A high-throughput behavioral screening platform for measuring chemotaxis by C. elegans.

Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multiwell plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

An efficient behavioral screening platform classifies natural products and other chemical cues according to their chemosensory valence in C. elegans.

Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction, and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multi-well plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals, but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs.

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT­1d, UNC­8d, and MEC­4d formed homomeric channels and that, unlike MEC­4d and UNC­8d, DEGT­1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT­1d and UNC­8d channels. Unexpectedly, MEC­4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.

A family of hyperpolarization-activated channels selective for protons.

Proton (H+) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few proton channels have been identified so far. Here, we identify a family of voltage-gated "pacemaker" channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpolarization and conducts protons into the cytosol. Surprisingly, protons permeate through the channel's voltage-sensing domain, whereas the pore domain is nonfunctional. Key to proton permeation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional domains, HCNL1 evolutionarily adapted to a low-Na+ freshwater environment to conserve sperm's ability to depolarize.

Parallel Processing of Two Mechanosensory Modalities by a Single Neuron in C. elegans.

Neurons convert synaptic or sensory inputs into cellular outputs. It is not well understood how a single neuron senses, processes multiple stimuli, and generates distinct neuronal outcomes. Here, we describe the mechanism by which the C. elegans PVD neurons sense two mechanical stimuli: external touch and proprioceptive body movement. These two stimuli are detected by distinct mechanosensitive DEG/ENaC/ASIC channels, which trigger distinct cellular outputs linked to mechanonociception and proprioception. Mechanonociception depends on DEGT-1 and activates PVD's downstream command interneurons through its axon, while proprioception depends on DEL-1, UNC-8, and MEC-10 to induce local dendritic Ca2+ increase and dendritic release of a neuropeptide NLP-12. NLP-12 directly modulates neuromuscular junction activity through the cholecystokinin receptor homolog on motor axons, setting muscle tone and movement vigor. Thus, the same neuron simultaneously uses both its axon and dendrites as output apparatus to drive distinct sensorimotor outcomes.

Synaptic Communication upon Gentle Touch.

Gentle touch sensation in mammals depends on synaptic transmission from primary sensory cells (Merkel cells) to secondary sensory neurons. Hoffman et al. (2018) identify norepinephrine and ß2-adrendergic receptors as the neurotransmitter-receptor pair responsible for sustained touch responses. The findings may deepen understanding of how drugs affect touch and pain sensation.

Microfluidics for mechanobiology of model organisms.

Mechanical stimuli play a critical role in organ development, tissue homeostasis, and disease. Understanding how mechanical signals are processed in multicellular model systems is critical for connecting cellular processes to tissue- and organism-level responses. However, progress in the field that studies these phenomena, mechanobiology, has been limited by lack of appropriate experimental techniques for applying repeatable mechanical stimuli to intact organs and model organisms. Microfluidic platforms, a subgroup of microsystems that use liquid flow for manipulation of objects, are a promising tool for studying mechanobiology of small model organisms due to their size scale and ease of customization. In this work, we describe design considerations involved in developing a microfluidic device for studying mechanobiology. Then, focusing on worms, fruit flies, and zebrafish, we review current microfluidic platforms for mechanobiology of multicellular model organisms and their tissues and highlight research opportunities in this developing field.

Physiological evidence of sensory integration in the electrosensory lateral line lobe of Gnathonemus petersii.

Mormyrid fish rely on reafferent input for active electrolocation. Their electrosensory input consists of phase and amplitude information. These are encoded by differently tuned receptor cells within the Mormyromasts, A- and B-cells, respectively, which are distributed over the animal's body. These convey their information to two topographically ordered medullary zones in the electrosensory lateral line lobe (ELL). The so-called medial zone receives only amplitude information, while the dorsolateral zone receives amplitude and phase information. Using both sources of information, Mormyrid fish can disambiguate electrical impedances. Where and how this disambiguation takes place is presently unclear. We here investigate phase-sensitivity downstream from the electroreceptors. We provide first evidence of phase-sensitivity in the medial zone of ELL. In this zone I-cells consistently decreased their rate to positive phase-shifts (6 of 20 cells) and increased their rate to negative shifts (11/20), while E-cells of the medial zone (3/9) responded oppositely to I-cells. In the dorsolateral zone the responses of E- and I-cells were opposite to those found in the medial zone. Tracer injections revealed interzonal projections that interconnect the dorsolateral and medial zones in a somatotopic manner. In summary, we show that phase information is processed differently in the dorsolateral and the medial zones. This is the first evidence for a mechanism that enhances the contrast between two parallel sensory channels in Mormyrid fish. This could be beneficial for impedance discrimination that ultimately must rely on a subtractive merging of these two sensory streams.

