Use of adjuvant chemotherapy remains a complex decision in the treatment of early stage non-small cell lung cancer (NSCLC), with risk of recurrence being the primary indicator . However, although several clinical and pathological factors are typically considered when assessing the risk of recurrence, none are significantly associated with clinical outcome with the exception of tumor size. GeneFx® Lung  is a multi-gene RNA expression signature that classifies early stage NSCLC patients as high-risk or low-risk for disease recurrence. GeneFx Lung risk category has been shown to be significantly associated with overall survival in several independent clinical studies. The published literature regarding the analytical validity, clinical validity and clinical utility of GeneFx Lung is summarized herein. Despite successful surgery, 50-70% of early stage (stages I and II) non-small cell lung cancer (NSCLC) patients die within 5 years.GeneFx® Lung is a 15 gene signature that can predict risk of recurrence in early stage NSCLC, independent of other, relevant clinicopathologic factors. RNA is isolated from NSCLC tissue, containing a minimum of 20% tumor which has been preserved in RNAlater® (an RNA stabilization reagent) or by being ‘fresh frozen’, using a phenol-chloroform method. Extracted RNA is reverse transcribed into cDNA and amplified using single primer isothermal amplification (SPIA) chemistry. The amplified cDNA is chemically and enzymatically fragmented and labelled with biotin. The cDNA targets are hybridized to an Affymetrix Human Genome U133 Plus 2.0 microarray, followed by a wash and stain procedure that binds streptavidin-phycoerythrin (SAPE) stain to the biotinylated cDNA molecules. Scanning identifies the number of cDNA transcripts that are present. The resulting data are processed using a proprietary algorithm to generate a risk score which is dichotomized into “high-risk” (risk score ≥ -0.10) or “low-risk” (risk score ˂ -0.10) categories. Patients whose tumors are categorized as high-risk should be considered, in concert with other clinical factors, for treatment with adjuvant chemotherapy.GeneFx Lung distinguishes itself from other prognostic gene expression signatures for early stage NSCLC in a number of ways:GeneFx Lung is applicable to the two major histological subtypes of NSCLC, adenocarcinoma and squamous cell carcinoma.GeneFx Lung results are dichotomized into 2 categories , making results interpretation intuitive.GeneFx Lung is clinically validated across several independent patient cohorts.GeneFx Lung is clinically validated on multiple technology platforms .,GeneFx Lung shows promise as a predictive signature for response to adjuvant chemotherapy.,,,,Lung cancer is the leading cause of cancer death. In 2014, it is estimated that there will be more than 225,000 new lung cancer cases in North America, 85% of which are NSCLC.Recommendations by independent groupsThe Wadsworth Center of the State of New York performed an independent review of the technology, standard operating procedures, quality measures, and analytical and clinical validation results of GeneFx Lung, resulting in full approval and licensure in the state of New York. GeneFx Lung testing is performed exclusively in the Helomics™ Corporation laboratory in Pittsburgh, Pennsylvania, which is certified to comply with the Centers for Medicare & Medicaid Services Clinical Laboratory Improvement Amendments (CLIA) program. The Helomics laboratory is licensed by CLIA, with independent licenses in the states of New York, California, Florida, Maryland, Pennsylvania and Rhode Island.Guidelines by professional groupsThe American Society of Clinical Oncology (ASCO) and The Cancer Care Ontario Program in Evidence-Based Care (CCO) jointly reviewed and provided recommendations regarding the role of adjuvant chemotherapy and radiation therapy in the treatment of patients with early stage NSCLC.Independent review articlesIn 2009, Zhu et al. reviewed the concepts and methodologies involved in identifying, developing and validating multi-gene signatures in lung cancer. Analytical ValidityThe precision, sensitivity and specificity of GeneFx Lung were recently evaluated, confirming the robust and reliable nature of the test.The accuracy of GeneFx Lung was evaluated in a study of 34 NSCLC samples in which biological replicates were assayed in the commercial laboratory  as well as an accredited reference laboratory .Although originally developed and validated using fresh frozen tissue, GeneFx Lung has been shown to perform equivalently in tissue preserved in RNAlater.GeneFx Lung maintains performance across various gene expression technology platforms. Although developed and validated on the Affymetrix U133 microarray platform, GeneFx Lung maintained statistical significance when using the Agilent 44K platform  as well as RT-qPCR .Clinical ValidityIn a prospective evaluation of 181 untreated early stage NSCLC patients, GeneFx Lung-designated risk of recurrence (high-risk vs. low-risk) was significantly correlated with overall survival, with a multivariate HR of 1.95 . The prognostic ability of GeneFx Lung was corroborated in subgroup analysis for stage I  and stage IA  patients, with 92% of low-risk, but only 61% of high-risk, stage IA patients achieving survival at 5 years. Furthermore, the signature’s prognostic ability is independent of histology, with utility in both adenocarcinoma and squamous cell carcinoma cases.Independent, published microarray datasets were used to validate the prognostic property of GeneFx Lung, which had been derived from 62 untreated early stage NSCLC patients in the JBR.10 clinical trial. GeneFx Lung was shown to be a significant prognostic factor  in three datasets totaling 308 early stage NSCLC patients, with HRs of 2.26, 2.27 and 3.57, spanning both adenocarcinoma and squamous cell carcinoma, as well as other less common histological subtypes.Clinical Utility and Other Supportive Studies,Initial studies have suggested that the GeneFx Lung signature may also be predictive of improved overall survival in early stage NSCLC patients treated with adjuvant chemotherapy. In two separate datasets, patients designated as high-risk by GeneFx Lung and treated with adjuvant chemotherapy experienced significantly improved overall survival compared to those left untreated.GeneFx Lung has been shown to perform equivalently in both fresh frozen and RNAlater-preserved tissue formats, however, performance in formalin-fixed paraffin embedded (FFPE) tissue has yet to be explored. FFPE represents a more stable and clinically accessible tissue format for clinical studies as well as commercial use.Clinical validations performed to date clearly indicate the prognostic significance of GeneFx Lung in designating the risk of recurrence in early stage NSCLC tumors. Because risk of recurrence may play a large role in making treatment decisions, the predictive significance of the signature should also be considered. Exploratory analyses have been promising, and additional, independent validations of GeneFx Lung as a predictive marker are warranted and should consider use of tumor recurrence-based endpoints .Although numerous factors are considered when staging NSCLC, there remains a clinical unmet need to delineate risk of recurrence in early stage patients as stage alone does not fully elucidate which patients may benefit from adjuvant chemotherapy. Clinicopathological factors currently employed are not significantly associated with treatment effectiveness, with the exception of tumor size. GeneFx Lung is a robust gene signature that has been validated in several independent cohorts to estimate risk of recurrence in early stage NSCLC, with overall survival being significantly associated with GeneFx Lung risk category. The studies reviewed herein support use of GeneFx Lung to assess risk of recurrence in both adenocarcinoma and squamous cell carcinoma cases of early stage NSCLC, thereby facilitating adjuvant chemotherapy treatment decisions in these patients.SB and AU are paid employees of Helomics Corporation and hold stock options. These competing interests do not affect our adherence to the PLOS Currents policies on sharing data and materials.


It is thought that Tregs utilize the cytotoxic proteins perforin and granzyme to directly lyse target immune cells. While this might be a major effector in preventing the anti-tumor response, little has been studied on the importance this pathway is in the context of glioma.Glioblastoma multiforme (GBM), is the most common adult brain tumor and despite many new therapies, remains largely incurable. Regulatory T cells (Treg) are highly immunosuppressive cells that accumulate within glioma microenvironment, and reduction of their function or trafficking has been previously shown to enhance survival in pre-clinical models of GBM. One of the principal mechanisms of Treg suppression An agonistic antibody against GITR (glucocorticoid induced TNF family receptor) has been demonstrated to negatively influence Tregs in a number of animal models. However, the role of this therapy in GBM has not yet been explored. We hypothesized that anti-GITR treatment suppresses granzyme expression by Tregs, which results in stronger anti-tumor immune responses against glioma.5 cells were stereotactically injected in the right hemisphere of 6-8 week old mice. Mice received anti-GITR or its isotype control directly into the tumor site (2ug) or i.p. (500ug) at both 9 and 14 days post implantation Mice were separately assessed for survival, and flow cytometric analyses were performed to determine the influence of the antibody on immune response in the brain.Using the GL261 murine model of GBM, 4 × 10+ Tregs with peripheral treatment. However, with intratumoral treatment, we saw both a decrease in granzyme expression and Treg number.Analysis revealed that many Tregs specifically within tumor tissue express granzyme B, which increases over time. Animals treated with anti-GITR antibody caused a significant increase in animal survival only when injected directly into the tumor site, while peripheral treatment only had a modest effect. Antibody treatment caused a significant decrease in granzyme BOur data suggests that within the glioma microenvironment, Tregs gain the ability to produce granzyme B, which can be suppressed by administration of anti-GITR antibody treatment, but this mechanism is not alone sufficient to enhance animal survival. However, intratumoral anti-GITR treatment dramatically reduced Tregs and their granzyme B expression, suggesting another mechanism in this therapeutic modality. This study shows that the site of anti-GITR administration could be critical in its efficacy when taken into the clinic.


