The Chloroplast Envelope of Angiosperms Contains a Peptidoglycan Layer.

Plastids in plants are assumed to have evolved from cyanobacteria as they have maintained several bacterial features. Recently, peptidoglycans, as bacterial cell wall components, have been shown to exist in the envelopes of moss chloroplasts. Phylogenomic comparisons of bacterial and plant genomes have raised the question of whether such structures are also part of chloroplasts in angiosperms. To address this question, we visualized canonical amino acids of peptidoglycan around chloroplasts of Arabidopsis and Nicotiana via click chemistry and fluorescence microscopy. Additional detection by different peptidoglycan-binding proteins from bacteria and animals supported this observation. Further Arabidopsis experiments with D-cycloserine and AtMurE knock-out lines, both affecting putative peptidoglycan biosynthesis, revealed a central role of this pathway in plastid genesis and division. Taken together, these results indicate that peptidoglycans are integral parts of plastids in the whole plant lineage. Elucidating their biosynthesis and further roles in the function of these organelles is yet to be achieved.

Hybrid Chemoenzymatic Synthesis of C7 -Sugars for Molecular Evidence of in†vivo Shikimate Pathway Inhibition.

The design of distinctive chemical synthesis strategies aims for the most efficient routes towards versatile compounds in drug target studies. Here, we establish a powerful hybrid synthetic approach of total chemical and chemoenzymatic synthesis to efficiently obtain various 7-deoxy-sedoheptulose (7dSh, 1) analogues, unique C7 sugars, for structure-activity relationship studies. 7dSh (1) is a rare microbial sugar with in planta herbicidal activity. As natural antimetabolite of 3-dehydroquinate synthase (DHQS), 7dSh (1) inhibits the shikimate pathway, which is essential for the synthesis of aromatic amino acids in bacteria, fungi, and plants, but absent in mammals. As glyphosate, the most used chemical herbicide faces restrictions worldwide, DHQS has gained more attention as valid target of herbicides and antimicrobial agents. In vitro and in vivo analyses of the C7 -deoxysugars confirm DHQS as enzymatic target, highlight the crucial role of uptake for inhibition and add molecular aspects to target mechanism studies of C7 -sugars as our contribution to global efforts for alternative weed-control strategies.

Flashing lights and epileptic spasms: should we be routinely performing intermittent photic stimulation in infants?

Abnormal cortical excitation in response to photic stimulation (photosensitivity) has historically been associated with generalized epilepsies, in patients outside of infancy. At our tertiary centre, we encountered a patient with infantile spasms secondary to a mutation in ALG13 (c320A>G) who had photic stimulation-induced epileptic spasms over a broad range of frequencies on multiple EEGs, which were worse without treatment and decreased as treatment was escalated. This is the first reported case of epileptic spasms triggered by photic stimulation and it is unclear whether the phenomenon is unique to this patient, to those with this mutation or whether it is present in a broader group of patients with infantile spasms.

Longitudinally extensive transverse myelitis with positive aquaporin-4 IgG associated with dengue infection: a case report and systematic review of cases.

BACKGROUND: Neuromyelitis Optica Spectrum Disorder can be associated with parainfectious and post-infectious triggers. Dengue virus infection is one of the most common arbovirus infections in the world, and may present with neurological manifestations. OBJECTIVES: We present a case of DENV-associated with LETM and positive aquaporin-4 IgG, and a systematic review of published cases. METHODS: Medline (Ovid) and PubMed were search through June 2021, for case reports, series and observational studies that described patients with DENV-associated LETM and/or NMOSD. RESULTS: An adolescent girl who had recently immigrated from a Dengue-endemic region presented with a LETM with high positive AQP4-IgG titer and seropositive DENV IgM/IgG antibodies. She responded well to steroids and subsequently started maintenance rituximab for her NMOSD diagnosis. LITERATURE REVIEW: 22 publications describing 27 patients met inclusion criteria. In addition to this case, three published cases met current criteria for NMOSD with serological evidence of acute DENV infection. CONCLUSIONS: It is unknown whether there is a pathophysiological association between DENV infection and NMOSD. Regardless, if an immune-mediated event is suspected, particularly NMOSD, appropriate immunotherapy should be considered early. Decision regarding long term immunotherapy may depend on index of suspicion of true NMOSD, and this is where AQP4-IgG status and follow-up is helpful.

