One year follow-up on a randomized study investigating serratus anterior muscle and pectoral nerves type I block to reduced neuropathic pain descriptors after mastectomy.

Breast cancer is the second most common diagnosed type of cancer in women. Chronic neuropathic pain after mastectomy occurs frequently and is a serious health problem. In our previous single-center, prospective, randomized controlled clinical study, we demonstrated that the combination of serratus anterior plane block (SAM) and pectoral nerve block type I (PECS I) with general anesthesia reduced acute postoperative pain. The present report describes a prospective follow-up study of this published study to investigate the development of chronic neuropathic pain 12 months after mastectomy by comparing the use of general anesthesia alone and general anesthesia with SAM + PECS I. Additionally, the use of analgesic medication, quality of life, depressive symptoms, and possible correlations between plasma levels of interleukin (IL)-1 beta, IL-6, and IL-10 collected before and 24 h after surgery as predictors of pain and depression were evaluated. The results showed that the use of SAM + PECS I with general anesthesia reduced numbness, hypoesthesia to touch, the incidence of patients with chronic pain in other body regions and depressive symptoms, however, did not significantly reduce the incidence of chronic neuropathic pain after mastectomy. Additionally, there was no difference in the consumption of analgesic medication and quality of life. Furthermore, no correlation was observed between IL-1 beta, IL-6, and IL-10 levels and pain and depression. The combination of general anesthesia with SAM + PECS I reduced the occurrence of specific neuropathic pain descriptors and depressive symptoms. These results could promote the use of SAM + PECS I blocks for the prevention of specific neuropathic pain symptoms after mastectomy.Registration of clinical trial: The Research Ethics Board of the Hospital Sirio-Libanes/Brazil approved the study (CAAE 48721715.0.0000.5461). This study is registered at Registro Brasileiro de Ensaios Clinicos (ReBEC), and ClinicalTrials.gov, Identifier: NCT02647385.

Population Coding of Capsaicin Concentration by Sensory Neurons Revealed Using Ca2+ Imaging of Dorsal Root Ganglia Explants from Adult pirt-GCaMP3 Mouse.

BACKGROUND/AIMS: Nociceptors detect noxious capsaicin (CAPS) via the transient receptor potential vanilloid 1 (TRPV1) ion channel, but coding mechanisms for relaying CAPS concentration [CAPS] remain obscure. Prolonged (up to 1h.) exposure to CAPS is used clinically to desensitise sensory fibres for treatment of neuropathic pain, but its signalling has typically been studied in cultures of dissociated sensory neurons employing low cell numbers and very short exposure times. Thus, it was pertinent to examine responses to longer CAPS exposures in large populations of adult neurons. METHODS: Confocal fluorescence microscopy was used to monitor the simultaneous excitation by CAPS of neuronal populations in intact L3/4 dorsal root ganglia (DRG) explants from adult pirt-GCaMP3 mice that express a cytoplasmic, genetically-encoded Ca2+ sensor in almost all primary sensory neurons. Peak analysis was performed using GraphPad Prism 9 to deconstruct the heterogenous and complex fluorescence signals observed into informative, readily-comparable measurements: number of signals, their lag time, maximum intensity relative to baseline (Max.) and duration. RESULTS: Exposure for 5 min. to CAPS activated plasmalemmal TRPV1 and led to increased fluorescence due to Ca2+ entry into DRG neurons (DRGNs), as it was prevented by capsazepine or removal of extracellular Ca2+. Increasing [CAPS] (0.3, 1 and 10 µM, respectively) evoked signals from more neurons (123, 275 and 390 from 5 DRG) with shorter average lag (6.4 ± 0.4, 3.3 ± 0.2 and 1.9 ± 0.1 min.) and longer duration (1.4 ± 0.2, 2.9 ± 0.2 and 4.8 ± 0.3 min.). Whilst raising [CAPS] produced a modest augmentation of Max. for individual neurons, those with large increases were selectively expedited; this contributed to a faster onset and higher peak of cumulative fluorescence for an enlarged responding neuronal population. CAPS caused many cells to fluctuate between high and low levels of fluorescence, with consecutive pulses increasing Max. and duration especially when exposure was extended from 5 to 20 min. Such signal facilitation counteracted tachyphylaxis, observed upon repeated exposure to 1 µM CAPS, preserving the cumulative fluorescence over time (signal density) in the population. CONCLUSION: Individual neurons within DRG differed extensively in the dynamics of response to CAPS, but systematic changes elicited by elevating [CAPS] increased signal density in a graded manner, unveiling a possible mechanism for population coding of responses to noxious chemicals. Signal density is sustained during prolonged and repeated exposure to CAPS, despite profound tachyphylaxis in some neurons, by signal facilitation in others. This may explain the burning sensation that persists for several hours when CAPS is used clinically.

Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse.

Nociceptors sense hazards via plasmalemmal cation channels, including transient receptor potential vanilloid 1 (TRPV1). Nerve growth factor (NGF) sensitises TRPV1 to capsaicin (CAPS), modulates nociceptor excitability and induces thermal hyperalgesia, but cellular mechanisms remain unclear. Confocal microscopy was used to image changes in intracellular Ca2+ concentration ([Ca2+]i) across neuronal populations in dorsal root ganglia (DRG) explants from pirt-GCaMP3 adult mice, which express a fluorescent reporter in their sensory neurons. Raised [Ca2+]i was detected in 84 neurons of three DRG explants exposed to NGF (100 ng/mL) and most (96%) of these were also excited by 1 µM CAPS. NGF elevated [Ca2+]i in about one-third of the neurons stimulated by 1 µM CAPS, whether applied before or after the latter. In neurons excitable by NGF, CAPS-evoked [Ca2+]i signals appeared significantly sooner (e.g., respective lags of 1.0 ± 0.1 and 1.9 ± 0.1 min), were much (>30%) brighter and lasted longer (6.6 ± 0.4 vs. 3.9 ± 0.2 min) relative to those non-responsive to the neurotrophin. CAPS tachyphylaxis lowered signal intensity by ~60% but was largely prevented by NGF. Increasing CAPS from 1 to 10 µM nearly doubled the number of cells activated but only modestly increased the amount co-activated by NGF. In conclusion, a sub-population of the CAPS-sensitive neurons in adult mouse DRG that can be excited by NGF is more sensitive to CAPS, responds with stronger signals and is further sensitised by transient exposure to the neurotrophin.

Fish skin pigmentation in aquaculture: The influence of rearing conditions and its neuroendocrine regulation.

Skin pigmentation pattern is a species-specific characteristic that depends on the number and the spatial combination of several types of chromatophores. This feature can change during life, for example in the metamorphosis or reproductive cycle, or as a response to biotic and/or abiotic environmental cues (nutrition, UV incidence, surrounding luminosity, and social interactions). Fish skin pigmentation is one of the most important quality criteria dictating the market value of both aquaculture and ornamental species because it serves as an external signal to infer its welfare and the culture conditions used. For that reason, several studies have been conducted aiming to understand the mechanisms underlying fish pigmentation as well as the influence exerted by rearing conditions. In this context, the present review focuses on the current knowledge on endocrine regulation of fish pigmentation as well as on the aquaculture conditions affecting skin coloration. Available information on Iberoamerican fish species cultured is presented.

Infective Aortic Valve Endocarditis Causing Embolic Consecutive ST-Elevation Myocardial Infarctions.

ST-elevation myocardial infarction (STEMI) is a rare and potentially fatal complication of infective endocarditis. We report the ninth case of embolic native aortic valve infective endocarditis causing STEMI and the first case to describe consecutive embolisms leading to infarctions of separate coronary territories. Through examination of this case in the context of the previous eight similar documented cases in the past, we find that infective endocarditis of the aortic valve can and frequently affect more than a single myocardial territory and can occur consecutively. Further, current treatment modalities for embolic infective endocarditis causing acute myocardial infarction are limited and unproven. This index case illustrates the potential severity of complications and the challenges in developing standardized management for such patients.

Morphological relationship between GnIH and GnRH neurons in the brain of the neotropical cichlid fish Cichlasoma dimerus.

