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Volumen 9, Asunto 4 (2015)

Artículo de investigación

Glucosamine-induced ER Stress Accelerates Atherogenesis: A Potential Link between Diabetes and Cardiovascular Disease

Vi T Dang, Daniel R Beriault, Arlinda Deng, Yuanyuan Shi and Geoff H Werstuck

Background: Cardiovascular disease is the leading cause of death worldwide and is responsible for three out of four deaths in diabetic individuals. Our lack of understanding of the molecular mechanisms linking diabetes and atherosclerosis impedes the development of effective treatment strategies. Hyperglycemia and glucosamine-supplementation have been shown to induce endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in murine models of atherosclerosis. We hypothesize that diabetes/hyperglycemia promotes atherosclerosis by a mechanism involving glucosamine-induced ER stress/UPR activation and that attenuation of ER stress, using the chemical chaperone 4- phenylbutyric acid (4PBA), will slow the accelerated development of atherosclerosis.

Methods: Hyperglycemia was induced in female Apolipoprotein E-deficient (ApoE-/-) mice by multiple low-dose streptozotocin injections or by the introduction of the Ins2+/Akita mutation. Glucosamine-supplementation was achieved by adding different concentrations of glucosamine (0.625-5% w/v) to the drinking water of ApoE-/- mice. Subsets of mice from each group were also treated with 4PBA. The development of atherosclerosis was evaluated based on atherosclerotic lesion area and volume at the aortic sinus. Levels of protein O-linked N-acetylglucosamine (O-GlcNAc) and ER stress markers were determined in atherosclerotic lesions using immunohistochemistry and immunofluorescence staining.

Results: Hyperglycemic and glucosamine-supplemented mouse models showed similar increases in O-GlcNAc and ER stress/UPR activation levels in atherosclerotic lesions. Lesion area was not significantly different between the three models of accelerated atherosclerosis. Glucosamine supplementation at ≥ 2.5% (w/v) significantly increased lesional O-GlcNAc, UPR activation and atherosclerotic lesion area/volume, independent of changes in any measured metabolic parameters. 4PBA mitigated ER stress and attenuated accelerated atherosclerosis in both hyperglycemic and glucosamine-supplemented mouse models.

Conclusion: These findings suggest that hyperglycemia promotes accelerated atherosclerosis by a mechanism involving glucosamine-induced ER stress. Accelerated atherosclerosis can be attenuated in hyperglycemic ApoE-/- mice by reducing ER stress levels.

Artículo de investigación

Twenty Two New Mutations in Mitochondrial tRNA Genes in Patients with Alzheimer's Tabriz, Iran

Shahin Asadi, Ali Nazirzadeh and Saeideh Habibi

Alzheimer's disease is the main form of memory, and memory loss in the elderly is the interplay of genes and environment play a role in its formation. The role of mitochondrial mutations in various neurological diseases, has effectively proven that some of these mutations of Alzheimer's disease in a non-Mendelian maternal mode of inheritance that are inherited.

All mitochondrial tRNA genes in 24 patients and 50 healthy controls using nucleotide sequences, was tested. Mitochondrial tRNA genes were found in fifteen change. The polymorphisms were eleven of them. Four changes T1633A, C1631A (in tRNA parents), T14723T, Q14704C, were classified as pathogenic mutations, such as heteroplasmy observed in patients, mutations of nucleotide sequences in different organisms has been identified. Polymorphism A12308G, eight patients were found in tRNA leucine. This change in various neurological diseases, as well as control samples has been reported.

We believe that these changes may influence the pathogenesis of Alzheimer's disease or the disease process act as a secondary injury. The percentage of heteroplasmy may be involved in the development of symptoms or onset of the disease.

Artículo de investigación

Poly(N–Isopropylacrylamide–Co–Acrylic Acid) Smart Nanocarriers for Drug Release: A Study of Theophylline Delivery

Álvarez–Bautista A, Mendizábal E, Duarte CMM and Katime I

Copolymeric nanoparticles of poly(N–isopropylacrylamide–co–acrylic acid) have been synthesized by inverse microemulsion polymerization. The obtained nanoparticles have been characterized by FTIR and DSC, to detect her molecular structure and glass transition temperatures respectively, by transmission electron microscopy (TEM) and quasielastic light scattering (QLS) to determine the sizes and size distribution of gels synthesized. The swelling of these nanogels has been studied in order to know their response at different pH and crosslinking agent concentration. These nanoparticles have subsequently been charged with a vasodilator drug (theophylline) to study the release–kinetics of the drug release at different pH. These nanogels have shown a controlled release at basic environment. After further biological viability studies this system could be used as smart carriers in nanomedicine.

Artículo de revisión

Single Cell Transcriptomics for Autoimmune Disorders

Bhawna Gupta and Sunil Kumar Raghav

The interplay of genetic, immunological and environmental factors is the driving force towards autoimmunity and each of these branches of biological science is necessary to identify the cause and progress of autoimmune disorders. Differential transcript abundance as an effect of environmental or epigenetic modifications may directly regulate emergence while a sustained copy number increase may drive disease progression. A precise evaluation of these transcript level differences could be the key to understand the mechanism of development and progression of autoimmune diseases however it is imperative to quantitate the subtle changes at the highest resolution. This review summarizes the studies that have explored the importance of analyzing differential transcriptome at single cell resolution, further to emphasize the importance of this approach for enhanced understanding and to identify more sensitive and specific biomarkers for autoimmune diseases.

