Increased abdominal imaging has led to an increase in the detection of the incidental small renal mass (SRM). With increasing recognition that the malignant potential of SRMs is heterogeneous, ranging from benign (15%-20%) to aggressive (20%), enthusiasm for more conservative management strategies in the elderly and infirmed, such as active surveillance (AS), have grown considerably. As the management of the SRM evolves to incorporate ablative techniques and AS for low risk disease, the role of renal mass biopsy (RMB) to help guide individualized therapy is evolving. Historically, the role of RMB was limited to the evaluation of suspected metastatic disease, renal abscess, or lymphoma. However, in the contemporary era, the role of biopsy has grown, most notably to identify patients who harbor benign lesions and for whom treatment, particularly the elderly or frail, may be avoided. When performing a RMB to guide initial clinical decision making for small, localized tumors, the most relevant questions are often relegated to proof of malignancy and documentation (if possible) of grade. However, significant intratumoral heterogeneity has been identified in clear cell renal cell carcinoma (ccRCC) that may lead to an underestimation of the genetic complexity of a tumor when single-biopsy procedures are used. Heterogeneous genomic landscapes and branched parallel evolution of ccRCCs with spatially separated subclones creates an illusion of clonal dominance when assessed by single biopsies and raises important questions regarding how tumors can be optimally sampled and whether future evolutionary tumor branches might be predictable and ultimately targetable. This work raises profound questions concerning the genetic landscape of cancer and how tumor heterogeneity may affect, and possibly confound, targeted diagnostic and therapeutic interventions. In this review, we discuss the current role of RMB, the implications of tumor heterogeneity on diagnostic accuracy, and highlight promising future directions. However, increasing detection of incidental SRMs, development of treatment alternatives in select patients, and the discovery of several effective biologically targeted drugs for metastatic disease have raised the awareness that pretreatment tumor histology can be useful and necessary to individualize treatment decisions,,-,,-The role of RMB has expanded to include the evaluation of complex cystic lesions, SRMs <4 cm, and determination of tumor subtype  were highly inaccurate, which limited their clinical utility,-,,-,,,,,,,,,,,,RMB is a relatively safe procedure with minimal morbidity. Contemporary series reveal overall complications rates ranging from 1.4%The ultimate goal of RMB is to appropriately match tumor treatment with tumor biology, so its utility becomes dependent upon the clinical scenario encountered and the accuracy of RMB for determination of malignancy and tumor grade. The diagnostic accuracy of RMB is currently estimated at >95%,,,,,,,,,,-,,,,-Historically, many non-informative biopsies were inappropriately considered “false-negative”, representing a major criticism of RMB, in that missed malignancies would potentially remain untreated,,,,,,Inaccurate RMB, including true false-negative and false-positive results, represents the most concerning outcome for clinicians. Concern is justified, as false-negative results could lead to surveillance of a malignancy with metastatic potential. Fortunately, the rate of false-negative RMB (excluding non-informative RMB) among modern series ranges from 0% to 3.8%,,,,Concern for a coexisting malignancy in otherwise benign tumors is also a significant barrier to routine RMB, undermining the validity of RMB and likely deterring its routine use,,,,,,,,,Histological subtype and grade are known prognostic factors in renal carcinoma and potentially important in the staging and management of small renal cancers-in situ hybridization to evaluate chromosomal abnormalities increased the diagnostic accuracy of ex vivo RMB from 87% with histopathology alone to 94%Utilization of molecular characteristics and refined RMB techniques has the potential to improve the accuracy of conventional RMB. Use of larger (18 gauge) needles is safe and allows acquisition of sufficient tissue for accurate diagnosis and may increase the diagnostic accuracy of RMBet al.A potential limitation in the imagined future of oncology is its underestimation of tumor heterogeneity—not just heterogeneity between tumors, which is a central feature of the new image of personalized medicine, but heterogeneity within an individual tumor  and detection of many allelic imbalances at the chromosomal level, in which one allele of a gene pair is lostIncorporation of RMB results could allow clinicians to reduce the treatment burden for patients without compromising disease specific survival, and incorporation of RMB into risk stratified management algorithms has been proposed as the next refinement in RMB-Patients with bilateral synchronous (BSRT) or unilateral multifocal renal tumors pose multiple prognostic and therapeutic challenges, similar to those of patients with hereditary RCCBetween 5% and 25% of patients who undergo surgery for a presumed single renal mass are found to have multifocal diseaseIn patients presenting with a renal tumor and clinical evidence of metastatic disease, RMB of the primary tumor and/or metastatic lesion can be done to obtain a tissue diagnosis. Indications for primary tumor RMB include inability to make a tissue diagnosis from a metastatic site, atypical appearance of the primary tumor on preoperative imaging, suspicion of multiple primary neoplasms and/or the need to make a histological diagnosis to guide treatmentThe addition of molecular and genetic tests on RMB specimens has exciting potential to provide prognostic information useful for treatment decisions,,,,Cytogenetic alterations in clear cell RCC, such as loss of 9p, are known to correlate with a significantly worse 5-year cancer-specific survival,,,,,,,While the current renaissance in RMB is exciting, several notable limitations deserve mention. A definite assessment of RMB accuracy remains difficult given most series are small, single institutional, and use varying definitions for biopsy successAdvances in the understanding of the limited biological potential of many SMRs, expanding treatment and surveillance options for RCC, improved biopsy techniques, and the integration of molecular factors into prognostic and therapeutic algorithms have renewed interest in RMB. Current indications for RMB include diagnostic work-up of renal tumors that are indeterminate on imaging, assessment of the primary renal tumor prior to initiation of systemic therapy for metastatic RCC, and of incidentally detected radiologically suspicious SRMs in patients at high surgical risk to support treatment decisions and avoid unnecessary surgery. Intratumoral heterogeneity, sampling error, and inconsistent classification of RMB failures in published studies make a precise determination of RMB accuracy difficult. Uniform reporting of RMB safety and efficacy in the literature as well as further studies addressing tumor heterogeneity and sampling error are needed. Differentiation of oncocytoma from oncocytic neoplasms poses a diagnostic dilemma, but incorporation of more sophisticated molecular analyses into enhanced RMB has promising potential.Despite these limitations, RMB has a definite and expanding role in the evaluation and treatment of renal masses, but remains significantly underutilized. While the ability to differentiate between high and low grade malignancies remains the chief limitation of RMB, we anticipate the further integration of percutaneous biopsy into clinical algorithms will guide patient counseling and inform personalized decision making. Future studies will focus on the role of repeat biopsy and the use of biomarkers and molecular fingerprinting in order to facilitate a more rational approach to the management of renal masses.


The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containing interneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals. in vivo have questioned the critical role of synaptic inhibition in these regions, as well as the importance of the BötC, which contradicts previous physiological and pharmacological studies. To further evaluate the roles of synaptic inhibition and the BötC, we bilaterally microinjected the GABAA receptor antagonist gabazine and glycinergic receptor antagonist strychnine into the pre-BötC or BötC in anesthetized adult rats in vivo and in perfused in situ brainstem–spinal cord preparations from juvenile rats. Muscimol was microinjected to suppress neuronal activity in the pre-BötC or BötC. In both preparations, disrupting inhibition within pre-BötC or BötC caused major site-specific perturbations of the rhythm and disrupted the three-phase motor pattern, in some experiments terminating rhythmic motor output. Suppressing BötC activity also potently disturbed the rhythm and motor pattern. We conclude that inhibitory circuit interactions within and between the pre-BötC and BötC critically regulate rhythmogenesis and are required for normal respiratory motor pattern generation.The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containing interneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals. Current models postulate that both generation of the rhythm and coordination of the inspiratory-expiratory pattern involve inhibitory synaptic interactions within and between these regions. Both regions contain glycinergic and GABAergic neurons, and rhythmically active neurons in these regions receive appropriately coordinated phasic inhibition necessary for generation of the normal three-phase respiratory pattern. However, recent experiments attempting to disrupt glycinergic and GABAergic postsynaptic inhibition in the pre-BötC and BötC in adult rats  Aergic and glycinergic receptor-mediated inhibition in the pre-BötC and BötC in rats. Our results demonstrate large, site-specific perturbations of respiratory rhythm and motor pattern including disruption of rhythmic motor output and thus confirm the critical role of inhibitory circuit interactions.Defining functional roles of postsynaptic inhibition in respiratory and other mammalian central pattern generation circuits is a longstanding problem. Inhibitory circuit interactions within and between the brainstem respiratory pre-BötC and BötC have been proposed to be critically involved in normal rhythm and motor pattern generation. A fundamental role of postsynaptic inhibition in these regions has been questioned in recent experiments attempting to pharmacologically disrupt this inhibition. To resolve this contradiction, we applied similar approaches of microinjecting selective pharmacological antagonists of GABARhythmic movements such as breathing and locomotion are produced by central pattern generator (CPG) networks containing interacting excitatory and inhibitory circuits. These circuits are the neural substrates for producing motor behavior  and defiin vitro, generates inspiratory bursting activity  both the intrinsic bursting of pre-BötC inspiratory neurons and inhibitory interactions between the neural populations in pre-BötC and BötC are fundamentally involved in generating the normal rhythmic respiratory pattern. Disruption of inhibition in these circuits would lead either to switching to the intrinsic rhythmic activity originating within the pre-BötC, or to sustained or apneustic-like activity.To resolve this problem, more complicated models have been developed  hypothesThis concept was challenged by a recent study in the anesthetized rat using targeted pharmacological blockade of fast inhibitory neurotransmission  from whiin situ brainstem–spinal cord preparation of juvenile rat. Specific pharmacological blockers of glycinergic (strychnine) and GABAAergic (gabazine) receptor-mediated inhibition were selectively microinjected into the pre-BötC or BötC and perturbations of the respiratory frequency and phases were evaluated. In addition, microinjections of the GABAA receptor agonist muscimol were used to inhibit neural activity in each compartment. Our results were not consistent with those reported by The present study was focused on resolving this contradiction and further evaluating roles of inhibitory interactions in pre-BötC and BötC in rhythm generation and shaping respiratory pattern. Our experiments were performed using two distinct preparations, the anesthetized, vagotomized adult rat, and the arterially perfused All experimental procedures used in this