Post-ischemic reorganization of sensory responses in cerebral cortex.

Introduction: Sensorimotor integration is critical for generating skilled, volitional movements. While stroke tends to impact motor function, there are also often associated sensory deficits that contribute to overall behavioral deficits. Because many of the cortico-cortical projections participating in the generation of volitional movement either target or pass-through primary motor cortex (in rats, caudal forelimb area; CFA), any damage to CFA can lead to a subsequent disruption in information flow. As a result, the loss of sensory feedback is thought to contribute to motor dysfunction even when sensory areas are spared from injury. Previous research has suggested that the restoration of sensorimotor integration through reorganization or de novo neuronal connections is important for restoring function. Our goal was to determine if there was crosstalk between sensorimotor cortical areas with recovery from a primary motor cortex injury. First, we investigated if peripheral sensory stimulation would evoke responses in the rostral forelimb area (RFA), a rodent homologue to premotor cortex. We then sought to identify whether intracortical microstimulation-evoked activity in RFA would reciprocally modify the sensory response. Methods: We used seven rats with an ischemic lesion of CFA. Four weeks after injury, the rats' forepaw was mechanically stimulated under anesthesia and neural activity was recorded in the cortex. In a subset of trials, a small intracortical stimulation pulse was delivered in RFA either individually or paired with peripheral sensory stimulation. Results: Our results point to post-ischemic connectivity between premotor and sensory cortex that may be related to functional recovery. Premotor recruitment during the sensory response was seen with a peak in spiking within RFA after the peripheral solenoid stimulation despite the damage to CFA. Furthermore, stimulation in RFA modulated and disrupted the sensory response in sensory cortex. Discussion: The presence of a sensory response in RFA and the sensitivity of S1 to modulation by intracortical stimulation provides additional evidence for functional connectivity between premotor and somatosensory cortex. The strength of the modulatory effect may be related to the extent of the injury and the subsequent reshaping of cortical connections in response to network disruption.

Post-Ischemic Reorganization of Sensory Responses in Cerebral Cortex.

Sensorimotor integration is critical for generating skilled, volitional movements. While stroke tends to impact motor function, there are also often associated sensory deficits that contribute to overall behavioral deficits. Because many of the cortico-cortical projections participating in the generation of volitional movement either target or pass-through primary motor cortex (in rats, caudal forelimb area; CFA), any damage to CFA can lead to a subsequent disruption in information flow. As a result, the loss of sensory feedback is thought to contribute to motor dysfunction even when sensory areas are spared from injury. Previous research has suggested that the restoration of sensorimotor integration through reorganization or de novo neuronal connections is important for restoring function. Our goal was to determine if there was crosstalk between sensorimotor cortical areas with recovery from a primary motor cortex injury. First, we investigated if peripheral sensory stimulation would evoke responses in the rostral forelimb area (RFA), a rodent homologue to premotor cortex. We then sought to identify whether intracortical microstimulation-evoked activity in RFA would reciprocally modify the sensory response. We used seven rats with an ischemic lesion of CFA. Four weeks after injury, the rats' forepaw was mechanically stimulated under anesthesia and neural activity was recorded in the cortex. In a subset of trials, a small intracortical stimulation pulse was delivered in RFA either individually or paired with peripheral sensory stimulation. Our results point to post-ischemic connectivity between premotor and sensory cortex that may be related to functional recovery. Premotor recruitment during the sensory response was seen with a peak in spiking within RFA after the peripheral solenoid stimulation despite the damage to CFA. Furthermore, stimulation evoked activity in RFA modulated and disrupted the sensory response in sensory cortex, providing additional evidence for the transmission of premotor activity to sensory cortex and the sensitivity of sensory cortex to premotor cortex's influence. The strength of the modulatory effect may be related to the extent of the injury and the subsequent reshaping of cortical connections in response to network disruption.

[Not all low back pain is renal colic]. / No todo dolor lumbar es un cólico nefrítico.

Low back pain is one of the most frequent reasons for consultation in primary care. Sometimes it is not easy to make a good differential diagnosis, because the number of pathologies that can express themselves with such symptoms is very wide and a complementary test or referral to the reference hospital is not always indicated. The following is a clinical case of a 59-year-old female patient with a history of dyslipidemia treated with ezetimibe, who consulted again for breakthrough low back pain, which was finally diagnosed as a penetrating ulcer of the abdominal aorta, treated endovascularly by the vascular surgery service. The clinical case aims to provide an overview of the acute treatment of aortic syndrome, showing the differences compared to the management of other common pathologies in primary care with the same clinical expression.

