By tracking the phosphorylation of S6 ribosomal protein as a marker of neuronal activity, we show that S6 becomes phosphorylated (pS6) in mouse VSNs stimulated by intraspecific and heterospecific pheromonal cues. We observed that female scent induces pS6 immunoreactivity in the apical VSNs of male vomeronasal epithelium, whereas male cues stimulate S6 phosphorylation in both the basal and apical VSNs of females. We also show that this dimorphic pattern of pS6 immunoreactivity is reproduced when heterospecific stimuli are used. Moreover, we found that a consistent proportion of VSNs is activated by both heterospecific and intraspecific pheromones. Additionally, we have evidence of adaptive responses to S6 phosphorylation when stimulation with cues of the same and opposite sex and of different species is sustained.In most mammals, the vomeronasal system has a pivotal role in mediating socio-sexual behaviours. The vomeronasal organ senses pheromones through the activation of specific receptors. Pheromone binding to cognate receptors activates Ca-influx via the gating of a cation channel that generates membrane depolarisation. The  The mouse VNO is equipped with chemosensory neurons (VSNs) expressing two large superfamilies of pheromone receptors, namely type-I (V1Rs) and type-II (V2Rs) vomeronasal receptors, and a small family of formyl peptide receptors (FPRs) that are believed to detect pathogenic molecules emitted by sick animals8. The expression pattern of these receptor families is complementary as V1Rs and FPRs are preferentially expressed in the apical chemosensory neurons of the VNO, whereas V2Rs are in the basal neurons. V1Rs and FPR reportedly bind small molecules whereas V2Rs bind polypeptides13. All of these receptors are believed to be equally expressed in males and females, thus excluding a consistent sexual dimorphism of the VNO14. In contrast, differences between sexes primarily occur in the content of male and female pheromonal secretions20. VSN responses initiate with the binding of pheromones to their cognate receptors and the resulting stimulation of two specific G-proteins, Gαi2 (co-expressing with V1Rs) and Gαo (co-expressing with V2Rs). This results in activation of the phospholipase-C signalling cascade with the production of diacylglycerol, which causes opening of the transient receptor potential channel (TrpC2), an increase in intracellular calcium and the generation of action potentials22. The electrical signal is thus propagated to dedicated central areas of the nervous system, specifically committed to elicit neuroendocrine and behavioural responses23. Hence, whereas the signal transduction mechanisms underlying desensitization to odour stimuli are quite well defined in the main olfactory epithelium24, the existence of adapting mechanisms to pheromonal stimuli that are physiologically relevant are still controversial in the VNO26. On the one hand, adapting mechanisms would be disadvantageous to sustain a complete or prolonged behavioural response, while, on the other hand, if absent, they would endure VSNs with a continuous background stimulation elicited by self, sex and strain odours, thus limiting the range of signal detection and discrimination, in vivo. However, in vivo, VSN activity, upon pheromonal stimulation, is difficult to track by the immunological detection of the traditional neuronal markers c-Fos or Egr 1 because they are poorly expressed29, although an exhaustive study, using in situ hybridisation to detect the up-regulation of RNA expression of the Egr 1 gene, has clarified many aspects of the pheromonal transduction12. In particular, this study focused on the association of vomeronasal receptors with cognate hetero- or intraspecific stimuli establishing the molecular basis of pheromonal information coding.Chemosensory perception is based on the detection of molecules that trigger physiological, reproductive and social responses including food search and avoidance, escape from predators, mating, aggressiveness and attractiveness. The vomeronasal organ of Jacobson (VNO) is an olfactory structure that is specifically responsible for converting chemical signals, pheromones, stemming from hetero- and conspecific animals, into neuroendocrine responses, thus modulating the social life of many speciesin-vivo activation of neurons in the central nervous system30. They found that the S6 ribosomal protein is phosphorylated in response to a wide variety of stimuli; this phosphorylation is comparable to the up-regulation of immediate early gene expression, such as c-Fos and Egr 1. Following this study, Jiang and colleagues reported that S6 phosphorylation, in-vivo, occurs in the olfactory neurons in response to the binding of specific odorants to their cognate receptors31. While this manuscript was under revision, Tsunoda and colleagues reported the activation of a subpopulation of rat and mouse vomeronasal neurons by S6 phosphorylation in response to rat tears32. This suggests that pS6 is also a reliable marker to determine neuronal activation in the peripheral olfactory and sub-olfactory systems.Few years ago, Knight and colleagues developed a method to determine in-vivo stimulation with different pheromonal cues. We confirm that this method is time saving, reliable and sensitive for any given pheromonal stimulus. Here, we show that phosphorylation of the S6 ribosomal subunit markedly and rapidly increases in the VSNs following exposure to intraspecific and heterospecific pheromones. Interestingly, complete dephosphorylation of pS6 occurs if stimulation with intraspecific and heterospecific cues is sustained, leaving VSN unable to respond to the same stimuli, via S6 rephosphorylation, for several hours. Thus, this work opens the question as to whether long-term filtering mechanisms of pheromonal stimuli exist in the VNO.Based on these previous observations, we have tracked VSN activity via the immunological detection of the phosphorylated S6 ribosomal subunit following In preliminary experiments to assess the temporal profile of pS6 expression in the VNO upon contact to pheromonal stimulation, CD1 male mice were introduced in a cage containing bedding mix of male and female Balb/c mice or to clean bedding for an hour and then sacrificed 0′, 60′ and 120′ after removal of the stimulus . Our results indicated that pS6-immunoreactive cells dramatically increased compared to the control (clean bedding) in the VNO immediately after 1 hr exposure to the stimulus (0′)  and the immunological signal remained unchanged for the remaining 120′  gene, to visualise pS6 expression in mature VSNs. Our results indicated that about 90% of pS6 positive neurons also expressed GFP  in which the expression of GFP (green fluorescent protein) is driven by the promoter of the 33. Our data showed that, for both female and male stimuli, only a very low percentage of V2R-expressing neurons co-expressed with pS6; however, male cues proportionally activated more V2R-expressing neurons in female VNO (13%) than the opposite condition (6%)  , C57/Bl6  and CD1  strains was employed as stimuli Fig. . In geneWhen female or male mice were exposed to bedding of the same sex, we observed a lower VNO activation than that generated by a stimulus of the opposite sex  Fig. . In addiTo better investigate this issue and assess whether an adaptive process also occurs with opposite sex stimuli, we exposed male mice to female bedding for different time intervals prior to sacrifice Fig. . As a coWe next assayed pS6 immunoreactivity in the VNO of mice exposed to heterospecific stimuli of both predator and non-predator species Fig. . In this12, we stimulated male mice with rat, hamster and mouse bedding and counterstained pS6-positive neurons with an antibody recognising all members of subfamily-D V2Rs (panD)34. Unexpectedly, we could not observe a preferential co-expression of pS6 with subfamily-D V2Rs in response to heterospecific stimuli  exclusively responded to heterospecific stimuliWe also studied the pS6 expression in a more physiological and restricted context. Therefore, in a series of experiments, we paired an isolated CD1 mouse with a Balb/c mouse of the opposite sex for an hour in a neutral cage containing fresh bedding. The experiment was performed taking into account the oestrous phase (dioestrous and oestrus) of both stimulated and stimulating females Fig. . We foun35. This method has been employed in detecting the neuronal activity in the central nervous system and in the olfactory system. Very recently, Tsunoda and colleagues reported pS6 phosphorylation of VSNs activity in response to rat tear stimulation32. In this report, we have confronted the activation in VSNs of female and male CD1 mice in response to intraspecific and heterospecific stimuli. In our hands, male VNO responded to female stimuli with a higher density of pS6-positive neurons than female VNO to male stimuli, confirming the observations made by He and colleagues in VNO slices of transgenic mice expressing the calcium indicator G-CaMP2, stimulated with urinary cues36. This result may reflect a more complex pheromonal blend constituting female excretions. We were able to confirm these data with stimuli from all mouse strains that we have tested, although both female and male bedding collected from the C57/Bl6 strain retained a slightly lower pheromonal activity than the other strains. Interestingly, this greater sensitivity of the male VNO in detecting female cues than the opposite condition is completely abolished if animals were not allowed to physically contact stimuli, suggesting that a significant proportion of female pheromonal molecules are chemically non-volatile. This finding seems to support the results reported by Nodari and colleagues, who showed that sulphated steroids, a group of barely volatile molecules which are specifically contained in female urine, account for a large fraction of the electrical activity triggered by this stimulus17. Sulphated steroids have been reported to selectively bind specific classes of V1Rs but not V2Rs37. Consistent with this observation, we found that female cues activate a large population of neurons mainly located to the apical layer of male VNO that showed a low co-expression with V2Rs. In contrast, male scent seems to activate neurons throughout the sensory VNO.In this work, we have studied the response of vomeronasal neurons of male and female mice to a large array of pheromonal stimuli (soiled beddings or encounters with conspecifics) via the immunological detection of the S6 ribosomal protein phosphorylation. We found that this method of analysing the neuronal activity of the vomeronasal organ in living animals is fast and more sensitive than the immunological detection of other neuronal markers reported in literature 36, we found that the VNO of both male and female mice responds to the same sex cues with lower neuronal activation than to the opposite sex cues. This observation is also confirmed when the source of the stimulus was the living animal rather than the soiled bedding. According to the consensus, mouse pheromone receptor genes do not show an evident sexual dimorphism based on both sequence and expression patterns6. Hence, and in contrast to our data, it would be expected that a given male or female stimulus activates the same number of chemosensory neurons in both male and female VNO. Thus, our results suggest the existence of a filtering mechanism to adapt the VNO to a persistent stimulation with pheromonal cues originating