AgRP-IRES-Cre mice. In free-feeding mice, we observed a fivefold increase in AgRP neuron firing with mounting caloric deficit in afternoon vs morning recordings. In food-restricted mice, as food became available, AgRP neuron firing dropped, yet remained elevated as compared to firing in sated mice. The rapid drop in spiking activity of AgRP neurons at meal onset may reflect a termination of the drive to find food, while residual, persistent spiking may reflect a sustained drive to consume food. Moreover, nearby neurons inhibited by AgRP neuron photostimulation, likely including satiety-promoting pro-opiomelanocortin (POMC) neurons, demonstrated opposite changes in spiking. Finally, firing of ARC neurons was also rapidly modulated within seconds of individual licks for liquid food. These findings suggest novel roles for antagonistic AgRP and POMC neurons in the regulation of feeding behaviors across multiple timescales.Agouti-related-peptide (AgRP) neurons—interoceptive neurons in the arcuate nucleus of the hypothalamus (ARC)—are both necessary and sufficient for driving feeding behavior. To better understand the functional roles of AgRP neurons, we performed optetrode electrophysiological recordings from AgRP neurons in awake, behaving DOI:http://dx.doi.org/10.7554/eLife.07122.001 Appetite is controlled in part by the opposing actions of the ‘hunger hormone’  and the ‘satiety hormone’ . Ghrelin is released by the stomach when empty and stimulates appetite, whereas leptin is released by fat stores and induces feelings of fullness. Both hormones travel via the bloodstream and are detected by a region of the brain called the hypothalamus.Ghrelin and leptin act specifically on a group of cells in the hypothalamus that contains at least two major cell types: AgRP neurons and POMC neurons. Electrode recordings from slices of mouse brain show that AgRP neurons fire more rapidly at night—when mice normally feed—than during the day, whereas POMC neurons do the opposite. This suggests that the activity of AgRP neurons drives food-seeking behavior, whereas POMC firing inhibits it. However, the absence of circulating hormones such as leptin and ghrelin in brain slices makes it difficult to draw firm conclusions about the role of these cells in controlling appetite.Mandelblat-Cerf, Ramesh, Burgess et al. have addressed this issue by performing the first recordings of spiking activity in individual AgRP neurons and other cells that are likely to be POMC neurons in awake mice. Consistent with the results of slice experiments, the firing rate of AgRP neurons increased steadily over the course of the day, suggesting that their activity signals an increasing need for food. Furthermore, as soon as food became available, the firing rate of the AgRP neurons suddenly dropped—even though the animals' energy reserves would still have been low. These results are consistent with the findings of two recent studies reported earlier this year that used different methods to indirectly measure neuronal activity in awake mice. Notably, even after the drop in activity, the firing rates of AgRP neurons remained above those recorded in fully sated mice—which possibly reflects the fact that the animals' energy reserves were still low. The putative POMC neurons generally showed opposite effects to the AgRP neurons.The results of these electrode recordings in awake mice thus suggest that AgRP and POMC neurons together maintain a drive to seek out food sources as energy reserves fall, and to refrain from doing so when energy reserves are plentiful. Moreover, the seemingly paradoxical drop in AgRP firing and increase in POMC firing upon receiving food may act as a signal to temporarily stop searching for food, so that feeding itself can begin. Alternatively, since the release of satiety hormones after eating a meal is slow, these rapid changes in firing may provide more immediate feedback to the neuronal circuits that regulate the drives to seek and consume food.DOI:http://dx.doi.org/10.7554/eLife.07122.002 The homeostatic drive to feed is at least partially driven by agouti-related-peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARC). These neurons have privileged access to slow hormonal signals of energy balance, such as ghrelin and leptin , and alsPrevious attempts to directly record spiking activity of AgRP neurons have been restricted to in vitro approaches, due to the technical challenges of extracellular electrophysiological recordings in the ARC in living animals, and the fact that AgRP neurons are intermingled with pro-opiomelanocortin (POMC) neurons with opposing effects on food intake . Consistinh neurons). Because the only ARC neurons currently known to be inhibited by AgRP neurons are POMC neurons . Because AgRP neurons are densely packed in the ARC  in the ARC of  the ARC , we used the ARC  from neu the ARC . We veriinh neurons’) by laser photostimulation . All recordings were performed in mice habituated to head restraint , as this enabled recordings with greater stability from a larger number of electrodes.We separated ARC neurons into classes by matching spikes with near-identical waveforms obtained during ongoing activity and periods of photostimulation and identifying neurons that were either driven  or suppressed  show the opposite trend, with short ISIs occurring more often in the morning than in the afternoon (p = 0.13). Taken together, these in vivo increases in AgRP firing and decreases in ARCinh firing from morning to afternoon recordings are consistent with the homeostatic roles proposed for AgRP and POMC neurons in feeding behavior . As described in h neuron .10.7554/inh neuron firing, were also observed at the population level. We visualized firing changes at the population level as follows: because neurons in our sample exhibited a range of baseline firing rates and firing rate variability  in three windows of time: (i) between 0–5 min post-feeding onset, a time that likely precedes most nutrient absorption and counter-regulatory visceral and hormonal changes e.g., , (ii) beinh neurons demonstrated opposite changes during this early pre-feeding period, as 66% (8/12) of ARCinh neurons showed a significant increase in firing, while only 17% (2/12) showed a significant decrease , mean firing rates were significantly different than baseline in 82% (40/49) of all ARC neurons . In partinh neurons, generally persisted across later feeding periods . Indeed, almost half of all ARC neurons recorded  showed significant changes in mean firing in the 5-min period following lickspout placement, but prior to feeding. As with post-feeding changes, 32% (7/22) of AgRP neurons showed a significant ‘anticipatory’ drop in firing, while only 5% (1/22) showed a significant increase. By contrast, 50% (6/12) of ARCinh neurons showed significant anticipatory increases in firing, while none showed a significant drop. Similar effects were evident in the auROC change index, plotted in inh neurons showed the opposite effect. These findings suggest that AgRP neurons decrease their firing upon identification of an upcoming source of food, either to decrease the drive to continue the search for food (see ‘Discussion’), or in anticipation of future meal-induced restoration of caloric deficit.We next assessed the possibility of even earlier changes in firing, immediately after presentation of the food-predicting cue (lickspout placement near the snout) but prior to onset of feeding. As shown in While we ensured that neurons recorded across daily sessions were distinct , in two cases, we did record from what appeared to be the same AgRP neuron in back-to-back sessions  is clearly evident in the example neuron in inh neurons. These findings remained intact when considering only epochs with or without lick events . These data further support the notion that additional circuits downstream of AgRP neurons may prevent additional feeding, even in contexts where AgRP neurons continue to fire at intermediate spike rates. Moreover, these data suggest that the rapid decrease in AgRP firing, associated with food cues and meal-initiation  as compared to low levels of spiking observed in AgRP neurons recorded from ad libitum-fed mice in the early stages of the light cycle  and to feeding onset, and that such decreases were sustained at later periods . In contrast to AgRP neurons, ARC signals . HoweverIn the case of AgRP neurons, initial evidence of non-homeostatic influences was suggested based on observations of a decrease in AgRP neuron c-Fos expression 2 hr after consumption of a calorie-free meal , though reductions in firing of vasopressin-secreting supraoptic neurons during drinking of water  must penetrate approximately 5.5 mm into the brain to gain access to the relatively small ARC nucleus, likely resulting in lower yield. Similar to previous optetrode studies e.g., , some acentation , we onlyentation . Alternaentation  and singentation  provide entation  across mIn summary, our recordings of spiking activity from identified classes of ARC neurons in awake mice provide direct support for previous hypotheses, based on in vitro recordings and in vivo manipulations, of slow, opposite changes in AgRP and POMC neuron spiking across hours, during slow changes in energy balance. In addition, our data suggest that AgRP and POMC neurons can be modulated on timescales inconsistent with a purely homeostatic role in feeding. In particular, the rapid drop in spiking activity of AgRP neurons at meal onset may reflect a termination of the drive to seek sources of food, while residual, persistent spiking in these neurons may reflect a sustained drive to consume food.Agrp gene  into the arcuate nucleus of the hypothalamus . 3 weeks after viral injection, mice were prepared for awake, head-fixed electrophysiology recordings by surgical implantation of a head post and an optetrode microdrive  and placed into a stereotaxic apparatus  on a heating pad (CWE). Ophthalmic ointment (Vetropolycin) was applied to the eyes. Using procedures identical to those described previously (To selectively express channelrhodopsin-2 (ChR2) in AgRP neurons, we injected eviously , a two-pFollowing recovery from surgery, mice were habituated to tolerate 1–2 hr of head restraint . Note that physiology studies in mice and primates commonly employ head-restraint, which enables more precise control and monitoring of feeding behavior and other behavioral and neurophysiological parameters . It was ad-libitum feeding weight. In a typical session, recordings began with spontaneous activity for ∼5 min followed by a laser stimulation protocol (∼5–10 min). We then recorded spontaneous activity as long as recordings were stable. To minimize stress, head-fixation of habituated mice was restricted to <3 hr. Therefore, we restricted our daily recordings to either morning or afternoon, and advanced the electrodes between recordings to ensure that different neurons were recorded in each daily session.A cohort of 9 mice, used to investigate diurnal rhythms of ARC neurons , had fread-libitum weight . Mice were trained to consume a high-calorie liquid meal replacement (Ensure) from a lickspout, while head-fixed on a spherical treadmill. Licking was detected via disruption of an infrared beam positioned in front of the lickspout. Upon detection of a lick, a 10 µl drop of Ensure was released using a solenoid and MonkeyLogic software , though it is likely that such cues nevertheless