Ependymal cells undergo astrocyte-like reactivity in response to neuroinflammation.

Ependymal cells form a specialized brain-cerebrospinal fluid (CSF) interface and regulate local CSF microcirculation. It is becoming increasingly recognized that ependymal cells assume a reactive state in response to aging and disease, including conditions involving hypoxia, hydrocephalus, neurodegeneration, and neuroinflammation. Yet what transcriptional signatures govern these reactive states and whether this reactivity shares any similarities with classical descriptions of glial reactivity (i.e., in astrocytes) remain largely unexplored. Using single-cell transcriptomics, we interrogated this phenomenon by directly comparing the reactive ependymal cell transcriptome to the reactive astrocyte transcriptome using a well-established model of autoimmune-mediated neuroinflammation (MOG35-55 EAE). In doing so, we unveiled core glial reactivity-associated genes that defined the reactive ependymal cell and astrocyte response to MOG35-55 EAE. Interestingly, known reactive astrocyte genes from other CNS injury/disease contexts were also up-regulated by MOG35-55 EAE ependymal cells, suggesting that this state may be conserved in response to a variety of pathologies. We were also able to recapitulate features of the reactive ependymal cell state acutely using a classic neuroinflammatory cocktail (IFNγ/LPS) both in vitro and in vivo. Taken together, by comparing reactive ependymal cells and astrocytes, we identified a conserved signature underlying glial reactivity that was present in several neuroinflammatory contexts. Future work will explore the mechanisms driving ependymal reactivity and assess downstream functional consequences.

Pubococcygeal Line Versus H-line as MR Defecography Reference for Bladder Descent.

INTRODUCTION AND HYPOTHESIS: Magnetic resonance defecography imaging techniques have been used widely to study pelvic floor function and diagnose pelvic organ prolapse (POP). The aim of this study was to investigate the diagnostic accuracy of the H-line to detect bladder descent compared with the current landmark, the pubococcygeal line (PCL). METHODS: In this retrospective cohort study, patients who underwent MR defecography in our medical center and were diagnosed with moderate to severe cystocele by radiological measurements were recruited. One rest image and one maximum evacuation image for each subject were used for the following measurements: bladder base perpendicular distance from the genital hiatus (GH), indicative of clinically significant bladder descent, PCL as the current radiological reference line, and the H-line, or minimal levator hiatus line, indicative of pelvic floor muscle and connective tissue support. Subjects were categorized as having clinically significant cystocele if the "bladder base" reached within 1 cm or lower of the GH (stage II or higher cystocele). A comparison was performed to assess differences and predictive capabilities of the reference lines relative to the GH measure. RESULTS: Seventy subjects were included, 30 with clinically significant bladder descent based on distance to GH. Women with bladder descent were older (64.0 ± 11.8 vs 51.2 ± 15.6, p < 0.001), had increased parity (3 [1-7] vs 2 [0-5], p = 0.009), and had a bladder that descended lower than the H-line at rest (1.9 ± 0.5 vs 2.2 ± 0.4, p = 0.003) and evacuation (-2.4 ± 1.6 vs -0.7 ± 1.1, p < 0.001). Multivariate regression analysis confirmed that age, length of the H-line at evacuation, the perpendicular distances between the H-line and the lowest bladder point at rest, and the PCL to the lowest bladder point at evacuation significantly correlated with bladder descent. Receiver operating characteristic analysis was used to identify a measurement threshold to diagnose clinically significant cystocele for both measurements, bladder base to the H-line: -1.2 (80.0, 72.5) area under the curve (AUC) 0.82, and bladder base PCL: -3.3 (77.8, 79.5) AUC 0.86. CONCLUSION: Our data support the application of using the minimal levator hiatus plane and specifically the H-line as a reliable landmark to diagnose bladder descent using MR defecography imaging.

A systematic comparison between FEBio and PolyFEM for biomechanical systems.

