Lineage-associated tracts defining the anatomy of the Drosophila first instar larval brain.

Fixed lineages derived from unique, genetically specified neuroblasts form the anatomical building blocks of the Drosophila brain. Neurons belonging to the same lineage project their axons in a common tract, which is labeled by neuronal markers. In this paper, we present a detailed atlas of the lineage-associated tracts forming the brain of the early Drosophila larva, based on the use of global markers (anti-Neuroglian, anti-Neurotactin, inscuteable-Gal4>UAS-chRFP-Tub) and lineage-specific reporters. We describe 68 discrete fiber bundles that contain axons of one lineage or pairs/small sets of adjacent lineages. Bundles enter the neuropil at invariant locations, the lineage tract entry portals. Within the neuropil, these fiber bundles form larger fascicles that can be classified, by their main orientation, into longitudinal, transverse, and vertical (ascending/descending) fascicles. We present 3D digital models of lineage tract entry portals and neuropil fascicles, set into relationship to commonly used, easily recognizable reference structures such as the mushroom body, the antennal lobe, the optic lobe, and the Fasciclin II-positive fiber bundles that connect the brain and ventral nerve cord. Correspondences and differences between early larval tract anatomy and the previously described late larval and adult lineage patterns are highlighted. Our L1 neuro-anatomical atlas of lineages constitutes an essential step towards following morphologically defined lineages to the neuroblasts of the early embryo, which will ultimately make it possible to link the structure and connectivity of a lineage to the expression of genes in the particular neuroblast that gives rise to that lineage. Furthermore, the L1 atlas will be important for a host of ongoing work that attempts to reconstruct neuronal connectivity at the level of resolution of single neurons and their synapses.

Effect of Focal Zone Size on Treatment Outcomes and Renal Injury Following Extracorporeal Shockwave Lithotripsy of Renal Calculi: A Prospective Randomized Study.

Background: The narrower focal zone (FZ) size of modern lithotripter was considered as one of the factors that resulted in suboptimal treatment result of extracorporeal shockwave lithotripsy (SWL). Therefore, we investigate the efficacy and safety of standard narrow or extended (FZ) sizes in SWL for patients with renal stones. Materials and Methods: In this prospective study conducted between April 2018 and October 2022, patients with renal stones were randomized to receive SWL with either standard or extended FZ. Treatment was delivered using a Modulith SLX-F2 lithotripter with a maximum of 3000 shocks at 1.5 Hz. The primary outcome was treatment success 12 weeks after a single SWL session, defined as the absence of a stone or stone fragment <4 mm on computed tomography. Secondary outcomes included the incidence of perinephric hematoma, stone-free rate (SFR), and changes in the urinary levels of acute renal injury markers. Results: A total of 320 patients were recruited, and 276 patients were randomized into the two groups. The two groups had similar baseline parameters. The treatment success rate was significantly better for standard FZ (74.3%) than the extended FZ group (59.3%) (p = 0.009). Standard FZ also had a significantly better SFR (Grade-A, 36.8% vs 23.0%, p = 0.013) and less pain after treatment. Both groups had similar perinephric hematoma formation rates, unplanned hospital admission rates, and changes in urinary acute renal injury markers. Conclusions: The standard narrow FZ has better treatment efficacy and similar safety compared with the extended FZ during SWL for renal stones. This clinical trial has been registered in the public domain (CCRBCTR) under trial number CUHK_CCRB00510.

In vitro assessment of intra-operative and post-operative environment in reducing bladder cancer recurrence.

Urinary bladder cancer is a common cancer worldwide. Currently, the modality of treating and monitoring bladder cancer is wide. Nonetheless, the high recurrence rate of non-muscle-invasive bladder cancer after surgical resection is still unsatisfactory. Hereby, our study demonstrated whether the intra-operative and post-operative environments will affect bladder cancer recurrence utilizing in vitro cell line model. Bladder cancer cell lines were submerged in four different irrigating fluids for assessing their tumorigenic properties. Our results showed that sterile water performed the best in terms of the magnitude of cytotoxicity to cell lines. Besides, we also investigated cytotoxic effects of the four irrigating agents as well as mitomycin C (MMC) in normothermic and hyperthermic conditions. We observed that sterile water and MMC had an increased cytotoxic effect to bladder cancer cell lines in hyperthermic conditions. Altogether, our results could be translated into clinical practice in the future by manipulating the intra-operative and post-operative conditions in order to lower the chance of residual cancer cells reimplant onto the bladder, which in turns, reducing the recurrence rate of bladder cancers.