induced ‘predictive’ changes in firing.A cohort of 5 mice was used to investigate whether optogenetic activation of AgRP neurons induced food consumption under head-fixed conditions . Mice weWe recorded extracellularly from multiple neurons simultaneously using a custom-built, 200-μm diameter optic fiber-coupled microdrive (an ‘optetrode’) with between four and eight manually constructed tetrodes (comprising of 4 twisted strands of electrode wire) attached to the sides of the fiber . In the Waveform spikes were then sorted offline using Neuralynx spike sorting software (SpikeSort) as follows: first, each spike waveform consisted of a 1-ms window surrounding its peak amplitude. Second, for each spike, we defined two features, amplitude of the peak and amplitude of the valley, for each of the four electrodes within a tetrode . Clusters were then defined according to these feature distributions, manually selecting the dimensions that best separated different clusters. We used several criteria to include a neuron in our data set. First, we inspected the ISI distribution. A histogram of the ISI distribution for the spikes within each cluster is expected to show a refractory period, that is, a dearth of spikes that occur within milliseconds of each other . Therefo2), with 3 s between pulse trains . The laser beam was passed through a Pockels cell to ensure accurate control of laser pulse shape (<0.2 ms timing accuracy) and amplitude . To ensure that spontaneous and light-evoked waveforms originated from the same cell, we validated that the correlation coefficients of the cross-correlations between spontaneous and light-evoked waveforms were above 0.95 and were not significantly different than the correlation between pairs of spontaneous spike waveforms , the initial trial of a 1-s laser pulse train in the series of 1-s trains could lead to a sharp increase or decrease in firing that did not return to pre-photostimulation baseline until 0–2 min after the end of the final laser photostimulation trial. We reasoned that these effects were clearly laser-evoked (two-sample Kolmogorov–Smirnov (KS) test, p < 0.05), and thus, we also used this information in our classification. This additional criterion only changed the cell classification in 7% (2/33) of AgRP neurons and 12% (3/25) of ARCinh neurons and did not affect the main conclusions of the study. While histology showed reasonably high penetrance of ChR2 expression in AgRP neurons, the ARCother category may include a small subset of AgRP neurons lacking sufficient or any ChR2 expression, or that an insufficient intensity of light reached the tetrode on which the unit was recorded. Note that ARCother and ARCinh neurons were only included from recordings during or subsequent to identification of a putative AgRP neuron, to ensure that no neurons from regions dorsal to the ARC were included.To determine whether a neuron showed a significant light-evoked response, we used a paired sample t-test comparing firing rates in the 2 s prior to a 20-Hz pulse train with the first half or the second half of the pulse train . This method was chosen because some clearly driven neurons showed more pronounced excitation after a delay of several 100's of milliseconds  of each ISI (To quantify the changes in firing rate (estimated in 5-s bins) during the feeding paradigm, auROC timecourses  were calinh, and ARCother neurons modulated by placement of the lickspout or feeding, we compared the distribution of firing rates (estimated in 5-s bins) before lickspout placement (up to 5 min prior to lickspout placement) with the distribution of firing rates post-lickspout placement  average firing rate histograms is that it assesses the impact of each lick or lick bout, irrespective of the occurrence of other prior or future licks or bouts. The analysis determines the linear ‘kernel’ —a set of coefficients that adjust the firing rate up or down at each moment in time relative to the occurrence of each individual lick or lick bout. If the relationship is indeed linear, then one should be able to perfectly predict the moment-by-moment firing of the neuron as the sum of the following three terms: (i) a fixed constant firing rate (in spikes/s), (ii) the convolution of the ‘lick’ kernel , an electrolytic lesion was made 400 µm above the final recording location by passing a mild current between two electrodes (25 mA for 30 s). Mice were given an overdose of tribromoethanol, perfused with 10% formalin, and brains were cut in 50-µm coronal sections. Sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Recording sites, identified by the presence of the fiber tract and/or electrolytic lesion, were all verified to be among ChR2-mCherry-expressing AgRP neurons.


Pulmonary contusion and acute respiratory distress syndrome (ARDS) is a common manifestation in polytraumatic patients. Although mechanical ventilation is still the first choice of treatment, a group of patients are still unable to maintain their oxygenation. The role of extracorporeal membrane oxygenation (ECMO) has been more clarified when the lung is extensively damaged and when conventional modality failed. ECMO provides the lung an opportunity to rest by permitting reduced ventilator settings and limiting further barotraumas. However, ECMO is still considered contraindicated in polytramatic patients combining pulmonary contusion and other organ hemorrhage because of systemic anticoagulation during the treatment. We herein report a patient who successfully survive a multitrauma combining pulmonary contusion and grade IV liver laceration using non-heparinized venovenous extracorporeal membrane oxygenation (vv-ECMO). The associated literature were reviewed. Acute pulmonary failure is a common manifestation in polytraumatic patients. The mechanism included pulmonary contusion, acute respiratory distress syndrome (ARDS) resulting from any inflammation process such as aspiration pneumonia, or fat embolism because of long bone fracture . The manExtracorporeal membrane oxygenation (ECMO) serves as the final method when conventional mechanical ventilation fails to maintain the oxygenation. It helps maintain systemic tissue oxygenation via extracorporeal circuit when the lung function is compromised ,3. HowevThe introduction of heparin-free ECMO seems to be the possible solution for such dilemma. We report our experience of using heparin-free vv-ECMO to help a patient survive a trauma combining acute pulmonary failure and severe liver subcapsular laceration. The literature regarding the application of ECMO in polytraumatic patients combining acute pulmonary failure and other vital organ damage were reviewed.A 19-year-old man suffered from a multi-trauma after a traffic accident when he was riding a motorcycle and collided into a car. Upon arrival at our tertiary trauma center, he initially presented with a Glasgow Coma Score of 8 and severe hypoxia. He was intubated immediately. Large amount of food content was sucked from the endotracheal tube. Primary chest computed tomography (CT) reported right lung consolidation with patchy opacities, which was consistent with a combination of blunt chest contusion and aspiration pneumonia Figure . The gasWith regards of a polytraumatic patient combining liver laceration, ECMO is contraindicated because the need of systemic anticoagulation may induce further internal bleeding. A heparin-free, vv-ECMO was thus suggested. An extracorporeal circuit was constructed via a venous access through internal jugular vein and femoral vein, using Seldinger technique. The blood flow was set at the rate of 2.42 L/min, and the FiO2 was set at 45%. The oxygenation status improved dramatically after the introduction of ECMO Table . The livWe searched the PubMed (2000–2013) database for case reports about the launch of ECMO regardless of heparin-containing or heparin-free in multi-trauma patients. The abstracts of all articles published in English were screened. The full texts of articles published in other languages but with an abstract in English were analyzed. Articles were selected for review if they included the following patient data: age, sex, clinical presentation, combined injury besides acute pulmonary failure, the details of ECMO treatment, and the outcome.There were six case reports containing 11 patients described in detail, and one clinical paper containing 10 cases found in the literature, which are listed in Table Despite of the various mechanical ventilation technique and the improved knowledge of the adjustment of ventilation parameters, a group of patients with traumatic pulmonary contusion or ARDS are still unable to benefit from these technique. ECMO has been proved to be an rescue therapy when conventional methods are ineffective. ECMO was also reported to be effective in polytraumatic patients combining pulmonary contusion and other organ damage including bronchial rupture ,4, endobThe application of heparin-free ECMO has been proposed recently to overcome the dilemma. Muellenbach et al. reported three successful cases of heparin-free vv-ECMO on a patient with traumatic lung failure and severe traumatic brain injury . MatthiaThe improvement of ECMO technique including centrifugal pumps and heparin-coated circuits reduced the amount of heparin needed. However, with regards to a patient who had spontaneous hemostasis on liver laceration, we still chose the heparin-free method to reduce the risk of rebleeding. Based on the hemodynamic stability and the daily improvement of lung condition, we did not used additional method to prevent clotting of the circuit except the close monitor of ACT. The duration of ECMO was five days, which is comparable to other report.ECMO can serve as a rescue method to provide the traumatic lung to rest. Although it was previously regarded to be contraindicated in polytraumatic patient with coexisting organ hemorrhage, there are growing successful experiences reported recently. We report a heparin-free, vv-ECMO method for patients combining acute pulmonary failure and nonoperative liver laceration, which may extend the feasibility of ECMO in polytraumatic patients.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


