Don't shake it! Mechanical stress testing of mRNA-lipid nanoparticles.

Shaking stress studies are typically performed during formulation development to test the liability of a drug product towards interfacial stress occurring during transport, especially if a liquid formulation is desired. We evaluated various shaking procedures using a polyA-surrogate solution and verified our findings by eGFP-LNP cell-expression experiments. Shaking on an orbital shaker in vertical and horizontal orientations at increasing speeds from 300 to 600 rpm resulted in decreasing levels of encapsulated nucleic acid content, larger LNP sizes, and decreasing PDI. We report that vertical and horizontal shaking of both polyA- and eGFP-LNPs led to white deposits on the inner glass vial surface, depending on time, rpm, and temperature. Increasing the fill volume/smaller headspace (0.3 versus 0.9 mL fill) did not mitigate this phenomenon in the studied configuration, and the use of hydrophobic primary packaging even accelerated the formation of white deposits. In contrast, we demonstrated that a lyophilized polyA-LNP dosage form was less susceptible to shaking and maintained cake integrity and product properties. Multiple vortexing steps resulted in an increase in LNP size, PDI, and a decrease in encapsulated polyA content. We conclude that shaking experiments of nucleic acid-loaded LNPs in their final configuration at intended transport conditions need to be considered during technical development.

Chip for dielectrophoretic microbial capture, separation and detection II: experimental study.

In our previous paper we have modelled a dielectrophoretic force (DEP) and cell particle behavior in a microfluidic channel (Weber MUet al2023 Chip for dielectrophoretic microbial capture, separation and detection I: theoretical basis of electrode designNanotechnologythis issue). Here we test and confirm the results of our modeling work by experimentally validating the theoretical design constraints of the ring electrode architecture. We have compared and tested the geometry and particle capture and separation performance of the two separate electrode designs (the ring and dot electrode structures) by investigating bacterial motion in response to the applied electric field. We have quantitatively evaluated the electroosmosis (EO) to positive DEP (PDEP) transition in both electrode designs and explained the differences in capture efficiency of the ring and dot electrode systems. The ring structure shows 99% efficiency of bacterial capture both for PDEP and for EO. Moreover, the ring structure shows an over 200 faster bacterial response to the electric field. We have also established that the ring electrode architecture, with appropriate structure periodicity and spacing, results in efficient capture and separation of microbial cells. We have identified several critical design constraints that are required to achieve high efficiency bacterial capture. We have established that the spacing between consecutive DEP traps smaller than the length of the depletion zone will ensure that the DEP force dominates bacterial motion over motility and Brownian motion.

Chip for dielectrophoretic microbial capture, separation and detection I: theoretical basis of electrode design.

We model the dielectrophoretic response ofE. colibacterial cells and red blood cells, upon exposure to an electric field. We model the separation, capture, and release mechanisms under flow conditions in a microfluidic channel and show under which conditions efficient separation of different cell types occurs. The modelling work is aimed to guide the separation electrode architecture and design for experimental validation of the model. The dielectrophoretic force is affected both by the geometry of the electrodes (the gradient of the electric field), the Re{CM(ω)} factor, and the permittivity of the medium ϵm. Our modelling makes testable predictions and shows that designing the electrode structure to ensure structure periodicity with spacing between consecutive traps smaller than the length of the depletion zone ensures efficient capture and separation. Such electrode system has higher capture and separation efficiency than systems with the established circular electrode architecture. The simulated, modelled microfluidic design allows for the separated bacteria, concentrated by dedicated dielectrophoretic regions, to be subsequently detected using label-free functionalized nanowire sensors. The experimental validation of the modelling work presented here and the validation of the theoretical design constraints of the chip electrode architecture is presented in the companion paper in the same issue (Weber MUet al2022 Chip for dielectrophoretic Microbial Capture, Separation and Detection II: Experimental Study).

A Thermosensitive, Chitosan-Based Hydrogel as Delivery System for Antibacterial Liposomes to Surgical Site Infections.