Reproductive biology of wild and domesticated Ensete ventricosum: Further evidence for maintenance of sexual reproductive capacity in a vegetatively propagated perennial crop.

Loss of sexual reproductive capacity has been proposed as a syndrome of domestication in vegetatively propagated crops, but there are relatively few examples from agricultural systems. In this study, we compare sexual reproductive capacity in wild (sexual) and domesticated (vegetative) populations of enset (Ensete ventricosum (Welw.) Cheesman), a tropical banana relative and Ethiopian food security crop. We examined floral and seed morphology and germination ecology across 35 wild and domesticated enset. We surveyed variation in floral and seed traits, including seed weight, viability and internal morphology, and germinated seeds across a range of constant and alternating temperature regimes to characterize optimum germination requirements. We report highly consistent floral allometry, seed viability, internal morphology and days to germination in wild and domesticated enset. However, seeds from domesticated plants responded to cooler temperatures with greater diurnal range. Shifts in germination behaviour appear concordant with a climatic envelope shift in the domesticated distribution. Our findings provide evidence that sexual reproductive capacity has been maintained despite long-term near-exclusive vegetative propagation in domesticated enset. Furthermore, certain traits such as germination behaviour and floral morphology may be under continued selection, presumably through rare sexually reproductive events. Compared to sexually propagated crops banked as seeds, vegetative crop diversity is typically conserved in living collections that are more costly and insecure. Improved understanding of sexual propagation in vegetative crops may have applications in germplasm conservation and plant breeding.

Efficacy of combinations of colistin with other antimicrobials involves membrane fluidity and efflux machinery.

OBJECTIVE: Despite its use was abandoned several decades ago, the polycationic peptide colistin has become the last hope to treat severe infections caused by multidrug-resistant Gram-negative bacteria. Thus, the development of colistin resistance may seriously compromise the efficacy of treatment. Moreover, colistin has high toxicity being dose dependent. A potentially effective strategy to avoid resistance may be to combine colistin with other antimicrobials. This may help in the rescue of old antimicrobials and in reducing toxic undesired effects. METHODS: Antimicrobial susceptibility determination, efflux machinery function measurements in different conditions and measurement of inhibition of the extrusion by colistin were performed. Moreover, modifications of anisotropy of the membranes by using fluorescent dyes was accomplished. RESULTS: Sub-inhibitory concentrations of colistin have a synergistic effect with several antimicrobials that act intracellularly (targeting protein synthesis and DNA replication). This effect was demonstrated through the uptake increases of acridine orange. in Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii but also in an intrinsically colistin-resistant species as Serratia marcescens. Measurements of the anisotropy of bacterial membranes, as a measure of membrane fluidity, showed significant changes indicative of colistin activity. CONCLUSION: The alterations in the cellular efflux machinery that resulted in higher intracellular concentrations of acridine orange, and likely of other antimicrobials combined with data of membrane fluidity and measured synergism in vitro allow us to envisage the use of these combinations to fight infections caused by multidrug-resistant bacteria.

Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma.

BACKGROUND: Pharmacological inhibition of B cell receptor (BCR) signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton's kinase (Btk) and spleen tyrosine kinase (Syk) inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents. METHODS: We evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn), in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma. RESULTS: IQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest. CONCLUSIONS: These results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.

Combined force spectroscopy, AFM and calorimetric studies to reveal the nanostructural organization of biomimetic membranes.

In this work we studied a binary lipid matrix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), a composition that mimics the inner membrane of Escherichia coli. More specifically, liposomes with varying fractions of POPG were analysed by differential scanning calorimetry (DSC) and a binary phase diagram of the system was created. Additionally, we performed atomic force microscopy (AFM) imaging of supported lipid bilayers (SLBs) of similar compositions at different temperatures, in order to create a pseudo-binary phase diagram specific to this membrane model. AFM study of SLBs is of particular interest, as it is conceived as the most adequate technique not only for studying lipid bilayer systems but also for imaging and even nanomanipulating inserted membrane proteins. The construction of the above-mentioned phase diagram enabled us to grasp better the thermodynamics of the thermal lipid transition from a gel-like POPE:POPG phase system to a more fluid phase system. Finally, AFM force spectroscopy (FS) was used to determine the nanomechanics of these two lipid phases at 27°C and at different POPG fractions. The resulting data correlated with the specific composition of each phase was calculated from the AFM phase diagram obtained. All the experiments were done in the presence of 10 mM of Ca(2+), as this ion is commonly used when performing AFM with negatively charged phospholipids.

