Surface display of functional moieties on extracellular vesicles using lipid anchors.

Extracellular vesicles (EVs) are efficient natural vehicles for intercellular communication and are under extensive investigation for the delivery of diverse therapeutics including small molecule drugs, nucleic acids, and proteins. To understand the mechanisms behind the biological activities of EVs and develop EV therapeutics, it's fundamental to track EVs and engineer EVs in a customized manner. In this study, we identified, using single-vesicle flow cytometry and microscopy, the lipid DOPE (dioleoyl phosphatidyl ethanolamine) as an efficient anchor for isolated EVs. Notably, DOPE associated with EVs quickly, and the products remained stable under several challenging conditions. Moreover, conjugating fluorophores, receptor-targeting peptides or albumin-binding molecules with DOPE enabled tracking the cellular uptake, enhanceing the cellular uptake or extending the circulation time in mice of engineered EVs , respectively. Taken together, this study reports an efficient lipid anchor for exogenous engineering of EVs and further showcases its versatility for the functionalization of EVs.

Performance Modeling of Spherical Capsules during Mixing of Self-Consolidating Concrete.

Autonomous healing is a very promising technique in self-healing concrete systems. For capsules to achieve their anticipated performance, they should be able to survive the harsh mixing conditions of concrete, yet rupture upon concrete cracking. At present, there are no standard test methods, either experimental or analytical, for determining the capsule survival rate during concrete mixing. This study investigates the correlation between the capsules' shell properties, concrete rheological properties, the capsules' external forces, and capsule survival rate during concrete mixing. Finite element and statistical modeling techniques were employed to evaluate the capsule performance and predict the survival rate of capsules during concrete mixing, with 68% confidence. The results revealed that the capsules' survivability during concrete mixing is highly influenced by the capsule's radius-to-thickness ratio, the rheological properties of the fresh concrete, the average-paste-thickness (APT) of the concrete mix, the aggregate content and angularity, and the speed of the mixer. In brief, capsules with a radius-to-thickness ratio between 30 and 45 are likely to survive concrete mixing and yet still rupture upon concrete cracking.

Cell-specific targeting of extracellular vesicles through engineering the glycocalyx.

Extracellular vesicles (EVs) are promising carriers for the delivery of a variety of chemical and biological drugs. However, their efficacy is limited by the lack of cellular specificity. Available methods to improve the tissue specificity of EVs predominantly rely on surface display of proteins and peptides, largely overlooking the dense glycocalyx that constitutes the outermost layer of EVs. In the present study, we report a reconfigurable glycoengineering strategy that can endogenously display glycans of interest on EV surface. Briefly, EV producer cells are genetically engineered to co-express a glycosylation domain (GD) inserted into the large extracellular loop of CD63 (a well-studied EV scaffold protein) and fucosyltransferase VII (FUT7) or IX (FUT9), so that the engineered EVs display the glycan of interest. Through this strategy, we showcase surface display of two types of glycan ligands, sialyl Lewis X (sLeX) and Lewis X, on EVs and achieve high specificity towards activated endothelial cells and dendritic cells, respectively. Moreover, the endothelial cell-targeting properties of sLeX-EVs were combined with the intrinsic therapeutic effects of mesenchymal stem cells (MSCs), leading to enhanced attenuation of endothelial damage. In summary, this study presents a reconfigurable glycoengineering strategy to produce EVs with strong cellular specificity and highlights the glycocalyx as an exploitable trait for engineering EVs.

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Performance of Capsules in Self-Healing Cementitious Material.

Encapsulation is a very promising technique that is being explored to enhance the autonomous self-healing of cementitious materials. However, its success requires the survival of self-healing capsules during mixing and placing conditions, while still trigger the release of a healing agent upon concrete cracking. A review of the literature revealed discontinuities and inconsistencies in the design and performance evaluation of self-healing cementitious material. A finite element model was developed to study the compatibility requirements for the capsule and the cementing material properties while the cement undergoes volume change due to hydration and/or drying. The FE results have provided insights into the observed inconsistencies and the importance of having capsules' mechanical and geometrical properties compatible with the cementitious matrix.