from the same animal or from animals of the same or opposite sex or from heterospecifics. This is also confirmed by experiments performed on castrated male mice. These mice do not produce most masculine pheromones and therefore they respond to intact male cues with a much stronger activation of the vomeronasal neurons, being less adapted to these stimuli. In contrast to the main olfactory system, where desensitization of chemosensory neurons upon odorant stimulation is unequivocally demonstrated38, adaptive mechanisms in the vomeronasal neurons are indeed an open question. Spehr and colleagues provided evidence that pheromone-evoked field potentials in intact VNO and in individual voltage-clamped VSNs undergo sensory adaptation, manifested by a time-dependent decline in sensitivity during prolonged or repetitive stimulation25. They also demonstrated that the complex Ca++-Calmodulin is responsible for this effect by inhibition of the diacylglycerol activated TrpC2 current25. However, other electrophysiological studies also failed to observe VSN adaptation when individual stimuli were delivered over a period of several hours36. Our in-vivo results show that pS6 immunoreactivity, employed as a marker of neuronal activity39, returns to background level in animals that are exposed to prolonged stimulation. However, electrophysiological adaptation occurs with a short latency after stimulus application, whereas the complete dephosphorylation of the S6 protein by a prolonged stimulation is a process that lasts several hours before it is fully accomplished. Indeed, after the removal of a short stimulus, S6 protein phosphorylation displays a similar time course. Similarly, several hours are required before VSNs, adapted to a given stimulus, respond to the same stimulus by S6 protein phosphorylation. This evidence suggests that biochemical and electrophysiological adaptation differs in terms of a temporal profile. Indeed, it remains possible that in our in-vivo experimental conditions, given the high sensitivity of the VNO to picomolar concentration of pheromones40, the temporal scale of the desentisization/resensitization process is dramatically amplified by the fact that the complete removal of the stimulus might be difficult to accomplish . Electrophysiological measurements are exquisitely reliable in determining neuronal activation; however, ex vivo experiments require organ removal and extensive washing and conditioning in physiological solutions. Thus, it is possible that resensitization of the VSNs may occur much faster than observed in the in-vivo experiments, here reported.Intriguingly, and in contrast to previous physiological studies based on electrophysiological recordings and Ca-imaging readouts31, reflects neuronal activation in the VNO, we largely ignore the adaptive mechanisms here reported, their potential role and to what extent the induction of pS6 is related to electrophysiological changes. In recent work, von der Weit and colleagues41 demonstrated that odour stimulation induces a reversible decrease in the transcription of genes corresponding to activated receptors. It is then possible that S6 phosphorylation may also be correlated to the mechanisms underlying this effect. Interestingly, the temporal scale of the RNA downregulation in the olfactory neurons appears approximately similar to the sensitization/desensitization effects that we observe in the vomeronasal neurons.Nevertheless, it should be considered that, although it is likely that S6 protein phosphorylation, as for the olfactory system43.In this context, it is interestingly to note that the chemosensory neurons of the Grueneberg ganglion, a peripheral olfactory organ that respond to stress odorants, express vomeronasal receptors and seem to undergo adaptation to c-Fos expression following a prolonged exposure to stimuliin vivo study employing a riboprobe to detect the expression of the proto-oncogene Egr 112. In our study, we found that the rat bedding mix  represents a very strong stimulus for the mouse VNO as up to 30% of the VSNs showed activation. However, when mice were exposed to female and male rat bedding, respectively, we identified a distribution of the pS6 immunoreactive cells that reflects the situation observed for mouse stimuli. In fact, female rat stimuli clearly activate apical neurons, whereas male stimuli activate neurons throughout the VNO. A similar pattern of expression is also observed with hamster stimuli, although they are generally less effective in activating VNO neurons (not shown). Interestingly, male mice adapted to female mouse scent showed a 30% reduction of response to female rat stimuli, suggesting that a significant proportion of VSNs that respond to mouse also respond to rat cues. This may indicate the presence of classes of pheromonal molecules that are shared between species for social communication. In line with our observation, Tsunoda and colleagues32 have recently demonstrated that a rat lacrimal protein, namely CRP1, activates VSNs of both rats and mice affecting specific behavioural performances. According to these observations, in future works, it will be interesting to investigate if the modality of communication across species may be extended to individuals belonging to different animal orders including humans.In this work, we have also tested heterospecific stimuli and, in contrast to intraspecific cues, observed no difference in the number of pS6 immunoreactive neurons between sexes. This is in line with that which was reported in a previous Mus musculus, CD1 strains) were housed under a 12 h light/dark cycle. All experiments were carried out between 9:00 a.m. and 12:00 a.m.Mice  and hamster (Mesocricetus auratus) were freshly collected from at least three cages and pooled before each experimental session.Bedding samples from mice , rat (Fox and bobcat urine were purchased from PredatorPee Store and sprayed over clean bedding before the experiments.Natrix natrix) were collected from the roads and the skin removed and cut into pieces.Freshly dead snakes  which varies over time, we sampled materials from cages housing several animals for over 5–7 days. Bedding, when necessary, was stored at −80 °C. When bedding stimuli were used, these were added to a clean cage just before introducing the animals. Single colonies of subject animals  were moved from the animal house and isolated for 3–4 days in a clean room before each experiment. During the experimental session with soiled bedding, an individual animal was introduced in a cage containing the stimulus and left for 1 hr unless indicated in the text. Control experiments were conducted using clean bedding under identical conditions.In behavioural experiments where CD1 mice were paired before VNO collection and processing, animals of the same sex were singly housed for 3 days in the same clean room under a fume hood to minimize the contact with volatiles molecules. Mice for pairing were then introduced, at the same time, in a cage containing clean bedding. Animals were allowed to investigate each other for 1 hr prior to sacrifice. Using this procedure, male mice could be paired without displaying aggressive behaviour.Mice were sacrificed by cervical dislocation and the VNO was then dissected, fixed for 4 hrs at 4 °C with a solution of 4% phosphate-buffered paraformaldehyde, and cryoprotected overnight at 4 °C in 30% sucrose. Subsequently, tissues were included in OCT embedding solution  and frozen in liquid nitrogen -cooled pentane.All animal protocols complied with the ethical guidelines for the care and use of laboratory animals issued by the Italian Government and were approved by the Animal Welfare Committee  of the University of Parma. All methods were carried out in accordance with the relevant guidelines and regulations.Cryostat-cut sections (20 μm) were blocked in 1% albumin, 0.3% Triton X-100 for 20 min and incubated with the anti-pS6 antibody  in the same blocking solution for 40 hrs at 4 °C. Sections were revealed with an anti-rabbit IgG conjugated with Alexa488 (or Alexa568) (1:350) (Invitrogen).44 (used at 1: 100 dilution) in the presence of 1% normal rabbit serum for 24 hrs at 4 °C. The immune-complexes were visualised with streptavidin-Alexa488 (or streptavidin-Alexa568) (1: 350).For double label immunohistochemistry, after pS6 development, sections received treatment with 3% normal rabbit serum for 1 hr at room temperature and were then incubated with the biotinylated anti-V2R (panC or panD) antibodyFluorescent images were obtained using a Zeiss LSM 510 Meta System scan integrated with Axiovert 200 M inverted microscope .Cryostat cut, coronal VNO sections to be analysed were collected in the central part of the VNO to obtain a better definition of the boundaries between the apical and basal layers of the neuroepithelium. Immunolabelled sections were counterstained with DAPI to display all nuclei. To count cells, sections were analysed with a 25× objective and images were processed with the Photoshop CS6 Extended program. A Nikon DS-2Mv Camera was used and the exposure time was set at 300 msec for images subjected to cell counting. The number of positive cells was calculated per selected area and as a percentage of total VNO cells. Given the very low background of the anti-pS6 antibody . Eight to 10 sections per animal were normally counted. Five to 10 animals for each experimental point were used. Mice living in three different colonies composed each experimental group. The experimenter who counted sections (corresponding author) was blinded to the experimental conditions (except for the experiment in Supplementary Fig. Sample size was determined to the conventional practice for behavioural tests. All data are expressed as means ± s.d. Two-way ANOVA t-test followed by Bonferroni post-hoc test was used for evaluation of immunostaining.The datasets generated and analyzed during the current study are available from the corresponding author.Supplementary Figures


