Objective: To assess the safety and effectiveness of amphotericin B cholesteryl sulfate complex for injection in the context of empirical and diagnostic antifungal therapy for patients with hematological malignancies in addition to invasive fungal illness. Methods: This single-arm clinical study enrolled 30 patients who received empirical and diagnostic-driven antifungal therapy for hematological malignancies combined with invasive fungal disease. The primary endpoint was safety. Response rate, fever duration, and treatment completion rate were all considered secondary objectives. Results: 30 participants were eventually enrolled in the study, and the treatment completion rate was 80.0% . Most adverse events were in grades 1-2. Infusion response was the most frequent adverse event (24/30, 80% ) . The overall response rate was 80.0% (24/30) . In 24 patients (80.0% ) , the fever persisted for 1 day. Conclusions: Treatment of invasive fungal illness in conjunction with hematological malignancies showed good efficacy and safety with amphotericin B cholesteryl sulfate complex for injection.

Acanthamoeba castellanii causes granulomatous amoebic encephalitis, an uncommon but severe brain infection and sight-threatening Acanthamoeba keratitis. Most of the currently used anti-amoebic treatments are not always effective, due to persistence of the cyst stage, and recurrence can occur. Here in this study we synthesize cinnamic acid and lactobionic acid-based magnetic nanoparticles (MNPs) using co-precipitation technique. These nanoformulations were characterized by Fourier transform infrared spectroscopy and Atomic form microscopy. The drugs alone (Hesperidin, Curcumin and Amphotericin B), magnetic NPs alone, and drug-loaded nano-formulations were evaluated at a concentration of 100 μg/mL for antiamoebic activity against a clinical isolate of A. castellanii. Amoebicidal assays revealed that drugs and conjugation of drugs and NPs further enhanced amoebicidal effects of drug-loaded nanoformulations. Drugs and drug-loaded nanoformulations inhibited both encystation and excystation of amoebae. In addition, drugs and drug-loaded nanoformulations inhibited parasite binding capability to the host cells. Neither drugs nor drug-loaded nanoformulations showed cytotoxic effects against host cells and considerably reduced parasite-mediated host cell death. Overall, these findings imply that conjugation of medically approved drugs with MNPs produce potent anti-Acanthamoebic effects, which could eventually lead to the development of therapeutic medications.

The development of stimuli-responsive synthetic channels that open and close in response to physical and chemical changes in the surrounding environment has attracted attention because of their potential bioapplications such as sensing, drug release, antibiotics, and molecular manipulation tools to control membrane transport in cells. Metal coordination is ideal as a stimulus for stimuli-responsive channels because it allows for reversible gating behavior through the addition and removal of metal ions and fine-tuning of channel structure through coordination geometry defined by the type of the metal ion and ligand. We have previously reported on transition metal-ion dependent ion permeability control of Amphotericin B (AmB) modified with a metal coordination site, 2,2'-bipyridine ligand (bpy-AmB). AmB is one of the polyene macrolide antibiotics, and it is known that the interaction between AmB and ergosterol molecules is required for AmB channel formation. In contrast, the Cu2+ coordination to the bpy moiety of bpy-AmB induces formation of Ca2+ ion-permeable channels in the ergosterol-free POPC membrane. However, the details of bpy-AmB properties such as channel stability, ion selectivity, pore size, and the effect of ergosterol on channel formation remain unclear. Here, we investigate bpy-AmB channels triggered by transition metal coordination in POPC or ergosterol-containing POPC liposomes using an HPTS assay, electrophysiological measurements, and time-resolved UV-vis spectral measurements. These analyses reveal that bpy-AmB channels triggered by Cu2+ ions are more stable and have larger pore sizes than the original AmB channels and enable efficient permeation of various cations. We believe that our channel design will lead to the construction of metal coordination-triggered synthetic ion channels.

