Dynamics of virological and immunological markers of HIV persistence after allogeneic haematopoietic stem-cell transplantation in the IciStem cohort: a prospective observational cohort study.

BACKGROUND: Allogeneic haematopoietic stem-cell transplantation (allo-HSCT) markedly reduces HIV reservoirs, but the mechanisms by which this occurs are only partly understood. In this study, we aimed to describe the dynamics of virological and immunological markers of HIV persistence after allo-HSCT. METHODS: In this prospective observational cohort study, we analysed the viral reservoir and serological dynamics in IciStem cohort participants with HIV who had undergone allo-HSCT and were receiving antiretroviral therapy, ten of whom had received cells from donors with the CCR5Δ32 mutation. Participants from Belgium, Canada, Germany, Italy, the Netherlands, Spain, Switzerland, and the UK were included in the cohort both prospectively and retrospectively between June 1, 2014 and April 30, 2019. In the first 6 months after allo-HSCT, participants had monthly assessments, with annual assessments thereafter, with the protocol tailored to accommodate for the individual health status of each participant. HIV reservoirs were measured in blood and tissues and HIV-specific antibodies were measured in plasma. We used the Wilcoxon signed-rank test to compare data collected before and after allo-HSCT in participants for whom longitudinal data were available. When the paired test was not possible, we used the Mann-Whitney U test. We developed a mathematical model to study the factors influencing HIV reservoir reduction in people with HIV after allo-HSCT. FINDINGS: We included 30 people with HIV with haematological malignancies who received a transplant between Sept 1, 2009 and April 30, 2019 and were enrolled within the IciStem cohort and included in this analysis. HIV reservoirs in peripheral blood were reduced immediately after full donor chimerism was achieved, generally accompanied by undetectable HIV-DNA in bone marrow, ileum, lymph nodes, and cerebrospinal fluid, regardless of donor CCR5 genotype. HIV-specific antibody levels and functionality values declined more slowly than direct HIV reservoir values, decaying significantly only months after full donor chimerism. Mathematical modelling suggests that allogeneic immunity mediated by donor cells is the main viral reservoir depletion mechanism after massive reservoir reduction during conditioning chemotherapy before allo-HSCT (half-life of latently infected replication-competent cells decreased from 44 months to 1·5 months). INTERPRETATION: Our work provides, for the first time, data on the effects of allo-HSCT in the context of HIV infection. Additionally, we raise the question of which marker can serve as the last reporter of the residual viraemia, postulating that the absence of T-cell immune responses might be a more reliable marker than antibody decline after allo-HSCT. FUNDING: amfAR (American Foundation for AIDS Research; ARCHE Program), National Institutes of Health, National Institute of Allergy and Infectious Diseases, and Dutch Aidsfonds.

A review of the current status of lenalidomide maintenance therapy in multiple myeloma in 2022.

INTRODUCTION: Recent advances in the diagnosis and management of multiple myeloma (MM) have improved patient outcomes. This progress in our understanding of MM has resulted in continuous suppressive therapy concepts, including induction, high dose chemotherapy with autologous stem cell transplantation (ASCT), consolidation, and maintenance therapy. The foundation of maintenance therapy has been with lenalidomide. Other novel immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), and targeted monoclonal antibodies have also contributed to this evolution. AREAS COVERED: This review summarizes the outcomes from phase II/III trials with long-term lenalidomide maintenance therapy alone or in combination with other agents in post-ASCT and non-transplant settings for newly diagnosed patients with MM. We review recent data considering a combination with newer medications and ongoing trials. We also review the optimal duration, MRD negativity rate, and safety and tolerability aspects of lenalidomide maintenance therapy. This review aims to present the current and emerging clinical evidence that supports using lenalidomide as a backbone for maintenance therapy in patients with MM. EXPERT OPINION: There is increasing evidence to support lenalidomide as the backbone of combination therapy in the maintenance setting.

