Spontaneous regression of lung metastases after transarterial chemoembolization for hepatocellular carcinoma.

Spontaneous regressions of primary and/or metastatic lesions have been rarely reported in hepatocellular carcinoma (HCC). Herein, we report the case of a 71-year-old man with HCC, focusing on shape changes of lung metastases over time. Lung metastasis of HCC was histologically diagnosed by percutaneous computed tomography (CT)-guided needle biopsy after the treatment of primary HCC lesion. Lung lesions had been observed on enhanced contrast computed tomography for >3 years without any local or systemic treatment for them. During this period, treatments including surgical procedure for relapsed bladder cancer and transarterial chemoembolization for HCC were performed. Metastatic lung lesions immediately regressed after these treatments. Therefore, accumulation of such cases may help elucidate spontaneous regression mechanisms in primary HCC or its lung metastases.

Regional differences in the prevalence of sensitization to environmental allergens: Analysis on IgE antibody testing conducted at major clinical testing laboratories throughout Japan from 2002 to 2011.

BACKGROUND: Identification of sensitized allergens for patients with respiratory allergy is an important step in disease care and environmental allergen control. The Japanese archipelago belongs to various climate categories due to its length from north to south which transverse the subarctic in the north to the subtropical in the south, suggesting substantial regional differences in dominant environmental allergens. However, few studies have assessed the regional differences in the prevalence of sensitization to environmental allergens. METHODS: We requested three major clinical testing laboratories to provide us with summarized results of antigen-specific IgE-antibody (Ab) measurements. These measurements were collected for clinical purposes throughout Japan from 2002 through 2011. The prevalence of positivity for IgE-Ab against 19 environmental allergens was calculated for each prefecture in order to evaluate regional differences. RESULTS: Test data on specific IgE-Ab of 19,969,753 orders were analyzed. The prevalence of positivity for house dust mites was high and the regional difference was low, whereas apparent regional differences were found for pollen, insects, and fungi. The prevalence of positivity for Japanese cedar was low in Hokkaido and Okinawa, while those to alder was highest in Hokkaido. Higher prevalence for insects was observed in southern areas (Okinawa and prefectures in Kyusyu). CONCLUSIONS: Findings of this study clearly demonstrated regional differences in the prevalence of sensitization to environmental allergens in Japan and the study also provides useful information for the clinician when deciding which allergens should preferentially be measured for IgE-Ab after considering regional difference.

[Analysis of Post-Recurrence Survival of Resected Lung Cancer with Brain Metastasis].

METHODS: We retrospectively evaluated the post-recurrence survival of 37 cases with brain metastases out of 439 consecutive resected cases of primary lung cancer between 2001 and 2017. FINDINGS: There was no difference in survival according to tumor size but survival was significantly shorter in patients with larger numbers of tumors. Patients initially treated with stereotactic radiosurgery(SRS)or surgical resection survived longer than those with whole-brain irradiation(WBI)(median survival: 23 months for SRS, 17 months for surgical resection, and 4 months for WBI: p<0.001 between SRS and WBI). CONCLUSIONS: As SRS is recommended for 1-4 tumors with maximum diameters ofC3 cm and surgical resection is recommended for tumors larger than 3 cm, these effective locoregional therapies should be aggressively adopted for local control of brain metastases with the aim of improved QOL and prolonged survival. Due to the deterioration of neurocognitive function, WBI should be avoided as initial treatment for brain metastases when effective locoregional therapy or systemic chemotherapy is available and reserved for leptomeningeal dissemination or miliary metastases.

Overexpression of Larp4B downregulates dMyc and reduces cell and organ sizes in Drosophila.

Regulation of cell and organ sizes is fundamental for all organisms, but its molecular basis is not fully understood. Here we performed a gain-of-function screen and identified larp4B whose overexpression reduces cell and organ sizes in Drosophila melanogaster. Larp4B is a member of La-related proteins (LARPs) containing an LA motif and an adjacent RNA recognition motif (RRM), and play diverse roles in RNA metabolism. However, the function of Larp4B has remained poorly characterized. We generated transgenic flies overexpressing wild-type Larp4B or a deletion variant lacking the LA and RRM domains, and demonstrated that the RNA-binding domains are essential for Larp4B to reduce cell and organ sizes. We found that the larp4B-induced phenotype was suppressed by dMyc overexpression, which promotes cell growth and survival. Furthermore, overexpression of larp4B decreased dMyc protein levels, whereas its loss-of-function mutation had an opposite effect. Our results suggest that Larp4B is a negative regulator of dMyc.

Deficiency of succinyl-CoA synthetase α subunit delays development, impairs locomotor activity and reduces survival under starvation in Drosophila.

