Cyclic neutropenia and concomitant IgA nephropathy: a case report.

BACKGROUND: IgA nephropathy (IgAN) is universally recognized as one of the most common primary glomerular diseases in all ages. Cyclic neutropenia (CN) is a rare haematologic disorder that is associated with mutations of the ELANE gene. The co-occurrence of IgAN and CN is extremely rare. This is the first case report of a patient with IgAN and genetically confirmed CN. CASE PRESENTATION: We report a case of a 10-year-old boy who presented with recurrent viral upper respiratory tract infections accompanied by several episodes of febrile neutropenia, haematuria, proteinuria and acute kidney injury. Upon first admission, his physical examination was unremarkable. His kidney function was impaired, whereas his urine microscopy showed evidence of macroscopic haematuria and proteinuria. Further workup showed elevated IgA. The renal histology was consistent with mesangial and endocapillary hypercellularity with mild crescentic lesions, while immunofluorescence microscopy showed IgA-positive staining, which was characteristic of IgAN. Moreover, genetic testing confirmed the clinical diagnosis of CN, therefore Granulocyte colony-stimulating factor (G-CSF) was initiated to stabilize the neutrophil count. Regarding proteinuria control, the patient was initially treated with an Angiotensin-converting-enzyme inhibitor for approximately 28 months. However, due to progressive proteinuria (> 1 g/24 h), Corticosteroids (CS) were added for a period of 6 months according to the revised 2021 KDIGO guidelines with favorable outcome. CONCLUSIONS: Patients with CN are more susceptible to recurrent viral infections, which can trigger IgAN attacks. In our case CS induced remarkable proteinuria remission. The use of G-CSF contributed to the resolution of severe neutropenic episodes, viral infections and concomitant AKI episodes, contributing to better prognosis of IgAN. Further studies are mandatory to determine whether there is a genetical predisposition for IgAN in children with CN.

Application of 89Zr-DFO*-immuno-PET to assess improved target engagement of a bispecific anti-amyloid-ß monoclonal antibody.

PURPOSE: The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used 89Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the 89Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders. METHODS: Adu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with 89Zr. APP/PS1 mice were injected with 89Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-ß plaques. Additionally, [89Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease. RESULTS: A 7-fold higher brain uptake was observed for [89Zr]Zr-DFO*-Adu-8D3 compared to [89Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [89Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [89Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [89Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice. CONCLUSION: 89Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aß plaque pathology. Here, we demonstrate that 89Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.

Lower limb joint angle variability and dimensionality are different in stairmill climbing and treadmill walking.

The present study tested if the quadratic relationship which exists between stepping frequency and gait dynamics in walking can be generalized to stairmill climbing. To accomplish this, we investigated the joint angle dynamics and variability during continuous stairmill climbing at stepping frequencies both above and below the preferred stepping frequency (PSF). Nine subjects performed stairmill climbing at 80, 90, 100, 110 and 120% PSF and treadmill walking at preferred walking speed during which sagittal hip, knee and ankle angles were extracted. Joint angle dynamics were quantified by the largest Lyapunov exponent (LyE) and correlation dimension (CoD). Joint angle variability was estimated by the mean ensemble standard deviation (meanSD). MeanSD and CoD for all joints were significantly higher during stairmill climbing but there were no task differences in LyE. Changes in stepping frequency had only limited effect on joint angle variability and did not affect joint angle dynamics. Thus, we concluded that the quadratic relationship between stepping frequency and gait dynamics observed in walking is not present in stairmill climbing based on the investigated parameters.

Reduced vertical displacement of the center of mass is not accompanied by reduced oxygen uptake during walking.