BACKGROUND AND OBJECTIVES: Finite element simulations are widely employed as a non-invasive and cost-effective approach for predicting outcomes in biomechanical simulations. However, traditional finite element software, primarily designed for engineering materials, often encountered limitations in contact detection and enforcement, leading to simulation failure when dealing with complex biomechanical configurations. Currently, a lot of model tuning is required to get physically accurate finite element simulations without failures. This adds significant human interaction to each iteration of a biomechanical model. This study addressed these issues by introducing PolyFEM, a novel finite element solver that guarantees inversion- and intersection-free solutions with completely automatic collision detection. The objective of this research is to validate PolyFEM's capabilities by comparing its results with those obtained from a well-established finite element solver, FEBio. METHODS: To achieve this goal, five comparison scenarios were formulated to assess and validate PolyFEM's performance. The simulations were reproduced using both PolyFEM and FEBio, and the final results were compared. The five comparison scenarios included: (1) reproducing simulations from the FEBio test suite, consisting of static, dynamic, and contact-driven simulations; (2) replicating simulations from the verification paper published alongside the original release of FEBio; (3) a biomechanically based contact problem; (4) creating a custom simulation involving high-energy collisions between soft materials to highlight the difference in collision methods between the two solvers; and (5) performing biomechanical simulations of biting and quasi-stance. RESULTS: We found that PolyFEM was capable of replicating all simulations previously conducted in FEBio. Particularly noteworthy is PolyFEM's superiority in high-energy contact simulations, where FEBio fell short, unable to complete over half of the simulations in Scenario 4. Although some of the simulations required significantly more simulation time in PolyFEM compared to FEBio, it is important to highlight that PolyFEM achieved these results without the need for any additional model tuning or contact declaration. DISCUSSION: Despite being in the early stages of development, PolyFEM currently provides verified solutions for hyperelastic materials that are consistent with FEBio, both in previously published workflows and novel finite element scenarios. PolyFEM exhibited the ability to tackle challenging biomechanical problems where other solvers fell short, thus offering the potential to enhance the accuracy and realism of future finite element analyses.

Sacrum and Coccyx Shape Changes During Pregnancy and After Delivery.

Remodeling of the sacrum and coccyx to accommodate pregnancy and delivery has been hypothesized but not directly quantified. This study aimed to quantify the remodeling of the sacrum and coccyx by comparing midsagittal lengths, angles, curvature, and shape between nulliparous, pregnant, and parous women using both 2 and 3 dimensional measures. Ninety pelvic magnetic resonance images of the pelvis were retrospectively collected and segmented. Twelve length, angle, and curvature measurements were made using definitions from previous literature on the midsagittal plane to define the sacrum, coccyx, and combined sacrum-coccyx shape. These measures were followed by a statistical shape analysis, which returned modes of variation and principal component scores. A separate MANCOVA analysis was conducted for both the 2D and 3D measures. The 2D and 3D analyses agreed that pregnant women had a significantly straighter coccyx and combined sacrum/coccyx than nulliparous (9.1% and 5.6%, respectively) and parous (7.5% and 2.7%, respectively) subjects. All comparisons showed that, on average, a pregnant woman's sacrum and coccyx were significantly straighter than their nulliparous counterparts. Then after delivery, the sacrum/coccyx returned, but not completely back to a more curved configuration.

Quantifying the effect of an endo-vaginal probe on position of the pelvic floor viscera and muscles.

INTRODUCTION AND HYPOTHESIS: Endovaginal ultrasound has long been hypothesized to have a significant effect on locations of what it visualizes. However, little work has directly quantified its effect. This study aimed to quantify it. METHODS: This cross-sectional study consisted of 20 healthy asymptomatic volunteers who underwent both endovaginal ultrasound and MRI. The urethra, vagina, rectum, pelvic floor, and pubic bone were segmented in both ultrasound and MRI using 3DSlicer. Then, using 3DSlicer's transform tool the volumes were rigidly aligned based on the posterior curvature of the pubic bone. The organs were then split into thirds along their long axis to compare their distal, middle, and proximal sections. Using Houdini, we compared the location of the centroid of each of the urethra, vagina, and rectum and the surface-to-surface difference of the urethra and rectum. The anterior curvature of the pelvic floor was also compared. Normality of all variables was assessed by Shapiro-Wilk test. RESULTS: The largest amount of surface-to-surface distance was observed in the proximal region for the urethra and rectum. Across all three organs, the majority of the deviation was in the anterior direction for geometries obtained from ultrasound versus those from MRI. For each subject, the trace defining the midline of the levator plate was more anterior for ultrasound compared to MRI. CONCLUSIONS: While it has often been assumed that placing a probe in the vagina probably distorts the anatomy, this study quantified the distortion and displacement of the pelvic viscera. This allows for better interpretation of clinical and research findings based on this modality.

Effect of Squeeze, Cough, and Strain on Dynamic Urethral Function in Nulligravid Asymptomatic Women: A Cross-Sectional Cohort Study.

