Giant Larvae Research Articles

Silencing of LLGL2 Suppresses the Estradiol-Induced BPH-1 Cell Proliferation through the Regulation of Autophagy.

Lethal giant larvae (Lgl) is an apical-basal polarity gene first identified in Drosophila. LLGL2 is one of the mammalian homologs of Lgl. However, little is known about its function in the prostate. In this study, to explore the new role of LLGL2 in the prostate, we examined the proliferative activity of a BPH-1 cell line, a well-established model for the human prostate biology of benign prostatic hyperplasia (BPH). The expression of LLGL2 was dose-dependently increased in BPH-1 cells after treatment with 17β-estradiol (E2). Additionally, E2 treatment increased the proliferation of the BPH-1 cells. However, the knockdown of LLGL2 with siRNA significantly suppressed the proliferation of the E2-treated BPH-1 cells. Moreover, si-llgl2 treatment up-regulated the expression of LC-3B, ATG7, and p-beclin, which are known to play a pivotal role in autophagosome formation in E2-treated BPH-1 cells. Overexpression of LLGL2 was able to further prove these findings by showing the opposite results from the knockdown of LLGL2 in E2-treated BPH-1 cells. Collectively, our results suggest that LLGL2 is closely involved in the proliferation of prostate cells by regulating autophagosome formation. These results provide a better understanding of the mechanism involved in the effect of LLGL2 on prostate cell proliferation. LLGL2 might serve as a potential target in the diagnosis and/or treatment of human BPH.

Evaluation of hatching rate and survival rate of giant freshwater prawn (Macrobrachium rosenbergii) in Installation of Brackish Water Culture Mayangan, Probolinggo, East Java, Indonesia

Giant freshwater prawn (Macrobrachium rosenbergii) is a fisheries commodity that has a high economical value. The purpose of this study is to determine hatching rate and survival rate of giant freshwater prawn in Installation of Brackish Water Culture Mayangan. This research was carried out at Installation of Brackish Water Culture Mayangan from December 2018 until January 2019. The work method used in this research is descriptive method. The giant freshwater prawn hatchery technique is carried out using a natural spawning system. Newly hatched larvae still have egg yolk as food reserves which will run out in 3 days. After that, the larvae will be fed with natural feed, namely Artemia sp. The results showed that the hatching rate and survival rate of giant freshwater prawn were 54.7% and 40.6%, respectively. Water quality parameters of giant freshwater prawn larvae for pH, temperature, dissolved oxygen were 7-8.7, 27-28°C, and 7-8 mg/l, respectively. Ammonia, nitrite, and nitrate for this study were 0.14-0.32 mg/l, 0.006-0.007 mg/l, and 0.2-0.4 mg/l.

Low water pH depressed growth and early development of giant freshwater prawn Macrobrachium rosenbergii larvae

Macrobrachium rosenbergii is one of the shellfish species with high aquaculture value due to its increasing market demand. However, the comparatively low production volume compared to demand coupled with the rapid decline of the natural environment, consequently, drives the potential depletion of the wild population. The decrease in water pH related to anthropogenic pollution is one of the most critical factors affecting the early life performances of M. rosenbergii. Therefore, this study was designed to examine the effect of low water pH on feeding, growth and development of M. rosenbergii early life stages. Experimental water pH was set as neutral (7.7 ± 0.4); mild-acidic (6.4 ± 0.5) and acidic (5.4 ± 0.2) with triplication at a stocking density of 2 larvae/L for 30 days. As expected, M. rosenbergii larvae were highly sensitive to acidic pH with no larvae survived beyond 48 h of exposure. Feeding, survival and growth of larvae were adversely affected by mild-acidic pH exposure as compared to neutral pH. Larvae exposed to mild-acidic water pH experienced a prolonged larval period and only metamorphosed to the post-larval stage at day-30. Whilst under neutral water pH, larval that metamorphosed to post-larval was first observed on day-23. The negative impact of decreased pH, even in mild-acidic pH exposure, on the feeding, survival, growth and development of M. rosenbergii larvae highlights the urgency of periodic pH monitoring during M. rosenbergii larviculture.