The six determinants of gait proposed that the goal of gait is to minimize vertical displacement of the body's center of mass (CoM) with the objective to optimize energy expenditure. On the contrary, recent investigations suggest that reduced vertical displacement leads to an increase in energy expenditure. However, these investigations had the included subjects deliberately changing their gait, which could bias the endpoint measures. The present study investigated the effect of reduced vertical displacement of the CoM on oxygen uptake and walking economy without imposing altered gait patterns. This was accomplished by having subjects walk on a curved treadmill and on a flat treadmill. Vertical displacement of the CoM (sacrum marker displacement), oxygen uptake, walking economy, stride characteristics and lower limb joint angles were measured. There were significant differences in stride characteristics and phase dependent differences in lower limb movement pattern between the two conditions which in size were comparable to the changes observed between different speeds. The vertical displacement of the CoM was significantly reduced on the curved treadmill. This was accompanied by an increase in oxygen uptake and walking economy. These results support recent assertions that the six determinants of gait do not serve to improve walking economy.

Posture and gaze tracking of a vertically moving target reveals age-related constraints in visuo-motor coupling.

Previously we have demonstrated that the effect of aging on posture and gaze active tracking of a visual target moving in the horizontal direction is dependent on target's complexity. In this study, we asked whether a similar phenomenon is present when tracking a visual target moving with varying complexity in the vertical direction. Ten young (22.98±2.9years) and 10 older adults (72.45±4.72years) tracked for 120s, a visual target moving vertically by shifting their bodyweight in the anterior-posterior direction. Three target motions were tested: a simple periodic (sine wave), a more complex (Lorenz attractor) and an ultra-complex random (Surrogated Lorenz attractor) pattern. Cross-spectral analysis revealed lower sway-target coherence as a function of age, regardless of target motion's complexity. This age effect was significant for the sway-target gain but not for the phase index. Gaze-target analysis revealed age related differences only when tracking the more complex targets. Regardless of age, tracking of the complex target was associated with lower cross Approximate Entropy. It is concluded that tracking of visual targets oscillating in the vertical direction reveals age related constraints that are independent of visual motion's complexity. These constraints are evident in the spatial and not temporal aspects of visuo-motor coupling, which suggests the presence of neuromuscular deficiencies in controlling visually guided postural sway in the anterior-posterior direction.

Aging affects postural tracking of complex visual motion cues.

Postural tracking of visual motion cues improves perception-action coupling in aging, yet the nature of the visual cues to be tracked is critical for the efficacy of such a paradigm. We investigated how well healthy older (72.45 ± 4.72 years) and young (22.98 ± 2.9 years) adults can follow with their gaze and posture horizontally moving visual target cues of different degree of complexity. Participants tracked continuously for 120 s the motion of a visual target (dot) that oscillated in three different patterns: a simple periodic (simulated by a sine), a more complex (simulated by the Lorenz attractor that is deterministic displaying mathematical chaos) and an ultra-complex random (simulated by surrogating the Lorenz attractor) pattern. The degree of coupling between performance (posture and gaze) and the target motion was quantified in the spectral coherence, gain, phase and cross-approximate entropy (cross-ApEn) between signals. Sway-target coherence decreased as a function of target complexity and was lower for the older compared to the young participants when tracking the chaotic target. On the other hand, gaze-target coherence was not affected by either target complexity or age. Yet, a lower cross-ApEn value when tracking the chaotic stimulus motion revealed a more synchronous gaze-target relationship for both age groups. Results suggest limitations in online visuo-motor processing of complex motion cues and a less efficient exploitation of the body sway dynamics with age. Complex visual motion cues may provide a suitable training stimulus to improve visuo-motor integration and restore sway variability in older adults.

Enhanced immunogenicity of multivalent MUC1 glycopeptide antitumour vaccines based on hyperbranched polymers.

Enhancing the immunogenicity of an antitumour vaccine still poses a major challenge. It depends upon the selected antigen and the mode of its presentation. We here describe a fully synthetic antitumour vaccine, which addresses both aspects. For the antigen, a tumour-associated MUC1 glycopeptide as B-cell epitope was synthesised and linked to the immunostimulating T-cell epitope P2 derived from tetanus toxoid. The MUC1-P2 conjugate is presented multivalently on a hyperbranched polyglycerol to the immune system. In comparison to a related vaccine of lower multivalency, this vaccine exposing more antigen structures on the hyperbranched polymer induced significantly stronger immune responses in mice and elicited IgG antibodies of distinctly higher affinity to epithelial tumour cells.