Patterns of Recording Epileptic Spasms in an Electronic Seizure Diary Compared With Video-EEG and Historical Cohorts.

BACKGROUND: Use of electronic seizure diaries (e-diaries) by caregivers of children with epileptic spasms is not well understood. We describe the demographic and seizure-related information of children with epileptic spasms captured in a widely used e-diary and explore the potential biases in how caregivers report these data. METHODS: We analyzed children with epileptic spasms in an e-diary, Seizure Tracker, from 2007 to 2018. We described variables including sex, time of seizure, percentage of spasms occurring as individual spasms (versus in clusters), cluster duration, and number of spasms per cluster. We compared seizure characteristics in the e-diary cohort with published cohorts to identify biases in caregiver-reported epileptic spasms. We also reviewed seizure patterns in a small cohort of children with epileptic spasms monitored on overnight video-electroencephalography (vEEG). RESULTS: There were 314 children in the e-diary cohort and nine children in the vEEG cohort. The e-diary cohort was more likely than expected to report counts divisible by five. The e-diary cohort had a lower proportion of nighttime spasms than expected based on data from published cohorts. The e-diary cohort had a significantly lower percentage of spasms as individual spasms, a greater number of spasms per cluster, and a greater cluster duration relative to the vEEG cohort. CONCLUSIONS: Caregivers using e-diaries for epileptic spasms may miss individual spams, be more likely to report long clusters, round counts to the nearest five, and underreport nighttime spasms. Clinicians should be aware of these reporting biases when using e-diary data to guide care for children with epileptic spasms.

Familial neonatal seizures caused by the Kv7.3 selectivity filter mutation T313I.

OBJECTIVE: A spectrum of seizure disorders is linked to mutations in Kv7.2 and Kv7.3 channels. Linking functional effects of identified mutations to their clinical presentation requires ongoing characterization of newly identified variants. In this study, we identified and functionally characterized a previously unreported mutation in the selectivity filter of Kv7.3. METHODS: Next-generation sequencing was used to identify the Kv7.3[T313I] mutation in a family affected by neonatal seizures. Electrophysiological approaches were used to characterize the functional effects of this mutation on ion channels expressed in Xenopus laevis oocytes. RESULTS: Substitution of residue 313 from threonine to isoleucine (Kv7.3[T313I]) likely disrupts a critical intersubunit hydrogen bond. Characterization of the mutation in homomeric Kv7.3 channels demonstrated a total loss of channel function. Assembly in heteromeric channels (with Kv7.2) leads to modest suppression of total current when expressed in Xenopus laevis oocytes. Using a Kv7 activator with distinct effects on homomeric Kv7.2 vs heteromeric Kv7.2/Kv7.3 channels, we demonstrated that assembly of Kv7.2 and Kv7.3[T313I] generates functional channels. SIGNIFICANCE: Biophysical and clinical effects of the T313I mutation are consistent with Kv7.3 mutations previously identified in cases of pharmacoresponsive self-limiting neonatal epilepsy. These findings expand our description of functionally characterized Kv7 channel variants and report new methods to distinguish molecular mechanisms of channel mutations.

Sensory input from the osphradium modulates the response to memory-enhancing stressors in Lymnaea stagnalis.

