The serotonin receptor 3E variant is a risk factor for female IBS-D.

Irritable bowel syndrome (IBS) is a gut-brain disorder of multifactorial origin. Evidence of disturbed serotonergic function in IBS accumulated for the 5-HT3 receptor family. 5-HT3Rs are encoded by HTR3 genes and control GI function, and peristalsis and secretion, in particular. Moreover, 5-HT3R antagonists are beneficial in the treatment of diarrhea predominant IBS (IBS-D). We previously reported on functionally relevant SNPs in HTR3A c.-42C > T (rs1062613), HTR3C p.N163K (rs6766410), and HTR3E c.*76G > A (rs56109847 = rs62625044) being associated with IBS-D, and the HTR3B variant p.Y129S (rs1176744) was also described within the context of IBS. We performed a multi-center study to validate previous results and provide further evidence for the relevance of HTR3 genes in IBS pathogenesis. Therefore, genotype data of 2682 IBS patients and 9650 controls from 14 cohorts (Chile, Germany (2), Greece, Ireland, Spain, Sweden (2), the UK (3), and the USA (3)) were taken into account. Subsequent meta-analysis confirmed HTR3E c.*76G > A (rs56109847 = rs62625044) to be associated with female IBS-D (OR = 1.58; 95% CI (1.18, 2.12)). Complementary expression studies of four GI regions (jejunum, ileum, colon, sigmoid colon) of 66 IBS patients and 42 controls revealed only HTR3E to be robustly expressed. On top, HTR3E transcript levels were significantly reduced in the sigma of IBS patients (p = 0.0187); more specifically, in those diagnosed with IBS-D (p = 0.0145). In conclusion, meta-analysis confirmed rs56109847 = rs62625044 as a risk factor for female IBS-D. Expression analysis revealed reduced HTR3E levels in the sigmoid colon of IBS-D patients, which underlines the relevance of HTR3E in the pathogenesis of IBS-D.

Serotonin type 3 receptor subunit gene polymorphisms associated with psychosomatic symptoms in irritable bowel syndrome: A multicenter retrospective study.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) of the serotonin type 3 receptor subunit (HTR3) genes have been associated with psychosomatic symptoms, but it is not clear whether these associations exist in irritable bowel syndrome (IBS). AIM: To assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS. METHODS: In this retrospective study, 623 participants with IBS were recruited from five specialty centers in Germany, Sweden, the United States, the United Kingdom, and Ireland. Depressive, anxiety, and somatization symptoms and sociodemographic characteristics were collected. Four functional SNPs - HTR3A c.-42C>T, HTR3B c.386A>C, HTR3C c.489C>A, and HTR3E c.*76G>A - were genotyped and analyzed using the dominant and recessive models. We also performed separate analyses for sex and IBS subtypes. SNP scores were calculated as the number of minor alleles of the SNPs above. The impact of HTR3C c.489C>A was tested by radioligand-binding and calcium influx assays. RESULTS: Depressive and anxiety symptoms significantly worsened with increasing numbers of minor HTR3C c.489C>A alleles in the dominant model (F depressive = 7.475, P depressive = 0.006; F anxiety = 6.535, P anxiety = 0.011). A higher SNP score (range 0-6) was linked to a worsened depressive symptoms score (F = 7.710, P-linear trend = 0.006) in IBS. The potential relevance of the HTR3C SNP was corroborated, showing changes in the expression level of 5-HT3AC variant receptors. CONCLUSION: We have provided the first evidence that HTR3C c.489C>A is involved in depressive and anxiety symptoms in individuals with IBS. The SNP score indicated that an increasing number of minor alleles is linked to the worsening of depressive symptoms in IBS.

The alternative serotonin transporter promoter P2 impacts gene function in females with irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a gut-brain disorder in which symptoms are shaped by serotonin acting centrally and peripherally. The serotonin transporter gene SLC6A4 has been implicated in IBS pathophysiology, but the underlying genetic mechanisms remain unclear. We sequenced the alternative P2 promoter driving intestinal SLC6A4 expression and identified single nucleotide polymorphisms (SNPs) that were associated with IBS in a discovery sample. Identified SNPs built different haplotypes, and the tagging SNP rs2020938 seems to associate with constipation-predominant IBS (IBS-C) in females. rs2020938 validation was performed in 1978 additional IBS patients and 6,038 controls from eight countries. Meta-analysis on data from 2,175 IBS patients and 6,128 controls confirmed the association with female IBS-C. Expression analyses revealed that the P2 promoter drives SLC6A4 expression primarily in the small intestine. Gene reporter assays showed a functional impact of SNPs in the P2 region. In silico analysis of the polymorphic promoter indicated differential expression regulation. Further follow-up revealed that the major allele of the tagging SNP rs2020938 correlates with differential SLC6A4 expression in the jejunum and with stool consistency, indicating functional relevance. Our data consolidate rs2020938 as a functional SNP associated with IBS-C risk in females, underlining the relevance of SLC6A4 in IBS pathogenesis.