The Effect of Walking Speed on Gait Variability in Healthy Young, Middle-aged and Elderly Individuals.

Previous research with healthy young adults has suggested that the temporal structure of gait variability is not random but shows self-similarity that is dependent on speed. Specifically, the strength of the long-range correlation of stride intervals follows a quadratic relationship with the minimum values at the respective preferred walking speed (PWS). The purpose of this study was to investigate if this relationship is affected by increasing age. Ten healthy young, seven healthy, middle-aged and seven healthy, elderly adults completed five-minute walking trials at 80%, 90%, 100%, 110% and 120% of their PWS on a treadmill. We investigated the temporal structure of gait variability by using detrended fluctuation analysis. In addition, we computed the Coefficient of Variation (CV) to identify effects on amount of gait variability. Our results revealed a significant quadratic relationship between the temporal structure of gait variability and speed for all groups extending the previously reported existence of such a relationship in healthy young adults to older individuals. However, only significant negative linear relationships were found between amount of variability and speed providing support that this relationship is not quadratic but linear across individuals of different ages. In addition, we found that the examination of the temporal structure of gait variability is more sensitive in differentiating middle-age and younger individuals. If middle-age is where the aging process starts, then measures of the temporal structure of gait variability are essential as prognostic and diagnostic tools of aging.

Atypical hemolytic-uremic syndrome (HUS) with recovery after a long-lasting anuria: a case report.

We report a case of a seven-year-old girl who suffered from atypical Hemolytic Uremic Syndrome (aHUS) complicated by septicaemia, central nervous system involvement, and cholangiitis. She remained anuric requiring treatment with peritoneal dialysis (PD) for a five-month period. In addition to conventional therapeutic measures including fresh frozen plasma (FFP) and blood cells transfusions she also underwent to plasma exchange (PE) treatment. Following a stormy hospitalization period of 17 weeks, the patient finally regained renal function and three years later she remains well on antihypertensive treatment and free of dialysis.

Endurance markers are related with local neuromuscular response for the intact but not for the ACL reconstructed leg during high intensity running.

AIM: It has been demonstrated that the local neuromuscular response during high intensity exercise has a strong relationship with endurance markers. However, a diminished neuromuscular response has been reported for the operated leg in athletes having undergone anterior cruciate ligament reconstruction (ACLR). The purpose of the present study was to examine the relationships between endurance markers and the EMG response during high intensity running in ACLR athletes. METHODS: Fourteen ACLR soccer players underwent a GXT test to volitional exhaustion and a 10-min bout of high intensity running. During the 10-min bout, EMG data were recorded at the 3rd and 10th minute from the vastus lateralis bilaterally using a telemetric system. The final EMG levels were expressed as a percentage of the initial values. Pearson moment product correlations were used to assess the relationship between the endurance markers of VO2max, velocity at lactate threshold (vLT), velocity at 4mM (V4) and the final EMG levels. RESULTS: Final EMG levels for the intact leg had a very strong relationship with vLT (r=0.77, P=0.001) and a strong relationship with V4 (r=0.68, P=0.008). Final EMG levels for the reconstructed leg had moderate relationship with vLT (r=0.47, P=0.09) and V4 (r=0.52, P=0.06). CONCLUSION: The neuromuscular response of the intact leg during high intensity running shows strong to very strong relationships with endurance markers. Failure of the ACLR leg to present relationships of similar strength may indicate that chronic perturbations modify the ability of the local muscular environment to tolerate sustained high intensity efforts.

A virtual reality training program for improvement of robotic surgical skills.