study were approved by either the NINDS Animal Care and Use Committee, or the Drexel University Institutional Animal Care and Use Committee, which oversees Drexel University's AAALAC International-accredited animal program. All electrophysiological recording and pharmacological microinjection experiments were performed via a surgically exposed ventral brainstem for access to the ventrolateral medullary pre-BötC and BötC regions  via a snout mask. Anesthetic depth was maintained at a level at which withdrawal reflexes, as well as changes in heart rate and blood pressure in response to pinching the distal hind limbs, were absent. After tracheotomy with a glass tube, animals were artificially ventilated with the same gas mixture . Electrocardiogram was measured via three small subcutaneous electrodes using conventional amplification and filtering  and monitored using an audio amplifier  and oscilloscope (Tektronix). One femoral artery and vein were cannulated for measurement of arterial pressure and infusion of drugs/saline, respectively. During all surgical procedures, rectal temperature was maintained at 37.0 ± 0.1°C via a servo-controlled heating blanket coupled to a rectal thermometer (Harvard Apparatus). The phrenic nerve (PN) was prepared for recording by dissecting the nerves free from the surrounding tissue. Ventral neck muscles , infrathyroid portions of the trachea and esophagus were removed. The body of the 1st neck vertebra (atlas) and base portion of occipital bone were removed to expose the ventral medulla and the axo-occipital membrane was cut. The dura was then opened using iridectomy scissors and residual bleeding from lateral epidural sinus was arrested by applying small pieces of gelfoam  soaked with thrombin solution  dissolved in artificial CSF (aCSF).Spontaneously breathing, adult male Sprague-Dawley rats (340–380 g) were anesthetized with isoflurane vaporized in O2O was maintained to prevent lung atelectasis during expiration. Animals were paralyzed by an intravenous bolus injection (2 mg/kg), followed by continuous infusion (3–4 mg/kg/h), of vecuronium bromide (Abbott Laboratories) dissolved in Ringer–Locke solution. End-tidal CO2 was maintained between 5.0% and 5.5%  by adjusting frequency of ventilation. If necessary, animals were continuously infused with Ringer–Locke solution (1.0–1.25% body weight or 10.0–12.5 ml/kg/h) to maintain a stable mean arterial pressure of 85–95 mmHg.All animals were vagotomized and baro- and chemoreceptor denervated via bilateral transection of the carotid sinus nerves to prevent cardiorespiratory reflex influences on motor nerve outputs . A bilatin situ arterially perfused brainstem–spinal cord preparations from juvenile rats (3–5 weeks old) as previously described  and the rat was decerebrated at a precollicular level. The descending aorta, PN, and cVN were surgically isolated. As with the in vivo preparations, ventral neck muscles, infrathyroid portions of the trachea, the esophagus, and then the 1st neck vertebra and base portion of the occipital bone were removed to expose the ventral medulla. The axo-occipital membrane was cut, and the dura was then cut open. The preparation was transferred to a recording chamber and secured in a stereotaxic head frame ventral side up. The descending aorta was cannulated with a double-lumen catheter for perfusion and recording of perfusion pressure with a pressure transducer (Micron Instruments). Vecuronium bromide was added to the perfusate to block neuromuscular transmission . The perfusate was gassed with 95% O2/5% CO2 and maintained at 31°C. Vasopressin  was added to the perfusate to raise and maintain perfusion pressure between 70 and 80 mmHg  juvenile rats  were anaesthetized deeply with 5% isoflurane and the portion of the body caudal to the diaphragm was removed. The head and thorax were immersed in ice-chilled carbogenated aCSF solution (in m 80 mmHg . Unless m NaCl and 2% pontamine sky blue. The pre-BötC is readily identified by a characteristic pattern of pre-inspiratory/inspiratory (pre-I/I) activity  and displayed continuously with software .With the rat in the supine position, the central ends of the cut PN were placed on bipolar silver hook electrodes for recording  and immersed in a mineral oil pool formed by skin flaps. To identify precise locations of the BötC and pre-BötC the activity of medullary expiratory and (pre)inspiratory neurons was recorded  by a ventral approach and glass (WPI) microelectrodes  filled with 0.5 in situ perfused brainstem–spinal cord preparations, we recorded with fire-polished glass suction electrodes inspiratory activity from PN, and cVN inspiratory and post-inspiratory activity. Signals were amplified , band-pass filtered (0.3–2 kHz), digitized (10 kHz sampling rate) with an AD converter [Cambridge Electronics Design (CED)], and then rectified and integrated digitally with Spike 2 software (CED). Extracellular population activity from pre-BötC or BötC respiratory neurons in the perfused in situ preparations was also recorded with a fine glass electrode (3–5 MΩ) filled with 0.5 m Na+ acetate.To monitor respiratory network activity and motor output in the A  and glycine  receptor antagonists (in aCSF) was pressure injected  into the BötC or pre-BötC bilaterally and simultaneously (1–1.5 nl/s) with small diameter  polished micropipettes (Drummond Scientific). In another set of experiments, the GABAA agonist muscimol was injected  into the BötC or pre-BötC bilaterally and simultaneously. Control injections were made at the same sites with aCSF alone. All injection volumes were measured by microscopically observing the change in level of the meniscus within the micropipette. Microinjections were done after electrophysiological recording to map the extracellular single unit/neuronal population activities characteristic of the pre-BötC or BötC regions as described above, and also in some in vivo experiments after pharmacological probing by microinjection of 5 nl of 10 mm l-glutamate (l-Glu) to further confirm locations of the pre-BötC or BötC regions, which have site-specific responses to brief (500 ms) local l-Glu microinjections  for the BötC region, and tachypnea with decreasing ABP for the pre-BötC region; see l-Glu microinjections based on the neuronal activity maps, BötC and pre-BötC in isoflurane anesthetized adult male rats (340–380 g) occupy restricted areas: 1.8–2.1 mm lateral, 550–850 µm depth from the ventral surface, and extending ∼400 µm in the rostrocaudal dimension  for pre-BötC, and for BötC 1.9–2.2 mm lateral, 450–750 µm depth, and 600–700 µm in the rostrocaudal dimension . At the end of each experiment fluorescent marker yellow-green microbeads  were injected via a pipette  into the site of microinjections for post hoc morphological verification , we performed tests for back leakage of injected solution along the microinjection pipette track. Pipettes with different outer diameters (12.5–50 µm) were filled with 2% of pontamine sky blue and only large diameter pipettes (>30 µm) showed substantial leak of dye back to the ventral surface during microinjections. Accordingly, for drug microinjections in vivo, we bilaterally microinjected a cocktail of gabazine and strychnine and in some experiments muscimol  with micropipettes (ID = 20 µm) positioned in the pre-BötC or BötC through the ventral medullary surface with microdrives (Marzhauser) after mapping locations of these regions with extracellular recording of pre-inspiratory (pre-BötC) or expiratory (BötC) neuronal population activities. Slow, continuous microinjection was accomplished by applying low pressure (30 mm Hg) to the pipettes with a pressure control and measurement system. In preliminary experiments we determined that, compared with the in vivo experiments, lower concentrations of drugs (30 µm of gabazine and strychnine or 10 µm muscimol) bilaterally injected into either of these regions caused rapid and large disturbances of respiratory motor output and hence all experiments with the perfused brainstem–spinal cord preparations were conducted at these lower drug concentrations. In some experiments, extracellular recordings of neuronal population activity in pre-BötC (during injections in BötC) or BötC (during injections in pre-BötC) regions were made during drug microinjections. Fluorescent microbeads were microinjected to mark the sites of drug microinjection  with heparin (1000 U/ml) followed by 500 ml of 4% paraformaldehyde (wt/vol) in 0.1 m PBS . A similar procedure was followed  for perfusion fixation of the in situ brainstem–spinal cord preparations. For both preparations, the brainstem was removed, postfixed in the same fixative for 24 h at 4°C, and subsequently cryoprotected by sequential incubation in 15% and 30% sucrose in PBS and stored overnight at 4°C in 30% sucrose, 0.1 m PBS solution. The medulla oblongata was sectioned parasagittaly or coronally at 30- or 50-µm-thick sections with a freezing microtome. For fluorescence immunohistochemistry, floating sections were incubated with 10% donkey serum in PBS with Triton X-100 (0.3%) and subsequently incubated for 48–72 h at room temperature with primary antibodies for choline acetyltransferase  to label motoneurons. Sections were then rinsed with PBS and incubated for 2 h with secondary antibodies for ChAT . Individual sections were mounted on slides and covered with an anti-fading medium . Fluorescent labeling was visualized with a laser-scanning confocal imaging system (Zeiss LSM 510).Immediately after marking locations of drug microinjection sites, animals I) and expiratory time (TE) were measured. TI was measured as the original integrated burst width at 20% of the peak height above baseline; TE was calculated as IBI – TI. Inspiratory amplitude (amp) was calculated by subtracting the local baseline from the peak value. The endpoint of the parameter quantification was defined when the perturbation during or after microinjections reached it maximum or the signals declined to noise level and the program started missing peak detections (which could appear as a quantum jump in IBI).All automated analyses of respiratory parameters from digitized nerve or neuronal population activities were performed with IDL software (Exelis VIS). Inspiratory events were detected from digitally rectified and integrated phrenic nerve signals via a 200 ms window moving average and peak detection algorithm that calculated a threshold-based zero derivative (positive peak) point. Following peak detection, interburst interval , inspiratory time (T14) and the SEM of the preceding normalized parameter values for the group time series were represented by a gray band. To determine statistical significance, the control values were compared with the endpoint values for each experiment within a group using a two-sided Wilcoxon signed-rank test .The time courses of the changes in the above parameters for a given experimental group were variable, which we assume resulted from variability in the times required for drug diffusion to affect a sufficient number of neurons to produce the perturbations. To represent group data, we computed the mean time courses of the parameter values by the following procedures. For each experiment, the time courses of the parameters were extracted by a 30 s window moving median up to the defined endpoint, and the parameter values for each time series were normalized to the computed mean values during the control period (from 120 to 0 s before start of microinjection). Each time course was divided into 100 time points representing 1% increments from the start of microinjection (time 0) to the endpoint. We then computed the group mean time, and also mean values of the normalized parameters, at each of these points, which were plotted and connected by lines to represent the mean time series. The mean endpoint was plotted with its ±SEM represented by crossbars (see in situ) from integrated, cycle phase-triggered (peri-event) neurograms aligned at the onset of the inspiratory phase defined by PN activity. Successive cycle-triggered traces were either overlaid, or represented as a colored raster plot to depict temporal profiles of activity intensity before and during/after drug injection periods  to establish characteristic regional excitatory perturbations. These in vivo experiments demonstrate that activation of pre-BötC (n = 5) by brief (500 ms) l-Glu microinjections always produced a transient increase of respiratory frequency  due to a significant reduction of expiratory phase duration  always transiently prolonged the respiratory period  to identify the pre-BötC region , primarily