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Cresyl violet and cresyl red, components of commercial cresyl violet acetate, were separated and purified using preparative column liquid chromatography. The stationary phase was silica gel and gradient elution was carried out using chloroform:methanol. The purified dyes were obtained in high yield; 51% of the original lot was recovered as cresyl violet and 40% as cresyl red. Separated materials were characterized by nuclear magnetic resonance and mass spectroscopy; UV-visible and Fourier-transform infrared spectra also were obtained for samples of pure cresyl violet and cresyl red. The colored constituents of the commercial dye lot were identified using thin layer chromatography and reverse phase high performance liquid chromatography. Both methodologies were suitable for routine testing; reverse phase high performance liquid chromatography is an appropriate tool for quality control and high resolution identification of these compounds. [ABSTRACT FROM AUTHOR]

A simple low pressure liquid chromatographic method is reported that can separate the basic fuchsine homologues, rosaniline, magenta II and new fuchsine from an impure commercial dye. The chromatographic purity of the separated dyes is > 90%. All homologues were obtained in multi-milligram amounts per chromatographic run; precise yields depend on the composition of the starting material and potentially may be greater. This is a useful preparative procedure for generating chromatographically pure samples of basic fuchsine homologues, especially those that cannot be obtained in pure form by direct synthesis. [ABSTRACT FROM AUTHOR]

Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

This is a paired prospective comparative cohort study with 58 patients, in order to analyze the clinical LD-WLI in patients with moderate or severe COVID19 pneumonia. The results of this study show that the Radiotherapy could be an option to improve the clinical response for patients with COVID-19., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Nasopharyngeal (NP) specimen testing by reverse transcriptase polymerase chain reaction (RT-PCR) is the standard of care for detecting SARS-CoV-2. Data comparing the sensitivity and specificity of the NP specimen to the less invasive, mid-turbinate (MT) nasal specimen in children are limited., Methods: Paired clinical NP and research MT specimens were collected from children <18 years with respiratory symptoms and tested by molecular assays to detect SARS-CoV-2 RNA. Sensitivity, specificity, and agreement (Cohen's kappa [κ]) were calculated for research MT specimens compared to the clinical NP specimens., Results: Out of 907 children, 569 (62.7%) had parental consent and child assent when appropriate to participate and provided paired MT and NP specimens a median of 4 days after symptom onset (range 1-14 days). 16.5% (n = 94) of MT specimens were positive for SARS-CoV-2 compared with 20.0% (n = 114) of NP specimens. The sensitivity of research MT compared to clinical NP specimens was 82.5% (95% CI: 74.2%, 88.9%), specificity was 100.0% (95% CI: 99.2%, 100.0%), and overall agreement was 96.1% (κ = 0.87). The sensitivity of MT specimens decreased with time from 100% (95% CI: 59.0%, 100.0%) on day 1 of illness to 82.1% (95% CI: 73.8%, 88.7%) within 14 days of illness onset; sensitivity was generally >90% when specimens were collected within the first week of illness., Conclusion: MT specimens, particularly those collected within the first week of illness, have moderately reduced sensitivity and equivalent specificity to less-tolerated NP specimens in pediatric outpatients. MT specimen use in children may represent a viable alternative to NP specimen collection., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1 . Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1 . Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments., Competing Interests: The authors declare no conflict of interest.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 μm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

We report herein the physicochemical properties and antimicrobial activity of a new monobrominated derivative of Azure B and its parent compound. These dyes are used as photosensitizers for photodynamic therapy and photodynamic antimicrobial chemotherapy. Relevant pharmaceutical properties (pKa, chemical and photochemical stability, and in vitro antimicrobial activity) were determined. A UV-visible spectrophotometry method was developed and validated according to the International Conference on Harmonization (ICH) guidelines for use in stability indicating studies and determination of the acid dissociation constant of Azure B and its monobrominated derivative. The results showed that both dyes were chemically stable. In addition, bromination of the phenothiazine dye decreased its photochemical stability and pKa value without affecting the ionization rate at physiological pH. The analytical parameters for validation of the method were linearity (r2 > 0.9981), limit of detection (LOD) (0.2-0.9 μM), limit of quantification (LOQ) (0.6-2.7 μM), and intra-day precision (0.76-1.40%) expressed as relative standard deviation (RSD). Recoveries ranging from 99.5 to 100.9% were obtained for the two dyes. Thus, this method provides a simple, sensitive, accurate, and precise assay for the determination of all compounds. The effect of photosensitizer concentration and visible irradiation time on lethal photosensitization against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was investigated. Both photosensitizers were active against the evaluated bacteria. However, the new monobrominated derivative was more effective than its predecessor and managed to eradicate these microorganisms by using different doses of the dye and light. In other words, a lower concentration of AzBBr and irradiation time were required to cause bacterial death equal to or greater than its precursor. The photodynamic efficacy of the two photosensitizers presented the following order: S. aureus > E. coli > P. aeruginosa. These studies indicated that the tested dyes satisfy the conditions of potential photosensitizers in terms of physicochemical and antimicrobial properties.