In vitro cytotoxicity of human urine and its potential toxic parameters towards bladder cancer cells.

BACKGROUND: Bladder cancer (CaB) has a high recurrence rate despite surgery. As bladder is constantly filled with urine, it is worthwhile to investigate whether it could have any detrimental effects on bladder cancer cells. METHODS: We investigated the cytotoxicity of urine samples from CaB patients and normal controls on four CaB cell lines and tested the percentage of cell death, proliferation, adhesion, invasion and colonies formation ability. In order to identify the potential components involving in urine cytotoxicity, we evaluated some basic physiochemical parameters of urines, such as pH, osmolarity, creatinine (Cr), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and phosphate (PO4). We further compared the pH values of urine samples between CaB who developed recurrence versus those who did not. A more in-depth analysis on inflammatory markers was performed for two representative urine samples which demonstrated opposite cytoxic effects. RESULTS: 23 CaB patients and 20 normal controls were recruited into this study. According to in vitro experiments, both CaB and non-CaB urines had comparable effect on cell toxicity, proliferation, adhesion, invasion and colonies formation ability in four cell lines, HTB9, RT4, T24 and UMUC3, while RT4 was the most sensitive to urine toxicity. After evaluating the relationship between basic physiochemical parameters and cytotoxicity, we found out that there were strong negative correlations between pH value and 24 hours death rate for the 4 CaB cell lines (HTB9 r = -0.6651, p<0.001; RT4 r = -0.8335, p<0.001; T24 r = -0.4924, p<0.001; UMUC3 r = -0.7066, p<0.001). Osmolarity, urine Cr and PO4 all had weakly or moderately positive correlations with CaB cells on 24 hours death rate. CaB patients who developed recurrence had more alkaline urine than those who did not develop recurrence. In the urine sample with the highest cytoxicity, high concentrations of IL-6 and IFN-gamma were found. CONCLUSIONS: Our study confirmed that there was not statistically significant difference in cytotoxicity between CaB and non-CaB urines. However, we identified some parameters that could have an impact on cytotoxicity towards CaB cells. Modifying certain urine characteristics peri-operatively may induce cytotoxicity, avoid tumour re-implantation, and reduce the chance of cancer recurrence.

Urinary Cell-Free DNA in Bladder Cancer Detection.

Urinary bladder cancer is a common urological cancer. Although flexible cystoscopy is widely employed in bladder cancer detection, it is expensive, invasive, and uncomfortable to the patients. Recently, urinary cell-free DNA (ucfDNA) isolated from urine supernatant has been shown to have great potential in bladder cancer detection and surveillance. Molecular features, such as integrity and concentration of ucfDNA, have been shown to be useful for differentiating bladder cancer patients from healthy controls. Besides, bladder cancer also exhibits unique genetic features that can be identified from sequencing and expression of ucfDNA. Apart from bladder cancer detection, ucfDNA is also useful for molecular classification. For example, ucfDNA exhibits significant differences, both molecularly and genetically, in non-muscle-invasive and muscle-invasive bladder cancers. There is no doubt that ucfDNA is a very promising tool for future applications in the field of bladder cancer.

Roles of peroxisome proliferator-activated receptor-alpha and -gamma in the development of non-small cell lung cancer.

Peroxisome proliferator-activated receptor (PPAR)-α and PPARγ participate in cell proliferation and apoptosis. Few studies have simultaneously investigated both PPARα and PPARγ in lung cancers in vivo. The roles of PPARα and -γ were investigated in the development of pulmonary tumors induced in the adult A/J mouse by treatment with 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone (NNK). Compared with the normal lung tissues, PPARγ expression was much higher in the NNK-induced lung tumor tissues. However, PPARγ transcriptional activity, and the levels of two major endogenous PPARγ ligands, 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, were significantly lower in the NNK-treated lung tissues. The ligand changes in mice were confirmed in human lung cancer tissues. Along with the alteration of PPARγ and its endogenous ligands, the level of PPARα and its activity were increased in the NNK-induced mouse lung tumors. Treatment of mice with the synthetic PPARγ ligand, pioglitazone, significantly inhibited the formation of mouse lung tumors induced by NNK. Our study demonstrated that the reduction of endogenous PPARγ ligands and increased PPARα occurred before the formation of lung tumors, indicating that the molecular changes play a role in lung carcinogenesis. The results suggest that the enhancement of PPARγ activity with its ligands, and the suppression of PPARα with its inhibitors, may prevent the formation of lung tumors, as well as accelerate the therapy of lung cancer. Our findings may also reveal the possibility of using the level of endogenous PPARγ ligands and the activities of PPARγ or PPARα as tumor markers for lung cancer.

