Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding.

The impact of psychiatric pathology on the prognosis and survival of men with prostate cancer undergoing radical prostatectomy.

INTRODUCTION AND OBJECTIVE: Cancer-specific anxiety is the most frequently reported psychological response after radical prostatectomy (RP). We evaluated the prevalence of pretreatment psychiatric pathology in patients with prostate cancer undergoing RP and identified the effects of psychiatric diagnoses on their survival and prognosis. MATERIAL AND METHODS: Retrospective multicenter observational study including 1078 men treated with RP for organ-confined prostate cancer. Groups: GP: patients with psychiatric pathology prior to RP; GNP: patients without psychiatric pathology prior to RP. Urological, oncological and psychiatric variables, descriptive statistics and multivariate analysis were included. RESULTS: 37.94% of patients presented a psychiatric diagnosis. Adjuvant radiotherapy was required in 27.83% and hormone therapy in 23.38%; being more frequent in GP. Cancer-specific survival was higher in GNP. Anxiety, depression, insomnia, smoking, psychosis and alcoholism were the most frequent. Low TNM and low presence of LUTS and SUI increased the probability of absence of psychiatric pathology. Fatigue, erectile dysfunction and cognitive impairment after RP with RT and/or HT were higher in GP. Older age and higher PSA at diagnosis increased the relative risk of psychiatric pathology and worse outcome. The most frequently related factors were RP, PSA, age and survival time. CONCLUSIONS: Psychiatric pathology is present in patients undergoing radical prostatectomy for prostate cancer, with a high impact on survival and prognostic outcomes.

Altered materno-fetal transfer of 13C-polyunsaturated fatty acids in obese pregnant women.

BACKGROUND & AIMS: Maternal obesity at conception is considered a major predictor of offspring obesity. This could by driven at least in part by an altered placental fat transfer. However, the pathophysiological mechanisms involved are not fully understood. We investigated the in vivo materno-fetal transfer of fatty acids (FAs) in obese pregnant women using stable isotopes. METHODS: Ten obese and ten normo-weight pregnant women (control) received orally a bolus of 13C-labeled FAs 12 h before elective caesarean section: oleic acid (13C-OA), linoleic acid (13C-LA) and docosahexaenoic acid (13C-DHA). Maternal blood samples were collected at -12 (basal), -8, -4, -2, 0 h relative to the time of cesarean section. At the time of birth, arterial and venous cord bloods as well as placental tissue were collected. FAs composition was determined by gas-liquid chromatography and isotopic enrichment by gas chromatography-combustion-isotope ratio mass spectrometry. RESULTS: Maternal plasma insulin and placental weight tended to higher values in obese pregnant women although they did not present serum hyperlipidemia. Higher concentrations of 13C-LA and 13C-DHA were found in non-esterified FAs fraction in maternal plasma of obese mothers. The ratio of placental uptake for 13C-LA and 13C-DHA was lower in obese women compared to normal weight pointing toward a limited capacity of FA placental transfer, especially of essential FAs. Maternal insulin was associated to this lower placenta/maternal plasma ratio for both 13C-LA (R = -0.563, P = 0.012) and 13C-DHA (R = -0.478, P = 0.033). In addition, the ratio cord/maternal plasma of 13C-LA was significantly lower in obese women compared to controls. CONCLUSIONS: In conclusion, obese mothers without hyperlipidemia showed a reduced materno-fetal transfer of polyunsaturated FAs which could affect fetal development. This affect dietary recommendation for obese pregnant women. TRIAL REGISTRY NUMBER: ISRCTN69794527.

The influence of smoking on bacterial resistance after vaccine or antibiotic prophylaxis against recurrent urinary tract infections. / Influencia del tabaquismo en la resistencia bacteriana después de la profilaxis frente a infecciones urinarias recurrentes con antibiótico o con vacuna.