Probability Characteristics of a Crack Hitting Spherical Healing Agent Particles: Application to a Self-Healing Cementitious System.

A geometric model is developed to statistically study the probability characteristics of crack intersecting self-healing capsules with a structured random distribution in a cement paste mix. To evaluate the probability of a crack intersecting encapsulated particles, the fill ratio of the crack, and the depth of the first-hit capsule, Monte Carlo simulations are performed. The variables are the crack geometry, i.e., width, length, depth, orientation, skewness, and so on; the size and mass fraction of healing capsules; and the agglomeration of capsules. Models based on statistical analyses for hit probability Ph, crack fill ratio Rf-95 at 95% confidence level, and first hit depth h0-95 at 95% confidence level are expressed as functions of capsule size and mass fraction, as well as crack geometry. The model assumptions and results are evaluated using data reported in the literature. The data include results from experimental and theoretical studies.

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Imaging the External Ear: Practical Approach to Normal and Pathologic Conditions.

The external ear (EE) is an osseous-cartilaginous structure that extends from the auricle to the tympanic membrane. It is divided into two parts: the auricle (or pinna) and the external auditory canal (EAC). Given the ease of access to the EE, imaging studies are not always needed to make a diagnosis. However, when lesions block visual access to areas deep to the EE abnormality, complications are suspected, or there is lack of response to treatment, imaging becomes essential. A basic understanding of the embryologic development and knowledge of the anatomy of the auricle and EAC are useful for accurate diagnosis of EE lesions. Congenital, traumatic, inflammatory, neoplastic, and vascular conditions can affect the EE. An overview of the anatomy and embryologic development of the EE is presented, with discussion and illustrations of common and uncommon conditions that affect EE structures and a focus on the CT and MRI features that are of interest to radiologists. CT is usually the first diagnostic modality used to evaluate the EAC and is the superior method for demonstrating bone changes. MRI provides excellent tissue characterization and enables one to better define lesion extension and perineural tumor spread. In addition, a flowchart to facilitate the differential diagnosis of EE abnormalities is provided. Online supplemental material is available for this article. ©RSNA, 2022.

High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.

Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50-150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)-labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence-conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application-specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Preparation and Isolation of siRNA-Loaded Extracellular Vesicles.

RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAi-mediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent reports have suggested that extracellular vesicles (EVs) form a natural transport system through which biomolecules, including RNA, is exchanged between cells. Therefore, EVs are increasingly being considered as potential therapeutic siRNA delivery systems.In this chapter we describe a method for preparing siRNA-loaded EVs, including a robust, scalable method to isolate them from cell culture supernatants.

Pseudoradial Nerve Palsy Caused by Acute Ischemic Stroke.

Pseudoperipheral palsy has been used to characterize isolated monoparesis secondary to stroke. Isolated hand nerve palsy is a rare presentation for acute cerebral stroke. Our patient presented with clinical features of typical peripheral radial nerve palsy and a normal computed tomography scan of the head, which, without a detailed history and neurological examination, could have been easily misdiagnosed as a peripheral nerve lesion deferring further investigation for a stroke. We stress the importance of including cerebral infarction as a critical differential diagnosis in patients presenting with sensory-motor deficit in an isolated peripheral nerve pattern. A good history and physical exam can differentiate stroke from peripheral neuropathy as the cause of radial nerve palsy.

Optimization of permeate flux produced by solar energy driven membrane distillation process using central composite design approach.