Harmless commensal Candida species, especially uncommon and rare ones may rarely cause a serious infection. Candida metapsilosis is a recently described yeast that is phenotypically indistinguishable from Candida parapsilosis and molecular methods are essential for its identification. We report the first case of Candida conjunctivitis due to C. metapsilosis obtained from the eye discharge of a 40-day-old girl with congenital heart disease admitted to the cardiac intensive care unit (CICU). The yeast isolate was identified by sequencing the entire ITS1-5.8 rRNA-ITS2 region. Antifungal susceptibility testing performed according to the CLSI M27-A3 showed that the isolate was susceptible to amphotericin B, fluconazole, itraconazole, voriconazole, clotrimazole, nystatin, terbinafine, 5-fluorocytosine, and caspofungin. Differentiation of the fungal new species allows us the accurate diagnosis and treatment, and a better understanding the microbial epidemiology.

Rhino-orbital cerebral mucormycosis has been commonly seen during the coronavirus disease 2019 (COVID-19) pandemic. Several factors responsible for etiology and pathophysiology have been identified, among which corticosteroids and diabetes have contributed to the lion's share of the outbreak of mucormycosis. In this report, we discuss a case of a 41-year-old non-diabetic male with a recent convalescence from COVID-19 infection presented with gradual vision loss and loss of sensations in his right eye. He was found to have periorbital swelling, restriction of extraocular movements in all gazes, chemosis, ptosis of the right eye, and right maxillary sinus tenderness. His serum investigations, radiologic findings, and blood culture were indicative of rhino-orbital cerebral mucormycosis. He was started on systemic liposomal amphotericin B immediately and underwent aggressive surgical debridement. A high index of clinical suspicion, aggressive multifaceted management, and follow-up are needed to have successful outcomes, thereby lowering the morbidity of coronavirus-associated mucormycosis.

We report for the first time a case of disseminated infection caused by Peziza ostracoderma, a mold not previously associated with invasive infections in humans. P. ostracoderma occurs in natural and sterilized soil and may cause hypersensitivity pneumonitis in greenhouse workers. The immunocompromised patient presented with neutropenic fever that did not respond to broad-spectrum antibiotics and developed multiple skin and lung lesions. A skin biopsy demonstrated an angioinvasive mold, identified as Peziza ostracoderma by culture and DNA sequencing. Minimum inhibitory concentration (MIC) for amphotericin B was 0.125 mg/L, for isavuconazole 0.125 mg/L, for voriconazole 0.06 mg/L, and for posaconazole 0.03 mg/L. The skin lesions have resolved completely, and the lung lesions have decreased significantly in size after 14 months of mold-active antifungal therapy, mostly isavuconazole. In conclusion, Peziza species can be opportunistic pathogens causing considerable morbidity in immunocompromised hosts. The infection may be successfully treated with mold-active antifungal drugs.

Disseminated candidiasis is a life-threatening disease and remains the most common bloodstream infection in hospitalized patients in the United States. Despite the availability of modern antifungal therapy, the crude mortality rate in the last decade has remained unacceptably high. Novel approaches are urgently needed to supplement or replace current antifungal therapies. In our study, we show that human intravenous immunoglobulin (IVIG) can provide protection against Candida auris and Candida albicans disseminated infections in A/J and C57BL/6 mouse models. The protective efficacy of IVIG is evidenced by the prolonged survival of mice with invasive candidiasis that were treated with human IVIG alone or in combination with amphotericin B. Our previous studies have led to the identification of a panel of Candida cell surface peptide and glycan epitopes, which are targeted by protective mouse monoclonal antibodies (mAbs) against invasive candidiasis. Of interest, the peptide- and glycan-specific IgGs could be detected in all 18 human IVIG samples. In particular, the specific IVIG lots with the highest protective peptide- and glycan-related IgGs provided the best protection. The combination of IVIG and amphotericin B had enhanced efficacy in protection compared to monotherapy against both multidrug-resistant (MDR) C. auris and C. albicans, with evidence of significantly prolonged survival and lower fungal burdens in targeted organs. This study provides evidence that the protective effects of IVIG were associated with the protective antibodies found in normal human donor sera against pathogenic Candida, and IVIG can be a novel therapy or adjunctive therapy with modern antifungal drugs against disseminated candidiasis. IMPORTANCE Since current antifungal treatments are ineffective in the immunocompromised population and no vaccine is available for humans, hope remains that antibody preparations selected for specific fungal antigens may make it possible to reduce the incidence and mortality of invasive candidiasis. Intravenous immunoglobulin (IVIG) has long been approved as a standard treatment for patients with immunodeficiency disorders who are also susceptible to fungal infection. IVIG has been widely used as prophylaxis or supplemental treatment for sepsis and septic shock; however, this form of adjunctive therapy lacks convincing data to establish its efficacy. In this study, 18 samples from commercial IVIG preparations were screened and evaluated by enzyme-linked immunosorbent assays (ELISAs); Candida peptide- and glycan-specific IgGs were detected with various titers among all IVIG lots. Importantly, significantly reduced organ fungal burdens and mortality were demonstrated in IVIG-treated mouse models of invasive candidiasis. IVIG lots with higher titers of Candida-specific IgGs provided better protection. These findings are important in (i) selecting Candida-specific IVIG therapy that may overcome several shortcomings of conventional IVIG therapy by targeting specific antigens responsible for disease pathogenesis, (ii) enhancing protective efficacy, and (iii) validating data from our previous studies and those of others showing that antibodies combined with conventional antifungal drugs provided enhanced resistance to disease. To our knowledge, this study is the first to demonstrate that human IVIG samples contain protective IgGs targeting Candida cell surface antigens and can be a novel therapy or adjunctive therapy with modern antifungal drugs against disseminated candidiasis.