Reproduction is regulated by the hypothalamic-pituitary-gonadal axis. The first neuropeptide identified that regulates this function was the decapeptide gonadotropin-releasing hormone (GnRH). Nowadays, in gnatostomates, a number of GnRH variants have been identified and classified into three different types: GnRH1, GnRH2, and GnRH3. Almost 30 years later, a new peptide that inhibits gonadotropin synthesis and secretion was discovered and thus named as gonadotropin-inhibitory hormone (GnIH). In avians and mammals, the interaction and regulation between GnRH and GnIH neurons has been widely studied; however, in other vertebrate groups there is little information about the relationship between these neurons. In previous works, three GnRH variants and a GnIH propeptide were characterized in Cichlasoma dimerus, and it was demonstrated that GnIH inhibited gonadotropins release in this species. Because no innervation was detected at the pituitary level, we speculate that GnIH would inhibit gonadotropins via GnRH. Thus, the aim of the present study was to evaluate the anatomical relationship between neurons expressing GnIH and the three GnRH variants by double labelling confocal immunofluorescence in adults of C. dimerus. Our results showed no apparent contacts between GnIH and GnRH1, fiber to fiber interactions between GnIH and GnRH2, and co-localization of GnIH and GnRH3 variant in neurons of the nucleus olfacto-retinalis. In conclusion, whether GnIH regulates the expression or secretion of GnRH1 in this species, an indirect modulation seems more plausible. Moreover, the present results suggest an interaction between GnIH and GnRH2 systems. Finally, new clues were provided to investigate the role of nucleus olfacto-retinalis cells and putative GnIH and GnRH3 interactions in the modulation of the reproductive network in teleost fish.

Optical saturation effects in intracavity Faraday modulation spectroscopy (INFAMOS).

We report on the observation of saturation effects in Intracavity Faraday Modulation Spectroscopy (INFAMOS). A quantum cascade laser operating at ∼5.3 µm is used to probe the 2Π3/2 and 2Π1/2 R(3.5) transitions in the fundamental band of nitric oxide. With average intracavity intensities up to 450 W cm-2, the saturation of these molecular transitions is observed up to a total pressure of ∼240 Torr. The experimental data are interpreted by incorporating saturation into a model for the INFAMOS line shape in the homogeneously broadened limit.

Ontogeny of Gonadotropin-Inhibitory Hormone (GnIH) in the cichlid fish Cichlasoma dimerus.

RFamide peptides are expressed in the early stages of development in most vertebrates. Gonadotropin-inhibitory hormone (GnIH) belongs to the RFamide family, and its role in reproduction has been widely studied in adult vertebrates, ranging from fish to mammals. As only three reports evaluated GnIH during development, the aim of this study was to characterise the ontogeny of GnIH in a fish model, Cichlasoma dimerus. We detected the presence of two GnIH-immunoreactive (GnIH-ir) cell clusters with spatial and temporal differences. One cluster was observed by 3 days post-hatching (dph) in the nucleus olfacto-retinalis (NOR) and the other in the nucleus posterioris periventricularis by 14 dph. The number of GnIH-ir neurons increased in both nuclei, whereas their size increased only in the NOR from hatchling to juvenile stages. These changes occurred from the moment larvae started feeding exogenously and during development and differentiation of gonadal primordia. We showed by double-label immunofluorescence that only GnIH-ir neurons in the NOR co-expressed GnRH3 associated peptide. In addition, GnIH-ir fibre density increased in all brain regions from 5 dph. GnIH-ir fibres were also detected in the retina, optic tract and optic tectum, suggesting that GnIH acts as a neuromodulator of photoreception and the integration of different sensory modalities. Also, there were GnIH-ir fibres in the pituitary from 14 dph, which were in close association with somatotropes. Moreover, GnIH-ir fibres were observed in the saccus vasculosus from 30 dph, suggesting a potential role of GnIH in the modulation of its function. Finally, we found that gnih was expressed from 1 dph, and that the pattern of variation of its transcript levels was in accordance with that of cell number. Present results are the starting point for the study of new GnIH roles during development. This article is protected by copyright. All rights reserved.

Broadening the optical bandwidth of quantum cascade lasers using RF noise current perturbations.

