Nitro-oxidative response to internalized multi-walled carbon nanotubes in Brassica napus and Solanum lycopersicum.

In addition to their beneficial effects on plant physiology, multi-walled carbon nanotubes (MWCNTs) are harmful to plants in elevated concentrations. This study compared the effects of two doses of MWCNT (10 and 80 mg/L) in Brassica napus and Solanum lycopersicum seedlings focusing on nitro-oxidative processes. The presence of MWCNTs was detectable in the root and hypocotyl of both species. Additionally, transmission electron microscopy analysis revealed that MWCNTs are heavily transformed within the root cells forming large aggregates. The uptake of MWCNTs negatively affected root viability and root cell proliferation of both species, but more intense toxicity was observed in S. lycopersicum compared to B. napus. The presence of MWCNT triggered more intense protein carbonylation in the relative sensitive S. lycopersicum, where increased hydrogen peroxide levels were observed. Moreover, MWCNT exposure increased the level of physiological protein tyrosine nitration which was more intense in S. lycopersicum where notable peroxynitrite accumulation occurred. These suggest for the first time that MWCNT triggers secondary nitro-oxidative stress which contributes to its toxicity. Moreover, the results indicate that the extent of the nitro-oxidative processes is associated with the extent of MWCNT toxicity.

Drosophila Rab39 Attenuates Lysosomal Degradation.

Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, we found no failure in the early steps of endocytosis and autophagy. On the contrary, we found that Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. Our data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.

[Investigation of laparoscopic bimanual technic education with laparoscopic training box]. / Kétkezes technika elsajátításának vizsgálata laparoszkópos training boxon.

INTRODUCTION: In some surgical wards residents start to do laparoscopic operations using both hands, while in other places they only use their dominant hand, and only start to use both hands later. There are no data at the moment about which method is more effective. METHODS: We divided 20 students with no laparoscopic experience into 2 groups: one group practised one hand at a time (1K), the other group used both hands (2K) during the 5 days. On the last day both groups had to do every exercise with one hand and two hands as well, then they had to do 3 new exercises, which needed both hands. We measured the time taken, and gave points for the videos taken inside the training box based on OSATS. For statistical analysis we used t-tests, p < 0.05 being significant. RESULTS: On the first day, there was no significant difference between the 1K and 2K groups considering the time taken (518/500 s) and the OSATS points (87/84; 54/55 points). Both groups improved in the mean time and points (1K: 52%, 77% 2K: 50%, 70%) as well, but there was no significant difference between them. In the case of new exercises on the last day, there was no difference between mean time (1K: 425 s, 2K: 411 s) and points (53/59 and 56/52), but there was a significant difference considering the points given for bimanuality. CONCLUSION: Based on our study, we cannot exactly state that the bimanuality needed for expert laparoscopic surgery would be easier to learn with immediately practising with both hands.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

[Measuring residents' and specialists' laparoscopic technique with the MENTOR® training box]. / Rezidensek és szakorvosok laparoscopos technikájának felmérése MENTOR® tréningboksz segítségével.

INTRODUCTION: Operating room is not the ideal place to acquire laparoscopic skills since patients can be put at risk and it is also relatively expensive. Using training boxes seems to be a more appropriate way of teaching and learning the technique, but there is little data about measuring the technique of experienced specialists and comparing their results with residents. METHODS: At the 1st Department of Surgery, Semmelweis University we tested 30 residents and 25 specialists in general surgery and urology on MENTOR® training box. Before training, all participants completed a questionnaire on professional experience, previous usage of training boxes, virtual simulators, and video games, and whether they played a musical instrument earlier. Subjects were asked to complete in a defined time limit 3 of the Fundamentals of Laparoscopic Surgery tasks (which is required for American surgical residents for surgical board examination), and 3 tasks decided by us. Linear regression analysis (ANOVA table) was used to evaluate the data. RESULTS: 16% of the specialists and 6.66% of the residents completed all tasks within time limit. Statistically significant correlation (p < 0.05) was demonstrated between the number of previous laparoscopic surgeries and task completion time, while there were no significant correlations between other factors, which may influence laparoscopic technique and task completion time. CONCLUSIONS: Training boxes are suitable for developing eye-hand coordination and bimanuality, as well as for learning instrument handling. Nonetheless, residents acquire most of their laparoscopic surgical skills on patients in Hungary, yet. For this reason there is a need for organized training opportunities.