Membrane distillation (MD) is considered as a relatively high-energy requirement. To overcome this drawback, it is recommended to couple the MD process with solar energy as the renewable energy source in order to provide heat energy required to optimize its performance to produce permeate flux. In the present work, an original solar energy driven direct contact membrane distillation (DCMD) pilot plant was built and tested under actual weather conditions at Jeddah, KSA, in order to model and optimize permeate flux. The dependency of permeate flux on various operating parameters such as feed temperature (46.6-63.4°C), permeate temperature (6.6-23.4°C), feed flow rate (199-451L/h) and permeate flow rate (199-451L/h) was studied by response surface methodology based on central composite design approach. The analysis of variance (ANOVA) confirmed that all independent variables had significant influence on the model (where P-value <0.05). The high coefficient of determination (R(2) = 0.9644 and R(adj)(2) = 0.9261) obtained by ANOVA demonstrated good correlation between experimental and predicted values of the response. The optimized conditions, determined using desirability function, were T(f) = 63.4°C, Tp = 6.6°C, Q(f) = 451L/h and Q(p) = 451L/h. Under these conditions, the maximum permeate flux of 6.122 kg/m(2).h was achieved, which was close to the predicted value of 6.398 kg/m(2).h.

Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER.

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.

LAPAROSCOPIC MANAGEMENT OF DISTAL URETERIC STONE IN BILHARZIAL URETER: RESULTS OF A SINGLE CENTER PROSPECTIVE STUDY.

No doubt, Bilharzial ureters are complicated by distal stricture due to precipitation of Bilharzial ova in distal ureter. These cases are associated with poorly functioning and grossly hydronephroic kidneys that hinder endoscopic manipulation of the coexistent distal, high burden, long standing impacted stones. Thus, laparoscopic uretrolithotomy was performed in 51 bilharzial patients with distal ureteric stones 4 trocars were used. The ureter was opened directly over the stone and the stone was extracted. A double-J stent was inserted into the ureter which was closed by 4-0 polyglactin running suture. The results showed that among 51 cases 33 males and 18 females; the mean age was 40.13 years. the mean stone size was 2.73 cm. Conversion to open surgery was in only one case; the mean operative time 92.05 (range 75-120 minutes); postoperative pain score ranged from 20 to 60, the mean number of PO analgesic request was 1.72 (range 1-3); it was once in 21, twice in 23 and thrice in 7 cases. Hospital stay ranged from 2 to 5 with a mean of 2.74 days; total duration of follow up ranged from 7 to 12 with a mean of 9.68. Stone recurrence reported in 4 cases; ureteric stricture reported in 2 cases. Stone free rate was reported to be 100%.

Laparoscopic management of distal ureteric stones in a bilharzial ureter: Results of a single-centre prospective study.

OBJECTIVE: To determine the efficacy and safety of the laparoscopic management of an impacted distal ureteric stone in a bilharzial ureter, as bilharzial ureters are complicated by distal stricture caused by the precipitation of bilharzial ova in the distal ureter. These cases are associated with poorly functioning and grossly hydronephrotic kidneys that hinder the endoscopic manipulation of the coexistent distal high burden of, and long-standing, impacted stones. PATIENTS AND METHODS: We used laparoscopic ureterolithotomy, with four trocars, to manage 51 bilharzial patients (33 men and 18 women; mean age 40.13 years) with distal ureteric stones. The ureter was opened directly over the stone and the stone was extracted. A JJ stent was inserted into the ureter, which was then closed with a 4-0 polyglactin running suture. RESULTS: The mean stone size was 2.73 cm. Conversion to open surgery was required in only one patient. The mean operative duration was 92 min, the postoperative pain score was 20-60, the mean (range) number of analgesic requests after surgery was 1.72 (1-3), comprising once in 21 patients, twice in 23 and thrice in seven. The mean hospital stay was 2.74 days, and the total duration of follow-up was 7-12 months. The stone recurred in four patients and a ureteric stricture was reported in two. All patients were rendered stone-free. CONCLUSION: Laparoscopy is a safe and effective minimally invasive procedure for distal ureteric stones in a bilharzial ureter with hydronephrosis.