We report on the broadening of the optical bandwidth of a distributed feedback quantum cascade laser (QCL) caused by the application of radio frequency (RF) noise to the injection current. The broadening is quantified both via Lamb-dip spectroscopy and the frequency noise power spectral density (PSD). The linewidth of the unperturbed QCL (emitting at ∼5.3 µm) determined by Lamb-dip spectroscopy is 680±170 kHz, and is in reasonable agreement with the linewidth of 460±40 kHz estimated by integrating the PSD measured under the same laser operating conditions. Measurements with both techniques reveal that by mixing the driving current with broadband RF noise the laser lineshape was reproducibly broadened up to ca 6 MHz with an increasing Gaussian contribution. The effects of linewidth broadening are then demonstrated in the two-color coherent transient spectra of nitric oxide.

LentiPro26: novel stable cell lines for constitutive lentiviral vector production.

Lentiviral vectors (LVs) are excellent tools to promote gene transfer and stable gene expression. Their potential has been already demonstrated in gene therapy clinical trials for the treatment of diverse disorders. For large scale LV production, a stable producer system is desirable since it allows scalable and cost-effective viral productions, with increased reproducibility and safety. However, the development of stable systems has been challenging and time-consuming, being the selection of cells presenting high expression levels of Gag-Pro-Pol polyprotein and the cytotoxicity associated with some viral components, the main limitations. Hereby is described the establishment of a new LV producer cell line using a mutated less active viral protease to overcome potential cytotoxic limitations. The stable transfection of bicistronic expression cassettes with re-initiation of the translation mechanism enabled the generation of LentiPro26 packaging populations supporting high titers. Additionally, by skipping intermediate clone screening steps and performing only one final clone screening, it was possible to save time and generate LentiPro26-A59 cell line, that constitutively produces titers above 106 TU.mL-1.day-1, in less than six months. This work constitutes a step forward towards the development of improved LV producer cell lines, aiming to efficiently supply the clinical expanding gene therapy applications.

Intracavity Faraday modulation spectroscopy (INFAMOS): A tool for radical detection.

We present the intra-cavity Faraday modulation spectroscopy technique, whereby optical feedback cavity-enhanced spectroscopy is coupled with Faraday modulation spectroscopy to greatly enhance the interaction path length of a laser beam with a paramagnetic sample in a magnetic field. We describe a first prototype based upon a cw quantum cascade laser targeting a selection of fundamental rovibrational R-branch transitions of nitric oxide (1890 cm-1), consisting of a linear cavity (finesse F=6300) and a water-cooled solenoid. We demonstrate a minimum detectable Verdet constant of Vmin=4.7×10-14 rad cm-1 G-1 Hz-1/2 (at SNR = 1), corresponding to a single-pass rotation angle of 1.6×10-10 rad Hz-1/2 and a limit of detection of 0.21 ppbv Hz-1/2 NO.

Photothermal Transport of DNA in Entropy-Landscape Plasmonic Waveguides.

The ability to handle single, free molecules in lab-on-a-chip systems is key to the development of advanced biotechnologies. Entropic confinement offers passive control of polymers in nanofluidic systems by locally asserting a molecule's number of available conformation states through structured landscapes. Separately, a range of plasmonic configurations have demonstrated active manipulation of nano-objects by harnessing concentrated electric fields. The integration of these two independent techniques promises a range of sophisticated and complementary functions to handle, for example, DNA, but numerous difficulties, in particular, conflicting requirements of channel size, have prevented progress. Here, we show that metallic V-groove waveguides, embedded in fluidic nanoslits, form entropic potentials that trap and guide DNA molecules over well-defined routes while simultaneously promoting photothermal transport of DNA through the losses of plasmonic modes. The propulsive forces, assisted by in-coupling to propagating channel plasmon polaritons, extend along the V-grooves with a directed motion up to ≈0.5 µm·mW-1 away from the input beam and λ-DNA velocities reaching ≈0.2 µm·s-1·mW-1. The entropic trapping enables the V-grooves to be flexibly loaded and unloaded with DNA by variation of transverse fluid flow, a process that is selective to biopolymers versus fixed-shape objects and also allows the technique to address the challenges of nanoscale interaction volumes. Our self-aligning, light-driven actuator provides a convenient platform to filter, route, and manipulate individual molecules and may be realized wholly by wafer-scale fabrication suitable for parallelized investigation.

