Longitudinal Progression of Patients with Long COVID Treated in a Post-COVID Clinic: A Cross-Sectional Survey.

BACKGROUND: In addition to the morbidity and mortality associated with acute infection, COVID-19 has been associated with persistent symptoms (>30 days), often referred to as Long COVID (LC). LC symptoms often cluster into phenotypes, resembling conditions such as fibromyalgia, postural orthostatic tachycardiac syndrome (POTS), and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). LC clinics have been established to best address the needs of LC patients and continuity of care. We developed a cross-sectional survey to assess treatment response through our LC Clinic (LCC). METHODS: A 25-question survey (1-10 Likert scale) was expert- and content-validated by LCC clinicians, patients, and patient advocates. The survey assessed LC symptoms and the helpfulness of different interventions, including medications and supplements. A total of 852 LCC patients were asked to complete the survey, with 536 (62.9%) responding. RESULTS: The mean time from associated COVID-19 infection to survey completion was 23.2 ± 6.4 months. The mean age of responders was 52.3 ± 14.1 (63% females). Self-reported symptoms were all significantly improved (P < .001) from the initial visit to the LCC (baseline) to the time of the follow-up survey. However, only 4.5% (24/536) of patients rated all symptoms low (1-2) at the time of the survey, indicating low levels of full recovery in our cohort. The patients rated numerous interventions as being helpful, including low-dose naltrexone (45/77; 58%), vagal nerve stimulation (18/34; 53%), and fisetin (28/44; 64%). CONCLUSIONS: Patients report general improvements in symptoms following the initial LCC visit, but complete recovery rates remain low at 23.2 ± 6.4 months.

Improving Operating Room Efficiency: Relocating a Surgical Oncology Program Within a Health Care System.

Background: To meet increased community and regional needs for quality services, our hospital system concluded that its established surgical oncology program-consisting of gynecologic oncology (4 physicians), surgical oncology (2 physicians), and otolaryngologic oncology (2 physicians)-would be best served by the transition of the comprehensive surgical oncology program to a new oncology-naive hospital. We describe the overall strategy and approach involved with this move, its implementation, operating room efficiency results, and physician satisfaction associated with the relocation. Methods: The purpose of the systematic plan for relocation, which was developed and refined during the 2 years preceding the move, was to facilitate a collective awareness and understanding of important patient-centered concepts and essential workflow. All parties involved in direct patient cancer care participated in multiple workgroups to successfully transition the surgical oncology practice. Following the transition to the oncology-naive hospital, components of the operative cases and surgical data were prospectively collected for the initial 6 weeks and compared to retrospective data from the last 8 weeks at the established hospital. The surgical day for each surgeon was deconstructed, and measured variables included total surgical cases, total surgical hours, surgical minutes per case, total anesthesia hours, first case on-time surgical starts, surgical stretcher wheels out to surgical stretcher wheels in, surgical stretcher wheels out to next case start, case end to postanesthesia care unit (PACU), and case end to case start. Results: Five hundred twenty-nine surgical cases encompassing 1,076 anesthesia hours and 710 surgical hours were completed during the 14-week evaluation period. The gynecologic oncologists completed the majority of surgical procedures in both settings. The percentage of first case on-time surgical starts initially decreased during the 6-week interval at the oncology-naive hospital, but interval subset analysis suggested a return to the pre-move norm. Surgical stretcher wheels out to surgical stretcher wheels in had a wide range (9 minutes to 305 minutes) for all surgical sections, but no statistically significant difference was seen overall or for any surgical section. Case end to PACU significantly increased for gynecologic oncology but not for surgical oncology or otolaryngologic oncology. Overall case end to case start times decreased nonsignificantly (63.7 ± 3.1 mean minutes vs 60.3 ± 1.7 mean minutes) following the move. A physician survey found that physicians' expectations were met in terms of the move occurring smoothly without major issues, surgical scheduling and accommodation, anesthesia services, and surgical personnel. Physicians indicated less satisfaction with quality and availability of instrumentation. Conclusion: The transfer of established surgical oncology services to an oncology-naive hospital was associated with early surgeon and operating room staff support, as well as process and programmatic alignment among stakeholders. The success of this transition required transparency, open and honest communication, and problem solving at all levels. The move of a surgical oncology program to an oncology-naive hospital was deemed successful without deterioration of time-related variables associated with operating room efficiency and physician satisfaction. The breakdown and analysis of key components of the surgical day offered additional opportunities for quality improvement in operating room efficiency.