BACTERIAL INFECTION IN THE INDUSTRY OF Giant Tiger Prawn (Penaeus monodon) Larvae in Tarakan City

Tarakan City is a hatchery center for giant tiger prawn larvae production. However, the quality of the larvae produced is still far from expectations, namely free of pathogens free and capable of being resistant to disease. Observation of bacterial infection in the growth phase of giant tiger prawn larvae was carried out to determine the cause and proper treatment in the hatchery. The results showed that the isolation of bacteria in tiger prawns post larvae using 3% TSA media obtained 8 isolates in the Amal Hatchery and 8 isolates in the Beringin Tiga Hatchery with the characteristics of the colonies being cream and yellow in color and the colony shape was irregular, entire, and law conversion. Based on the gram test including gram negative and biochemical tests identified the type; Alcaligenes denitrificans, Pseudomonas diminuta, Shewanella putrefaciens, Bordetella bronchiseptica, Vibrio spp, Alcaligenes faecalis, Flavobacterium breve, Flavobacterium thalpophilum, Flavobacterium multivorum, Pseudomonas pseudoalcaligenes, Pasteurella Pseudomonas.Keywords: Tiger prawn larvae, Bacteria, Hatchery

Schistosoma japonicum proteins that interact with the gynecophoral canal protein identified using a yeast two-hybrid system

The large amount of schistosome eggs produced by mature female worms not only induce major pathological damage to the host but also lead to the transmission of schistosomiasis. Mature female schistosome worms need constant pairing contact with a male partner as male signaling is indispensable to female growth, development, and reproduction. The gynecophoral canal protein (GCP), a cell-surface glycoprotein, plays a potential role in the interaction between males and females and in stimulating female development and maturation. In this study, a yeast two-hybrid cDNA library of Schistosoma japonicum (Sj) parasites 18 days post-infection (dpi) was constructed; the Sjgcp gene was inserted into a pGBKT7-BD bait plasmid and used as a bait protein to screen for its molecular interactions using a yeast mating procedure. Twenty-four prey proteins that interacted with the SjGCP were selected after excluding false positives; the interactions between S.japonicum lethal giant larvae (SjLGL) and SjGCP, S.japonicum type V collagen (SjColV) and SjGCP, were verified by co-immunoprecipitation. The RNA interference against SjGCP, SjColV and SjGCP + SjColV led to severe underdevelopment of tegument in male worms and vitelline globules in female worms as well as reduced reproductive capacity of the females. Collectively, SjGCP and its interacting proteins may play pivotal roles in growth and development. The findings also suggested that SjGCP and its interacting protein partners might represent new candidate targets for drug development against schistosomiasis.

EFFECTS OF REPLACEMENT OF FARM- MADE FEED WITH LANSY PL ON GROWTH AND SURVIVAL RATE OF THE GIANT FRESHWATER PRAWN (Macrobrachium rosenbergii) LARVAE

This study aimed to research the effects of replacing farm-made feed with Lansy PL. The experiment consisted of 4 treatments in which giant freshwater prawn larvae were fed with different feeding regimes. Treatment 1(Control) consisted of 03 feeding times of farmmade feed and 02 times of Artermia per day; Treatment 2 consisted of 2 times of farm-made feed, 1 time of Lansy PL and 2 times of Artermia per day; Treatment 3 consisted of 1 time of farmmade feed, 2 times of Lansy PL and 2 times of Artermia per day; and Treatment 4 consisted of 3 times of Lansy PL and 2 times of Artermia per day. Each container for larval rearing was made of plastic and had a volume of 120 liters, and larvae were stocked at a density of 60 inds/l with water salinity at 12o=oo. After 30 days of rearing, Treatment 3 reached the highest metamorphosis index of 10.73 ± 0.06, whichwas not statisticaly different (p > 0.05) than the treatment substituting Lanzy PL 1 time per day, but was statistically different from the other two treatments. The length of Postlarvae 15 was 9.83 ± 0.09 mm, and the survival rate was 62.78 ± 0.66%. Its yield of 37.67 ± 0.40 fish/L was also the best and was statistically different compared to the rest of the treatments. Therefore, the results of the study conclude that replacing farm-madefeed with Lansy PL for 2 meals per day gives the best results when rearing giant freshwater prawn larvae.