INTRODUCTION: The influence of tobacco on the microbiological spectrum, resistance-sensitivity pattern and evolution in patients with recurrent urinary tract infections (RUTI) is analyzed. Evaluation of the effect of polyvalent bacterial vaccine on the prevention of RUTI and smoking status. MATERIAL AND METHODS: Retrospective multicenter study of 855 women with RUTI receiving suppressive antibiotic treatment or bacterial vaccine between 2009 and 2013. Group A (GA): Antibiotic (n=495); Subgroups: GA1 non-smoker (n=417), GA2 smoker (n=78). Group B (GB): Vaccine (n=360); Subgroups: GB1 non-smoker (n=263), GB2 smoker (n=97). VARIABLES: Age, pre-treatment UTI, disease-free time (DFT), microbial species, sensitivity and resistance. Follow-up at 3, 6 and 12 months with culture and SF-36 questionnaire. RESULTS: Mean age 56.51 years (18-75), similar between groups (P=.2257). No difference in the number of pretreatment UTIs (P=.1329) or in the distribution of the bacterial spectrum (P=.7471). DFT was higher in subgroups B compared with A. Urine cultures in GA1: E. coli 62.71% with 8.10% resistance (33% quinolones; 33% cotrimoxazole; 33% quinolones + cotrimoxazole); in GA2 E. coli 61.53% with 75% resistance (16.66% quinolones; 33.33% quinolones + cotrimoxazole; 16.66% amoxicillin-clavulanate; 16.66% erythromycin + phosphomycin + clindamycin) (P=.0133). There were no differences between patients of GA treated with cotrimoxazole and nitrofurantoin (P=.8724). Urine cultures in GB1: E. coli 47.36% with 22.22% resistance (5.55% ciprofloxacin; 5.55% cotrimoxazole; 5.55% ciprofloxacin + cotrimoxazole; 5.55% amoxicillin/clavulanic acid). In GB2 E. coli 70.02% with 61.90% resistances (30.76% quinolones; 30.76% cotrimoxazole; 30.76% quinolones + cotrimoxazole; 17.69% amoxicillin-clavulanic acid) (P=.0144). CONCLUSIONS: The development of bacterial resistance is more frequent among women with smoking habits and recurrent urinary infections. This could influence a worse response to preventive treatments, either with antibiotics or vaccines.

Impact of organ confined prostate cancer treatment on quality of life. / Impacto en la calidad de vida del tratamiento del cáncer de próstata organoconfinado.

INTRODUCTION: Prostate cancer (PCa) is the second most common male cancer in the world. Its incidence is estimated to grow to 1.7 million new cases and 499,000 new deaths by 2030. Treatment of OCPC can affect patients physically and mentally, as well as their close relationships and their job or career, which conditions health-related quality of life (QoL). OBJECTIVE: Evaluate the impact on QoL attributable to the treatment for Organ Confined Prostate Cancer (OCPC). MATERIALS AND METHODS: Prospective multicenter observational study of 406 patients with OCPC treated from January 2015 to June 2018. The sample was divided into four study groups, according to the type of treatment: radical prostatectomy (RP) (GA), external radiotherapy (ERT) (GB), brachytherapy (BT) (GC) and other treatments different from monotherapy with RP, ERT or BT (GD). RESULTS: The age in GC was lower, the mean Prostate Specific Antigen (PSA) of all patients was 8.13 ng/ml, the group with the highest mean PSA was GB with a mean of 10.43 ng/dL, the mean Tumor Stage (TNM) was 3.82, and GD had the lowest post treatment quality of life. CONCLUSION: OCPC treatment affects QoL. Curative monotherapies, specifically RP and BT, have less effect on QoL than external radiotherapy or other therapeutic alternatives. Urinary incontinence and fistulas secondary to OCPC have the highest impact on QOL impairment. The internationally validated SF 36 questionnaire is a useful cross-sectional measure of QOL to compare the impact of OCPC treatment modalities.

Tyrosine kinase A, C and fibroblast growth factor-2 receptors in bovine embryos cultured in vitro.

Neurotrophins and basic fibroblast growth factor are ligands of tyrosine kinase receptors, though they bind to different tyrosine kinase receptor classes. Neurotrophins bind to receptor tyrosine kinase class VII, Trk receptor family, while basic fibroblast growth factor binds to receptor tyrosine kinase class IV, FGF receptor family. The mammalian uterine tract immunolocalizes neurotrophins and bFGF; therefore their cognate receptors might exert a role during embryonic development. Using RT-PCR, we found mRNA for p75(NTR) TrkA, TrkC and FGFr2 throughout the early bovine embryonic development in vitro. Immunofluorescent staining, assessed by confocal microscopy, showed the expression of TrkA and TrkC proteins in oocytes and all embryonic stages analyzed. We have provided a novel description of TrkA and TrkC proteins, and TrkA, TrkC, p75(NTR) and FGFr2 mRNA expression throughout mammalian embryonic development. This work may help to design future research with neurotrophins in bovine embryo culture and embryonic stem cells.

