DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence.

BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.

Effect of Brown Seaweed (Macrocystis pyrifera) Addition on Nutritional and Quality Characteristics of Yellow, Blue, and Red Maize Tortillas.

The objective of this study was to analyze the effect of incorporating Macrocystis pyrifera into yellow, blue, and red maize masa and tortillas. The nutritional composition and mineral content of tortillas was determined, and the color, texture, total phenolic compounds (TPC), and antioxidant capacity of masas and tortillas were measured. The addition of seaweed led to a significant decrease in moisture and a significant increase in ash, protein, and fiber, while no differences were observed in the lipid and carbohydrate content. There was a significant increase in all analyzed minerals (Na, Ca, P, K, and Mg). Tortillas weighed 24.54 ± 1.02 g, had a diameter of 11.00 ± 0.79 cm, and a thickness of 0.32 ± 0.09 cm. All color parameters were significantly affected by seaweed concentration. The hardness of the masas was 2.18-22.32 N, and the values of the perforation test of the tortillas were 1.40-4.55 N. The TPC of the masas and tortillas was measured in water and methanol:water extracts. Results were higher in the water extracts (1141.59-23,323.48 mg GAE/100 g masa and 838.06-2142.34 mg GAE/100 g tortilla). Antioxidant capacity (ORAC) was higher for methanol:water extracts (14,051.96-44,928.75 µmol TE/100 g masa and 14,631.47-47,327.69 µmol TE/100 g tortilla).

TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells.

A conditional knock-in mouse was generated in which the TAK1 catalytic subunit was largely replaced by the kinase-inactive TAK1[D175A] mutant in immune cells. The activation of p38α MAP kinase, c-Jun N-terminal kinases 1 and 2 (JNK1/2) and the canonical IKK complex induced by stimulation with several TLR-activating ligands was reduced in bone marrow-derived macrophages (BMDM) from TAK1[D175A] mice. TLR signalling in TAK1[D175A] BMDM was catalysed by the residual wild-type TAK1 in these cells because it was abolished by either of two structurally unrelated TAK1 inhibitors (NG25 and 5Z-7-oxozeaenol) whose off-target effects do not overlap. The secretion of inflammatory mediators and production of the mRNAs encoding these cytokines induced by TLR ligation was greatly reduced in peritoneal neutrophils or BMDM from TAK1[D175A] mice. The Pam3CSK4- or LPS-stimulated activation of MAP kinases and the canonical IKK complex, as well as cytokine secretion, was also abolished in TAK1 knock-out human THP1 monocytes or macrophages. The results establish that TAK1 protein kinase activity is required for TLR-dependent signalling and cytokine secretion in myeloid cells from mice. We discuss possible reasons why other investigators, studying myeloid mice with a conditional knock-out of TAK1 or a different conditional kinase-inactive knock-in of TAK1, reported TAK1 to be a negative regulator of LPS-signalling and cytokine production in mouse macrophages and neutrophils.

Comparative Study of Physicochemical Properties and Biocompatibility (L929 and MG63 Cells) of TiN Coatings Obtained by Plasma Nitriding and Thin Film Deposition.

Ti6Al4V is one of the most lightweight, mechanically resistant, and appropriate for biologically induced corrosion alloys. However, surface properties often must be tuned for fitting into biomedical applications, and therefore, surface modification is of paramount importance to carry on its use. This work compares the interaction between two different cell lines (L929 fibroblasts and osteoblast-like MG63) and medical grade Ti6Al4V after surface modification by plasma nitriding or thin film deposition. We studied the adhesion of these two cell lines, exploring which trends are consistent for cell behavior, correlating with osseointegration and in vivo conditions. Modified surfaces were analyzed through several physicochemical characterization techniques. Plasma nitriding led to a more pronounced increase in surface roughness, a thicker aluminum-free layer, made up of diverse titanium nitride phases, whereas thin film deposition resulted in a single-phase pure titanium nitride layer that leveled the ridged topography. The selective adhesion of osteoblast-like cells over fibroblasts was observed in nitrided samples but not in thin film deposited films, indicating that the competitive cellular behavior is more pronounced in plasma nitrided surfaces. The obtained coatings presented an appropriate performance for its use in biomedical-aimed applications, including the possibility of a higher success rate in osseointegration of implants.

White matter disturbances in phenylketonuria: Possible underlying mechanisms.