The purpose of this study was to use a simulated virtual reality environment for training of surgical skills and then to identify if the learning that occurred was transferable to a real world surgical task. The virtual surgical tasks consisted of bimanual carrying, needle passing and mesh alignment. In this ongoing study, the experimental group (n = 5) was trained by performing four blocks of the virtual surgical tasks using the da Vinci surgical robot. Pre and post training, all subjects were tested by performing a suturing task on a "life-like" suture pad. The control group (n = 5) performed only the suturing task. Significantly larger pre and post differences were revealed in time to task completion (p < 0.05) and total distance travelled by the dominant side instrument tip (p < 0.01) in the experimental group as compared to the control group. These differences were specific to the suture running aspect of the surgical task. In conclusion, virtual reality surgical skills training may produce a significant learning effect that can transfer to actual robot-assisted laparoscopic procedures.

Consistency of performance of robot-assisted surgical tasks in virtual reality.

The purpose of this study was to investigate consistency of performance of robot-assisted surgical tasks in a virtual reality environment. Eight subjects performed two surgical tasks, bimanual carrying and needle passing, with both the da Vinci surgical robot and a virtual reality equivalent environment. Nonlinear analysis was utilized to evaluate consistency of performance by calculating the regularity and the amount of divergence in the movement trajectories of the surgical instrument tips. Our results revealed that movement patterns for both training tasks were statistically similar between the two environments. Consistency of performance as measured by nonlinear analysis could be an appropriate methodology to evaluate the complexity of the training tasks between actual and virtual environments and assist in developing better surgical training programs.

The effects of a plyometric training program on the latency time of the quadriceps femoris and gastrocnemius short-latency responses.

AIM: The purpose of this study was to determine if a plyometric training program can affect the latency time of the quadriceps femoris and gastrocnemius short-latency responses (SLRs) of the stretch reflex. METHODS: Sixteen healthy subjects (12 female and 4 male) were randomly assigned to either a control or a plyometric training group. Maximum vertical jump height (VJ) and SLRs of both quadriceps femoris and gastrocnemius were measured before and after a four week plyometric training program. RESULTS: Plyometric training significantly increased VJ (mean+/-SEM) by 2.38+/-0.45 cm (P<0.05) and non-significantly decreased the latency time of the quadriceps femoris SLR (mean+/-SEM) 0.363+/-0.404 ms (P>0.05) and gastrocnemius SLR (mean+/-SEM) 0.392+/-0.257 ms (P>0.05). VJ results support the effectiveness of plyometric training for increasing VJ height. CONCLUSIONS: The non-significant changes in the latency time of the quadriceps femoris and gastrocnemius SLRs seen in the training group suggest that performance improvements following a four-week plyometric training program are not mediated by changes in the latency time of the short-latency stretch reflex.

Anterior cruciate ligament reconstruction with a quadrupled hamstrings tendon autograft does not restore tibial rotation to normative levels during landing from a jump and subsequent pivoting.

AIM: Recent research suggested that the anterior curciate ligament (ACL) reconstruction does not restore tibial rotation to normal levels when a bone patellar tendon bone (BPTB) graft is used during high demanding activities. Our goal was to determine if the usage of an alternative graft, as the quadrupled semitendinosus-gracilis (ST/G), restore tibial rotation to normal values in a population of athletically active individuals while performing a usual for their sport activity. METHODS: Eleven subjects, all reconstructed with an ST/G graft, were assessed in vivo, 9 months postoperatively, while they jumped off a 40 cm platform, landed on the ground and subsequently pivoted at 90 degrees. The evaluation period was identified from initial foot contact with the ground, included the pivoting of the ipsilateral leg, and was completed upon touchdown of the contralateral leg. By that time the patients had already returned to their sports activities. RESULTS: The maximum range of motion of the tibial rotation for the pivoting leg, during the evaluation period was found significantly (P=0.0001) larger in the reconstructed leg as compared to the intact contralateral, although both clinical and arthrometer assessments revealed restoration of anterior translation. CONCLUSIONS: It was concluded that ACL reconstruction with an ST/G graft does not restore tibial rotation to normal levels during this high demanding activity. It seems that new surgical techniques are needed to better replicate the actual anatomy and function of the natural ACL in order to address this problem.

Physiological demands of competitive basketball.