Ontogeny of sensorimotor gating and immune impairment induced by prenatal immune challenge in rats: implications for the etiopathology of schizophrenia.

It has been hypothesized that the maternal immune response to infection may influence fetal brain development and lead to schizophrenia. Animal experimentation has supported this notion by demonstrating altered sensorimotor gating (prepulse inhibition, PPI) in adult rats prenatally exposed to an immune challenge. In the present study, pregnant rats were exposed to the bacterial endotoxin lipopolysaccharide (LPS) throughout gestation and the offspring were examined by evaluating the PPI, dopaminergic function, brain protein expression and cytokine serum levels from weaning to late adulthood. Prenatal LPS exposure induced a deficit in PPI that emerged at 'puberty' and that persisted throughout adult life. This prenatal insult caused age-specific changes in accumbal dopamine levels and in synaptophysin expression in the frontal cortex. Moreover, serum cytokine levels were altered in an age- and cytokine-dependent manner. Here we show that prenatal LPS administration throughout pregnancy causes maturation-dependent PPI deficits and age-dependent alterations in dopamine activity, as well as in synaptophysin expression and cytokine levels.

A cannabinoid agonist interferes with the progression of a chronic model of multiple sclerosis by downregulating adhesion molecules.

Adhesion molecules are critical players in the regulation of transmigration of blood leukocytes across the blood-brain barrier in multiple sclerosis (MS). Cannabinoids (CBs) are potential therapeutic agents in the treatment of MS, but the mechanisms involved are only partially known. Using a viral model of MS we observed that the cannabinoid agonist WIN55,212-2 administered at the time of virus infection suppresses intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in brain endothelium, together with a reduction in perivascular CD4+ T lymphocytes infiltrates and microglial responses. WIN55,212-2 also interferes with later progression of the disease by reducing symptomatology and neuroinflammation. In vitro data from brain endothelial cell cultures, provide the first evidence of a role of peroxisome proliferator-activated receptors gamma (PPARgamma) in WIN55,212-2-induced downregulation of VCAM-1. This study highlights that inhibition of brain adhesion molecules by WIN55,212-2 might underline its therapeutic effects in MS models by targeting PPAR-gamma receptors.

Porphycenes: facts and prospects in photodynamic therapy of cancer.

The photodynamic process induces cell damage and death by the combined effect of a photosensitizer (PS), visible light, and molecular oxygen, which generate singlet oxygen ((1)O(2)) and other reactive oxygen species that are responsible for cytotoxicity. The most important application of this process with increasing biomedical interest is the photodynamic therapy (PDT) of cancer. In addition to hematoporphyrin-based drugs, 2nd generation PSs with better photochemical properties are now studied using cell cultures, experimental tumors and clinical trials. Porphycene is a structural isomer of porphyrin and constitutes an interesting new class of PS. Porphycene derivatives show higher absorption than porphyrins in the red spectral region (lambda > 600 nm, epsilon > 50000 M-(1)cm(-1)) owing to the lower molecular symmetry. Photophysical and photobiological properties of porphycenes make them excellent candidates as PSs, showing fast uptake and diverse subcellular localizations (mainly membranous organelles). Several tetraalkylporphycenes and the tetraphenyl derivative (TPPo) induce photodamage and cell death in vitro. Photodynamic treatments of cultured tumor cells with TPPo and its palladium(II) complex induce cytoskeletal changes, mitotic blockage, and dose-dependent apoptotic or necrotic cell death. Some pharmacokinetic and phototherapeutic studies on experimental tumors after intravenous or topical application of lipophilic alkyl-substituted porphycene derivatives are known. Taking into account all these features, porphycene PSs should be very useful for PDT of cancer and other biomedical applications.

[Cannabinoid system and neuroinflammation:therapeutic perspectives in multiple sclerosis]. / El sistema cannabinoide en situaciones de neuroinflamación: perspectivas terapéuticas en la esclerosis múltiple.