White matter pathologies, as well as intellectual disability, microcephaly, and other central nervous system injuries, are clinical traits commonly ascribed to classic phenylketonuria (PKU). PKU is an inherited metabolic disease elicited by the deficiency of phenylalanine hydroxylase. Accumulation of l-phenylalanine (Phe) and its metabolites is found in tissues and body fluids in phenylketonuric patients. In order to mitigate the clinical findings, rigorous dietary Phe restriction constitutes the core of therapeutic management in PKU. Myelination is the process whereby the oligodendrocytes wrap myelin sheaths around the axons, supporting the conduction of action potentials. White matter injuries are implicated in the brain damage related to PKU, especially in untreated or poorly treated patients. The present review summarizes evidence toward putative mechanisms driving the white matter pathology in PKU patients.

Novel Alkyl(aryl)-Substituted 2,2-Difluoro-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides: Synthesis, Antimicrobial Activity, and CT-DNA Binding Evaluations.

The synthesis, antimicrobial activity evaluations, biomolecule-binding properties (DNA), and absorption and emission properties of a new series of (Z)-1,1,1-trichloro-4-alkyl(aryl)amino-4-arylbut-3-en-2-ones (4, 5) and 2,2-difluoro-3-alkyl(aryl)amino-4-aryl-6-(trichloromethyl)-2H-1,3,2-oxazaborinin-3-ium-2-uides (6, 7) in which 3(4)-alkyl(aryl) = H, Me, iso-propyl, n-butyl, C6H5, 4-CH3C6H4, 4-CH3OC6H4, 4-NO2C6H4, 4-FC6H4, 4-BrC6H4, 2-naphthyl, is reported. A series of ß-enaminoketones (4, 5) is synthesized from the O,N-exchange reaction of some amines (3) with (Z)-1,1,1-trichloro-4-methoxy-4-aryl-but-3-en-2-ones (1, 2) at 61-90% yields. Subsequently, reactions of the resulting ß-enaminoketones with an appropriate source of boron (BF3.OEt2) gave the corresponding oxazaborinine derivatives (6, 7) at 50-91% yields. UV-Vis and emission properties of biomolecule-binding properties for the DNA of these new BF2-ß-enamino containing CCl3 units were also evaluated. Some compounds from the present series also exhibited potent antimicrobial effects on various pathogenic microorganisms at concentrations below those that showed cytotoxic effects. Compounds 4d, 4e, 6e, and 6f showed the best results and are very significant against P. zopfii, which causes diseases in humans and animals.

Hollow cathode plasma nitriding of medical grade Ti6Al4V: A comprehensive study.

Ti6Al4V used in biomedical applications still has several surface-related problems, such as poor bone compatibility and low wear resistance. In this work, the formation of a protective layer of titanium nitride obtained by plasma treatment in hollow cathode was studied, and the best experimental conditions were verified by a statistical factorial design of experiments. The samples were characterized in terms of their physical and chemical properties, correlating the effects of time (min) and temperature (°C). An achieved ideal condition was further analysed in terms of in vitro cytotoxicity, micro-abrasion, and electrochemical properties. The carried-out assessment has shown that nitrided condition has an improvement in wettability, microhardness, along with TixNy formation and roughness increment, when compared to pristine condition.

Investigation of plasma treatment on UHMWPE surfaces: Impact on physicochemical properties, sterilization and fibroblastic adhesion.

Ultra-high molecular weight polyethylene (UHMWPE) is a prevailing bearing material applied in joint arthroplasty. Despite not being a novel biomaterial, its debris as consequence of long application and surface properties usually still lead to short lifespan. Many of the drawbacks are associated with sterilization methods that degrade the surface properties of UHMWPE. This work aims at improving the sterilizing treatment and also increasing material wettability, without losing bulk properties, which are essential for an orthopedic bearing. Cold plasma in hollow cathode setting was used for the material surface functionalization. Samples were characterized through contact angle (WCA), x-ray diffraction (XRD), optical microscopy, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and profilometry. Optimal points based on immediate surface wettability, shelf time and sterilization efficacy were chosen for biocompatibility evaluation. When comparing cell viability through MTT among treated samples (OP1, OP2 and UV), a slight reduction in OP2 viability could be seen after 7 days incubation, which is also observed in Giemsa staining and SEM images. In late incubation, OP1 loses its hydrophilic character and displays higher cell adhesion than its counterparts UV and OP2. At the end, OP2 showed less cells growing over the biomaterial after 7 days exposition compared to OP1 and UV. OP1 presented a more hydrophobic surface and improved cell adhesion, differently from OP2 and UV, which maintained their wettability conditions in late incubation. Cell analysis results indicate that surface wetting influences cell morphology and consequent cell adhesion, in which more hydrophobic surfaces are shown to favor fibroblast adhesion properties.

Amniotic Fluid Cells, Stem Cells, and p53: Can We Stereotype p53 Functions?