Succinyl-CoA synthetase/ligase (SCS) is a mitochondrial enzyme that catalyzes the reversible process from succinyl-CoA to succinate and free coenzyme A in TCA cycle. SCS deficiencies are implicated in mitochondrial hepatoencephalomyopathy in humans. To investigate the impact of SCS deficiencies in Drosophila, we generated a null mutation in Scs alpha subunit (Scsα) using the CRISPR/Cas9 system, and characterized their phenotype. We found that the Drosophila SCS deficiency, designated ScsαKO, contained a high level of succinyl-CoA, a substrate for the enzyme, and altered levels of various metabolites in TCA cycle and glycolysis, indicating that the energy metabolism was impaired. Unlike SCSα deficiencies in humans, there was no reduction in lifespan, indicating that Scsα is not critical for viability in Drosophila. However, they showed developmental delays, locomotor activity defects, and reduced survival under starvation. We also found that glycogen breakdown occurred during development, suggesting that the mutant flies were unable to produce sufficient energy to promote normal growth. These results suggested that SCSα is essential for proper energy metabolism in Drosophila. The ScsαKO flies should be useful as a model to understand the physiological role of SCSα as well as the pathophysiology of SCSα deficiency.

Evolution of sex-peptide in Drosophila.

The Drosophila sex-peptide (SP) has been identified as a seminal fluid component that induces post-mating responses (PMRs) in the inseminated females, such as inhibition of remating and stimulation of egg-laying. SP has been thought to play a central role in sexual conflict and sexually antagonistic co-evolution. Most of the sequenced Drosophila genomes contain SP orthologs, but their functions have been poorly characterized. Recently, we have investigated cross-species activity of D. melanogaster SP by means of injection into virgin females of other species. Among 11 species examined, SP response was observed in 6 species belonging to the D. melanogaster species group only. These species females express SP receptor (SPR) in their oviducts at relatively high levels, which was visualized by using a GFP-tagged SP. Furthermore, females of this species group responded to their own SP orthologs. However, females of the species outside the group did not respond to their own SP orthologs, even though all of them were potent inducers of SP-response in D. melanogaster. Our results suggested that the SP/SPR-mediated PMR was established in the lineage of the D. melanogaster species group.

The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila.

Misexpression Suppressor of Ras 4 (MESR4), a plant homeodomain (PHD) finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. Here it is shown that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. P-element insertions in the MESR4 locus, which were homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation was identified. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene was greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remained low. However, in the context of wing vein formation, genetic interaction experiments suggested that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggested that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development. genesis 53:701-708, 2015. © 2015 Wiley Periodicals, Inc.

Visualizing Molecular Functions and Cross-Species Activity of Sex-Peptide in Drosophila.

The Drosophila melanogaster sex-peptide (melSP) is a seminal fluid component that induces postmating responses (PMR) of females via the sex-peptide receptor (SPR) . Although SP orthologs are found in many Drosophila species, their functions remain poorly characterized. It is unknown whether SP functions are conserved across species or rather specific to each species. Here, we developed a GFP-tagged melSP (G-SP) and used it to visualize cross-species binding activity to the female reproductive system of various species. First we demonstrated that ectopically expressed G-SP induced PMR in D. melanogaster females and bound to the female reproductive system, most notably to the common oviduct. No binding occurred in the females lacking SPR, indicating that G-SP binding was dependent on SPR. Next we tested whether G-SP binds to the common oviducts from 11 Drosophila species using dissected reproductive tracts. The binding was observed in six species belonging to the D. melanogaster species group, but not to those outside the group. Injection of melSP reduced the receptivity of females belonging to the D. melanogaster species group, but not of those outside the group, being consistent with the ability to bind G-SP. Thus the SP-mediated PMR appears to be limited to this species group. SPR was expressed in the oviducts at high levels in this group; therefore, we speculate that an enhanced expression of SPR in the oviduct was critical to establish the SP-mediated PMR during evolution.

Identification of a novel role for Drosophila MESR4 in lipid metabolism.

Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose-like organ called the fat body, which plays a crucial role in energy homeostasis. Here, we conducted a fat body-specific misexpression screen to identify genes involved in lipid metabolism. We found that over-expression of a nuclear protein with nine C2 H2 type zinc-finger motifs and a PHD-finger, Misexpression suppressor of ras 4 (MESR4), reduces lipid accumulation in the fat body, whereas MESR4 knockdown increases it. We further show that MESR4 up-regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.

Overexpression of dilp2 causes nutrient-dependent semi-lethality in Drosophila.

