Bombykol receptors in the silkworm moth and the fruit fly

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the “empty neuron” system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor.     

Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell

The myelodysplastic syndromes (MDS) comprise a group of premalignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it is well established that many malignancies can be transplanted, there is little evidence to demonstrate that a premalignant disease entity, such as MDS or colonic polyps, can be transplanted and subsequently undergo malignant transformation in vivo. Using mice that express a NUP98-HOXD13 (NHD13) transgene in hematopoietic tissues, we show that a MDS can be transplanted to WT recipients. Recipients of the MDS bone marrow displayed all of the critical features of MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML. Even when transplanted with a 10-fold excess of WT cells, the NHD13 cells outcompeted the WT cells over a 38-week period. Limiting-dilution experiments demonstrated that the frequency of the cell that could transmit the disease was ≈1/6,000–1/16,000 and that the MDS was also transferable to secondary recipients as a premalignant condition. Transformation to AML in primary transplant recipients was generally delayed (46–49 weeks after transplant); however, 6 of 10 secondary transplant recipients developed AML. These findings demonstrate that MDS originates in a transplantable, premalignant, long-term repopulating, MDS-initiating cell.     

A fluorescent reporter of caspase activity for live imaging

There is a growing interest in the mechanisms that control the apoptosis cascade during development and adult life. To investigate the regulatory events that trigger apoptosis in whole tissues, we have devised a genetically encoded caspase sensor that can be detected in live and fixed tissue by standard confocal microscopy. The sensor comprises two fluorophores, mRFP, monomeric red fluorescent protein (mRFP) and enhanced green fluorescent protein (eGFP), that are linked by an efficient and specific caspase-sensitive site. Upon caspase activation, the sensor is cleaved and eGFP translocates to the nucleus, leaving mRFP at membranes. This is detected before other markers of apoptosis, including anti–cleaved caspase 3 immunoreactivity. Moreover, the sensor does not perturb normal developmental apoptosis and is specific, as cleavage does not occur in Drosophila embryos that are unable to activate the apoptotic cascade. Importantly, dying cells can be recognized in live embryos, thus opening the way for in vivo imaging. As expected from the high conservation of caspases, it is also cleaved in dying cells of chick embryos. It is therefore likely to be generally useful to track the spatiotemporal pattern of caspase activity in a variety of species.     

Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV═O species

Iron oxygenases generate elusive transient oxygen species to catalyze substrate oxygenation in a wide range of metabolic processes. Here we resolve the reaction sequence and structures of such intermediates for the archetypal non-heme Fe^II and α-ketoglutarate-dependent dioxygenase TauD. Time-resolved Raman spectra of the initial species with ¹⁶O¹⁸O oxygen unequivocally establish the Fe^IV═O structure. ¹H/²H substitution reveals direct interaction between the oxo group and the C1 proton of substrate taurine. Two new transient species were resolved following Fe^IV═O; one is assigned to the ν_FeO mode of an Fe^III─O(H) species, and a second is likely to arise from the vibration of a metal-coordinated oxygenated product, such as Fe^II─O─C₁ or Fe^II─OOCR. These results provide direct insight into the mechanism of substrate oxygenation and suggest an alternative to the hydroxyl radical rebinding paradigm.     

THE EFFECT OF HOOKWORM INFECTION AND URINARY SCHISTOSOMIASIS ON BLOOD HEMOGLOBIN CONCENTRATION OF SCHOOLCHILDREN LIVING IN NORTHERN MOZAMBIQUE