Ambient fine particulate matter inhibits 15-lipoxygenases to promote lung carcinogenesis.

BACKGROUND: Epidemiological observations have demonstrated that ambient fine particulate matter with dp < 2.5 µm (PM2.5) as the major factor responsible for the increasing incidence of lung cancer in never-smokers. However, there are very limited experimental data to support the association of PM2.5 with lung carcinogenesis and to compare PM2.5 with smoking carcinogens. METHODS: To study whether PM2.5 can contribute to lung tumorigenesis in a way similar to smoking carcinogen 4-methylnitrosamino-l-3-pyridyl-butanone (NNK) via 15-lipoxygenases (15-LOXs) reduction, normal lung epithelial cells and cancer cells were treated with NNK or PM2.5 and then epigenetically and post-translationally examined the cellular and molecular profiles of the cells. The data were verified in lung cancer samples and a mouse lung tumor model. RESULTS: We found that similar to smoking carcinogen NNK, PM2.5 significantly enhanced cell proliferation, migration and invasion, but reduced the levels of 15-lipoxygenases-1 (15-LOX1) and 15-lipoxygenases-2 (15-LOX2), both of which were also obviously decreased in lung cancer tissues. 15-LOX1/15-LOX2 overexpression inhibited the oncogenic cell functions induced by PM2.5/NNK. The tumor formation and growth were significantly higher/faster in mice implanted with PM2.5- or NNK-treated NCI-H23 cells, accompanied with a reduction of 15-LOX1/15-LOX2. Moreover, 15-LOX1 expression was epigenetically regulated at methylation level by PM2.5/NNK, while both 15-LOX1 and 15-LOX2 could be significantly inhibited by a set of PM2.5/NNK-mediated microRNAs. CONCLUSION: Collectively, PM2.5 can function as the smoking carcinogen NNK to induce lung tumorigenesis by inhibiting 15-LOX1/15-LOX2.

Patterns of growth and tract formation during the early development of secondary lineages in the Drosophila larval brain.

The Drosophila brain consists of a relatively small number of invariant, genetically determined lineages which provide a model to study the relationship between gene function and neuronal architecture. In following this long-term goal, we reconstruct the morphology (projection pattern and connectivity) and gene expression patterns of brain lineages throughout development. In this article, we focus on the secondary phase of lineage morphogenesis, from the reactivation of neuroblast proliferation in the first larval instar to the time when proliferation ends and secondary axon tracts have fully extended in the late third larval instar. We have reconstructed the location and projection of secondary lineages at close (4 h) intervals and produced a detailed map in the form of confocal z-projections and digital three-dimensional models of all lineages at successive larval stages. Based on these reconstructions, we could compare the spatio-temporal pattern of axon formation and morphogenetic movements of different lineages in normal brain development. In addition to wild type, we reconstructed lineage morphology in two mutant conditions. (1) Expressing the construct UAS-p35 which rescues programmed cell death we could systematically determine which lineages normally lose hemilineages to apoptosis. (2) so-Gal4-driven expression of dominant-negative EGFR ablated the optic lobe, which allowed us to conclude that the global centrifugal movement normally affecting the cell bodies of lateral lineages in the late larva is causally related to the expansion of the optic lobe, and that the central pattern of axonal projections of these lineages is independent of the presence or absence of the optic lobe.

Estrogen receptor alpha promotes smoking-carcinogen-induced lung carcinogenesis via cytochrome P450 1B1.

