Valorization of Coffee Silverskin Using Extraction Cycles and Water as a Solvent: Design of Process.

Coffee silverskin is a byproduct of the coffee industry, appearing in large quantities during the roasting step. In this work, a sober and simple water process is proposed, using extractions cycles, to produce valuable products including (a) an extract rich in caffeine, (b) possibly pure caffeine, and (c) insoluble fibers. The hypothetical number of necessary cycles was calculated and compared to the number of cycles used experimentally. Two types of cycles, with and without water compensation, were compared for their water consumption and the amount of caffeine extracted. The use of cycles, with the resulting product from a previous extraction as a solvent for fresh biomass, drove a significant rise in the content of caffeine determined by a UV-visible detector with a spectrophotometer and ultra-performance liquid chromatography (UPLC). After 11 extraction cycles with water compensation, we obtained an extract 4.5 times more concentrated in caffeine (4.25 mg/mL) than after a single extraction (1.03 mg/mL).

Synaptotagmin 1-triggered lipid signaling facilitates coupling of exo- and endocytosis.

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.

Mechanical properties and chemical stability of alginate-based anisotropic capillary hydrogels.

Anisotropic capillary hydrogels are formed by ionotropic gel formation of an aqueous sodium alginate solution during unidirectional diffusion and complexation with divalent cations. The type of cation used for gel formation dictates the size of the resulting capillary structure which might facilitate applications as biomaterial scaffolds in tissue engineering and regenerative medicine. Until now, such capillary hydrogel structures have not been characterized regarding their mechanical properties, and we hypothesise that both, the macroscopic capillary structure and the concentration of a chemical crosslinking agent, influence the mechanical properties and the stability of the hydrogels against degradation and dissolution in physiological environment. We prepared anisotropic gels with capillary sizes ranging between 12 and 100 µm using electrolyte solutions containing Ba2+, Sr2+, Zn2+ or Ni2+ cations. They were chemically crosslinked with hexamethylene diisocyanate in concentrations varying between 1 and 100 mmol L-1. Hydrogel properties were determined by swelling experiments, oscillatory rheometry and microindentation analysis and monitored during incubation in phosphate-buffered solution at 37 °C for up to three months. We found, that the mechanical strength generally decreases with increasing capillary diameter. The higher the concentration of the crosslinking agent, the higher is the mechanical strength. The resistance of the alginate hydrogels against degradation is positively correlated with the concentration of the crosslinking agent. Furthermore, microindentation experiments revealed a microscopic anisotropy of the mechanical properties resulting from a perpendicular orientation of the polymer fibres relative to the capillary axis.

Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission.

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration.

Membrane-Permeant, Bioactivatable Coumarin Derivatives for In-Cell Labelling.

The delivery of small molecule fluorophores with minimal compartmentalization is currently one of the most critical technical problems in intracellular labelling. Here we introduce sulfonated and phosphonated coumarin dyes, demonstrate rapid cell entry via a prodrug approach, and show a lack of interaction with membranes, organelles, or other compartments. The dyes show no specific localization and are evenly distributed in the cells. Our fluorogenic, clickable phosphonate derivatives successfully tagged model targets in intact cells and the increase in brightness upon click reaction was around 60-fold.

Dizziness and benign paroxysmal positional vertigo among retirement home residents: a cross-sectional descriptive and interventional study.