Molecular evidence of undescribed Ceratonova sp. (Cnidaria: Myxosporea) in the freshwater polychaete, Manayunkia speciosa, from western Lake Erie.

We used PCR to screen pooled individuals of Manayunkia speciosa from western Lake Erie, Michigan, USA for myxosporean parasites. Amplicons from positive PCRs were sequenced and showed a Ceratonova species in an estimated 1.1% (95% CI=0.46%, 1.8%) of M. speciosa individuals. We sequenced 18S, ITS1, 5.8S, ITS2 and most of the 28S rDNA regions of this Ceratonova sp., and part of the protein-coding EF2 gene. Phylogenetic analyses of ribosomal and EF2 sequences showed the Lake Erie Ceratonova sp. is most similar to, but genetically distinct from, Ceratonova shasta. Marked interspecific polymorphism in all genes examined, including the ITS barcoding genes, along with geographic location suggests this is an undescribed Ceratonova species. COI sequences showed M. speciosa individuals in Michigan and California are the same species. These findings represent a third parasite in the genus Ceratonova potentially hosted by M. speciosa.

Activation of serotonergic neurons during salicylate-induced tinnitus.

HYPOTHESIS: Serotonergic neurons are activated during salicylate-induced tinnitus and modulate the cochlea during tinnitus. BACKGROUND: During salicylate-induced tinnitus in the gerbil, neurons in the dorsal raphe nucleus were activated. Because approximately half of the neurons in this region are serotonergic, this indicates that serotonin (5-HT) might play a role in the mechanisms of central tinnitus. The goal of this study was to determine if serotonergic neurons are activated during salicylate-induced tinnitus. Furthermore, to determine if the same neurons might modulate the cochlea during tinnitus, neuroanatomic tract-tracing with 5-HT immunohistochemistry was used to determine if serotonergic neurons project to the gerbil cochlea. METHODS: A randomized, prospective study was performed. Six gerbils were injected with salicylate (saline for controls). Four hours later, the gerbils were euthanized and perfused, and their brains were collected for immunohistochemical labeling of 5-HT and c-fos. For the tract-tracing, FluoroGold was injected into the cochleae of 3 gerbils. The gerbils were euthanized and perfused 4 to 11 days later, and the brains immunohistochemically were processed for 5-HT. RESULTS: More serotonergic neurons expressed c-fos in the salicylate-injected animals compared with the controls. The increase was significant for 3 of the 8 major serotonergic cell groups including B7, B9, and the caudal linear nucleus. Despite robust labeling of olivocochlear and vestibular efferents with FluoroGold, 5-HT-labeled neurons containing FluoroGold were lacking. CONCLUSION: Salicylate-induced tinnitus activates serotonergic neurons in rostral cell groups. Activation of these neurons is not likely to influence cochlear function directly but is likely to influence a number of auditory and nonauditory regions known to be involved with tinnitus.

Upper thoracic postsynaptic dorsal column neurons conduct cardiac mechanoreceptive information, but not cardiac chemical nociception in rats.

Postsynaptic dorsal column (PSDC) neurons transmit noxious visceral information from the lower thoracic and lumbosacral spinal cord. Cuneothalamic neurons in the PSDC pathway and upper thoracic (T(3)-T(4)) spinal neurons ascending through the ventrolateral funiculus (VLF) have been shown to transmit nociceptive cardiac information. Therefore, we hypothesized that upper thoracic PSDC neurons transmit noxious cardiac information. Neuronal responses to intrapericardially injected mechanical (1.0 ml saline) and noxious chemical (0.2 ml algogenic chemicals) stimuli were recorded from antidromically activated PSDC and VLF neurons in the T(3)-T(4) spinal cord of anesthetized Sprague-Dawley rats. Of the PSDC neurons, 43% responded to mechanical stimulation, but only one responded to noxious chemical stimuli. Fifty-eight percent of VLF neurons responded to mechanical stimulation and all responded to noxious chemical stimulation. Fluoro-Ruby (FR)-labeled PSDC neurons in the T(3)-T(4) spinal cord of Sprague-Dawley rats were processed for c-fos immunohistochemistry following intrapericardial stimulation with mechanical, chemical, or control stimuli. Sections were viewed under epifluorescence and light microscopy to detect FR-labeled neurons containing a c-fos immunoreactive (IR) nucleus. An average of 6 PSDC neurons per rat was found in the T(3) and T(4) spinal segments. The average number of c-fos-IR neurons per segment varied by type of stimulus: 12 (control), 67 (chemical) and 85 (mechanical) for T(3) and 8 (control), 37 (chemical) and 62 (mechanical) for T(4). None of the 200 PSDC neurons examined expressed c-fos-IR regardless of stimulus. Together, these results suggest that thoracic PSDC neurons transmit mechanical cardiac information, but they play a minimal role in cardiac nociception.