due to shortening of TE, as well as a decrease of the amplitude of integrated PN activity. The group (n = 6) in vivo data are summarized in p = 0.03), TI decreased to 71.3 ± 4.5% (p = 0.03), and TE was reduced to 52.6 ± 3.2% (p ≤ 0.03) of control values. The amplitude of integrated PN activity decreased to 32.1 ± 3.9% (p ≤ 0.03) of pre-injection control values. These parameter values, all of which are statistically significant, were obtained at 95.83 ± 10.03 s after onset of the microinjections when the perturbations approached maximum values. The average injection time for the group was 57.66 ± 22.09 s. After these perturbations, the time required for recovery of inspiratory activity with a pattern resembling the control activity was variable, but usually a period of minimally 40 min was required in vivo.Microinjections of the gabazine-strychnine cocktail were performed after initially identifying the pre-BötC region electrophysiologically by recording pre-I/I neuronal population activity and/or also after microinjected m gabazine-strychnine within the pre-BötC of in situ perfused juvenile rat brainstem–spinal cord preparations , TI slightly increased to 110.4 ± 4.0% (p = 0.03), and TE decreased to 43.7 ± 8.0% (p = 0.03) of control values  when maximum changes in parameter values obtained. Recovery of control patterns of nerve activity typically occurred 15–20 min after terminating the bilateral microinjections in these in situ experiments.Similarly rapid and large perturbations were caused by bilateral microinjections of 30 µin situ and in vivo, and in some (n = 3/6) of the in vivo experiments in this group, after confirming the characteristic suppression of recorded phrenic discharge and pressor blood pressure responses by l-Glu microinjections. In contrast to the results obtained with microinjections in the pre-BötC, bilateral microinjections of 110 nl of 250 µm gabazine and strychnine into the BötC in vivo (n = 6), and also the microinjections in the in situ experiments with the lower concentrations of the inhibitory receptor antagonists (n = 6), significantly reduced the frequency of integrated PN discharge due primarily to a prolongation of TE, accompanied by a reduction of TI and the amplitude of integrated PN activity. For the averaged group in vivo data (p ≤ 0.03), TI was reduced to 88.4 ± 5.3% (p ≤ 0.06), and TE increased to 257.2 ± 48.0% (p ≤ 0.03) of control values. The amplitude of integrated phrenic discharge decreased to 39.9 ± 13.8% (p ≤ 0.03) of control values , and TI decreased to 56.2 ± 13.0%, (p = 0.03) of pre-injection control values. Integrated PN discharge amplitude was reduced to 15.4 ± 7.0%  or BötC (n = 6) in situ disrupted the three-phase respiratory motor output pattern as analyzed from simultaneous recordings of PN and cVN activity, the latter of which always exhibited prominent post-I discharge. Cycle-triggered averages and time-series raster plots of integrated PN and cVN shown in Aergic and glycinergic inhibition is attenuated in either region. This loss of post-I activity as the drug-induced perturbation develops represents a transformation from a three-phase to a two-phase motor output pattern (The microinjections of gabazine-strychnine in either the pre-BötC ( pattern .A and glycine receptor antagonists in the BötC (frequency decrease) versus the pre-BötC (frequency increase) demonstrate site-specificity of the perturbations. To further test for site-specific perturbations by manipulating local inhibition, we regionally inhibited/attenuated neuronal activity by bilateral microinjection of the GABAA receptor agonist muscimol. In these experiments, the pre-BötC and BötC regions were first identified electrophysiologically by extracellular recording and/or by microinjection of l-Glu in vivo. As illustrated in Figure 11, bilateral microinjection of 25–30 nl of 100 µm muscimol in the pre-BötC in vivo progressively reduced the frequency and amplitude of phrenic discharge and eliminated inspiratory motor output within 20 s in all experiments (n = 7). The suppression of inspiratory activity persisted for 40.1 ± 26.8 s during which rhythmic inspiratory activity could be restored immediately by subsequent bilateral microinjection of 110 nl of 250 µm gabazine at the same site  in the pre-BötC in the in situ preparations (n = 5) also rapidly suppressed phrenic inspiratory activity , bilateral microinjection of the same volume and concentration of muscimol progressively increased discharge frequency, due primarily to a reduction of TE, and reduced the amplitude of integrated phrenic inspiratory discharge , inspiratory frequency increased to 175.19 ± 8.4% (p = 0.015), TE decreased to 51.4 ± 3.3% (p = 0.015), and TI decreased to 83.4 ± 3.3% (p = 0.015) of pre-injection control values (m muscimol in the in situ preparations (n = 6). At 139 ± 20 s following the onset of the microinjections, the group averaged inspiratory frequency increased to 148.1 ± 16.88% (p = 0.03), TE decreased to 53.1 ± 8.0% (p = 0.03), and TI was essentially unchanged  as recorded from neuronal populations/nerve motor output activity, as well as the durations, amplitudes, and shapes of integrated activity within phases.Brainstem respiratory networks consist of excitatory and inhibitory circuits distributed within interacting regions of the medulla and pons including the medullary pre-BötC and BötC, which are proposed to contain core circuits involved in respiratory rhythm and pattern generation. Although there is general agreement that excitatory pre-BötC circuits generate rhythmic inspiratory activity transmitted to and driving spinal and cranial inspiratory motor outputs, functional roles of inhibitory pre-BötC and BötC circuits continue to be debated . It has in vivo, most recently in adult rats with conflicting results   results . In the E, reduced inspiratory discharge amplitude, and eliminated post-I activity thereby disrupting the normal three-phase respiratory pattern. In some cases, rhythmic motor output was completely terminated.(1) Blocking inhibition in the pre-BötC augmented inspiratory discharge frequency due to a shortening of TE, reduced the amplitude of inspiratory discharge, disrupted the three-phase pattern due to a loss of rhythmic post-I activity, and in some cases transiently terminated inspiratory motor output accompanying a loss of pre-BötC inspiratory activity.(2) Blocking inhibition in the BötC reduced inspiratory discharge frequency associated with a lengthened TAergic inhibition in pre-BötC and BötC circuits are fundamentally involved in dynamical regulation of respiratory rhythm generation and are required for generating the normal three-phase respiratory pattern. Our studies also confirm a fundamental role of BötC inhibitory circuits and their interactions with pre-BötC inspiratory rhythm generating circuits.Therefore, we conclude that ongoing glycinergic and GABAAergic and glycinergic inhibition led to sustained tonic discharge on phrenic nerves, indicating disruption of rhythmic inspiratory motor output. Furthermore, results obtained in anesthetized vagotomized adult rabbits by Aergic and glycinergic antagonists in several cases. Moreover, in these studies in rabbits, bilateral microinjection of muscimol in the BötC potently augmented inspiratory discharge frequency and caused loss of rhythmic inspiratory activity that was replaced by tonic discharge of phrenic nerves.Our results, obtained in juvenile and adult rats, are consistent with previous studies in anesthetized, non-vagotomized adult cats  demonstrA and glycine receptor-mediated postsynaptic inhibition in the pre-BötC and BötC have a major role in controlling the frequency and amplitude of inspiratory circuit activity during eupneic breathing in vivo. Other results obtained by pharmacologically blocking glycinergic inhibition with systemically applied strychnine in perfused rat and mouse brainstem–spinal cord preparations in situ, disturbing synaptic inhibition throughout the respiratory network, also demonstrate major disturbances of respiratory rhythm and disruption of the three-phase respiratory pattern at cellular and circuit levels , supported by their results showing that the BHIR is blocked by antagonizing synaptic inhibition in the pre-BötC. Other results presented suggested that block of inhibition in the pre-BötC and BötC does not disturb generation of laryngeal post-I activity in vagus-intact rats and thus they concluded that inhibitory circuit interactions do not participate in normal three-phase respiratory pattern generation. This result conflicts with our present and previous results  or archaerhodopsin in spontaneously breathing adult mice function  and theiAergic postsynaptic inhibition. As known, pre-BötC contains inhibitory neurons with each type of transmission, as well as neurons coexpressing both GABA and glycine (in vivo (Another important consideration for interpreting the significance of these results is that silencing pre-BötC glycinergic neurons may not be analogous to pharmacologically blocking even postsynaptic glycinergic receptors, particularly because relevant glycinergic synapses may originate from neurons outside of the pre-BötC such as BötC neurons not transduced in sufficient numbers with viral vectors targeted to the pre-BötC. These optogenetic results also cannot be readily compared with the effects of pharmacological blockade of both glycinergic and GABA glycine , so all  glycine  as discu(in vivo . These oAergic and glycinergic inhibition in the intact system. The former is a technical problem that is difficult to solve. This would require extensive local intracellular recordings to analyze endogenous postsynaptic inhibitory potentials simultaneously with the pharmacological perturbations. In our approach, we limited the microinjection volumes of the pipette solution containing inhibitory antagonists, in an attempt to achieve site-specificity of the perturbations. This may have precluded sufficient inhibitory receptor block throughout the local circuits to cause complete disruption of inspiratory rhythm generation in these cases. On the other hand, pre-BötC excitatory neurons and circuits have intrinsic rhythmogenic properties (in vitro.A continuing debate is whether a normal eupneic inspiratory rhythm and respiratory pattern can be generated without synaptic inhibition in the pre-BötC in the intact system where the pre-BötC interacts with the BötC and other sources of phasic/tonic synaptic inhibition. We show that disrupting pre-BötC synaptic inhibition leads either to higher frequency oscillations accompanied by a large reduction in the amplitude of pre-BötC/phrenic inspiratory activity or to tonic phrenic nerve activity in some causes, indicating that a normal eupneic inspiratory rhythm generally does not occur in the absence of synaptic inhibition. The variable persistence of inspiratory rhythmic activity in other cases, albeit with large disturbances of the inspiratory rhythm and inspiratory–expiratory pattern, indicates that either we did not sufficiently block postsynaptic inhibition in the pre-BötC with our targeted pharmacological approach in these cases, or some form of inspiratory rhythm generation occurs after disruption of GABAoperties , so it iOur models of excitatory and inhibitory pre-BötC circuits and their interactions with BötC inhibitory neurons in the intact system incorporate intrinsic rhythmogenic properties in the excitatory pre-BötC population . These min situ was disrupted accompanying the large disturbances of inspiratory rhythm by blocking inhibition in the pre-BötC or BötC. This confirms that the normal eupneic breathing pattern with appropriately coordinated spinal and cranial motoneuron activity relies on synaptic inhibition in BötC and pre-BötC circuits. These inhibitory circuits normally operate with pontine and other excitatory inputs (The three-phase organization of the respiratory pattern at least y inputs . The pony inputs  so the oy inputs . Previouy inputs  and thisy inputs . This aly inputs  after iny inputs , and shoA fundamental role for BötC neurons in respiratory pattern generation has been questioned by We note that the projections and postsynaptic targets of BötC and pre-BötC inhibitory interneurons, particularly mutual inhibitory connections between these neurons, have not been mapped structurally. Establishing these connections remains an important problem. Nevertheless, our results are consistent with mutual interactions and a fundamental role of the BötC and its expiratory inhibitory neurons in three-phase respiratory pattern generation and control of inspiratory rhythm.


