RESUMO

The aim of this study was to determine whether X-ray fluorescence spectrometry (XRF) could be used to detect changes in hair zinc concentration in response to a modest daily increase in zinc from the consumption of zinc biofortified wheat flour. This study was conducted as part of an effectiveness trial (BiZiFED2) exploring the potential for zinc biofortified wheat to alleviate zinc deficiency in adolescent girls aged 10-16 years in Pakistan (trial registration ID ISRCTN17107812). A randomized controlled design was used. Participants received either control flour or zinc biofortified flour for 6 months. Consumption of biofortified flour resulted in an average daily increase in dietary zinc intake of 1.5 mg per day above that of the control flour. At baseline and at the end of the intervention, individual hair samples (control: n = 59, intervention: n = 64) were analyzed for zinc and sulfur content by XRF. Data were analyzed using linear mixed effects models to contrast between trial groups the changes from baseline to end point and also to compare baseline and end point values within each trial group. Increases from baseline to endpoint in both sulfur and zinc were significantly greater in the intervention group compared to control (sulfur counts. CONTROL: baseline = 119.87 ± 20.33 and endpoint = 121.58 ± 23.58/intervention: baseline = 122.67 ± 24.19 and endpoint = 131.60 ± 21.34); (Zinc counts. CONTROL: baseline = 50.88 ± 14.33 and endpoint = 54.82 ± 14.61/intervention: baseline = 49.61 ± 10.77 and endpoint = 58.79 ± 12.20). For these parameters, there were also significant increases from baseline to endpoint in the intervention group but not in control. Furthermore, for Zn:S count ratio there were no differences in terms of the magnitude of the change from baseline to endpoint in the control group, although significant increases from baseline to endpoint were evident in the intervention group (Zn:S count ratio. CONTROL: baseline = 0.42 ± 0.10 and endpoint = 0.45 ± 0.08/intervention: baseline = 0.41 ± 0.08 and endpoint = 0.45 ± 0.08). A modest increase in dietary zinc over 6 months resulted in a detectable increase in both sulfur and zinc counts in individual hairs measured using XRF. This offers a sensitive, non-invasive method to monitor changes within subjects in response to dietary zinc interventions.

RESUMO

BACKGROUND: Novel and emerging biomarkers of zinc status are being developed to help study and address zinc deficiency around the world. Two potential biomarkers, nail and hair, involve the measurement of zinc from easily accessible keratin-based components of the body. Portable X-ray fluorescence (XRF) is a relatively new approach to the assessment of zinc in nail or hair, and has a number of compelling advantages compared with other techniques. The aim of the current study was to test the ability of XRF to measure zinc in keratinized reference materials (RMs) designed to simulate nail and hair. METHODS: Four Keratin Matrix RMs were prepared and characterized for numerous trace elements by the New York State Department of Health's Wadsworth Center. The Keratin Matrix RMs consisted of powdered samples of caprine (goat) horns pooled from several animals. Concentrations of zinc, as assessed by inductively coupled plasma mass spectrometry (ICP-MS), were similar to what would be expected from human nail or hair tissues. Repeat measurements of the RMs were made using a portable XRF system. The XRF zinc results were compared with the ICP-MS zinc concentrations. Three different approaches to quantifying the zinc content by XRF were performed: (1) zinc signal to total signal ratio, (2) zinc signal to sulfur signal ratio, and (3) system output zinc concentration. RESULTS: The portable XRF results from a given RM were found to be consistent across repeat trials under all three approaches to XRF quantitation. Precision, calculated as the relative standard deviation of repeat measurements ranged from an average result of 0.8 % (using the system output zinc concentration method) to 6.1 % (using the zinc signal to sulfur signal ratio method). Measurement of the RMs provided XRF zinc results which scaled well with ICP-MS zinc concentration, particularly when using the XRF zinc to total and system zinc concentration methods. A Bland-Altman plot showed that the XRF system zinc concentration output exceeded the ICP-MS zinc concentration by, on average, 10.2 % ± 1.2 %. CONCLUSION: Overall, both accuracy and precision of measurement were found to be promising for portable XRF, provided appropriate conversions to concentration are introduced. The results of this study indicate that portable XRF is an effective and dependable method of assessing zinc concentration in keratinized tissue RMs. This will have implications for the future use of portable XRF to monitor zinc status in humans through measurements of nail and hair.

Assuntos

RESUMO

The invasiveness of late-stage cutaneous squamous cell carcinoma (cSCC) is associated with poor patients' prognosis and linked to strong upregulation of the glycoprotein Podoplanin (PDPN) in cancer cells. However, the function of PDPN in these processes in cSCC carcinogenesis has not been characterized in detail yet. Employing a CRISPR/Cas9-based loss-of-function approach on murine cSCC cells, we show that the loss of Pdpn results in decreased migration and invasion in vitro. Complementing these in vitro studies, labelled murine control and Pdpn knockout cells were injected orthotopically into the dermis of nude mice to recapitulate the formation of human cSCC displaying a well-differentiated morphology with a PDPN-positive reaction in fibroblasts in the tumor stroma. Smaller tumors were observed upon Pdpn loss, which is associated with reduced tumor cell infiltration into the stroma. Utilizing Pdpn mutants in functional experiments in vitro, we provide evidence that both the intra- and extracellular domains are essential for cancer cell invasion. These findings underline the critical role of PDPN in cSCC progression and highlight potential therapeutic strategies targeting PDPN-dependent cancer cell invasion, especially in late-stage cSCC patients.