Cross-organ sensitization of thoracic spinal neurons receiving noxious cardiac input in rats with gastroesophageal reflux.

Gastroesophageal reflux (GER) frequently triggers or worsens cardiac pain or symptoms in patients with coronary heart disease. This study aimed to determine whether GER enhances the activity of upper thoracic spinal neurons receiving noxious cardiac input. Gastric fundus and pyloric ligations as well as a longitudinal myelotomy at the gastroesophageal junction induced acute GER in pentobarbital-anesthetized, paralyzed, and ventilated male Sprague-Dawley rats. Manual manipulations of the stomach and lower esophagus were used as surgical controls in another group. At 4-9 h after GER surgery, extracellular potentials of single neurons were recorded from the T3 spinal segment. Intrapericardial bradykinin (IB) (10 microg/ml, 0.2 ml, 1 min) injections were used to activate cardiac nociceptors, and esophageal distensions were used to activate esophageal afferent fibers. Significantly more spinal neurons in the GER group responded to IB compared with the control group (69.1 vs. 38%, P < 0.01). The proportion of IB-responsive neurons in the superficial laminae of GER animals was significantly different from those in deeper layers (1/8 vs. 46/60, P < 0.01); no difference was found in control animals (7/25 vs. 20/46, P > 0.05). Excitatory responses of spinal neurons to IB in the GER group were greater than in the control group [32.4 +/- 3.5 impulses (imp)/s vs. 13.3 +/- 2.3 imp/s, P < 0.01]. Forty-five of 47 (95.7%) neurons responded to cardiac input and ED, which was higher than the control group (61.5%, P < 0.01). These results indicate that acute GER enhanced the excitatory responses of thoracic spinal neurons in deeper laminae of the dorsal horn to noxious cardiac stimulus.

Activation of serotonergic neurons during salicylate-induced tinnitus.

OBJECTIVES/HYPOTHESIS: In gerbils in which tinnitus was induced by salicylate, neurons in the dorsal raphe nucleus were activated. Since about half the neurons in this region are serotonergic, this indicates that serotonin (5-HT) might play a role in the mechanisms of central tinnitus. The goal of this study was to determine if serotonergic neurons are activated during salicylate-induced tinnitus. Further, to determine if the same neurons might modulate the cochlea during tinnitus, neuroanatomical tract-tracing with 5-HT immunohistochemistry was used to determine if serotonergic neurons project to the gerbil cochlea. STUDY DESIGN: Randomized, prospective, gerbils. METHODS: Six gerbils were injected with salicylate (saline for controls). Four hours later, the gerbils were euthanized, perfused, and their brains collected for immunohistochemical labeling of 5-HT and c-fos. For the tract-tracing, FluoroGold was injected into the cochleae of 3 gerbils. The gerbils were euthanized and perfused 4-11 days later and the brains immunohistochemically processed for 5-HT. RESULTS: More serotonergic neurons expressed c-fos in the salicylate-injected animals compared to controls. The increase was significant for 3 of the 8 major serotonergic cell groups including B7, B9, and the caudal linear nucleus. Despite robust labeling of olivocochlear and vestibular efferents with FluoroGold, 5-HT-labeled neurons containing FluoroGold were lacking. CONCLUSIONS: Salicylate-induced tinnitus activates serotonergic neurons in rostral cell groups. Activation of these neurons is not likely to influence cochlear function directly, but is likely to influence a number of auditory and non-auditory regions known to be involved with tinnitus.