While rapamycin and the “rapalogs” Everolimus and Temsirolimus have been approved for clinical use in the treatment of a number of forms of cancer, they have not met overarching success. Some tumors are largely refractory to rapamycin treatment, with some even undergoing an increase in growth rates. However the mechanisms by which this occurs are largely unknown. The results presented here reveal novel cell-signaling mechanisms that may lead to this resistance. The absence of TGFβ signaling results in resistance to rapamycin. Additionally, we observed that treatment of some cancer cell lines with rapamycin and its analogs not only potentiates mitogenic signaling and proliferation induced by HGF, but also stimulates the pro-survival kinase Akt. Together, the data show that the effectiveness of rapamycin treatment can be influenced by a number of factors and bring to light potential biomarkers for the prediction of responsiveness to treatment, and suggest combination therapies to optimize rapalog anticancer efficacy. Many human cancers have overactive mechanistic Target of Rapamycin Complex 1 (mTORC1), which contains mTOR, Raptor, and GβL, and functions as a protein kinase. Rapamycin and its analogs  are allosteric inhibitors of this complex and are approved by the Food and Drug Administration for use against mantle cell lymphoma Here we present new mechanisms that may explain tumor resistance to rapalogs and a new way in which mTORC1 signaling interfaces with cell cycle control. Previous studies indicated that rapamycin potentiates TGFβ-mediated cell cycle arrest We also find that in some cancer cell lines rapamycin increases cell proliferation. One mechanism responsible for this is the potentiation of HGF/c-Met driven mitogenesis by mTORC1 inhibition. In other cancer lines such as the HCT116 colon cancer cell line, rapalogs and the mTORC1/2 inhibitor Torin increase tyrosine phosphorylation of a subset of cellular proteins and enhance the phosphorylation of proteins with Akt and PKC consensus phosphorylation sites. These effects parallel inhibitor-induced increases in the levels of IRS1, IGF-IRβ, phospho-Erk, and phospho-Akt[T308].In summary, the data presented here provide new insights into mechanisms by which cancer responsiveness to rapamycin and rapalogs is determined, and these results may lead to future diagnostic analyses to predict which patients will benefit from these agents. Further, these observations suggest that rapalogs and c-Met inhibitors may function in a synergistic manner against some cancers. However loss of TGFβ signaling, as frequently occurs in human cancers, could suppress tumor responsiveness to mTORC1 inhibitors.2. Unless otherwise noted, cell lines were obtained from the American Type Culture Collection (ATCC) . The TβRIIflx/flx and TβRII-/- hepatocyte cell lines were a gift from Dr. W. Grady flx/flx cell line was provided by Dr. H.L. Moses EMT, CL2 cell lines were previously described Cell lines were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum in a humidified 37°C incubator with 5% COCompounds and growth factors used to treat cells were: Rapamycin, TGFβ, HGF , Activin , BMP4, Nodal, Torin1 , Insulin, Transferrin, and Selenium (ITS) , SU11274, AG490 , and CGP57380 . U0126 was obtained from Promega . A TGFβ Type I Receptor kinase inhibitor (616451) was purchased from EMD Millipore .Lentiviral vectors used to construct the HCC1954 shScramble, HCC1954 shMet, and HCC1954 shBeclin cell lines were generated by co-transfecting shRNA constructs  along with viral packaging plasmids pMD2.G and psPAX2 obtained from Addgene  into the 293T cell line using Lipofectamine Reagent . Medium from the transfected 293T cell line was then used to infect the target cell line, which was subsequently selected using 5 µg/mL Puromycin. The MDA-MB-361/TPR-Met cell line was generated using the pBABE-puro TPR-Met retroviral vector (#10902) from Addgene.Immunoblotting was performed as described Biotin-tagged oligonucleotides encoding wild type and mutant E2F promoter regions as described previously 3H]Thymidine incorporation assays were carried out as previously described 3H]Thymidine pulse.[P-values were calculated using Student's t-test with GraphPad Prism, GraphPad Software, Inc., La Jolla, CA, and P-values less than 0.05 were considered statistically significant.All results presented were obtained in at least three independent experiments. Error bars represent the standard deviation of triplicate determinations. flx/flx cells  were found to express higher levels of the Hepatocyte Growth Factor (HGF) receptor c-Met. Therefore we examined the effect of rapamycin on the proliferation of neuTEMT, CL2 cells induced by HGF or Fetal Bovine Serum (FBS). Rapamycin increased the proliferation of the post-EMT cells in both the presence and absence of HGF when grown in medium without serum, but supplemented with Insulin, Transferrin, and Selenium (ITS) , and that EMT is associated with dramatic changes in mitogenic signaling pathways and cell responsiveness to targeted therapeutics um (ITS) . Howeverignaling . In bothignaling , arrows.EMT, CL2 cells, although the proliferative responses were lower in magnitude. Further examination of the properties of HCC1954 cells demonstrated that their proliferation was weakly stimulated by both HGF and rapamycin, but more strongly increased by combined treatment with HGF + rapamycin  or both EGFR and Her2, reduced the proliferative effects of HGF and rapamycin on HCC1954 cells  473 primarily by mTORC2 473 can also be phosphorylated by other kinases   . Low connockdown .389 (308 at the 2, 8, and 24 hour time points, while 100 nM Torin blocked Akt phosphorylation on Ser473 at all time points examined. To assess whether the MEK/Erk pathway might be the pathway that renders S6 phosphorylation insensitive to inhibition by rapamycin, we examined whether the MEK inhibitor U0126 altered S6 phosphorylation either alone or in combination with rapamycin or Torin. Interestingly, S6 phosphorylation was only blocked by combined treatment with U0126 + rapamycin or U0126 + Torin, but was not significantly decreased by any of these agents when applied alone (473. U0126 also partially suppressed Torin- and rapamycin-induced phosphorylation of Akt on Thr308. Together, the results in Immunoblot analyses employing antibodies recognizing proteins phosphorylated on tyrosine, or proteins phosphorylated on Akt or PKC consensus recognition sequences showed that the tyrosine phosphorylation of a subset of proteins is increased by mTOR inhibitors . Interes389 , this died alone . U0126 uRapamycin inhibition of cell proliferation was shown to occur through suppression of E2F-dependent transcription 308.These results likely have important implications for mTORC1/2-targeted therapy. First, the findings indicate that the effects of mTORC1/2 inhibitors may in some cases be strongly concentration dependent with low and high concentrations producing differing responses. Second, in some cell lines, mTORC1/2 inhibitors increase phosphorylation of Akt on ThrRapamycin and the rapalogs have found a number of uses in the clinic. mTORC1 inhibitors are utilized not only in anti-cancer therapy, but also in anti-rejection therapy for transplant patients as well as in a coating on stents in order to prevent restenosis. In all of their uses, the antiproliferative effects of these drugs contribute to their clinical efficacy. However, these interesting compounds have yielded inconsistent results, leading to the need to better understand the molecular mechanisms by which they block cell division. The results reported here indicate that cross talk exists between the mTORC1 and TGFβ signaling pathways that might influence the therapeutic efficacy of mTORC1 inhibitors against specific tumors.In some situations, rapamycin potentiation of TGFβ-mediated cell cycle arrest may play an important role in the cytostatic effects of mTORC1 inhibitors. The magnitude of this effect will depend on whether the cell type involved undergoes TGFβ-induced cytostasis. Some mesenchymal cell types have intact TGFβ signaling pathways and mount transcriptional responses to TGFβ, but TGFβ does not significantly affect their proliferation. Further, many cancer cells lack expression of the TGFβ receptors or the TGFβ-activated transcriptional regulators Smads 2, 3, and 4. In addition, the levels of autocrine or paracrine TGFβ to which cells are exposed, or the levels of basal ligand-independent TGFβ signaling may vary. The MMTV-PyMT transgenic mice provide a useful model of human breast cancer because the steps in their progression are similar to those of human tumors MMTV-Her2/neu transgenic mouse tumors are known to undergo EMT in vivo, which is associated with downregulation of Her2 and upregulation of other receptor tyrosine kinases upon which the post-EMT cells become dependent 308 in the rapalog resistant HCT116 colon cancer cell line. Additional studies will be required to determine how common this effect is. Thr308 is phosphorylated by PDK1 308 kinase other than PDK1, which is activated in response to mTORC1/2 inhibitors. Unfortunately, PDK1 inhibitors may produce confusing results with respect to effects on Akt phosphorylation on Thr308308 phosphorylation was enhanced with increasing Torin concentrations, while Torin had a biphasic effect on the levels of IGF1R and IRS1, suggesting that these are two mechanistically distinct effects through which mTOR inhibitors can increase Akt signaling.Rapamycin and the mTORC1/2 inhibitor Torin increased Akt phosphorylation on the PDK1 site Thr473 is first phosphorylated by mTORC2, and this in turn facilitates PDK1 mediated phosphorylation of Thr308 (reviewed in 308 phosphorylation more effectively than Torin, while the opposite is observed. A recent study 473 and Thr308 including various tyrosine phosphorylation sites (reviewed in 473 phosphorylation Novel mechanisms of cross talk have been revealed which may have bearing on these results. mTORC1 activation of S6K1 followed by Sin1 phosphorylation inhibits mTORC2, preventing Akt activation 308 phosphorylation suggests an important role for cross talk between the MEK/Erk and Akt/mTOR pathways in this effect. The finding that both U0126 and Torin suppress Akt Ser473 phosphorylation indicates cross talk between the MEK/Erk and Akt/mTOR pathways in HCT116 cells in the control of this site as well. The observation that inhibition of both of these signaling axes is required to block S6 phosphorylation, but that only inhibition of mTORC1 is required to inhibit S6K phosphorylation, suggests the existence of at least two kinases capable of mediating S6 phosphorylation in these cells. Our findings may provide an explanation for results showing that PI3K and MEK inhibitors exhibit enhanced efficacy in HCT116 and HT29 xenograft tumor studies when used in combination as compared with monotherapy The observation that MEK inhibition partially downregulates rapamycin- and Torin-induced Akt ThrThus, the effect of mTOR inhibitors on the proliferation of a given cell will depend on many factors including the condition of the TGFβ signaling pathway and TGFβ levels, the status of the c-Met signaling pathway and HGF levels, in the case of Torin, the concentration of the drug, and finally, cross-talk and functional redundancy between the MEK/Erk and mTORC2/Akt and Akt/mTORC1 signaling axes. Further studies are needed to determine to what extent each of the signaling effects described above influences the efficacy of rapalogs in animal models and in the clinic. Importantly, such observations may explain how in some instances rapalogs are ineffective or, worse, inadvertently promote cancer metastasis