The aim of this study was to assess physiological demands of competitive basketball by measuring oxygen consumption (VO2) and other variables during practice games. Each of 12 players (20.4 +/- 1.1 years) was monitored in a 20-min practice game, which was conducted in the same way as actual games with the presence of referees and coaches. VO2 was measured by a portable system during the game and blood lactate concentration (LA) was measured in brief breaks. Subjects were also videotaped for time-motion analysis. Female and male players demonstrated respective VO2 of 33.4 +/- 4.0 and 36.9 +/- 2.6 mL/kg/min and LA of 3.2 +/- 0.9 and 4.2 +/- 1.3 mmol/L in the practice games (P>0.05). They spent 34.1% of play time running and jumping, 56.8% walking, and 9.0% standing. Pre-obtained VO(2max) was correlated to VO(2) during play (r=0.673) and to percent of duration for running and jumping (r=0.935 and 0.962 for females and males, respectively). This study demonstrated a greater oxygen uptake for competitive basketball than that estimated based on a previous compendium. The correlation between aerobic capacity and activity level suggests the potential benefit of aerobic conditioning in basketball.

Validating advanced robot-assisted laparoscopic training task in virtual reality.

The purpose of this study was to validate a complex robotic surgical task, mesh alignment, in virtual reality. Nine subjects unrolled and aligned a mesh onto an inanimate template for the mesh alignment task in both an actual (the da Vinci Robotic Surgical System) and a virtual environment. Data analysis included time to task completion, distance traveled, and speed, of the surgical instrument, as well as electromyography of the extensors and flexors of the dominant arm of the subject. Paired t-tests were used to compare the dependent variables between the actual and virtual environments. The virtual mesh alignment task was statistically similar for all variables except the flexor activity as compared to the actual task. In conclusion, virtual reality could be used as an effective environment to train the next generation of robot-assisted laparoscopic surgeons.

Intraductal ultrasonography in the diagnosis of Mirizzi syndrome.

BACKGROUND AND STUDY AIMS: Common bile duct (CBD) compression can be caused by stones in the cystic duct (Mirizzi syndrome) which can be difficult to diagnose even with endoscopic retrograde cholangiopancreatography (ERCP). Conventional imaging often gives insufficient information and endoscopic ultrasonography (EUS) and magnetic resonance imaging may improve diagnostic accuracy, but often the final diagnosis is made during exploratory surgery. PATIENTS AND METHODS: All patients undergoing ERCP during a 3-year period were prospectively analyzed if they fulfilled the inclusion criteria: gallbladder in situ; obstructive jaundice with CBD stenosis, demonstrated at endoscopic retrograde cholangiography (ERC), but unexplained at ultrasonography; and inability to demonstrate the cystic duct during ERC. Intraductal ultrasonography (IDUS) was carried out over a guide wire using a 20-MHz probe. Prior to ERCP, patients were evaluated with abdominal ultrasonography and computed tomography (CT), as well as by magnetic resonance cholangiopancreatography (MRCP) or EUS in some. RESULTS: 74 patients out of 2089 undergoing ERCP fulfilled the entry criteria. Final diagnoses, from surgical exploration (n = 41), cytology (n = 21), or endoscopic extraction of stones from the cystic duct (n = 12), were Mirizzi syndrome (type I) in 30 patients and other causes in 44 patients (gallbladder carcinoma [n = 16], pancreatic carcinoma [n = 9], metastatic compression [n = 9], other [n = 10]). CT had shown suspected Mirizzi syndrome in 1/30 cases (3 %) and MRCP in 12/19 evaluated cases (63 %). EUS allowed a correct diagnosis in 11 of 15 evaluated cases (73 %). IDUS required an additional 8 +/- 3 min and showed a sensitivity of 97 % and specificity of 100 %. CONCLUSION: IDUS is a sensitive and specific method for the diagnosis of Mirizzi syndrome.

Robotic surgery and training: electromyographic correlates of robotic laparoscopic training.