Cryptococcosis is an opportunistic mycosis that mainly affects immunosuppressed patients. The treatment is a combination of three antifungal agents: amphotericin B, 5-flucytosine and fluconazole. However, these drugs have many disadvantages, such as high nephrotoxicity, marketing bans in some countries and fungal resistance. One of the solutions to find possible new drugs is pharmacological repositioning. This work presents repositioned drugs as an alternative for new antifungal therapies for cryptococcosis. All the studies here were performed in vitro or in animal models, except for sertraline, which reached phase 3 in humans. There is still no pharmacological repositioning approval for cryptococcosis in humans, though this review shows the potential of repurposing as a rapid approach to finding new agents to treat cryptococcosis.

We report a rare case of concurrent pulmonary and cerebral mucormycosis initially misdiagnosed as a metastatic tumor. A 66-year-old man with a complaint of progressive right-sided limb weakness for 3 days. Head MRI showed a left parietal occupying lesion with severe edema, and a chest CT scan showed a parenchymal mass with speculation and pleural invasion in his left lung. The patient was initially diagnosed with brain metastases from lung cancer and underwent a craniotomy. Many fungal hyphae were found in the left parietal lesion, and the final pathological diagnosis of intracranial mucormycosis. After craniotomy and an entire course of treatment with liposomal amphotericin B, the patient was completely cured of both intracranial and pulmonary occupying lesions. We hope that this case experience will help expand neurosurgeons' differential diagnosis and treatment of such diseases.

To study clinicoepidemiology and surgical complications in acute invasive fungal rhinosinusitis. Retrospective observational study carried in GMC Akola from February 2021 to April 2022. Detailed history and clinical examination, nasal endoscopic biopsy or swab for KOH and fungal culture was taken. CECT/MRI PNS + Orbit + Brain was done. All patients underwent surgery and tissue sample send for histopathological examination. Total 146 patients included in study with M:F ratio 1.7:1. Most affected age group was between 40 and 60 years. 107 (78.6%) patients had history of COVID-19.Mucorale is most commonly found fungal species (90.4%) followed by aspergillus (2.7%) & mixed species (6.8%). Diabetes Mellitus is most common comorbidity. Intraoperative complications were bleeding (72.60%), CSF leak (4.1%), orbital hematoma (0.68%), nasolacrimal duct trauma (2.05%), periorbital hematoma (0.68%). Post operative complications like synechiae (56.16%), OAF (45.89%), hypoesthesia (25.34%), decreased vision (16.43%), facial pain (20.54%), facial deformity (20.54%), diplopia (6.8%), headache (30.13%), anosmia (39.72%), dental pain (20.54%), earache (9.58%), hyposmia (45.89%), periorbita ecchymosis (0.68%), residual disease (16.10%), recurrence (2.05%), death (2.05%) was observed. Prompt surgical debridement of devitalized tissue and early adequate dosage of antifungal (inj. Amphotericin-b) treatment are necessary as delay in surgical debridement and treatment can worsen the prognosis of disease. Among all complications faced maximum were manageable with early interventions but few of them were inevitable due to extensive nature of disease.