Serotonin-immunoreactive neurons in the postnatal MAO-A KO mouse lateral superior olive project to the inferior colliculus.

During development, serotonin (5-HT) accumulates in thalamic, noradrenergic, and auditory brainstem neurons that are non-serotonergic in the adult. As demonstrated in somatosensory thalamocortical projections, this accumulation of 5-HT is necessary for the precise organization of afferent terminal arborizations. Accumulation of 5-HT in the auditory brainstem appears to be most robust in the lateral superior olive (LSO) and as demonstrated in the MAO-A knockout mouse, is present at birth and begins to taper off at postnatal day 7 (P7). During the same developmental period, 5-HT-positive terminal endings in the inferior colliculus (IC) have been reported to be more numerous than in the adult [O. Cases, C. Lebrand, B. Giros, T. Vitalis, E. De Maeyer, M. Caron, D. Price, P. Gaspar, I. Seif, Plasma membrane transporters of serotonin, dopamine and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase A knock-outs, J. Neurosci. 18 (1998) 6914-6927]. It has been hypothesized that the serotonergic terminal fibers in the IC belong to neurons whose cell bodies reside in the LSO. Here, we provide evidence based on morphological and tract-tracing data that LSO neurons containing serotonin in the perinatal mouse, project to the IC. These data suggest that, similar to thalamocortical projections in other sensory systems, 5-HT may play a role in regulating development of LSO terminal arbors in the IC.

Experimental evidence that the serotonin transporter mediates serotonin accumulation in LSO neurons of the postnatal mouse.

During the same postnatal period of development when their terminal projection patterns in the midbrain are maturing, lateral superior olivary (LSO) neurons are immunoreactive for serotonin (5-HT). As there is no evidence that LSO neurons synthesize 5-HT, it is likely that they accumulate 5-HT via the 5-HT transporter. To determine if the 5-HT transporter is responsible for 5-HT inside postnatal mouse LSO neurons, pups (postnatal ages 5-6) were treated with fluoxetine and LSO neurons examined for 5-HT. We also evaluated whether LSO neurons containing 5-HT expressed the 5-HT transporter. To further rule out any potential synthesis of 5-HT, brainstem sections of mice at postnatal ages when 5-HT staining is the most robust were stained for the rate-limiting enzyme in the synthesis of 5-HT, tryptophan hydroxylase. Fluoxetine treatment reduced or in most cases, completely eliminated the number of neurons in the LSO stained for 5-HT. Postnatal LSO neurons containing 5-HT were immunoreactive for the 5-HT transporter; in older animals in which 5-HT was no longer observed in the LSO neurons, 5-HT transporter expression was similarly absent. Further, LSO neurons in mice at any age did not stain for tryptophan hydroxylase. These results indicate that LSO neurons express the functional 5-HT transporter to internalize 5-HT; this mechanism may serve to regulate extracellular 5-HT levels during maturation of their terminal endings in the inferior colliculus.

Serotonin immunoreactivity in auditory brainstem neurons of the postnatal monoamine oxidase-A knockout mouse.

Altered levels of extracellular serotonin (5-HT) during development cause structural abnormalities in the neural projections of sensory systems. To better understand the potential role of 5-HT in the development of auditory system projections, we examined 5-HT immunoreactivity (IR) in auditory brainstem nuclei of postnatal mice. We previously observed 5-HT-IR in the lateral superior olive (LSO) of wild type mice. In the current study, we used a genetic model (monoamine oxidase-A knockout mouse) in which brain 5-HT levels are abnormally high to improve detection of 5-HT. In the cochlear nucleus of this knockout, 5-HT-IR cell bodies were observed in the dorsal cochlear nucleus (DCN), a primary relay to the inferior colliculus (IC). In the superior olivary complex, 5-HT-IR somata were observed in the LSO, another relay to the IC. Labeled somata were also observed within the IC itself. The 5-HT immunostaining in all 3 regions was transient and was not observed beyond postnatal day 8. These results suggest that 5-HT may play a role in the branching and refinement of DCN and LSO axon collaterals within the IC, as well as IC axon collaterals within the medial geniculate body. The pattern of expression indicates that 5-HT has a developmental role in select populations of neurons of the ascending auditory pathway prior to any influences of sound-evoked activity.