In the freshwater environment species often rely on chemosensory information to modulate behavior. The pond snail, Lymnaea stagnalis, is a model species used to characterize the causal mechanisms of long-term memory (LTM) formation. Chemical stressors including crayfish kairomones and KCl enhance LTM formation (≥24 h) in Lymnaea; however, how these stressors are sensed and the mechanism by which they affect the electrophysiological properties of neurons necessary for memory formation are poorly understood. Here, we assessed whether the osphradium, a primary chemosensory organ in Lymnaea, modulates LTM enhancement. To test this we severed the osphradial nerve proximal to the osphradium, using sham-operated animals as controls, and assessed the behavioral and electrophysiological response to crayfish kairomones and KCl. We operantly conditioned aerial respiratory behavior in intact, sham and osphradially cut animals, and tested for enhanced memory formation after exposure to the chemical stressors. Sham-operated animals displayed the same memory enhancement as intact animals but snails with a severed osphradial nerve did not show LTM enhancement. Extracellular recordings made from the osphradial nerve demonstrate that these stressors evoked afferent sensory activity. Intracellular recordings from right pedal dorsal 1 (RPeD1), a neuron necessary for LTM formation, demonstrate that its electrophysiological activity is altered by input from the osphradium following exposure to crayfish kairomones or KCl in sham and intact animals but no response is seen in RPeD1 in osphradially cut animals. Therefore, sensory input from the osphradium is necessary for LTM enhancement following exposure to these chemical stressors.

Differences in neuronal activity explain differences in memory forming abilities of different populations of Lymnaea stagnalis.

The ability to learn and form long-term memory (LTM) can enhance an animal's fitness, for example, by allowing them to remember predators, food sources or conspecific interactions. Here we use the pond snail, Lymnaea stagnalis, to assess whether variability between natural populations (i.e., strains) in memory forming capabilities correlates with electrophysiological properties at the level of a single neuron, RPeD1. RPeD1 is a necessary site of LTM formation of aerial respiratory behaviour following operant conditioning. We used strains from two small, separate permanent ponds (TC1 and TC2). A comparison of the two populations showed that the TC1 strain had enhanced memory forming capabilities. Further, the behavioural phenotype of enhanced memory strain was explained, in part, by differences in the electrophysiology of RPeD1. Compared to RPeD1 from the naive TC2 strain, RPeD1 from the TC1 strain has both a decreased resistance and decreased excitability. Moreover, 24h after a single 0.5h training session, those membrane properties, as well as the firing and bursting rate, decrease further in the TC1 strain but not in the TC2 strain. The initial differences in RPeD1 properties in the TC1 strain coupled with their ability to further change these properties with a single training session suggests that RPeD1 neurons from the TC1 strain are "primed" to rapidly form memory.

Intermediate and long-term memory are different at the neuronal level in Lymnaea stagnalis (L.).

Both intermediate-term memory (ITM) and long-term memory (LTM) require novel protein synthesis; however, LTM also requires gene transcription. This suggests that the behavioural output of the two processes may be produced differently at the neuronal level. The fresh-water snail, Lymnaea stagnalis, can be operantly conditioned to decrease its rate of aerial respiration and, depending on the training procedure, the memory can last 3h (ITM) or >24h (LTM). RPeD1, one of the 3 interneurons that form the respiratory central pattern generator (CPG) that drives aerial respiration, is necessary for memory formation. By comparing RPeD1's electrophysiological properties in naïve, 'ITM-trained', 'LTM-trained' and yoked control snails we discovered that while the behavioural phenotype of memory at 3 and 24h is identical, the situation at the neuronal level is different. When examined 3h after either the 'ITM' or 'LTM' training procedure RPeD1 activity is significantly depressed. That is, the firing rate, input resistance, excitability and the number of action potential bursts are all significantly decreased. In snails receiving the ITM-training, these changes return to normal 24h post-training. However, in snails receiving the 'LTM-training', measured RPeD1 properties (firing rate, excitability, membrane resistance, and the number of action potential bursts fired) are significantly different at 24h than they were at 3h. Additionally, 24h following LTM training RPeD1 appears to be functionally "uncoupled" from its control of the pneumostome as the link between RPeD1 excitation and pneumostome opening is weakened. These data suggest that the behavioural changes occurring during LTM are due to more widespread neuronal reorganization than similar behavioural changes occurring during ITM. Thus ITM and LTM are not just distinct in a chronological and transcriptional manner but are also distinct at the level of neuronal properties.

