Multi-site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system.

High-dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30-marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2-week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay-specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter-site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

Profilin induces lamellipodia by growth factor-independent mechanism.

Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.

Analysis of the role of the Rac/Cdc42 GTPases during planar cell polarity generation in Drosophila.

Initial genetic studies in Drosophila suggested that several members of the Rho subfamily (RhoA, Rac1 and Cdc42) are involved in planar cell polarity (PCP) establishment. However, analyses of Rac1, Rac2 and Mtl loss-of-function (LOF) mutants have argued against their role in this process. Here, we investigate in detail the role of the Rho GTPases Mtl, Cdc42, Rac1 and Rac2 in PCP generation. These functional analyses were performed by overexpressing Mtl in eyes and wings, by performing genetic interaction assays and by using a combination of triple and quadruple mutant LOF clones. We found that Mtl overexpression caused PCP phenotypes and that it interacted genetically with other Rho GTPases, such as Rac1 and Cdc42 as well as with several PCP genes, such as stbm, pk and aos. However, Mtl was not found to interact with Rac2, RhoA and other members of the Fz/PCP pathway. Triple mutant clones of Rac1, Rac2 and Mtl were found to exhibit mild PCP defects which were enhanced by reduction of Cdc42 function with a hypomorphic Cdc42 allele. Taken together, these and previous results suggest that Rho GTPases may have partially overlapping functions during PCP generation. Alternatively, it is also possible that the mild PCP phenotypes observed could indicate that they are required at low levels in that process. However, since not all of them function upstream of a JNK cassette, we propose that they may act in at least two parallel pathways.

Hypotensive effect of profilin on rabbit intraocular pressure.

Ocular hypertension is a negative process that occurs within the eye and is the main risk factor to develop glaucoma, a progressive loss of vision due to degeneration of retinal ganglion cells. The protein transduction technique allows a cargo to cross biological membranes. Using this technique we have previously shown that a membrane permeable version of profilin I (PTD4-profilin) increased aqueous humour outflow facility. Here we have investigated if a topical application of PTD4-profilin was able to modify intraocular pressure in rabbits. 10 microM PTD4-profilin (10 microL), reduced intraocular pressure by 20% compared to the control vehicle, this value being in the range of other commercial drugs, which produced intraocular pressure reductions between 18 and 35%. The mean-time effect for PTD4-profilin was 6.8 h and was also in the same range as commercial products that provided values between 4.3 and 5.5 h. According to the results presented here we propose PTD4-profilin as a new approach for the treatment of ocular hypertension and PTD4 as a new strategy to facilitate the penetration of molecules into the eye.

Use of transduction proteins to target trabecular meshwork cells: outflow modulation by profilin I.

PURPOSE: Fusion proteins containing a protein transduction domain (PTD4) are able to cross biological membranes. We tested the applicability of the protein transduction method for study of the aqueous humor trabecular outflow pathway by targeting the actin cytoskeleton, which is known to be involved in outflow facility regulation. METHODS: Expression vectors useful for generating fusion proteins with the PTD4 domain and the actin-binding protein Profilin I were constructed. The transductional and functional properties of these proteins were tested in bovine trabecular meshwork cells in culture. The effects of PTD4-Profilin I on outflow facility were evaluated in perfused bovine anterior segments. PTD4-beta-galactosidase was used to visually check correct delivery of fusion proteins to trabecular meshwork cells. RESULTS: The fusion proteins generated were characterized by western blot. Immunocytochemistry experiments showed intracellular staining for PTD4-Profilin I in trabecular meshwork cells in culture. The fusion protein was found in the cytoplasm associated with actin filaments and in the leading edge of the cellular membrane. In contrast, control Profilin I, without the PTD4 domain, was unable to cross the cell membrane. In perfused anterior segments, 2 microM PTD4-Profilin I increased trabecular outflow facility in a reversible manner, while Profilin I had no significant effect. Anterior segments perfused with PTD4-beta-galactosidase showed positive staining in the trabecular meshwork tissue. CONCLUSIONS: Protein transduction technology is a valuable tool for targeting trabecular meshwork tissue, not only for performing physiological studies, but also as a potential drug-delivery method. Profilin I action on the actin cytoskeleton further reinforces the importance of this structure in outflow facility regulation.