"Non-serotonergic" lateral superior olivary neurons of the neonatal mouse contain serotonin.

In mutant mice which have high serotonin (5-HT) levels perinatally, neurons in the lateral superior olive (LSO, which are not serotonergic), contain 5-HT transiently in the neonatal period of development. We determined that the same phenomenon occurs in non-genetically altered mice: LSO neurons were 5-HT-immunoreactive at postnatal days 1 and 8. These findings suggest accumulation of 5-HT in by LSO neurons during postnatal development.

Inferior colliculus projections to pontine nuclei in guinea pig.

The present study examined the neural projection from the inferior colliculus to the pontine nuclei in guinea pig. This projection has been reported in other animals, and our goal was to establish the projection in guinea pig, a commonly used auditory model. Ultimately, we wanted to determine if the pontine nuclei could be a component of the descending auditory system from the inferior colliculus to the cochlear nucleus. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into one inferior colliculus of 10 animals and the pontine nuclei examined under a light microscope to detect PHA-L-labeled fibers. PHA-L-labeled fibers were observed in the ipsilateral pontine nuclei in 70% of the animals. While the majority of labeled fibers were smooth in appearance, a few fibers with en passant type varicosities (indicating synapses) were observed in the dorsolateral area of the pontine nuclei, adjacent to the lateral lemniscus. These findings do not support a robust projection from the inferior colliculus to the pontine nuclei in guinea pig. This is in opposition to findings in bat in which the projection may play a major role in modulating responses to sound.

Dense serotonergic innervation of principal nuclei of the superior olivary complex in mouse.

To evaluate species differences in the serotonergic innervation of the superior olivary complex, serotonergic fibers and varicosities were labeled with immunohistochemistry in mouse. Many immunoreactive fibers and varicosities were observed in two of the three principal nuclei, in addition to some periolivary nuclei. This pattern of staining differs greatly from that observed in other mammals in which periolivary, but not principal nuclei are richly innervated by serotonin (5-HT). These results indicate a functional relationship between the 5-HT system and both the ascending and descending auditory systems in the mouse.

A medullary source of norepinephrine in cat cochlear nuclear complex.

Norepinephrine is believed to modulate important functions of the cochlear nuclear complex (CNC) such as the detection of signals in noise and the processing of timing cues. To better understand the impact of the noradrenergic system in the CNC, we used neurotransmitter immunohistochemistry combined with retrograde tract-tracing to identify the noradrenergic cell groups that project to the CNC. Here we present data showing that the CNC receives noradrenergic inputs from the A1 cell group located in the ventrolateral medulla. The projection from A1 to the CNC may be part of a system-wide modulation by the noradrenergic system based on stress and arousal level, or it may be part of a separate circuit that modulates its targets during survival behaviors.

Pontine sources of norepinephrine in the cat cochlear nucleus.

In the current study, the distribution of noradrenergic neurons in the pontine tegmentum that project to the cochlear nucleus was determined with retrograde tract tracing combined with neurotransmitter immunohistochemistry in the cat. Double-labeled neurons were observed in all noradrenergic cell groups, in both the dorsolateral and the ventrolateral tegmentum. Half of the double-labeled cells were located in the locus coeruleus complex. Most of these were situated in its ventral division. Most other double-labeled cells were located in peribrachial regions, especially lateral to the brachium conjunctivum. Relatively few double-labeled cells were observed in both the A4 and the A5 cell groups, 2% and 0.4%, respectively, of the total. Except for neurons in A5, which projected only contralaterally, the projections were bilateral, with an ipsilateral preponderance. The results indicate that neurons located in the ipsilateral dorsolateral tegmentum, namely, in the locus coeruleus complex and the peribrachial region, are the primary source of pontine noradrenergic afferents to the cochlear nucleus of the cat.

Aminergic projections to cochlear nucleus via descending auditory pathways.