Pattern recognition receptors (PRRs) expressed on immune cells are crucial for the early detection of invading pathogens, in initiating early innate immune response and in orchestrating the adaptive immune response. PRRs are activated by specific pathogen-associated molecular patterns that are present in pathogenic microbes or nucleic acids of viruses or bacteria. However, inappropriate activation of these PRRs, such as the Toll-like receptors (TLRs), due to genetic lesions or chronic inflammation has been demonstrated to be a major cause of many hematological malignancies. Gain-of-function mutations in the TLR adaptor protein MYD88 found in 39% of the activated B cell type of diffuse large B cell lymphomas and almost 100% of Waldenström’s macroglobulinemia further highlight the involvement of TLRs in these malignancies. MYD88 mutations result in the chronic activation of TLR signaling pathways, thus the constitutive activation of the transcription factor NFκB to promote cell survival and proliferation. These recent insights into TLR pathway driven malignancies warrant the need for a better understanding of TLRs in cancers and the development of novel anti-cancer therapies targeting TLRs. This review focuses on TLR function and signaling in normal or inflammatory conditions, and how mutations can hijack the TLR signaling pathways to give rise to cancer. Finally, we discuss how potential therapeutic agents could be used to restore normal responses to TLRs and have long lasting anti-tumor effects. Pattern recognition receptors (PRRs) are germline-encoded receptors with the ability to relay “danger signals” to the host in order to mediate an early innate immune response. The term “pattern recognition receptors” comes from their ability to recognize specific pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) , 2. PRRsOne of the most well characterized PRR is the TLR . TLRs arThis review will focus on TLRs and malignancies associated with the dysregulation of TLR signaling pathways. TLR activation by somatic MYD88 mutations and chronic inflammations has been implicated in a number of hematological malignancies. Targeting the TLR signaling network has gained increasing attention from researchers and clinicians seeking strategies to achieve long lasting anti-tumor outcomes. Here, we discuss the signal transduction and immune regulation by TLRs and the immunological malignancies that manifest from dysregulation of TLR pathways, including how targeting these pathways could be an attractive therapeutic regime.Drosophila protein Toll, which is best known for its essential role in establishing dorsoventral polarity during embryogenesis in insects , a particularly aggressive subtype of DLBCL whose pathogenesis relies on constitutively active NFκB, frequently accumulates MYD88 mutations. 39% of tumor samples contain mutations in MYD88, and strikingly, 29% of those mutations result in a single nucleotide change from leucine into proline at position 265 (L265P) . shRNA kin vitro and in vivo , 2–10% of chronic lymphocytic leukemia (CLL), 69% of cutaneous diffuse large B cell lymphoma (CBCL), and 38% of primary central nervous system lymphoma (PCNSL) (previously reviewed in Ref. ). Howeve in vivo . Neverthin vitro . Other sin vitro . Given tin vitro . The useRemarkably, inflammation enables most of the key cellular and molecular capabilities that are required for carcinogenesis, such as genomic instability, proliferative abnormality, and reprograming of the stromal environment . AlthougHelicobacter pylori infection is associated with gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma, infections with hepatitis B and C viruses are associated with hepatocellular carcinoma, and infections with Bacteroides species are linked to colon cancer , an enzyme that promotes immunoglobulin gene class switching by catalyzing deamination of cytosines in DNA . In addiH. pylori (gastric lymphoma), Chlamydia psittacii , and Borrelia burgdorferi (cutaneous lymphoma) are a prime example of lymphoid malignancies associated with chronic inflammation  and Pephinh-MYD88 have also been developed to target MYD88 dimerization in the treatment of lymphoma patients with MYD88 mutations , and CLL B cells that selectively express high levels of TLR9 undergo profound apoptosis by the activation of STAT1, reduction of Bcl-xl pro-survival protein, and elevation of Fas and Fas ligand . TLR9 trPattern recognition receptors protect us from danger and damage associated signals, however, inappropriate activation of these pathways can cause cancer. TLRs can also use ubiquitously available self-ligands such as our own DNA to drive aberrant cell growth when the adaptor protein MYD88 is mutated. This recent finding is one of the many pieces of supportive evidence for Virchow’s hypothesis that chronic inflammation is linked with cancer development. Studies into mutations in the TLR signaling pathways have significantly advanced our understanding on the involvement of TLRs in cancer. However, the potential for targeting TLRs as anti-cancer therapy remains an area that is not yet fully understood. Often TLRs act as a “double-edged sword” in cancer, over active TLR signal provides a microenvironment that is necessary for malignant cell proliferation; on the other hand, TLR agonists can also be used to inhibit cancer cell growth. A better understanding of the involvement of TLRs in cancer would help in tipping the balance between tumor stimulatory and inhibitory effects and the development of novel anti-cancer agents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA-editing is a tightly regulated, and essential cellular process for a properly functioning brain. Dysfunction of A-to-I RNA editing can have catastrophic effects, particularly in the central nervous system. Thus, understanding how the process of RNA-editing is regulated has important implications for human health. However, at present, very little is known about the regulation of editing across tissues, and individuals.Here we present an analysis of RNA-editing patterns from 9 different tissues harvested from a single mouse. For comparison, we also analyzed data for 5 of these tissues harvested from 15 additional animals. We find that tissue specificity of editing largely reflects differential expression of substrate transcripts across tissues. We identified a surprising enrichment of editing in intronic regions of brain transcripts, that could account for previously reported higher levels of editing in brain. There exists a small but remarkable amount of editing which is tissue-specific, despite comparable expression levels of the edit site across multiple tissues. Expression levels of editing enzymes and their isoforms can explain some, but not all of this variation.Together, these data suggest a complex regulation of the RNA-editing process beyond transcript expression levels.The online version of this article (doi:10.1186/s12864-015-2291-9) contains supplementary material, which is available to authorized users. RNA-editing is an enzyme-mediated post-transcriptional modification whereby individual nucleotide residues are altered in the nascent RNA transcript molecule . In contThe most common RNA-editing alteration is enzymatic deamination of adenosine nucleotides to inosine, by the family of ADAR enzymes , 3. ThesThere are 3 members of the ADAR family in vertebrates: Adar, Adarb1, and Adarb2 . A number of substrates are reported to be edited by Adar and Adarb1 –6. HowevThe gene for Adar produces two main protein isoforms. Expression of the longer Adar isoform, p150, is inducible by interferon stimulation  and has Owing to the role of interferon in defense against microbial infection, the p150 isoform of Adar has been thought to function in the editing of double stranded viral RNA. In humans, double stranded RNA structures often form in the long 3’ UTR regions of genes when juxtaposed Alu repeat elements are present. These inverted transposable elements, which are remnants of ancient viral insertions, thus form the secondary structure substrate for hyper-editing [In contrast to the hyper-editing of many adenosine residues in close proximity, site-selective editing tends to occur consistently at specific residues, and even within the coding sequence of transcripts. Aside from the propensity of RNA substrate to form hairpin structures, little is known about other mechanisms regulating this more selective form of editing. The importance of site-selective editing events is highlighted by the necessity of an Adarb1 mediated edit in the neurotransmitter receptor protein GluR-B for developmental viability in mice .The ADAR gene family is highly conserved across metazoans, and highly expressed in the nervous system , 17, 18.RNA-editing has also been implicated in functions and disease outside the nervous system. Site-specific editing of AZIN1 is associated with more aggressive tumor-initiating potential in hepatocellular carcinoma , while aIt is clear from these examples that RNA-editing is functionally important, and must require tight regulation to prevent dysfunction. However very little is known about how this regulation occurs. We do not have a thorough understanding of the variability in editing between individuals, nor the intra-individual variability across tissues to properly address this question. Furthermore, there has not yet been a genome-wide study to address, in an unbiased manner, the scope of functionally relevant RNA-editing in healthy individuals.Previous high throughput RNA-editing surveys have been limited in the range of tissues studied, with a bias towards central nervous system (CNS) tissues, or in their genomic breadth. Transcriptome-wide RNA-seq analyses of RNA-editing in mice have largely been limited to whole brain and cerebral cortex , 27.A valuable study of RNA-editing was performed across seven distinct tissues from a single human individual. However, the study used specific padlock probes, not transcriptome wide RNA-seq, and resulted in only 569 putative RNA-editing sites .In order to investigate the mechanisms underlying the regulation of RNA-editing, we present an analysis of a novel transcriptome wide RNA-seq dataset from nine tissues in a single individual mouse. Additionally we present a comparison and combined analysis of 20 previously published RNA-seq samples from mice of the same strain across a variety of tissues. To the best of our knowledge, this is the only dataset for which we can address in a genome-wide fashion, questions both on the variability of RNA-editing across tissues in a single individual and within specific tissues across individuals. We then use these data to investigate the underlying mechanisms regulating the important process of RNA-editing.We obtained a high confidence set of RNA-DNA differences (RDDs) by analyzing the RNA and DNA from various tissues of a single adult mouse Fig. . In paraWe identified 13,798 RDDs within our dataset, including 5053 novel A-to-G changes not previously reported in the Darned or Radar databases of RNA-editing sites , 30. In 22% of A-to-I RDDs fall within coding exons, and only 159 would result in a non-synonymous change. Further inspection of the genic context of editing sites revealed consistent results across samples within the same tissue. For example, all eight brain tissue samples showed a much higher proportion of intronic RDDs to coding exon RDDs than any of the other tissues. Likewise, the two heart samples had the highest coding exon RDD to intronic RDD ratio. Therefore RNA editing preferentially occurs within intronic sequence, and the amount of intronic substrate available appears to be tissue dependent.To investigate the functional relevance of the A-to-G RDDs identified from our analysis, we used RefSeq gene annotations to determine the genic context and predicted functional consequence  of each RDD. Across all tissues, most of the RDD sites occurred within introns Fig. . This is%. However, the same six edited genes  are found to recur in most of the significant GO categories for the brain samples. Likewise for the testis results, three edited genes  account for the results. Thus there are few ontologies enriched for protein altering RNA-editing, and these are driven by a very small number of edited genes.We next considered whether certain classes of genes were preferentially edited within our samples as this might reveal a regulatory aspect of the editing process. Within each tissue, we collected genes containing protein altering RDDs and tested for enrichment of any functional category amongst the genes normally expressed within that tissue. We performed a Gene Ontology analysis for each tissue