The main aim of the present study was to synthetize polyacrylamide nanoparticles and to use them as photosensitizer carriers. The new monobrominated derivatives (monobrominated neutral red and monobrominated azure B) were the photosensitizers used for antimicrobial photodynamic therapy. They were loaded into the nanocarriers and their antibacterial and oxidative activities were evaluated. The polyacrylamide nanoparticles were evaluated and prepared by inverse microemulsion polymerization. The nanoparticles obtained were characterized by size, polydispersity index, and zeta potential analysis. The Dynamic Light Scattering indicated that the diameter of the particle (z-average) was optimal, with an acceptable polydispersity index. The antibacterial activity of the polyacrylamide nanoparticles loaded with photosensitizers was evaluated against Staphylococcus aureus. Both photosensitizers loaded into the nanoparticles showed great potential as antibacterial agents since they suppressed the bacterial growth. The maximum percentage of growth reduction was 35.5% (>2 Log CFU/mL), with the monobrominated azure B loaded into the nanocarrier with 2 hydroxyethyl methacrylate against methicillin resistant S. aureus. The improved physicochemical and photophysical properties of these photosensitizers were accompanied by a significant increase in the photoantimicrobial action, in conventional-sensitive and-methicillin resistant S. aureus. The results obtained clearly suggest that polyacrylamide nanoparticles loaded with photosensitizers have great potential for further application in antimicrobial photodynamic therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Locally severe outbreaks of Fusarium wilt of cotton (Gossypium spp.) in South Georgia raised concerns about the genotypes of the causal pathogen, Fusarium oxysporum f. sp. vasinfectum. Vegetative complementation tests and DNA sequence analysis were used to determine genetic diversity among 492 F. oxysporum f. sp. vasinfectum isolates obtained from 107 wilted plants collected from seven fields in five counties. Eight vegetative complementation groups (VCG) were found, with VCG 01117B and VCG 01121 occurring in 66% of the infected plants. The newly recognized VCG 01121 was the major VCG in Berrien County, the center of the outbreaks. All eight VCG resulted in significant increases in the percentages of wilted leaves (27 to 53%) and significant reductions in leaf weight (40 to 67%) and shoot weight (33 to 60%) after being stem punctured into Gossypium hirsutum 'Rowden'. They caused little or no significant reductions in shoot weight and height or increases in foliar symptoms and vascular browning in a soil-infestation assay. Soil infestation with Meloidogyne incognita race 3 (root-knot nematode) alone also failed to cause significant disease. When coinoculated with M. incognita race 3, all VCG caused moderate to severe wilt. Therefore, the VCG identified in this study belong to the vascular-competent pathotype, and should pose similar threats to cotton cultivars in the presence of the root-knot nematode. Use of nematode-resistant cultivars, therefore, is probably the best approach to control the disease in Georgia.

A highly virulent race 4 (Cal race 4) of Fusarium oxysporum f. sp. vasinfectum was identified in California cotton fields in 2001, and has since been found in increasing numbers of fields. Cal race 4 isolates contain a unique Tfo1 transposon insertion in the PHO gene that was not found in other F. oxysporum f. sp. vasinfectum genotypes. Based on this insertion, a multiplex polymerase chain reaction method was developed to detect the Cal race 4 pathogen. A panel of F. oxysporum f. sp. vasinfectum isolates representing different vegetative compatibility groups (VCG) and DNA sequence types was assembled to test the specificity of the detection method. In all, 16 of 17 Cal race 4 isolates produced a 583-bp amplicon; the other isolate produced a 396-bp amplicon reflecting the absence of the Tfo1 insertion. This isolate was a moderately virulent pathogen among Cal race 4 isolates. In total, 80 other F. oxysporum isolates associated with cotton and 11 other formae speciales of F. oxysporum produced only the 396-bp amplicon. The method also distinguished Cal race 4 isolates from India race 4 isolates and China race 7 isolates, which did not possess the unique Tfo1 insertion but otherwise had identical DNA sequences, and all belong to VCG0114. The method is capable of detecting the pathogen directly from infected stem tissues even before external symptom appears and, thus, provides an effective tool for timely identification of infested fields and seed lots, and should help reduce dissemination of Cal race 4 in the U.S. Cotton Belt.

