Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes

Optical imaging of individual vesicle exocytosis is providing new insights into the mechanism and regulation of secretion by cells. To study calcium-triggered secretion from astrocytes, we used acridine orange (AO) to label vesicles. Although AO is often used for imaging exocytosis, we found that imaging vesicles labeled with AO can result in their photolysis. Here, we define experimental and analytical approaches that permit us to distinguish unambiguously between fusion, leakage, and lysis of individual vesicles. We have used this approach to demonstrate that lysosomes undergo calcium-triggered exocytosis in astrocytes.     

Population Pharmacokinetics of Flucloxacillin In Bone and Soft Tissue– Standard Dosing is Not Sufficient to Achieve Therapeutic Concentrations

Pharmaceutical Research - Flucloxacillin is a β-lactam penicillin commonly used in the treatment of bone and soft tissue infections. In a recent porcine study, we found surprisingly low time...     

Maturational changes in laminin, fibronectin, collagen IV, and perlecan in germinal matrix, cortex, and white matter and effect of betamethasone

Germinal matrix is selectively vulnerable to hemorrhage in premature infants, and use of prenatal betamethasone is associated with a lower occurrence of germinal matrix hemorrhage. Because the major components of extracellular matrix of the cerebral vasculature-laminin, fibronectin, collagen IV, and perlecan-provide structural stability to blood vessels, we examined whether the expression of these molecules was decreased in the germinal matrix and affected by betamethasone. In both human fetuses and premature infants, fibronectin was significantly lower in the germinal matrix than in the cortical mantle or white matter anlagen. Conversely, laminin alpha1 gene expression was greater in the human germinal matrix compared with the cortical mantle or white matter. Expression of alpha1- and alpha2(IV) collagen chains increased with advancing gestational age. Low-dose prenatal betamethasone treatment enhanced fibronectin level by 1.5-2-fold whereas a high dose reduced fibronectin expression by 2-fold in rabbit pups. Because fibronectin provides structural stability to the blood vessels, its reduced expression in the germinal matrix may contribute to the fragility of germinal matrix vasculature and the propensity to hemorrhage in premature neonates.     

Intercellular calcium signaling mediated by point-source burst release of ATP

Calcium signaling, manifested as intercellular waves of rising cytosolic calcium, is, in many cell types, the result of calcium-induced secretion of ATP and activation of purinergic receptors. The mechanism by which ATP is released has hitherto not been established. Here, we show by real-time bioluminescence imaging that ATP efflux is not uniform across a field of cells but is restricted to brief, abrupt point-source bursts. The ATP bursts emanate from single cells and manifest the transient opening of nonselective membrane channels, which admits fluorescent indicators of < or = 1.5 kDa. These observations challenge the existence of regenerative ATP release, because ATP efflux is finite and restricted to a point source. Transient efflux of cytosolic nucleotides from a subset of cells may represent a conserved pathway for coordinating local activity of electrically nonexcitable cells, because identical patterns of ATP release were identified in human astrocytes, endothelial cells, and bronchial epithelial cells.     

A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid β

Because it lacks a lymphatic circulation, the brain must clear extracellular proteins by an alternative mechanism. The cerebrospinal fluid (CSF) functions as a sink for brain extracellular solutes, but it is not clear how solutes from the brain interstitium move from the parenchyma to the CSF. We demonstrate that a substantial portion of subarachnoid CSF cycles through the brain interstitial space. On the basis of in vivo two-photon imaging of small fluorescent tracers, we showed that CSF enters the parenchyma along paravascular spaces that surround penetrating arteries and that brain interstitial fluid is cleared along paravenous drainage pathways. Animals lacking the water channel aquaporin-4 (AQP4) in astrocytes exhibit slowed CSF influx through this system and a ~70% reduction in interstitial solute clearance, suggesting that the bulk fluid flow between these anatomical influx and efflux routes is supported by astrocytic water transport. Fluorescent-tagged amyloid β, a peptide thought to be pathogenic in Alzheimer's disease, was transported along this route, and deletion of the Aqp4 gene suppressed the clearance of soluble amyloid β, suggesting that this pathway may remove amyloid β from the central nervous system. Clearance through paravenous flow may also regulate extracellular levels of proteins involved with neurodegenerative conditions, its impairment perhaps contributing to the mis-accumulation of soluble proteins.     