IMD-0354 targets breast cancer stem cells: a novel approach for an adjuvant to chemotherapy to prevent multidrug resistance in a murine model.

Although early detection of breast cancer improved in recent years, prognosis of patients with late stage breast cancer remains poor, mostly due to development of multidrug resistance (MDR) followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability and other stem cell-like properties, are concentrated in a side population (SP) of cells, which were proposed to be responsible for MDR and tumor repopulation that cause patients to succumb to breast cancer. Therefore, targeting of CSCs as an adjuvant to chemotherapy should be able to provide a more effective treatment of this disease. Here, we used IMD-0354, an inhibitor of NF-κB, identified for targeting CSCs, in a combination therapy with doxorubicin encapsulated in targeted nanoparticles. IMD-0354 did target CSCs, evidenced by a decrease in the SP, demonstrated by the inhibition of the following: dye/drug efflux, reduction in ABC transporters as well as in colony formation in soft agar and low attachment plates. Decrease of stem-like gene expression of Oct4, Nanog and Sox2, and apoptosis resistance related to the Survivin gene also was observed after treatment with this compound. In addition, IMD-0354 targeted non-CSCs as indicated by reducing viability and increasing apoptosis. Targeted drug delivery, achieved with a legumain inhibitor, proved to enhance drug delivery under hypoxia, a hallmark of the tumor microenvironment, but not under normoxia. Together, this allowed a safe, non-toxic delivery of both anticancer agents to the tumor microenvironment of mice bearing syngeneic metastatic breast cancer. Targeting both bulk tumor cells with a chemotherapeutic agent and CSCs with IMD-0354 should be able to reduce MDR. This could eventually result in decreasing tumor recurrences and/or improve the outcome of metastatic disease.

Three possible cases of cranioclasis indicating obstetric extraction of the fetus in a Spanish rural population of the late 17th and early 18th Centuries

Cranioclasis is a technique that was formerly used to extract the fetus during births that were complicated due to different causes. This procedure was usually resorted to once the fetus was confirmed to be dead. This technique was substituted by the caesarean section in the mid-twentieth century. The aim of this study is to analyze osseous lesions observed in the crania of three neonates buried in the period between the end of the 17th century and the beginning of the 18th century in the church-fortress known as Iglesia Fortaleza de Nuestra Señora de los Ángeles in Castielfabib, Rincón de Ademuz, province of Valencia, Spain. The instrumental incisions found in the occipital bones of the three neonates, as well as the overlap of their neurocranial bones, are compatible with cranioclasis. The cranial lesions in the three neonate occipital bones discovered in Castielfabib in Ademuz-Valencia, Spain could confirm the practice of cranioclasis in this region of Spain at the end of the 17th century and the beginning of the 18th century

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Analysis of the cervical double transverse foramen in present Spanish population

The aim of our study was to investigate the prevalence and morphometry of double transverse foramina in cervical vertebrae in a living population and to discuss their clinical importance. This is a retrospective single-center study. 253 (84.3%) computed tomography scan images of the cervical spine were collected from a total sample of 300 Spanish subjects that underwent a computed tomography study, 173 from men (68.3%) and 80 from women (31.6%), aged between 18 and 90 years old. The presence or absence of a double transverse foramen of each cervical vertebra was recorded, and the maximum right-left diameter, maximum antero-posterior diameter and area of each transverse foramen were measured. The applied statistics were multivariate models for repeated measures, Student t test and Pearson’s chi-squared test.Double transverse foramina in C4, C5, C6 and C7 were observed, the most prevalent being in C6 (45.8%), followed by C5 (23.5%), C4 (4.7%) and C7 (4.3%). The unilateral formation was significantly the most frequent. No differences were found based on sex. In the vertebrae with a double transverse foramen, the principal transverse foramen was significantly larger than the accessory transverse foramen. However, in these vertebrae the principal transverse foramen was significantly smaller when compared with the transverse foramen of normal vertebrae.C6 presents the greatest prevalence of double transverse foramina, although they are also observed in C4, C5 and C7. The double transverse foramen causes the principal transverse foramen to be smaller when compared with normal vertebrae, thus it should be taken into account in clinical practice

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