BACKGROUND: The prevalence of dizziness increases with age. We aimed to determine the point prevalence of dizziness and, in particular, of benign paroxysmal positional vertigo (BPPV) among retirement home residents. Furthermore, we aimed to evaluate the efficacy of a 2-axis turntable based BPPV treatment. METHODS: We contacted all large retirement homes in or around the city of Zurich (Switzerland). 10 retirement homes (with a total of 536 residents) agreed to participate in this study. 83 rejected inquiries by residents led to a potential study population of 453 residents. After a structured interview evaluating the presence and characteristics of dizziness, all willing patients were tested for positional vertigo and nystagmus on a portable and manually operated 2-axis turntable that was transported to the retirement home. Testing consisted of the Dix-Hallpike and supine roll maneuvers to both sides. Participants were immediately treated with the appropriate liberation maneuver whenever BPPV was diagnosed. Otherwise, taking the resident's medical history, a neuro-otological bedside examination, and a review of the available medical documentation was used to identify other causes of dizziness. RESULTS: Out of the 453 residents, 75 (16.6%; average age: 87.0 years; 68% female) were suffering from dizziness presently or in the recent past and gave their consent to participate in this study. Among the participants tested on the turntable (n = 71), BPPV was present in 11.3% (point prevalence). Time-related properties, triggering factors and qualitative attributes of vertigo or dizziness were not significantly different between the dizzy participants with and those without BPPV. In all BPPV patients, appropriate liberation maneuvers were successful. CONCLUSIONS: BPPV could be demonstrated in about one tenth of retirement home residents with dizziness or recent dizziness. Such point prevalence of BPPV translates to a much higher yearly prevalence if one assumes that BPPV is not present on every day. Our finding suggests that retirement home residents suffering from dizziness should be regularly tested for BPPV and treated with appropriate liberation maneuvers, ideally on turntable to reduce strain. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03643354 .

Monitoring the cellular metabolism of a membrane-permeant photo-caged phosphatidylinositol 3,4,5-trisphosphate derivative.

To deliver charged lipid derivatives to the cell interior, bioactivatable and photo-activatable protecting groups are frequently used. The intracellular metabolism of the protecting groups, as well as the lipid itself, are key factors that determine biological activity. Here we followed the cellular metabolism of cell-permeant photo-activatable ("caged") and non-caged membrane-permeant analogs of dioctanoyl phosphatidylinositol 3,4,5-trisphosphate (diC8PIP3) carrying biodegradable protecting groups by mass spectrometry. After successful cell entry, the photo-activatable group can be removed on demand by a light pulse. Hence, UV irradiation acts as a switch to expose the cellular metabolism to a bolus of active compound. To investigate lipid metabolites and to capture a more complete metabolome, we adapted standard extraction methods and employed multi-reaction monitoring mass spectrometry (MRM-MS). This required a previously developed permethylation method that stabilized metabolites and enhanced volatility of the phosphoinositide metabolites. The mass spectrometric analysis allowed for the monitoring of the intracellular removal of photo-activatable caging as well as biodegradable protecting groups from the membrane-permeant phosphoinositides along with cellular turnover, namely by dephosphorylation. We found that phosphate masking groups, namely acetoxymethyl esters, were rapidly removed by endogenous enzymes while butyrates masking hydroxy groups showed a longer lifetime, giving rise to trapped intermediates. We further identified key intermediate metabolites and demonstrated the beneficial effect of caging groups and their removal on the formation of favorable metabolites. Surprisingly, caging and protecting groups were found to influence each other's stability.

Maturation of the matrix and viral membrane of HIV-1.

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

Sub-50 nm ultra-small organic drug nanosuspension prepared by cavi-precipitation and its brain targeting potential.

The purpose of this study was to show whether it is possible to prepare sub 100 nm or preferably sub-50 nm drug nanosuspension (NS) of suitable quality for intravenous administration. Furthermore, we have studied how the brain targeting potential of such small size organic NS differs from relatively bigger size NS. Two combination technologies (cavi-precipitation, H96) and a standard high-pressure homogenization (HPH) technology were used to prepare drug NS of different sizes. The cavi-precipitation process generated the smallest AmB NS, i.e., 27 nm compared to 79 nm by H96 technology and 252 nm by standard HPH technology. Dialysis of the nanosuspension in the original dispersion media was found to be the most efficient solvent removal method without negatively affecting particle size. The removal of organic solvent was found to drastically improve the stability of the formulations. The protein adsorption pattern shows that the small size NS particles obtained by the cavi-precipitation process have the potential to circulate longer in the bloodstream and have the potential to be taken up by the blood-brain barrier. The cavi-precipitation process generated ultrafine NS particles, which fulfilled the quality requirements for intravenous administration and offer a potential solution for brain targeting.