[Diagnosis of urethritis in men. A 3-year review]. / Diagnóstico microbiológico en varones. Revisión de 3 años.

OBJECTIVES: The aim of this study is to know the prevalence and tendency of microorganisms producing urethritis, in men, in the City Centre of Madrid. METHODS: Cross-sectional study. The urethral samples of 1.248 men were analyzed, for 3 years. The samples were studied for: GRAM stain, when secretion exists; culture in habitual plates; detection of C. trachomatis, U. urealyticum and M. hominis, when there was suspicious, study of T. vaginalis and when suspicious injuries exist, study of virus Herpes simplex. RESULTS: The percentage of positive samples was 22.60%. The isolated microorganisms were: U. urealyticum 7.61%, N. gonorrhoeae 6.33%, C. trachomatis 4.81%, M. hominis 0.24%, H. parainfluenzae 1.76%, H. influenzae 1.12%, Candida spp 0.48%, S. pyogenes 0.16% and Herpes virus simplex (2) 0.08%. Two or more microorganisms were isolated in 1.68%. The percentage of positive samples in 2003 was 17.41% and N. gonorrhoeae the most frequent microorganism (6.22%). In 2004 was 25.57% and the most frequent U. urealyticum (10.18%). In 2005 the 24.50% of the samples were positive and U. urealyticum the most frequent (7.92%). The 79.41% of N. gonorrhoeae were susceptible to all antibiotics tested. It is not found resistance to ceftriaxone, claritromicine and amoxicilline/clavulanic acid. The 11.76% were betalactamase- producing. The 26.47% of Haemophilus spp. were betalactamase- producing and all strains were susceptible to cefotaxime. CONCLUSIONS: The isolated microorganisms most frequently were: U. urealyticum, N. gonorrhoeae and C. trachomatis. There is an increase of 7% of prevalence between the years 2003 and 2005. Ceftriaxone, claritromicine and amoxicilline/clavulanic acid were susceptible to all the strains studied and cefotaxime to all Haemophilus spp.

Disrupted IRF6-NME1/2 Complexes as a Cause of Cleft Lip/Palate.

Mutations and common polymorphisms in interferon regulatory factor 6 ( IRF6) are associated with both syndromic and nonsyndromic forms of cleft lip/palate (CLP). To date, much of the focus on this transcription factor has been on identifying its direct targets and the gene regulatory network in which it operates. Notably, however, IRF6 is found predominantly in the cytoplasm, with its import into the nucleus tightly regulated like other members of the IRF family. To provide further insight into the role of IRF6 in the pathogenesis of CLP, we sought to identify direct IRF6 protein interactors using a combination of yeast 2-hybrid screens and co-immunoprecipitation assays. Using this approach, we identified NME1 and NME2, well-known regulators of Rho-type GTPases, E-cadherin endocytosis, and epithelial junctional remodeling, as bona fide IRF6 partner proteins. The NME proteins co-localize with IRF6 in the cytoplasm of primary palatal epithelial cells in vivo, and their interaction with IRF6 is significantly enhanced by phosphorylation of key serine residues in the IRF6 C-terminus. Furthermore, CLP associated IRF6 missense mutations disrupt the ability of IRF6 to bind the NME proteins and result in elevated activation of Rac1 and RhoA, compared to wild-type IRF6, when ectopically expressed in 293T epithelial cells. Significantly, we also report the identification of 2 unique missense mutations in the NME proteins in patients with CLP (NME1 R18Q in an IRF6 and GRHL3 mutation-negative patient with van der Woude syndrome and NME2 G71V in a patient with nonsyndromic CLP). Both variants disrupted the ability of the respective proteins to interact with IRF6. The data presented suggest an important role for cytoplasmic IRF6 in regulating the availability or localization of the NME1/2 complex and thus the dynamic behavior of epithelia during lip/palate development.

Basic fibroblast growth factor protects cerebellar neurons in primary culture from NMDA and non-NMDA receptor mediated neurotoxicity.