Insulin/insulin-like growth factor (IGF) plays an important role as a systemic regulator of metabolism in multicellular organisms. Hyperinsulinemia, a high level of blood insulin, is often associated with impaired physiological conditions such as hypoglycemia, insulin resistance, and diabetes. However, due to the complex pathophysiology of hyperinsulinemia, the causative role of excess insulin/IGF signaling has remained elusive. To investigate the biological effects of a high level of insulin in metabolic homeostasis and physiology, we generated flies overexpressing Drosophila insulin-like peptide 2 (Dilp2), which has the highest potential of promoting tissue growth among the Ilp genes in Drosophila. In this model, a UAS-Dilp2 transgene was overexpressed under control of sd-Gal4 that drives expression predominantly in developing imaginal wing discs. Overexpression of Dilp2 caused semi-lethality, which was partially suppressed by mutations in the insulin receptor (InR) or Akt1, suggesting that dilp2-induced semi-lethality is mediated by the PI3K/Akt1 signaling. We found that dilp2-overexpressing flies exhibited intensive autophagy in fat body cells. Interestingly, the dilp2-induced autophagy as well as the semi-lethality was partially rescued by increasing the protein content relative to glucose in the media. Our results suggest that excess insulin/IGF signaling impairs the physiology of animals, which can be ameliorated by controlling the nutritional balance between proteins and carbohydrates, at least in flies.

[A case of a septic pulmonary embolism-related implanted central venous port].

We report a case of a 32-year-old woman with a septic pulmonary embolism-related implanted central venous port. She was treated with S-1/cisplatin(CDDP)chemotherapy for recurrent gastric cancer. Her disease was progressive after five courses of S-1/CDDP combination therapy. Because of peritonitis carcinomatosa, her oral intake was poor, so we placed an implanted central venous port in her right subclavian vein. We administered 5-FU/Leucovorin/paclitaxel combination therapy and total parenteral nutrition from the port. Chemotherapy was effective, so we stopped total parenteral nutrition after one month. Two months later, multiple nodular shadows appeared in her left lung fields without apparent symptoms. Because we suspected septic pulmonary embolism related to the venous port, we removed the venous port promptly and administered antibiotics with a broad spectrum. Pulmonary shadows disappeared immediately, and no recurrence was observed afterward.

Impaired energy metabolism in a Drosophila model of mitochondrial aconitase deficiency.

Aconitase catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, and its deficiency in humans is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. Here we investigated the effect of gene knockout and knockdown of the mitochondrial aconitase Acon in Drosophila. Acon-knockout flies were homozygous lethal, indicating that Acon is essential for viability. RNA interference-generated Acon-knockdown flies exhibited a variety of phenotypes, such as reduced locomotor activity, a shortened lifespan, and increased cell death in the developing brain. Metabolomic analysis revealed that acetyl-CoA, citrate/isocitrate, and cis-aconitate were significantly increased, while most metabolites of glycolysis and the TCA cycle were reduced. Reduced triacylglyceride and increased acetyl-CoA suggested that lipids were used as an energy source because of the impaired glycolysis and TCA cycle. The Acon-knockdown model should facilitate further understanding of the pathophysiology of m-aconitase deficiency in humans.

Impaired fatty acid oxidation in a Drosophila model of mitochondrial trifunctional protein (MTP) deficiency.

Mitochondrial trifunctional protein (MTP), which consists of the MTPα and MTPß subunits, catalyzes long-chain fatty acid ß-oxidation. MTP deficiency in humans results in Reye-like syndrome. Here, we generated Drosophila models of MTP deficiency by targeting two genes encoding Drosophila homologs of human MTPα and MTPß, respectively. Both Mtpα(KO) and Mtpß(KO) flies were viable, but demonstrated reduced lifespan, defective locomotor activity, and reduced fecundity represented by the number of eggs laid by the females. The phenotypes of Mtpα(KO) flies were generally more striking than those of Mtpß(KO) flies. Mtpα(KO) flies were hypersensitive to fasting, and retained lipid droplets in their fat body cells as in non-fasting conditions. The amount of triglyceride was also unchanged upon fasting in Mtpα(KO) flies, suggesting that lipid mobilization was disrupted. Finally, we showed that both Mtpα(KO) and Mtpß(KO) flies accumulated acylcarnitine and hydroxyacylcarnitine, diagnostic markers of MTP deficiencies in humans. Our results indicated that both Mtpα(KO) and Mtpß(KO) flies were impaired in long-chain fatty acid ß-oxidation. These flies should be useful as a model system to investigate the molecular pathogenesis of MTP deficiency.

Calcineurin and its regulator sra/DSCR1 are essential for sleep in Drosophila.