Prophylaxis and the treatment of surgical site infections (SSIs) with antibiotics frequently fail due to the antibiotic resistance of bacteria and the ability of bacteria to reside in biofilms (i.e., bacterial clusters in a protective matrix). Therefore, alternative antibacterial treatments are required to combat biofilm infections. The combination of diethyldithiocarbamate (DDC-) and copper ions (Cu2+) exhibited antibiofilm activity against the staphylococci species associated with SSIs; however, the formation of a water-insoluble Cu(DDC)2 complex limits its application to SSIs. Here, we describe the development and antibiofilm activity of an injectable gel containing a liposomal formulation of Cu(DDC)2 and Cu2+ (lipogel). Lyophilized liposomes were incorporated into a mixture of chitosan (CS) and beta-glycerophosphate (ßGP), and the thermosensitive gelling properties of CS-ßGP and the lipogel were determined. The liposomes remained stable after lyophilization over six months at 4-6 °C and -20 °C. The sol-gel transition of the gel and lipogel occurred between 33 and 39 °C, independently of sterilization or storage at -20 °C. CS-ßGP is biocompatible and the liposomes were released over time. The lipogel prevented biofilm formation over 2 days and killed 98.7% of the methicillin-resistant Staphylococcus aureus and 99.9% of the Staphylococcus epidermidis biofilms. Therefore, the lipogel is a promising new prophylaxis and treatment strategy for local application to SSIs.

Mitotane Nanocarriers for the Treatment of Adrenocortical Carcinoma: Evaluation of Albumin-Stabilized Nanoparticles and Liposomes in a Preclinical In Vitro Study with 3D Spheroids.

Adrenocortical carcinoma (ACC) is a heterogeneous malignancy related to poor prognosis and limited treatment options. The orphan drug mitotane (MT) is still a cornerstone in ACC therapy, however, its application is characterized by low aqueous solubility, poor bioavailability, and unfavorable pharmacokinetics, often resulting in below-target plasma concentrations or toxic side effects. Throughout the last decades, nanoparticulate formulations have become attractive carriers to improve anticancer therapy. In this study, injectable MT liposomes (DOPC-MT) and albumin-stabilized MT nanoparticles (BSA-MT) were investigated in depth with respect to their physicochemical properties, and their colloidal and therapeutical stability upon storage. Furthermore, in vitro cytotoxicity was evaluated using the ACC model cell line NCI-H295R for preparing multicellular tumor spheroids, and was compared to non-malignant human dermal fibroblasts. Our results clearly demonstrate that BSA-MT, unlike DOPC-MT, represents a stable and storable MT formulation with a high drug concentration in an aqueous medium. Dual centrifugation was established as a reproducible method for nanoparticle preparation. Although an efficient cytotoxic effect on ACC tumor spheroids was demonstrated, concomitant low toxicity to fibroblasts suggests that higher drug concentrations may be tolerated in vivo. Consequently, BSA-MT is a novel and promising therapeutical approach to address key challenges in MT treatment.

Fluid-Screen as a real time dielectrophoretic method for universal microbial capture.

Bacterial culture methods, e.g. Plate Counting Method (PCM), are a gold standard in the assessment of microbial contamination in multitude of human industries. They are however slow, labor intensive, and prone to manual errors. Dielectrophoresis (DEP) has shown great promise for particle separation for decades; however, it has not yet been widely applied in routine laboratory setting. This paper provides an overview of a new DEP microbial capture and separation method called Fluid-Screen (FS), that achieves very fast, efficient, reliable and repeatable capture and separation of microbial cells. Method verification experiments demonstrated that the FS system captured 100% of bacteria in test samples, a capture efficiency much higher than previously reported for similar technology. Data generated supports the superiority of the FS method as compared to the established Plate Counting Method (PCM), that is routinely used to detect bacterial contamination in healthcare, pharmacological and food industries. We demonstrate that the FS method is universal and can capture and separate different species of bacteria and fungi to viruses, from various sample matrices (i.e. human red blood cells, mammalian cells).

Preclinical In Vitro Studies with 3D Spheroids to Evaluate Cu(DDC)2 Containing Liposomes for the Treatment of Neuroblastoma.

Preclinical in vitro studies of drug candidates for anticancer therapy are generally conducted on well-established 2D cell models. Unfortunately, these models are unable to mimic the properties of in vivo tumors. However, in vitro 3D models (spheroids) have been proven to be superior in reflecting the tumor microenvironment. Diethyldithiocarbamate (DDC-) is the active metabolite of Disulfiram, an approved drug for alcoholism and repurposed for cancer treatment. DDC- binds copper in a molar ratio of 2:1 resulting in a water-insoluble Cu(DDC)2 complex exhibiting anticancer activities. Delivery of the Cu(DDC)2 complex using nanoparticulate carriers provides decisive advantages for a parental application. In this study, an injectable liposomal Cu(DDC)2 formulation was developed and the toxicity was compared with a 2D neuroblastoma and a 3D neuroblastoma cell model. Our results indicate that Cu(DDC)2 liposomes complied with the size requirements of nanoparticles for intravenous injection and demonstrated high drug to lipid ratios as well as colloidal stability upon storage. Furthermore, an efficient cytotoxic effect on neuroblastoma 2D cell cultures and a very promising and even more pronounced effect on 3D cell cultures in terms of neuroblastoma monoculture and neuroblastoma co-culture with primary cell lines was proven, highly encouraging the use of Cu(DDC)2 liposomes for anticancer therapy.