INTRODUCTION: The endocannabinoid system consists of cannabinoid receptors, endogenous ligands and the enzymatic elements involved in their synthesis and breakdown. AIM: To report on currently held knowledge about the functioning of the system as a modulator of the neuroinflammatory processes associated with chronic diseases such as multiple sclerosis. DEVELOPMENT: Cannabinoids are synthesised and released on demand and their production increases in times of neuroinflammation and neural damage. In this context then, their actions in the microglial cells and in the astrocytes are characterised by a lowered expression of inflammatory mediators and pro-inflammatory cytokines. Furthermore, cannabinoids can play a role as neuroprotectors by means of different types of mechanisms and, in experimental models of multiple sclerosis, they slow down the symptoms, reduce inflammation and can favour remyelination. CONCLUSIONS: The clinical use of cannabinoids or pharmacological agents that affect the endogenous cannabinoid system during inflammation of the central nervous system and in multiple sclerosis is currently under consideration and subject to debate. Detailed analysis of the results obtained over the past decade has made it possible to establish the existence of several mechanisms of action of cannabinoids in pathologies affecting the central nervous system that are accompanied by chronic inflammation. Likewise, they also clearly show that the cannabinoid system is an interesting proposal as a new therapeutic tool.

Recent advances in combinatorial chemistry applied to development of anti-HIV drugs.

A compilation of combinatorial chemistry techniques applied to anti-HIV drug development is presented in this review. This synthetic strategy together with high throughput screening assays has allowed the discovery and optimization of novel lead anti-HIV compounds.

Necrotic cell death induced by photodynamic treatment of human lung adenocarcinoma A-549 cells with palladium(II)-tetraphenylporphycene.

In this study we describe photodamaging and photokilling effects of palladium(II)-tetraphenylporphycene (PdTPPo) (previously incorporated into dipalmitoylphosphatidylcholine liposomes) on the human lung adenocarcinoma A-549 cell line. No dark cytotoxicity was found when the drug was applied at 10(-6) M or 5 x 10(-7) M for 1 or 18 h, respectively. After 1-h treatment with 10(-7) M or 5 x 10(-7) M PdTPPo followed by red light irradiation for variable times, dose-dependent lethal effects were observed in A-549 cells. Apoptosis was not found after the above photodynamic treatments or under even milder sublethal conditions. In contrast to HeLa cells subjected to PdTPPo photosensitization where either apoptosis or necrosis were induced, morphological analysis and electrophoretical DNA pattern of A-549 cells always revealed a clearly necrotic death mechanism. However, A-549 cells died by apoptosis after serum and L-glutamine deprivation, indicating that only the photodynamically induced apoptosis was inhibited. Immunofluorescent labeling revealed that microtubules and actin microfilaments were immediately and strongly damaged by photodynamic treatments with PdTPPo. No metaphase arrest and/or mitotic alterations were observed after phototreatments. Present results show that the cell type plays a fundamental role in relation to the apoptotic or necrotic response to photosensitization, and that cytoskeletal components are important targets implicated in cell death processes.

Selective hydrolysis of 2,4-diaminopyrimidine systems: a theoretical and experimental insight into an old rule.

Hydrolysis of the amino groups in condensed 2,4-diaminopyrimidine systems (1) has been used as a common method for the synthesis of oxo-substituted pyrimidines. In particular, the treatment with 6 M HCl usually yields exclusively the 2-amino-4-oxopyrimidine isomer (2). During our work, we found that the hydrolysis of the amino groups present in some condensed 2,4-diaminopyrimidine systems unexpectedly afforded exclusively the 4-amino-2-oxopyrimidine isomer (3). In this paper, we present the experimental work and ab initio calculations carried out to understand this discrepancy. As a part of such study, eight compounds containing a 2,4-diaminopyrimidine moiety were calculated in gas phase and in aqueous solution, and some acid hydrolyses were reexamined. Results showed that the presence of an electron-donating nitrogen linked to C6 of the 2,4-diaminopyrimidine ring changes the preferred hydrolysis site to yield the 4-amino-2-oxopyrimidine isomer.

Synthesis and biological activity of 7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF) and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).

