliary TB had CNS involvement and MRI demonstrated outstanding potential over other methods. Therefore, a routinely screening of CNS TB should be strongly suggested in miliary TB and MRI could be used as the initial approach in resources rich settings.

The within-host population dynamics of Mycobacterium tuberculosis vary with treatment efficacy.

BACKGROUND: Combination therapy is one of the most effective tools for limiting the emergence of drug resistance in pathogens. Despite the widespread adoption of combination therapy across diseases, drug resistance rates continue to rise, leading to failing treatment regimens. The mechanisms underlying treatment failure are well studied, but the processes governing successful combination therapy are poorly understood. We address this question by studying the population dynamics of Mycobacterium tuberculosis within tuberculosis patients undergoing treatment with different combinations of antibiotics. RESULTS: By combining very deep whole genome sequencing (~1000-fold genome-wide coverage) with sequential sputum sampling, we were able to detect transient genetic diversity driven by the apparently continuous turnover of minor alleles, which could serve as the source of drug-resistant bacteria. However, we report that treatment efficacy has a clear impact on the population dynamics: sufficient drug pressure bears a clear signature of purifying selection leading to apparent genetic stability. In contrast, M. tuberculosis populations subject to less drug pressure show markedly different dynamics, including cases of acquisition of additional drug resistance. CONCLUSIONS: Our findings show that for a pathogen like M. tuberculosis, which is well adapted to the human host, purifying selection constrains the evolutionary trajectory to resistance in effectively treated individuals. Nonetheless, we also report a continuous turnover of minor variants, which could give rise to the emergence of drug resistance in cases of drug pressure weakening. Monitoring bacterial population dynamics could therefore provide an informative metric for assessing the efficacy of novel drug combinations.

The phosphatidyl inositol 3-kinase pathway is central to the pathogenesis of Kit-activated melanoma.

Mouse Kit L575P, the ortholog of human KIT L576P, a common KIT mutation found in human melanoma was expressed in an immortalized but non-transformed mouse Ink4a-Arf-deficient melanocyte cell line. The resultant Ink4a-Arf-deficient Kit L575P-expressing melanocytes exhibited increased proliferation, the ability to grow in soft agar, and increased migration. When these cells were injected subcutaneously into NOD/SCID/gamma(c) mice, melanomas arose in 5 of 7 (71%) mice. One of seven mice (14%) injected with these cells developed metastatic disease. Evaluation of signal transduction pathways downstream of constitutively activated Kit L575P revealed striking activation of the phosphatidyl inositol 3-kinase (PI3K) pathway. Inhibition of the PI3K pathway pharmacologically or genetically abolished the transformation phenotypes gained by the L575P single mutant. These studies validate this Kit L575P-activated model of melanoma and establish the PI3K pathway as a dominant signaling pathway downstream of Kit in melanoma.

PC-1/PrLZ contributes to malignant progression in prostate cancer.

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.

Identification and characterization of the novel human prostate cancer-specific PC-1 gene promoter.

Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5'-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5' deletion analysis identified the -1579 bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; -4939 bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6 kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between -345 and -359 bp of the PC-1 5'-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy.

[Impact of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2].

OBJECTIVE: To study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2. METHODS: Recombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells. RESULTS: PC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells. CONCLUSION: RNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.

[Malignant transformation of NIH3T3 cells induced by ectopic expression of PC-1 gene].

OBJECTIVE: To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. METHODS: Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression. RESULTS: NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice. CONCLUSION: Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.

[Effects of 6-BA on cluster root formation and organic acid exudation in white lupin grown under phosphorus deficiency].

Phosphorus deficiency results in cluster root formation and increased organic acid exudation. The regulatory mechanisms for these processes, however, are not yet clear. In the present study, influences of 6-BA (6-benzyl aminopurine) on cluster root formation, exudation of citrate and malate and their concentrations in the root clusters of P-deficient white lupin plants were studied by using non-destructive localized collection method and high-performance liquid chromatography (HPLC) technique. The results showed that application of exogenous 6-BA to P-deficient plants did not influence plant growth and P distribution within the plant (Tables 2,3), whereas the cluster root formation (Fig. 1, Table 1) and organic acid exudation (Table 4) were inhibited. The inhibitory effects could be reversed by omitting 6-BA from the growth medium, and even some stimulatory effects was observed, when lower concentration of 6-BA (10(-8) mol/L) was applied (Fig. 1, Tables 1 and 4). The inhibitory effects of higher concentration of 6-BA (10(-7) mol/L) were not reversible. (Table 4). Treatment with 6-BA also had some influence on organic acid concentration in the tissue of cluster roots (Table 5). The possible reasons for the effects on cluster root formation and organic acid exudation by 6-BA are discussed.