individually, where the universe of genes was simply the set of genes with an RPKM expression level ≥ 10 within that tissue. Additional file % of sites are shared between any two tissues, and even fewer (0.7 %) are shared across all 9 tissues. These numbers both increase slightly when only RDDs within coding exons are considered . This might suggest that more functionaly relevant RDDs, such as those within coding regions, are more likely to be shared across tissues. However, for RDDs predicted to be non-synonymous, and thus more likely to cause a functional change in the resulting protein product, the proportion that are shared across tissues decreases to 8 %. Thus, the recurrence of editing sites across tissues is low, but increases modestly when considering only synonymous editing events within coding exons.After examining aggregated trends in RNA-editing across tissues and individuals, we next surveyed the variation in editing per site. This finer scale analysis is driven by the assumption that functionally relevant editing sites should be recurrently edited across individuals, and possibly across tissues. We first looked at the number of A-to-I RDD sites that were shared among the 9 tissue samples from our single male mouse Fig. a. An ediWe next looked at the inter-individual variability of calling RDDs. If RDDs are functionally important, we assume that their presence will be consistent across individuals, when looking in the same type of tissue. Here we included the previously published RNA-seq samples  due to insufficient sequencing depth), to serve as biological replicates for four of our tissues. We had 7 adult brain samples, and 2 samples each for the heart, kidney, and liver tissues.Within the 7 brain samples we found a much higher proportion of RDD sites that were shared  than across tissues within a single individual Fig. b. Thus tWe next wanted to see if this pattern held true in the other tissues. Since we only had 2 samples per tissue for the heart, kidney, and liver, for consistency we subset our brain samples to include only 2 samples ). The brain samples had the highest consistency in RDD recurrence within coding and non-synonymous sites . Our RNA-seq data provides us with both the overall expression level estimates for the Adars, and isoform level estimates based on splice junctions Fig. .Fig. 6EAdar is most highly expressed in the brain and spleen samples, which is consistent with brain and spleen having higher number of observed RDDs after accounting for sequencing depth see Fig. a. Adarb1From the literature, it is expected that there are differences in Adar isoform usage across tissues, with the shorter p110 isoform more highly expressed in CNS tissues. Therefore, it is important to study gene expression at the isoform level as well. When we examined the data across our samples, we confirmed the elevated expression of the p110 isoform within brain samples Fig. b. The loWhile spleen and thymus have Adar expression levels similar to those found in brain, the difference in isoform usage may result in differential regulation of editing between these tissues. There are some reported motifs prefered by Adar and Adarb1, but little is known about the specificity differences between the p110 and p150 isoforms of Adar. Both isoforms contain a z-alpha DNA binding domain at their N-terminal end, but the alternative 5’ UTR and splice of the p150 isoform encodes a second z-alpha domain upstream of the first. This extra z-alpha domain could potentially direct the editing enzymes to prefer a subset of nascent transcripts with proximity to a particular DNA sequence that interact with the z-alpha domains. Thus differences in the resulting protein isoforms may provide a mechanism for different selectivity of editing substrates.We also discovered a correlation of the p110 (J1 junction) with an internal in-frame alternative splicing event (J9) that results in the addition of 26 amino acids between the second double stranded RNA-binding domain and the deamination domain. This region includes four potential phosphorylation sites. The functional effect of this change, in altering the relative position of the deamination domain to the bound RNA transcript, could play a role in the sequence context of RNA-editing events. Thus isoform usage may influence and correlate with specific motifs surrounding editing sites within a sample.To test this hypothesis, we analyzed the neighboring nucleotides surrounding edited sites across tissues to look for tissue specific patterns that might elucidate editing specificity of the Adar isoforms , and RPKM estimates for the mutually exclusive 5‘ exons that distinguish the p110 and p150 isoforms of Adar. These expression levels were used to look for correlations with per site editing frequencies across samples.Within coding exons we found 16 RDD sites whose variable editing frequencies had significant positive correlations with at least one of the editing enzymes or isoforms tested. After multiple testing correction the editing site within the gene Tmem63b remains significant. While the other 15 sites are not statistically significant after correcting for multiple testing, interesting trends were observed. Two distinct patterns may explain the regulation of editing Fig. . The firPrevious studies suggest that RNA-editing is prevalent within transcripts of the central nervous system. In fact, the most well known A-to-I editing sites occur within coding regions of neuronal signalling genes. Among these, the transcripts of several genes involved with ionotropic glutamate signalling are typically edited at 100 % frequency. This begs the question of why these sites are not simply changed in the genome rather than edited in the transcript. Several studies report the importance of these editing events in modulating receptor activity in a developmental manner , 32–36 aHere we have quantified that the number of sites which undergo A-to-I editing is remarkably higher in transcripts derived from the brain. While the ability to detect editing events increases with sequencing depth , 37, we Interestingly, in our study, the vast majority of edited sites within brain samples occurred in intronic regions of genes. This enrichment for intronic editing accounted for the abnormally high amount of editing sites in brain tissues. The abundance of editing events within intronic sequence is intriguing as we did not expect high levels of sequencing coverage over introns. The RNA-sequencing library prep used in this study was a standard poly-d(T) protocol which should enrich for fully mature, poly-adenlyated mRNA transcripts that have already undergone splicing. However, intron retention appears to be a more common phenomenon within neural transcripts . The recPrevious studies have found that RNA editing in mice occurs less frequently than in humans . This isRNA-editing patterns varied across all tissues examined and sites edited in multiple tissues were not common. We demonstrate that tissue specific editing is largely a consequence of the tissue specific expression of the edited transcript. While our data do not argue against the existence of tissue specific editing cofactors, it appears that a major regulator of editing is the expression of the edited transcripts.However, we did find evidence for a small number of transcripts expressed in multiple tissues but with variable levels of editing. In several of these cases a change in the expression or isoform usage of the editing enzymes correlates with the observed difference in editing. This suggests that the editing frequency is regulated by expression of a particular editing enzyme. In fact, the editing sites in both Flnb and Copa have been previously studied in the context of human hepatocellular carcinoma . Using bWhile the gene Tmem63b is expressed in multiple tissues, editing is only observed within brain. This simpler, binary editing status is consistent with the brain having the highest expression of both Adarb1 and nearly exclusive expression of the Adar p110 isoform. However, Adars can dimerize to form homo- and heterodimers –49. ThisThis study within mouse tissues is largely consistent with genome wide patterns of editing described in human datasets , and furAn 8 week old C57BL/6J male mouse was obtained from The Jackson Laboratory and housed according to the animal use guidelines of Genentech, conforming to California State legal and ethical practices. This animal was euthanized by exsanguination with phosphate buffered saline (PBS) and tissues were collected, flash frozen and stored at –80 °C until further processing. DNA and RNA were extracted with either the All Prep DNA/RNA mini kit , the RNeasy Fibrous tissue mini kit (heart and skin) or the RNeasy Lipid tissue mini kit (brain) according to manufacturers protocol (Qiagen).We generated 100 bp, paired-end standard RNA-seq libraries from the 9 tissues  of the single adult male mouse described above. Each RNA-seq library resulted in 28 million to 77 million uniquely mapped reads.Additionally, from this same mouse, we extracted genomic DNA and generated 75 bp paired end whole genome sequence libraries from the brain and spleen tissue. These produced 586 million and 445 million uniquely mapped reads, respectively.For biological replicates of our dataset from a single mouse, we analyzed previously published RNA-seq data from 3 relevant datasets , totalling 20 additional samples from 15 animals. The first dataset included 76 bp single-end RNA-seq data from 6 tissues  of adult C57BL/6 mice . This exThe second dataset included 80 bp single-end RNA-seq data from the cerebral cortex of embryonic (E17) and adult (3 - 4 months old) C57BL/6J female mice . This exFinally, the third dataset included whole brain derived 76 bp paired-end RNA-seq data from two adult (ages 2 – 3 months) C57BL/6NJ female mice . These d% of the cycles had Phred scores ≥ 23), and ribosomal RNA contamination. The remaining reads were then aligned to the mouse reference genome (mm9) using the GSNAP alignment tool [Our nine RNA-seq samples derived from a single individual mouse, and two whole genome DNA samples were sequenced on an Illumina HiSeq machine. The fastq sequence files for all RNA-seq samples, including the 20 previously published samples, were then filtered for read quality  were first called using the Bioconductor package VariantTools . To incrTo infer the strand of a transcript, we considered only RNA-editing events that occurred within annotated genes, and discarded SNVs that were located in more than one gene on opposite strands.To exclude spurious variants arising from regions of the genome that are problematic for short read alignment, we also discarded SNVs within homopolymer tracts, UCSC repeat masker track elements , UCSC multi-mappability regions, and self-chain regions  was then produced by stringent filtering of the SNVs from RNA using the whole genome sequence data. After all the filtering above, an SNV in the RNA was considered an RDD if the genomic data showed zero reads supporting the edited base, and had > 20X total coverage at the site in question. Although highly stringent, this gave us confidence in calling the remaining RDD sites RNA-editing events .RNA-seq data generated for this study are available from NCBI’s Gene Expression Omnibus (GSE74747). Whole genome sequence data generated for this study are available from the European Nucleotide Archive (ERP010840).