This study aims to assess the association between schistosomiasis and hookworm infection with hemoglobin levels of schoolchildren in northern Mozambique. Through a cross-sectional survey, 1,015 children from five to 12 years old in the provinces of Nampula, Cabo Delgado and Niassa were studied. Hookworm infection and urinary schistosomiasis were diagnosed, through Ritchie and filtration methods, with a prevalence of 31.3% and 59.1%, respectively. Hemoglobin levels were obtained with a portable photometer (Hemocue^®). The average hemoglobin concentration was 10.8 ± 1.42 g/dL, and 62.1% of the children presented levels below 11.5 g/dL, of which 11.8% of the total number of children had hemoglobin levels below 9 g/dL. A multiple linear regression analysis demonstrated negative interactions between hemoglobin levels and ancylostomiasis, this being restricted to the province of Cabo Delgado (β = -0.55; p < 0.001) where an independent interaction between hemoglobin levels and urinary schistosomiasis was also observed (β = -0.35; p = 0.016). The logistical regression model indicated that hookworm infection represents a predictor of mild (OR = 1.87; 95% CI = 1.17-3.00) and moderate/severe anemia (OR = 2.71; 95% CI = 1.50 - 4.89). We concluded that, in the province of Cabo Delgado, hookworm and Schistosoma haematobium infections negatively influence hemoglobin levels in schoolchildren. Periodical deworming should be considered in the region. Health education and improvements in sanitary infrastructure could achieve long-term and sustainable reductions in soil-transmitted helminthiases and schistosomiasis prevalence rates.     

An ex vivo swine tracheal organ culture for the study of influenza infection

Background  The threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. Ex vivo organ culture (EVOC) systems have been successfully used in the study of both human and animal respiratory pathogens. Objectives  We aimed to develop an air interface EVOC using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. Methods  Tracheal explants were maintained in the air interface EVOC system for 7 days. Histological characteristics were analysed with different staining protocols and co‐ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. Explants were infected with a swine H1N1 influenza virus. Influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real‐time RT‐PCR. Results  Histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. Challenge with swine H1N1 influenza virus showed that the EVOC tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. Conclusion  The air interface EVOC system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection.     

A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway

Mutant larvae for the Drosophila gene lethal giant larva (lgl) develop neoplastic tumors in imaginal discs. However, lgl mutant clones do not form tumors when surrounded by wild-type tissue, suggesting the existence of a tumor-suppressing mechanism. We have investigated the tumorigenic potential of lgl mutant cells by generating wing compartments that are entirely mutant for lgl and also inducing clones of various genetic combinations of lgl⁻ cells. We find that lgl⁻ compartments can grow indefinitely but lgl⁻ clones are eliminated by cell competition. lgl mutant cells may form tumors if they acquire constitutive activity of the Ras pathway (lgl⁻ UAS-ras^V12), which confers proliferation advantage through inhibition of the Hippo pathway. Yet, the majority of lgl⁻ UAS-ras^V12 clones are eliminated in spite of their high proliferation rate. The formation of a tumor requires in addition the formation of a microenvironment that allows mutant cells to evade cell competition.     

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate “MitoMax,” a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.Specialized biological processes are carried out in specific organelles and subcellular compartments. For example, mitochondria are the site of oxidative respiration, neurons pass electrical or chemical signals to others through synapses, and apical and basolateral domains of epithelial cells are critical for their polarized functions. Understanding how these structures underlie specialized functions requires the comprehensive identification of proteins within spatially defined cellular domains.A common strategy to study the localization of a particular protein is to generate green fluorescent protein (GFP) fusion proteins. However, it is time-consuming and labor-intensive to investigate protein localization at a large scale using GFP tagging, especially in vivo. Therefore, highly sensitive mass spectrometry (MS) approaches have been developed to systematically characterize the proteome of subcellular compartments. However, using MS approaches to characterize the proteome of subcellular domains has been limited by purification methods and is commonly associated with numerous false positives and false negatives due to contamination and loss of components during purification, respectively. For example, mitochondria are composed of an outer membrane and an inner membrane, generating two subcompartmental regions: the intermembrane space and the matrix located within the inner membrane. Because the ultrastructure of mitochondria is often disrupted during isolation processes, the isolation of specific subcompartmental regions of mitochondria is prone to contamination.Recently, a method based on an engineered ascorbate peroxidase (APEX) has been developed and shown to function in cultured mammalian cells for proteomic mapping (1). Upon activation, the APEX enzyme turns a biotin-phenol substrate into a highly reactive radical that covalently tags neighboring proteins on electron-rich amino acids such as tyrosine. Biotinylated endogenous proteins can then be isolated and identified by MS. Thus, APEX labeling can be applied to bypass organelle purification steps, offering an alternative approach for systematic proteomic characterization in live cells. Here we report that the approach can be applied to characterize the subcellular proteome in live tissues and map the mitochondrial matrix proteome of Drosophila muscle. In addition to characterizing a number of uncharacterized putative mitochondrial proteins, we establish MitoMax, a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation.     

Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains,facilitating B cell activation

In a previous study, we demonstrated that β1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc₃Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5^−/−) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc₃Cer synthase activity was detected in the tissues of these mice. These results demonstrate that β3GnT5 is the sole enzyme synthesizing Lc₃Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5^−/− B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5^−/− resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5^−/− B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5^−/− B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5^−/− B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.     

Characterization of an electron conduit between bacteria and the extracellular environment

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.     

Chemotherapy-Induced Cardiotoxicity: Subclinical Cardiac Dysfunction Evidence Using Speckle Tracking Echocardiography

Objectives:

In our study, we aimed to identify early markers of cardiac dysfunction in patients treated with mitoxantrone. We also looked at cardiac functional changes during therapy by analyzing longitudinal deformation and by measuring left ventricular (LV) and left atrial (LA) global strain.

Materials and Methods:

LA and LV global longitudinal strain were analyzed in 20 patients affected by multiple sclerosis and treated with mitoxantrone. Patients underwent echocardiography before treatment, after every drug administration during the 12-months treatment period, and finally after 6 and 12 months of drug discontinuation.

Results:

Compared with baseline values, patients showed a significant reduction of both LA and LV longitudinal global strain at the end of treatment with mitoxantrone (LA_GS% T10 vs. T0 values: 15,2 ± 12,5 vs. 20,2 ± 11,1; LV_GS%: ─16,4 ± 2,5 vs. ─17,4 ± 3,8). Strain reduction reverted after treatment discontinuation (LA_GS% FU vs. T0 values: 20,4 ± 15,7 vs. 20,2 ± 11,1; LV_GS%: ─17,3 ± 3,3 vs. ─17,4 ± 3,8).

Conclusions:

Impairment of longitudinal deformation during mitoxantrone therapy may indicate a dysfunction related to early myocardial damage. These findings appear to be reversible after treatment discontinuation.     

