CDC7 inhibition induces replication stress-mediated aneuploid cells with an inflammatory phenotype sensitizing tumors to immune checkpoint blockade.

Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.

Virus purification highlights the high susceptibility of SARS-CoV-2 to a chlorine-based disinfectant, chlorous acid.

Chlorous acid water (HClO2) is known for its antimicrobial activity. In this study, we attempted to accurately assess the ability of chlorous acid water to inactivate SARS-CoV-2. When using cell culture supernatants of infected cells as the test virus, the 99% inactivation concentration (IC99) for the SARS-CoV-2 D614G variant, as well as the Delta and Omicron variants, was approximately 10ppm of free chlorine concentration with a reaction time of 10 minutes. On the other hand, in experiments using a more purified virus, the IC99 of chlorous acid water was 0.41-0.74ppm with a reaction time of 1 minute, showing a strong inactivation capacity over 200 times. With sodium hypochlorite water, the IC99 was 0.54ppm, confirming that these chlorine compounds have a potent inactivation effect against SARS-CoV-2. However, it became clear that when using cell culture supernatants of infected cells as the test virus, the effect is masked by impurities such as amino acids contained therein. Also, when proteins (0.5% polypeptone, or 0.3% BSA + 0.3% sheep red blood cells, or 5% FBS) were added to the purified virus, the IC99 values became high, ranging from 5.3 to 76ppm with a reaction time of 10 minutes, significantly reducing the effect. However, considering that the usual usage concentration is 200ppm, it was shown that chlorous acid water can still exert sufficient disinfection effects even in the presence of proteins. Further research is needed to confirm the practical applications and effects of chlorous acid water, but it has the potential to be an important tool for preventing the spread of SARS-CoV-2.

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

Physiologically based pharmacokinetic modeling of ponatinib to describe drug-drug interactions in patients with cancer.

PURPOSE: This study aimed to investigate the drug-drug interactions of ponatinib with strong, moderate, or weak CYP3A4 inhibitors/inducers by developing physiologically based pharmacokinetic (PBPK) models. METHODS: Simcyp® Ver 20.1 (Certara Inc., Sheffield, UK) was used to construct a PBPK model for ponatinib and to predict its interaction with strong, moderate, or weak CYP3A4 inhibitors/inducers. The constructed model was validated by comparing predicted values with actual observed values. Inhibitors or inducers that increased or decreased the area under the plasma concentration curve of ponatinib by more than two-fold when used in combination were considered significant. RESULTS: The PBPK model of ponatinib accurately represented its oral pharmacokinetics. It also reasonably predicted its pharmacokinetics when combined with ketoconazole and rifampicin. No weak to strong CYP3A4 inhibitor combinations significantly increased the AUC of ponatinib. However, the strong CYP3A4 inducers rifampicin (oral, 600 mg QD) and phenytoin (oral, 100 mg TID) decreased AUC by 60-70% and 50%, respectively. CONCLUSIONS: The PBPK model predicted a significant drug interaction when ponatinib was combined with a strong CYP3A4 inducer. Conversely, the combination with weak-to-strong CYP3A4 inhibitors did not suggest a drug interaction with ponatinib.

Combination treatment with a PI3K/Akt/mTOR pathway inhibitor overcomes resistance to anti-HER2 therapy in PIK3CA-mutant HER2-positive breast cancer cells.

Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) are observed in 15-20% of breast cancers (HER2+ breast cancers), and anti-HER2 therapies have significantly improved prognosis of patients with HER2+ breast cancer. One resistance mechanism to anti-HER2 therapies is constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway. Combination therapy with small-molecule inhibitors of AKT and HER2 was conducted in HER2+ breast cancer cell lines with or without PIK3CA mutations, which lead to constitutive activation of the PI3K pathway. PIK3CA mutations played important roles in resistance to single-agent anti-HER2 therapy in breast cancer cell lines. Combination therapy of a HER2 inhibitor and an AKT inhibitor, as well as other PI3K pathway inhibitors, could overcome the therapeutic limitations associated with single-agent anti-HER2 treatment in PIK3CA-mutant HER2+ breast cancer cell lines. Furthermore, expression of phosphorylated 4E-binding protein 1 (p4EBP1) following the treatment correlated with the antiproliferative activities of the combination, suggesting that p4EBP1 may have potential as a prognostic and/or efficacy-linking biomarkers for these combination therapies in patients with HER2+ breast cancer. These findings highlight potential clinical strategies using combination therapy to overcome the limitations associated with single-agent anti-HER2 therapies in patients with HER2+ breast cancer.