At present, polyacrylamide nanoparticles are attractive to drug delivery. However, some physicochemical characteristics of these nanoparticles still need to be further improved in practice. Polyacrylamide nanoparticles with an average size of 80nm and a zeta potential of -30mV were synthesized and used as photosensitizer carriers. The new monobrominated derivatives and parent compounds were the photosensitizers for the photodynamic therapy loaded in the nanocarrier. The physicochemical characterization of the prepared nanoparticles, drug loading, the ability to generate singlet oxygen and chemical stability were investigated. The novel tested nanoparticles exhibited a loading percentage of between 80 and 99%, higher generation of singlet oxygen and good stability in comparison with the corresponding starting reagent. According to these results, the novel polyacrylamide nanoparticles are excellent candidates for drug vehiculization., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aggregation phenomenon of Azure B, monobrominated Azure B, Thionine and Methylene Blue was studied by UV-Visible spectrophotometry in different media as a function of dye concentration and temperature variations. The tests carried out in organic solvents allowed the identification of monomeric species of these compounds, which have not been reported in literature and have been wrongly assigned for years. The results obtained in water allowed demonstrating that different kinds of aggregates are present in this medium. In addition, the aggregation tendency of these dyes in organic solvent and aqueous media was established. Several parameters such as lipophilicity, effect of bulky substituents and interactions with media were considered to interpret the aggregation behavior of thiazine dyes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report herein the synthesis, and the physicochemical and pharmacokinetic properties of N-acyloxymethyl prodrugs of allopurinol (Allop) (2a-f). Allop is a compound with activity against Trypanosoma cruzi, a causative agent of Chagas disease. Its pathology leads to a huge number of infections and deaths per year, because in addition to many sufferers only having limited access to health services only an inefficient chemotherapy is available. Relevant pharmaceutical properties (pKa, stability, solubility, lipophilicity, in vitro permeability, binding protein, xanthine oxidase binding) were also determined. The results obtained showed that derivatives behave as prodrugs of Allop, since they exhibit improved physicochemical and pharmacokinetic properties relative to their precursor. This behavior turns these compounds into active reservoirs of Allop, and reduces its unfavorable characteristics, so 2a-f compounds are excellent candidates for the treatment of Chagas disease. This work is therefore an important contribution leading to the suppression of Chagas disease.

Maize is the dominant cereal crop produced in the US. One of the main fungal pathogens of maize is Fusarium verticillioides, the causative agent of ear and stalk rots. Significantly, the fungus produces a group of mycotoxins - fumonisins - on infested kernels, which have been linked to various illnesses in humans and animals. Nonetheless, durable resistance against F. verticillioides in maize is not currently available. In Texas, over 2.1 million acres of maize are vulnerable to fumonisin contamination, but understanding of the distribution of toxigenic F. verticillioides in maize-producing areas is currently lacking. Our goal was to investigate the genetic variability of F. verticillioides in Texas with an emphasis on fumonisin trait and geographical distribution. A total of 164 F. verticillioides cultures were isolated from 65 maize-producing counties. DNA from each isolate was extracted and analyzed by PCR for the presence of FUM1- a key fumonisin biosynthesis gene - and mating type genes. Results showed that all isolates are in fact F. verticillioides capable of producing fumonisins with a 1:1 mating-type gene ratio in the population. To further study the genetic diversity of the population, isolates were analyzed using RAPD fingerprinting. Polymorphic markers were identified and the analysis showed no clear correlation between the RAPD profile of the isolates and their corresponding geographical origin. Our data suggest the toxigenic F. verticillioides population in Texas is widely distributed wherever maize is grown. We also hypothesize that the population is fluid, with active movement and genetic recombination occurring in the field.

The photodynamic activity of brominated derivatives of New Fuchsin and Azure B was studied in solution and in cell suspensions of Candida albicans. The spectroscopic and photodynamic properties of these photosensitizers were compared with those of Crystal Violet and Azure B, which represent active photosensitizer related to each family of compounds. Triarylmethane derivatives absorb intensely with a band centered at ∼ 570 nm, while the phenothiazinium dyes at ∼ 650 nm. Photooxidation of 9,10-dimethylanthracene was observed using phenothiazinium compounds indicating the formation of singlet molecular oxygen, while it was not detected using triarylmethane agents. However, triarylmethane dyes were able to photooxidize l-tryptophan. In yeast cell suspensions, the photosensitized inactivation of C. albicans increases with photosensitizer concentration, causing a ∼ 5 log decrease of cell survival, when the cultures are treated with 20 μM of Crystal Violet and irradiated for 60 min. Under these conditions, the photodynamic activity of 50 μM Azure B induced a ∼ 3 log decrease of cell survival. Studies of photodynamic action mechanism indicated that photoinactivation of C. albicans cells induced by triarylmethane compounds involves mainly type I photoprocess. Although, phenothiazinium derivatives produce singlet molecular oxygen, a contribution of other reactive oxygen species cannot be discarded in the photoinactivation of C. albicans., (Copyright © 2014 Elsevier B.V. All rights reserved.)