Synchronous Infra-Slow Oscillations Organize Ensembles of Accessory Olfactory Bulb Projection Neurons into Distinct Microcircuits.

The accessory olfactory system controls social and sexual behavior. In the mouse accessory olfactory bulb, the first central stage of information processing along the accessory olfactory pathway, projection neurons (mitral cells) display infra-slow oscillatory discharge with remarkable periodicity. The physiological mechanisms that underlie this default output state, however, remain controversial. Moreover, whether such rhythmic infra-slow activity patterns exist in awake behaving mice and whether such activity reflects the functional organization of the accessory olfactory bulb circuitry remain unclear. Here, we hypothesize that mitral cell ensembles form synchronized microcircuits that subdivide the accessory olfactory bulb into segregated functional clusters. We use a miniature microscope to image the Ca2+ dynamics within the apical dendritic compartments of large mitral cell ensembles in vivo We show that infra-slow periodic patterns of concerted neural activity, indeed, reflect the idle state of accessory olfactory bulb output in awake male and female mice. Ca2+ activity profiles are distinct and glomerulus-specific. Confocal time-lapse imaging in acute slices reveals that groups of mitral cells assemble into microcircuits that exhibit correlated Ca2+ signals. Moreover, electrophysiological profiling of synaptic connectivity indicates functional coupling between mitral cells. Our results suggest that both intrinsically rhythmogenic neurons and neurons entrained by fast synaptic drive are key elements in organizing the accessory olfactory bulb into functional microcircuits, each characterized by a distinct default pattern of infra-slow rhythmicity.SIGNIFICANCE STATEMENT Information processing in the accessory olfactory bulb (AOB) plays a central role in conspecific chemosensory communication. Surprisingly, many basic physiological principles that underlie neuronal signaling in the AOB remain elusive. Here, we show that AOB projection neurons (mitral cells) form parallel synchronized ensembles both in vitro and in vivo Infra-slow synchronous oscillatory activity within AOB microcircuits thus adds a new dimension to chemosensory coding along the accessory olfactory pathway.

The suitability of liposomes for the delivery of hydrophobic drugs - A case study with curcumin.

Liposomes are a popular formulation strategy for the delivery of anticancer drugs. While their benefits for formulating hydrophilic anticancer drugs have been clearly shown during the last decades, the suitability of liposomes for the delivery of hydrophobic drugs is questionable. Curcumin is a diphenolic plant compound that is extensively researched for its anticancer properties. It was chosen as a hydrophobic model drug in this study. Due to its low bioavailability, poor solubility and instability in aqueous media it is a highly problematic compound and requires particular formulation techniques. Curcumin liposomes with lipids of different rigidities were comprehensively investigated in respect to their physicochemical properties, their storage and serum stability. In vitro experiments were conducted with common 2D cell models and additionally with multicellular tumor spheroids (MCTS) as a more sophisticated tool to represent the physiology of avascular solid tumors. Our results indicate that liposomes containing the fluid phospholipid dioleoylphosphatidylcholine (DOPC) represent an excellent formulation to enhance the solubility and stability of curcumin. However, in presence of serum or cells, curcumin is rapidly released from the protecting and stabilizing lipid bilayer. Thus, improvement of the in vivo efficacy of curcumin is probably not achieved by using liposomes. Cytotoxicity and uptake experiments showed clearly a reduced effectivity of curcumin liposomes in the 3D cell model in comparison to the 2D model. This not only illustrates the limitations of monolayer cultures in predicting drug and nanocarrier interactions with solid tumors, but also further questions the use of liposomes as a formulation strategy in the treatment of solid tumors with curcumin.

Screening for Hazardous Drinking in Nursing Home Residents: Evaluating the Validity of the Current Cutoffs of the Alcohol Use Disorder Identification Test-Consumption Questions by Using Ethyl Glucuronide in Hair.