Previous long-term observational studies found that Tai Chi practitioners had better knee-joint proprioceptive acuity versus controls in an older population. We evaluated the effects of Tai Chi for knee-joint proprioception in knee osteoarthritis (OA) in a randomized controlled trial.2 with knee pain on most days of the previous month and tibiofemoral OA (K/L grade ≥2) to Tai Chi  or to an attention control (stretching and wellness education). The 60 minute intervention sessions occurred twice-weekly for 12 weeks. The knee joint proprioception was measured using a Biometrics™ electrogoniometer with an ADU301 angle display unit during each assessment visit. Three test angles  were evaluated with each subject in a sitting position taken as neutral (0 degrees). The mean error (absolute angle error) between the actual and replicated angles was calculated for each of the three test angles. The Tai Chi and control groups were compared by intention-to-treat using t-tests.We randomized 40 eligible individuals , and median K/L grade 4; 75% were female, 70% were white. The participants in the Tai Chi intervention exhibited significantly improved proprioception at 30 degrees, but not at 45 or 60 degrees, at 12 weeks. Patients who continued Tai Chi practice after 12 weeks also reported no significant improvements in knee proprioception at 24 and 48 weeks.The participants had a mean age of 65 y (SD 7.8), a mean disease duration of 10 y (SD 7.6), a mean BMI of 30.0 kg/mTai Chi appears to be beneficial for knee proprioception in people with severe knee OA at a 30 degree test angle immediately following 12 weeks of practice. However, we were unable to demonstrate that Tai Chi has any long-term effects on knee proprioception, nor were we able to find any effects on proprioception at larger test angles (45 and 60 degrees). Standardized and reproducible measures for knee proprioception should be explored in future research. Proprioception - or the perception of limb position in space - is critical to the preservation of joint stability. Impaired proprioception may play a role in the progression of knee OA by causing abnormal stresses on the knee joint. These stresses can produce changes in the articular structures, which may result in decreased confidence in postural stability and, consequently, impaired performance in daily activities. Proprioception is well-documented to decrease with the natural ageing process, but decreases are more profound in patients with knee OA.13 Hurley and Scott found that exercise significantly improved quadriceps muscle strength and knee joint position sense in 52 patients with knee OA completing an exercise regime. Moreover, these improvements were maintained in the exercise group after six months of follow-up.11 Tai Chi, with an emphasis on balance, muscle strengthening and the integration of the mind and body, may be an ideal proprioceptive exercise for older individuals with knee OA.13 Tai Chi has been shown to act on a number of sensorimotor systems that contribute to postural control. A longitudinal study conducted by Tsang and Hui-Chan reported that 21 elderly individuals practicing Tai Chi for at least three years had better knee-joint proprioceptive acuity (less absolute angle errors in passive knee joint repositioning testing) than the 21 controls.12 The research group went on to report that in a group of 68 elderly subjects, those practicing Tai Chi regularly for a minimum of four years showed significantly better knee joint proprioception than those subjects who were long-term swimmers or runners or sedentary controls.12 Despite limited observational evidence, these results generally support the view that long-term Tai Chi practice led to better knee-joint proprioceptive acuity and neuromuscular activities in an older population.Research suggests that exercise may be an effective therapy for promoting proprioceptive acuity.Thus, we aimed to evaluate the effectiveness of Tai Chi on knee proprioception in a group of older individuals with knee OA. We hypothesized that participants receiving Tai Chi would show greater improvement in knee joint proprioception than participants treated with an attention control intervention consisting of wellness education and stretching.14 Patients with knee OA were recruited from the greater Boston area and written consent was obtained from all eligible participants prior to enrolment.The study was conducted at Tufts Medical Centre, an urban tertiary care academic hospital in Boston, USA. The detailed methodology of the full study protocol was published in a previous publication.To ensure the adequate enrolment of underrepresented groups, we placed advertisements in local media. We also used the rheumatology clinic patient database at Tufts Medical Centre to identify patients with knee OA. For interested respondents, we determined eligibility through a brief, scripted interview which posed questions whose predictive values for knee OA were known from population-based data. Applicants who screened positive on the telephone interview were scheduled for eligibility visits, whereby written informed consent was obtained.2; WOMAC pain subscale score >4015 (with a range 0–100); fulfilment of the American College of Rheumatology criteria for knee OA16 with radiographic knee OA grade ≥ 2.17 We excluded individuals with prior Tai Chi training or experience with similar types of alternative medicine, such as Qi Gong or yoga; individuals with serious medical conditions limiting their ability for full participation as determined by their primary care physicians; individuals with intra-articular steroid injections given in the previous three months; individuals with reconstructive surgery on the affected knee or any intra-articular hyaluronate injections in the previous six months; and individuals unable to pass the Mini-Mental examination with a score < 24.18 For individuals with knee OA in both knees, the knee reported as the most painful at the baseline was designated as the study knee. If both knees were equally affected (occurring in two participants) one knee was chosen at random as the affected knee.The eligibility criteria were: age ≥ 55 years; body mass index (BMI) ≤ 40 kg/mThe participants were randomly assigned to Tai Chi (n=20) or an Attention Control group (n=20). Randomization assignments were designated by the statistician (CS) using computer-generated numbers to randomize permuted blocks of size two and four so that each block was complete. They were provided in sealed, opaque envelopes and opened upon the participant’s agreement to participate. The block size was randomly assigned to minimize the correct prediction of assignments while preserving approximate balance between groups. Outcomes that required the analysis of knee strength were based on the evaluation of the knee reported as most painful at the baseline. If both knees were equally affected (which occurred in two participants), one knee was chosen at random as the affected knee.19 with minor modifications that were suitable for people with knee pain. The modifications involved eliminating stances that required greater than 90 degrees of knee flexion and which could cause excess knee joint stress. The patients were instructed to practice Tai Chi daily at home and to maintain their usual physical activities throughout the study.Subjects in the Tai Chi group participated in 60 minute Tai Chi sessions twice weekly for 12 weeks. The sessions were instructed by a Tai Chi master with more than 20 years of teaching experience and included: (1) 10 minutes of self-massage and a review of Tai Chi principles; (2) 30 minutes of Tai Chi movements; (3) 10 minutes of breathing technique; (4) 10 minutes of relaxation. The programme consisted of ten forms from classical Yang Style Tai Chi20 The control group attended 60 minute sessions twice weekly for 12 weeks. Every session included 40 minutes of didactic lessons on: (1) knee OA as a disease; (2) diet and nutrition; (3) therapies to treat knee OA; (4) physical and mental health education . The final 20 minutes consisted of stretching exercises involving the upper body, trunk and lower body, with each stretch being held for 10 to 15 seconds. The participants were instructed to practice at least 20 minutes of stretching exercises per day at home and to maintain their usual physical activities.Subjects in the attention control group received a wellness education and stretching programme.Knee joint proprioception was measured at each assessment visit  with a non-weight-bearing passive knee joint repositioning test using a biometrics electrogoniometer with an ADU301 angle display unit. Three test angles  were evaluated with each participant in a sitting position taken as neutral (0 degrees). The participant was instructed to sit on the edge of an examination table with their lower leg dangling freely over the edge of the table. The electrogoniometer was then placed longitudinally in alignment with the participant’s femur and tibia and was secured to the skin with double-sided medical tape. With the participant’s eyes closed, the examiner first moved the participant’s leg into one of the test angles and held it for five seconds before returning the leg to the freely dangling position. With their eyes still closed, the participant was asked to attempt to reproduce the test angle. The practiced and replicated angle was repeated for all three test angles. The mean error (absolute angle error) between the actual and replicated angles was calculated for each of the three test angles.The electrogoniometer was placed longitudinally in alignment with the femur and tibia with double-sided medical tape and used to determine each of the three test angles. The participants were first shown one of the angles, which was held for a few seconds, and were then asked to close their eyes and attempt to reproduce the angle; this was repeated for all three test angles. We also repeated these assessments at 24 and 48 weeks to test the durability of the response.We analysed the data on an intent-to-treat basis and compared between the groups any changes in proprioception across all times at the baseline, 12, 24, and 48 weeks with mixed models, using ‘time’ and ‘group’ as categorical fixed factors with random intercepts and first-order autocorrelation of the errors. The mean error (absolute angle error) between the actual and replicated angles was calculated for each of the three test angles. The Tai Chi and control groups were compared by intention-to-treat using t-tests. We evaluated potential effects of confounding factors or interaction with treatment by covariates including age, gender, BMI, disease duration, disease severity , co-morbidities, health status, effusion and function, modified Tai Chi style, changes in joint position and body weight, and use of pain medications. A two-sided p-value < 0.05 was considered to indicate statistical significance. The results were presented as between group differences.21Between October 2005 and February 2006, 366 individuals were screened by telephone and 62 were identified for further evaluation. Forty participants (65%) were found eligible and randomized to the Tai Chi or Attention Control group See . The rem2 (± 4.8 kg/m2), and a median K/L grade of 4; 75% were female, 70% were white. No significant differences were seen at the baseline demographics, radiographic score or outcome measures. The attendance rate was 85% for Tai Chi versus 89% for the attention control.At the 24 week follow-up, 13 out of 20 participants (65%) in the Tai Chi group continued to practice Tai Chi while 12 out of 20 (60%) in the control group continued to practice stretching exercises. At the 48 week follow-up, these rates were 9 out of 20 (45%) for Tai Chi and 8 out of 20 (40%) for the control.14 Our results are partially consistent with the results of two observational studies examining the impact of Tai Chi on knee-joint proprioception and neuromuscular activities.13 However, it is important to note that our results were likely limited by problems we encountered in measuring proprioception. These problems included limitations with the instrument used to measure proprioception itself, with the methods we employed in using the instrument to measure proprioception, and with the length of the Tai Chi intervention that study participants received. In this section, we aim to explain the methodological challenges in measuring knee proprioception in order to improve these measurements for future research. Our findings suggest that Tai Chi may be beneficial for knee proprioception in patients with severe knee OA at a 30 degree test angle immediately following 12 weeks of practice. However, we were unable to demonstrate that these effects are maintained at follow-up. There have been several previous trials testing the effects of Tai Chi for joint proprioception in patients with knee OA.The first methodological challenge may be attributed to the use of an electrogoniometer - rather than a custom-built device - to measure proprioception. Measurement