MADS-box transcription factors (TFs) regulate functionally diverse gene targets in eukaryotes. In select ascomycetes, MADS-box TFs have been shown to play a role in virulence, and vegetative and sexual development. Here, we characterized Fusarium verticillioides MADS-box TFs, Mads1 and Mads2, in terms of their roles in secondary metabolism and sexual mating. Sequence analyses showed that MADS1 and MADS2 encode TFs with a SRF-type dimerization domain and a MEF2-type dimerization domain, respectively. The MADS1 and MADS2 knockout mutants (Fmt1 and Fmt2 strains, respectively) exhibited decreased vegetative growth and FB1 production when compared to the wild-type. Fmt1 showed reduced expression of 14 polyketide synthase (PKS) genes present in the organism, whereas Fmt2 did not display a change in PKS gene expression. Significantly, the deletion of MADS1 and MADS2 in the MAT1-2 genotype (Fmt4 and Fmt5 strains, respectively) led to strains that failed to produce perithecia and ascospores when crossed with the MAT1-1 wild-type strain. Notably, deletion of either gene did not have an effect on the ability of the fungus to colonize maize stalk or kernels. FB1 production and PKS expression data suggest that Mads1 is a broad regulator of secondary metabolism in F. verticillioides, and may target regulons upstream of Mads2 to influence FB1 production. In addition, MADS-box TFs in F. verticillioides play a critical role in the perithecia development.

Objective: This study utilized a combination of HPV self-sampling, iFTA elute specimen cards, and long distance transport for centralized processing of specimens to determine the feasibility of large-scale screening in remote and transient populations., Methods: This study was performed in two locations in Peru (Manchay and Iquitos). The "Just For Me" cervico-vaginal brush and iFTA elute cards were used for the collection and transport of specimens. Samples were shipped via FedEx to China and tested for 14 types of high-risk HPV using PCR based MALDI-TOF. HPV positive women were treated with cryotherapy after VIA triage, and followed-up with colposcopy, biopsy, ECC, and repeat HPV testing at 6 months., Results: Six hundred and forty three women registered, and 632 returned a sample over a 10 day period. Within 2 weeks, specimens were shipped, samples tested, and results received by study staff. Sixty-eight women (10.8%) tested positive, and these results were delivered over 4 days. Fifty-nine HPV positive women (87%) returned for evaluation and treatment, and 2 had large lesions not suitable for cryotherapy. At 6 months, 42 women (74%) returned for follow-up, and 3 had CIN 2 (all positive samples from the endocervical canal). Ninety eight percent of participants reported that they would participate in this type of program again., Conclusions: Utilizing HPV self-sampling, solid media specimen cards for long distance transport, and centralized high throughput processing, we achieved rapid delivery of results, high satisfaction levels, and low loss to follow-up for cervical cancer screening in remote and transient populations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Triarylmethane and thiazine dyes have attracted attention as anticancer and antimicrobial agents, due to their structural features and selective localizations. Although these dyes have been initially explored in the context of photodynamic therapy, some of these such as New Fuchsin and Azure B have still not been extensively investigated. For this reason, we evaluated the chemical stability, aggregation effect, and lipophilicity, as well as the photodynamic activity against LM-2 murine mammary carcinoma cells of five new brominated dyes of triarylmethane and thiazine. These cationic compounds were obtained at high purities and unequivocally characterized by conventional techniques. The introduction of bromine atoms into the chromophoric system of New Fuchsin and Azure B dyes gave rise to a moderate bathochromic shift and increased the lipophilicity, thereby improving their photophysical and photochemical properties for biomedical applications. Moreover, the in vitro photodynamic activity demonstrated that, as the degree of bromination increased, the phototoxicity remained unchanged or decreased. The lower efficiency to inactivate cultured tumor cells may be attributed to the formation of the colorless carbinol pseudobase and aggregation effects for triarylmethane and thiazine dyes, respectively. A promising strategy to reverse the biological activity decrease observed might be the design of third-generation photosensitizers., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Cervical cancer is a preventable disease which causes significant morbidity and mortality, particularly in developing countries. Although technology for early detection continues to improve, prevention programs suffer from significant barriers. Community-based participatory research is an approach to research which focuses on collaboration with the community to surmount these barriers. The objective of this study was to evaluate the utility of community-based participatory research techniques in a mother-child screen/treat and vaccinate program for cervical cancer prevention in Manchay, Peru., Materials and Methods: Human papillomavirus (HPV) self-sampling and cryotherapy were used for the screen/treat intervention, and the Gardasil vaccine was used for the vaccine intervention. Community health workers from Manchay participated in a 3-day educational course, designed by the research team. The community health workers then decided how to implement the interventions in their community. The success of the program was measured by (1) the ability of the community health workers to determine an implementation plan, (2) the successful use of research forms provided, (3) participation and retention rates, and (4) satisfaction of the participants., Results: (1) The community health workers used a door-to-door approach through which participants were successfully registered and both interventions were successfully carried out; (2) registration forms, consent forms, and result forms were used correctly with minimal error; (3) screen/treat intervention: 97% of registered participants gave an HPV sample, 94% of HPV-positive women were treated, and 90% returned for 6-month follow-up; vaccine intervention: 95% of registered girls received the first vaccine, 97% of those received the second vaccine, and 93% the third; (4) 96% of participants in the screen/treat intervention reported high satisfaction., Conclusions: Community-based participatory research techniques successfully helped to implement a screen/treat and vaccinate cervical cancer prevention program in Manchay, Peru. These techniques may help overcome barriers to large-scale preventive health-care interventions.