Synthesis and Cellular Labeling of Multifunctional Phosphatidylinositol Bis- and Trisphosphate Derivatives.

We synthesized the first multifunctionalized phosphoinositide polyphosphate derivatives featuring a photo-removable protecting group ("cage"), a photo-crosslinkable diazirine group, and a terminal alkyne group useful for click chemistry. We demonstrate that the lipid derivatives readily enter cells. After photo-crosslinking, cell fixation and fluorescent tagging via click chemistry, we determined the intracellular location of the lipid derivatives before and after uncaging of the lipids. We find that there is rapid trafficking of PI(3,4)P2 and PI(3,4,5)P3 derivatives to the plasma membrane, opening the intriguing possibility that there is active transport of these lipids involved. We employed the photo-crosslinking and click chemistry functions to analyze the proteome of PI(3,4,5)P3 -binding proteins. From the latter, we validated by RNAi that the putative lipid binding proteins ATP11A and MPP6 are involved in the transport of PI(3,4,5)P3 to the plasma membrane.

Can the cavi-precipitation process be exploited to generate smaller size drug nanocrystal?

OBJECTIVE: Cavi-precipitation has the potential to generate drug nanocrystals very efficiently. Achieving smaller than 100 nm particle size for organic drug substances still remained a challenge. The objective of this study was to demonstrate if cavi-precipitation technology can be used to generate smaller than 100 nm drug nanocrystal particle. SIGNIFICANCE: This study demonstrates that cavi-precipitation process can be used to generate drug nanocrystals of the model compound resveratrol (RVT) consists of crystallites of 30-50 nm size. METHOD: RVT was dissolved in different organic solvents to prepare the solvent phase (S-phase). Several stabilizers were tested for the organic phase. A combination of SDS and PVP was used stabilizer system in the aqueous anti-solvent phase (AS-phase). The S-phase was added to the AS-phase inside the Emulsiflex C5 homogenizer. Nanosuspension was characterized by laser diffractometry (LD), photon correlation spectroscopy (PCS) and scanning electron microscopy (SEM). The solid state of the suspended particles was investigated by powder X-ray diffractometry (PXRD) and differential scanning calorimetry (DSC). RESULTS: It was found that DMSO, alone or in combination with acetone in the S-Phase generated the smallest size RVT nanocrystals. The optimum solvent (S) antisolvent (AS) ratio (S:AS) was found to be 3.6:56.4 (v:v). Span 20 was identified as the best stabilizer for the organic phase at a ratio (w:w) of 1:3 (Span 20:RVT). The particles precipitated from different solvents were predominantly crystalline. CONCLUSIONS: The best sample had a mean particle size (LD) of 167 nm [d(0.5)] which was composed of smaller crystallites having 30-50 nm size (SEM).

Enhancement of Intracellular Calcium Ion Mobilization by Moderately but Not Highly Positive Material Surface Charges.

Electrostatic forces at the cell interface affect the nature of cell adhesion and function; but there is still limited knowledge about the impact of positive or negative surface charges on cell-material interactions in regenerative medicine. Titanium surfaces with a variety of zeta potentials between -90 mV and +50 mV were generated by functionalizing them with amino polymers, extracellular matrix proteins/peptide motifs and polyelectrolyte multilayers. A significant enhancement of intracellular calcium mobilization was achieved on surfaces with a moderately positive (+1 to +10 mV) compared with a negative zeta potential (-90 to -3 mV). Dramatic losses of cell activity (membrane integrity, viability, proliferation, calcium mobilization) were observed on surfaces with a highly positive zeta potential (+50 mV). This systematic study indicates that cells do not prefer positive charges in general, merely moderately positive ones. The cell behavior of MG-63s could be correlated with the materials' zeta potential; but not with water contact angle or surface free energy. Our findings present new insights and provide an essential knowledge for future applications in dental and orthopedic surgery.

Treatment and management of primary antibody deficiency: German interdisciplinary evidence-based consensus guideline.