Systemic Therapy De-Escalation in Early-Stage Triple-Negative Breast Cancer: Dawn of a New Era?

Early-stage triple negative breast cancer (TNBC) has been traditionally treated with surgery, radiation, and chemotherapy. The current standard of care systemic treatment of early-stage II and III TNBC involves the use of anthracycline-cyclophosphamide and carboplatin-paclitaxel with pembrolizumab in the neoadjuvant setting followed by adjuvant pembrolizumab per KEYNOTE-522. It is increasingly clear that not all patients with early-stage TNBC need this intensive treatment, thus paving the way for exploring opportunities for regimen de-escalation in selected subgroups. For T1a tumors (≤5 mm), chemotherapy is not used, and for tumors 6-10 mm (T1b) in size with negative lymph nodes, retrospective studies have failed to show a significant benefit with chemotherapy. In low-risk patients, anthracycline-free chemotherapy may be as effective as conventional therapy, as shown in some studies where replacing anthracyclines with carboplatin has shown non-inferior results for pathological complete response (pCR), which may form the backbone of future combination therapies. Recent advances in our understanding of TNBC heterogeneity, mutations, and surrogate markers of response such as pCR have enabled the development of multiple treatment options in the (neo)adjuvant setting in order to de-escalate treatment. These de-escalation studies based on tumor mutational status, such as using Poly ADP-ribose polymerase inhibitors (PARPi) in patients with BRCA mutations, and new immunotherapies such as PD1 blockade, have shown a promising impact on pCR. In addition, the investigational use of (bio)markers, such as high levels of tumor-infiltrating lymphocytes (TILs), low levels of tumor-associated macrophages (TAMs), and complete remission on imaging, also look promising. In this review, we cover the current standard of care systemic treatment of early TNBC and review the opportunities for treatment de-escalation based on clinical risk factors, biomarkers, mutational status, and molecular subtype.

Small and medium-sized enterprises' contribution in digital technology.

Researchers have mentioned the importance of digitization in improving efficiency and productivity in Small and Medium Enterprises (SME). Fortunately, there is no proof that Digitization can be used to deal with the outcome of severe incidents like COVID-19. The research paper suggested that the increased rate of SMEs has increased significantly. This was entirely due to the advent of Digital Technology (DT). In this way, both product and the process become more automated in digitalization, resulting in increased quality and demand. Considering the high scope for higher development, India's SME sector still has much space for new digital technologies to be integrated. This paper addresses the main scenario of SMEs in India and their benefit in GDP. Also, the research includes a brief analysis of CRM applications and digital payment options in SMEs.

Activation of the Erk/MAPK signaling pathway is a driver for cadmium induced prostate cancer.

PURPOSE: Cadmium (Cd) is reported to be associated with carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer (PCa) remain elusive. MATERIALS AND METHODS: RWPE1, PWR1E and DU 145 cells were used. RT2 Profiler Array, real-time-quantitative-PCR, immunofluorescence, cell cycle, apoptosis, proliferation and colony formation assays along with Gene Set Enrichment Analysis (GSEA) were performed. RESULT: Chronic Cd exposure of non-malignant RWPE1 and PWR1E cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. Even a two-week Cd exposure of PCa cell line (DU 145) significantly increased the proliferation and decreased apoptosis. RT2 profiler array of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis in both Cd-RWPE1 and Cd-PWR1E cell lines. GSEA showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from Erk/MAPK pathway and performed profile analysis in a PCa data set from the TCGA/GDC data base. We observed upregulation of these genes in PCa compared to normal samples. An increase in phosphorylation of the Erk1/2 and Mek1/2 was observed in Cd-RWPE1 and Cd-PWR1E cells compared to parental cells, confirming that Cd-exposure induces activation of the Erk/MAPK pathway. CONCLUSION: This study demonstrates that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate cells. Understanding these dominant oncogenic pathways may help develop optimal therapeutic strategies for PCa.