India is well known as the diabetes "capital" of the world but now it is also becoming the mucormycosis "capital" of the world. Indian Council of Medical Research has formed an "Evidence-Based Advisory in The Time of COVID-19 on Screening, Diagnosis, and Management of Mucormycosis." As per this advisory, an oral and maxillofacial surgeon forms an integral part of the team dedicated to fight this epidemic of mucormycosis. Also, there are other fungal infections such as aspergillosis which are getting reported in these patients affecting the paranasal sinuses and the jaws. Aggressive surgical debridement and a thorough knowledge of anti-fungal therapy are must in treating these fungal infections. The aim of this article is to give an overview on the available anti-fungal therapy required to manage the ever-increasing rise in fungal infections faced by maxillofacial surgeons in post-COVID-19 patients.

The sudden epidemic of mucormycosis amid COVID-19 pandemic has significantly challenged our understanding of the disease while affecting the whole medical and surgical management. Overzealous use of steroids in the management of covid-19 and uncontrolled diabetes mellitus has resulted in a tremendous rise in mucormycosis cases further burdening the already strained health care infrastructure and health care workers, especially the anesthesiologists. While working in a tertiary care institute of the country, we have been facing multiple challenges in its anesthetic management on a daily basis. This article is a case series involving four different patients who were operated for rhino-orbito-cerebral mucormycosis with a brief discussion on various aspects of this multisystem epidemic.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes (voriconazole, amphotericin) or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94-1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain (MIC 50%, 2 and 4 µg/mL, respectively). Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.

In spite of its high effectiveness in the treatment of both leishmaniasis as well as a range of fungal infections, the free form of the polyene antibiotic amphotericin B (AmB) does not entertain the status of the most preferred drug of choice in clinical settings. The high intrinsic toxicity of the principal drug could be considered the main impedance in the frequent medicinal use of this otherwise very effective antimicrobial agent. Taking into consideration this fact, the pharma industry has introduced many novel dosage forms of AmB to alleviate its toxicity issues. However, the limited production, high cost, requirement for a strict cold chain, and need for parenteral administration are some of the limitations that explicitly compel professionals to look for the development of an alternate dosage form of this important drug. Considering the fact that the nano-size dimensions of drug formulation play an important role in increasing the efficacy of the core drug, we employed a green method for the development of nano-assemblies of AmB (AmB-NA). The as-synthesized AmB-NA manifests desirable pharmacokinetics in the treated animals. The possible mechanistic insight suggested that as-synthesized AmB-NA induces necrosis-mediated cell death and severe mitochondrial dysfunction in L. donovani promastigotes by triggering depolarization of mitochondrial membrane potential. In vivo studies demonstrate a noticeable decline in parasite burden in the spleen, liver, and bone marrow of the experimental BALB/c mice host. In addition to successfully suppressing the Leishmania donovani, the as-formed AmB-NA formulation also modulates the host immune system with predominant Th1 polarization, a key immune defender that facilitates the killing of the intracellular parasite.