Background: It is estimated that 15% of couples living in industrialized countries are infertile, ie have failed to conceive, reproductive age, after 12 months ormore of regular intercourse without contraception. During the past decade has increased the demand for fertility treatments because they believe are moreeffective now., Objective: To unify the therapeutic approach and service to patients and set a precedent for a Mexican Official Standard respect and support for the legislation of these procedures., Method: Consensus by technical experts group panel with the participation of 34 national centers accredited for use in assisted reproduction. He organized seven workshops with the following themes: 1) selection of patients for assisted reproduction treatment, 2) schemes controlled ovarian stimulation for assisted reproduction techniques of high complexity, 3) preparation and egg retrieval technique, 4) transferembryo; 5) luteal phase supplementation; 6) indications and techniques of cryopreservation and 7) informed consent. Each table had a coordinator who wrote and presented the findings to the full, it made a number of observations until they reached unanimity of criteria, which are reflected in this document., Results: Patient selection for assisted reproduction techniques is the first step of the process. Proper selection lead to success, in the same way that a bad pick up for failure. In the case of egg donation the most important recommendation is that only one to two embryos transferred in order to reduce multiple pregnancy rates and maintaining high pregnancy rates.

Functional activity of voltage-gated sodium channels (VGSC) has been associated to the invasion and metastasis behaviors of prostate, breast and some other types of cancer. We previously reported the functional expression of VGSC in primary cultures and biopsies derived from cervical cancer (CaC). Here, we investigate the relative expression levels of VGSC subunits and its possible role in CaC. Quantitative real-time PCR revealed that mRNA levels of Na(V) 1.6 α-subunit in CaC samples were ∼40-fold higher than in noncancerous cervical (NCC) biopsies. A Na(V) 1.7 α-subunit variant also showed increased mRNA levels in CaC (∼20-fold). All four Na(V) β subunits were also detected in CaC samples, being Na(V) β1 the most abundant. Proteins of Na(V) 1.6 and Na(V) 1.7 α-subunits were immunolocalized in both NCC and CaC biopsies and in CaC primary cultures as well; however, although in NCC sections proteins were mainly relegated to the plasma membrane, in CaC biopsies and primary cultures the respective signal was stronger and widely distributed in both cytoplasm and plasma membrane. Functional activity of Na(V) 1.6 channels in the plasma membrane of CaC cells was confirmed by whole-cell patch-clamp experiments using Cn2, a Na(V) 1.6-specific toxin, which blocked ∼30% of the total sodium current. Blocking of sodium channels VGSC with tetrodotoxin and Cn2 did not affect proliferation neither migration, but reduced by ∼20% the invasiveness of CaC primary culture cells in vitro assays. We conclude that Na(V) 1.6 is upregulated in CaC and could serve as a novel molecular marker for the metastatic behavior of this carcinoma., (Copyright © 2011 UICC.)