Gathering, evaluating and managing customer feedback during aircraft production.

This paper proposes a systematic approach for gathering requirements during production through customers' remote access to the partially and fully assembled aircraft and its modules. The paper also proposes an evaluation and management of these recorded requirements and their utilization in the development of an aircraft. Modular product architecture is used for the modular organization of the product, product-service, and production system for the gathering, evaluation and management of feedback for product development perspectives. A mobile and wearable augmented reality system is used to virtually walk through the partially or fully manufactured product and to compare the status of the production with the product model to be produced. Change requests are captured as customer feedback. The knowledge thus acquired can be overlaid (augmented) on the real product, i.e. the aircraft. This approach is able to record the dynamic requirements of targeted customers. These changes can be carried out in the current version of the aircraft, and also incorporated into future versions. The implementation using case studies is presented for gathering feedback during assembly as well as for evaluating and managing the recorded feedback for exemplary modules (cabin and galley) of an aircraft. The use of the evaluation results in the development of an aircraft is also presented in the paper.

Rhl quorum sensing affects the virulence potential of Pseudomonas aeruginosa in an experimental urinary tract infection.

Pseudomonas aeruginosa quorum sensing systems (QSS) las, rhl and PQS play a significant role in pathogenicity. Although multiple studies have explored the role of the las QSS in various types of infections, including urinary tract infections (UTIs), the role of the rhl QSS is still poorly understood. In this study, an attempt was made to explore the role of the rhl QSS in the pathogenesis of UTI. The wild-type strain P. aeruginosa PAO1 and its isogenic rhl QSS mutant strains, PDO-100 and PDO-111, were used. Strains were studied for the expression of extra-cellular virulence factors and the ability to induce experimental UTI in a mouse model. Evaluation was based on in vitro estimation of virulence factors (elastase, protease, rhamnolipid) and in vivo bacteriological and histopathological analysis of renal and bladder tissue. The cytokine levels were determined in the renal tissue of infected mice. Wild-type PAO1 showed the highest level of virulence traits. The tissue bacteriology and histopathology were indicative of establishment of UTI. The level of inflammatory cytokines was consistent with the bacteriology and pathology results. The rhl QSS mutant strains showed significantly reduced pathogenicity. On supplementation with C4-homoserine lactone, strain PDO-100 showed a significant increase in pathogenicity. The results indicate that the rhl QSS affects the virulence potential of P. aeruginosa and that, despite the presence of the las QSS, mutant strains exhibited reduced pathology. Therefore, the rhl QSS may be exploited in more detail for future drug intervention tactics.

Functional outcome after surgical treatment of perilunate injuries: A series of 12 cases.

PURPOSE: Perilunate injuries (PLIS) are complex injuries, which are frequently missed in the initial setting, and delayed presentation leads to poor functional outcomes. In this study, we are presenting our experience of treating these injuries by surgical treatment and the effect of neglect on their outcome. MATERIALS AND METHODS: In this retrospective study, 11 patients with 12 PLIS, which presented to our center from January 1, 2000 to December 31, 2012 were included. They were managed surgically as open reduction and internal fixation. Function was documented by using Mayo score. To ascertain the effect of the delay/neglect on the outcome, the patients were divided into two groups according to time between day of injury and final surgery (group I - operated within 6 weeks of injury and group II - treated after 6 weeks of injury). RESULTS: Average postoperative Mayo score was 76.4. Average Mayo score in group I was 93.7 (90-95). Average Mayo score in group II was 67.1 (60-75). Although functional result as Mayo score was significantly better in group I (p value <0.5) even chronic or delayed group patients also had good to fair results. CONCLUSION: Early diagnosis and treatment of such injuries should be emphasized as delay in treatment leads to progressive poor results. Well-planned surgical management gives good functional results even in delayed cases.

Neglected anterior dislocation of shoulder with large Hillsach's lesion & deficient glenoid: Treated by autogenous bone graft & modified Latarjet procedure.