Leishmaniasis is a worldwide infectious parasitic disease caused by different species of protozoa of the genus Leishmania, which are transmitted to animals and humans through the bite of insects of the Psychodidae family. In the present work, the antileishmanial activity of an alkaloid extract of the bulbs of Clinanthus milagroanthus S. Leiva & Meerow (Amaryllidaceae) was evaluated in vitro, in vivo, and in silico against the parasite Leishmania braziliensis, and the chemical profile of the sample was determined by GC-MS analysis. At concentrations of 1, 10, and 100 µg·mL-1, the alkaloid extract presented inhibition percentages of 8.7%, 23.1%, and 98.8%, respectively, against L. braziliensis with a p < 0.05, and IC50 values of 18.5 ± 0.3 µg·mL-1. Furthermore, at a dose of 1.0 mg·kg-1, a greater decrease in lesion size was observed (90%) for in vivo assays, as well as a decrease in infection (96%), finding no significant differences (p > 0.05) in comparison with amphotericin B (92% and 98%, respectively). Eleven alkaloids were identified in C. milagroanthus bulbs: galanthamine, vittatine/crinine, 8-O-demethylmaritidine, anhydrolycorine, 11,12-dehydroanhydrolycorine, hippamine, lycorine, 2-hydroxyanhydrolycorine, 7-hydroxyclivonine, 2α-hydroxyhomolycorine, and 7-hydroxyclivonine isomer. A molecular model of Leishmania braziliensis trypanothione reductase (TRLb) was built using computational experiments to evaluate in silico the potential of the Amaryllidaceae alkaloid identified in C. milagroanthus toward this enzyme. The structures galanthamine, 7-hydroxyclivonine isomer, and crinine showed better estimated free energy of binding than the reference compound, amphotericin B. In conclusion, this is the first in vitro, in vivo, and in silico report about the antileishmanial potential and alkaloid profiling of the extract of C. milagroanthus bulbs, which could become an interesting source of bioactive molecules.

The liposomal amphotericin B (AmB) formulation, AmBisome®, still represents the best therapeutic option for cutaneous and visceral leishmaniasis. However, its clinical efficacy depends on the patient's immunological status, the clinical manifestation and the endemic region. Moreover, the need for parenteral administration, its side effects and high cost significantly limit its use in developing countries. This review reports the progress achieved thus far toward the understanding of the mechanism responsible for the reduced toxicity of liposomal AmB formulations and the factors that influence their efficacy against leishmaniasis. It also presents the recent advances in the development of more effective liposomal AmB formulations, including topical and oral liposome formulations. The critical role of the AmB aggregation state and release rate in the reduction of drug toxicity and in the drug efficacy by non-invasive routes is emphasized. This paper is expected to guide future research and development of innovative liposomal formulations of AmB.

(1) Background: Although invasive fungal infections are a major cause of neonatal morbidity and mortality, data on the incidence and outcomes of localized abscesses in solid organs due to fungal infections are scarce. The aim of this study was to consolidate evidence and enhance our understanding on neonatal liver abscesses due to invasive fungal infections. (2) Methods: An electronic search of the PubMed and Scopus databases was conducted, considering studies that evaluated fungal liver abscesses in the neonatal population. Data on the epidemiology, clinical course, treatment, and outcome of these infections were integrated in our study. (3) Results: Overall, 10 studies were included presenting data on 19 cases of neonatal fungal liver abscesses. Candida spp. were the most common causative pathogens (94.7%). Premature neonates constituted the majority of cases (93%), while umbilical venous catheter placement, broad spectrum antibiotics, and prolonged parenteral nutrition administration were identified as other common predisposing factors. Diagnosis was established primarily by abdominal ultrasonography. Medical therapy with antifungal agents was the mainstay of treatment, with Amphotericin B being the most common agent (47%). Abscess drainage was required in four cases (21%). Eradication of the infection was achieved in the majority of cases (80%). (4) Conclusions: Even though fungal liver abscess is a rare entity in the neonatal population, clinicians should keep it in mind in small, premature infants who fail to respond to conventional treatment for sepsis, particularly if an indwelling catheter is in situ. A high index of suspicion is necessary in order to achieve a timely diagnosis and the initiation of the appropriate treatment.