Lethal giant larvae gene is required for normal nymphal development and midgut morphogenesis in Locusta migratoria.

Disruption of morphogenesis, an essential process in organismal development, can lead to disruption of biological processes, reduction in fitness, or even death of an organism. The roles of lethal giant larvae (Lgl) protein in maintaining tissue organization have been studied extensively in mammals, but little is known about this gene's roles in promoting correct tissue morphogenesis in insects. In this study, we identified an Lgl ortholog in Locusta migratoria. RT-qPCR results revealed that LmLgl was constitutively expressed during third, fourth, and fifth instar nymphs. Furthermore, LmLgl showed highest expression in the ovary followed by wing pads, midgut, hindgut, Malpighian tubules, and foregut of the third-instar nymphs. To examine the role of LmLgl in L. migratoria development, RNA interference was performed during nymphal stages. Silencing of LmLgl increased body size but decreased bodyweight by 9.0%. Histological sections of the midgut revealed abnormal large masses of disordered epithelial cells in dsLmLgl-injected nymphs. In addition, downregulation of LmLgl transcript levels significantly altered the morphological structure in midgut, resulting in the formation of tumor-like structures. Our results indicated that LmLgl may act as a tumor-suppressor gene, which plays an essential role in maintaining a normal morphological structure in the midgut of L. migratoria. Our results also suggest that LmLgl may be explored as a potential target for developing dsRNA-based biological pesticides for managing insect pests.

Deletion of Smooth Muscle Lethal Giant Larvae 1 Promotes Neointimal Hyperplasia in Mice.

Vascular smooth muscle cell (VSMC) proliferation and migration contribute to neointimal hyperplasia after injury, which causes vascular remodeling related to arteriosclerosis, hypertension, and restenosis. Lethal giant larvae 1 (LGL1) is a highly conserved protein and plays an important role in cell polarity and tumor suppression. However, whether LGL1 affects neointimal hyperplasia is still unknown. In this study, we used smooth muscle-specific LGL1 knockout (LGL1SMKO) mice generated by cross-breeding LGL1flox/flox mice with α-SMA-Cre mice. LGL1 expression was significantly decreased during both carotid artery ligation in vivo and PDGF-BB stimulation in vitro. LGL1 overexpression inhibited the proliferation and migration of VSMCs. Mechanistically, LGL1 could bind with signal transducer and activator of transcription 3 (STAT3) and promote its degradation via the proteasomal pathway. In the carotid artery ligation animal model, smooth muscle-specific deletion of LGL1 accelerated neointimal hyperplasia, which was attenuated by the STAT3 inhibitor SH-4-54. In conclusion, LGL1 may inhibit neointimal hyperplasia by repressing VSMC proliferation and migration via promoting STAT3 proteasomal degradation.

Effect of meal size on the postprandial metabolic response in Chinese giant salamander Andrias davidianus larvae

The digestive physiological characteristics of Chinese giant salamander Andrias davidianus have not been reported in the existing literature, and improving our knowledge in this area could help in the artificial culture of the species. The effect of meal size on the specific dynamic action (SDA) of A. davidianus larvae was assessed in this study at a water temperature of 20°C. The Chinese giant salamander was fed by test diets at meal sizes of 0.5, 1, 2, 4, 8 and 12% of its body mass (4.41 ± 0.07 g), and the postprandial oxygen consumption rate (MO2) was measured at 2 h intervals until the rate returned to its preprandial level. Peak MO2 (MO2peak) increased with meal size but levelled off at larger meal sizes within the studied range (8-12% body mass). The factorial metabolic scope increased from 1.27 to 2.11 when meal size increased from 0.5 to 12%. Time to reach MO2peak was not affected by the relative meal size. The SDA duration increased from 8.89 to 47.33 h as the relative meal size increased from 0.5 to 12%. The amount of energy expended during SDA increased linearly with increasing meal size (R2 = 0.842, p < 0.001, n = 60). There was no significant difference in the SDA coefficient among the different relative meal size groups. These data suggest that (1) A. davidianus larvae increase their energy demands during digestion by prolonging the SDA duration rather than increasing the MO2peak as meal size increases, and (2) A. davidianus showed a smaller factorial metabolic scope in the process of digesting large meal sizes, which may be related to the limited MO2peak of the species.