The typical presentation of early stage lung cancers on low-dose CT screening are non-calcified pulmonary nodules. However, there is a wide spectrum of unusual focal abnormalities that can be early presentations of lung cancer. These abnormalities include, for example, cancers associated with ‘cystic airspaces’ or scar-like cancers. The detection of lung cancer with low-dose CT can be affected by the absence of intravenous contrast medium. As a consequence, endobronchial and central lesions can be difficult to recognize, raising the potential for missed cancers. Focal lesions arising within pre-existing lung disease, such as lung fibrosis or apical scars, can also be early lung cancer manifestations and deserve particular consideration as recognition of these lesions may be hindered by the underlying disease. Furthermore, the unpredictable growth rate of lung cancer, which ranges from indolent to aggressive cancers, necessitates attention to the wide spectrum of progression in lung cancer appearance on serial low-dose CT scans. In this pictorial review we discuss the spectrum of early lung cancer presentation in low-dose CT screening, highlighting typical as well as unusual radiological features and the varied growth rates of early lung cancer.Teaching PointsThere is a wide spectrum of early presentations of lung cancer on LDCT.• Low radiation dose and the absence of contrast medium injection can affect lung cancer detection.• Lung cancer growth shows various behaviours, ranging from indolent to aggressive cancers.• Familiarity with LDCT technique can improve CT screening effectiveness and avoid missed diagnosis.•  Lung cancer is the most commonly diagnosed cancer, and was the leading cause of cancer death globally in males in 2008; among females, it was the fourth most commonly diagnosed cancer and the second leading cause of cancer death .The National Lung Cancer Screening Trial (NLST) has recently demonstrated that low-dose computed tomography (LDCT) reduces lung cancer mortality by 20 % in high-risk subjects compared to chest X-ray screening –6.LDCT of the lung would seem to be a simple radiological interpretation but the absence of contrast material injection and low signal-to-noise ratio due to the low radiation exposure can lead to misinterpretation and missed significant findings.Moreover, the distinction between different types of pulmonary nodules, awareness of the behaviours of early stage lung cancer and the interpretation of serial LDCT scans are fundamental in the management of the findings.In this pictorial review, we discuss the wide spectrum of early LDCT findings in lung cancer screening, highlighting typical as well as unusual radiological features and the varied growth rates of early stage lung cancer.LDCT for screening purposes is an unenhanced, single breath-hold scan, conducted from the lung apices through the lung bases.Low-dose protocols of the chest can be achievable due to a high contrast resolution between the air and lung nodules, gaining a low radiation dose while maintaining good diagnostic quality.There is no consensus on which level of dose is considered a ‘low-dose’ CT, however, the major screening trials used tube voltages between 90 kVp and 140 kVp and tube current in the range of 30–80 mAs . Both paA gantry rotation time of <0.5 seconds is recommended by the ACR : faster LDCT collimation should be set with the purpose of achieving thin-section reconstruction images, of at least ≤1.5 mm . Thin-seMost LDCT screening studies have used the filtered back projection technique for image reconstruction; nevertheless, iterative reconstruction algorithms have been shown to improve image quality while reducing radiation dose –13. ReceVarious methods and tools are available in reporting chest low-dose CT. Beside the evaluation of axial thin-section images, the detection rate of pulmonary nodules can be improved using maximum intensity projection (MIP) which consists of projecting the voxel with the highest attenuation value on every view throughout the volume onto a 2D image. Bastarrika et al.  showed tThe use of CAD systems can improve radiologists detection rate for small pulmonary nodules . The accHowever, to date, there is no consensus on the optimal reading technique; further investigations are needed to assess which method should be used to improve the detection rate of pulmonary nodules on low-dose CT.Low-dose CT can identify small pulmonary nodules in heavy smokers and thus improve the detection of lung cancer at an earlier and potentially more curable stage , 23. NonAccording to their attenuation, lung nodules can be classified as solid or subsolid, the latter further divided into part-solid or non-solid nodules .The spectrum of early stage lung cancer presentation at low-dose CT includes solid nodules as well as part-solid nodules and ground-glass nodules Fig. .Fig. 1PrA pulmonary nodule is defined as solid when it completely obscures the entire lung parenchyma within it. Non-solid nodules, or pure ground-glass nodules (pGGN), are defined as focal nodular areas of increased attenuation through which lung parenchymal structures, such as the pulmonary vessels or airway structures, can be visualized. Part-solid nodules are those having both ground-glass and solid components.Lung nodule attenuation is, therefore, a key feature in lung cancer screening, with particular emphasis on subsolid nodules. Subsolid nodules are complex lesions both for interpretation and management, and recently the Fleischner society has published recommendations for their management . Of subsAn increase in pulmonary nodule attenuation at LCDT follow-up, the development or increment of a solid component within the pulmonary nodule should raise suspicions of malignancy, even if the nodule size is stable Figs.  and 3.FiThe pulmonary nodule size is another important predictor for malignancy in heavy smokers, as good evidence exists for a strong correlation between nodule size and risk of malignancy , 29: in Besides the typical presentation of lung cancer, e.g. pulmonary nodule, there is a wide spectrum of focal abnormalities seen on low-dose CT that could not truly be defined as a pulmonary nodules but, nevertheless, are early presentations of primary lung cancer. These kinds of abnormalities, which are unusual forms compared to pulmonary nodules, include cancers associated with ‘cystic airspaces’ or ‘non-nodular’ cancers presenting, for example, as fibrotic changes or scar-like lesions. Such focal abnormalities are initial signs of lung cancer which usually develop over time in a lung nodule or suspicious pulmonary opacities and, therefore, should be checked at follow-up or annual repeat LDCT.There is increasing evidence that early stage lung cancer could arise from pre-existing pulmonary ‘cystic airspaces’ –35, inclp < 0.0001). In a recent analysis aimed at evaluating findings of 22 interval and post-screen carcinomas in lung cancer screening, the authors showed that 22 % (5/22) of missed cancers presented as bulla wall thickening on LDCT . Wh. Wh26]. Part-solid nodules, especially those in which the solid component is larger than 5 mm, should be considered malignant until proven otherwise, provided either growth or no change is seen at the 3-month LDCT follow-up . For these complex lesions, the growth of the solid portion is more important and reliable in assessing the malignancy of the nodule Fig. .Solid nodules have a wide range of growth but usually present faster growth compared to subsolid nodules , 44 Fig.Fig. 7FaIt is also important to consider that care should be taken not to assume that all lesions that partially decrease in size at short-term LDCT follow-up are necessarily benign. It is documented that adenocarcinomas can decrease temporarily in size after antibiotic therapy, owing to fibrosis or atelectasis ; in thisThe detection of lung cancer with low-dose CT may be hindered by the absence of intravenous contrast medium and the low signal-to-noise ratio. As a consequence, endobronchial lesions, pulmonary-hilum lung cancers, and parenchymal abnormalities at lung apices or within lung fibroses are difficult to recognize, raising the potential for missed cancers.Even if small lung cancer detected by screening are more frequently localised in peripheral lung areas, nevertheless, a small amount of lung cancers are also centrally located or endobronchial.The majority of screen-detected lung cancers are localised in the periphery of the lungs, represented by the large amount of adenocarcinomas more often localised peripherally , while sCompared to peripherally located carcinomas, such lung cancers could be difficult to detect because low-dose CT images are unenhanced scans and possibly because screening a population at this location is far less common. In a recent publication by Scholten and colleagues  focused Additional lung regions that deserve specific consideration in lung cancer screening are the lung apices. The detection and interpretation of parenchymal abnormalities within lung apices could be a challenge in lung cancer screening, as apical regions are a frequent location of fibrotic scars and differential diagnosis between lung cancer and fibrotic changes could be complicated. Indeed, as reported by Scholten and colleagues , an apicLung cancer diagnosis should, therefore, be taken into account when a new lesion arises in a previously detected apical scar or when a focal apical abnormality shows progressive enlargement at annual repeat low-dose CT scans. Any change over time within an apical scarring should be carefully evaluated to detect potential lung cancer Fig. .Fig. 11RThe detection of lung cancer could also be affected by the location of the lesion when arising within previous lung abnormalities, such as interstitial lung disease. Sverzellati and colleagues  showed tThere is a wide spectrum of low-dose CT findings in lung cancer screenings. Awareness of the various radiological features of early lung cancers and the familiarity with the low-dose CT technique can improve the CT screening effectiveness and avoid missed diagnosis.


Peripheral neuroinflammation is characterized by hematogenous mononuclear leukocyte infiltration into peripheral nerves. Despite significant clinical knowledge, advancements in molecular biology and progress in developing specific drugs for inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, there are currently no specific therapies that modulate pathogenic peripheral nerve inflammation. Modeling leukocyte trafficking at the blood-nerve barrier using a reliable human in vitro model and potential intravital microscopy techniques in representative animal models guided by human observational data should facilitate the targeted modulation of the complex inflammatory cascade needed to develop safe and efficacious therapeutics for immune-mediated neuropathies and chronic neuropathic pain.The online version of this article (doi:10.1186/s12974-015-0469-3) contains supplementary material, which is available to authorized users. Peripheral neuropathies are a very common group of disorders that currently affect an estimated 20 million people in the USA alone, and hundreds of millions of patients worldwide. An estimated 20–30 % of cases are cryptogenic or idiopathic, for which no known cause has been determined despite extensive laboratory evaluation. A major consequence of peripheral neuropathies is the development of neuropathic pain, which affects over 1 % of the US population .For acquired neuropathies where it is intuitively expected that pathologic interactions between the systemic circulation and peripheral nerves play an important role in disease initiation or progression or both, aberrant or compromised function of the blood-nerve barrier (BNB) could be key to fundamentally understanding how peripheral neuropathies and neuropathic pain develop. Relatively little is known with regards to the function, significance, and mechanisms of pathogenic alterations at the BNB. When functioning properly, the BNB regulates the movement of solutes, macromolecules, and leukocytes from the circulating blood to the innermost aspects of peripheral nerves and nerve roots, known as the endoneurium –7. StrucImmune-mediated peripheral neuropathies, such as Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), have unknown or incompletely understood triggers; however, the presence of hematogenously derived monocytes/macrophages and T-lymphocytes in affected patient nerve biopsies implies a pathogenic role for leukocyte infiltration across the BNB , 13–15. Neutrophil infiltration has been shown to correlate with increased pain perception in a rat model of partial sciatic nerve transection . ChemokiGuided by these observations, we can infer that inhibition of leukocyte infiltration at onset or earlier stages of disease progression could reduce the extent of demyelination and axonal injury in immune-mediated neuropathies and development of chronic pain in inflammatory and traumatic neuropathies, resulting in improved patient outcomes in these disorders. This review article will explore recent advances of in vitro leukocyte trafficking models at the BNB and discuss the potential application of intravital microscopy for in vivo study in animal models. Emerging knowledge from these models based on hypothesis-driven