IMPORTANCE: In the past, urethral shape, mobility, and urodynamics have been used to retrospectively demonstrate correlations with stress urinary incontinence. Our previous work has shown a relationship between urethral function and shape in symptomatic women. OBJECTIVE: This study aimed to characterize the effect of pelvic floor squeeze and strain maneuvers on urethral shapes and pressure in a cohort of patients without pelvic floor disorders. STUDY DESIGN: In this cross-sectional study, volunteers underwent dynamic pelvic floor ultrasound examination, and a modified urodynamic study. Urethral length, thickness, and proximal and distal swing angles were measured at rest, squeeze, and strain. The midsagittal urethral walls were traced so that a statistical shape model could be performed. Means and standard deviations of imaging and urodynamic measures were calculated. RESULTS: Data from 19 participants were analyzed. On average during squeeze compared with rest, urethral length increased by 6%, thickness decreased by 42% (distal, P < 0.001), 10% (middle), and urethral pressure increased by 14%. Opposite shape changes-length decreased by 10% ( P = 0.001), thickness increased by 57% (distal, P < 0.001), 20% (middle, P < 0.001)-and increased urethral mobility were observed during strain, with larger pressure increases occurring (29%, P < 0.001). Fifty-one percent of the total shape variance described the differences between maneuvers. These differences were statistically different between groups ( P < 0.001 for comparisons, all others P > 0.05). CONCLUSIONS: Dynamic ultrasound and urodynamics allow for the establishment of baseline ranges in urethral metrics (2-dimensional measures, shape, and pressure) and how they are altered during maneuvers. These data can allow for a more objective identification of incontinence via ultrasound and urodynamic testing.

Multimodal measurements of levator bowl volume in nulligravid asymptomatic women: endovaginal ultrasound versus MRI.

INTRODUCTION AND HYPOTHESIS: Measurements of levator bowl volume using advanced imaging, may be predictive of pelvic floor muscle function. The aim of this study was to compare the volume of the levator bowl using both magnetic resonance imaging (MRI) and endovaginal ultrasound (EVU) of healthy asymptomatic women. METHODS: All participants underwent a comprehensive interview including completion of the Pelvic Floor Distress Inventory Questionnaire-20 questionnaire, pelvic examination with a pelvic organ prolapse quantification evaluation, MRI, and EVU. The pelvic floor was segmented using Slicer and the MRI segmentations were trimmed using two methods: soft-tissue landmarks and the field of view (FOV) of the ultrasound volume. The levator bowl volume of the 3D segmented shapes was measured using Blender's 3D printing toolkit. Normality was tested using the Shapiro-Wilks test and comparisons were made using self-paired t tests. RESULTS: The final analysis included 19 patients. Levator bowl volume measured via MRI was larger than that measured in EVU (46.1 ± 7.9 cm3 vs 27.4 ± 5.9 cm3, p<0.001). Reducing the FOV of the MRI to that of EVU caused the MRI volume to be much closer to the EVU volume (35.5 ± 3.3 cm3 vs 27.4 ± 5.9 cm3, p<0.001); however, it remained significantly larger. CONCLUSION: Levator bowl volume measured using MRI was larger than that measured using EVU no matter the method of delineation of the levator muscles. Although EVU is safe, cheap, and easy to perform, it captures a smaller volume of levator bowel than MRI.

Quantifying the physiologic motions of the pelvic viscera during evacuation in nulligravid asymptomatic women.

INTRODUCTION AND HYPOTHESIS: Pelvic organ prolapse (POP) is often diagnosed during an in-office examination, which looks for motion of the vaginal wall while performing a strain maneuver. It is believed that the pelvic organs in adequately supported women are relatively stationary. This study was aimed at investigating the physiological displacements of pelvic organs using MR defecography. METHODS: This prospective cohort study included 19 volunteers. Midsagittal slices representing rest and the maximum movement of the posterior vaginal fornix during three maneuvers were identified. Normalized axes for analysis were defined as the x' (line connecting the inferior-posterior-most point on the pubic symphysis to the anterior edge of the sacrococcygeal joint) and the y' (line orthogonal to the x axis that passed through the sacral promontory). The positions of the posterior vaginal fornix, mid-vagina, bladder neck, anorectal junction, and hymen were recorded. These subjects were then analyzed using the current radiological grading system of POP to determine any overlap between asymptomatic subjects and diagnostic ranges of POP. RESULTS: Evacuation caused the most motion in the landmarks. The majority of the motion of the landmarks was along the y axis. The posterior vaginal fornix experienced significant descent (125% of the initial distance) without much anterior-posterior translation (7% of the initial distance) during defecation. All landmarks experienced similar trends. CONCLUSIONS: We have shown that there is significant rotational motion of the pelvic organs around the pubic bone in adequately supported women. This motion when described using radiological grading is likely to be considered mild or moderate prolapse, which may contribute to overdiagnosis of POP.