ZAK Inhibitor PLX4720 Promotes Extrusion of Transformed Cells via Cell Competition.

Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine.

Surface contamination of the outer and blister packages of oral anticancer drugs: A multicenter study.

INTRODUCTION: All guidelines necessitate wearing personal protective equipment during dispensing of oral anticancer drugs. This study aims to measure the degree of contamination on the press-through-package strips of oral anticancer drugs in Japan. METHOD: Surface contamination of the external packaging of anticancer drugs was examined by performing wipe tests at four hospitals and two community pharmacies. The following commercially available drugs were examined: Xeloda®, TS-1®, and methotrexate tablets and SA-1® and Rheumatrex® capsules. RESULTS: The wipe tests' results revealed that the contamination levels of Xeloda® and TS-1® tablets and SA-1® capsules were within their detection limits. In some facilities, the contamination levels on the press-through-package strips of Rheumatrex® capsules were 3.27 × 10-1, which is close to its detection limit. However, across all facilities, the contamination level of methotrexate tablets was above its detection limit. CONCLUSION: The results of this study suggested that adherence to oral anticancer drugs may not occur during manufacture or transportation. However, it may be due to the presence of pollutants in the facilities. Prevention of pollution in facilities might eliminate the need to wear personal protective equipment during dispensing of oral anticancer drugs.

Salient Features and Outline of the Joint Japanese Guidelines for Safe Handling of Cancer Chemotherapy Drugs.

The purpose of this paper is to introduce the outline and describe the salient features of the "Joint Guidelines for Safe Handling of Cancer Chemotherapy Drugs" (hereinafter, "Guideline"), which were published in July 2015. The purpose of this Guideline is to provide guidance to protect against occupational exposure to hazardous drugs (HDs) to all medical personnel involved in cancer chemotherapy, including physicians, pharmacists, and nurses and home health-care providers. The Guideline was developed according to the Medical Information Network Distribution Service guidance for developing clinical practice guidelines, with reference to five authoritative guidelines used worldwide. PubMed, Cumulative Index to Nursing and Allied Health Literature, Ichushi-Web, and Cochrane Central Register of Controlled Trials were used for a systematic search of the literature. Eight clinical questions (CQs) were eventually established, and the strength of recommendation for each CQ is presented based on 867 references. The salient features of the Guideline are that it was jointly developed by three societies (Japanese Society of Cancer Nursing, Japanese Society of Medical Oncology, and Japanese Society of Pharmaceutical Oncology), contains descriptions including the definition of HDs and the concept of hierarchy of controls, and addresses exposure control measures during handling of chemotherapy drugs. Our future task is to collect additional evidence for the recommended exposure control measures and to assess whether publication of the Guideline has led to adherence of measures to prevent occupational exposure.

Validation of a Liquid Chromatography-Tandem Mass Spectrometric Assay for Quantitative Analysis of Lenvatinib in Human Plasma.

Toward conducting clinical pharmacokinetic studies of an antineoplastic agent, lenvatinib, we developed a liquid chromatography-tandem mass spectrometric assay for its quantitative analysis in human plasma. Analyte (lenvatinib) and internal standard (IS, propranolol) in the plasma were extracted by using acetonitrile and chromatographically separated by using a XTerra MS C18 column with 0.2 mL/min flow and mobile phase starting with 0.1% formic acid in water, followed by increasing percentage of acetonitrile. Detection was performed by using combined reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS-MS) with positive ion electrospray ionization. MS-MS ion transitions used were 427.602>371.000 for lenvatinib and 260.064>116.005 for IS. This study was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification, recovery, and matrix effect according to the Guideline on Bioanalytical Method Validation in Pharmaceutical Development in Japan. Calibration curve was plotted by using lenvatinib concentrations ranging within 9.6-200 ng/mL, and correlation coefficients (r2) were in excess of 0.997. Intra- and interday accuracy ranged within 95.8-108.3% with mean recoveries of 66.8% for lenvatinib, and precision was <6.7% at all quality control concentration levels. Matrix effect analysis showed extraction efficiency of 15.7% for lenvatinib. Collectively, these findings demonstrate the feasibility of this method to evaluate kinetic disposition of lenvatinib.

Autophagy-dependent regulation of tumor metastasis by myeloid cells.