BACKGROUND: Robotic laparoscopic surgery has been shown to decrease task completion time, reduce errors, and decrease training time, as compared with manual laparoscopic surgery. However, current literature has not addressed the physiologic effects, in particular muscle responses, to training with a robotic surgical system. The authors seek to determine the frequency response of electromyographic (EMG) signals of specific arm and hand muscles with training using the da Vinci Surgical System. METHODS: Seven right-handed medical students were trained in three tasks with the da Vinci Surgical System over 4 weeks. These subjects, along with eight control subjects, were tested before and after training. Electromyographic (EMG) signals were collected from four arm and hand muscles during the testing sessions, and the median EMG frequency and bandwidth were computed. RESULTS: The median frequency and frequency bandwidth both were increased after training for two of the three tasks. CONCLUSION: The results suggest that training reduces muscle fatigue as a result of faster and more deliberate movements. These changes occurred predominantly in muscles that were the dominant muscles for each task, whereas the more demanding task recruited more diverse motor units. An evaluation of the physiologic demands of robotic laparoscopic surgery using electromyography can provide us with a meaningful quantitative way to examine performance and skill acquisition.

Robotic surgery training and performance: identifying objective variables for quantifying the extent of proficiency.

BACKGROUND: To understand the process of skill acquisition in robotic surgery and to allow useful real-time feedback to surgeons and trainees in future generations of robotic surgical systems, robotic surgical skills should be determined with objective variables. The aim of this study was to assess skill acquisition through a training protocol, and to identify variables for the quantification of proficiency. METHODS: Seven novice users of the da Vinci Surgical System engaged in 4 weeks of training that involved practicing three bimanual tasks with the system. Seven variables were determined for assessing speed of performance, bimanual coordination, and muscular activation. These values were compared before and after training. RESULTS: Significant improvements were observed through training in five variables. Bimanual coordination showed differences between the surgical tasks used, whereas muscular activation patterns showed better muscle use through training. The subjects also performed the surgical tasks considerably faster within the first two to three training sessions. CONCLUSIONS: The study objectively demonstrated that the novice users could learn to perform surgical tasks faster and with more consistency, better bimanual dexterity, and better muscular activity utilization. The variables examined showed great promise as objective indicators of proficiency and skill acquisition in robotic surgery.

Reduction of miss rates of colonic adenomas by zoom chromoendoscopy.

BACKGROUND AND AIMS: The aim of this study was to determine the detection rate of polyps using zoom chromoendoscopy (ZE) compared with standard video colonoscopy. PATIENTS AND METHODS: End-to-end colonoscopies were performed in 50 patients by two different endoscopists blinded for each other's results. Lesions detected during initial standard colonoscopy (C1) were biopsied or removed by snare resection. The second colonoscopy (C2) was done with a zoom colonoscope spraying the whole colon with indigocarmine (0.4%). In addition, detected mucosal lesions were documented prior to ZE and then classified according to the pit pattern classification before biopsy or removal. The retrieval time for each procedure was determined. RESULTS: The average retrieval time for C1 was 13+/-9 min (9-24) and 28+/-11 min (16-38, p<0.05) for ZE. During C1, 56 lesions were detected in 26 of 50 patients (34 hyperplastic and 22 adenomatous). During C2, 19 additional polyps were documented prior to ZE (15% tandem miss rate), and 20 further lesions were detected with ZE (21% additional polyp detection rate compared to C1 and C2 without ZE). Of the 39 additional lesions removed during C2 after ZE, 29 were hyperplastic and 10 were adenomatous. Most adenomas detected during the second investigation were found in patients in whom adenomatous polyps had already been removed during the initial colonoscopy (9 of 26 patients vs 1 of 24 patients, p<0.02). No carcinoma was detected. The pit pattern classification allowed a correct differentiation between hyperplastic and adenomatous polyps (accuracy 93%, sensitivity 90%, specificity 97%). CONCLUSION: Using zoom chromoendoscopy, the rate of detecting colonic polyps can be increased at the cost of a longer retrieval time.