Assuntos

RESUMO

Neuronal intracellular Cl- concentration ([Cl-]i) influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pHi) or cell volume. Up to date, neuronal [Cl-]i has predominantly been studied in model systems of reduced complexity. Here, we implemented the genetically encoded ratiometric Cl- indicator Superclomeleon (SCLM) to estimate the steady-state [Cl-]i in cortical neurons from anesthetized and awake mice using 2-photon microscopy. Additionally, we implemented superecliptic pHluorin (SE-pHluorin) as a ratiometric sensor to estimate the intracellular steady-state pH (pHi) of mouse cortical neurons in vivo. We estimated an average resting [Cl-]i of 6 ± 2 mM with no evidence of subcellular gradients in the proximal somato-dendritic domain and an average somatic pHi of 7.1 ± 0.2. Neither [Cl-]i nor pHi were affected by isoflurane anesthesia. We deleted the cation-Cl- co-transporter KCC2 in single identified neurons of adult mice and found an increase of [Cl-]i to approximately 26 ± 8 mM, demonstrating that under in vivo conditions KCC2 produces low [Cl-]i in adult mouse neurons. In summary, neurons of the brain of awake adult mice exhibit a low and evenly distributed [Cl-]i in the proximal somato-dendritic compartment that is independent of anesthesia and requires KCC2 expression for its maintenance.

RESUMO

Mover, a member of the exquisitely small group of vertebrate-specific presynaptic proteins, has been discovered as an interaction partner of the scaffolding protein Bassoon, yet its function has not been elucidated. We used adeno-associated virus (AAV)-mediated shRNA expression to knock down Mover in the calyx of Held in vivo. Although spontaneous synaptic transmission remained unaffected, we found a strong increase of the evoked EPSC amplitude. The size of the readily releasable pool was unaltered, but short-term depression was accelerated and enhanced, consistent with an increase in release probability after Mover knockdown. This increase in release probability was not caused by alterations in Ca(2+) influx but rather by a higher Ca(2+) sensitivity of the release machinery, as demonstrated by presynaptic Ca(2+) uncaging. We therefore conclude that Mover expression in certain subsets of synapses negatively regulates synaptic release probability, constituting a novel mechanism to tune synaptic transmission.

Assuntos

RESUMO

PURPOSE: In this study, we assessed the outcome of penetrating keratoplasties using organ-cultured corneal tissues at the University Eye Hospital, Ludwig-Maximilians-University, Munich, Germany. The goal was to identify perioperative and postoperative risk factors that may affect graft survival. PATIENTS AND METHODS: The medical records of 377 patients who underwent a penetrating keratoplasty between 2001 and 2011 were reviewed. Organ-cultured corneal tissue was obtained from the eye bank of Ludwig-Maximilians-University. Perioperative and postoperative risk factors for graft failure were evaluated by univariate and multivariate analyses. RESULTS: The 5-year overall survival rate of penetrating keratoplasties was 68%. Graft failure occurred in 26% of patients. High-risk keratoplasties, such as repeat penetrating keratoplasties and emergency penetrating keratoplasties, as well as postoperative conditions, such as glaucoma, retinal surgery, suture problems, persistent epithelial defect, infectious keratitis, and graft rejection, were significantly associated with graft failure in the multivariate analyses. CONCLUSION: This study showed a similar graft-survival rate as demonstrated in previous studies. In addition, a number of perioperative and postoperative risk factors were identified in this specific patient population.

RESUMO

PURPOSE: The aim of this study was to evaluate the outcome of penetrating keratoplasties, at the University Eye Hospital, Ludwig-Maximilians-University, Munich, Germany, using organ-cultured donor corneas and to identify preoperative risk factors, which may influence the event of graft failure. METHODS: In this study, 377 medical records of patients, who underwent penetrating keratoplasty between 2001 and 2011, were reviewed. Organ-cultured donor corneas were obtained from the eye bank, Ludwig-Maximilians-University, Munich, Germany. Donor-related and preoperative recipient-related risk factors for graft failure were analyzed by univariate and multivariate analyses. RESULTS: Graft failure occurred in 26% of patients. The following preoperative factors were significantly associated with graft failure by multivariate analyses: high donor age, low donor endothelial cell density, high patient age, indications of infectious keratitis, acute perforation of noninfectious keratitis, prior graft failure, chemical burn, trauma, glaucoma-associated corneal decompensation, high-risk graft indications, corneal edema, anterior chamber lens, diabetes mellitus, atopy, and autoimmune diseases. CONCLUSIONS: This study demonstrated a success rate of 74%, which is consistent with previous studies. Various preoperative recipient-related factors seem to influence the outcome of penetrating keratoplasties, whereas few donor-related factors have a significant association with graft failure.

Assuntos

RESUMO

Collybistin (Cb) is a brain specific guanine nucleotide exchange factor that interacts with the inhibitory postsynaptic scaffold protein gephyrin. Cb is essential for the postsynaptic clustering of gephyrin and major GABA(A) receptor subtypes during the formation and maintenance of GABAergic synapses in the hippocampus and other areas of the forebrain. In the rat, four distinct splice variants (Cb1, Cb2(SH3-), Cb2(SH3+) and Cb3), have been described, which differ in their C-termini (Cb1-3) and in respect of the SH3-domain that is absent in Cb2(SH3-). In the human brain, only a single isoform (hPEM2) corresponding to Cb3, was found to be expressed. This has been implicated in neurological defects such as hyperekplexia, epilepsy, anxiety, aggression and mental retardation. In this study, we address the functional significance of the differentially spliced Cb isoforms by generating a shRNA-mediated knock-down of endogenous Cb in hippocampal cultured neurons that is subsequently rescued by the expression of distinct Cb isoforms. We found that the Cb knock-down induced impairment in GABAergic neurotransmission could be rescued by the expression of any of the Cb isoforms, independent of their C-termini or the presence of the SH3-domain in the N-terminal region. Thus, the different Cb isoforms all confer basic functionality.

Assuntos