In recent years, great interest has been devoted to finding alternative sources for human stem cells which can be easily isolated, ideally without raising ethical objections. These stem cells should furthermore have a high proliferation rate and the ability to differentiate into all three germ layers. Amniotic fluid, ordinarily discarded as medical waste, is potentially such a novel source of stem cells, and these amniotic fluid derived stem cells are currently gaining a lot of attention. However, further information will be required about the properties of these cells before they can be used for therapeutic purposes. For example, the risk of tumor formation after cell transplantation needs to be explored. The tumor suppressor protein p53, well known for its activity in controlling Cell Prolif.eration and cell death in differentiated cells, has more recently been found to be also active in amniotic fluid stem cells. In this review, we summarize the major findings about human amniotic fluid stem cells since their discovery, followed by a brief overview of the important role played by p53 in embryonic and adult stem cells. In addition, we explore what is known about p53 in amniotic fluid stem cells to date, and emphasize the need to investigate its role, particularly in the context of cell tumorigenicity.

p53 Is Active in Human Amniotic Fluid Stem Cells.

Despite increasing interest in human amniotic fluid cells, very little is known about the regulation and function of p53 in this cell type. In this study, we show that undifferentiated human amniotic fluid cells express p53, yet at lower levels than in cancer cells. The p53 protein in amniotic fluid cells is mainly localized in the nuclei, however, its antiproliferative activity is compromised in these cells. Igf2, a maternal imprinted gene, and c-jun, a proto-oncogene, are regulated by p53 in these cells. DNA damage leads to an increase in p53 abundance in human amniotic fluid cells and to transcriptional activation of its target genes. Interestingly, cell differentiation toward the neural lineage leads to p53 induction as differentiation progresses.

Beta-glucuronidase activity in dried blood spots: Reduced technique with biochemical parameters determined.

INTRODUCTION: Mucopolysaccharidoses (MPS) occur due to deficiency in the activity of enzymes that catalyze the breakdown of glycosaminoglycans. MPS VII is caused by deficiency of the beta-glucuronidase enzyme (GUSB). OBJECTIVES: This study aimed to enhance the technique to measure GUSB activity by reducing the amount of reagents and the size of the DBS, as well as to determine some biochemical parameters of enzyme of healthy individuals. METHODS: The measurement of GUSB in 3 and 1.2mm DBS (with reagents reduced 2.5- and fourfold) was correlated and the precision of the technique was tested. Optimal pH, Km and Vmax, and thermostability parameters were determined and time and temperature of sample storage were established. RESULTS: The correlations among the techniques were significant. Although the correlation coefficient was similar, fourfold reduction was selected. pH4.4 had the highest enzyme activity. GUSB's Km was 1.25mM, while Vmax was 594.48nmol/h/mL. After pre-incubation of the sample at 60°C, its activity dropped from 100% to 15.8% at 120min. GUSB activity significantly decreased after 45days of storage at 4, 25, and 37°C. CONCLUSIONS: This research allowed a previously described technique for MPS VII diagnosis to be adapted for smaller amounts of sample and reagents. That will facilitate the use of smaller amounts of samples, which may be used for other techniques and to save material. Given the importance of early MPS VII diagnosis due to the severity of the disease, using reliable diagnostic techniques in DBS is essential.

A new case of "de novo" BRCA1 mutation in a patient with early-onset breast cancer.

Most of the de novo BRCA1/2 mutations have been identified in patients with early-onset breast cancer and without family history of the disease. The identification of these alterations could play a prominent role in the prevention and treatment strategies and may influence clinical management of patients.

Synthesis of novel trifluoromethyl-substituted spiro-[chromeno[4,3-d]pyrimidine-5,1'-cycloalkanes], and evaluation of their analgesic effects in a mouse pain model.

Herein we report the synthesis of twelve 2,5-substituted 4-(trifluoromethyl)-spirochromeno[4,3-d]pyrimidines (7-10), as well as an evaluation of their analgesic effect in a mouse pain model. The nine new chromeno[4,3-d]pyrimidines (7-9) were synthesized from the cyclocondensation reactions of three 2,2,2-trifluoro-1-(4-methoxyspiro[chromene-2,1'-cycloalkane]-3-yl)ethanones (3) containing 5-, 6- and 7-membered spirocycloalkanes, with some well-known amidine salts (4-6) [NH2CR(NH)]-in which R=Me, Ph, and NH2-at yields of 60-95%. Subsequently, three new 2-(pyrrol-1-yl)-4-(trifluoromethyl)-chromeno[4,3-d]pyrimidines (10) were obtained through a Clauson-Kaas reaction between the respective 2-(amino)-4-(trifluoromethyl)-chromeno[4,3-d]pyrimidines (9) and 2,5-dimethoxy-tetrahydrofuran. The analgesic evaluation showed that these 4-(trifluoromethyl)chromeno[4,3-d]pyrimidines (100mg/kg, p.o.) and Ketoprofen (100mg/kg, p.o.) significantly reduced capsaicin-induced spontaneous nociception. Moreover, the 2-pyrrolyl-spirocyclohexane derivative 10b (100 and 300mg/kg, p.o.) had an anti-allodynic effect comparable to Ketoprofen (100 and 300mg/kg, p.o.) in the arthritic pain model, without causing locomotor alterations in the mice. These results suggest that the compound 10b is a promising molecule for new analgesic drugs in the treatment of pathological pain, such as in arthritis.