Accuracy and spread of nest search behaviour in the Saharan silver ant, Cataglyphis bombycina, and in the salt pan species, Cataglyphis fortis.

Desert ants of the genus Cataglyphis are renowned for their navigation abilities, especially for their beeline homing after meandering foraging excursions reaching several hundreds of meters in length. A spiralling nest search is performed when an ant misses the nest entrance upon completing its homebound travel. We examined the nest search behaviours of two desert ant species dwelling in different habitats-Cataglyphis bombycina living in the dunes of the Sahara and Cataglyphis fortis found in the salt pans of North Africa. The two species show distinct differences in walking behaviour. C. bombycina performs a strict tripod gait with pronounced aerial phases, high stride frequencies, and extremely brief ground contact times. In view of these peculiarities and the yielding sand dune substrate, we hypothesised that homing accuracy, and namely distance measurement by stride integration, should be lower in C. bombycina, compared to the well-studied C. fortis with less specialised walking behaviour. We tested this hypothesis in ants' homebound runs from a feeding site in a linear channel setup. Surprisingly, the accuracies of nest searches were similar in the two ant species, and search accuracy was also independent of the walking substrate, soft dune sand or a hard floor. The spread of the nest search, by contrast, differed significantly between the two species, C. bombycina exhibiting a larger search spread. This may be interpreted as an increased path integration uncertainty due to the above locomotor specialisations, or as a compensation strategy accounting for the silver ants' particular environmental and behavioural situation.

High-speed locomotion in the Saharan silver ant, Cataglyphis bombycina.

The diurnal thermophilic Saharan silver ant, Cataglyphis bombycina, is the fastest of the North African Cataglyphis desert ant species. These highly mobile ants endure the extreme temperatures of their sand dune environment with outstanding behavioural, physiological and morphological adaptations. Surprisingly, C. bombycina has comparatively shorter legs than its well-studied sister species Cataglyphis fortis from salt pan habitats. This holds despite the somewhat hotter surface temperatures and the more yielding sand substrate. Here, we report that C. bombycina employs a different strategy in reaching high running speeds, outperforming the fastest known runs of the longer-legged C. fortis ants. Video analysis across a broad range of locomotor speeds revealed several differences to C. fortis Shorter leg lengths are compensated for by high stride frequencies, ranging beyond 40 Hz. This is mainly achieved by a combination of short stance phases (down to 7 ms) and fast leg swing movements (up to 1400 mm s-1). The legs of one tripod group exhibit almost perfect synchrony in the timings of their lift-offs and touch-downs, and good tripod coordination is present over the entire walking speed range (tripod coordination strength values around 0.8). This near synchrony in leg movement may facilitate locomotion across the yielding sand dune substrate.

Abnormalities of mucosal serotonin metabolism and 5-HT3 receptor subunit 3C polymorphism in irritable bowel syndrome with diarrhoea predict responsiveness to ondansetron.