by the electrogoniometer can be affected by the skin and body compositions of the study population. Furthermore, the device is dependent upon proper placement by the examiner, highlighting the importance of extensive training for the testers placing the markers to limit variability.22 More importantly, the resting position may change over time with each participant, particularly while undergoing an intervention such as Tai Chi, which focuses on joint alignment. The test angles measured were 30, 45 and 60 degrees. Patients, especially those with severe knee OA, may have a full knee extension of less than 60 degrees and, thus, would be limited in their testing. Therefore, test angles, along with the resting position angle, ought to be calculated on an individual basis from an anatomical position designated as ‘zero degrees’. Furthermore, only one trial was tested for the each of the three test angles. Increasing the number of trials tested would ensure more accurate calculations.A second methodological challenge may be attributed to the protocol design, in particular the knee selection, reference joint angle and testing angle. The study knee, designated by the presence of knee OA or a higher pain index, was tested when in fact the ‘kicking knee’ is most commonly tested for proprioception acuity. Other neuromuscular research suggests testing proprioception in the ‘stabilizing leg’ because it is the postural support limb. Our testing procedures defined the natural resting position of the participants to be zero degrees and neglected to account for variability in the resting positions of the participants, particularly among Knee OA patients who experience varying degrees of structural changes.Thirdly, our proprioception outcomes may have been influenced by the procedures implemented. To begin testing, the examiner moved the participant’s leg to the test angle, held the angle for three seconds, and then lowered the leg to rest. Next, the participant was instructed to close their eyes and reproduce the test angle. The discrepancy between passive positioning and active repositioning, as well as between the opened and closed eyes, raises concerns for the accuracy of measurement. Strong proprioception research relies on passive positioning and repositioning with closed eyes throughout the entirety of the measurement procedures in order to minimize confounding variables and participant distractions. Our results may have been strengthened if the participants were given the opportunity for practice trials, if they were tested both with their eyes opened and closed, and if more trials were performed.13 In addition, the small sample size may have limited the data’s interpretation and the evaluation of the results.Finally, we cannot rule out the possibility that the Tai Chi performed in this clinical trial may have constituted an insufficient amount of exercise in influencing proprioceptive acuity. Proprioceptive gains are specific to the joint angles trained, but participants in our programme practiced a modified Yang style Tai Chi that eliminated deep knee bends in order to project the knees. By limiting the joint movements at the knee during the training, it is possible that we also limited the benefits to proprioceptive acuity at the knee. Additionally, the participants practised Tai Chi for just 12 weeks and had no prior experience with Tai Chi or similar disciplines prior to the intervention. In contrast, previously mentioned observational studies found significant effects of Tai Chi on proprioception in participants who were termed ‘Tai Chi practitioners’ with a minimum of three years’ experience.In conclusion, while we were able to demonstrate that Tai Chi may benefit knee proprioception in patients with severe Knee OA under certain conditions, we were unable to replicate the results of several previous clinical trials finding that proprioceptive deficits in older patients with knee OA consistently improved following an exercise intervention. As impaired proprioception may play a role in the progression in knee OA, finding feasible measures to improve proprioceptive acuity is of critical interest. We conclude that further research should focus on identifying standardized and reproducible methods for the accurate measurement of knee proprioception.


The aim of this study was to examine the potential association between intrauterine exposure to maternal diabetes and attention deficits in the offspring.Adolescent offspring of a prospectively followed cohort of women with type 1 diabetes (n = 269) and a control group from the background population (n = 293) participated in a follow-up assessment in 2012–2013. We used scores from Conners Continuous Performance Test II to assess attention and based on a principal component analysis we evaluated scores on five different attention factors: focused attention, vigilance, hyperactivity/impulsivity, sustained attention and response style.A higher frequency of the exposed offspring had a parent/self-reported use of Attention Deficit Hyperactivity Disorder (ADHD) medication compared to the control group . Clinical significant differences between adolescents exposed to maternal diabetes and unexposed controls were not found in either single scores on Conners Continuous Performance Test or on any of the five attention factors identified.Exposure to maternal type 1 diabetes did not seem to increase the risk of attention deficits in the adolescent offspring. However, a higher self-reported use of ADHD medication in the exposed group could suggest a difference in attention not revealed by the applied test. Attention-deficit hyperactivity disorder is a common neurodevelopmental disorder characterized by persistent hyperactivity, impulsivity and inattention with a worldwide prevalence of 3–4% –3. The eWe recently reported impaired long-term cognitive function in adolescent offspring of a well-characterized cohort of Danish women with type 1 diabetes . FurtherAnimal studies as well as clinical studies have shown adverse effects of maternal diabetes on the development of the brain and behavior in the offspring –23, ThusWe performed a follow-up examination of offspring of a well-characterized cohort of Danish women with type 1 diabetes to assess if exposure to maternal diabetes can affect the risk of attention deficits in adolescence.EPICOM  is a Danish prospective nation-wide follow-up study of the long-term effects of exposure to maternal diabetes.During 1993–1999 all pregnant women with type 1 diabetes in Denmark were prospectively followed and data on maternal demography, diabetes status and pregnancy outcome were reported to a central register in the Danish Diabetes Association (n = 1215) . The womAt the age of 13 to 19 years offspring of women in the register were invited to a follow-up examination (singletons and one child per mother) (n = 746). A control group matched according to gender, age and postal code (as a marker of socioeconomic status) was identified through the Danish Central Civil Registration System. At inclusion all control participants and/or their parents were asked about a prior history of gestational diabetes, and in addition all available obstetric records were scrutinized for information on gestational diabetes.We excluded 16 diabetes exposed offspring due to: maternal diabetes diagnosis had been reclassified to MODY or type 2 diabetes (n = 12), no contact between mother and child (n = 2), drug abuse (n = 1), pregnancy at the time of recruitment (n = 1).Reasons for exclusion of control offspring were: adopted child (n = 4), the obstetric record revealed a maternal diagnosis of gestational diabetes (n = 3), birth place outside of Denmark (n = 1).The EPICOM study examined 278 diabetes exposed offspring  and 303 unexposed offspring . Participants were all born in Denmark and mainly of white European ethnicity (98.8%). A total of 269 (36.1% of invited) exposed and 293 (15.3% of invited) control offspring completed the computer based attention test . All nonThe protocol was in accordance with the Helsinki Declaration and approved by the regional ethical committee of Central Denmark (M-20110239). We obtained written informed consent from all participants or their parents if the participant was younger than 18 years.Two diabetes exposed participants had known diabetes (type 1), and one was diagnosed with type 1 diabetes in our study sample. All participants, both diabetes exposed and controls, had an oral glucose tolerance test (OGTT) as a part of the examination program. Data from the OGTT has been published recently .The participants were examined at one of three Danish University Hospitals  in the period April 2012 to October 2013.We used Conners Continuous Performance Test version II   to evaluIn Conners CPT-II the participants had to press the spacebar for all letters, displayed on a computer screen, except when the letter X appeared. Administration time for the test is 14 minutes with six blocks of trials with various inter stimulus intervals (ISIs). ISIs are 1, 2 or 4 seconds and make it possible to test the ability to adjust to changing demands. All participants were given standardized instructions and completed a short practice session before the actual test, conducted by exposure blinded testers. The test was conducted after a light meal and in a quiet room without disturbances. Only the participant and the tester were present in the room during the test, while parents waited outside.Conners CPT-II provides a large number of individual test measures. To reduce the number of variables and to obtain a clearer picture of components of attention, we followed Egeland & Kovalik-Gran  and subjOnly three factors had eigenvalues larger than 1, but varimax rotation with three, four and five retained components showed a pattern similar to that obtained by Egeland & Kovalic for only five components. Thus, the first five components explained 83% of the variance, and the rotated matrix of loading showed the following attention factors:Factor 1, Focused attention: defined by the loadings of omissions, hit reaction time standard error, variability and perseverations. Factor 1 explained 24.0% of the variance.Factor 2, Vigilance: defined by the loadings of hit reaction time by differing inter stimulus intervals and hit reaction time standard error by inter stimulus interval. Factor 2 explained 17.5% of the variance.Factor 3, Hyperactivity/impulsivity: defined by the loading of commissions and hit reaction time. Factor 3 explained 16.1% of the variance.Factor 4, Sustained attention: defined by the loadings of hit reaction time by block, hit reaction time standard error by block. Factor 4 explained 15.0% of the variance.Factor 5, Response style: defined by the loading of response style. Factor 5 explained 10.3% of the variance.The first four factors clearly corresponded to four of the Egeland & Kovalic factors with the same test scores having high loadings on the same factors except for the response style test score. Egeland & Kovalic called their fifth factor change in control and this fifth factor reflected the measures of change in omissions and commissions which were not included in our analysis. In their analysis Response Style loaded together with commissions and hit reaction time on the hyperactivity/impulsivity factors, but this was not the case in our analyses with rotation of 4 or 5 factors.Exposure was maternal type 1 diabetes in fetal life.Our primary outcomes were indications of attention deficits