Objective: To compare short-term complication rates in dogs and cats undergoing cystotomy closure by use of an inverting double-layer pattern (group I) versus cystotomy closure by use of an appositional single-layer pattern (group A)., Design: Retrospective case series., Animals: 144 client-owned dogs and cats that underwent cystotomy between 1993 and 2010., Procedures: Information on signalment, reason for cystotomy, method of cystotomy closure, complications that developed during hospitalization, and duration of hospitalization were obtained from the medical record. The effect of closure technique on short-term complication rate and duration of hospitalization was examined., Results: 2 of the 144 animals developed dehiscence and uroabdomen following cystotomy closure: 1 from group A and 1 from group I. Of group A animals, 29 of 79 (37%) developed minor complications such as hematuria and dysuria. Of group I animals, 33 of 65 (50%) developed the same complications. Group A and group I animals did not differ significantly with regard to prevalence of minor or major complications. The mean duration of hospitalization was 4.1 days and did not differ significantly between groups., Conclusions and Clinical Relevance: An appositional single-layer suture pattern for cystotomy closure was a safe and effective procedure with minimal risk of urine leakage and a short-term complication rate of 37%. The appositional single-layer suture pattern for cystotomy closure may be recommended for clinical use because the inverting double-layer suture pattern offered no clear advantage.

Human ether à-go-go 1 (Eag1) potassium channels are potential tumor markers and therapeutic targets for several types of malignancies, including cervical cancer. Estrogens and human papilloma virus oncogenes regulate Eag1 gene expression, suggesting that Eag1 may already be present in pre-malignant lesions. Therefore, Eag1 could be used as an early marker and/or a potential risk indicator for cervical cancer. Consequently, we studied Eag1 protein expression by immunochemistry in cervical cancer cell lines, normal keratinocytes, cervical cytologies from intraepithelial lesions, biopsies from cervical intraepithelial neoplasias (CIN 1, 2 and 3) and in normal smears from patients taking or not taking estrogens. Two hundred and eighty-six samples obtained by liquid-based cytology and fifteen CIN biopsies were studied. We observed Eag1 protein expression in the cervical cancer cell lines, as opposed to normal keratinocytes. Eag1 was found in 67% of the cervical cytologies from low-grade intra-epithelial lesions and in 92% of the samples from high-grade intraepithelial lesions, but only in 27% of the normal samples. Noteworthy, morphologically normal cells obtained from dysplastic samples also exhibited Eag1 expression. In CIN biopsies we found that the higher the grade of the lesion, the broader the Eag1 protein distribution. Almost 50% of the normal patients taking estrogens displayed Eag1 expression. We suggest Eag1 as a potential marker of cervical dysplasia and a risk indicator for developing cervical lesions in patients taking estrogens. Eag1 detection in cervical cancer screening programs should help to improve early diagnosis and decrease mortality rates from this disease.

New Fuchsin (NF), also known as Magenta III, has potential applications in photodynamic therapy. The commercial product labeled NF contains two other dye components in different proportions, Magenta II and Magenta I (Rosaniline). The proportions of NF, Magenta II, and Magenta I determined by reversed-phase high-performance liquid chromatography (RP-HPLC) in the commercial sample used were 71.6 +/- 0.4%, 25.2 +/- 0.2%, and 2.8 +/- 0.1% (n = 7), respectively. The isolation, purification, and characterization of commercial NF dye components were carried out applying different techniques, such as preparative column liquid chromatography (PCLC), thin layer chromatography (TLC), RP-HPLC, absorption spectrophotometry, nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry (ESI-MS), and tandem electrospray ionization mass spectrometry (ESI-MS-MS). After separation and isolation, the degree of purity obtained for NF compound was higher than 95% and 92% for Magenta II and Magenta I compounds, respectively. Therefore, it is essential to ensure a high degree of purity of these dyes as raw material to obtain new drugs intended for therapeutic treatments.

Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-à-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention., (Copyright 2009 Elsevier Inc. All rights reserved.)

The urinary tract infection is a worldwide health problem, with a ratio of 9 to 1 in women compared with men. In 80% of the cases, the causing bacteria is Escherichia coli. During reproductive life this infection represents a great amount of work disabilities, hence the need to insist on its prophylaxis. The red cranberry juice is an option to prevent urinary tract infection, a quality demonstrated in several recent publications which emphasize that its mechanism of action lies in the effect exerted by proantocyanidines, especially those of type A, in the urothelium that prevent Escherichia coli from adhering to this and exerts its antibacterial action, which is achieved with the ingestion of at least 300 mL of juice every day.

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation.