Autophagy is a vital process controlling the lysosomal degradation of cellular organelles and thereby regulating tissue homeostasis in an environment-dependent fashion. Recent studies have unveiled the critical role of tumor cell-derived autophagy in regulating pro-tumor and anti-tumor processes depending on different stages and tumor microenvironments. However, the precise mechanism whereby autophagy regulates tumor progression remains largely unclear. Since myeloid cells contribute to tumor progression and metastasis, we evaluated the role of myeloid cell-specific autophagy in the regulation of tumor progression. We found that the number and size of metastatic lesions were smaller in myeloid cell-specific autophagy-deficient mice. Furthermore, autophagy-mediated regulation of TGF-ß in myeloid cells was associated with the induction of epithelial-mesenchymal transition (EMT), which increases the invasive and metastatic potentials of tumor cells. Myeloid-derived autophagy also plays a critical role in impairing antitumor immune responses and promoting the survival and accumulation of M2 macrophages in tumor tissues in a CSF-1 and TGF-ß-dependent manner. Taken together, our findings elucidate previously unrecognized mechanisms by which myeloid cells promote tumor progression through autophagy-mediated regulation of malignancy and immune tolerance.

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

Plectin is a novel regulator for apical extrusion of RasV12-transformed cells.

Several lines of evidence have revealed that newly emerging transformed cells are often eliminated from the epithelium, though the underlying molecular mechanisms of this cancer preventive phenomenon still remain elusive. In this study, using mammalian cell culture systems we have identified plectin, a versatile cytoskeletal linker protein, as a novel regulator for apical extrusion of RasV12-transformed cells. Plectin is accumulated in RasV12 cells when they are surrounded by normal epithelial cells. Similarly, cytoskeletal proteins tubulin, keratin, and Epithelial Protein Lost In Neoplasm (EPLIN) are also accumulated in the transformed cells surrounded by normal cells. Knockdown or functional disruption of one of these molecules diminishes the accumulation of the others, indicating that the accumulation process of the individual protein mutually depends on each other. Furthermore, plectin-knockdown attenuates caveolin-1 (Cav-1) enrichment and PKA activity in RasV12 cells and profoundly suppresses the apical extrusion. These results indicate that the plectin-microtubules-EPLIN complex positively regulates apical elimination of RasV12-transformed cells from the epithelium in a coordinated fashion. Further development of this study would open a new avenue for cancer preventive medicine.

Novel chemoimmunotherapeutic strategy for hepatocellular carcinoma based on a genome-wide association study.

Pharmacotherapeutic options are limited for hepatocellular carcinoma (HCC). Recently, we identified the anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) gene as a susceptibility gene for hepatitis C virus-induced HCC in a genome-wide association study (GWAS). To prove the concept of HCC immunotherapy based on the results of a GWAS, in the present study, we searched for drugs that could restore MICA expression. A screen of the FDA-approved drug library identified the anti-cancer agent vorinostat as the strongest hit, suggesting histone deacetylase inhibitors (HDACis) as potent candidates. Indeed, the HDACi-induced expression of MICA specific to HCC cells enhanced natural killer (NK) cell-mediated cytotoxicity in co-culture, which was further reinforced by treatment with an inhibitor of MICA sheddase. Similarly augmented anti-tumor activity of NK cells via NK group 2D was observed in vivo. Metabolomics analysis revealed HDACi-mediated alterations in energy supply and stresses for MICA induction and HCC inhibition, providing a mechanism for the chemoimmunotherapeutic actions. These data are indicative of promising strategies for selective HCC innate immunotherapy.

Natural courtship song variation caused by an intronic retroelement in an ion channel gene.

Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

Stability of lenalidomide suspension after preparation by a simple suspension method for enteral tube administration.