High-throughput analysis of yeast replicative aging using a microfluidic system

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.Aging and age-associated diseases are becoming the fastest-growing area of epidemiology in most developed countries (1–4). Identification of molecular mechanisms that lead to the development of interventions to delay the onset of age-associated diseases could have tremendous global impacts on public health (5). The budding yeast Saccharomyces cerevisiae was the first eukaryotic genome to be sequenced, and has been instrumental in discovering molecular pathways involved in all aspects of eukaryotic cells (6–9). S. cerevisiae is an important model for discovering evolutionarily conserved enzymes that regulate aging, such as Sir2 and Tor1 (10).Yeast replicative lifespan (RLS) is determined by manually separating the daughter cells from a mother cell on a Petri dish with a microscope-mounted glass needle, and counting the number of divisions throughout the life of the cell. Tens or hundreds of cells per strain have to be dissected and counted to determine whether the lifespans of two strains are statistically different (11–14). This method has not changed appreciably since the initial discovery of yeast replicative aging in 1959 (15). A well-trained yeast dissector can monitor and handle no more than 300 cells at once, and a typical lifespan experiment usually thus lasts ∼4 wk. Most lifespan experiments include an overnight 4 °C incubation everyday throughout the experiment for practical purposes, adding another factor that can complicate data interpretation. This tedious and low-throughput procedure has substantially hindered progress. Therefore, new strategies are required to take advantage of the power of yeast genetics and apply high-throughput unbiased genetic screen approaches to yeast aging research.Microfluidic devices have been developed to capture yeast cells for high-resolution imaging analysis during vegetative growth (16–20). Recently, such devices have been designed that enable the tracking of yeast cells throughout their lifespan, making it possible to record and study cellular phenotypic changes during aging (21–23). However, many issues prevent the use of microfluidic devices in a high-throughput manner for lifespan screens. First, although the time required to monitor the entire lifespan of the yeast cell has been dramatically reduced, the throughput is limited to 1–4 channels per device (21–23). Second, mother cells were immobilized underneath soft elastomer [polydimethylsiloxane (PDMS)] micropads (21, 22). Although several hundred trapping micropads can be assembled for each microfluidic channel, this trap design suffers from a low retention rate of ∼30% by the end of the lifespan; this seriously limits the number of usable cells in the lifespan calculation to ∼100, which restricts statistical significance of the lifespan analysis. Third, the ability for trapping micropads to retain old cells depends on the larger size of old cells compared with young cells (21, 22). However, old cells often generate large daughter cells that also become trapped by the micropads. Fourth, the micropad design often allows more than one cell to be trapped; multiple cells can be trapped underneath one micropad, whereas no cells are trapped under others. Finally, in one of the designs, cell-surface labeling and chemical modification of the device are required, which has proven to be technically challenging for fabrication and to introduce adverse effects on replicative lifespan (23).Here, we present a microfluidic platform called high-throughput yeast aging analysis chip (HYAA-Chip), which solves all of the described challenges and limitations. This innovative design can trap up to 8,000 individual yeast cells in cup-shaped PDMS structures evenly distributed to 16 discrete channels; captured cells are cultivated and aged as fresh medium continuously flows through, which removes newly budded daughter cells. The HYAA-Chip provides automated whole-lifespan tracking with fine spatiotemporal resolution and large-scale data quantification of single yeast cell aging by combining simple fabricated microfluidics with high-resolution time-lapse microscopy. The HYAA-Chip is label-free, independent of size differences between mother and daughter cells, has up to 96% single-cell trapping efficiency, and up to 92% retention rate for the initially trapped mother cells.     

Role of the translocation partner in protection against AID-dependent chromosomal translocations

Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt’s lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCδ are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igβ, both of which are somatically mutated by AID. Igβ/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igβ/IgH translocations. Instead, B cells are protected against Igβ/IgH translocations by a BAFF- and PKCδ-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.     

Induction and rescue of Nod2-dependent Th1-driven granulomatous inflammation of the ileum

Mutations in the NOD2 gene are strong genetic risk factors for ileal Crohn’s disease. However, the mechanism by which these mutations predispose to intestinal inflammation remains a subject of controversy. We report that Nod2-deficient mice inoculated with Helicobacter hepaticus, an opportunistic pathogenic bacterium, developed granulomatous inflammation of the ileum, characterized by an increased expression of Th1-related genes and inflammatory cytokines. The Peyer’s patches and mesenteric lymph nodes were markedly enlarged with expansion of IFN-γ–producing CD4 and CD8 T cells. Rip2-deficient mice exhibited a similar phenotype, suggesting that Nod2 function likely depends on the Rip2 kinase in this model. Transferring wild-type bone marrow cells into irradiated Nod2-deficient mice did not rescue the phenotype. However, restoring crypt antimicrobial function of Nod2-deficient mice by transgenic expression of α-defensin in Paneth cells rescued the Th1 inflammatory phenotype. Therefore, through the regulation of intestinal microbes, Nod2 function in nonhematopoietic cells of the small intestinal crypts is critical for protecting mice from a Th1-driven granulomatous inflammation in the ileum. The model may provide insight into Nod2 function relevant to inflammation of ileal Crohn’s disease.     

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.     

