Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma

Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.     

Modelling the correlation between EGFr expression and tumour cell radiosensitivity, and combined treatments of radiation and monoclonal antibody EGFr inhibitors

ABSTRACT: Purpose To estimate the effects of heterogeneity on tumour cell sensitivity to radiotherapy combined with radiosensitizing agents attributable to differences in expression levels of Epidermal Growth Factor Receptor (EGFr). Materials and methods Differences in radiosensitivity are not limited to cells of different cancer histotypes but also occur within the same cancer, or appear during radiotherapy if radiosensitizing drugs are combined with ionizing radiation. A modified biologically effective dose (MBED), has been introduced to account for changes in radiosensitivity parameters (alpha and alpha/beta) rather than changes in dose/fraction or total dose as normally done with standard biologically effective dose (BED). The MBED approach was applied to cases of EGFr over-expression and cases where EGFr inhibitors were combined with radiation. Representative examples in clinical practice were considered. RESULTS: Assuming membrane EGFr over-expression corresponds to reduced radiosensitivity (alphaH = 0.15 Gy-1 and alphaH/betaH = 7.5 Gy) relative to normal radiosensitivity (alpha = 0.2 Gy-1 and alpha/beta = 10 Gy), an increased dose per fraction of 2.42 Gy was obtained through the application of MBED, which is equivalent to the effect of a reference schedule with 30 fractions of 2 Gy. An equivalent hypo-fractionated regime with a dose per fraction of 2.80 Gy is obtained if 25 fractions are set. Dose fractionations modulated according to drug pharmacokinetics are estimated for combined treatments with biological drugs. Soft and strong modulated equivalent hypo-fractionations result from subtraction of 5 or 10 fractions, respectively. CONCLUSIONS: During this computational study, a new radiobiological tool has been introduced. The MBED allows the required dose per fraction to be estimated when tumour radiosensitivity is reduced because EGFr is over-expressed. If radiotherapy treatment is combined with EGFr inhibitors, MBED suggests new treatment strategies, with schedules modulated according to drug pharmacokinetics.     

Salvinorin A reduces mechanical allodynia and spinal neuronal hyperexcitability induced by peripheral formalin injection

ABSTRACT: BACKGROUND: Salvinorin A (SA), the main active component of Salvia Divinorum, is a non-nitrogenous kappa opioid receptor (KOR) agonist. It has been shown to reduce acute pain and to exert potent antinflammatory effects. This study assesses the effects and the mode of action of SA on formalin-induced persistent pain in mice. Specifically, the SA effects on long-term behavioural dysfuctions and changes in neuronal activity occurring at spinal level, after single peripheral formalin injection, have been investigated. Moreover, the involvement of microglial and glial cells in formalin-induced chronic pain condition and in SA-mediated effects has been evaluated. RESULTS: Formalin induced a significant decrease of mechanical withdrawal threshold at the injected and contralateral paw as well as an increase in the duration and frequency, and a rapid decrease in the onset of evoked activity of the nociceptive neurons 7 days after formalin injection. SA daily treatment significantly reduced mechanical allodynia in KOR and cannabinoid receptor 1 (CB1R) sensitive manner. SA treatment also normalized the spinal evoked activity. SA significantly reduced the formalin-mediated microglia and astrocytes activation and modulated pro and anti-inflammatory mediators in the spinal cord. CONCLUSION: SA is effective in reducing formalin-induced mechanical allodynia and spinal neuronal hyperactivity. Our findings suggest that SA reduces glial activation and contributes in the establishment of dysfunctions associated with chronic pain with mechanisms involving KOR and CB1R. SA may provide a new lead compound for developing anti-allodynic agents via KOR and CB1R activation.     