Background: PhoD family enzymes liberate phosphate from organic compounds.Results: The structure of PhoD reveals an active site with one Fe3+ and two Ca2+ ions.Conclusion: PhoD represents a new class of phosphatase related to purple acid phosphatases.Significance: The requirement of PhoD for iron ions may limit bacterial phosphate acquisition in low iron environments. Bacillus subtilis and the phospholipase D of Streptomyces chromofuscus. We present the crystal structure of B. subtilis PhoD. PhoD is most closely related to purple acid phosphatases (PAPs) with both types of enzyme containing a tyrosinate-ligated Fe3+ ion. However, the PhoD active site diverges from that found in PAPs and uses two Ca2+ ions instead of the single extra Fe2+, Mn2+, or Zn2+ ion present in PAPs. The PhoD crystals contain a phosphate molecule that coordinates all three active site metal ions and that is proposed to represent a product complex. A C-terminal helix lies over the active site and controls access to the catalytic center. The structure of PhoD defines a new phosphatase active site architecture based on Fe3+ and Ca2+ ions.The PhoD family of extra-cytoplasmic phosphodiesterases are among the most commonly occurring bacterial phosphatases. The exemplars for this family are the PhoD protein of  Under conditions of phosphate deficiency bacteria attempt to mobilize phosphate from organic molecules by synthesizing extra-cytoplasmic alkaline phosphatase enzymes. Analysis of the Global Ocean Survey metagenomic data set  suggestsEscherichia coli contains two Zn2+ and one Mg2+ ion, and the catalytic mechanism involves an enzyme-bound phosphoserine intermediate  charge-transfer transition , which gives PAPs their characteristic purple color. The occurrence of a tyrosinate-ligated Fe3+ ion is highly characteristic for PAPs, with the only other currently identified examples being found in the structurally unrelated diferric DNA ligase from Ferroplasma acidiphilum . Withine enzyme , 11. Thediphilum . It is, eriments .B. subtilis PhoD. Unexpectedly, the active site contains three metal ions, these being identified as a Fe3+ ion and two Ca2+ ions. Our structural data show that the PhoD active site is distinct from that found in PAPs but that these two enzyme families conserve the position and coordination environment of their Fe3+ ions. A phosphate molecule bound to the PhoD active site gives insight into the mechanism of catalysis.In an effort to define the active site architecture of the PhoD phosphatase family we have determined the structure of B. subtilis strain 168 chromosomal DNA using the primers 5′-CCAGTGGGTCTCAGGTGGTGCGCCTAACTAAGC-3′ and 5′-CGGCGTCGACTTAATTCGTGATTTTTGCACG-3′. The resulting amplicon was digested with BsaI and SalI and cloned into the same sites in plasmid pCA597 . Site-specific mutations were introduced into the phoD coding sequence in plasmid pCA597-mphoD using the QuikChangeTM method (Stratagene).The sequence coding for the mature domain of PhoD (codons 57–593) was amplified from d pCA597  to produE. coli strain BL21 (DE3)(mphoD and pRARE (Novagen) was grown aerobically at 37 °C in 2×YT medium (Sigma-Aldrich) supplemented with 50 μg/ml kanamycin. Protein expression was induced at an A600 nm = 0.6 with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside, and the culture grown was then grown for 15 h at 20 °C.L21 (DE3) containim Hepes NaOH, pH 7.4, 200 mm NaCl, 1 mm CaCl2), with EDTA-free complete protease inhibitors (Roche Applied Science) and DNase I (Sigma-Aldrich). The cells were broken by three passages through a French press at 8000 p.s.i. The lysate was clarified by successive centrifugation at 10,000 × g for 20 min at 4 °C and at 45,000 × g for 1 h at 4 °C, and then applied to a 5-ml self-packaged Strep-Tactin column . Recombinant PhoD was eluted from the column with resuspension buffer containing 2.5 mm desthiobiotin and then supplemented with 1 mg of His6-Ulp1 protease  or bis-p-nitrophenyl phosphate (bis-pNPP). Assays were started by the addition of 1 μg/ml PhoD to 100 mm Tris-HCl, pH 8.0, 2 mm CaCl2, and 1 mm pNPP or bis-pNPP at 25 °C. Reactions were stopped after 5 min by the addition of 1 m final concentration NaOH. p-Nitrophenol production was quantified using ϵ405 nm = 18,000 m−1 cm−1.Alkaline phosphatase activity was assayed spectrophotometrically by measuring the release of m Tris-HCl, pH 8.0, 2 mm CaCl2, 1 mm test substrate, and 1 μg of PhoD. After a 5-min incubation at 25 °C the reaction was terminated by the addition of 200 μl of malachite green reagent (AMS Biotechnology) followed by incubation at room temperature for 30 min. Production of phosphate was obtained from absorbance at 620 nm and quantified using a phosphate standard curve.The substrate specificity of PhoD was assessed by measuring phosphate release from test substrates. Each 800-μl reaction contained 100 mm NaCl, 0.1 m Na2HPO4/KH2PO4, 54% PEG 200, pH 5.6, with protein at 7.8 mg/ml, a total drop volume of 400 nl, and a 50:50 protein-to-mother liquor ratio. The crystals were cryo-protected in mother liquor containing 10% (w/v) ethylene glycol and flash-cooled in liquid nitrogen. X-ray data were collected at beamlines ID23_1 (European Synchrotron Radiation Facility (ESRF), Grenoble, France) and I03 . The data were processed in space group I4122 using Process  (3KBP). Input of this partial model for a second round of Buccaneer building placed 524 residues of a monomer into the map. This was used as the search model for molecular replacement via MOLREP  . The mulng Ltd.)  and to png Ltd.) . The hanng Ltd.)  built a a MOLREP , placinga MOLREP . The quam Tris-HCl, pH 7.2, 200 mm potassium acetate. Measurements were taken at the Ion Beam Centre (University of Surrey).The sample for proton-induced x-ray emission (micro-PIXE) analysis was purified in a calcium-free buffer composed of 20 mEPR spectra were collected on an X-band CW EPR spectrometer (Bruker Biospin GmbH) using an X-band Super High Sensitivity Probehead (Bruker) and equipped with a low temperature helium flow cryostat (Oxford Instruments CF935). Data were analyzed using the program EasySpin .B. subtilis PhoD was heterologously expressed in the cytoplasm of E. coli. Purified recombinant PhoD protein was a monomer in solution as judged by size-exclusion multi-angle laser light scattering (A). The recombinant PhoD exhibited phosphomonoesterase activity with the colorigenic substrate pNPP and exhibited phosphodiesterase activity with the colorigenic substrate bis-pNPP (B). However, the specific activity of the recombinant enzyme was very much lower than previously reported for the native enzyme (2+ supplementation (B), and so Ca2+ was subsequently included in all buffers used for the purification and analysis of PhoD. At pH 8.0 and a temperature of 25 °C, the Ca2+-supplemented recombinant PhoD protein had a mK for pNPP of 50 μm and a kcat of 1.2 s−1. PhoD was able to release phosphate from compounds containing phosphoester bonds but was not able to cleave phosphorus-nitrogen or phosphorus-carbon bonds (A).attering A. The rebis-pNPP B. Howevee enzyme . We obseentation B, and soon bonds A.2+ was purple in color with a broad absorbance band in the visible spectrum, indicating the presence of a prosthetic group (B). The addition of the Ca2+-selective chelator EGTA caused a shift in the visible absorbance maximum from around 540 nm to ∼560 nm (B), suggesting that Ca2+ is associated with the prosthetic group. Proton-induced x-ray emission spectroscopy of PhoD purified in the absence of added Ca2+ ions detected iron and calcium but no cobalt, magnesium, manganese, nickel, or zinc. The presence of iron in PhoD was confirmed by EPR spectroscopy, which detected the g = 4.3 and weak g = 9.6 peaks characteristic of the middle and lower Kramer's doublets of an isolated high spin rhombic Fe3+ ion (C). No further signals were elicited by oxidation with hydrogen peroxide. All EPR signals disappeared on reduction with sodium dithionite, as expected of reduction of a monomeric Fe3+ ion to the EPR silent Fe2+ state (C). The Fe3+ signal was unperturbed by the addition of 50 mm EDTA (C), showing that the iron in PhoD is too tightly bound to be removed by chelator treatment. The presence of iron in PhoD has not previously been reported. However, iron is found in the homologous S. chromofuscus PLD enzyme  anomalous Fourier maps at three wavelengths allowed the assignment of the three active site metal ions as one Fe3+ ion and two Ca2+ ions, which we label CaA and CaB . Substitution of individual amino acids involved in metal ion coordination at each of the three metal sites either abolished  or drastically reduced (D207A and D266A variants) PhoD activity with pNPP as a substrate (B), showing that all three metal ions are required for the phosphomonoesterase activity of PhoD.The core of the PhoD structure is a sandwich of two long β-sheets flanked by α-helices of varying lengths A and 4. ubstrate B, showinB). Phosphate is a known competitive inhibitor of PhoD     with a DDB 1XZW)  due to t3+ site the sole difference in amino acid ligand sets between PhoD and the PAPs is a cysteine-for-aspartate substitution in PhoD (Cys-180). Cysteine is an unusual ligand to a Fe3+ ion. Indeed, to the best of our knowledge, the structurally unrelated alkaline phosphatase PhoX , consistent with the different coordination preferences of Ca2+ and the divalent metal ions found in the PAPs. Specifically, the CaA site has irregular 8-fold coordination with all amino acid ligation being provide by Asp residues.The Ca protein B, consisB site in PhoD has no analogue in the PAPs. The CaB ion has regular octahedral ligation with Asp and Asn ligands.The CaB and 5A). One oxygen atom of the phosphate ligates the iron, one oxygen atom bridges CaA and the iron, and one oxygen atom bridges the two calcium ions. The interactions between the phosphate and the CaA-Fe pair replicate the tripodal μ–η2–η2-geometry observed between phosphate and the two metal ions of sweet potato PAP in PDB structure 1XZW (B). A similar mode of bridging tripodal coordination has also been seen for phosphate bound to the dimetal centers of Sporosarcina pasteurii urease .All three metal ions in PhoD have bonding interactions with the phosphate group present in the active site B and 5A.ure 1XZW Fig. 3B)BB and 5Ai urease  and an oiobacter . In addiB. subtilis PhoD protein forms an α-helix (residues 560–577), which lies over the active site . The C-terminal helix is held in position by a network of salt bridges, which include interactions with the phosphate ion located in the active site, and with Asp-436, which bridges the Fe and CaA ions (C).The C-terminal region of the ive site A and 6. the PAPs A. The C-CaA ions C.2) relative to the whole protein average (28.2 Å2), showing that the C-terminal helix is more mobile than the rest of the PhoD structure (D). This suggests that the helix is relatively weakly bound to the main body of PhoD and could plausibly be released at some stage of the catalytic cycle. Attempts to observe the predicted movement of the C-terminal helix by co-crystallization with the bulky substrate analogues phosphonoacetic acid and phosphonohexanoic in place of phosphate were unsuccessful.The C-terminal helix blocks access to the catalytic site. Furthermore, as judged by modeling studies with pNPP substituted for the phosphate molecule seen in the crystal structure, steric clashes with the helix would also prevent substrates binding to the active site. As a consequence, the C-terminal helix must move from the position seen in the crystal structure in order for substrates to bind to PhoD. The main chain crystallographic B-factor for the C-terminal helix is high . In this context it is worth noting that PhoD family proteins can be divided on sequence grounds into two subfamilies, one of which possesses the cysteine Fe3+ ligand seen in B. subtilis PhoD and also S. chromofuscus PLD, whereas the second has the aspartate ligand found in the PAPS site.The structure of gestions  that Pho3+ ion. Although the PAPs bind an additional Fe2+, Mn2+, or Zn2+ ion, B. subtilis PhoD binds two Ca2+ ions. Other members of the PhoD family almost certainly bind the same two Ca2+ ions because the coordinating amino acids are close to invariant within the family . Although the position of the CaA ion in PhoD is similar to that of the divalent ion in the PAPs, the ligand sets provided by the two protein families are completely distinct. CaA in PhoD has five coordination bonds from Asp residues, whereas the divalent ion in PAPs is ligated by an invariant His2Asp1Asn1 set  it is possible that other PhoD family members also have specialist biological roles rather than operating as general phosphatases like members of the PhoA and PhoX families.Notwithstanding their similarities, the PhoD and PAPs families clearly form distinct sequence groups and differ in their active site metal complement beyond the tyrosine-ligated FeAsn1 set . It shouAsn1 set . A few bAsn1 set , and theB. subtilis PhoD has a flexible C-terminal helix overlaying the active site (–ive site . Althougive site –36. In mve site –. An equiB), suggesting that PhoD possesses a nucleophilic hydroxide between the Fe and CaA ions and that the PhoD structure represents the initial product complex formed by the reaction involving this ion.The way in which phosphate is bound to the active site of PhoD in our crystal structure is likely to mimic the substrate-enzyme interaction at some point in the catalytic reaction. We can get considerable insight into the likely mechanism of PhoD catalysis by interpreting the phosphate complex in the light of the extensive body of work on the mechanism of PAPs. In PAPs the two active site metal ions are bridged by a hydroxide ion. The current view is that phosphomonoester substrates coordinate both PAP metal ions. The bridging hydroxide ion then undertakes a nucleophilic attack on the phosphorus atom in-line with the bond to the leaving group. This results in the hydroxide ion being incorporated into the phosphate product . A strucFig. 33+-coordinated terminal hydroxide group followed by a second nucleophilic attack by the bridging hydroxide. A similar mechanism is plausible for PhoD with the phosphate complex corresponding to the initial product of the second hydrolytic reaction.PhoD is a phosphodiesterase and so must be capable of catalyzing two successive hydrolysis reactions. This process could involve repeating the reaction involving the bridging hydroxide, with the initial product rotating in the active site before the second reaction. Alternatively, the two reactions could utilize distinct nucleophiles. Comparisons with the PAPs are again potentially informative in distinguishing between these two possibilities. Although PAPs have historically been considered not to catalyze phosphodiesterase reactions due to their inability to hydrolyze bis-pNPP  recent wB) in PhoD. Because PAPs are able to catalyze phosphodiesterase reactions at a similar rate to phosphomonoester hydrolysis with only two metal ions .A striking difference between the PhoD and PAP families  is the presence of a third metal ion (Catal ions  it is un3+ and Ca2+ ions used by PhoD is unusual and to the best of our knowledge has only otherwise been seen in the structurally unrelated PhoX alkaline phosphatase (2+ as the divalent cation may simply reflect the high availability of Ca2+ ions in most terrestrial and marine environments (–3+- and Ca2+-dependent phosphatase.The combination of Fesphatase . Remarkaronments . By contronments  and may ronments . Export ronments –43. The


Melaleucaalternifolia, also known as tea tree essential oil (TTO). This essential oil has antiseptic properties and can represent a natural-product alternative for hand hygiene in health-care settings which currently use mainly products based on triclosan and chlorhexidine.The antimicrobial effects of essential oils have been reported in the scientific literature, especially regarding the essential oil of Melaleucaalternifolia essential oil; 0.5% triclosan; 2.0% chlorhexidine.To evaluate the antimicrobial efficacy in hand hygiene performed using three distinct soaps containing 2.0% Escherichia coli K12 and then the hands were washed with each of the products being assessed or the reference soap (soft soap). The number of microorganisms was counted before  and after  each procedure and microbial logarithmic reduction was performed for each of the participants in each procedure. Data were analyzed using two non-parametric tests: Wilcoxon test and Friedman test. Level of significance p=0.01 one-sided.Was applied the methodology of the European Committee for Standardization, EN1499 version April 2013, indicated to evaluate the efficacy of antiseptics for hand hygiene. The hands of 15 healthy volunteers were artificially contaminated with When the Wilcoxon test was applied, the three test products showed to be superior to soft soap , whereas the soaps containing triclosan or TTO were superior in efficacy to soap containing chlorhexidine. When Friedman’s test was applied, products that showed superior efficacy when compared to the soft soap were those containing 0.5% triclosan (p<0.001) and 2.0% TTO (p<0.001) and when efficacy was compared between the products, the three were equivalent, with no superiority between them.When the Wilcoxon test is used, the three soaps can be considered antimicrobial, with the soaps containing 2.0% TTO and 0.5% triclosan being superior to the soap with 2.0% CHX. If Friedman’s test is used, only the 0.5% triclosan soap and the soap containing 2.0% TTO can be considered antimicrobial and both showed similar antimicrobial efficacy.None declared.


Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.The predatory bacterium  Bdellovibrio bacteriovorus HD100 preys on other Gram-negative bacteria by inserting into the host's periplasm where B. bacteriovorus grows and replicates, taking advantage of the nutrient rich environment of the host cell B. bacteriovorus is being studied as a living antibiotic for therapeutic, agriculture, and waste treatment purposes B. bacteriovorus protein synthesis dependent on host degradation products B. bacteriovorus encodes all twenty aminoacyl-tRNA synthetases (aaRSs) Asn, Asn is synthesized on tRNAAsn using an indirect two-step pathway by taking advantage of an aspartyl-tRNA synthetase (AspRS) with relaxed tRNA specificity, a non-discriminating AspRS (ND-AspRS) Asn, which is then amidated by the amidotransferase GatCAB to Asn-tRNAAsnGln formation in bacteria lacking a GlnRS, about two-thirds of all known bacteria For translation, B. bacteriovorus, like other δ-proteobacteria, also encodes GatCAB Escherichia coli that encode AsnRS and GlnRS, GatCAB is typically absent B. bacteriovorus could use GatCAB for tRNA-dependent Asn synthesis given that it lacks both asparagine synthetases (AsnA and AsnB) B. bacteriovorus to use the two-step Asn-tRNAAsn synthetic pathway, it also must encode a ND-AspRS despite having an AsnRS. Previously, two bacteria were known to encode GlnRS and both routes for Asn-tRNAAsn formation: Deinococcus radiodurans and Thermus thermophilusDespite coding for both AsnRS and GlnRS, B. bacteriovorus codes for only one AspRS B. bacteriovorus AspRS is non-discriminating in order to facilitate GatCAB synthesis of Asn on tRNAAsn. We demonstrate that the B. bacteriovorus AspRS can readily form Asp-tRNAAsn as the first step in tRNA-dependent Asn biosynthesis. By analyzing bacterial genomes, we found a significant number of bacteria may also encode both routes for Asn-tRNAAsn synthesis including additional species with a second AspRS. However, only a limited number of bacteria encode AsnRS, GlnRS, GatCAB, and only one AspRS but neither Asn synthetase like B. bacteriovorus.However, B. bacteriovorus HD100 genomic DNA was a gift from Dr. John Tudor (Saint Joseph's University). Samples were sequenced at the Yale DNA Analysis Facility on Science Hill . Nuclease P1 and amino acids were from Sigma-Aldrich . Phenol, ATP, and chloroform were from Fisher Scientific . [α-32P]ATP (10 mmol/µCi) was from Perkin Elmer . Polyethylenimine (PEI)-cellulose thin layer chromatography (TLC) glass plates were from EMD Millipore . Restriction enzymes, Escherichia coli BL21(DE3) and NEB10β strains, OneTaq DNA Polymerase, and T4 DNA ligase were from New England Biolabs . E. coli JF448 was from the Yale Coli Genetic Stock Center . E. coli trpA34 was a gift from the Söll Laboratory at Yale University .Oligonucleotides were from Integrated DNA Technologies . B. bacteriovorus aspS (Bd3311) was cloned between the NdeI and BamHI restriction sites in pET28a to be N-terminally His6-tagged. B. bacteriovorus AspRS was overproduced using the autoinduction method S. aureus AspRS following manufacturer's protocols (Qiagen) Legionella pneumophila aspS was chemically synthesized  and then subcloned between the NdeI and BamHI sites in pET28a and overproduced and purified as described for the S. aureus AspRS B. bacteriovorus asnC (Bd1054) was chemically synthesized with optimized codons for overproduction in E. coli (Life Technologies GeneArt). The optimization increased the number of codons, 52% to 90%, in the GeneArt's top codon class (90–100) based on frequency of codon usage in E. coliNdeI and BamHI sites to be N-terminally His6-tagged. AsnRS was over-produced as described previously for the S. aureus homolog 2 and 300 mM NaCl. The purified AsnRS was dialyzed in 50 mM Tris-HCl, pH 7.6 with 10 mM MgCl2, 30 mM NaCl, and 50% glycerol, and then concentrated and stored as described The in vitro transcribed and the resultant tRNA was purified by chromatography as described 2 added to a final concentration of 5 mM at 65°C. Samples were stored at −20°C and 32P-labeled as described previously using the E. coli CCA-adding enzyme Methanothermobacter thermautotrophicus tRNAGln was in vitro transcribed, purified, and folded as described previously 32P-labeled as described previously using the E. coli CCA-adding enzyme The tRNA genes were 32P-based assay 2, 5 mM DTT, 4 mM L-Asp, and 4 mM ATP. The AsnRS reactions contained 50 mM HEPES-KOH, pH 7.5, 30 mM KCl, 15 mM MgCl2, 5 mM DTT, 4 mM L-Asn, and 4 mM ATP. For plateau aminoacylation of tRNA, reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNA, 11.0 µM tRNA, and 3.0 µM enzyme. Steady-state kinetic studies with 5 nM AspRS were carried out at 37°C with 0.055–1.0 µM 32P-labeled tRNA, and 0–10.0 µM tRNA over 6 min. Steady-state kinetic studies with 5 nM AsnRS were carried out at 37°C with 0.055–1.0 µM 32P-labeled tRNAAsn, and 0–12.0 µM tRNAAsn over 6 min. Reaction mixtures and enzymes were pre-incubated for 30 sec at 37°C. Reactions were started by the addition of enzyme and repeated three to four times. Time points were quenched, digested, separated by TLC, processed and analyzed as described previously L. pneumophila AspRS was measured in the presence of 0.1 µM 32P-labelled tRNA, 50 mM HEPES-KOH, pH 7.2, 30 mM KCl, 15 mM MgCl2, 5 mM DTT, 4 mM L-Asp, and 4 mM ATP over 5 minutes. Reactions were started by the addition of L. pneumophila AspRS to a final concentration of 10 nM at 37°C. Time points were quenched, digested, separated by TLC, processed and analyzed as described previously The aminoacylation activities of the aaRSs were monitored using the established B. bacteriovorus aspS was cloned into pCBS2 between the NdeI and BglII restriction sites (pCBS2-Bb-aspS) L. pneumophila aspS was subcloned into pCBS2 (pCBS2-Lp-aspS). Following transformation into E. coli trpA34 cells, the cultures were grown and assayed as described previously on M9 minimal media agar plates with or without Trp with minor adjustments 600 by diluting with M9 minimal media before 5 mL of adjusted culture was spun down at 1,500×g for 5 min, and washed three times with M9 minimal media supplemented with ampicillin (100 µg/ml). After