UNLABELLED: Smoking carcinogen N-nitrosamines such as 4-methylnitrosamino-l-3-pyridyl-butanone (NNK) require metabolic activation to exert their genotoxicity. The first activation step is mainly catalyzed by cytochrome P450 (CYP) family. Estrogen receptor α (ERα) plays a role in lung pathology. The association between them is unknown. In this study, we explored the relationship and function of CYP1B1 and ERα in NNK-induced lung tumorigenesis. CYP1B1 and ERα expression was analyzed in human lung cancer tissues and NNK-induced lung tumor of A/J mice. Cell lines NCI-H23 and NCI-H460 were employed to further study the responsible mechanisms using various cellular and molecular approaches. Our in vivo experiments demonstrated that CYP1B1 and ERα were over-expressed at the early stage of NNK-induced lung tumorigenesis. Microarray analysis found that ERα was involved in the extracellular-signal-regulated kinase (ERK)/MAPK pathway. NNK activated RAS/ERK/AP1 as it remarkably increased the levels of p-ERK, c-Fos, and c-Jun but inhibited multiple negative regulators of Ras/ERK/AP1, Pdcd4, Spry1, Spry2, and Btg2 through up-regulating miR-21. Both CYP1B1 siRNA and ERK-specific inhibitor U0126 suppressed NNK-mediated ERα up-regulation, suggesting that ERα was downstream of CYP1B1 and ERK. ERK inactivation led to the accumulation of CYP1B1, indicating that CYP1B1 was upstream of ERK activation. Inhibition of ERK or ERα decreased NNK-induced cell proliferation. Blockage of CYP1B1 or ERα induced apoptosis of lung cancer cells. Collectively, NNK-mediated ERα induction is via CYP1B1 and ERK and contributes to the lung carcinogenesis. The inhibition of CYP1B1, ERK, or ERα may arrest the lung cancer cell growth, implicating a pivotal strategy for the treatment of lung cancer. KEY MESSAGES: Smoking carcinogen NNK requires metabolic activation to exert their genotoxicity. CYP1B1 is the enzyme to catalyze NNK. NNK activates CYP1B1 and ERK to induce ERα. Inhibition of CYP1B1, ERK, or ERα arrests the lung cancer cell growth.

Thromboxane A2 receptor α promotes tumor growth through an autoregulatory feedback pathway.

Tobacco smoking can cause a number of cancers. The role of thromboxane synthase (TxAS) in smoking-related cancers is largely unknown. In this study, 37 pairs of tumor and non-tumor lung tissues of non-small-cell lung cancer, 5 lung cancer cell lines, and a mouse tumor model were used to study TxAS and its related molecules. A mouse model of smoking carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK)-induced lung tumor showed an increase in TxAS. Thromboxane A2 receptor (TP) was aberrant in lung cancer tissues of smokers. TxAS and TP were increased in lung tissues of NNK-treated mice. The in vitro studies showed that TPα rather than TPß promoted tumor growth, and NNK increased TPα. NNK-induced TxAS, which depended on activation of cyclooxygenase-2 (COX-2), ERK and NF-κB, could be inhibited by miR-34b/c. TPα played a positive role in NNK-induced COX-2/ERK/NF-κB activation, leading to the upregulation of TxAS expression and thromboxane A2 (TxA2) synthesis. The newly synthesized TxA2 could further activate TPα, forming an autoregulatory feedback loop for TPα activation. Collectively, NNK promotes lung tumor growth via inducing TxAS and TPα, which constitutes an auto-positive feedback loop to exaggerate the growth. This study suggests that TPα and TxAS are the ideal targets against smoking-related lung cancer.

Anticancer efficacy of 5F in NNK-induced lung cancer development of A/J mice and human lung cancer cells.

The mechanism responsible for the apoptotic effect induced by ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) is not fully understood and its in vivo effect has not been tested. In this study, the effect and mechanism of 5F was investigated in cigarette smoking carcinogen 4-methylnitrosamino-1-3-pyridyl-butanone (NNK)-induced mouse lung tumor model and in cultured lung cancer cells NCI-H23 and CRL-2066. 5F were given to mice after they were treated with NNK for 18 weeks. The effect of 5F on the lung tumor formation was examined, and its side effect was monitored. Cell proliferation and apoptosis were determined through expression of PCNA, Bcl-2, Bax, and TUNEL assay in in vivo animal model. 5F significantly inhibited the NNK-induced lung tumors by inducing apoptosis and suppressing cell proliferation in vivo with minimal side effects. Cell culture experiments showed that 5F translocated Bax into the mitochondria, downregulated Bcl-2, activated caspase-9 and caspase-3, released cytochrome c into the cytosol, and translocated AIF from the mitochondria to the nucleus, which leading to G2-M cell cycle arrest and cell apoptosis. 5F also activated ERK1/2 and the inhibition of ERK1/2 suppressed 5F-mediated changes in apoptotic molecules. In addition to ERK1/2, 5F activated Akt. The inhibition of Akt further facilitated the apoptosis induced, suggesting that Akt activation was anti-apoptotic rather than pro-apoptotic. Collectively, 5F is effective against lung cancer in vivo with minimal side effects. It induces apoptosis in lung cancer through the mitochondrial-mediated pathway, in which the activation of ERK is critical.