The cochlear nucleus (CN) receives descending input from a variety of auditory nuclei. Descending inputs from the superior olive in particular have been well described, especially those of olivocochlear neurons, which terminate ultimately in the cochlea. It has been demonstrated that olivocochlear neurons receive serotonergic and noradrenergic inputs and thus form a route by which the aminergic system may modulate cochlear mechanisms. Since olivocochlear neurons send collaterals into the CN, it is possible that they also from a route by which the aminergic systems modulate CN processes. The goal of the current study was to determine if neurons in the superior olive that projected to the CN received serotonergic or noradrenergic inputs. The retrograde tracer WGAapoHRP-Au was injected into the CN of cats. The brainstems were silver-enhanced to visualize the tracer and then immunohistochemically processed with antibodies raised against serotonin or dopamine-beta-hydroxylase (DBH) to label serotonergic or noradrenergic fibers, respectively. The sections were viewed with high power light microscopy to determine if the retrogradely labeled neurons were contacted by serotonin- or DBH-immunoreactive varicosities. Retrogradely labeled cells were observed in auditory brainstem nuclei known to project to the CN including the superior olivary complex and inferior colliculus bilaterally and the opposite CN. In these regions, retrogradely labeled neurons were closely associated with serotonin- and/or DBH-immunoreactive varicosities. Assuming a synaptic relationship between the projection neurons and varicosities, these results indicate that the serotonergic and noradrenergic systems innervate the descending pathways to the CN. Since the serotonergic and noradrenergic systems modulate their targets based on level of arousal, these results support the theory that descending systems are involved in selective attention.

Serotonin in the inferior colliculus.

It has been recognized for some time that serotonin fibers originating in raphe nuclei are present in the inferior colliculi of all mammalian species studied. More recently, serotonin has been found to modulate the responses of single inferior colliculus neurons to many types of auditory stimuli, ranging from simple tone bursts to complex species-specific vocalizations. The effects of serotonin are often quite strong, and for some neurons are also highly specific. A dramatic illustration of this is that serotonin can change the selectivity of some neurons for sounds, including species-specific vocalizations. These results are discussed in light of several theories on the function of serotonin in the IC, and of outstanding issues that remain to be addressed.

High-grade esophageal dysplasia: long-term survival and quality of life after esophagectomy.

BACKGROUND: Esophagectomy for high-grade dysplasia in Barrett's esophagus has been advocated. Although long-term survival data exist, little is known about functional outcome and quality of life in this particular subset of patients. METHODS: The records of all patients who underwent esophageal resection for high-grade dysplasia from June 1991 through July 1997 were reviewed. Long-term functional outcome and quality of life were assessed using a two-part written survey. RESULTS: There were 54 patients (48 men, 6 women). Median age was 64 years (range, 36 to 83 years). Ivor Lewis esophagogastrectomy was performed in 34 patients (63%), transhiatal esophagectomy in 10 (18%), extended esophagectomy in 8 (15%), and other in 2 (4%). Invasive carcinoma was found in 19 patients (35%). Five patients (9%) were stage 0, 7 (13%) stage I, 3 (6%) stage IIA, 1 (2%) stage IIB, and 3 patients (6%) stage III. There was one operative death (1.8%). Complications occurred in 31 patients (57%). Median hospitalization was 13 days (range, 11 to 44 days). Follow-up was complete in all patients and ranged from 6 months to 9 years (median, 63 months). Overall 5-year survival was 86% and did not differ significantly from a population matched for age and gender. Five-year survival for patients with only high-grade dysplasia was 96% and 68% for patients with cancer (p = 0.017). Quality of life was measured by the Medical Outcomes Study 36-Item Short-Form Health Survey. For patients with only high-grade dysplasia, the role-physical and role-emotional scores were better than for the control population (p < 0.03). For patients with cancer, the health perception score was worse than for the control population (p < 0.03). Scores measuring physical-function, social function, mental health, bodily pain, and energy/fatigue were similar. CONCLUSIONS: Although perioperative morbidity is significant, surgical resection of high-grade dysplasia in Barrett's esophagus provides excellent long-term survival with acceptable function and quality of life.

5-HT(1A) receptor subtype mRNA expression in cochlear nucleus.

Previous studies indicate expression of various serotonin receptor subtypes, including the 5-HT(1A) receptor subtype, in rodent cochlear nucleus. Our long-term goal is to identify the types of cochlear nucleus neurons, which are well described in cat, that express 5-HT receptors. In the current study, the reverse transcriptase/polymerase chain reaction and the in situ hybridization method were used to detect the mRNA encoding a portion of the 5-HT(1A) receptor subtype in the cochlear nucleus of the cat.