Synthesis and antinociceptive activity of new 2-substituted 4-(trifluoromethyl)-5,6-dihydrobenzo[h]quinazolines.

A useful synthetic route for an initial new series of 2-substituted 4-(trifluoromethyl)-5,6-dihydrobenzo[h]quinazolines (3), as well as an evaluation of their analgesic effect in a mice pain model, is reported. Five new quinazolines were formed from the cyclocondensation reactions of 2,2,2-trifluoro-1-(1-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)ethanone (1) with some well-known amidine salts [NH2CR(=NH)] (2), in which R=H, Me, Ph, NH2 and SMe, at a 40-70% yield. Subsequently, due to the importance of the pyrrole nucleus, a 2-(pyrrol-1-yl)quinazoline (4) was obtained through a Clauson-Kaas reaction from the respective 2-(amino)quinazoline, in a reaction with 2,5-dimethoxy-tetrahydrofuran. The analgesic evaluation demonstrated that four 5,6-dihydrobenzo[h]quinazolines (compounds of 3c (R=Ph), 3d (R=NH2), 3e (R=SMe), and 4 (R=pyrrol-1-yl); 100mg/kg, p.o.) and ketoprofen (100mg/kg, p.o.) significantly reduced the spontaneous nociception in a capsaicin-induced test. Moreover, in comparison with ketoprofen (100 and 300mg/kg, p.o.), compound 3c (30-300mg/kg, p.o.) showed an anti-hyperalgesic action in an arthritic pain model without locomotor alterations in the mice, suggesting that quinazoline 3c is a promising prototype scaffold for new analgesic drugs in the treatment of pathological pain such as that in arthritis.

Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases.

In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers "in vitro". In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases.

Amniotic membrane mesenchymal cells-derived factors skew T cell polarization toward Treg and downregulate Th1 and Th17 cells subsets.

We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4(+) T-helper (Th) and CD8(+) cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO(+)CD62L(+)) and effector memory (CD45RO(+)CD62L(-)) subsets. We evaluated the phenotype of CD4(+) cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet(+)CD119(+)) and Th17 (RORγt(+)CD161(+)) populations, while having no effect on the Th2 population (GATA3(+)CD193(+)/GATA3(+)CD294(+)cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1ß), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4(+)FoxP3(+) cells, and an increase of CD25(+)FoxP3(+) and CD39(+)FoxP3(+) Treg in the CD4(+) population. Induction of Treg cells was corroborated by the increased secretion of TGF-ß. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.

Antinociceptive effects of ethanolic extract from the flowers of Acmella oleracea (L.) R.K. Jansen in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, Acmella oleracea (L.) R.K. Jansen, popularly known as "jambu", has been used by some communities from Amazon region to treat toothache. In this study we examined the antinociceptive effect of the ethanolic extract obtained from the flowers of Acmella oleracea (EEAO) in animal models of nociceptive (chemical and thermal) and neuropathic (partial sciatic nerve ligation) pain. MATERIALS AND METHODS: Adult male mice were treated by intraperitoneal route (i.p.) with EEAO before the induction of nociceptive response by formalin, capsaicin and cinnamaldehyde, thermal heat hyperalgesia (hot plate test) and mechanical allodynia (traumatic sciatic nerve injury). Acute toxicity and non-specific sedative effects were evaluated. RESULTS: EEAO (10, 30 and 100 mg/kg) reduced both neurogenic and inflammatory phases of the formalin- and also capsaicin- and cinnamaldehyde-induced orofacial nociception. Interestingly, EEAO at 100mg/kg (i.p.) also reversed capsaicin-induced heat hyperalgesia assessed as the latency to paw withdrawal in the hot plate test. Also in the hot plate test, paw withdrawal latency was increased by EEAO (100 mg/kg) and this response was only partially reversed by naloxone. Furthermore, EEAO (100 mg/kg) also reduced mechanical allodynia caused by partial sciatic nerve ligation for 3 h. The estimated LD50 value was 889.14 mg/kg and EEAO did not alter the locomotion of animals in the open-field test. CONCLUSION: Taken together, our data show that EEAO produces prevalent antinociceptive effects and does not cause adverse effects. The presence of N-alkylamides, including spilanthol, suggests that the therapeutic effect of EEAO is related to its highest anesthetic activity.