Low environmental calcium blocks long-term memory formation in a freshwater pulmonate snail.

The freshwater snail Lymnaea stagnalis (L.) is considered a calciphile and exhibits reduced growth and survival in environments containing less than 20 mg/l environmental calcium. Although it has no apparent effect on survival at 20 mg/l, reducing environmental calcium increases metabolic demand, and as such we consider that this level of calcium acts as a stressor on the snail. We exposed snails to acute periods of low environmental calcium and tested their ability to form intermediate-term memory (ITM) and long-term memory (LTM) following one trial operant conditioning (1TT) to reduce aerial respiratory activity in hypoxic conditions. We also assessed whether there were changes in the electrophysiological properties of a single neuron, right pedal dorsal 1 (RPeD1), which has been demonstrated to be necessary for LTM formation. Following training in high (80 mg/l) environmental calcium, L. stagnalis formed ITM and LTM lasting 24 h and demonstrated a significant reduction in all activity measured from RPeD1; however when snails were exposed to low (20 mg/l) environmental calcium they were able to form ITM but not LTM. Although no behavioral LTM was formed, a partial reduction in RPeD1 activtiy measured 24 h after training was observed, indicating a residual effect of training. The strong effect that environmental calcium concentration had on physiology and behavior in response to training to reduce aerial respiration in L. stagnalis suggests that it is an element of gastropod husbandry that needs to be carefully considered when studying other traits. This study also indicates that L. stagnalis found naturally in low calcium environments may be less able to adapt to novel stressors than populations found in harder waters.

The consequences of reversible gill remodelling on ammonia excretion in goldfish (Carassius auratus).

Goldfish acclimated to cold water (e.g. 7°C) experience a marked reduction in functional lamellar surface area owing to the proliferation of an interlamellar cell mass (ILCM), a phenomenon termed gill remodelling. The goal of the present study was to assess the consequences of the reduced functional surface area on the capacity of goldfish to excrete ammonia. Despite the expected impact of ambient temperature on functional surface area, fish acclimated to 7°C and 25°C exhibited similar rates of ammonia excretion (J(net,amm)); the Q10 values for fed and starved fish were 1.07 and 1.20, respectively. To control for possible temperature-related differences in rates of endogenous ammonia production, J(net,amm) was determined at the two acclimation temperatures after loading fish with 1.12 µmol g₋1 of NH4Cl. In the 3 h post-injection period, J(net,amm) was elevated to a greater extent in the 25°C fish. To estimate the potential contribution of increased ventilation and cardiac output to ammonia clearance in the warmer fish, the ammonia loading experiment was repeated on the 7°C fish immediately after they were exercised to exhaustion. The rate of excretion of ammonia was significantly increased in the exercised 7°C fish (presumably experiencing increased ventilation and cardiac output for at least some of the measurement period) suggesting that differences in external and internal convection may at least partially explain the enhanced capacity of the 25°C fish to clear the ammonia load. To more specifically assess the contribution of the different functional surface areas on the differing rates of ammonia clearance at the two acclimation temperatures, the 7°C fish were exposed for 7 days to hypoxia (P(O2)=10 mmHg=1.33 kPa), a treatment known to cause the disappearance of the ILCM. The results demonstrated that the hypoxia-associated loss of the ILCM was accompanied by a significant increase in the rate of ammonia clearance in the 7°C fish when returned to normoxic conditions. To determine whether compensatory changes in the ammonia transporting proteins might be contributing to sustaining J(net,amm) under conditions of reduced functional lamellar surface area, the relative expression and branchial distribution of four Rh proteins were assessed by western blotting and immunocytochemistry. Although the relative expression of the Rh proteins was unaffected by acclimation temperature, there did appear to be a change in the spatial distribution of Rhag, Rhbg and Rhcg1. Specifically, these three Rh proteins (and to a lesser extent Rhcg2) appeared to localize in cells on the outer edge of the ILCM that were enriched with Na(+)/K(+)-ATPase. Thus, we suggest that despite the impediment to ammonia excretion imposed by the ILCM, goldfish acclimated to 7°C are able to sustain normal rates of excretion owing to the redistribution of ammonia transporting cells.

Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii: Evidence for the involvement of Rh and UT proteins.

The nature of ammonia and urea excretion was examined in the Pacific hagfish (Eptatretus stoutii), which, under resting conditions, excreted similar quantities of nitrogen as either ammonia or urea. In the presence of high external ammonia (HEA) concentrations, ammonia was taken up at high rates and then excreted at similarly high rates upon return to normal water. However, although elevated by HEA, plasma ammonia levels were maintained at approximately 1-4 µmolNg⁻¹, reflecting time-dependent decreases in the rates of ammonia uptake, the possible conversion of ammonia to urea, and the potential active excretion of ammonia against a gradient. Internal injections of NH4Cl caused marked increases in the rate of ammonia excretion and a delayed increase in urea excretion that may have resulted from increasing urea levels in the plasma. Conversely, when the rate of urea excretion was reduced in the presence of 0.1 mM phloretin, ammonia excretion was significantly elevated. Rates of urea excretion were initially increased by approximately 1000-fold following internal urea injections while the presence of high external urea levels (5-100 mM final concentration) resulted in associated linear increases in plasma urea levels. Using hagfish skin mounted in Ussing chambers, the rate of diffusion of ammonia across the skin exceeded that of urea by approximately four times when equivalent gradients were imposed. Based on western blotting and immunocytochemistry, hagfish gill appears to possess Rh proteins (Rhag, Rhbg and Rhcg1) and urea transporter proteins. Despite the tolerance of hagfish to high levels of ammonia and urea, it is suggested that the presence of ammonia and urea transporter proteins may be required during the period of time hagfish spend in burrows or while feeding, when conditions of high ammonia and/or urea might be encountered.

Do zebrafish Rh proteins act as dual ammonia-CO2 channels?

Transfer of the small gaseous molecules CO2 and NH3 across biological membranes, long thought to occur solely by simple diffusion, is now known to be facilitated by members of two multigene families: aquaporins (AQP) and rhesus (Rh) proteins. Although it is accepted that AQP1 and Rh proteins are involved in CO2 and NH3 transfer, respectively, the idea that a single channel can exhibit selectivity for both gases is controversial. Indeed, studies using the same in vitro model (human red blood cells) have provided evidence both for and against a role for Rh proteins as CO2 channels. Thus, this study was initiated to provide in vivo evidence for a dual function of Rh proteins as ammonia and CO2 channels. Here, we show that in zebrafish (Danio rerio), direct ammonia-CO2 competition experiments in adults or translational knockdowns of Rh proteins in larvae affects both ammonia and CO2 excretion. These results suggest that Rh proteins in zebrafish may be common pathways for transport of ammonia and CO2.

Acid-base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost.

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid-base regulation during respiratory acidosis either by facilitating or constraining HCO(3)(-) accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid-base status during exposure to increasing levels of hypercapnia (nominally 1-5% CO(2)). Fish exhibited a well-developed ability to increase plasma HCO(3)(-) levels with values of 39.8 ± 2.8 mmol l(-1) being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO(2) = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO(3)(-) during hypercapnia was associated with marked increases in branchial net acid excretion (J (NET)H(+)) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J (NET)H(+) was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na(+)/K(+)-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO(3)(-) concentrations of 92.2 ± 4.8 mmol l(-1). At the highest level of hypercapnia, rectal fluid HCO(3)(-) levels were increased significantly to 141.8 ± 7.4 mmol l(-1) but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l(-1)). Thus, the impressive accumulation of plasma HCO(3)(-) to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO(3)(-) excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO(3)(-) concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO(3)(-). The apparent inability of P. notatus to effectively excrete HCO(3)(-) following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.

The responses of zebrafish (Danio rerio) to high external ammonia and urea transporter inhibition: nitrogen excretion and expression of rhesus glycoproteins and urea transporter proteins.