Therapeutic Potential of Multipotent Mesenchymal Stromal Cells and Their Extracellular Vesicles.

The therapeutic potential of mesenchymal stromal cells (MSCs) is evident by the number of new and ongoing trials targeting an impressive variety of conditions. In bone and cartilage repair, MSCs are expected to replace the damaged tissue, while in other therapies they modulate a therapeutic response by the secretion of bioactive molecules. MSCs possess a phenotypic plasticity and harbor an arsenal of bioactive molecules that can be released upon sensing signals in the local milieu either directly or packaged in extracellular vesicles (EVs). The reported paracrine effects comprise many of the important functions of MSCs, including supporting hematopoietic stem cells in the bone marrow, promoting angiogenesis, and modulating the immune system. The major drawback in MSC therapy is the incomplete understanding of cell fate following systemic administration as well as the mechanisms by which these cells correct disease. In this review we discuss what is known about MSC engraftment, hemocompatibility, and immunomodulation, as well as the potential of bringing the MSC-EV field toward a clinical translation.

Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides.

Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, "ZEN™") to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2'-O-methyl (2'OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2'OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2'OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2'OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

A three-step approach for the derivation and validation of high-performing predictive models using an operational dataset: congestive heart failure readmission case study.

BACKGROUND: The aim of this study was to propose an analytical approach to develop high-performing predictive models for congestive heart failure (CHF) readmission using an operational dataset with incomplete records and changing data over time. METHODS: Our analytical approach involves three steps: pre-processing, systematic model development, and risk factor analysis. For pre-processing, variables that were absent in >50% of records were removed. Moreover, the dataset was divided into a validation dataset and derivation datasets which were separated into three temporal subsets based on changes to the data over time. For systematic model development, using the different temporal datasets and the remaining explanatory variables, the models were developed by combining the use of various (i) statistical analyses to explore the relationships between the validation and the derivation datasets; (ii) adjustment methods for handling missing values; (iii) classifiers; (iv) feature selection methods; and (iv) discretization methods. We then selected the best derivation dataset and the models with the highest predictive performance. For risk factor analysis, factors in the highest-performing predictive models were analyzed and ranked using (i) statistical analyses of the best derivation dataset, (ii) feature rankers, and (iii) a newly developed algorithm to categorize risk factors as being strong, regular, or weak. RESULTS: The analysis dataset consisted of 2,787 CHF hospitalizations at University of Utah Health Care from January 2003 to June 2013. In this study, we used the complete-case analysis and mean-based imputation adjustment methods; the wrapper subset feature selection method; and four ranking strategies based on information gain, gain ratio, symmetrical uncertainty, and wrapper subset feature evaluators. The best-performing models resulted from the use of a complete-case analysis derivation dataset combined with the Class-Attribute Contingency Coefficient discretization method and a voting classifier which averaged the results of multi-nominal logistic regression and voting feature intervals classifiers. Of 42 final model risk factors, discharge disposition, discretized age, and indicators of anemia were the most significant. This model achieved a c-statistic of 86.8%. CONCLUSION: The proposed three-step analytical approach enhanced predictive model performance for CHF readmissions. It could potentially be leveraged to improve predictive model performance in other areas of clinical medicine.

First use of a drug-eluting balloon in the treatment of acute renal artery occlusion and in-stent restenosis.

In-stent restenosis in a renal artery (RA) of a solitary functioning kidney is a serious complication of RA stenting. Drug-eluting balloons (DEB) have emerged as a novel way to manage restenosis. In this paper, the authors reported the first use of a DEB in the treatment of severe in-stent restenosis and thrombosis of a drug-eluting stent deployed in a RA. The patient presented with oligo-anuria and a serum creatinine (Scr) of 9 mg/dL that improved back to baseline of 2 mg/dL after the successful procedure. The optimal use of DEB in similar cases will have to be determined by larger clinical trials.