assessed by individual test measures in Conners CPT-II and by five attention factors derived from principal component analysis of variables from Conners CPT-II: focused attention, vigilance, hyperactivity/impulsivity, sustained attention and response style.Parental educational level was estimated from a questionnaire completed by the offspring and their parents. It was calculated as the sum of maternal and paternal years in school and in higher education.Maternal age at delivery, parity and information about complications in pregnancy were retrieved from the register in the Danish Diabetes Association (exposed group) or from obstetric medical records (control group). Pregnancy complications were defined as occurrence of hydramnios  or preeclampsia (blood pressure>140/90 mmHg and proteinuria).Information on breastfeeding, smoking in pregnancy and parental intelligence was not available. Sufficient data on delivery mode was not available in the control group and therefore not included in our analysis.Information about gestational age, birth weight, congenital malformations and neonatal complications were provided from the Danish Diabetes Association register (exposed group) or retrospectively from obstetric medical records (control group).We combined and defined neonatal complications as hypoglycemia , jaundice (need of treatment with phototherapy), respiratory distress (need of assisted ventilation or continuous positive airway pressure>1 hour), Apgar 5min<7 and systemic infections (need of systemic antibiotics treatment).Birth weight standard deviation scores (bwSDS) were calculated using intrauterine growth curves adjusted for gestational age and gender .We considered offspring gender, age at follow-up, parity, parental educational length and maternal age at delivery potential confounders.Gestational age, bwSDS, pregnancy complications and neonatal complications were considered potential mediators between maternal diabetes and offspring attention deficits.Continuous data with normal distribution are presented as mean and standard deviation , while cWe adjusted for confounders and mediators on each principal component factor in multivariate linear regression analyses with maternal diabetes as the independent variable and each of the principal component factors as outcome. The regression coefficient β with 95% confidence interval corresponds to the mean difference between the diabetes exposed and the control group.Statistical analyses were performed with IBM statistics SPSS version 22, with a significance level of 0.05. Correction for multiple testing with the Bonferroni method was applied in analysis of our five principal components and in these tests the level of significance was 0.05/5 = 0.01.Mean maternal diabetes duration at conception in the exposed group was 12.5 (SD 8.2) years, and the frequencies of pregestational macroalbuminuri and proliferative retinopathy were 5.6% and 8.0%, respectively.Participants and non-participants in the exposed group differed at baseline according to bwSDS  and maternal peri-conceptional HbA1C (7.3% (56mmol/mol) vs. 7.5% (58 mmol/mol), p = 0.02), but were similar with respect to parity, maternal pre-pregnancy age, maternal pre-pregnancy BMI, duration of diabetes and HbA1C in second and third trimester.Differences between exposed and control offspring were not found in any of the individual test measures derived from Conners CPT II . SimilarNo interaction between gender and exposure was found. Gender differences were evaluated in a model controlling for exposure group and a higher male score was found for factor 3 (hyperactivity/impulsivity) , but no significant differences in the remaining factors.We did not observe any significant interaction between maternal diabetes and low parental education on the risk of attention deficits in the offspring.We found low correlations between age at follow up and each of the five factors: correlation coefficients ranged from -0.13 to 0.16.In this large prospectively followed cohort we found a higher self-reported use of ADHD medication, but no increase in attention deficits in adolescent offspring exposed to maternal type 1 diabetes in utero compared to a control group from the background population. We used Conners CPT-II to assess attention deficits and did not find any clinical significant differences on individual test measures or in crude or adjusted analysis of attention scores derived from principal component analysis. Attention scores were analyzed because combination of test scores may be more reliable and sensitive to attention deficits.Focused attention (factor 1) is the ability to concentrate on a specific stimulus and exclude others. Vigilance (factor 2) is the ability to remain alert, when stimuli are less frequent. A high score on the factor hyperactivity/impulsivity (factor 3) are obtained with many commissions and short reaction time and would be expected in individuals with ADHD, while sustained attention (factor 4) describes the capacity to stay focused throughout the test and could be affected in individuals with depression . A diffeWe performed analysis of the effects of gender and age, owing to a well-documented gender specific difference in attention deficits and remission of symptoms during adolescence . Males hIn a recently published article on the EPICOM cohort , offspriOne of the major strength of this study is the use of a large, well-characterized and prospectively followed cohort of adolescent children of women with type 1 diabetes and a control group identified from the background population, both with detailed baseline information available.In order to describe different aspects of attention we applied a high-signal-to-noise continuous performance test, a widely used test of attention deficits, in particular ADHD, and we identified attention factors in principal component analyses. Unfortunately, other sources of information on attention performance were not available. Impaired executive function and cognitive flexibility are known to be present in individuals with ADHD , and perWe did consider some of the potential problems with the use of Conners CPT II in our analyses, and chose not to include the overall confidence index. The identification of attention factors was done to focus on the different aspects of attention and to have a basis for deriving composite measures presumably more reliable than the individual test scores.A limitation to our study was the risk of selection bias owing to the limited and different proportions of participation in our two groups. We have previously shown that both groups had intelligence scores above the population mean indicating selection from the better performing part of the population , which aLow socioeconomic status is known to have a strong effect on the risk of development of attention deficits in the offspring through different pathways . The effFinally, statistical power should be sufficient with 269 participants in the exposed group and 293 in the control group. Thus, the statistical power would be 84% for detecting a group difference of one quarter of a standard deviation.In a nested case-control study, including individuals from the Norwegian Birth and Prescription Databases, only 3.0% n = 88) of diabetes exposed offspring had been prescribed ADHD medication in the period 2004–2012 [8 of diabIncreased risk of ADHD symptoms in offspring exposed to maternal gestational diabetes (GDM) has been described previously , 10, in In line with this, a register-based study on the EPICOM cohort found that offspring born to mothers with poor glycemic control in pregnancy attained lower grades in primary school compared to matched controls, while those born to mother with good glycemic control obtained better grades than their matched controls in an analysis adjusted for parental educational level . AchieveIt is possible that not only intrauterine exposure to diabetes but also a shared genetic susceptibility to both type 1 diabetes and ADHD could influence attention in the offspring. A German register study found an increased frequency of ADHD in children with type 1 diabetes compared to those without, while a Norwegian study found no association between type 1 diabetes and ADHD , 37. OffIn conclusion, we found a higher self-reported use of ADHD medication among diabetes exposed offspring compared to the control group. However, no increased risk of attention deficits in adolescence was demonstrated in this large, prospective and well-characterized cohort exposed to maternal Type 1 diabetes during fetal life.


E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases.Uropathogenic  Escherichia coli, or E. coli for short, is a type of bacteria commonly found in the guts of people and animals. Certain types of E. coli can cause urinary tract infections (UTIs): they travel from the digestive tract up to the bladder (and sometimes to the kidneys) where they provoke painful symptoms. To cause the infection, the bacteria must become solidly attached to the lining of the bladder; otherwise they will get flushed out whenever urine is expelled.E. coli has many different types of pili, but one seems particularly important in UTIs: type 1 pili. These pili are formed of subunits that assemble into a long coil-shaped rod, which is tipped by adhesive molecules that can stick to body surfaces. The current hypothesis is that the pili act as shock absorbers: when the bladder empties, the pili’s coil-like structure can unwind into a flexible straight fiber. This would take some of the forces off the adhesive molecules that are attached to the bladder, and help the bacteria to remain in place when urine flows out. However, the exact structure of type 1 pili is still unclear, and the essential role of their coil-like shape unconfirmed.Pili are hair-like structures that cover a bacterium and allow it to attach to surfaces. E. coli to invade and colonize the bladder.Here, Spaulding, Schreiber, Zheng et al. use a microscopy method called cryo-EM to reveal the structure of the type 1 pili at near atomic-level, and identify the key units necessary for their coiling properties. The experiments show that pili with certain mutations in these units unwind much more easily when the bacteria carrying them are ‘tugged on’ with molecular tweezers. The bacteria with mutant pili are also less able to cause UTIs in mice. The coiling ability of the type 1 pili is therefore essential for Every year, over 150 million people worldwide experience a UTI; for 25% of women, the infection regularly returns. Antibiotics usually treat the problem but bacteria are becoming resistant to these drugs. New treatments could be designed if scientists understand what roles pili play in the infection mechanisms. Escherichia, Klebsiella, Pseudomonas, Haemophilus, Salmonella and Yersiniae genera  pili. Notably, CUP pili are critical virulence factors in a wide range of pathogenic bacteria, including e genera . To date habitat . Interes E. coli .E. coli (UPEC), which is the causative agent of the majority of urinary tract infections (UTIs). UTIs affect 150 million people annually worldwide and are associated with significant morbidity and economic impact (4 cells (4 CFU/ml) and high bacterial bladder burdens (>104 CFU) two or more weeks after inoculation  based on their genetic relatedness , and a C-terminal pilin domain, which connects the adhesin to the bulk of the pilus (via a reaction known as donor strand exchange (DSE). In DSE, the strand donated to a nascent subunit by the