This evidence-based clinical guideline provides consensus-recommendations for the treatment and care of patients with primary antibody deficiencies (PADs). The guideline group comprised 20 clinical and scientific expert associations of the German, Swiss, and Austrian healthcare system and representatives of patients. Recommendations were based on results of a systematic literature search, data extraction, and evaluation of methodology and study quality in combination with the clinical expertise of the respective representatives. Consensus-based recommendations were determined via nominal group technique. PADs are the largest clinically relevant group of primary immunodeficiencies. Most patients with PADs present with increased susceptibility to infections, however immune dysregulation, autoimmunity, and cancer affect a significant number of patients and may precede infections. This guideline therefore covers interdisciplinary clinical and therapeutic aspects of infectious (e.g., antibiotic prophylaxis, management of bronchiectasis) and non-infectious manifestations (e.g., management of granulomatous disease, immune cytopenia). PADs are grouped into disease entities with definitive, probable, possible, or unlikely benefit of IgG-replacement therapy. Summary and consensus-recommendations are provided for treatment indication, dosing, routes of administration, and adverse events of IgG-replacement therapy. Special aspects of concomitant impaired T-cell function are highlighted as well as clinical data on selected monogenetic inborn errors of immunity formerly classified into PADs (APDS, CTLA-4-, and LRBA-deficiency).

[S2k guideline for the diagnosis and therapy of zoster and post-zoster neuralgia]. / S2k-Leitlinie zur Diagnostik und Therapie des Zoster und der Postzosterneuralgie.

This guideline is aimed at registrars and consultants in dermatology, ophthalmology, ENT, pediatrics, neurology, virology as well as infectiology, anaesthesia and generell medicine as well as policymakers and payers and purchasers of care. It was developed by dermatologists, virologists, ophthalmologists, ENT physicians, neurologists, pediatrician and anesthetists using a formal consensus process (S2k).The guideline provides an overview of clinical and molecular diagnostics as well as antigen detection, antibody culture and viral culture. Diagnostic special situations and complicated courses of the disease are also considered. The antiviral therapy of zoster and postzoster neuralgia is presented in general and for special situations. Detailed information on the treatment of pain is mentioned and presented in an overview. Likewise, the local therapeutic measures are discussed.

Calculated parenteral initial treatment of bacterial infections: Infections in the ear, nose, throat and mouth and jaw area.

This is the sixth chapter of the guideline "Calculated initial parenteral treatment of bacterial infections in adults - update 2018" in the 2nd updated version. The German guideline by the Paul-Ehrlich-Gesellschaft für Chemotherapie e.V. (PEG) has been translated to address an international audience. The chapter deals with the antibacterial treatment of more severe infections of the ear, the nose, the throat and the maxillofacial region, including odontogenic and salivary gland infections.

ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia.

A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.

S2k guidelines for the diagnosis and treatment of herpes zoster and postherpetic neuralgia.

The present guidelines are aimed at residents and board-certified specialists in the fields of dermatology, ophthalmology, ENT, pediatrics, neurology, virology, infectious diseases, anesthesiology, general medicine and any other medical specialties involved in the management of patients with herpes zoster. They are also intended as a guide for policymakers and health insurance funds. The guidelines were developed by dermatologists, virologists, ophthalmologists, ENT physicians, neurologists, pediatricians and anesthesiologists/pain specialists using a formal consensus process (S2k). Readers are provided with an overview of the clinical and molecular diagnostic workup, including antigen detection, antibody tests and viral culture. Special diagnostic situations and complicated disease courses are discussed. The authors address general and special aspects of antiviral therapy for herpes zoster and postherpetic neuralgia. Furthermore, the guidelines provide detailed information on pain management including a schematic overview, and they conclude with a discussion of topical treatment options.

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway.

Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P2 by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P2-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis and is undetectable in endocytic clathrin-coated pits. Instead, we found that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase and mTOR complex 1. These results reveal a function for INPP4-mediated PI(3,4)P2 hydrolysis in local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model whereby synthesis and turnover of PI(3,4)P2 are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.