Resection of the Tracheobronchial Bifurcation With Complete Preservation of Lung Parenchyma.

BACKGROUND: The resection of the tracheobronchial bifurcation with complete preservation of lung parenchyma remains a challenge owing to the limited indications for surgery, anesthesiologic management, operative technique, and postoperative course. The aim of this retrospective study was to evaluate factors influencing the perioperative course and long-term survival. METHODS: Between 1989 and 2014, 19 patients underwent a resection of the distal trachea and carina with complete preservation of lung tissue, 16 for malignant tumors (7 adenoid cystic carcinomas, 3 carcinoid tumors, 3 mucoepidermoid tumors, 2 squamous cell carcinomas, and 1 small cell carcinomas), 2 for inflammatory stenosis, and 1 after a complex traumatic rupture. RESULTS: Surgical approach was posterolateral thoracotomy in 17 patients and median sternotomy in 2. In 16 patients, end-to-end anastomosis was performed, and in 3 patients, combined end-to-end and side-to-end anastomosis were performed. The operative mortality was 0%, the perioperative complication rate was 26.3%. Six patients with adenoid cystic carcinoma and all patients with lung carcinoma received adjuvant radiotherapy; only 1 patient with small cell lung cancer had chemotherapy before surgery. Long-term results are excellent in patients with benign disease, typical and atypical carcinoid tumor, mucoepidermoid carcinoma, and in most patients with adenoid cystic carcinoma. Two patients with lung cancer died 28 and 45 months after surgery, and 1 patient with adenoid cystic carcinoma died 75 months after surgery. CONCLUSIONS: Resection of the tracheobronchial bifurcation with complete preservation of lung indicated for selected patients with local tumor growth at the distal trachea and carina provides low perioperative mortality and complications and results in long-term survival rates.

A K(+)-selective CNG channel orchestrates Ca(2+) signalling in zebrafish sperm.

Calcium in the flagellum controls sperm navigation. In sperm of marine invertebrates and mammals, Ca(2+) signalling has been intensely studied, whereas for fish little is known. In sea urchin sperm, a cyclic nucleotide-gated K(+) channel (CNGK) mediates a cGMP-induced hyperpolarization that evokes Ca(2+) influx. Here, we identify in sperm of the freshwater fish Danio rerio a novel CNGK family member featuring non-canonical properties. It is located in the sperm head rather than the flagellum and is controlled by intracellular pH, but not cyclic nucleotides. Alkalization hyperpolarizes sperm and produces Ca(2+) entry. Ca(2+) induces spinning-like swimming, different from swimming of sperm from other species. The "spinning" mode probably guides sperm into the micropyle, a narrow entrance on the surface of fish eggs. A picture is emerging of sperm channel orthologues that employ different activation mechanisms and serve different functions. The channel inventories probably reflect adaptations to species-specific challenges during fertilization.

Remediastinoscopy in restaging of lung cancer after induction therapy.

OBJECTIVES: The aim of the present study was to evaluate the feasibility and diagnostic value of repeat mediastinoscopy as part of the response-evaluation protocol of 2 phase II multimodality studies for either stage IIIA/B non-small cell lung cancer or small cell lung cancer. METHODS: From January 1991 through December 1998, 104 patients (79 men and 25 women) with stage IIIA/B non-small cell lung cancer (84 patients) or small cell lung cancer (17 patients) were enrolled in 2 different multimodality trials and underwent remediastinoscopy after induction chemoradiotherapy. The median age was 56 years (range, 34-72 years). Sensitivity, specificity, accuracy, and predictive values of remediastinoscopy were calculated by using standard definitions. RESULTS: Remediastinoscopy was feasible in 98% of cases. Mortality was nil, and morbidity very low (1.9%). Lymph node downstaging (N0) was observed in 84 patients, persisting N2 disease was observed in 15 patients, and N3 disease was observed in 5 patients. Sensitivity was 61%, specificity was 100%, and accuracy was 88%. Positive predictive and negative predictive values reached 100% and 85%, respectively. According to the results of remediastinoscopy, 81 patients underwent surgical intervention, 3 refused the operation, and an unnecessary thoracotomy could be avoided in the remaining 20. CONCLUSIONS: Remediastinoscopy provides a histologic proof of mediastinal downstaging with high diagnostic accuracy, is technically feasible with low morbidity, and still remains a valuable tool, even in an era of highly sophisticated imaging and endoscopic procedures. Persisting nodal disease at repeat mediastinoscopy carries a poor survival in the majority of cases because of occult metastases, so that indication for surgical intervention in such an unfavorable group of patients should be evaluated very carefully.