Synovial fluid mesenchymal progenitor cells from patients with juvenile idiopathic arthritis demonstrate limited self-renewal and chondrogenesis.

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases affecting joints with a prevalence of one in a thousand children. There is a growing body of literature examining the use of mesenchymal stem/progenitor cells (MPCs) for the treatment of adult and childhood arthritis, however, we still lack a clear understanding of how these MPC populations are impacted by arthritic disease states and how this could influence treatment efficacy. In the current study we examined the immunophenotyping, self-renewal ability and chondrogenic capacity (in vitro and in vivo) of synovial derived MPCs from normal, JIA and RA joints. Synovial MPCs from JIA patients demonstrated reduced self-renewal ability and chondrogenic differentiation capacity. Furthermore, they did not induce cartilage regeneration when xenotransplanted in a mouse cartilage injury model. Synovial MPCs from JIA patients are functionally compromised compared to MPCs from normal and/or RA joints. The molecular mechanisms behind this loss of function remain elusive. Further study is required to see if these cells can be re-functionalized and used in cell therapy strategies for these JIA patients, or if allogenic approaches should be considered.

pH-sensitive packaging of cationic particles by an anionic block copolymer shell.

Cationic non-viral vectors show great potential to introduce genetic material into cells, due to their ability to transport large amounts of genetic material and their high synthetic versatility. However, designing materials that are effective without showing toxic effects or undergoing non-specific interactions when applied systemically remains a challenge. The introduction of shielding polymers such as polyethylene glycol (PEG) can enhance biocompatibility and circulation time, however, often impairs transfection efficiency. Herein, a multicomponent polymer system is introduced, based on cationic and hydrophobic particles (P(nBMA46-co-MMA47-co-DMAEMA90), (PBMD)) with high delivery performance and a pH-responsive block copolymer (poly((N-acryloylmorpholine)-b-(2-(carboxy)ethyl acrylamide)) (P(NAM72-b-CEAm74), PNC)) as shielding system, with PNAM as alternative to PEG. The pH-sensitive polymer design promotes biocompatibility and excellent stability at extracellular conditions (pH 7.4) and also allows endosomal escape and thus high transfection efficiency under acidic conditions. PNC shielded particles are below 200 nm in diameter and showed stable pDNA complexation. Further, interaction with human erythrocytes at extracellular conditions (pH 7.4) was prevented, while acidic conditions (pH 6) enabled membrane leakage. The particles demonstrate transfection in adherent (HEK293T) as well as difficult-to-transfect suspension cells (K-562), with comparable or superior efficiency compared to commercial linear poly(ethylenimine) (LPEI). Besides, the toxicity of PNC-shielded particles was significantly minimized, in particular in K-562 cells and erythrocytes. In addition, a pilot in vivo experiment on bone marrow blood cells of mice that were injected with PNC-shielded particles, revealed slightly enhanced cell transfection in comparison to naked pDNA. This study demonstrates the applicability of cationic hydrophobic polymers for transfection of adherent and suspension cells in culture as well as in vivo by co-formulation with pH-responsive shielding polymers, without substantially compromising transfection performance.

Urethral support in female urinary continence part 2: a computational, biomechanical analysis of Valsalva.

INTRODUCTION AND HYPOTHESIS: In Part 1, we observed urethral mechanics during Valsalva that oppose current continence theories. In this study, we utilize a finite element model to elucidate the role of supportive tissues on the urethra during Valsalva. By determining the sensitivity of urethral motion and deformations to variations in tissue stiffnesses, we formulate new hypotheses regarding mechanisms of urethral passive closure. METHODS: Anatomy was segmented from a nulliparous, continent woman at rest. The model was tuned such that urethral motion during Valsalva matched that observed in that patient. Urethra and surrounding tissue material properties were varied using Latin hypercube sampling to perform a sensitivity analysis. As in Part 1, urethral length, proximal and distal swinging, and shape parameters were measured at peak Valsalva for 50 simulations, and partial rank correlation coefficients were calculated between all model inputs and outputs. Cumulative influence factors determined which tissue properties were meaningfully influential (≥ 0.5). RESULTS: The material properties of the urethra, perineal membrane, bladder, and paraurethral connective tissues meaningfully influenced urethral motion, deformation, and shape. Reduction of the urethral stiffness and/or the perineal membrane soft constraint resulted in simulated urethral motions and shapes associated with stress urinary incontinence in Part 1. CONCLUSIONS: The data from Parts 1 and 2 suggest that connective tissues guide the controlled swinging motion and deformation of the urethra needed for passive closure during Valsalva. The swinging and kinking quantified in Part 1 and simulated in Part 2 are inconsistent with current continence theories.