Neglected anterior dislocation of shoulder is rare in spite of the fact that the anterior dislocation of the shoulder is seen in around 90% of the acute cases. Most of the series of neglected dislocation describe posterior dislocation to be far more common.(1) (,2) We hereby report a case of the neglected anterior shoulder dislocation in a 15 year old boy who had a history of epilepsy. There was a large Hill Sachs lesion in humeral head which was impacted in glenoid inferiorly and glenoid was eburnated at that margin. The humeral head was reconstructed with a tricortical iliac graft. Glenoid was reconstructed by transfer of coracoids process of scapula to antero-inferior glenoid (modified Latarjet procedure). This case is unique because management of humeral head defect with bone graft is not mentioned in anterior dislocation.

Acetabular fractures labelled poor surgical choices: Analysis of operative outcome.

PURPOSE: We report the surgical outcome in 52 patients with acetabular otherwise considered as poor surgical choices. METHODS: 43 male and 9 female patients were operated at a mean age of 43 years and followed up for a mean duration of 60.3 months. There were 22 elementary fractures and 31 associated ones according to Letournal and Judet classification. Osteosynthesis was attempted in 48 patients whereas a primary total hip arthroplasty was performed in 4 patients. Outcome was assessed radiologically and functionally employing Harris Hip Score (HHS). RESULTS: Average HHS in osteosynthesis group was 82.56 ± 12.4 with excellent to good results in 59.6% of the cases. Symptomatic osteoarthritis occurred in 13.5% of cases, avascular necrosis and severe heterotopic ossification in 7.7% each, infection and nerve palsy in 11.5% each. CONCLUSION: Although the complication rates in this series is marginally more than that reported in literature, we recommend that the indications of surgical fixation in acetabular fractures need to be extended to those which were considered poor surgical choices.

Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India.

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

Attenuation of quorum sensing controlled virulence of Pseudomonas aeruginosa by cranberry.

BACKGROUND & OBJECTIVES: Emergence of antimicrobial resistance in Pseudomonas aeruginosa has led to the search for alternative agents for infections control. Natural products have been a good alternative to present antibiotics. The present study was undertaken to evaluate the effectiveness of cranberry in attenuation of virulence of P. aeruginosa in experimental urinary tract infection (UTI) in mouse model. Efforts were also directed to explore the action of cranberry towards virulence of P. aeruginosa through quorum sensing (QS) inhibition. METHODS: Efficacy of cranberry was evaluated in an experimental UTI mouse model and on production of QS signals, alginate, pyochelin, haemolysin, phospholipase-C, cell-surface hydrophobicity, uroepithelial cell-adhesion assay and biofilm formation by already standardized methods. RESULTS: Presence of cranberry showed significant decline in the production of QS signals, biofilm formation and virulence factors of P. aeruginosa in vitro (P<0.001). Further, cranberry was found to be useful in prevention of experimental UTI in mouse model as indicated by reduced renal bacterial colonization and kidney tissues destruction. INTERPRETATION & CONCLUSIONS: The findings of the present study indicated that cranberry inhibited QS and hence elaboration of virulence factors of P. aeruginosa. It also affected the adherence ability of this pathogen. This approach can lead to the discovery of new category of safe anti-bacterial drugs from dietary sources such as cranberry with reduced toxicity without the risk of antibiotic resistance.

Development of pH responsive novel emulsion adjuvant for oral immunization and in vivo evaluation.