Altered Expression of Par3, aPKC-λ, and Lgl1 in Hippocampus in Kainic Acid-Induced Status Epilepticus Rat Model

Introduction: Mossy fiber sprouting (MFS) is a frequent histopathological finding in temporal lobe epilepsy (TLE) and is involved in the pathology of TLE. However, molecular signals underlying MFS remain unclear. Partitioning defective 3(Par3), atypical protein kinase C-λ(aPKC-λ), and lethal giant larvae 1(Lgl1) were involved in the neuronal polarity and axon growth. The potential roles of those proteins in MFS and epileptogenesis of TLE were investigated.Material and Methods: The epileptic rat models were established by intracerebroventricular injection of kainic acid (KA). The degree of MFS was measured by using Timm staining, Neuronal loss and the expression aPKC-λ, Par3, and Lgl1 in hippocampus were measured by using immunohistochemistry and western blot analysis.Results: The neuronal loss in CA3 region was observed from 3 days to 8 weeks, while the neuronal loss in the hilar region was observed from 1 to 8 weeks in experimental group. The Timm score in the CA3 region in experimental group was significantly higher than that in the control group from 2 to 8 weeks. Compared with control group, the expressions of Par3 and Lgl1 were upregulated and the expression of aPKC-λ was downregulated in the experimental groups. Positive correlation between the Par3 expression and Timm scores, and the negative correlation between the aPKC-λ expression and Timm scores in CA3 region were discovered in experimental group.Conclusion: The findings of the present study indicated that aPKC-λ, Par3, and Lgl1 may be involved in MFS and in the epileptogenesis of temporal lobe epilepsy.

Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division.

A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, we have learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, we have performed a pilot RNAi screen in Drosophila larval brain NB lineages using RasV12 scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, we have tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with RasV12 scrib does cause tumor-like overgrowth. Based on these results, we have randomly screened 79 RNAi lines detecting 15 potential novel ACD regulators/tumor suppressor genes. We conclude that RasV12 scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process.

Eggs Immersion with Vitamin C on Hatching Rate, Growth and Mortality of Giant Gourami Larvae

<b>Background and Objective:</b> The giant gourami (<i>Osphronemus goramy</i>; Lacepede, 1801) is one of Indonesia's main freshwater fish of economic benefit for food security. This study evaluated the effects of vitamin C dosages used for immersion embryonic and larval of giant gourami. <b>Materials and Methods:</b> The vitamin C using Ascorbic acid standard (FLUKA, Sigma-Aldrich), each weighed 50, 100, 150 and 200 mg and dissolved in 1 L of fresh borehole water is referred to as P<sub>2</sub>, P<sub>3</sub>, P<sub>4</sub> and P<sub>5</sub>. At the same time, 1 L of fresh borehole water not added with vitamin C is called P<sub>1</sub> (Placebo). Each treatment was formulated with 5 L and poured into an incubator tank with a capacity of 10 L Then, as many as 150 eggs are incubating per experiment in each incubator tank. These larvae of each experiment were rearing for 20 days using live feed (tubifex) 40% per feed. Each treatment was three replicates. <b>Results:</b> In this study, larval hatching rates varied from 78.59-98.22%, with the survival of endogenous feeding periods ranging between 93.08 and 99.10%. The specific growth rate increased from 11.14 and 13.38% per day. The Coefficient of thermal growth increased from 1.49-1.70. Larval mortality after exogenous feeding for 20 days of the experiment decreased from 21.11-5.56%. The Coefficient of variation in weight was between 3.19 and 14.04%. <b>Conclusion:</b> Giant gourami eggs and larvae hatched and survived and grew well in immersion with a dose of vitamin C up to 200 mg L<sup>1</sup>.

Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.

LLGL2 Increases Ca2+ Influx and Exerts Oncogenic Activities via PI3K/AKT Signaling Pathway in Hepatocellular Carcinoma.