Inferior stabilization of cementless compared with cemented dual-mobility cups in elderly osteoarthrosis patients: a randomized controlled radiostereometry study on 60 patients with 2 years’ follow-up

Background and purpose — Elderly patients may benefit from a dislocation low-risk dual-mobility (DM) articulation in total hip arthroplasty, but the best cup fixation method is unknown. We compared cup migration for cemented and cementless DM cups using radiostereometry.Patients and methods — In a patient-blinded randomized trial, 60 patients (33 female) with osteoarthritis were allocated to cemented (n = 30) or cementless (n = 30) Avantage DM cup fixation. Criteria were age above 70 years, and T-score above –4. We investigated cup migration, periprosthetic bone mineral density (BMD), and patient-reported outcome measures (PROMs) until 24 months postoperative follow-up.Results — At 24 months mean proximal cup migration was 0.11 mm (95% CI 0.00–0.23) for cemented cups and 0.09 mm (CI –0.09 to 0.28) for cementless cups. However, cementless cups generally migrated more than cemented cups at 12 and 24 months. Cemented cups had no measurable migration from 3 months’ follow-up, while cementless cups had not yet stabilized at 24 months in all rotations. Cementless cups showed statistically significantly more maximum total point motion (MTPM) at 12- and 24-month follow-up compared with cemented cups in patients with low systemic BMD (p = 0.01). Periprosthetic BMD changes did not statisticially significantly correlate to proximal migration in either cup fixation group (p > 0.05). PROMs improved similarly in both groups.Interpretation — Cemented cups were well fixed at 3 months. The cementless cups migrated more in patients with low BMD, showed an inconsistent pattern of migration, and migrated in different directions during the first and second year without tendency to stabilization. Cemented fixation of the Avantage DM cup seems safer in elderly patients

The most common indication for revision of a conventional primary total hip arthroplasty (THA) is aseptic loosening of the components (SHAR 2016, NJR 2017, DHAR 2018).Implant fixation method (i.e., cemented or cementless) in primary THA seems mainly based on the surgeon’s preference and national trends. The Danish Hip Replacement Registry report shows a decrease in the use of cemented cup fixation in osteoarthrosis (OA) patients above 70 years (DHAR 2018). This trend has also been described in the United Kingdom (UK) and Australian Joint Registries, while in Sweden and Norway cemented cup fixation is still the preferred fixation method in elderly patients (SHAR 2016, NAR 2017, NJR 2017).The dual-mobility (DM) concept, with 2 articulation surfaces and increased jump distance, may decrease the dislocation rate and increase range of motion compared with standard single mobility (SM) THAs. The long-term survival and the best fixation method of the newer Avantage Reload DM cup in elderly patients is currently unknown but retrospective studies on other types of primary DM THAs suggest acceptable survival rates (Batailler et al. 2017) .Excessive early (2-year) implant micromotion measured with radiostereometric analysis (RSA) is a strong predictor for later implant loosening and poor survival (Karrholm et al. 1997, Nieuwenhuijse et al. 2012, Pijls et al. 2012), and our primary aim was to investigate the early RSA-measured migration of cemented and cementless Avantage DM cups in elderly (> 70 years old) OA patients until 24 months’ follow-up. Secondary endpoints included systemic and periprosthetic bone mineral density (BMD) measurements, and clinical outcome scores.     

Pulmonary mucormycosis in the aftermath of critical COVID-19 in an immunocompromised patient: Mind the diagnostic gap

Mucormycosis has recently been recognized as a severe complication of COVID-19 with high fatality rates. We report a fatal case of COVID-19 associated mucormycosis (CAM) in a non-diabetic immunocompromised patient, who was first misdiagnosed and treated for COVID-19 associated aspergillosis (CAPA). The risk factors and initial clinical presentation of CAPA and CAM are similar, but CAM has a more aggressive course and CAPA and CAM are treated differently. Dedicated diagnostic workup is essential to ensure early treatment of CAM with surgical debridement and targeted antifungal therapy.