Core-crosslinked, temperature- and pH-responsive micelles: design, physicochemical characterization, and gene delivery application.

Stimuli-responsive block copolymer micelles can provide tailored properties for the efficient delivery of genetic material. In particular, temperature- and pH-responsive materials are of interest, since their physicochemical properties can be easily tailored to meet the requirements for successful gene delivery. Within this study, a stimuli-responsive micelle system for gene delivery was designed based on a diblock copolymer consisting of poly(N,N-diethylacrylamide) (PDEAm) as a temperature-responsive segment combined with poly(aminoethyl acrylamide) (PAEAm) as a pH-responsive, cationic segment. Upon temperature increase, the PDEAm block becomes hydrophobic due to its lower critical solution temperature (LCST), leading to micelle formation. Furthermore, the monomer 2-(pyridin-2-yldisulfanyl)ethyl acrylate (PDSAc) was incorporated into the temperature-responsive PDEAm building block enabling disulfide crosslinking of the formed micelle core to stabilize its structure regardless of temperature and dilution. The cloud points of the PDEAm block and the diblock copolymer were investigated by turbidimetry and fluorescence spectroscopy. The temperature-dependent formation of micelles was analyzed by dynamic light scattering (DLS) and elucidated in detail by an analytical ultracentrifuge (AUC), which provided detailed insights into the solution dynamics between polymers and assembled micelles as a function of temperature. Finally, the micelles were investigated for their applicability as gene delivery vectors by evaluation of cytotoxicity, pDNA binding, and transfection efficiency using HEK293T cells. The investigations showed that core-crosslinking resulted in a 13-fold increase in observed transfection efficiency. Our study presents a comprehensive investigation from polymer synthesis to an in-depth physicochemical characterization and biological application of a crosslinked micelle system including stimuli-responsive behavior.

The impact of anionic polymers on gene delivery: how composition and assembly help evading the toxicity-efficiency dilemma.

Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymerization. The copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphology, interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addition of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.

Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

OBJECTIVES: Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. METHODS: Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. RESULTS: Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified. CONCLUSIONS: Understanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles.

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

Solely aqueous formulation of hydrophobic cationic polymers for efficient gene delivery.

Cationic polymers are promising gene delivery vectors due to their ability to bind and protect genetic material. The introduction of hydrophobic moieties into cationic polymers can further improve the vector efficiency, but common formulations of hydrophobic polymers involve harsh conditions such as organic solvents, impairing intactness and loading efficiency of the genetic material. In this study, a mild, aqueous formulation method for the encapsulation of high amounts of genetic material is presented. A well-defined pH-responsive hydrophobic copolymer, i.e. poly((n-butylmethacrylate)-co-(methylmethacrylate)-co-(2-(dimethylamino) ethylmethacrylate)), (PBMD) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Exploiting the pH-dependent solubility behavior of the polymer, stable pDNA loaded nanoparticles were prepared and characterized using analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). This novel formulation approach showed high transfection efficiencies in HEK293T cells, while requiring 5- to 10-fold less pDNA compared to linear polyethylenimine (LPEI), in particular at short incubation times and in serum-containing media. Furthermore, the formulation was successfully adopted for siRNA and mRNA encapsulation and the commercially approved polymer Eudragit® E(PO/100). Overall, the aqueous formulation approach, accompanied by a tailor-made hydrophobic polymer and detailed physicochemical and application studies, led to improved gene delivery vectors with high potential for further applications.

Carprofen elicits pleiotropic mechanisms of bactericidal action with the potential to reverse antimicrobial drug resistance in tuberculosis.