We have investigated the ability of bFGF to protect cerebellar neurons from neurotoxicity by excitatory amino acids. We have found that preincubation with 1-2.5 nM bFGF for 1-6 days significantly protected neurons from excitotoxic damage via NMDA receptors as well as ionotropic non-NMDA receptors. bFGF neuroprotection appeared not to be dependent upon neuronal differentiation and was not mimicked by other neurotrophins including BDNF, NT-3 and NGF. A greater rise in extracellular calcium-dependent cGMP formation, following either depolarization or excitatory amino acid receptor activation was observed in bFGF-pretreated neurons. We suggest that neuroprotection from excitotoxicity following bFGF treatment may be associated to the modulation of neurochemical pathways dependent upon extracellular calcium influx.

Inhibition of protein phosphatases induces IGF-1-blocked neurotrophin-insensitive neuronal apoptosis.

We have previously described the marine toxin okadaic acid (OKA) to be a potent neurotoxin for cultured rat cerebellar neurons. Here we show that OKA-induced neurodegeneration involves the DNA fragmentation characteristic of apoptosis and is protein synthesis-dependent. DNA fragmentation and neurotoxicity correlated with inhibition of protein phosphatase (PP) 2A rather than PP1 activity. Neurotrophins NT-3 and BDNF failed to protect from OKA-induced apoptotic neurotoxicity that was, however, totally prevented by insulin-like growth factor-1. Neuronal death by OKA was significantly reduced by protein kinase C inhibitors and by the L-type calcium channel agonist Bay K8644, while it was potentiated by the reduction of free extracellular calcium concentrations.

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine.

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity. Nefopam (100 microM) effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the VSSC activator veratridine. Delayed neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. In contrast, excitotoxicity following direct exposure of neurons to glutamate was not affected. Neuroprotection by nefopam was dose-dependent. 50% protection was obtained at 57 microM while full neuroprotection was achieved at 75 microM nefopam. Veratridine-induced sodium influx was completely abolished in nefopam-treated neurons. Intracellular cGMP and oxygen radical formation following VSSC stimulation by veratridine were also effectively prevented by nefopam. Our data are consistent with an inhibitory action of nefopam on VSSC and suggest that nefopam may modulate the release of endogenous glutamate following activation of these channels. This novel action of nefopam may be of great interest for the treatment of neurodegenerative disorders involving excessive glutamate release and neurotransmission.

Terfenadine prevents NMDA receptor-dependent and -independent toxicity following sodium channel activation.

Exposure of cultured cerebellar neurons to terfenadine prevented the N-methyl-D-aspartate (NMDA) receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. Delayed neurotoxicity by veratridine (24 h) occurring independently from NMDA receptor activation was also prevented by terfenadine. Terfenadine did not protect from excitotoxicity following direct exposure of neurons to glutamate. Our results suggest that terfenadine may modulate endogenous glutamate release following activation of VSSCs.

Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death.

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 microM) or glutamate (15 microM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 microM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25 microM), oxatomide (20 microM), and triprolidine (50 microM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (+)-alpha-tocopherol (200 microM) or the endogenous antioxidant melatonin (200 microM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.

Domoic acid-containing toxic mussels produce neurotoxicity in neuronal cultures through a synergism between excitatory amino acids.

In 1987, an intoxication by cultured mussels produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. The intoxication was attributed to the presence in mussels of domoic acid, a rare excitatory amino acid acting at the non-NMDA receptor. We now report that a domoic acid-containing mussel extract is more neurotoxic for cultured neurons than purified domoic acid. Moreover, we show that this increase in neurotoxicity is selectively due to domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high concentrations in mussel tissue. We also show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity, and we present evidence suggesting that the neurotoxic synergism may occur through a reduction of the voltage-dependent Mg2+ block at the NMDA receptor-associated channel, following activation of non-NMDA receptors by domoic acid. Thus, based on our results, we suggest that the contemporary presence in the brain of concentrations of domoic acid insufficient alone to be toxic, together with excitatory amino acids, of endogenous and eventually of diet-related origin, may have been relevant in the occurrence of the neurological problems reported.

Aluminum-induced degeneration of astrocytes occurs via apoptosis and results in neuronal death.