BackgroundLethal giant larvae (Lgl), scaffolding proteins, regulate the epithelial cell apicobasal polarity in Drosophila. They play important roles in asymmetric cell division, cell migration, and progenitor cells self-renewal as tumor suppressors. One of Lgl mammalian homologues proteins, LLGL2 overexpression has been reported in ER+ breast cancer and promotes tumor proliferation through regulating leucine uptake. Nonetheless, the role of LLGL2 in hepatocellular carcinoma (HCC) is still unknown.MethodsTCGA dataset mining, qRT-PCR, Western blot along with immunohistochemistry assays were employed to explore LLGL2 expression in human HCC samples and cell lines. Moreover, the clinical value of LLGL2 was investigated in 156 HCC patients. Furthermore, the role as well as the molecular mechanism of LLGL2 in the progression of HCC was explored through a series of in vitro and in vivo experiments.ResultsLLGL2 was up-regulated in HCC tissues, which was related with certain clinicopathological features including tumor number, vascular invasion as well as advanced stage. High expression of LLGL2 predicted poor prognosis after hepatectomy. LLGL2 promoted HCC cells proliferation, migration and invasion through PI3K/ATK signaling by promoting calcium ion influx.ConclusionOur study identified that LLGL2 is a tumor promoter in HCC for the first time, which could potentially be utilized as a new biomarker and a therapeutic target for HCC.

Endophytic Bacillus Bacteria Living in Sugarcane Plant Tissues and Telchin licus licus Larvae (Drury) (Lepidoptera: Castniidae): The Symbiosis That May Open New Paths in the Biological Control.

Bacteria of the genus Bacillus can colonize endophytically and benefit several crops including the control of some pest orders. In view of the benefits provided by these microorganisms and in order to find out an efficient biotechnological control for the giant borer, our interest in studying the microorganisms in symbiosis with sugarcane and the giant borer has arisen, since there is no efficient chemical or biological control method for this pest. Therefore, endophytic Bacillus strains were isolated from three sugarcane niches (apoplast fluid, central internode cylinder and roots) and also from the giant borer larvae living inside sugarcane varieties grown in the Northeast region of Brazil. The taxonomical characterization (16S rRNA) of 157 Gram-positive isolates showed that 138 strains belonged to the Bacillus genus. The most representative species were phylogenetically closely related to B. megaterium (11.5%) followed by B. safensis (10.8%), B. cereus (8.9%), B. oleronius (8.9%), B. amyloliquefaciens (7.0%), and B. pacificus (6.4%). BOX-PCR analyses showed very distinct band pattern profiles suggesting a great diversity of Bacillus species within the sugarcane niches and the digestive tract, while the B. cereus group remained very closely clustered in the dendrogram. According to XRE biomarker analysis, eleven strains (FORCN005, 007, 008, 011, 012, 014, 067, 076, 092, 093, and 135) correspond to B. thuringiensis species. Additional studies using conserved genes (glp, gmk, pta, and tpi) indicated that most of these strains were phylogenetically closely related to B. thuringiensis and may be considered different subspecies. In conclusion, this study suggests that the culturable Bacillus species are greatly diversified within the plant niches and showed Bacillus species in the digestive tract of the giant borer for the first time. These results open new perspectives to understand the role and functions played by these microorganisms in symbiosis with this pest and also the possibility of developing an efficient biological control method for the giant borer using strains identified as the B. thuringiensis species.

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults.

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

Melatonin improves cognitive behavior, oxidative stress, and metabolism in tumor‐prone lethal giant larvae mutant of Drosophila melanogaster