BACKGROUND: The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has elevated TB to a major global health priority. Repurposing drugs developed or used for other conditions has gained special attention in the current scenario of accelerated drug development for several global infectious diseases. In a similar effort, previous studies revealed that carprofen, a non-steroidal anti-inflammatory drug, selectively inhibited the growth of replicating, non-replicating and MDR clinical isolates of M. tuberculosis. OBJECTIVES: We aimed to reveal the whole-cell phenotypic and transcriptomic effects of carprofen in mycobacteria. METHODS: Integrative molecular and microbiological approaches such as resazurin microtitre plate assay, high-throughput spot-culture growth inhibition assay, whole-cell efflux inhibition, biofilm inhibition and microarray analyses were performed. Analogues of carprofen were also synthesized and assessed for their antimycobacterial activity. RESULTS: Carprofen was found to be a bactericidal drug that inhibited mycobacterial drug efflux mechanisms. It also restricted mycobacterial biofilm growth. Transcriptome profiling revealed that carprofen likely acts by targeting respiration through the disruption of membrane potential. The pleiotropic nature of carprofen's anti-TB action may explain why spontaneous drug-resistant mutants could not be isolated in practice. CONCLUSIONS: This immunomodulatory drug and its chemical analogues have the potential to reverse TB antimicrobial drug resistance, offering a swift path to clinical trials of novel TB drug combinations.

Antimicrobial and Efflux Pump Inhibitory Activity of Carvotacetones from Sphaeranthus africanus Against Mycobacteria.

Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.

Tuning of endosomal escape and gene expression by functional groups, molecular weight and transfection medium: a structure-activity relationship study.

The use of genetic material by non-viral transfer systems is still in its initial stages, but there are high expectations for the development of targeted therapies. However, nucleic acids cannot enter cells without help, they must be well protected to prevent degradation and overcome a variety of biological barriers, the endosomal barrier being one of the greatest cellular challenges. Herein, the structure-property-relationship was investigated in detail, using well-defined polymers. Polyacrylamides were synthesized via RAFT polymerization resulting in a polymer library of (i) different cationic groups as aminoethyl acrylamide (AEAm), dimethylaminoethyl acrylamide (DMAEAm), dimethylaminopropyl acrylamide (DMAPAm) and guanidinopropyl acrylamide (GPAm); (ii) different degree of polymerization; and investigated (iii) in different cell culture settings. The influence of molar mass and cationic moiety on complex formation with pDNA, cytotoxicity and transfection efficiency of the polymers were investigated. The systematic approach identified a pH-independent guanidinium-containing homopolymer (PGPAm89) as the polymer with the highest transfection efficiency and superior endosomal release under optimal conditions. Since PGPAm89 is not further protonated inside endosomes, common escape theories appear unsuitable. Therefore, the interaction with bis(monoacryloylglycerol)phosphate, a lipid specific for endosomal vesicles, was investigated. Our research suggests that the interactions between amines and lipids may be more relevant than anticipated.

Flavonoids as Novel Efflux Pump Inhibitors and Antimicrobials Against Both Environmental and Pathogenic Intracellular Mycobacterial Species.

Therapeutic treatment options for opportunistic non-tuberculous mycobacterial (NTM) infection and/or serious mycobacterial infections such as tuberculosis (TB) and leprosy are limited due to the spread of antimicrobial resistance mechanism. Plant-derived natural compounds as prospective efflux pump inhibitors may present a promising adjunct to conventional chemotherapy by enhancing mycobacterial susceptibility to antibiotics. This study served to evaluate the antimicrobial and resistance-modifying profile of a range of plant-derived flavonoids against the mycobacterial model strains: M. smegmatis, M. aurum, and M. bovis BCG. The minimum inhibitory concentrations (MICs) of the compounds against the mycobacterial strains were determined using both agar dilution and broth dilution assays, while their efflux inhibitory activity was investigated via an ethidium bromide-based fluorometric assay. All compounds were screened for their synergistic effects with ethidium bromide (EtBr) and rifampicin (RIF) against M. smegmatis. Skullcapflavone II (5,2'-dihydroxy-6,7,8,6'-tetramethoxyflavone, 1) exerted potent antimicrobial activity against M. aurum and M. bovis BCG and considerably increased the susceptibility of M. smegmatis to EtBr and RIF. Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone, 2) was determined to be the most potent efflux-inhibitor in M. aurum and M. smegmatis. However, a connection between strong modulatory and putative efflux activity of the compounds could not be observed. Nevertheless, the results highlight two polymethoxyflavones, skullcapflavone II and nobiletin, with potent antimycobacterial and antibiotic resistance modulating activities as valuable adjuvants in anti-mycobacterial therapies.