experiments focused on the determinants and signaling pathways for specific leukocyte subpopulation chemoattraction, arrest, transmigration (paracellular or transcellular), and local tissue migration could guide targeted anti-inflammatory drug therapies directed at pathogenic leukocyte trafficking in human peripheral nerve disorders.In order to effectively develop targeted molecular therapies for inflammatory neuropathies, one must first have an understanding of peripheral nerve structure and the BNB. Peripheral nerves consist of an external epineurium, inner perineurium, and innermost endoneurium Fig. . The endEndoneurial endothelial cells and the innermost layer of perineurial myofibroblasts regulate solute and macromolecular transport, as well as leukocyte entry into the endoneurium due to their specialized intercellular adherens and tight junctions and lack of fenestrae , 34, 35.These specialized microvascular endothelial cells also express selective mitogen receptors such as vascular endothelium growth factor receptor-2 and glial cell line-derived neurotrophic factor family receptor alpha-1 that drive proliferation, differentiation, response to injury and restoration of barrier function , 38; speThe mechanisms of leukocyte infiltration across the BNB are at the early stages of elucidation, guided by observations in other microvascular barriers. Leukocyte trafficking across the endothelium is a sequential, coordinated process driven by interactions between selectins (such as E- and P-selectin), chemokines, cell adhesion molecules (such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 [VCAM-1]) expressed on the endothelial cell luminal membrane, and ligands (such as P-selectin glycoprotein-1), chemokine receptors, and integrins expressed by circulating leukocytes. This is referred to as the multi-step paradigm for leukocyte trafficking. Leukocyte rolling, adhesion, and transmigration have been described by pathologists and vascular biologists for many years. However, additional steps have been identified that are crucial for leukocyte migration, dependent on the specific microvascular bed: tethering, slow rolling, adhesion strengthening and spreading, intravascular crawling, and paracellular and transcellular transmigration . Due to Mechanisms of leukocyte infiltration into the endoneurium are pathologically relevant to immune-mediated neuropathies, such as GBS and CIDP. There are currently no models that allow direct evaluation of leukocyte trafficking in peripheral nerves in vivo. Developing a near-physiological human in vitro BNB has been essential to understanding possible mechanisms of leukocyte-endothelial interactions in vivo . Static Ultrastructural examination of epineurial macrovessels in peripheral nerves demonstrates lack of electron dense intercellular tight junctions that are present in endoneurial microvessels , 29. EndLeukocyte trafficking across the BNB can be studied in real time using a flow-dependent leukocyte trafficking assay that entails a parallel plate flow chamber coupled to time-lapse video microscopy , 54–58 . Primary4 integrin) [The in vitro BNB may be treated with physiological concentrations of proinflammatory cytokines  up to 48 h prior to experiments to promote leukocyte trafficking without passively altering transendothelial resistance or inducing endothelial cell death . Studyinntegrin) . These cntegrin) . The repPeripheral blood mononuclear leukocytes (PBMLs) from untreated patients with immune-mediated neuropathies such as GBS or CIDP, or patients with chronic neuropathic pain can be infused across the BNB model at predefined flow rates or shear forces that mimic estimated capillary hemodynamics in peripheral nerves. In this model system, firmly adherent PBMLs become noticeable within seconds and start to form adherent clusters with transmigration after about 5 min, with an increase in the number of attached and transmigrated PBMLs over time, reaching a peak between 20 and 25 min . In ordeM integrin-ICAM-1 mediated signaling , firm adhesion concentrated at intercellular membranes, and transmigration via the paracellular route. Endothelial cell cytokine activation is a stronger stimulus for leukocyte trafficking than systemic leukocyte activation that occurs in GBS. GBS patient-derived mononuclear leukocytes preferentially adhere to the BNB in vitro  on GBS patient leukocyte subpopulations and its critical role in leukocyte transmigration via ICAM-1 at the BNB using this model [By using pHEndECs as well as patient-derived PBMLs with estimates of capillary flow rates and alterations in the endoneurial microenvironment in vivo, this in vitro human BNB model currently provides the most physiologic means to study human peripheral nerve inflammation pathogenesis as a tool to guide the discovery and potential efficacy of specific molecular-based target drugs to treat immune-mediated neuropathies such as GBS and CIDP. Our previous work demonstrated the differential expression αis model , suggestis model , 63 provLimitations of the described flow-dependent human in vitro BNB model include the use of a parallel plate system rather than a hollow, capillary-like, microtube chamber that allows more accurate application of physiological shear forces; the absence of an abluminal compartment to apply chemoattractant molecules and collect transmigrated leukocytes for downstream analyses; suspension of patient-derived leukocytes in medium with less viscosity than circulating blood, potential alterations  with cryopreserved patient-derived leukocytes ex vivo, potential changes in BNB endothelial biology with in vitro culture; and unknown effects of endothelial cell culture without potentially supportive Schwann cells and pericytes. Despite these limitations, this model provides an avenue to determine whether endothelial cytokine activation or leukocyte activation state influences leukocyte rolling velocities and probability of firm leukocyte arrest, determine which chemokine ligand-receptor pairs are crucial for leukocyte subpopulation arrest and integrin activation, as well as subsequent transmigration at the BNB, determine the differential roles of ICAM-1, VCAM-1, and fibronectin connecting segment-1 in specific leukocyte subpopulation adhesion as well as the signaling mechanisms involved in leukocyte docking at intercellular junctions and paracellular migration at the human BNB relevant to normal immune surveillance and pathogenic neuroinflammation.There are currently no models that permit direct assessment of leukocyte trafficking across the BNB in real time in vivo. However, there are various microscopic techniques that allow us to visualize leukocyte trafficking in vivo that may advance our understanding of the multi-step paradigm and provide technical considerations that could guide peripheral nerve leukocyte trafficking studies in the future. The structural organization of peripheral nerves provides a challenge to directly viewing leukocyte trafficking at the BNB in vivo as these low-density microvessels are located within the innermost endoneurium surrounded by axons and loose arrays of collagen fibers aligned in parallel with these microvessels. Furthermore, the endoneurium is surrounded by the multilayered perineurium (forming a fascicle or nerve bundle), and these nerve bundles are embedded within the epineurium consisting of longitudinal arrays of collagen fibers. Superficial extrinsic peripheral nerve blood vessels  and epineurial macrovessels are theoretically easier to visualize; however, these vessels lack tight junctions and possess fenestrations and as a consequence are not part of the restrictive BNB Fig. . VisualiIntravital microscopy (IVM) techniques have been used for decades with bright field transillumination to help scientists understand the biophysical conditions under which leukocytes are exposed to in microvessels –66. ThusBrightfield IVM is a basic technique used to observe leukocyte trafficking; however, ideal tissues must be translucent and cannot contain any internal structures that scatter or absorb light. This is not the case in peripheral nerves with the multilayered organization that consists of longitudinal arrays of collagen fibrils within the epineurium and less compact arrays within the endoneurium in which the microvessels reside with myelinated and unmyelinated axons. This along with the fact that at physiological flow rates, leukocytes may be obscured by rapidly moving red blood cells make this an impractical model to observe leukocyte infiltration at the BNB. Furthermore, only cells that are clearly rolling or stationary can be detected by this technique . More adA limitation of single beam two-photon IVM is the requirement of higher laser power to enhance sensitivity and detection speed which results in sample degradation. Multibeam two-photon IVM using a multifocal scan head that splits a laser beam into a line of 64 foci with charge-coupled device field detector allows real-time sample illumination at full power. This technique has been shown visualize tissues down to penetration depths of 200 μm at an image quality similar to surface detection after applying depth-correction point of spread function . HoweverEpi-luminescence fluorescent IVM can be applied to track leukocyte trafficking in vivo by injecting specific fluorophores or fluorescent dyes that facilitate the detection of microvessels. Using Rhodamine 6G to visualize real-time leukocyte-endothelial interactions in the brain, intraperitoneal administration was demonstrated to be as effective as the standard intravenous injection route . HoweverIVM and two-photon microscopy have been employed to visual leukocyte-endothelial interactions and elucidate determinants of leukocyte trafficking in mouse models of central nervous system inflammation such as experimental autoimmune encephalomyelitis  –83, endoAlthough it has been debated whether IVM adequately visualizes deeper-residing cerebral post-capillary venules that form the blood-brain barrier in addition to superficial vessels that do not, these studies have been useful in determining the role of proinflammatory signals: microglial and endothelial activation, Toll-like receptors, specific selectins and their counterligands, leukocyte integrins and endothelial cell adhesion molecules, and chemokines and chemokine receptors; as well as anti-inflammatory signals such as cannabinoid receptor-2 and annexin A1 interacting with its receptor FRR2/ALX in trafficking of total leukocyte or leukocyte subpopulation such as T-cells , neutrophils or dendritic cells in the central nervous system inflammation , 79–89.Hopefully, the advances made in IVM and two-photon microscopic techniques to visualize leukocyte trafficking in the central nervous system will guide approaches needed to study leukocyte trafficking across the BNB in the near future. These approaches if applicable to the mouse sciatic nerve may aid to elucidate the molecular determinants and signaling mechanisms relevant to T-cell immune surveillance in healthy nerves, monocyte, T-cell, and neutrophil trafficking into the endoneurium at different stages of acute and chronic immune-mediated demyelinating neuritis or peripheral neuropathy following nerve crush injury, as well as aid to determine the leukocyte subpopulations and molecular signals essential for peripheral nerve recovery following inflammation and trauma. These observations could translate to human peripheral nerves due to similarities in neural-immune responses between mice and humans.Since IVM approaches have not been developed for peripheral nerves, leukocyte trafficking at the BNB in vivo is currently studied indirectly using animal models of peripheral nerve inflammation. Animal models of acute and chronic demyelinating neuritis such as EAN, chronic-relapsing EAN, and spontaneous autoimmune peripheral polyneuropathy (SAPP) have been used to aid decipher relevant mediators and signaling mechanisms of pathogenic leukocyte trafficking that should be preferably guided by in situ observational data from peripheral nerves obtained from affected patients . DespiteCurrently available treatments for immune-mediated neuropathies and chronic neuropathic pain are partly efficacious or focus on symptom control. Recent advances in cell and molecular biology as well as medicinal chemistry and pharmacology should drive the development of specific therapies for these disorders. Although other pathologic aspects of peripheral nerve inflammation and nerve recovery after injury are important considerations, knowledge of aberrant leukocyte infiltration at the BNB has potential to halt the deleterious effects of hematogenously derived leukocytes in these disorders. In a similar vein, care would be required not to impede the trafficking of regulatory leukocyte subpopulations into the peripheral nervous system and be aware of the potential potent systemic immunosuppressive properties specific anti-inflammatory drugs could possess. Using in vitro and in vivo models designed to mimic essential aspects of human peripheral nerve inflammation guided by observational human peripheral nerve data, knowledge obtained will help further elucidate important signaling pathways and hopefully guide the development of more specific and efficacious treatments for pathologic peripheral nerve inflammation.


Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla. Drosophila larvae remain immersed in the food source and feed constantly until the mid-third instar (foraging stage), when they enter a wandering stage, characterized by cessation of eating, purging of the gut, and exiting the food source to search for a suitable dry pupation site4Drosophila melanogaster larvae in both foraging and wandering stages, and identified CG14686, whose expression was preferentially elevated in wandering stage larvaeCG14686 increased larval resistance to desiccation stress during the early foraging stage. CG14686 RNAi larvae lost more weight under desiccated conditions than control larvae, and subsequently their mortality rates significantly increased. Based on these data, we dubbed this gene Desiccate (Desi). Desi encodes a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif. Although the larval epidermis was initially identified as the primary tissue for Desi expression, our subsequent study of adults illustrated that gustatory sense organs of the labellum express Desi more robustly than the epidermis at this stage. Morphological analysis of Desi expression in the labellum roughly revealed that Desi was expressed in capsular layers surrounding the gustatory neuronsDesi-expressing cells caused drastic mortality of the transgenic fly pharate adults with malformation of the labellum. Although these results imply the importance of Desi-expressing cells and Desi itself in the adult labellum, the functional role of labellar Desi as well as its precise expression sites remain unknown.Regulation of their water concentration is a fundamental requirement for all organisms. In particular, small terrestrial arthropods such as insects have an extremely large surface-to-volume ratio and are in danger of desiccation by evaporation through the integument to the environment. The conservation of body water is therefore essential for their survival2Desi expression in the adult labellum and larval epidermis. Electron microscopic analyses of labellar Desi expression localized two different types of non-neuronal cells, epidermis and thecogen cells. Desi in the adult labellum epidermis showed similar localization as that in the larval body epidermis: Desi signals localized around the tips of microvilli on the apical surface of the epidermal cells and in the assembly zone between the epidermis and lamellate cuticle. Thecogen cells also produce Desi proteins and likely release them into the inner sensillum lymph sinus. The biological role of Desi expressing in the labellum was analyzed by manipulation as well as analyses of its expression levels. Labellar expression of Desi was elevated in Drosophila adults, which was accompanied by an increase in their water ingestion under arid conditions. This observation was consistent with the fact that Desi overexpression activated the water-seeking activity. In contrast, flies expressing RNAi against Desi significantly decreased their water ingestion due to desensitization of the labellar sensilla. These results indicate the essential role of Desi in regulating normal taste sensing by the gustatory organs, which is very important for animals to maintain an adequate water concentration by acceleration of water ingestion via elevation of the sensillar taste sensitivity, especially under arid conditions.In the present study, we primarily sought to reveal the localization of Desi expression in the region surrounding the gustatory neurons of Drosophila melanogaster adults. To identify the precise cell type expressing Desi in the labellum, we used a transgenic fly expressing GFP under the direction of the Desi-Gal4 driver. Strong GFP signals were detected in the capsular layers covering the proximal dendrites and nerve cell bodies in the labellum , Desi expression levels in the labellum increased linearly for 15 h to approximately five times the initial level, indicating that Desi expression in the gustatory organs is also elevated by desiccation stress (y w adults under arid (0% RH) and moist (100% RH) conditions. The amount of water consumed by the adults under 0% RH was much larger than that consumed under 100% RH, indicating that desiccation enhanced the water ingestion of the adults  (Desi expression in the gustatory organs repressed water ingestion by test flies (Desi in the labellum affect the water-sensing abilities of the labellar sensilla.To estimate the contribution of pression . Overexp (0% RH) . In contst flies , indicatDesi, we performed a different type of water ingestion assay, a CAFE assay, using several transgenic fly lines. When test flies were allowed to drink from a capillary tube, Desi RNAi flies consumed much less water than the control strain flies did (tetanus toxin light chain (TNT) and dsDesi are respectively expressed in Desi-expressing cells and sensory neurons, Desi-Gal4;UAS-TNT and pickpocket28 (ppk28)-Gal4;UAS-dsDesi, suggesting that neither neurotoxin overexpression in Desi-expressing cells nor Desi RNAi in sensory neurons caused any change in the water ingestion  or Desi knockdown under the direction of taste receptor gene drivers such as sucrose  or caffeine  receptor gene drivers, indicating that neurotoxin overexpression in Desi-expressing cells did not affect taste sensing and that Desi expression in Desi-expressing cells themselves is important in preserving normal taste recognition in test flies. The contribution of Desi to the detection of various tastants was further confirmed by showing that Desi overexpression and knockdown increased and decreased, respectively, feeding activities of test transgenic flies especially under arid conditions  , Gr5a-Gal4 , and Gr66a (caffeine)  test. When one prothoracic leg of water-deprived test transgenic flies was touched with water, the proportion of ol flies . Depress mM NaCl . Further mM NaCl  or 10 mMAi flies . The tasUAS-TNT)  or Desi affeine) . These rDesi RNAi, we initially measured water (1 mM KCl)-induced action potentials in the gustatory receptor neurons of the labellum sensillum by performing tip recordings. Consistent with the behavioral assays, the frequencies of action potentials were increased in control flies by application of water  encapsulated by thecogen accessory cells10111213Desi RNAi flies compared with control flies when test flies were soaked in CoCl2 ethanol solution after exposure to desiccation  affect taste perception and host plant preference: while Drosophila sechellia shows a preference for the ripe fruit of Morinda cirifolia, its closely related species, D. melanogaster, avoids the fruit. However, introduction of the odorant-binding protein genes, D. sechellia Obp57d and Obp57e, shifted the oviposition-site preference of D. melanogaster Obp57d/eKOflies to that of the original speciesStudies of the regulation of Desi encodes a 261-amino acid single-pass transmembrane protein points us to the future study concerning its functional properties as a membarane protein. Although we do not have any direct evidence that Desi regulates the water permeability of the epidermis and thecogen cell layer, the fact that immunoreactive Desi signals form small clusters around the tips of microvilli on the apical surface of the epidermal cells  and the inner sensillum lymph sinus. The fact that al cells  and in tal cells  enabled Desi contributes to protection of insects from desiccation risks due to preservation of body water in two different ways, protection of dehydration through the body integument and acceleration of water ingestion via elevation of the sensillar taste sensitivity.Finally, based on the present results together with the conservation of orthologous genes in a broad range of insect species, we propose that the gene UAS-Desi and Desi-Gal4 lines were generated as described previously6UAS-dsDesi (dsCG14684)) strains were obtained from the Vienna Drosophila RNAi Center . The Gr5a-Gal4 strain was provided by J. R. Carlson ppk28-Gal4 was provided by K. Scott Gr66a-Gal4 is available from the Bloomington Center. UAS-TNT strains were gifts from C. O’Kane . Mostly male flies were used experiments except as specifically described, and all analytical experiments were done at 25°C. All flies were kept in the culture tubes (inside humidities: over 80% RH) before using them as test animals. Arid condition (0% RH) was made in a Petri dish inside a desiccator containing dried silica gels, while moist condition (100% RH) was made in a Petri dish containing water-absorbed cotton.The amount of medium ingested by test flies was quantified by measuring the absorbance of the whole body extract at 630 nm after feeding them the test diet containing 1.0% blue dye as described previouslyDesi-Gal4 and UAS-dsDesi) and Desi RNAi flies were placed under 0% RH for indicated periods, and whole bodies were soaked in 1 M CoCl2 ethanol solution for 1 h. Labella of test flies were dissected, immediately rinsed in ethanol, and put on glass slides in glycerol. Sensilla of each labellum were observed using a confocal microscope . The percentage of bluish sensilla was scored by counting 10 sensilla from a randomly selected portion on each labellum. Sensilla were scored as bluish if they transmitted less than 30% of the visible light measured at 655 nm using the confocal software EZ-C1.The water concentration was measured basically according to the procedure of Ayres and GlanvilleFeeding behaviors were assayed by three different methods, a two-way choice assay38Drosophila Ringer’s solution into the abdomen, and a recording electrode (glass capillary with a 20-μm diameter tip) filled with a test taste solution covered the tip of a single taste sensillum. Test sensilla were stimulated up to 2 s with the recording electrode, and electrical signals were amplified and recorded after filtration. The electrophysiological responses to sugar, salt, and water were recorded on an l-type sensillum, and those to caffeine were recorded on an i-type sensillum. Data were then analyzed with DBWAVE softwareElectrophysiology was performed according to a procedure slightly modified from the tip-recording method42Fluorescences of GFP and Alexa Fluor dye were monitored by a laser scanning confocal microscope . Sensory neurons in test flies were visualized by immunohistochemistry using the monoclonal antibody anti-Futsch:22C10, which is specific for the neuronal microtubule protein. Detailed procedures of electron microscopy and scanning electron microscopy are described in Methods of the How to cite this article: Kawano, T. et al. Function of desiccate in gustatory sensilla of drosophilamelanogaster. Sci. Rep.5, 17195; doi: 10.1038/srep17195 (2015).