Distinct signaling of Drosophila chemoreceptors in olfactory sensory neurons

In Drosophila, olfactory sensory neurons (OSNs) rely primarily on two types of chemoreceptors, odorant receptors (Ors) and ionotropic receptors (Irs), to convert odor stimuli into neural activity. The cellular signaling of these receptors in their native OSNs remains unclear because of the difficulty of obtaining intracellular recordings from Drosophila OSNs. Here, we developed an antennal preparation that enabled the first recordings (to our knowledge) from targeted Drosophila OSNs through a patch-clamp technique. We found that brief odor pulses triggered graded inward receptor currents with distinct response kinetics and current–voltage relationships between Or- and Ir-driven responses. When stimulated with long-step odors, the receptor current of Ir-expressing OSNs did not adapt. In contrast, Or-expressing OSNs showed a strong Ca²⁺-dependent adaptation. The adaptation-induced changes in odor sensitivity obeyed the Weber–Fechner relation; however, surprisingly, the incremental sensitivity was reduced at low odor backgrounds but increased at high odor backgrounds. Our model for odor adaptation revealed two opposing effects of adaptation, desensitization and prevention of saturation, in dynamically adjusting odor sensitivity and extending the sensory operating range.From insects to mammals, the sense of smell begins with odor detection by olfactory sensory neurons (OSNs) (1–6). Recently, rapid advances have been made in understanding chemoreceptors in Drosophila OSNs (7–9). To date, Drosophila is the only model organism for which odor selectivity is known for most of its odorant receptors (Ors) (10, 11), and an Or expression pattern has been mapped to OSNs (12, 13). In addition, another family of chemoreceptors called ionotropic receptors (Irs) has been identified and characterized (14–16). These two types of chemoreceptors respond to different odors, thus endowing Drosophila OSNs with unique and complementary properties for odor detection (17). In contrast to the advanced molecular understanding of these two types of chemoreceptors, the mechanisms of their cellular signaling in native OSNs remain unclear, particularly hampered by the technical difficulty of carrying out patch-clamp recordings of Drosophila OSNs.Drosophila OSNs are encased in hair-like sensilla in the antennae and maxillary palps, with each sensillum containing the dendrites of one to four OSNs that are wrapped by sheath cells (18). The responses of native Drosophila OSNs to odors have traditionally been measured by electroantennogram (EAG) (19), which extracellularly measures the potentials across the entire antenna. In addition, single-sensillum recording (SSR) was developed to provide a higher spatial resolution by measuring the local field potentials (LFPs) from a single sensillum (20–24). These methods, especially SSR, have greatly advanced understanding of the odor selectivity of both Ors and Irs (10, 11, 14). However, because sheath cells and other OSNs also contribute to EAG and SSR signals (25), the response characteristics obtained by such measurements are often contaminated. Patch-clamp recordings of single OSNs could ideally overcome this issue while facilitating the experimental manipulations of a cell’s membrane potential; however, this standard method has unfortunately not yet been routinely applied to Drosophila OSNs.Here, we developed a Drosophila antennal preparation and succeeded in performing patch-clamp recordings of single identified OSNs. By using a fast solution change system to deliver liquid-phase odor stimuli, we investigated the response properties of odor-induced receptor currents of Drosophila OSNs. We found that OSNs expressing Ors exhibited slow response kinetics, outward receptor current rectification, and strong adaptation to odors. We further demonstrated that this adaptation was produced by a Ca²⁺ influx into OSNs because it could be eliminated by voltage clamping at positive holding potentials, by removing extracellular Ca²⁺, or by removing internal free Ca²⁺ with a Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Importantly, in contrast to the long-held view that adaptation simply increases sensitivity, we found that Or-mediated adaptation selectively reduced odor-signaling gain at low odor backgrounds but increased the gain at high odor backgrounds, thereby extending the dynamic odor-operating range. In contrast, odor-induced receptor currents in Ir-expressing OSNs showed fast response kinetics and, surprisingly, did not adapt.     

Neisseria infection of rhesus macaques as a model to study colonization,transmission, persistence,and horizontal gene transfer

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe–host interactions. We developed a rhesus macaque model for studying Neisseria–host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.