Notes about the uses of plants by one of the last healers in the Basilicata Region (South Italy)

Human societies utilize mollusks for myriad material and spiritual ends. An example of their use in a religious context is found in Brazil's African-derived belief systems. Candomblé, an Afro-Brazilian religion introduced during the 18^th-19^th centuries by enslaved Yoruba, includes various magical and liturgical uses of mollusks. This work inventoried the species utilized by adherents and to analyzed their symbolic and magical context. Data were obtained from Candomblé temples in two cities in the northeast of Brazil-Caruaru, in the state of Pernambuco, and Campina Grande, in the state of Paraíba. Questionnaires administered to eleven adepts revealed that at least nineteen mollusk species are being used. Shells from Monetaria moneta, M. annulus and Erosaria caputserpentis were cited by all of the interviewees. Three uses stood out: divination (jogo de búzios); utilization as ritual objects; and employment as sacrificial offerings (Igbin or Boi-de-Oxalá). The jogo de búzios (shell toss), employed in West Africa, Brazil and Cuba, is of fundamental importance to the cult, representing the means by which the faithful enter in contact with the divinities (Orixás) and consult people's futures (Odu). The utilization of mollusks in Candomblé is strongly influenced by ancient Yoruba myths (Itãs) which, having survived enslavement and generations of captive labor, continue to guide the lives of Brazil's African Diaspora.     

Neuropsychological profile and clinical effects of arginine treatment in children with creatine transport deficiency

ABSTRACT: BACKGROUND: SLC6A8, an X-linked gene, encodes the creatine transporter (CRTR) and its mutations lead to cerebral creatine (Cr) deficiency which results in mental retardation, speech and language delay, autistic-like behaviour and epilepsy (CRTR-D, OMIM 300352). CRTR-D represents the most frequent Cr metabolism disorder but, differently from Cr synthesis defects, that are partially reversible by oral Cr supplementation, does not respond to Cr treatment even if precociously administrated. The precursors of Cr are the non-essential amino acids Glycine (Gly) and Arginine (Arg), which have their own transporters at the brain-blood barrier level and, therefore, their supplementation appears an attractive and feasible therapeutic option aimed at stimulating Cr endogenous synthesis and, in this way, at overcoming the block of Cr transport within the brain. However, until now the effects of Arg and/or Gly supplementation on Cr brain levels and behaviour have been controversial. METHODS: In this study five Italian male patients affected by CRTR-D were supplemented with oral LArg at a dosage of 300 mg/kg/day divided into 3 doses, for 24-36 months. Biochemical and plasmatic amino acids examinations and thyroid hormone dosages were periodically performed. Moreover, Proton and Phosphorus Magnetic Resonance Spectroscopy (MRS) was monitored during follow-up in concurrence with neuropsychological evaluations. RESULTS: During L-Arg treatment a clinical improvement in motor skills and to a lesser extent in communication and attention was observed. In addition, all patients had a reduction in the number and frequency of epileptic seizures. Daily living skills appeared also to be positively influenced by L-Arg treatment. Moreover, Total Cr and especially PhosphoCr, evaluated byproton and phosphorus spectroscopy, showed a mild increase, although well below the normal range. CONCLUSION: This study provides information to support the effectiveness of L-Arg supplement treatment in CTRT-D patients; in fact the syndromic pattern of cognitive and linguistic deficit presented by CRTR-D patients was partially altered by L-Arg supplementation especially at a qualitative clinical level. Oral L-Arg may represent not only a protective factor towards a further cognitive decline, but can lead to the acquisition of new skills.     

A Novel Malate Dehydrogenase from Ceratonia siliqua L. Seeds with Potential Biotechnological Applications

A novel malate dehydrogenase (MDH; EC 3.1.1.1.37), hereafter MDHCs, from Ceratonia siliqua seeds, commonly known as Carob tree, was purified by using ammonium sulphate precipitation, ion exchange chromatography on SteamLine SP and gel-filtration. The molecular mass of the native protein, obtained by analytical gel-filtration, was about 65?kDa, whereas, by using SDS-PAGE analysis, with and without reducing agent, was 34?kDa. The specific activity of purified MDHCs (0.25?mg/100?g seeds) was estimated to be 188 U/mg. The optimum activity of the enzyme is at pH 8.5, showing a decrease in the presence of Ca²⁺, Mg²⁺ and NaCl. The N-terminal sequence of the first 20 amino acids of MDHCs revealed 95?% identity with malate dehydrogenase from Medicago sativa L. Finally, the enzymatic activity of MDHCs was preserved even after absorption onto a PVDF membrane. To our knowledge, this is the first contribution to the characterization of an enzyme from Carob tree sources.     

Longitudinal tracking of human fetal cells labeled with super paramagnetic iron oxide nanoparticles in the brain of mice with motor neuron disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.