We recently described the syntheses of 12a-c, 4-amino-7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF), and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), in six steps from commercially available p-substituted methyl benzoates in 20-27% overall yields. Such analogues were tested in vitro against CCRF-CEM leukemia cells and showed that they are completely devoid of any activity, the IC(50) being higher than 20 microg/mL for all cases. To clarify if the presence of the carbonyl group in position C7, the distinctive feature of our synthetic methodology, is the reason for this lack of activity, we have now obtained the 7-oxo substituted analogues of 5-DATHF and DDATHF, 18a-c, in 10-30% overall yield. Testing of 18a-c in vitro against CCRF-CEM leukemia cells revealed that these compounds are totally inactive. A molecular modeling study of 18b inside the active site of the complex E. coliGARTFase-5-DATHF-GAR pointed to an electronic repulsion between the atoms of the 7-oxo group and the carbonyl group of Arg90 as a possible explanation for the inactivity of 18a-c.

Determination of the partition coefficients of a homologous series of ciprofloxacin: influence of the N-4 piperazinyl alkylation on the antimicrobial activity.

Partitioning of a fluoroquinolone antibiotic, ciprofloxacin, and its N-piperazinyl alkyl derivatives, between octanol or Escherichia coli lipid membrane extract and aqueous buffer pH 7.4, was studied. The experimental partition coefficients (Pexp) were corrected at this pH using an expression that includes the microconstant values of each compound. The relationship between the corrected partition coefficients expressed as logP (thermodynamic partition coefficient) and the diffusion through the lipid bilayers ('hydrophobic pathway') of entry has been considered here. In this work, we have explored the possibility of using our model to provide physicochemical evidences to support such a via. The correlation between logP values and antibacterial activities (expressed as minimal inhibitory concentration (MIC) values) of the homologous series of antibiotics against different bacteria were studied. A parabolic behaviour was observed which evidenced that the only increase in lipophilicity does not result in an enhanced antimicrobial activity for the homologous family studied.

Determination by fluorimetric titration of the ionization constants of ciprofloxacin in solution and in the presence of liposomes.

A fluorescence titration method was applied for the determination of pKa of ciprofloxacin (CPX) in solution. Values of 6.18 +/- 0.05 and 8.76 +/- 0.03 were obtained for pKa1 and pKa2, respectively. The method was used to determine the ionization constants in the presence of liposomes of dipalmitoylphosphatidylcholine (DPPC) and DPPC with 10 mol% of dipalmitoylphosphatidylglycerol. A dependence on the surface charge of liposomes was found which supported the existence of a basic electrostatic interaction between CPX and the phospholipid bilayer. Both pK values for the N-4 butyl-piperazinyl derivative (BCPX) of the parent compound were also determined in solution and in the presence of liposomes. The competition of both drugs for the same binding site as 1-anilino-8-naphtalene sulfonate demonstrate that the interaction is governed by electrostatic forces.

Evidence of an efflux pump in Serratia marcescens.

Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10(-7) to 10(-9). Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug.

Role of the outer membrane in the accumulation of quinolones by Serratia marcescens.

Accumulation of four quinolones by Serratia marcescens was measured fluorometrically. The passage of quinolones through the outer membrane was studied in both lipopolysaccharide-deficient and porin-deficient mutants. The lipopolysaccharide (LPS) layer formed a partially effective barrier for highly hydrophobic quinolones such as nalidixic acid. Quinolones with a low relative hydrophobicity coefficient seemed to pass preferentially through the water-filled Omp3 porin channels. Results were confirmed when Omp3 was cloned in a porin-defective Escherichia coli.

Photosensitizing properties of palladium-tetraphenylporphycene on cultured tumour cells.

In this study we describe photokilling properties and effects on the mitotic index (MI) of cultured HeLa cells induced by palladium(II)-tetraphenylporphycene (PdTPP(0)). The drug was synthesized by refluxing tetraphenylporphycene (TPP(0)) and PdCl2 in dimethylformamide, followed by evaporation and purification by chromatography. Cells were treated with different concentrations of PdTPPo incorporated into dipalmitoylphosphatidylcholine liposomes, and red light irradiation (lambda > 600 nm) was performed at 21 mW/cm2. No dark toxicity was found when the drug was applied under our experimental conditions. Using lethal (LD(100)) treatments, cells showed the immediate occurrence of bubbles on the plasma membrane, whereas homogeneous nuclear condensation and loss of cytoplasm appeared 3-24 h later. An increased MI was found 6-8 h after sublethal LD(25) and LD(40) treatments as well as a high proportion of abnormal metaphases with altered spindle microtubules. Chromatin condensation and fragmentation were observed 8 h after LD(75) treatments. These results show that in comparison with TPP(0), the new sensitizer PdTPPo has more efficient photokilling properties which could be very valuable for the photodynamic therapy of cancer.