chaperone is replaced by the N-terminal extension (Nte) of an incoming subunit  and chaperone (FimC) help type 1 pili assemble into a composite pilus structure consisting of a short tip fibrillum made up of the adhesin protein (FimH) and two minor subunits (FimG and FimF) that is joined to the pilus rod, a homopolymer of ~1000 FimA subunits. Once extruded to the extracellular surface, the type 1 pilus coils into a rigid right-handed helical structure that is capable of unwinding into a flexible linear fiber . This abHere, using high-resolution cryo-electron microscopy (cryo-EM), we solved the structure of the type 1 pilus rod. Residues involved in critical FimA-FimA interactions were identified that when mutated reduced the force required to unwind the helix, despite not altering the ability of the FimA protein to be incorporated into the pilus rod. When introduced into the chromosome of UTI89, a human UPEC clinical cystitis isolate, these point mutations dramatically reduced the ability of UTI89 to establish an intestinal reservoir and cause acute and chronic cystitis. In contrast, these point mutant strains did not result in large scale differences in levels of piliation or ability to agglutinate mannose-expressing guinea pig red blood cells in vitro. Taken together our results show that the identity of residues within the FimA rod, are critical for the type 1 pili mediated virulence of UPEC in the urinary tract and suggest that the helical pilus rod has an important functional role, beyond serving as a platform to present FimH, in promoting colonization in the gut and infection of the bladder.E. coli K12 strain BW25113. As shown in fimA PCR product showed that the encoded amino acid sequence was identical to the FimA protein from BW25113 (GenBank AIN34588.1) and MG1655 (GenBank NP_418734.1). There was no possibility of cross-contamination of the FimA and T4P filament images, as each has a very different helical symmetry. Averaged power spectra from FimA filaments  from the ilaments  showed nilaments .0 interacts with six adjacent subunits   . The donN+2,N+3) .BW2511322–33, which are part of the interior surface of the rod helix, show structural differences between our cryo-EM rod structure and the previously solved crystal structures of FimA  for the type 1 pilus have been generated using the same solid-state NMR data (E. coli (UPEC)  , howevertructure  that is t models . The oveis 5.7 Å , while iis 5.7 Å . Between aligned .Comparison of our type 1 pilus model with the P pilus structure  shows anE. coli genes evolve slowly . To avoid confusion herein all residues are discussed using the BW25113 numbering system.To examine the importance of FimA-FimA interactions on bacterial pathogenesis, we chose to investigate how mutations in the fimA sequences (dN) to synonymous nucleotide mutations (dS) in each codon in the alignment (dN/dS) to estimate the selection pressures acting on each residue in the FimA protein using a fixed-effects likelihood measurement algorithm  while a dN/dS ratio <1 indicates conservation at the codon . Here, we found that 66 codons were under purifying selection, five codons were under adaptive selection, and an additional 27 codons were too conserved to be included in analysis ; including nearly every UPEC strain (96.3% or 26/27 strains). Importantly, the fimA sequences from UPEC strains were spread through the phylogenetic tree, indicating that there was not a single variant of FimA found in all UPEC strains . Here, wabeled C . This pafimA gene, which were subsequently cloned into the expression vector, pTRC99a  by altering the left inverted repeat necessary for promoter inversion . Most of our fimA mutants showed hemagglutination (HA) titers similar to the wild-type (WT) fimA  showed statistically significant evidence of strong purifying selection (We assessed the function of the type 1 pilus in each WT) fimA . HoweverWT) fimA . This suWT) fimA . Expresselection .BW25113 variants produced by the UTI89-LON∆fimA strain on pili force-extension responses, we chose six FimA mutant strains that did not disrupt pilus formation or produce severe bacterial clumping in vitro  and applied optical tweezers (0 and N+3), thus any changes in these interactions caused by mutations would shift the magnitude of the force plateau. To investigate this, we first measured unwinding force of single pili in the strain expressing WT FimA and subsequently examined the force response for each fimA mutant. The unwinding force of WT pili was 30.3 ± 0.2 pN (mean ±standard error (SE)) whereas all mutants showed a reduction in the force required for pilus unwinding . For these studies, we chose to investigate the effects of four FimA mutants that were examined via optical tweezers. Each of these mutations were made in the codon of a highly conserved amino acid positions of FimAUTI89 .In the bladder, type 1 pili are required for binding and invasion of UPEC into superficial facet cells that line the bladder lumen and for the formation of intracellular bacterial communities (IBCs) during the first 6–18 hr of acute infection. This has been shown to be critical for ongoing infection in both humans and mouse models of cystitis . To detefimA sequence, we found that all reintegrated FimA mutant strains demonstrated similar levels of GP-RBC agglutination, in vitro  bacteriuria and high titer (>104 cfu/ml) bladder bacterial burdens at sacrifice >4 weeks post-infection), strains producing FimAUTI89 variants with the D62R, D114R, and A22R substitutions were outcompeted by up to six logs by the reintegrated WT strain (4 cfu/ml at least once during the 4 week infection) in this experiment are shown in We next investigated how each mutant altered the kinetics of bladder infection during competitive infections with WT UTI89 over 28 days. In mice that developed chronic cystitis  significantly altered the ability of UTI89 to form IBCs at 6 hr post infection (hpi)  . Howeveron (hpi) , with 8/on (hpi) . The D62–14 days .UTI89 variants D62R, D114R, and A22R showed lower levels of intestinal colonization (by up to 2 logs) in the feces, cecum, and colon of mice compared to those colonized by WT fimA  in the feces and cecum than the WT strain  that expressed adhesive pili in vitro but reduced the ability of UTI89 to colonize the bladder, acutely and chronically. One of these mutants, FimAUTI89 A22R, was severely attenuated during acute infection, forming almost no IBCs and thus was unable to chronically infect the mouse bladder.FimA is critical for proper assembly of the type 1 pilus and, as the major subunit that makes up the pilus rod, is also critical for the proper display of the FimH adhesin. However, here we have uncovered a more complex and previously unappreciated role for FimA and the type 1 pilus rod in host-pathogen interactions. We identified three mutations in FimABW25113 protein require more than twice the force to unwind than the pili formed by the FimA BW25113 variant with an A22R mutation (~30 pN vs. 11pN), likely due to steric clashes caused by this substitution affecting the tightly packed interface between subunits. Thus, we hypothesize that the pilus formed by the FimA A22R mutant variant may not have the strength to absorb and withstand the shear forces experienced during urination and thus allow the bacteria to be swept out of the bladder, preventing infection. In the gut, the pilus rod may play a similar role during mucus shedding. A recent study suggests that type 1 pili may promote UPEC colonization of the upper crypts  that were attenuated in bladder colonization also displayed significantly reduced intestinal colonization. However, in general, the phenotypes of the UTI89fimA mutants in gut colonization were not as severe as in the bladder.We hypothesize that the identified FimA point mutants handicap UPEC pathogenicity in vivo by altering the properties of the pilus rod. The pilus rod is hypothesized to act as a ‘molecular spring’ transitioning between a flexible, linear fiber and a coiled helix. This spring-like property is thought to prevent the pilus from breaking or detaching from the host surface by temporarily expanding to a linear form after encountering shear forces, which can occur in the bladder during urine voiding or in the gut during fecal or mucus shedding. Such a transition between the helical and unwound form of the FimA homopolymer has been predicted to significantly dampen the force experienced by the adhesion-receptor complex at the tip . Since Fr crypts . These bUTI89 (D62R and D114R) resulted in a mild clumping phenotype when bacteria were grown in vitro. This clumping phenotype was also seen in the equivalent mutants in FimABW25113 . These residues are located on the exterior surface of the rod structure and do not have as strong an effect on the force needed for unwinding as FimABW2511 A22R. However, the clumping phenotype suggests that these mutations may alter side-to-side interactions between different pilus rods promoting pilus-pilus interactions within and between bacteria, thus inhibiting the pilus mediated interactions needed in vivo for attachment, invasion and/or IBC formation. Interestingly, we do not observe a defect in bladder colonization in mice infected with FimAUTI89 D62R until 10dpi, suggesting that normal rod function is needed during both chronic and acute infection.Two mutations in FimATogether, our data suggest that the type 1 pilus rod may mediate colonization phenotypes through damping of shear forces or through other mechanism(s) outside of scaffold support for FimH-binding. A possibility that we cannot exclude at the moment is that the lower uncoiling force of the A22R mutant exposes parts of the rod that would be largely buried in the wt filament to host proteases. This would still suggest that the mechanical properties of these rods have been ‘tuned’ to optimize for particular environments, as a wt rod that could never uncoil would be susceptible to breakage by the shear forces, while a rod that uncoils too easily would be unnecessarily exposed to digestion by proteases.Salmonella  from the selection pressure maintaining the codons that were necessary for pilus function (such as A22 in FimAUTI89). Given the intricacy of pilus assembly, one could expect that most of the codons under purifying selection would be related to pilus construction. Instead, we found that many codons under purifying selection are involved in keeping the force needed to unwind the type 1 rod within a narrow range. This leads to reasoning that a ‘weak’ or ‘loose’ FimA rod may be just as detrimental for E. coli as having no rod at all, at least in the eyes of evolution.Evolutionary and structural analysis of FimA, in combination with our in vitro and in vivo phenotyping, yielded several important insights into the selection pressures faced by UPEC as well as the evolutionary trajectories that pathogens follow to enhance their colonization of different host niches. Notably, we saw that different phenotypes caused by mutations in the FimA protein were associated with different classes of selection pressure. For example, the bacterial clumping phenotype is associated with mutation of three codons under purifying selection, but an equal number of the mutations that resulted in clumping are in codons with little to no evidence of selective pressure. In contrast, all mutations that failed to complement a E. coli pathogenesis and persistence and appear to have been carefully ‘tuned’ by evolution. Further studies of the hundreds of CUP pili