Tetraspanins displayed in retrovirus-derived virus-like particles and their immunogenicity.

Virus-like particles (VLPs) are a particular subset of subunit vaccines which are currently explored as safer alternatives to live attenuated or inactivated vaccines. VLPs derived from retrovirus (retroVLPs) are commonly used as scaffolds for vaccine candidates due to their ability to incorporate heterologous envelope proteins. Pseudotyping retroVLPs is however not a selective process therefore, host cellular proteins such as tetraspanins are also included in the membrane. The contribution of these host-proteins to retrovirus immunogenicity remains unclear. In this work, human cells silenced and not silenced for tetraspanin CD81 were used to produce CD81(-) or CD81(+) retroVLPs. We first analyzed mice immune response against human CD81. Despite effective silencing of CD81 in retroVLP producing cells, both humoral and cellular immune responses showed persistent anti-CD81 immunogenicity, suggesting cross reactivity to related antigens. We thus compared the incorporation of related tetraspanins in retroVLPs and showed that decreased CD81 incorporation in CD81(-) retro-VLPs is compensated by an increased incorporation of CD9 and CD63 tetraspanins. These results highlight the dynamic nature of host-derived proteins incorporation in retroVLPs membrane, which should be considered when retrovirus-based biopharmaceuticals are produced in xenogeneic cells.

Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells.

This article describes a novel method merging the cloning of viral vector producer cells with vector titer screening, allowing for screening 200-500 clones in 2 weeks. It makes use of a GFP separated into two fragments, S10 and S11 (Split GFP), fluorescing only upon transcomplementation. Producer cells carrying a S11 viral transgene are cloned in 96-well plates and co-cultured with target cells stably expressing S10. During the period of clone expansion, S11 viruses infect S10 target cells reconstituting the GFP signal. Transcomplemented fluorescence data provide direct estimation of the clone's productivity and can be analyzed in terms of density distribution, offering valuable information on the average productivity of the cell population and allowing the identification of high-producing clones. The method was validated by establishing a retrovirus producer from a nude cell line, in <3 months, inserting three vector constructs without clone selection or screening in between. Clones producing up to 10(8) infectious particles per ml were obtained, delivering optimal ratios of infectious-to-total particles (1 to 5). The method was additionally used to evaluate the production performance of HEK 293 and HEK 293T cell lines demonstrating that the latter sustains increased titers. Finally, it was used to study genetic manipulation of glutathione metabolism in retrovirus production showing that changing cell metabolism steers higher vector expression with titer increases of more than one order of magnitude.This method is a valuable tool not only for cell line development but also for genetic manipulation of viral vector and/or producer cells contributing to advancing the field of viral gene therapy.

Can different conditioning activities and rest intervals affect the acute performance of taekwondo turning kick?

This study compared the acute effect of strength, plyometric, and complex exercises (combined strength and plyometric exercise) in the countermovement jump (CMJ) and frequency speed of kick test (FSKT) and attempted to establish the best rest interval to maximize performance in the CMJ, number of kicks, and impact generated during FSKT. Eleven taekwondo athletes (mean ± SD; age: 20.3 ± 5.2 years; body mass: 71.8 ± 15.3 kg; height: 177 ± 7.2 cm) participated. One control and 9 experimental conditions were randomly applied. Each condition was composed of warm-up, conditioning activity (half-squat: 3 × 1 at 95% 1RM; jumps: 3 × 10 vertical jumps above 40-cm barrier; or complex exercise: half-squat 3 × 2 at 95% 1RM + 4 vertical jumps above 40-cm barrier), followed by different rest intervals (5-, 10-minute, and self-selected) before CMJ and FSKT. The conditions were compared using an analysis of variance with repeated measures, followed by Bonferroni's post hoc test. The alpha level was set at 5%. Significant difference was found in the number of kicks (F9,90 = 1.32; p = 0.239; and η2 = 0.116 [small]). The complex method with a 10-minute rest interval (23 ± 5 repetitions) was superior (p = 0.026) to the control (19 ± 3 repetitions), maximum strength with a self-selected rest interval (328 ± 139 seconds; 18 ± 2 repetitions) (p = 0.015), and plyometric with a 5-minute rest interval (18 ± 3 repetitions) (p < 0.001). Our results indicate that taekwondo athletes increased the number of kicks in a specific test by using the complex method when 10-minute rest interval was used.