The present work has been envisaged to develop a pH responsive multiple emulsion (W/O/W) adjuvant for successful antigen delivery via oral route. The formulation was prepared by double emulsion technique using squalene as oil phase and a combination of antigens and Quil A as internal aqueous phase. Squalene emulsion (O/W) adjuvants are well accepted for parenteral administration. Instead of using conventional surfactants, Quil A was incorporated in the internal aqueous compartment for the first time. The microscopic analysis confirmed the formation of the W/O/W multiple emulsions with globule diameter ranging from 7 to 12 µm. Considerably, addition of Quil A resulted in marked improvement in both in vitro stability and immune response. Furthermore, addition of pectin to the external aqueous phase resulted in improved in vitro stability. A formulation with gel like structure was formed when pectin was used as stabilizer and also accounted with a significant increase in viscosity (from 275.3 cP to 330.4 cP) in the simulated gastric fluid, which reverts back to 290.2 cP after exposure to simulated intestinal fluid. Induction of strong mucosal and systemic immune response suggested that the developed formulation could be useful for the delivery of multiple biomolecules via oral route.

Neglected post burns contracture of hand in children: Analysis of contributory socio-cultural factors and the impact of neglect on outcome.

BACKGROUND: No study has ever evaluated the causes and effect of neglect on the outcome of post burns contractures of hand in children. METHODS: 66 hands in 61 children (mean age 12.22 years) with a mean neglect of 11.6 years (range 5-17 years) were assessed for the causes of neglect and the outcome of surgery. Average follow up was 6.6 years. The results were assessed in two groups of 5-10 years neglect as group I and >10 years neglect as group II. RESULTS: In a total number of 134 contracted rays in 66 hands, the surgical procedures included local Z/V-Y flap (51 rays), cross finger flap (48 rays), full thickness graft (35 rays). Additional external fixator with a distracter was used in 3 patients treated at a delay of 14, 16 and 17 years. 50 (81.96%) patients belonged to rural and slum areas. The reasons for delayed treatment included poverty - 33 patients, lack of awareness of surgical treatment - 16 patients; and indifference of parents - 12 patients. 44 (72.13%) children were illiterates. With treatment the average DASH score improved from 65.10 to 36.90 (p < .000) and from 68.14 to 45.93 (p < .000) in group I and II respectively. The results were significantly superior in group I (p < .000). CONCLUSION: The main factors for neglect in treatment of post burns contracture include poverty, lack of awareness and illiteracy. All the patients showed significant improvement in function after the surgery. Contractures with higher neglect had significantly inferior outcome.

Pucker sign in proximal humeral fractures: implications on management.

Fracture of the surgical neck of humerus in young patients is a relatively rare injury. We reviewed the available material on the topic and identified puckering at the shoulder in high-energy fracture of the surgical neck as a finding which has been reported infrequently but signifies a need for open reduction. We present a review of the literature on the subject and our similar experience in two young males who had puckering and ecchymosis at the shoulder.

Detection and quantification of quinolone signalling molecule: a third quorum sensing molecule of Pseudomonas aeruginosa by high performance-thin layer chromatography.

Sophisticated network of quorum sensing involves the production of chemical signals which regulate the combined expression of virulence genes and biofilm formation in Pseudomonas aeruginosa. Two well-characterized acyl homoserine lactone based las and rhl systems together with alkyl quinolone based Pseudomonas quinolone signalling (PQS) are fundamental components of this network. Third signalling molecule, 2-heptyl-3-hydroxy-4-quinolone (PQS) is of paramount importance because of its interconnecting role in quorum sensing hierarchy in P. aeruginosa. Accurate detection of PQS molecule is very important to understand the involvement of this system in infection process of P. aeruginosa. In this study, high performance-thin layer chromatography (HP-TLC) method was developed for detection as well as quantification of PQS signal molecules in P. aeruginosa, which combines conventional method like TLC with sophisticated instrumentation. This method was validated using parameters like linearity, accuracy, precision, reproducibility and sensitivity. Intra- and inter-day accuracy and precision values were determined which were found to be within acceptable level and hence showed reproducibility. Measurement of PQS in the range of 0.01nmol indicated excellent sensitivity of this approach for quantifying PQS molecule. Automated sampling, rapid and simultaneous analysis of large number of samples and minimal errors make this method more suitable for analysis of PQS signalling molecules. Production of PQS was found to be strain dependent since variation in amount of PQS was observed among different P. aeruginosa isolates. Further, PQS production was also dependent on growth phase of P. aeruginosa with maximum production in late stationary phase.