encoded in Gram-negative bacteria are needed to further understand the unique and general aspects of the evolution of CUP pilus fibers. In addition, other bacterial pili, such as T4P, which have arisen independently of CUP pili but can play similar roles in pathogenesis, can also elongate under force . Mice were acquired from indicated vendors and randomly placed into cages (n = 5 mice/cage) by employees of Washington University’s Division of Comparative Medicine (DCM); no additional methods for randomization were used to determine how animals were allocated to experimental groups. Investigators were not blinded to group allocation during experiments.Animals were maintained in a single room in our vivarium. Prior to and after infection all animals received PicoLab Rodent Diet 20 (Purina) 8 CFU per 100 μL for intestinal infections and ~1×108 CFU per 50 μL for bladder infections. Bacteria were subsequently washed three times with PBS and then concentrated to ~1×108 CFU per 50 μL for bladder infections.For bladder infections, 6 week old female C3H/HeN mice were obtained from Envigo and were maintained in our vivarium for one week prior to infection. Bladder infections were performed via transurethral inoculation . UPEC st8 CFU UPEC in 100 μL phosphate-buffered saline (PBS) ) followed 24 hr later by an oral gavage of ∼10ne (PBS) . BacteriIn all cases, fecal and urine samples were collected directly from each animal at the indicated time points. Fecal samples were immediately weighed and homogenized in 1 mL PBS. Urine samples were immediately diluted 1:10 prior to plating. Mice were sacrificed via cervical dislocation under isofluorane anesthesia and their organs were removed and processed under aseptic conditions. Intestinal segments (cecum and colon) were weighed prior to homogenization and plating on LB supplemented with the appropriate antibiotic.Exclusion criteria for mice were pre-established; (i) both introduced strains in competitive infections became undetectable during the course of a 14 day experiment, and (ii) mice died or lost >20% of their body weight. No mice in this study met these criteria. Each experiment was conducted with both technical  and biological  replicates.fimA mutant strain. To accurately count the number of IBCs, mice were sacrificed 6 or 12 hr after infection. Bladders were removed aseptically, bi-sected, splayed on silicone plates and fixed in 4% (v/v) paraformaldehyde. IBCs, readily discernable as punctate violet spots, were quantified by LacZ staining of bladder wholemounts  of 1 and guinea pig erythrocytes normalized to an OD640 of 2. The experiment was conducted in parallel in PBS with 2% w/v methyl-α-D-mannopyranoside.Bacteria were grown under type 1 pilus-inducing conditions , with apElectron micrographs (EM) were taken of UTI89 or UTI89 isogenic mutants after growth under type 1 pilus-inducing conditions. A total of 300 bacterial cells were counted for each condition, and piliation on those cells was classified as bald (no pili), low (1 to 20 pili/cell), moderate (20 to 200 pili/cell), or abundant (>200 pili/cell).4 laser  operating at 1064 nm for trapping a single bacterium or microspheres. A probe laser (low power HeNe-laser operating at 632.8 nm) is merged with the trapping laser using a polarizing beam splitter cube (PBSC). The light from the probe laser is refracted by the trapped object and collected by the condenser and thereafter imaged onto a 2D position sensitive detector . The PSD convert the incoming light to a photocurrent and thereafter to a voltage that is sent to a programmable low pass filter , later collected by a computer and processed with an in-house LabVIEW program. We minimized the amount of noise in the setup and optimized the measured time series using the Allan variance method described in (For expression of type 1 pili the strains were grown in Luria Broth (LB) supplemented with carbenicillin (100 µg/mL) and IPTG (50 µM), at 37°C overnight. The optical tweezers (OT) setup is built around an inverted microscope  equipped with a high numerical aperture oil immersion objective  and a 1292 × 964 pixel camera with a cell size of 3.75 × 3.75 μm  Figure 4. The OT ribed in .600 = 1) suitable for single cell analysis using optical tweezers (OT). Surfactant-free 2.5 µm amidine polystyrene microspheres  were similarly suspended in Milli-Q water, these microspheres were trapped and used as force probes. To mount bacteria and reduce the influence of surface interactions we prepared a 1:500 suspension of 9.5 mm carboxylate-modified latex microspheres  in Milli-Q. We dropped ten microliters of the microsphere-water suspension onto 24 × 60 mm coverslips  and placed these in an oven for 60 min at 60°C to immobilize the microspheres to the surface. To firmly adhere bacteria to the microspheres, we added a solution of 20 mL of 0.01% poly-L-lysine  to the coverslips, which, after 45 min incubation at 60°C, were stored until use. A free-floating bacterium was trapped by the optical tweezers run at low power to avoid cell damage. The bacterium was thereafter mounted on a large 9.5 µm microsphere coated with poly-L-lysine. We trapped a small free-floating 2.5 µm microsphere by the optical tweezers with normal power (a few hundreds of mW) and brought it close to (within tens of µm) but not in direct contact with, the bacterium. To calibrate the trap stiffness we used the Power spectrum method by sampling the microspheres position at 131,072 Hz and average 32 consecutive data sets acquired for 0.25 s each (https://github.com/elifesciences-publications/ot-control).To prepare a sample we suspended bacteria in 1xPBS to a concentration  around the area containing the poly-L-lysine-coated microspheres on one of the coverslips. Carefully, we dropped a 3 mL suspension of bacteria and a 3 mL suspension of probe microspheres  onto the area and sealed the flow chamber by placing a 20 × 20 mm coverslip  on top. Thereafter, we mounted the sample in a sample holder that is fixed to a piezo-stage  in the OT instrumentation. To get a reliable OT calibration parameter values we measured the temperature using a thermocouple in the sample chamber, 23.0 ± 0.1°C and the suspension viscosity was assumed to only vary with temperature, thus, the viscosity was set to 0.932 mPas ± 0.002 mPas.E. coli strain BW25113  of the motion-corrected image stacks. The CTF was corrected by multiplying the images from the first two-chunk sums with the theoretical CTF, which is a Wiener filter in the limit of a very poor signal-to-noise ratio (SNR). This both corrects the phases which need to be flipped and improves the SNR. The e2helixboxer routine within EMAN2 . Then the grids were plunge-frozen using a Vitrobot Mark IV , and subsequently imaged in a Titan Krios at 300keV with a Falcon II direct electron detector (pixel size 1.05 Å/pixel). A total of 6803 images, each of which was from a total exposure of 2 s dose-fractionated into seven chunks, were collected at a range of underfocus between 0.5 ~ 3 μm. Images were motion corrected using MotionCorr , and thein EMAN2  was usedin EMAN2  reconstrWe used a previous FimA NMR model  as an initial template to dock into the cryo-EM map by rigid body fitting, and then manually edited the model in Chimera  and CootE. coli FimA, FimC, and FimH were obtained by individual searches the Ensembl Bacteria Genome database . The signal sequences were trimmed from the remaining homologs using Geneious v 6.1.7  using custom bash scripts  to synonymous (dS) mutation in each codon  using a fixed-effects likelihood test to identify statistical significance  Determination of unwinding force and correlation between unwinding force and in vivo observations are not convincing yet and need to be substantiated.2) When correlating the in vitro and in vivo results, there are several uncontrolled variables that need to be addressed including proteases, bacterial clumping, number of pili versus structure of pili for fimA mutants.Further detail can be found in the separate reviews below. If you have any questions about the revisions, please let us know.Reviewer #1:The only concern of this reviewer is that some leaps are made to correlate observations in vitro, with those in vivo. The suggestion that the spring-like qualities of the pilus rod are critical for pathogenesis is entirely possible, but is far from proven. Moreover, few specifics are provided that relate the actual shear forces of urine at various distances from the mucosal boundary to the forces measured for variations in the protein. A number of uncontrolled variables could explain why mutant variants of the protein are less effective in vivo, including increased susceptibility to tissue resident proteases, altered interactions with various elements of the mucosa, altered physical states imposed (such as the clumping that was observed), etc. While an interesting prospect, the mechanical properties proposed could be explored further for proof, or the discussion of this prospect modified in the text . Aside from that concern, this reviewer found the paper to be of considerable interest and well done.Reviewer #2:The authors determined the Type I pilus structure by cryo electron microscopy. The images in 1) The A22R mutant has a significant defect at 6 hours in the mouse. Does this mutant affect the ability of bacteria to invade the superficial facet cells?2) A few of the mutants were tested in vivo, but not in vitro. For example, E123R was tested in the mouse and displayed a defect in acute and chronic UTI (and a slight defect in gut colonization), but the force measurements for unwinding this pili were not determined.Reviewer #3:The manuscript by Caitlin Spaulding et al. reports on the relation between atomic structure, evolutionary conservation, biophysical properties, and host pathogen interactions of bacterial type I pili. Using electron microscopy, they solve the structure of the type I pilus at atomic resolution. Next, by genomic comparison of the T1P genes between different species, they find that the gene encoding of the major subunit of the pilus, fimA, shows a higher variability compared to other parts of the T1P machinery. In particular, residues at the exterior are prone to adaptive selection. Based on their positions in the atomic model, the authors investigate the effects of point mutations on pilus expression and agglutination. They state that the unwinding force of the pilus strongly depends on these mutations while functionality is unaffected. Finally, they investigate the effect of point mutations on the competitive fitness within the bladder of a mouse model, on their ability cause chronic infections, and on their ability to colonize the intestine. The authors conclude that reducing the unwinding force of T1P affects host-pathogen interactions.The idea to test the relevance of elastic properties of pili for host-pathogen interactions and in particular of bladder infections is wonderful. The general approach of starting with an atomic model of the pilus to identify promising candidates for amino acid substitutions is highly convincing. I have three major problems with this study and I will describe them in detail below. I would like to state that I don't have enough expertise in structural biology to assess the quality of the structural data.1) Measurement of the unwinding force.a) The authors state in the Materials and methods part they expect "attachment a few pili with the microsphere". Determination of the unwinding force, however, is only possible it the authors