Cervical cancer (CaC) is the third most frequent cause of death from cancer among women in the world and the first in females of developing countries. Several ion channels are upregulated in cancer, actually potassium channels have been suggested as tumor markers and therapeutic targets for CaC. Voltage-gated sodium channels (VGSC) activity is involved in proliferation, motility, and invasion of prostate and breast cancer cells; however, the participation of this type of channels in CaC has not been explored. In the present study, we identified both at the molecular and electrophysiological level VGSC in primary cultures from human cervical carcinoma biopsies. With the whole cell patch clamp technique, we isolated and identified a voltage-gated Na(+) current as the main component of the inward current in all investigated cells. Sodium current was characterized by its kinetics, voltage dependence, sensitivity to tetrodotoxin (TTX) block and dependence to [Na(+)](o). By analyzing the expression of mRNAs encoding TTX-sensitive Na(+) channel alpha subunits with standard RT-PCR and specific primers, we detected Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.7 transcripts in total RNA obtained from primary cultures and biopsies of CaC. Restriction enzyme analysis of PCR products was consistent with the molecular nature of the corresponding genes. Notably, only transcripts for Na(v)1.4 sodium channels were detected in biopsies from normal cervix. The results show for the first time the functional expression of VGSC in primary cultures from human CaC, and suggest that these channels might be considered as potential molecular markers for this type of cancer.

The aim of this research was to develop and validate a sensitive, rapid, easy, and precise reversed-phase liquid chromatography (LC) method for stability studies of bifonazole (I) formulated with tinctures of calendula flower (II). The method was especially developed for the analysis and quantitative determination of I and II in pure and combined forms in cream pharmaceutical formulations without using gradient elution and at room temperature. The influence on the stability of compound I of temperature, artificial radiation, and drug II used for the new pharmaceutical design was evaluated. The LC separation was carried out using a Supelcosil LC-18 column (25 cm x 4.6 mm id, 5 microm particle size); the mobile phase was composed of methanol-0.1 M ammonium acetate buffer (85 + 15, v/v) pumped isocratically at a flow rate of 1 mL/min; and ultraviolet detection was at 254 nm. The analysis time was less than 10 min. Calibration graphs were found to be linear in the 0.125-0.375 mg/mL (rI = 0.9991) and 0.639-1.916 mg/mL (rII = 0.9995) ranges for I and II, respectively. The linearity, precision, recovery, and limits of detection and quantification were satisfactory for I and II. The results obtained suggested that the developed LC method is selective and specific for the analysis of I and II in pharmaceutical products, and that it can be applied to stability studies.

A rapid, reliable and specific UV spectrophotometric method was developed to determine Phenilpropanolamine Hydrochloride (I), Caffeine (II) and Diazepam (III) formulated in tablets. This method was validated and compared with a liquid chromatography (LC) procedure used for the simultaneous quantitative analysis of the drugs. The established linearity ranges by both methods for compounds I, II and III were 0.36-0.88, 0.012-0.028 and 0.036-0.084 mg/ml, respectively. The correlation coefficients by HPLC were r(I)(2)=0.997, r(II)(2)=0.999, r(III)(2)=0.999 and by the UV spectrophotometric method were r(I)(2)=0.998, r(II)(2)=0.996, r(III)(2)=0.999. LC and UV methods showed excellent precision and accuracy. As regards precision, LC showed CV values range of 0.2-0.9 and UV 0.15-0.72. On the other hand, accuracy was obtained with CV values range of 0.1-1.8 and 0.32-1.11 for LC and UV, respectively. The recoveries of I, II and III were >98.04% for both methods over the linear range. The UV and HPLC methods have been successfully used to determine the I, II and III content in tablets of different origin.

The main objective of this study was to develop and test the applicability of a sensitive, accurate, and precise liquid chromatographic (LC) method for evaluating the stability characteristics of a new bisisoxazolylnaphthoquinone, 2-(3,5-dimethyl-4-isoxazolylamino)-N-(3,5-dimethyl-4-isoxazolyl)-1,4-naphthoquinone-4-imine compound 1. The method was shown to be selective and stability-indicating. Isocratic elution with a mobile phase of methanol-water (75 + 25, v/v) on a reversed-phase column with UV detection at ambient temperature completely resolved compound 1 from its degradation products. The LC system was calibrated by plotting peak responses versus known concentrations of a reference standard by using an internal standardization procedure. Complete elution occurred after 12 min with a peak symmetry factor of 0.95 for the drug peak. The kinetic degradation of compound 1 was studied over a pH range of 0.88-14.00 to determine the kinetic parameters involved in its decomposition path in aqueous solution.