BACKGROUND: Irritable bowel syndrome with diarrhoea (IBS-D) is a common condition, greatly reducing the quality of life with few effective treatment options available. AIM: To report the beneficial response shown in our trial with the 5-hydroyxtryptamine (5-HT) receptor 3 antagonist, ondansetron in IBS-D METHODS: A randomised, placebo-controlled, cross-over trial of 5 weeks of ondansetron versus placebo in 125 patients meeting modified Rome III criteria for IBS-D as previously described. Patients were compared to 21 healthy controls. 5-HT and 5-HIAA were measured in rectal biopsies. Whole gut transit time was assessed using a radio-opaque marker technique. Whole blood DNA was genotyped for an insertion polymorphism in the promoter region of the serotonin transporter gene SLC6A4, as well as single nucleotide polymorphisms (SNPs) of the tryptophan hydroxylase gene TPH1 and 5-HT3 receptor genes HTR3A, C and E. RESULTS: Patients' biopsies showed significantly higher 5-HIAA levels (2.1 (1.2-4.2) pmol/mg protein vs 1.1 (0.4-1.5) in controls, P < .0001). 39 patients used < 4 mg/d ("super-responders") while 55 required ≥ 4 mg/d. 5-HT concentrations in rectal biopsies were significantly lower in super-responders (21.3 (17.0-31.8) vs 37.7 (21.4-61.4), P = .0357) and the increase in transit time on ondansetron was significantly greater (15.6 (1.8-31) hours vs 3.9 (-5.1-17.9) hours). Stool consistency responders were more likely to carry the CC genotype of the SNP p.N163K rs6766410 of the HTR3C gene (33% vs 14%, P = .0066). CONCLUSION: IBS-D patients have significant abnormalities in mucosal 5-HT metabolism. Those with the lowest concentration of 5-HT in rectal biopsies showed the greatest responsiveness to ondansetron.

Naturalistic path integration of Cataglyphis desert ants on an air-cushioned lightweight spherical treadmill.

Air-cushioned spheres are widely used as treadmills to study behavioural and neurophysiological questions in numerous species. We describe an improved spherical treadmill design that reliably registers the path and walking behaviour of an animal walking on top of the sphere. The simple and robust set-up consists of a very light hollowed styrofoam ball supported by an air stream in a hollow half sphere and can be used indoors and outdoors. Two optical mouse sensors provided with lenses of 4.6 mm focal length detect the motion of the sphere with a temporal resolution of more than 200 frames s-1 and a spatial resolution of less than 0.2 mm. The treadmill can be used in an open- or closed-loop configuration with respect to yaw of the animal. The tethering allows animals to freely adjust their body posture and in the closed-loop configuration to quickly rotate around their yaw axis with their own moment of inertia. In this account, we present the first evidence of naturalistic homing navigation on a spherical treadmill for two species of Cataglyphis desert ants. We were able to evaluate with good precision the walking speed and angular orientation at any time. During homing the ants showed a significant difference in walking speed between the approach and search phases; moreover, they slowed down significantly as soon as they reached zero vector state, the fictive nest position.

How to find home backwards? Locomotion and inter-leg coordination during rearward walking of Cataglyphis fortis desert ants.

For insects, flexibility in the performance of terrestrial locomotion is a vital part of facing the challenges of their often unpredictable environment. Arthropods such as scorpions and crustaceans can switch readily from forward to backward locomotion, but in insects this behaviour seems to be less common and, therefore, is only poorly understood. Here we present an example of spontaneous and persistent backward walking in Cataglyphis desert ants that allows us to investigate rearward locomotion within a natural context. When ants find a food item that is too large to be lifted up and to be carried in a normal forward-faced orientation, they will drag the load walking backwards to their home nest. A detailed examination of this behaviour reveals a surprising flexibility of the locomotor output. Compared with forward walks with regular tripod coordination, no main coordination pattern can be assigned to rearward walks. However, we often observed leg-pair-specific stepping patterns. The front legs frequently step with small stride lengths, while the middle and the hind legs are characterized by less numerous but larger strides. But still, these specializations show no rigidly fixed leg coupling, nor are they strictly embedded within a temporal context; therefore, they do not result in a repetitive coordination pattern. The individual legs act as separate units, most likely to better maintain stability during backward dragging.

Carrier-based dry powder inhalation: Impact of carrier modification on capsule filling processability and in vitro aerodynamic performance.

This study aims to investigate the effect of carrier characteristics and dosator capsule filling operation on the in vitro deposition of mixtures containing salbutamol sulphate (SS) and lactose and mannitol as model carrier materials. The carrier surfaces of lactose and mannitol were modified via wet decantation. The impact of the decantation process on the properties of carriers was investigated by laser diffraction, density and powder flow measurements, N2 physisorption, small and wide angle X-ray scattering (SWAXS) and scanning electron microscopy (SEM). Differences in carrier type and untreated and decanted materials were identified and the SAXS measurements proved to be a promising technology confirming the successful removal of fines. Adhesive carrier API mixtures with carrier-to-API ratio of 99:1 wt% were prepared, mixture homogeneity was tested and subsequently the mixtures were filled into capsules at different process settings. Finally, the influence of the decantation process on the in vitro performance of the adhesive mixtures was tested with a next generation impactor. For lactose, the decantation decreased the fine particle fraction (FPF) of SS, whereas the FPF of mannitol as a carrier was only affected by the capsule filling process. In summary, the DPI formulation based on untreated lactose, especially by capsule filling using a dosing chamber to powder layer (compression) ratio of 1:2, proved to be superior in terms of the dosing accuracy (RSD<0.8%) and the in vitro aerodynamic performance (FPF of 12%).