Subfunctionalization of Parental Polyamine Oxidase (PAO) Genes in the Allopolyploid Tobacco Nicotiana tabacum (L.).

Polyamines play an important role in developmental and environmental stress responses in plants. Polyamine oxidases (PAOs) are flavin-adenine-dinucleotide-dependent enzymes associated with polyamine catabolism. In this study, 14 genes were identified in the tobacco genome that code for PAO proteins being named based on their sequence homology with Arabidopsis PAOs (AtPAO1-5): NtPAO1A-B; NtPAO2A-C, NtPAO4A-D, and NtPAO5A-E. Sequence analysis confirmed that the PAO gene family of the allopolyploid hybrid Nicotiana tabacum is not an exact combination of the PAO genes of the maternal Nicotiana sylvestris and paternal Nicotiana tomentosiformis ones. The loss of the N. sylvestris homeolog of NtPAO5E and the gain of an extra NtPAO2 copy, likely of Nicotiana othophora origin, was revealed. The latter adds to the few pieces of evidence suggesting that the paternal parent of N. tabacum was an introgressed hybrid of N. tomentosiformis and N. othophora. Gene expression analysis indicated that all 14 PAO genes kept their expression following the formation of the hybrid species. The homeologous gene pairs showed similar or opposite regulation depending on the investigated organ, applied stress, or hormone treatment. The data indicate that the expression pattern of the homeologous genes is diversifying in a process of subfunctionalization.

Absence of Arabidopsis Polyamine Oxidase 5 Influences the Cytokinin-Induced Shoot Meristem Formation from Lateral Root Primordia.

Lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems (SMs) by the application of exogenous cytokinin. Here, we report that Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) contributes to this process, since the rate of SM formation from LRPs was significantly lower in the pao5-2 knockout mutant. Furthermore, the presented experiments showed that AtPAO5 influences SM formation via controlling the thermospermine (T-Spm) level. Gene expression analyses supported the view that the pao5-2 mutation as well as exogenous T-Spm downregulate the expression of the class 3 haemoglobin coding genes AtGLB1 and AtGLB2. AtGLB1 and 2 have been reported to augment cytokinin sensitivity, indirectly inhibiting the expression of type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs). In agreement, the same ARR-coding genes were found to be upregulated in the pao5-2 mutant. Although GLB proteins might also control cytokinin-induced nitric oxide (NO) accumulation, we could not find experimental evidence for it. Rather, the negative effect of NO-donor treatment on AtPAO5 gene expression and SM formation was seen. Nevertheless, a hypothetical pathway is set up explaining how AtPAO5 may affect direct shoot meristem formation, controlling cytokinin sensitivity through T-Spm and GLBs.

Polyamine Oxidase-Generated Reactive Oxygen Species in Plant Development and Adaptation: The Polyamine Oxidase-NADPH Oxidase Nexus.

Metabolism and regulation of cellular polyamine levels are crucial for living cells to maintain their homeostasis and function. Polyamine oxidases (PAOs) terminally catabolize polyamines or catalyse the back-conversion reactions when spermine is converted to spermidine and Spd to putrescine. Hydrogen peroxide (H2O2) is a by-product of both the catabolic and back-conversion processes. Pharmacological and genetic approaches have started to uncover the roles of PAO-generated H2O2 in various plant developmental and adaptation processes such as cell differentiation, senescence, programmed cell death, and abiotic and biotic stress responses. Many of these studies have revealed that the superoxide-generating Respiratory Burst Oxidase Homolog (RBOH) NADPH oxidases control the same processes either upstream or downstream of PAO action. Therefore, it is reasonable to suppose that the two enzymes co-ordinately control the cellular homeostasis of reactive oxygen species. The intricate relationship between PAOs and RBOHs is also discussed, posing the hypothesis that these enzymes indirectly control each other's abundance/function via H2O2.

Evaluation of Effective Mass in InGaAsN/GaAs Quantum Wells Using Transient Spectroscopy.