The mechanisms by which aluminum interacts with the nervous system are only partly understood. In this study, we used cultured astrocytes and neurons to investigate the effects of long exposures to aluminum (1 mM). We found that aluminum accumulated both in neurons and astrocytes. After 8-12 days exposure, aluminum caused strong changes in the morphology of astrocytes including shrinkage of cell bodies and retraction of processes. Exposures over 15-18 days reduced astrocytes viability by 50%. Aluminum-induced degeneration of astrocytes involved the DNA fragmentation characteristic of apoptosis, and staining of aluminum-treated astrocytes with the DNA-binding fluorochrome Hoeschst 33258 revealed the typical apoptotic condensation and fragmentation of chromatin. Aluminum was also found to be neurotoxic, causing first (4-6 days) abnormal clustering and aggregation, and later (8-12 days) neuronal death. Interestingly, aluminum neurotoxicity occurred in neuroglial cultures containing approximately 10% astrocytes but not in near-pure neuronal cultures containing only 1% astrocytes. Staining of co-cultured cells with Hoeschst 33258 showed apoptotic condensation and fragmentation of chromatin in aluminum-treated astrocytes but not in co-cultured neurons. Our study demonstrates that aluminum can induce the apoptotic degeneration of astrocytes, and that this toxicity is critical in determining neuronal degeneration and death. Aluminum-mediated apoptosis of cultured astrocytes may be also a valuable model system to study the mechanisms underlying apoptosis in glial cells.

Two components in neurotoxicity by L-2-amino-3-phosphonopropionate in cultured cerebellar neurons.

Exposure of cultured cerebellar neurons to the putative metabotropic glutamate receptor antagonist L-2-amino-3-phosphonopropionate (L-AP3) for 24 h produced a neurotoxic effect which was prevented by the addition of the NMDA receptor antagonist (+)-10,11-dihydro-5-methyl-5-H-dibenzo-[a,d]-cyclohepten-5,1 0-imine hydrogen maleate (MK-801). MK-801 did also reduce neurotoxicity following 72 h exposure to L-AP3 neurotoxicity in the presence of MK-801 was antagonized by glutamate. Our results suggest that metabotropic glutamate receptors may play an important role in neuronal survival by controlling NMDA receptor-dependent as well as independent pathways.

Beta-amyloid precursor protein in human digital skin.

The occurrence and distribution of beta-amyloid precursor protein (beta APP) and of beta-amyloid peptide (beta/A4) was investigated using immunoblotting and immunohistochemical techniques in the digital skin of healthy adult subjects. beta APP-like proteic bands with apparent molecular masses between 55-60 kDa, 100-125 kDa (corresponding to the full-length beta APP isoforms), 145-150 kDa, and 200 kDa were found in pellets and supernatants of whole skin and dermis. The same proteins, except that of approximately 200 kDa, were also found in pellets from the epidermis, whereas epidermic supernatants were unreactive. beta/A4 was not found by immunoblotting. Light microscope immunohistochemistry showed beta APP immunoreactivity (IR) in: (a) dermal nerves; (b) lamellar cells of Meissner, as well as inner-core, outer-core and capsule of Pacinian corpuscles; and (c) dermal blood vessels, sweat glands and, occasionally, epidermis. The distribution of beta/A4 IR matched that of beta APP, and no evidence of extracellular beta/A4 IR was encountered. Present results demonstrate that beta APP, but not beta/A4, is normally present in human glabrous (digital) skin. The potential clinical relevance of these findings is discussed.

Expression of beta-amyloid precursor protein (APP) in human dorsal root ganglia.

The present study reports the occurrence and localization of beta-amyloid precursor protein (APP) immunoreactivity (IR) in human lumbar dorsal root ganglia of healthy adult subjects (age range 25-43 years). To ascertain that ganglionic cells displayed APP IR, neurofilament (NFP) and S-100 proteins (S100P) were studied in parallel. Immunoblotting revealed four or five major proteins with apparent molecular masses between 100-125 kDa, which corresponded with the different full-length APP isoforms. Moreover, an additional protein of approximately 55 kDa was detected. Selective APP IR was observed restricted to the satellite glial cell cytoplasms whereas neuron cell bodies resulted unlabeled. Moreover, some intraganglionic nerve fibers also displayed APP IR, apparently labelling Schwann cells. No individual differences among subjects were observed neither in the pattern of APP IR distribution, nor in the intensity of APP IR. Although it remains to be demonstrated whether or not human primary sensory neurons express APP, present results strongly suggest that supporting glial cells may be a primary source of APP or any related peptide, at least in adult healthy people. The functional and clinical relevance of these findings, if any, remain to be clarified.