Walking and running in the desert ant Cataglyphis fortis.

Path integration, although inherently error-prone, is a common navigation strategy in animals, particularly where environmental orientation cues are rare. The desert ant Cataglyphis fortis is a prominent example, covering large distances on foraging excursions. The stride integrator is probably the major source of path integration errors. A detailed analysis of walking behaviour in Cataglyphis is thus of importance for assessing possible sources of errors and potential compensation strategies. Zollikofer (J Exp Biol 192:95-106, 1994a) demonstrated consistent use of the tripod gait in Cataglyphis, and suggested an unexpectedly constant stride length as a possible means of reducing navigation errors. Here, we extend these studies by more detailed analyses of walking behaviour across a large range of walking speeds. Stride length increases linearly and stride amplitude of the middle legs increases slightly linearly with walking speed. An initial decrease of swing phase duration is observed at lower velocities with increasing walking speed. Then it stays constant across the behaviourally relevant range of walking speeds. Walking speed is increased by shortening of the stance phase and of the stance phase overlap. At speeds larger than 370 mm s(-1), the stride frequency levels off, the duty factor falls below 0.5, and Cataglyphis transitions to running with aerial phases.

The influence of residual water on the secondary structure and crystallinity of freeze-dried fibrinogen.

The purpose of this work was to investigate the influence of water content on the secondary structure of a freeze-dried protein (fibrinogen) after a storage period of two weeks. To that end, attenuated reflectance Fourier transformed infrared (ATR-FTIR) and Raman spectra were generated and evaluated and the crystalline state of the fibrinogen bulks was determined via X-ray diffraction. First, a PCA (principal component analysis) of the spectral data was performed. While the α-helix and ß-turn contents were increasing with the increasing water content, the ß-sheet content was decreasing. A partial least squares (PLS) model was developed to correlate the mid-infrared and Raman spectral changes with the degree of crystallinity. The obtained R(2) value of 0.953 confirmed a correlation between changes in the secondary structure and crystallinity of the samples. The results demonstrated that the combined ATR-FTIR and Raman approach could be used to predict the crystalline state in freeze-dried fibrinogen products.

The influence of residual water on the solid-state properties of freeze-dried fibrinogen.

The purpose of this work was to investigate the influence of residual water in freeze-dried protein powders on the dissolution behavior of the solid-state proteins. To that end, six freeze-dried fibrinogen powder lots were stored at four levels of relative humidity and analyzed with regard to the particle size and shape, the specific surface area, the solid state of protein and the inner surface. Furthermore, the dissolution behavior of the powders was investigated. We clearly identified differences in the specific surface area, specific inner surface area, crystallinity, particle size and shape, which we were able to correlate to the dissolution behavior. These differences were triggered due to the different levels of residual moisture during two weeks of storage. Thus, we were able to show that the storage conditions have significant impact on the processing of pharmaceutical protein materials.

Specific surface, crystallinity, and dissolution of lyophilized fibrinogen. A study by combined small- and wide-angle X-ray scattering (SWAXS).

The nano-structural properties of six different batches of lyophilized fibrinogen at various contents of residual humidity (6-20%) were studied by small- and wide-angle X-ray scattering (SWAXS) and related to the dissolution properties. As structural parameters, the specific surface and relative degree of crystallinity, from SAXS and WAXS, respectively, were used, and correlated to the dissolution rates. BET surface area and electron microscopy were used as ancillary methods. The results show a complex, biphasic behavior: above 9% water content the crystallinity increased, and the specific surface decreased with increasing water contents; at the lowest water contents (6%), however, where the WAXS patterns showed amorphous structure of the fibrinogens, the specific surface and dissolution rates diverged over a wide range of values. Systematic correlations could be established between specific surface and dissolution rates for the water contents below 13%: the dissolution rates were found to decrease with increasing specific surface, most notably in the amorphous form, in contrast to expectations from classical thermodynamics. Protein conformational changes and hydrophobic surface formation upon depletion of water could be possible causes. This is supported by the protective effect of the high-HLB surfactant PS-80, which was found to enlarge the specific surface.