While adult zebrafish, Danio rerio, possess ammonia and urea transporters (Rh and UT proteins, respectively) in a number of tissues, they are most heavily concentrated within the gills. UT has a diffuse expression pattern within Na+-K+-ATPase (NKA)-type mitochondrion-rich cells and Rh proteins form a network similar to the arrangement seen in pufferfish gills (Nakada et al., 2007b). Rhag expression appeared to be limited to the pillar cells lining the blood spaces of the lamellae while Rhbg was localized to the outer layer of both the lamellae and the filament, upon the pavement cells. Exposure to high external ammonia (HEA) or phloretin increased tissue levels of ammonia and urea, respectively, in adult and juvenile zebrafish; however, the responses to these stressors were age dependent. HEA increased mRNA levels for a number of Rh proteins in embryos and larvae but did not elicit similar effects in adult gills, which appear to compensate for the unfavourable ammonia excretory gradient by increasing expression of V-type H+-ATPase. Phloretin exposure increased UT mRNA levels in embryos and larvae but was without effect in adult gill tissue. Surprisingly, in both adults and juveniles, HEA increased the mRNA expression of UT and phloretin increased the mRNA expression of Rh proteins. These results imply that, in zebrafish, there may be a tighter link between ammonia and urea excretion than is thought to occur in most teleosts.

Renal expression and localization of SLC9A3 sodium/hydrogen exchanger and its possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss).

Experiments were performed to assess the possible involvement of the Na(+)/H(+) exchanger isoform 3 (NHE3; SLC9A3) in renal acid-base regulation in adult rainbow trout (Oncorhynchus mykiss). NHE3 mRNA was expressed at high levels in the kidney relative to its paralog, NHE2. The results of in situ hybridization demonstrated an abundance of NHE3 mRNA in renal tubules. The combination of immunocytochemistry and histological staining revealed that NHE3 was confined to the apical membrane of proximal tubules, where it was colocalized with the vacuolar-type H(+)-ATPase. Levels of NHE3 protein (assessed by Western blotting) were increased during hypercapnia, likely as a result of increased transcription, as indicated by increasing levels of NHE3 mRNA (as determined by real-time PCR). Plasma cortisol concentration was increased during hypercapnia, and administration of exogenous cortisol caused a marked increase in NHE3 mRNA and protein. Thus we speculate that the elevation of plasma cortisol during hypercapnia contributes to transcriptional activation of NHE3 that ultimately promotes acid-base regulation by stimulating H(+) secretion and HCO(3)(-) reabsorption.

Wave reflection effects in the central circulation of American alligators (Alligator mississippiensis): what the heart sees.

A large central compliance is thought to dominate the hemodynamics of all vertebrates except birds and mammals. Yet large crocodilians may adumbrate the avian and mammalian condition and set the stage for significant wave transmission (reflection) effects, with potentially detrimental impacts on cardiac performance. To investigate whether crocodilians exhibit wave reflection effects, pressures and flows were recorded from the right aorta, carotid artery, and femoral artery of six adult, anesthetized American alligators (Alligator mississippiensis) during control conditions and after experimentally induced vasodilation and constriction. Hallmarks of wave reflection phenomena were observed, including marked differences between the measured profiles for flow and pressure, peaking of the femoral pressure pulse, and a diastolic wave in the right aortic pressure profile. Pulse wave velocity and peripheral input impedance increased with progressive constriction, and thus changes in both the timing and magnitude of reflections accounted for the altered reflection effects. Resolution of pressure and flow waves into incident and reflected components showed substantial reflection effects within the right aorta, with reflection coefficients at the first harmonic approaching 0.3 when constricted. Material properties measured from isolated segments of blood vessels revealed a major reflection site at the periphery and, surprisingly, at the junction of the truncus and right aorta. Thus, while our results clearly show that significant wave reflection phenomena are not restricted to birds and mammals, they also suggest that rather than cope with potential negative impacts of reflections, the crocodilian heart simply avoids them because of a large impedance mismatch at the truncus.