PURPOSE: A simple suspension method has been developed for tube administration, in which tablets (and capsules) are disintegrated in hot water (55℃) without grinding (or opening) them. In the present study, we evaluated the feasibility of this simple suspension method for the preparation of lenalidomide (Celgene, Summit, New Jersey and USA) suspension by testing the stability of this drug at 55℃ and its adsorbability on the tube. METHODS: We examined, by high-performance liquid chromatography, the time-dependent changes in the concentration of lenalidomide in suspensions of the drug prepared by the simple suspension method. The high-performance liquid chromatography analyses of lenalidomide were performed on Prominence LC-20AB/SPD-20 A (Shimadzu, Kyoto, Japan) with a ZORBAX SB-C18 RR analytical column (Agilent Technologies, Santa Clara, California, USA; particle size: 2.1 × 100 mm, 3.5 µm) at a flow rate of 0.4 mL/min. A solvent system consisting of 10 mM ammonium acetate (pH 7.0)/acetonitrile was used as the eluent and the eluate was detected by UV at 254 nm. RESULTS: Lenalidomide was confirmed to remain stable in hot water at 55℃ for 24 h in the prepared suspension by the simple suspension method, and more than 99% of the drug could be recovered from the suspension. In addition, 94.5-98.0% of the drug amount could pass through a percutaneous endoscopic gastrostomy tube. Lenalidomide was scarcely adsorbed on to the percutaneous endoscopic gastrostomy tube made of polyurethane or polyvinyl chloride. CONCLUSION: Lenalidomide was found to be stable even in hot water and was not adsorbed on to the percutaneous endoscopic gastrostomy tube.

The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia.

Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.

The Coordinated Actions of TIM-3 on Cancer and Myeloid Cells in the Regulation of Tumorigenicity and Clinical Prognosis in Clear Cell Renal Cell Carcinomas.

Clear cell renal cell carcinoma (ccRCC) is one of most common cancers in urogenital organs. Although recent experimental and clinical studies have shown the immunogenic properties of ccRCC as illustrated by the clinical sensitivities to various immunotherapies, the detailed immunoregulatory machineries governing the tumorigenicity of human ccRCC remain largely obscure. In this study, we demonstrated the clinical significance and functional relevance of T-cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) expressed on tumor cells and myeloid cells in patients with ccRCC. TIM-3 expression was detected on cancer cells and CD204(+) tumor-associated macrophages (TAM), and higher expression level of TIM-3 was positively correlated with shorter progression-free survival (PFS) in patients with ccRCC. We found that TIM-3 expression was detected on a large number of tumors, and there was significant correlation between an increased number of TAMs and high expression level of TIM-3 in patients with ccRCC. Furthermore, TIM-3 rendered RCC cells with the ability to induce resistance to sunitinib and mTOR inhibitors, the standard regimen for patients with ccRCC, as well as stem cell activities. TIM-3 expression was induced on CD14(+) monocytes upon long-term stimulation with RCC cells, and TIM-3-expressing myeloid cells play a critical role in augmenting tumorigenic activities of TIM-3-negative RCC cells. More importantly, treatment with anti-TIM-3 mAb suppressed its tumorigenic effects in in vitro and in vivo settings. These findings indicate the coordinated action of TIM-3 in cancer cells and in myeloid cells regulates the tumorigenicity of human RCC.

Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila.

How do evolved genetic changes alter the nervous system to produce different patterns of behavior? We address this question using Drosophila male courtship behavior, which is innate, stereotyped, and evolves rapidly between species. D. melanogaster male courtship requires the male-specific isoforms of two transcription factors, fruitless and doublesex. These genes underlie genetic switches between female and male behaviors, making them excellent candidate genes for courtship behavior evolution. We tested their role in courtship evolution by transferring the entire locus for each gene from divergent species to D. melanogaster. We found that despite differences in Fru+ and Dsx+ cell numbers in wild-type species, cross-species transgenes rescued D. melanogaster courtship behavior and no species-specific behaviors were conferred. Therefore, fru and dsx are not a significant source of evolutionary variation in courtship behavior.

Cellular and behavioral functions of fruitless isoforms in Drosophila courtship.

BACKGROUND: Male-specific products of the fruitless (fru) gene control the development and function of neuronal circuits that underlie male-specific behaviors in Drosophila, including courtship. Alternative splicing generates at least three distinct Fru isoforms, each containing a different zinc-finger domain. Here, we examine the expression and function of each of these isoforms. RESULTS: We show that most fru(+) cells express all three isoforms, yet each isoform has a distinct function in the elaboration of sexually dimorphic circuitry and behavior. The strongest impairment in courtship behavior is observed in fru(C) mutants, which fail to copulate, lack sine song, and do not generate courtship song in the absence of visual stimuli. Cellular dimorphisms in the fru circuit are dependent on Fru(C) rather than other single Fru isoforms. Removal of Fru(C) from the neuronal classes vAB3 or aSP4 leads to cell-autonomous feminization of arborizations and loss of courtship in the dark. CONCLUSIONS: These data map specific aspects of courtship behavior to the level of single fru isoforms and fru(+) cell types-an important step toward elucidating the chain of causality from gene to circuit to behavior.