BACKGROUND: Because of physiological changes, elderly people are much more exposed to the adverse effects of alcohol. Therefore, hazardous drinking is defined at lower levels as compared to younger adults. This work aimed to evaluate the validity of the current cutoff levels of the Alcohol Use Disorder Identification Test-Consumption (AUDIT-C) questions to detect hazardous drinking in the elderly by using ethyl glucuronide in hair (HEtG). METHODS: In a border region between Austria and Germany, 344 nursing home residents were included from 33 of the 107 nursing homes. Residents were asked to answer the AUDIT-C questions, hair samples were obtained, and nursing staff members were asked for their assessments of the residents' alcohol consumption. Hair samples were analyzed for HEtG using gas chromatography-mass spectrometry. Receiver-operating characteristic (ROC) curve analysis was performed to determine the validity of cutoff values for the AUDIT-C to detect an alcohol consumption of ≥10 g of alcohol/d. RESULTS: A total of 11.3% of the nursing home residents (n = 344) drank ≥10 g of alcohol/d (4.9% >60 g of alcohol/d, 6.4% 10 to 60 g of alcohol/d, 88.7% <10 g of alcohol/d)). For the drinking limit of ≥10 g of alcohol/d, ROC curve analysis showed a balanced sensitivity and specificity, with an AUDIT-C cutoff of ≥4 for men (sensitivity: 70%, specificity: 83.6%; AUC = 0.823, CI = 0.718 to 0.928, p < 0.001) and ≥2 for women (sensitivity: 73.7%, specificity: 81.9%; AUC = 0.783, CI = 0.653 to 0.914, p < 0.001). Nursing staff (n = 274) underestimated alcohol consumption and evaluated 40% of the chronic-excessive alcohol consumers (>60 g of alcohol/d) as being abstinent. CONCLUSIONS: Our data suggest that an AUDIT-C cutoff of ≥4 for men and ≥2 for women can be recommended to detect the consumption of ≥10 g of alcohol/d in the elderly. Because the nursing staff to a large extent underestimates the alcohol consumption among nursing home residents, further teaching of the staff, improvement of screening instruments for the elderly, and the use of objective biomarkers might be helpful for recognizing hazardous drinking and can thus help improve the quality of life of the elderly.

Anxiety disorders and behavioral inhibition in preschool children: a population-based study.

This study assessed the prevalence of anxiety disorders in preschool children and their associations with behavioral inhibition as a temperamental precursor. A representative sample of 1,342 children aged 4­7 years (M = 6;1, SD = 4.80) was examined with a standardized parental questionnaire, including items referring to anxiety disorders at the current age and behavioral inhibition at the age of 2 years. The total prevalence of anxiety disorders was 22.2 %. Separation anxiety (SAD) affected 7 %, social phobia (SOC) 10.7 %, specific phobia (PHOB) 9.8 % and depression/generalized anxiety (MDD/GAD) 3.4 % of children. The prevalence of most types of anxiety was higher in girls except for separation anxiety, which affected more boys. Behavioral inhibition in the second year of life was associated with all types of anxiety. Anxiety disorders are common but frequently overlooked in preschool children. Different subtypes can be differentiated and are often preceded by behavioral inhibition. Assessment, prevention and treatment of anxiety disorders are recommended in preschool children.

Effect of viologen-phosphorus dendrimers on acetylcholinesterase and butyrylcholinesterase activities.

The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is the first step in checking whether new compounds can be considered as drugs for treating neurodegenerative diseases. The effect of viologen-phosphorus dendrimers on AChE and BChE activities was studied. The results show that the effects on the cholinesterase activities depend on dendrimer type and size. Viologen dendrimers can interact with the enzymes in two ways: they can bind either to a peripheral site of the enzyme or to amino acids located near the active site, inhibiting catalysis by both cholinesterases. All tested non-toxic viologen-phosphorus dendrimers inhibited the activities of both cholinesterases, showing their potential as new drugs for treating neurodegenerative diseases.

Biological properties of new viologen-phosphorus dendrimers.

Some biological properties of eight dendrimers incorporating both phosphorus linkages and viologen units within their cascade structure or at the periphery were investigated for the first time. In particular cytotoxicity, hemotoxicity, and antimicrobial and antifungal activity of these new macromolecules were examined. Even if for example all these species exhibited good antimicrobial properties, it was demonstrated that their behavior strongly depends on several parameters as their size and molecular weight, the number of viologen units and the nature of the terminal groups.

Modulation of skin pigmentation by the tetrapeptide PKEK: in vitro and in vivo evidence for skin whitening effects.

Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro-Lys-Glu-Lys) can exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB-stimulated mRNA expression of interleukin (IL)-6, IL-8 and TNF-α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation-inducing soluble mediator α- (α-MSH). (ii) PKEK treatment significantly inhibited UVB-induced upregulation of genes encoding for IL-1α, IL-6, IL-8, TNF-α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP-containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP-containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB-induced skin pigmentation and may be suited to serve as a skin tone-modulating agent in cosmetic products.

Rational design of an aryl-C-glycoside catalyst from a natural product O-glycosyltransferase.

Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O- versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O- and C-glycosylation.

Organisation of the biosynthetic gene cluster and tailoring enzymes in the biosynthesis of the tetracyclic quinone glycoside antibiotic polyketomycin.

Polyketomycin is a tetracyclic quinone glycoside produced by Streptomyces diastatochromogenes Tü6028. It shows cytotoxic and antibiotic activity, in particular against Gram-positive multi-drug-resistant strains (for example, MRSA). The polyketomycin biosynthetic gene cluster has been sequenced and characterised. Its identity was proven by inactivation of a alpha-ketoacyl synthase gene (pokP1) of the "minimal polyketide synthase II" system. In order to obtain valuable information about tailoring steps, we performed further gene-inactivation experiments. The generation of mutants with deletions in oxygenase genes (pokO1, pokO2, both in parallel and pokO4) and methyltransferase genes (pokMT1, pokMT2 and pokMT3) resulted in new polyketomycin derivatives, and provided information about the organisation of the biosynthetic pathway.

The Aspergillus nidulans enzyme TdiB catalyzes prenyltransfer to the precursor of bioactive asterriquinones.

The asterriquinones represent a class of ascomycete metabolic products whose significance stems from remarkable and useful pharmacological activities, among those antiretroviral (e.g., against the HI-virus), antitumor, and antidiabetes properties. Recently, the first genetic locus for an asterriquinone, the clustered terrequinone genes tdiA-E, was identified during a genome-wide screen in Aspergillus nidulans for "orphan" natural product biosynthesis loci. Here, we describe overexpression and characterization of TdiB, which catalyzes the reverse prenylation event during terrequinone A biosynthesis, which is the transfer of dimethylallyl diphosphate to carbon atom 2' of the intermediate didemethylasterriquinone D, to yield asterriquinone C-1. TdiB does not depend on the presence of divalent metal cations for catalysis and lacks the canonical prenyl diphosphate binding motif (D/N)DXXD.

A one-pot chemoenzymatic synthesis for the universal precursor of antidiabetes and antiviral bis-indolylquinones.

Bis-indolylquinones represent a class of fungal natural products that display antiretroviral, antidiabetes, or cytotoxic bioactivities. Recent advances in Aspergillus genomic mining efforts have led to the discovery of the tdiA-E-gene cluster, which is the first genetic locus dedicated to bis-indolylquinone biosynthesis. We have now genetically and biochemically characterized the enzymes TdiA (bis-indolylquinone synthetase) and TdiD (L-tryptophan:phenylpyruvate aminotransferase), which, together, confer biosynthetic abilities for didemethylasterriquinone D to Aspergillus nidulans. This compound is the universal intermediate for all bis-indolylquinones. In this biochemical study of a bis-indolylquinone synthetase and a fungal natural product transaminase, we present a one-pot chemoenzymatic protocol to generate didemethylasterriquinone D in vitro. As TdiA resembles a nonribosomal peptide synthetase, yet catalyzes carbon-carbon-bond formation, we discuss the implications for peptide synthetase chemistry.

Accurate prediction of the Aspergillus nidulans terrequinone gene cluster boundaries using the transcriptional regulator LaeA.

LaeA is a regulatory protein of the genus Aspergillus which controls global expression of secondary metabolism gene clusters. During a previous genome-wide transcriptional profiling screen, numerous novel gene clusters were found to be regulated by LaeA including a cluster required for the production of the antitumor agent terrequinone A. Beginnings and ends of secondary metabolite gene clusters are difficult to predict by gene sequence alone, but sharp demarcation of transcriptional control by LaeA suggested this protein might present a tool to identify cluster ends. To address this possibility in a first test case, we created null mutants of three genes, one regulated and two not regulated by LaeA, lying at the first, sixth and seventh position of a contiguous seven gene region where the third gene in this continuum was already shown to be required for terrequinone A production. Only the gene regulated by LaeA, tdiE, sixth in the series, was required for terrequinone A. In this pilot experiment, these findings experimentally validate LaeA-based predictions of the terrequinone biosynthetic locus boundaries. Further, this study identifies TdiE as a protein of unknown yet essential function for terrequinone A biosynthesis.