Quorum sensing signal molecules produced by Pseudomonas aeruginosa cause inflammation and escape host factors in murine model of urinary tract infection.

Quorum sensing (QS) is well established for its role in pathogenesis of various infections of Pseudomonas aeruginosa. However, its role in local tissue damage during urinary tract infection (UTI) is not yet fully established. To have insight in this, the present study was planned. UTI was established in mice using standard strain PAO1 and its isogenic QS mutant JP2. One group was challenged only with QS signals. Damage was assessed in terms of histopathology and pathology markers, malondialdehyde (MDA) and reactive nitrogen intermediates (RNI). Effect on pathogen motility, uroepithelial adhesion, and host serum sensitivity was also ascertained. PAO1-infected mice showed severe inflammation and tissue destruction, while mice infected with JP2 showed no significant destruction. JP2 was also unable to mount any tissue pathology markers, MDA and RNI, whereas PAO1 showed significantly higher levels of these two. Presence of only QS signals also showed considerable renal pathology. Strain JP2 also showed less motility, reduced uroepithelial cell adhesion, and increased serum sensitivity. Result highlights that QS signals induce local tissue pathology along with interference of host protective mechanisms during UTI.

Quorum sensing signal molecules cause renal tissue inflammation through local cytokine responses in experimental UTI caused by Pseudomonas aeruginosa.

Quorum sensing (QS) plays a pivotal role in the virulence of Pseudomonas aeruginosa. Urinary tract infections (UTI's) caused by P. aeruginosa leads to tissue destruction and inflammation. Interaction of host immune cells and pathogen at the epithelial surface are regulated by cytokines. Role of virulence factors has been implicated in the induction of renal cytokine responses. However, information regarding the role of QS, an important virulence factor of P. aeruginosa, is lacking. This study examined role of QS signal molecules for their ability to induce pro and anti-inflammatory renal cytokines and inflammation in experimental UTI. For this, both standard parent P. aeruginosa strain (PAO1) capable of producing QS signal molecules and its isogenic single and double mutant strains lacking this ability were used. Mice infected with QS producer strain PAO1, evoked significant renal tissue inflammation and cytokine response with high neutrophil migration and AHL production in vivo. Mild inflammation, low level of renal cytokines and other factors were observed in mice infected with QS single and double mutant strains. These results provide first evidence regarding the role of QS signal molecules and point towards the importance of AHLs in induction of renal cytokines and tissue inflammatory responses.

Acyl homoserine lactones from culture supernatants of Pseudomonas aeruginosa accelerate host immunomodulation.

The virulence of Pseudomonas aeruginosa is multifactorial and under the control of quorum sensing signals, such as acyl homoserine lactones (AHLs). The importance of these molecules in the establishment of infection has been previously reported. These molecules either improve the virulence potential of P. aeruginosa or modulate the host immune response. To establish the immune modulating potential of quorum sensing signal molecules, previous studies have only used synthetic AHLs. However, there can be differences in the biological properties of synthetic and natural AHLs. The use of naturally extracted AHLs from the culture supernatant of P. aeruginosa is likely to simulate natural conditions more than the use of synthetic AHLs. Therefore, in the present study, the immune modulating potential of synthetic and naturally extracted AHLs was compared using a thymidine uptake assay, immunophenotyping and sandwich ELISA in order to assess mouse T-cell proliferation and production of Th1 and Th2 cytokines. Natural AHLs were able to suppress T-cell proliferation, even at low concentrations, compared to synthetic AHLs. The majority of cells undergoing proliferation were CD4+, as revealed by immunophenotyping. The inhibition of T-cells was stronger with natural AHLs compared to synthetic AHLs. Moreover, the natural AHLs were also able to shift immune responses away from host protective Th1 responses to pathogen protective Th2 responses.