[Willingness to pay for human papillomavirus vaccine in Metropolitan Santiago, Chile]. / Disposición a pagar por vacunas contra el virus papiloma humano en la Región Metropolitana de Santiago de Chile.

BACKGROUND: Human papillomavirus (HPV) infection is a risk factor for cervical cancer and can be prevented with the HPV vaccine. AIM: To explore the willingness of parents to pay for HPV vaccine for their offspring. MATERIAL AND METHODS: A survey about the willingness to pay for HPV vaccine was answered by 386 individuals of the highest socioeconomic level who had a daughter aged between 12 and 18 years. The survey included information about the risks of HPV infection. RESULTS: Parents would pay a mean of US$ 758 for the vaccine. Twenty five percent of parents were not willing to pay for it. If the cost of the vaccine would be reduced by 50%, only 4% of parents would not pay for it. The willingness to pay is associated with the price of the vaccine, the income level of respondents and the size of the family. CONCLUSIONS: Most respondents would pay for HPV vaccine for their daughters, despite the relatively high cost.

Disposición a pagar por vacunas contra el virus papiloma humano en la Región Metropolitana de Santiago de Chile / Willingness to pay for human papillomavirus vaccine in Metropolitan Santiago, Chile

Background: Human papillomavirus (HPV) infection is a risk factor for cervical cancer and can be prevented with the HPV vaccine. Aim: To explore the willingness of parents to pay for HPV vaccine for their offspring. Material and Methods: A survey about the willingness to pay for HPV vaccine was answered by 386 individuals of the highest socioeconomic level who had a daughter aged between 12 and 18 years. The survey included information about the risks of HPV infection. Results: Parents would pay a mean of US$ 758 for the vaccine. Twenty five percent of parents were not willing to pay for it. If the cost of the vaccine would be reduced by 50%, only 4% of parents would not pay for it. The willingness to pay is associated with the price of the vaccine, the income level of respondents and the size of the family. Conclusions: Most respondents would pay for HPV vaccine for their daughters, despite the relatively high cost.

Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis.

Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

The effect of PAMAM dendrimers on mesenchymal stem cell viability and differentiation.

Stem cells and nanomaterials are two new and exciting fields of science that are evolving very fast and that are starting to establish ties. Nanomaterials should, however, be designed to interact with stem cells without compromising their biological characteristics, in other words, without affecting their viability and differentiation potential. In the present report and for the first time, the effects of poly(amidoamine) (PAMAM) dendrimers on the viability and differentiation ability towards the osteogenic and adipogenic lineages of human mesenchymal stem cells (hMSCs) are systematically evaluated. Studies were done as a function of the cell culture media composition and PAMAM dendrimer surface functionalization, generation, and concentration. hMSCs were exposed to amino and hydroxyl (generations 2, 4 and 6), and carboxylate (generations 1.5, 3.5 and 5.5) functionalized dendrimers, at two different concentrations (10 µg/mL and 0.5 µg/mL), for a period of 21 days. Overall, the results revealed that amino functionalized dendrimers can be severely cytotoxic, the extension of cell death being dependent on the concentration of amino groups in solution. However, in all cases, the differentiation of hMSCs towards the osteogenic and adipogenic phenotypes seems not to be affected as demonstrated by staining in in vitro cultures.

Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin.

A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in Escherichia coli and the purified protein was conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of botulinum neurotoxin type A (BoNT/A) via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) essential for neurotransmitter release. BoTIM-coupled liposomes, loaded with peptide inhibitors of proteases, yielded considerable attenuation of the neuroparalytic effects of BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer antiproteases to suppress the symptoms of botulism ex vivo. Delivery of the BoNT/A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.