Blastomycosis is caused by a thermally dimorphic fungus that thrives in moist acidic soil. Blastomyces dermatitidis is the species responsible for most infections in North America and is especially common in areas around the Great Lakes, the St. Lawrence Seaway, and in several south-central and southeastern United States. Other Blastomyces species have more recently been discovered to cause disease in distinct geographic regions around the world. Infection almost always occurs following inhalation of conidia produced in the mold phase. Acute pulmonary infection ranges from asymptomatic to typical community-acquired pneumonia; more chronic forms of pulmonary infection can present as mass-like lesions or cavitary pneumonia. Infrequently, pulmonary infection can progress to acute respiratory distress syndrome that is associated with a high mortality rate. After initial pulmonary infection, hematogenous dissemination of the yeast form of Blastomyces is common. Most often this is manifested by cutaneous lesions, but osteoarticular, genitourinary, and central nervous system (CNS) involvement also occurs. The diagnosis of blastomycosis can be made by growth of the mold phase of Blastomyces spp. in culture or by histopathological identification of the distinctive features of the yeast form in tissues. Detection of cell wall antigens of Blastomyces in urine or serum provides a rapid method for a probable diagnosis of blastomycosis, but cross-reactivity with other endemic mycoses commonly occurs. Treatment of severe pulmonary or disseminated blastomycosis and CNS blastomycosis initially is with a lipid formulation of amphotericin B. After improvement, therapy can be changed to an oral azole, almost always itraconazole. With mild to moderate pulmonary or disseminated blastomycosis, oral itraconazole treatment is recommended.

Itraconazole is the first choice for treating sporotrichosis. Amphotericin B is indicated for severe and disseminated forms. The aim of the study was to evaluate the antifungal susceptibility of Sporothrix brasiliensis strains isolated from patients with severe sporotrichosis treated with amphotericin B and correlate with clinical outcomes. Clinical and epidemiological data were obtained from severe sporotrichosis cases caused by S. brasiliensis. Antifungal susceptibility tests against amphotericin B, itraconazole, terbinafine, posaconazole, and 5-flucytosine were performed. Moreover, possible synergisms between amphotericin B and posaconazole or 5-flucytosine were assessed. Relationships between clinical and laboratorial data were then analyzed. Forty-six S. brasiliensis isolates from 37 patients were studied. Clinical forms included disseminated (94.6%) and disseminated cutaneous sporotrichosis (5.4%). The median treatment time was 784 days (range: 7 to 3115 days). Cure occurred in 45.9% of the cases and death due to sporotrichosis in 24.3%. Forty-three (93.5%) S. brasiliensis isolates were classified as wild-type for all the antifungals tested according to their in vitro antifungal susceptibility. There was no synergism for the combinations studied. Finally, we found no association between higher Minimal Inhibitory Concentration (MIC) values of amphotericin B or itraconazole with unfavorable outcomes; however, there were higher MIC values of itraconazole in strains isolated from alcoholic patients. Possibly, clinical factors, such as the extent of dissemination, immunosuppression, and late treatment onset, are the main determinants of patient outcomes, rather than antifungal resistance. The current study suggests that the need to use amphotericin B therapy is not associated with the emergence of S. brasiliensis resistant strains.

Meningeal sporotrichosis is rare and occurs predominantly in immunosuppressed individuals. This retrospective study explored clinical and laboratory characteristics, treatment, and prognosis of patients with disseminated sporotrichosis who underwent lumbar puncture (LP) at a Brazilian reference center from 1999 to 2020. Kaplan-Meier and Cox regression models were used to estimate overall survival and hazard ratios. Among 57 enrolled patients, 17 had meningitis. Fifteen (88.2%) had HIV infection, and in 6 of them, neurological manifestations occurred because of the immune reconstitution inflammatory syndrome (IRIS). The most frequent symptom was headache (88.2%). Meningeal symptoms at first LP were absent in 7/17 (41.2%) patients. Sporothrix was diagnosed in cerebrospinal fluid either by culture or by polymerase chain reaction in seven and four patients, respectively. All but one patient received prolonged courses of amphotericin B formulations, and seven received posaconazole, but relapses were frequent. Lethality among patients with meningitis was 64.7%, with a higher chance of death compared to those without meningitis (HR = 3.87; IC95% = 1.23;12.17). Meningeal sporotrichosis occurs mostly in people with HIV and can be associated with IRIS. Screening LP is indicated in patients with disseminated disease despite the absence of neurological complaints. Meningitis is associated with poor prognosis, and better treatment strategies are needed.