Sleep is a fundamental biological process for all animals. However, the molecular mechanisms that regulate sleep are still poorly understood. Here we report that sleep-like behavior in Drosophila is severely impaired by mutations in sarah (sra), a member of the Regulator of Calcineurin (RCAN) family of genes. Sleep reduction in sra mutants is highly correlated with decreases in Sra protein levels. Pan-neural expression of sra rescues this behavioral phenotype, indicating that neuronal sra function is required for normal sleep. Since Sra regulates calcineurin (CN), we generated and examined the behavior of knock-out mutants for all Drosophila CN genes: CanA-14F, Pp2B-14D, and CanA1 (catalytic subunits), and CanB and CanB2 (regulatory subunits). While all mutants show at least minor changes in sleep, CanA-14F(KO) and CanB(KO) have striking reductions, suggesting that these are the major CN subunits regulating sleep. In addition, neuronal expression of constitutively active forms of CN catalytic subunits also significantly reduces sleep, demonstrating that both increases and decreases in CN activity inhibit sleep. sra sleep defects are suppressed by CN mutations, indicating that sra and CN affect sleep through a common mechanism. Our results demonstrate that CN and its regulation by Sra are required for normal sleep in Drosophila and identify a critical role of Ca(2+)/calmodulin-dependent signaling in sleep regulation.

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila.

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.

POSH promotes cell survival in Drosophila and in human RASF cells.

In Drosophila, Eiger, a tumor necrosis factor α (TNFα) superfamily ligand, induces cell death by activating the c-Jun N-terminal kinase (JNK) pathway. Here, we report that overexpression of Plenty of SH3s (POSH) suppresses Eiger-induced cell death and produces highly deformed tissues. These results imply that high levels of POSH protect tissues from cell death. In humans, rheumatoid arthritis synovial fibroblasts (RASF) are generally resistant to apoptosis. We show that POSH is expressed at relatively high levels in RASF, and its reduction by RNAi sensitizes these cells to Fas-mediated apoptosis. Thus, we demonstrate that POSH promotes cell survival in Drosophila and in human RASF.

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila.

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.

Insulin-degrading enzyme antagonizes insulin-dependent tissue growth and Abeta-induced neurotoxicity in Drosophila.

Insulin-degrading enzyme (IDE) is implicated in the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer's disease (AD). Here we provide genetic evidence that Drosophila Ide (dIde) antagonizes the insulin signaling pathway and human Abeta-induced neurotoxicity in Drosophila. In this study, we also generated a dIde knockout mutant (dIde(KO)) by gene targeting, and found that loss of IDE increases the content of the major insect blood sugar, trehalose, thus suggesting a conserved role of IDE in sugar metabolism. Using dIde(KO) as a model, further investigations into the biological functions of IDE and its role in the pathogenesis of DM2 and AD can be made.

Overexpression of grappa encoding a histone methyltransferase enhances stress resistance in Drosophila.

Histone deacetylases, such as silent information regulator 2 (Sir2) and Rpd3 are involved in chromatin silencing and implicated in lifespan determination in several organisms. The yeast Dot1 gene encoding a histone methyltransferase affects localization of silencing proteins including Sir2, and plays an essential role in the repair of damaged DNA. However, it is not known whether an alteration of a histone methyltransferase activity influences lifespan or stress resistance, which is often associated with extended lifespan. Here we investigated whether the Drosophilagrappa (gpp) gene, a Dot1 homolog influences lifespan and stress resistance using transgenic flies overexpressing gpp and those bearing a partial loss-of-function mutation. Overexpression of gpp throughout the adult stage did not extend the lifespan, but significantly enhanced resistances when they were kept on medium containing 1% H(2)O(2), or those with poor nutrients. As well, gpp-overexpressing flies were behaviourally more active than control flies. We investigated whether gpp overexpression induced anti-oxidant genes, Catalase, Sod, Sod2, GstD2, dhd, TrxT and Trx-2. However, none of these genes was induced. A partial loss-of-function mutations in gpp dramatically reduced the lifespan under oxidative and caloric stresses. Taken together, these results demonstrated that gpp is required for normal lifespan and stress resistance, and that its overexpression increases stress resistance in Drosophila, without obvious induction of representative anti-oxidant genes.

MachiBase: a Drosophila melanogaster 5'-end mRNA transcription database.

MachiBase (http://machibase.gi.k.u-tokyo.ac.jp/) provides a comprehensive and freely accessible resource regarding Drosophila melanogaster 5'-end mRNA transcription at different developmental states, supporting studies on the variabilities of promoter transcriptional activities and gene-expression profiles in the fruitfly. The data were generated in conjunction with the recently developed high-throughput genome sequencer Illumina/Solexa using a newly developed 5'-end mRNA collection method.