The goal of this study was to determine the kinetic parameters involved in the decomposition of 2-(5-methyl-4-isoxazolylamino)-N-(5-methyl-4-isoxazolyl)-1,4-naphthoquinone-4-imine (1) in aqueous solution and to identify the main degradation products. An isocratic HPLC assay was used to study the degradation rate of 1. The products of hydrolysis were identified by comparison of their retention times with those of authentic samples. The amount of 1 and the two degradation products resulting from storage of 1 in various buffer solutions was followed in function of time by a reversed-phase HPLC stability-indicating method. The observed degradation rates followed pseudo-first-order kinetics at constant pH, temperature and ionic strength. The logk-pH-profile was constructed at 35 degrees C from the first-order rate constants obtained from studies at pH values ranging from 0.88 to 10.80 (mu=0.5 M). Hydrolysis in the acidic and alkaline media resulted in the formation of two degradation products in each case. The pH-rate profile of 1 in buffer solution was adequately described using a four-term rate equation. The obtained pH-rate profile indicated specific acid-base catalysis with a region of maximum stability between pH 6.40 and 7.40 which can be adequate for formulations of 1.

In the last years, the adherence adnexal disease has been increased due to pelvic infections. This may cause infertility problems depending, of the nature, extension and localization inside the pelvis. In this paper we inform the results obtained in 70 patients with adnexal adhesions to whom it was performed a salpingo-ovariolysis with microsurgery technology, with the purpose of promote the fertility. All the patients received pre, trans and postoperative support (Heparin, steroids and antimicrobials). The minimal time of postoperative observation was at least 6 months. In 27 (Group I) of the 70 cases (38.6%), the adherences were avascular (IA & IIA based on Hulka's classification); and in the 43 patients of the group II (61.4%), the lesions were dense and vascular (IB & IIB). In group I, 15 pregnancies were obtained (55.6%), 13 at term, 1 miscarriage and 1 ectopic pregnancy; in the group II we documented 9 pregnancies (20.9%), 5 at term, 4 miscarriage (I trimester).

The kinetics of enolization and degradation of N-(5-methyl-4-isoxazolyl)-4-amino-1,2-naphthoquinone (1) was investigated in aqueous solutions over a pH range of 7.30 to 12.25, at 35 degrees C and at constant ionic strength (mu = 0.5) using reversed-phase HPLC. Pseudo-first-order kinetics was observed throughout the pH range studied. The rate of enolization (ke), the keto-enol equilibrium constant (Kt), and specific base catalysis rate constant (kCH) were determined. Good agreement between the theoretical pH-rate profile and the experimental data supports the proposed transformation process. The average recovery for 1 and its tautomerization product 2-hydroxy-N-(5-methyl-4-isoxazolyl)-1,4-naphthoquinone 4-imine (2) from mixtures of different composition was evaluated.

The degradation kinetics of N-(5-methyl-4-isoxazolyl)-4-amino-1,2-naphthoquinone (1) was studied in aqueous solution over a pH range of 0.65-7.50, at 35 degrees C and at a constant ionic strength of 0.5. The degradation rates were determined by high-pressure liquid chromatography and were observed to follow pseudo-first-order kinetics with respect to the concentration of 1. The pH-rate profile was linear with slope -1 under acidic pH, becoming pH independent from 3.50 to 7.50. Good agreement between the theoretical pH-rate profile and the experimental data supports the proposed degradation process. The catalytic rate constants for hydrogen ion and water were kH = 0.901 M-1 h-1 and k0 = 1.34 x 10(-3)h-1, respectively. These data are appropriate to develop a stable dosage form of 1. The accuracy, peak sharpness, and asymmetry factor for the analytical method were determined.

This is a study using light microscopy and electronic microscopy of Fallopian tube subjected to sterilization; and compare it with a control group. A total of 60 samples from patients with different surgical procedures, whose age was from 20 to 40 years; they were amenorrheic, without antecedents of pelvic inflammatory disease nor endometriosis. Among the control group, there were lesions as isthsmical nodular salpingitis in 30.7%. There was chronic salpingitis in 15.3%; follicular salpingitis in 23%, and polyps in 15%. In the group with OTB antecedents, endosalpingiosis was found in 20%; follicular salpingitis in 30% and chronic salpingitis in the remaining 50%. By means of electronic, ultrastructural microscopy, were seen lesions at myosalpinx level, that is, alterations in myofilaments, formation, much fibrosis and descilliation in the adjacent to OTB. It may be concluded that these findings, never seen before, may condition an alteration of tubal motility in patients subjected to rechannelization procedures, that condition an alteration in gametes transportation, avoiding fecundation or a greater incidence ectopic pregnancy incidence.