Mutant lethal giant larvae (lgl) flies (Drosophila melanogaster) are known to develop epithelial tumors with invasive characteristics. The present study has been conducted to investigate the influence of melatonin (0.025 mM) on behavioral responses of lgl mutant flies as well as on biochemical indices (redox homeostasis, carbohydrate and lipid metabolism, transaminases, and minerals) in hemolymph, and head and intestinal tissues. Behavioral abnormalities were quantitatively observed in lgl flies but were found normalized among melatonin-treated lgl flies. Significantly decreased levels of lipid peroxidation products and antioxidants involved in redox homeostasis were observed in hemolymph and tissues of lgl flies, but had restored close to normalcy in melatonin-treated flies. Carbohydrates including glucose, trehalose, and glycogen were decreased and increased in the hemolymph and tissues of lgl and melatonin-treated lgl flies, respectively. Key enzymes of carbohydrate metabolism showed a significant increment in their levels in lgl mutants but had restored close to wild-type baseline levels in melatonin-treated flies. Variables of lipid metabolism showed significantly inverse levels in hemolymph and tissues of lgl flies, while normalization of most of these variables was observed in melatonin-treated mutants. Lipase, chitinase, transaminases, and alkaline phosphatase showed an increment in their activities and minerals exhibited decrement in lgl flies; reversal of changes was observed under melatonin treatment. The impairment of cognition, disturbance of redox homeostasis and metabolic reprogramming in lgl flies, and restoration of normalcy in all these cellular and behavioral processes indicate that melatonin could act as oncostatic and cytoprotective agents in Drosophila.

Fluctuating temperature regime impairs growth in giant gourami (Osphronemus goramy) larvae

Growth and physiology were compared in giant gourami (Osphronemus goramy) larvae reared in constant (25, 30, and 35 °C) and daily fluctuating (25–35 °C) temperature regimes. Larvae aged eight days post-hatching were reared indoors for 21 days in four closed recirculating systems (four aquaria per system) in the different temperature regimes. Samples of larvae were collected every week from each temperature regime for growth measurements. At the end of the experiment, cortisol and glucose concentrations, as well as oxygen consumption and ventilation rates, were determined in fish from each temperature regime. Results showed that the fluctuating temperature regime did not affect survival or abnormality, but significantly impaired growth in giant gourami larvae. Physiological responses, assessed using cortisol and glucose levels and oxygen consumption, corroborate previous findings, suggesting that a stable temperature of 30 °C is optimal for rearing giant gourami larvae. Our experimental approach demonstrated that temperature variations observed in larval rearing structures on small-scale fish farms are likely to impair the production of giant gourami fry.

A comprehensive in vivo screen for anti-apoptotic miRNAs indicates broad capacities for oncogenic synergy

microRNAs (miRNAs) are ~21–22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. We explored this concept in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensive in vivo phenotypic assays, we identify an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. We utilize secondary assays to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, we modeled this situation by asking whether activation of anti-apoptotic miRNAs could serve as “second hits”. Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, we find that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of our screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings.

Human giant larvae-1 promotes migration and invasion of malignant glioma cells by regulating N-cadherin.

Human giant larvae-1 (Hugl-1) is a human homologue of Drosophila tumor suppressor lethal (2)-giant larvae and has been reported to be involved in the development of human malignancies. Previous studies performed by our group demonstrated that Hugl-1 inhibits glioma cell proliferation in an intracranial model of nude mice. However, the exact molecular mechanisms underlying the participation of Hugl-1 in glioma invasion and migration, and in the depolarizing process remain largely unknown. Utilizing the U251-MG cells with stable expression of Hugl-1, the present study used wound healing, Transwell invasion and western blot assays to explore the role and specific mechanism of Hugl-1 in glioma invasion and migration. The results of the present study demonstrated that overexpression of Hugl-1 decreased cell-cell adhesion and increased cell-cell extracellular matrix adhesion. In addition, overexpression of Hugl-1 promoted pseudopodia formation, glioma cell migration and invasion. The molecular mechanism of action involved the negative regulation of N-cadherin protein levels by Hugl-1. Overexpression or knockdown of N-cadherin partially suppressed or enhanced the effects of Hugl-1 on glioma cell migration and invasion, respectively. Furthermore, Hugl-1 inhibited cell proliferation, while promoting cell migration, which suggests that it may serve a two-sided biological role in cellular processes. Taken together, these results suggest that Hugl-1 promotes the migration and invasion of malignant glioma cells by decreasing N-cadherin expression. Thus, Hugl-1 may be applied in the development of targeted and personalized treatment.