washing, the samples were resuspended in 0.2 mL of M9 minimal media supplemented with ampicillin (100 µg/ml) before spotting 2 µL of culture on M9 minimal media agar ampicillin (100 µg/ml) plates with or without L-Trp (20 µg/ml), and supplemented with the other 19 amino acids (20 µg/ml each).The B. bacteriovorus aspS and gatCAB  were fused into an artificial operon as described previously NdeI and BglII restriction sites (pCBS2-Bb-aspS-gatCAB) and transformed into E. coli JF448 cells. The cells were grown and assayed as described previously on M9 minimal media agar plates with or without Asn 600 of 0.45. The samples were then diluted 100-fold in M9 minimal media before spotting 2 µL of culture on M9 minimal media agar ampicillin (100 µg/ml) plates with or without L-Asn (20 µg/ml), and supplemented with the other 19 amino acids (20 µg/ml each).The asnS), GlnRS (glnS), GatCAB , AsnA (asnA), AsnB (asnB), and AspRS (aspS). Genes were searched either in the UniProt (http://www.uniprot.org) or KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) databases. Sequences were then compared to known relevant enzymes by BLAST to validate the presence of the relevant active sites and domain architecture. The E. coli CFT AsnRS (AAN79540), GlnRS (AAN79239), AsnA (AAN83104), and AsnB (AAN79222), the H. pylori J99 GatA (AAD06348) and GatB (AAD06184), and the Deinococcus radiodurans discriminating AspRS (AAF10918) and ND-AspRS (AAF10623) sequences were used for the analysis. This was of particular importance to distinguish AsnA, which lacks an anticodon-binding domain, from its orthologs, AspRS and AsnRS Asn isoacceptors of these bacteria were analyzed for the presence of a U1-A72 base pair. The tRNA isoacceptors sequences studied were either from the Genomic tRNA Database (http://lowelab.ucsc.edu/GtRNAdb/) or the KEGG database (http://www.genome.jp/kegg/). The results of the survey are detailed in Bacterial genomes representing 547 different genera were analyzed for genes encoding AsnRS  to synthesize Asn B. bacteriovorus AspRS aspartylates tRNAAsn to enable the bacterium to synthesize Asn in a tRNA-dependent manner.For B. bacteriovorus AspRS is non-discriminating, we overproduced the enzyme in E. coli and purified it to homogeneity Asp and tRNAAsn to similar levels  lack an AsnRS and the indirect route with GatCAB is the only means for Asn-tRNAAsn synthesis. Primarily, these bacteria are Actinobacteria in the order Actinomycetales. Of those with GatCAB and AsnRS, four are Firmicutes in the Clostridiaceae family and all encode at least one Asn synthetase. However, in the case of C. acetobutylicum, the AsnB is split into two halves and does not appear to be functional under normal physiological conditions with Asn synthesis being tRNA-dependent Amycolatopsis mediterranei RB, also encoded an additional AspRS with AsnRS and GatCAB. It also in its genome has four asnB genes encoding the glutamine-dependent Asn synthetase (AsnB).Beyond the Deinococcus-Thermus phylum, an additional 15 bacterial genera encode an extra AspRS . C. acetAsn biosynthetic pathways. AsnRS and GatCAB are encoded in 174 bacterial genera with 163 also coding for a tRNAAsn with a U1-A72 base pair  while AsnRS evolved from a duplication of an archaeal AspRS Asn formation in LUCA was via the indirect pathway using a ND-AspRS and GatCAB Asn formation Two distinct routes for Asn-tRNAL. delbruekii bulgaricus, the presence of AsnRS to aminoacylate tRNAAsn appears to have lessened the selective pressure for AspRS to recognize tRNAAsn as a substrate, facilitating the AspRS to evolve specicity for just tRNAAspL. delbruekii bulgaricus GatCAB is for only Gln-tRNAGln synthesis B. bacteriovorus, AspRS retained its relaxed tRNA specificity along with GatCAB after acquisition of AsnRS possibly for tRNA-dependent Asn biosynthesis as was found in S. aureusAsn formation before acquiring AsnRS to directly attach the Asn to tRNAAsn. As many δ-proteobacteria, like the predatory bacterium Myxococcus xanthusGeobacter metallireducensAsn formation as hypothesized for B. bacteriovorus, L. pneumophila, and S. aureusIn some bacterial species like Asn formation. Based on our genomic analysis, about 30% of bacterial genera surveyed could code for both routes as they encode GatCAB, AsnRS, and tRNAAsn isoacceptors with a U1-A72 base pair. In the few bacteria like B. bacteriovorus that also encode GlnRS but neither Asn synthetase, it is likely GatCAB was retained for tRNA-dependent Asn production. Similarly, those bacteria like S. aureus without a GlnRS and both Asn synthetases may also use GatCAB for tRNA-dependent Asn biosynthesis in addition to Gln-tRNAGln formation Asn formation as their AspRS enzymes might be specific for tRNAAsp. For example, L. delbruekii bulgaricus AspRS does not aspartylate tRNAAsn and uses GatCAB only for Gln-tRNAGln formation Asn formation, providing the foundation to better understand how Asn metabolism and protein synthesis are integrated into bacterial physiology and adaptation to certain environmental niches.It is unclear how many other bacteria encode both routes for Asn-tRNATable S1Asn, Gln-tRNAGln, and Asn synthesis.Genomic analysis of bacterial genomes from 547 different genera for genes related to Asn-tRNA(XLSX)Click here for additional data file.Table S2Asn, Gln-tRNAGln, and Asn synthesis.Genomic analysis of bacterial genomes encoding at least two AspRS enzymes for genes related to Asn-tRNA(XLSX)Click here for additional data file.


Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs) recorded in the neurons that initiate startle, the Mauthner-cells (M-cell). We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms) and rapidly (<50 ms) decaying (feed-forward) inhibitory process that contributes to PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI). Additionally we observed increases of the evoked postsynaptic potential (PSP) peak amplitude  and duration . In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested interstimulus intervals (ISIs) (20–500 ms). Bicuculline also increased PSP peak amplitude  and PSP duration . Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs) by 15.07 ± 3.21%, N = 7 and 16.23 ± 7.08%, N = 5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic) inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.Prepulse inhibition (PPI) is understood as a sensorimotor gating process that attenuates sensory flow to the startle pathway during early stages (20–1000 ms) of information processing. Here, we applied  Startle is a rapid, massive contraction of facial and skeletal muscles that is triggered by the onset of intense and/or abrupt visual, auditory, or tactile stimuli. Startle is thought to function as a protective mechanism that minimizes impacts to vulnerable areas  and/or facilitates collision avoidance or escape  contributing during the peak inhibitory response, and GABAB receptors adding to the longer lasting inhibition mediated by muscarinic receptors   circuit in teleost fish presents an alternative model system for studying PPI and startle plasticity that is accessible to 5A receptors modulate the excitability of goldfish M-cells, and linked these effects to changes in startle plasticity. Given these advances in our understanding of neuromodulatory processes contributing to startle plasticity, here we investigated the signaling mechanisms directly mediating PPI at the level of the M-cell.A growing number of studies indicate that PPI in the M-cell system is modulated by multiple pre- and post-synaptic mechanisms. Neumeister et al.  showed tARs in the mediation of PPI in the M-cells, the decision-making neurons of the goldfish auditory startle circuit. We targeted these receptor systems because they are densely expressed in the M-cell membrane  of either sex were used in these experiments. Adult fish 7–13 cm in standard body length were purchased from Hunting Creek Fisheries . Fish were socially housed, with 5–6 fish per 60L aquaria, in recirculating conditioned water  with a 12:12 light/dark photoperiod. Animals were housed and treated in accord with protocols established by the Institutional Animal Care and Use Committee (IACUC) of Hunter College, City University of New York.Sixteen common goldfish  electrical stimulation generated by an isolated stimulator . A visible muscular contraction (twitch) was elicited with spinal stimulation to confirm proper placement of the spinal electrode, then d-tubocurarine  was administered intramuscularly. When the twitch response was abolished, typically within 0–3 min of injecting the tubocurarine, a craniotomy was performed to expose the medulla for microelectrode placement and recordings. The water level in the recording chamber was maintained throughout these procedures (and subsequent recordings) at the height of the mid-body, below the spinal incision.The M-cell was localized by a characteristic negative extracellular potential (15–20 mV) generated in the axon cap during antidromic stimulation, which provides an unambiguous indicator of electrodes’ placement relative to the soma and axon cap , the classical GlyR antagonist, and bicuculline (Tocris Biosciences), the classical GABAAR antagonist, because both drugs have long and well-documented histories of use in the M-cell system. Drugs were dissolved in a 500 μL solution of physiological saline  and superfused directly to the exposed medulla, as in past studies  stimuli were used to activate orthodromic inputs to the M-cells with or without a preceding sound stimulus (prepulse), the latter at multiple interstimulus intervals (ISIs) ranging between 20–500 ms. Sound stimuli in all experimental conditions were 200 Hz single-cycle “pips” produced at 80 dB re: 20 μPa in air. This stimulus intensity was chosen to elicit subthreshold responses in the M-cell because PPI is by definition elicited by subthreshold sounds, and the use of an identical conditioning (prepulse) and test (pulse) stimuli allows a within-subjects comparison of prepulse-pulse relationships on a trial-by-trial basis that is less sensitive to changes in baseline excitability. Stimuli were generated by a function generator , and output to a shielded subwoofer  placed 30 cm from the recording chamber. A microphone placed 10 cm above the fish’s head recorded acoustic waveforms and encoded these in parallel with intracellular recordings. A hydrophone  was also used for sound calibration but was removed during experiments. In testing conditions that measured PPI, sound stimuli were produced at 6 inter-stimulus intervals  measured from the onset of each stimulus.Intracellular recordings were analyzed offline with custom and commercial software . To analyze the contribution of distinct inhibitory networks on sensory processing, we measured the peak depolarization and duration of sound-evoked post-synaptic potentials (PSPs) for comparison across treatment conditions. PSP duration was defined as the time between peak depolarization and the decline of excitation to 37% of peak, as per the calculation of tau (τ). We also compared peak depolarizations of early and later components of sound-evoked PSPs in order to compare how consistently and/or selectively each treatment condition acted on discrete temporal components of the sound response (see Section Results for details).PULSE/PSPPREPULSE* 100), i.e., as the normalized percentage change of the pulse response by a prepulse. Thus, higher PPI values reflect greater inhibition. Importantly, this relative measure allows the consistency of prepulse-pulse relationships to be tested across treatment conditions independently of possible changes in M-cell excitability  presented 20–500 ms prior to an identical stimulus (pulse), as in Lingenhöhl and Friauf , Neumeist analyses were appropriate for the within-subjects design of these experiments. The exception to this was the analysis of the pharmacological