can make sure that only a single pilus is attached at the time.b) I haven't found any information about how the mutations analysed in c) Only technical replicates from a single sample are inacceptable. Data should be acquired on biological replicates on at least three different days.2) Expression levels and functionality of pili with point mutations.The authors state that "FimA mutant strain produced minimal changes to the levels of type 1 piliation […] and to the function of the type 1 pilus". The experimental data supporting this statement is shown in 3) Comparison between different strains.a) The authors explain that they used different bacterial strains with different fimA sequences. This is probably fine, but the way the data is presented now, I get confused with the different notations of the point mutations. For example, is the A22R mutation in b) The authors look at the effects of amino acid substitutions at different levels of complexity (which is great) but I currently get lost in the details. Please explain the criteria for analysing specific mutations better within the individual parts of the Results section. Also, please explain the connections between the conclusions drawn from mutant analysis of conservation, unwinding, and host-pathogen interactions related to the structure in an explicit way. Essential revisions:There are two major points to be addressed:1) Determination of unwinding force and correlation between unwinding force and in vivo observations are not convincing yet and need to be substantiated.We thank the reviewers for bringing up this point. Determination of the unwinding force of the WT type 1 pili in this study correlates well with previously published studies . There have been numerous parallel-plate flow chamber studies mimicking the fluid conditions exposing bacteria attaching to host cells. These studies show indeed the need of pili for cell attachment (works by Wendy Thomas group). In such in vitro studies, it has been observed that pili do in fact unwind when the cell is exposed to fluid flow .Correspondingly, in this study we find that the unwinding force is significantly different for the WT and the A22R mutant and this mutant is severely attenuated in bladder and gut colonization.2) When correlating the in vitro and in vivo results, there are several uncontrolled variables that need to be addressed including proteases, bacterial clumping, number of pili versus structure of pili for fimA mutants.The reviewers are correct in pointing out that there are uncontrolled variables in our in vitro and in vivo experiments. in vitro bacterial clumping and pilus expression are variable between FimA mutants, however, we show via hemagglutination assays that this does not impact the function of these pili in vitro. We do find differences between WT and mutant FimA expressing strains in both two different niches within the urinary tract (the bladder and the kidney) and within the gut. In allin vivo mouse experiments, there are numerous uncontrolled variables present, including host and bacterial proteases. While we do not control for these proteins in our studies, we note that we use the same strain of mice for each experiment and biological replicates for experiments are performed in mice that are the same age, from the same vendor and batch, and have been exposed to the same conditions and diet in our vivarium. While host proteases might alter pathogenesis, our WT and mutant mice should be exposed to the same protease conditions. We have now added the possibility to the Discussion that the uncoiling of the A22R mutant might make sites accessible to host proteases that would not be available in the coiled wt rod. As we note, this does not change the conclusion of our paper that the mechanical properties of the rod have been “tuned” over evolution as an adaptation to specific environments. Further, while we feel that the appearance of phenotypes such as clumping or alterations in pilus expression highlights the role of residues in interactions within type 1 pili, the in vitro measured phenotype that correlates best with in vivo virulence is the optical tweezer experiments. Note, that we have now removed results of the mutant E121R, which had the most clumping phenotype (see below).Further detail can be found in the separate reviews below. If you have any questions about the revisions, please let us know.Reviewer #1:The only concern of this reviewer is that some leaps are made to correlate observations in vitro, with those in vivo. The suggestion that the spring-like qualities of the pilus rod are critical for pathogenesis is entirely possible, but is far from proven.Please see response to essential revision 1.Moreover, few specifics are provided that relate the actual shear forces of urine at various distances from the mucosal boundary to the forces measured for variations in the protein.Urine flow in the urinary tract is indeed complex and hash: boluses are transported from the kidney to the bladder via peristaltic motion; and high fluid flow exposes bacterial cells to high shear forces during expulsion of urine from the bladder. In addition, peristaltic motion creates shear forces that include reversal wall shear forces and normal forces. Bacteria need to attach and adhere in these conditions, which implies that they are not more than a “coiled-pili-length” away from the surface. Therefore, the shear forces, which increase more-or-less linearly with height for small displacement, will not be significantly different at various distances from the mucosal boundary. This implies also for flow chamber experiments.Several flow chamber experiments generating shear stress of similar magnitude as physiological conditions  have been performed ). Interestingly, these experiments exposed cells from low to high forces, that is, from 10 – 140 pN , which is in the range of WT type 1 pili uncoiling. Pili uncoiling for WT bacteria, leading to bacterial displacement of the order of a few micrometers, have also been demonstrated in flow chamber experiments. Thus experiments support that shear forces exposing bacteria within physiological conditions are sufficiently to uncoil WT pili . Since we measured lower uncoiling force for the mutants, we expect that these “softer-spring” pili attaching bacteria to cells will not be able to modulate and dampen the fluid forces as good as WT pili. This reduced damping capability will therefore make it harder for bacteria to attach and adhere.A number of uncontrolled variables could explain why mutant variants of the protein are less effective in vivo, including increased susceptibility to tissue resident proteases, altered interactions with various elements of the mucosa, altered physical states imposed (such as the clumping that was observed), etc. While an interesting prospect, the mechanical properties proposed could be explored further for proof, or the discussion of this prospect modified in the text .Please see response to essential revisions 1 and 2.Aside from that concern, this reviewer found the paper to be of considerable interest and well done.Reviewer #2:[…] 1) The A22R mutant has a significant defect at 6 hours in the mouse. Does this mutant affect the ability of bacteria to invade the superficial facet cells?This is an important point. To address this question we now include data regarding the ability of the A22R mutant to invade into bladder tissue at 1, 3, and 6 hours. This was done via gentamycin protection assays. We found that the A22R mutant does indeed have an invasion defect compared to WT starting at 1 hpi. This ability of this strain to persist in the lumen of the bladder is also attenuated compared to WT. We have added this invasion data to 2) A few of the mutants were tested in vivo, but not in vitro. For example, E123R was tested in the mouse and displayed a defect in acute and chronic UTI (and a slight defect in gut colonization), but the force measurements for unwinding this pili were not determined.Thank you for pointing this out. The E123R mutant was not included in the optical tweezer data due to difficulty in obtaining reading with single pili attached the beads. The attachment of a single pilus to a bead is essential for us to calculate an accurate unwinding force measurement. After multiple additional failed attempts to acquire this data for this FimA mutant, we have decided to remove all analysis of this mutant from the paper.Reviewer #3:[…] 1) Measurement of the unwinding force.a) The authors state in the Materials and methods part they expect "attachment a few pili with the microsphere". Determination of the unwinding force, however, is only possible it the authors can make sure that only a single pilus is attached at the time.We thank the reviewer for bringing this issue to our attention. Based on this comment, it is clear that our description of the molecular tweezers experiment in the Materials and methods section did not clearly explain how the force-spectroscopy measurements were taken. Therefore, we have now included additional text in the Materials and methods to clarify the experimental set up for this method. Further, we have included an additional figure  that betThe new text reads:“A free-floating bacterium was trapped by the optical tweezers run at low power to avoid cell damage. […] Finally, we controlled the piezo-stage and sampled the data using an in-house LabView program .”b) I haven't found any information about how the mutations analysed in Thank you for this comment, we have provided additional text in the Materials and methods section that explicitly states our use of only a single pilus to make any force measurements. Please see response above to point 1a.c) Only technical replicates from a single sample are inacceptable. Data should be acquired on biological replicates on at least three different days.This is an excellent and critical point made by the reviewer. We have included additional data into 2) Expression levels and functionality of pili with point mutations.The authors state that "FimA mutant strain produced minimal changes to the levels of type 1 piliation […] and to the function of the type 1 pilus". The experimental data supporting this statement is shown in In order to better characterize the variability that we observe in pilus expression with each mutant, we have altered the text in the manuscript to more explicitly reflect the data presented in This text now reads:“When compared to a strain with the reintegrated WT UTI89 fimA sequence, we found that all reintegrated FimA mutant strains demonstrated similar levels of GP-RBC agglutination, in vitro ,despite 3) Comparison between different strains.a) The authors explain that they used different bacterial strains with different fimA sequences. This is probably fine, but the way the data is presented now, I get confused with the different notations of the point mutations. For example, is the A22R mutation in This is an excellent point that was made by multiple reviewers. In order to prevent confusion, we have chosen to refer to all residues from both strains using the BW25113 number system.We have also addressed this change in the text:UTI89 differs from our cryo-EM rod FimABW25113 sequence in 16 residues, with all of the differences lying within highly variable residues located on the exterior of the pilus rod, including two additional amino acids at the N-terminus. […] To avoid confusion herein all residues are discussed using the BW25113 numbering system.”“Thus, we do note that FimA