Transient spectroscopies are sensitive to charge carriers released from trapping centres in semiconducting devices. Even though these spectroscopies are mostly applied to reveal defects causing states that are localised in the energy gap, these methods also sense-charge from quantum wells in heterostructures. However, proper evaluation of material response to external stimuli requires knowledge of material properties such as electron effective mass in complex structures. Here we propose a method for precise evaluation of effective mass in quantum well heterostructures. The infinite well model is successfully applied to the InGaAsN/GaAs quantum well structure and used to evaluate electron effective mass in the conduction and valence bands. The effective mass m/m0 of charges from the conduction band was 0.093 ± 0.006, while the charges from the valence band exhibited an effective mass of 0.122 ± 0.018.

Polyamine Metabolism Is Involved in the Direct Regeneration of Shoots from Arabidopsis Lateral Root Primordia.

Plants can be regenerated from various explants/tissues via de novo shoot meristem formation. Most of these regeneration pathways are indirect and involve callus formation. Besides plant hormones, the role of polyamines (PAs) has been implicated in these processes. Interestingly, the lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems by exogenous cytokinin application. In this system, no callus formation takes place. We report that the level of PAs, especially that of spermidine (Spd), increased during meristem conversion and the application of exogenous Spd improved its efficiency. The high endogenous Spd level could be due to enhanced synthesis as indicated by the augmented relative expression of PA synthesis genes (AtADC1,2, AtSAMDC2,4, AtSPDS1,2) during the process. However, the effect of PAs on shoot meristem formation might also be dependent on their catabolism. The expression of Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) was shown to be specifically high during the process and its ectopic overexpression increased the LRP-to-shoot conversion efficiency. This was correlated with Spd accumulation in the roots and ROS accumulation in the converting LRPs. The potential ways how PAO5 may influence direct shoot organogenesis from Arabidopsis LRPs are discussed.

Polyamines treatment during pollen germination and pollen tube elongation in tobacco modulate reactive oxygen species and nitric oxide homeostasis.

Several signaling pathways have been shown to be involved in the regulation of pollen germination and pollen tube elongation. Among others, exogenously applied polyamines were found to strongly affect pollen maturation, pollen tube emergence and elongation. In this study, our aim was to investigate the regulatory relation among exogenous polyamines, and endogenous reactive oxygen species and nitric oxide under pollen germination and the apical growth of pollen tube in tobacco plants. We have found that the various polyamines differentially affected the metabolism of nitric oxide and reactive oxygen species during the processes of pollen germination in the grain and the lengthening pollen tube. It is hypothesized that their differential effects might be related to their distinct influence on the endogenous nitric oxide and reactive oxygen species levels.

Sec20 is Required for Autophagic and Endocytic Degradation Independent of Golgi-ER Retrograde Transport.

Endocytosis and autophagy are evolutionarily conserved degradative processes in all eukaryotes. Both pathways converge to the lysosome where cargo is degraded. Improper lysosomal degradation is observed in many human pathologies, so its regulatory mechanisms are important to understand. Sec20/BNIP1 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 1) is a BH3 (Bcl-2 homology 3) domain-containing SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) protein that has been suggested to promote Golgi-ER retrograde transport, mitochondrial fission, apoptosis and mitophagy in yeast and vertebrates. Here, we show that loss of Sec20 in Drosophila fat cells causes the accumulation of autophagic vesicles and prevents proper lysosomal acidification and degradation during bulk, starvation-induced autophagy. Furthermore, Sec20 knockdown leads to the enlargement of late endosomes and accumulation of defective endolysosomes in larval Drosophila nephrocytes. Importantly, the loss of Syx18 (Syntaxin 18), one of the known partners of Sec20, led to similar changes in nephrocytes and fat cells. Interestingly. Sec20 appears to function independent of its role in Golgi-ER retrograde transport in regulating lysosomal degradation, as the loss of its other partner SNAREs Use1 (Unconventional SNARE In The ER 1) and Sec22 or tethering factor Zw10 (Zeste white 10), which function together in the Golgi-ER pathway, does not cause defects in autophagy or endocytosis. Thus, our data identify a potential new transport route specific to lysosome biogenesis and function.

Rab2 promotes autophagic and endocytic lysosomal degradation.

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.