effects on PPI, which by design considered three potential main effects , include two dimensions of repeated measures (ISI and drug treatment), and two axes that are better measured on continuous rather than categorical scales. Given those parameters, general linear mixed-models (GLMM) were used for those analyses. In these models subjects were treated as random effects  while ISI and drug-treatment were treated as fixed effects, and the dependent variable was the magnitude of PPI. Planned post hoc comparisons were made between comparable ISIs in drug and control conditions  with the Holmes-Bonferroni correction.Data were analyzed with JMP 10.0.0 (SAS) or Graphpad v5.0 (Prism). All data reported in figures and text reflect mean values and error bars illustrate SEM. All datasets were tested with the D’Agostino and Pearson Omnibus or K-S tests to confirm assumptions of normality were met for parametric statistical tests. In almost all cases, simple paired-N = 9), 5–10 responses in a single M-cell were measured at ISIs ranging from 20–500 ms in control and drug treatment conditions. The results show an overall attenuation of the PSP to the secondary stimulus (pulse) when compared to the PSP evoked by the lead stimulus (prepulse) in control conditions; in short, synaptic PPI  = 2.98, P = 0.088, N = 9), but our analysis identified a significant ISI X strychnine interaction  = 5.7276, P < 0.0001, N = 9). Post hoc tests (Holmes-Bonferonni) confirmed this effect was due to an ISI-specific reduction in the PPI effect at the 20 ms ISI (P < 0.001). These findings indicate that GlyRs mediate inhibition that contributes to PPI for as long as 20 ms, but this glycinergic component decays within 50 ms of the onset of prepulse stimuli.These experiments tested if treatment with strychnine, a GlyR antagonist, affects auditory PPI in the Mauthner cells (M-cells), the decision-making neurons of the goldfish startle circuit. Figures  Figures . After aN = 9; see Figure t test (t(8) = 6.08, P = 0.0003, N = 9). The duration of sound-evoked PSPs  was also significantly greater  = 6.11, P = 0.0003, N = 9; Figure As noted above, strychnine also affected the waveform of sound evoked M-cell PSPs, i.e., auditory processing. In order to analyze this more directly, we tested the effects of strychnine on M-cell sound-evoked PSPs evoked by a single sound pip . Figure N = 9) of peak depolarization during the initial EPSP, but a significantly greater enhancement of excitation during the mixed-PSP component of the response = 8.54, P = 0.0034; see Figure We next analyzed how the GlyR antagonist acted on different components of the overall sound response. The M-cell PSP reflects the integration of multiple excitatory and inhibitory inputs activated by primary auditory afferences. These include electrotonic and chemical excitation via mixed VIIIth nerve synapses at the M-cell lateral dendrite  sound-evoked excitation was measured in single M-cells for 5–10 trials at varying ISIs before and after treatment with an antagonist. Similar to the effects we observed with strychnine, we found that the GABAAR antagonist caused prominent changes in the amplitude and duration of sound evoked M-cell PSPs; these effects are analyzed in detail below. In contrast to the effects of strychnine, however, we found that treatment with bicuculline severely disrupted PPI. This was apparent in the overall reduction in PPI over the entire range of ISIs tested  = 89.3722, P < 0.0001, N = 5) and the interaction of bicuculline x ISI  = 7.4119, P < 0.0001, N = 5). Post hoc analyses (Bonferonni-Holm corrected) found the latter effect was attributable to ISI-specific reductions in PPI intensity at ISIs of 20 ms (P < 0.001), 50 ms (P < 0.001), 75 ms (P < 0.001), and 100 ms (P < 0.05). In sum, these results indicate that blockade of GABAARs causes a general disruption of PPI.We applied the same experimental approach as described above to test the effects of bicuculline (10 mM superfusion), a GABAAR antagonist, bicuculline on sound-evoked depolarization in separate trials using only a single sound pip (without prepulses) to determine how GABAARs contribute to auditory processing independently of PPI. We again approached our analysis by examining the effect of the GABAAR antagonist on the overall peak depolarization and duration of sound-evoked excitation. Figure t(4) = 28.12, P < 0.0001, N = 5). Similarly, the duration of sound-evoked PSPs increased by 284.95 ± 65.64% in drug conditions  = 3.07, P = 0.037, N = 5).In these experiments, we tested the effects of the GABAAR antagonist on initial (EPSP) and subsequent components (PSP) of the sound-response  = 1.116, P = 0.327, N = 5). The latter result is consistent with a drug induced increase in presynaptic excitation and/or by a decrease in inhibitory tone in the M-cell system which increases the neurons excitability (see below).As in our analysis of GlyR-mediated components of sound-evoked excitation, we also measured the potentially differential effects of the GABAAR antagonists enhance sound-evoked excitation in the M-cell  = 4.314, P = 0.005) consistent with a decrease in conductance. Importantly, RMP  was not affected by treatment with strychnine (t(9) = 0.4104, P = 0.69), indicating the disruption of a shunting inhibition rather than a persistent hyperpolarization.In prior experiments, we reported that GlyR and GABA Figures , 4. Name Figures . Figure t(4) = 3.09, P = 0.036, N = 5), but had no significant effect on RMP , consistent with a shunting inhibitory process.Similarly, bicuculline treatment increased the magnitude of APs by 16.23 ± 7.08% . Our primary findings indicate that GABAARs function as effector mechanisms mediating the onset and peak effect of PPI, corresponding to interstimulus-intervals (ISIs) ranging from 20–100 ms. GlyRs, in contrast, are primarily involved in the mediation of fast-onset feed-forward (sound-evoked) inhibitory processes that rapidly decay but overlap and contribute to the onset of PPI (20 ms). Independent of these distinct roles in sound-evoked inhibition, GABAARs and GlyRs both act as mediators of tonic inhibitory processes that modulate M-cell excitability, causing corresponding shifts in the magnitude and duration of sound-evoked excitation.The aim of this study was to determine if GlyRs and/or GABAAR antagonist was most prominent at ISIs from 20–100 ms, corresponding to the time-course of peak inhibition, i.e., where PPI is strongest. At longer ISIs, the reductions in PPI in bicuculline conditions relative to controls were not statistically significant (P > 0.05); however, this may be mainly due to the generally weak effect of PPI at long ISIs. We interpret the failure of PPI after pharmacological blockade as direct, in vivo evidence that GABAARs function as critical effector mechanisms mediating PPI at the level of the M-cells. The effects observed here can conceptually be understood as a form of long lasting GABAergic “feed-foward” inhibition that works in concert with the fast acting and fast decaying glycinergic feed-forward inhibition that has been previously described in the M-cell  tested, 20 ms. PPI was fully recovered and comparable to control conditions within 50 ms and for all longer ISIs. Further, even at the 20 ms ISI, the GlyR antagonist never fully abolished PPI effects, suggesting that the GlyR-dependent component of PPI acts in concert with another inhibitory process at this short latency from prepulse onset. Indeed, in experiments with the GABAAR antagonist, strychnine treatment significantly increased the peak magnitude and duration of sound-evoked excitation independently of PPI; importantly, some of these effects were time-dependent relative to the onset of the sound stimulus. That is, although strychnine increased the peak magnitude of sound-evoked excitation in the earliest phase of auditory processing (EPSP), prior to the onset of feed-forward inhibition, the enhancement of excitation during the mixed-PSP phase that includes inhibition was dramatically greater. The differential enhancement of the EPSP relative to the PSP reflects the disruption of distinct processes; namely, a tonic inhibition that persistently modulates excitability, and a sound-activated phasic inhibition that significantly influences the magnitude and time-course of the sound response, i.e., the temporal fidelity of auditory processing. In additional experiments we showed that the magnitude of antidromically-evoked APs increased after strychnine treatment, indicating a general increase in M-cell excitability consistent with the enhancement of the EPSP. Tonic glycinergic inhibition of the M-cell has been characterized in previous studies . Our results thus emphasize the likely contribution of multiple inhibitory pathways in the mediation of PPI. These findings should be interpretated cautiously, however, as the selective effects of the antagonists used are dependent on their effective concentrations, which could not be confirmed in the intact, in vivo preparations used in these experiments.Our results show that feed-forward inhibition and PPI are overlapping phenomena; however, they are functionally distinct and operate over different times-scales. Namely, feed-forward inhibition, like PPI, is recruited by weak auditory stimuli; unlike PPI, feed-forward inhibition attenuates sound-evoked excitation, generally, rather than selectively inhibiting responses to subsequent stimuli. Experiments testing auditory responses without prepulses showed that the onset of sound-evoked glycinergic inhibition occurs during sound-evoked excitation; further, in PPI experiments, we showed that sound-evoked glycinergic inhibition persists to 20 ms but is decayed within 50 ms. Thus, put plainly, sound-evoked glycinergic inhibition is recruited too early and decays too rapidly to mediate PPI, but this time-course is entirely consistent with the well-characterized primary (disynaptic) feed-forward inhibitory pathway (Korn et al., ex vivo brain-slice preparation. Moreover, the time-course of PPI mediated by GABAARs reported in rodents is similar to the ISI-specific effects we report here in the fish startle system.These results highlight some striking similarities with advances in rodent model systems. Yeomans et al.  showed tin vitro patch-clamp recordings to demonstrate that glycine directly inhibits PnC neurons in a rat brain slice preparation; however, they found no evidence that GlyRs were involved in phasic inhibitory processes, including feed-forward inhibition and short-term synaptic depression.Our findings are somewhat in contrast with studies of glycinergic inhibition in rodent preparations. Koch and Friauf  showed tin vivo synaptic processes in mature, awake goldfish, in vitro slice preparations used to record from PnC neurons were derived from embryonic rat brains (Yeomans et al., ARs during development, functional differences between mature and embryonic circuits may be expected (Ehrlich et al., In contrast, our experiments identified GlyR-dependent phasic inhibitory processes that attenuate sound-evoked excitation in multiple contexts Figures , 2. Thesin vivo synaptic signaling mechanisms that directly mediate the balance of excitation and inhibition at the sensorimotor interface of the startle circuit. Prior studies in the M-cell and other model systems have examined the role of neuromodulators, particularly monoaminergic transmitters (Medan and Preuss, in vivo electrophysiological methods can be applied to dissect overlapping inhibitory processes and effector mechanisms to directly test predictions drawn from advances in other model systems. Thus the M-cell presents an appropriate tool for dissecting the functional roles of synaptic processes as well as the effector mechanisms mediating their effects.In sum, this study characterized 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.