Artículo de investigación

Age of Salpingo-Oophorectomy and Risk of Peritoneal Carcinomatosis in Patients with a BRCA Mutation

Stjepanovic N, Carrasco E, Gadea N, Gil-Moreno A, Perez A, Diaz B, Franco S, Cabrera S, Garcia A, Castellvi J, Oaknin A, Díez O, Bonache S, Gutiérrez-Enríquez S, Vilaro M and Balmaña J

Background: Women with BRCA1/2 germline mutations who undergo bilateral salpingo-oophorectomy (BSO) are left with a residual risk of peritoneal serous carcinoma (PSC). We aimed to identify the incidence and risk factors for the development of PSC after BSO in BRCA1/2 mutation carriers.
Methods: One-hundred-seventeen BRCA1/2 mutation carriers who underwent BSO were evaluated for further development of PSC. The BSO specimens were evaluated for occult Fallopian tube carcinoma (FTC), ovarian carcinoma (OVC) and serous tubal intraepithelial carcinoma (STIC) in all patients. P53-signature was available in 58 patients. Clinical data was obtained from patients’ charts. We calculated the association between clinical, pathological and molecular risk factors of PSC after BSO.
Results: Analysis of BSO specimens revealed occult FTC in 1 woman (0.8%), STIC in 2 women (1.7%), and 6/58 women (10.3%) had a positive “p53 signature”. Older age at menopause (p=0.007) and shorter oral contraceptive use (p<0.001) were associated with FTC and STICs. The incidence of PSC after BSO was 1.7% (two patients). Both were BRCA2 mutation carriers and one had a history of STIC. The only risk factor identified for PSC after BSO was older age at surgery (63.5 years for patients with PSC vs. 48.6 years for patients without PSC, p<0.001).
Conclusions: The incidence of PSC after BSO in our series is 1.7% and it is associated with an older age at BSO. Earlier menopause and oral contraceptive use seem to be associated with a decrease in the prevalence of occult FTC and STICs.

Reporte de un caso

Prenatal and Postnatal Diagnostics of a Child with Bardet-Biedl Syndrome: Case Study

Magdalena Pasińska, Lech Dudarewicz, Lucjusz Jakubowski and Olga Haus

Bardet Biedl Syndrome (BBS) is a rare, genetically determined syndrome which can result from a mutation in one of 19 known genes (often called BBS complex), which play a vital role in building structures and cell functions of cilia. Phenotypic symptoms of the syndrome usually progress in the first decade of life, however, they are characterized by a high diversity which makes their diagnosis difficult, especially in the early stage of life. The diagnosis is most frequently based on clinical symptoms and established in late childhood or adult life. BBS is a disorder of the non-motile cilia, which play the role of antennae receiving and transmitting sensory signals to photoreceptors of the retina, hearing cells or olfactory cells. Clinical symptoms of the disorders affecting these structures are: retinal pigmentary degeneration, polydactyly, learning difficulties, and formation of kidney, liver and pancreas cysts. The inheritance mode of BBS is in 80% autosomal recessive, which is connected with both parents carrying a mutated allele, and usually only giving birth to a symptomatic child defines a family as being at genetic risk, whith 25% risk of having another child with the disease and 50% of having offspring that are asymptomatic carriers For families with an identified mutation there exists a possibility of conducting prenatal or preimplementation genetic diagnosis in subsequent pregnancies The article presents a case of a patient in whom prenatal ultrasonography and subsequent clinical trials in post-natal period resulted in a Bardet-Biedl syndrome diagnosis, which was later confirmed through molecular tests.

Artículo de opinión

On the Parental Origin of Trisomy 21 Downs Syndrome

Larsson L and Hulten MA

The most common form of Down’s syndrome is due to an extra chromosome No. 21, i.e. three instead of two chromosomes 21, Trisomy 21 (T21). This was discovered over 50 years ago. We now know with certainty that the extra chromosome usually comes from the mother (in approximately 19 out of 20 cases) and also that the probability of having a child with Down’s syndrome increases with maternal age. However, we still do not know how this comes about.

It is generally accepted that, in rare cases, the mother has carried the extra chromosome 21 since the time when her ovaries were developing, when she herself was a fetus. On the other hand, it is also generally accepted that, in the majority of cases, the extra chromosome is due to an error in the so-called reduction division (meiosis), when the number of chromosomes are halved in the formation of egg cells. The reduction division begins when the mother is a fetus and is not finished until her monthly ovulation, from puberty until menopause.

Maj Hulten, Professor of Clinical Genetics, believe in contrast to most other researchers that many women may be low-grade ovarian T21 mosaics. In other words their ovaries contain both cells with the normal chromosome number as well as those with an extra chromosome, trisomy 21. This in turn could explain both the origin of T21 Down's syndrome in children of younger mothers, and the so-called maternal age effect, i.e. that the probability of having a child with T21 Down’s syndrome increases with maternal age.

Artículo de revisión

The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence from the Literature with Analysis

Beecham JE and Stephanie Seneff

The causes of autism spectrum disorder (ASD) are not well understood. Only a minority of cases are explainable by specific abnormalities in DNA sequence, whereas the majority are widely assumed to be linked to epigenetic effects, and/or likely impacted by environmental factors. Here, we postulate autism causation via environmental and/or dietary sourced toxin acting intermittently in utero on human fetuses to disrupt neurodevelopment in a nondose dependent manner. Our theory is informed by a mini-review and correlation of selected studies from the research literature related to autism, including radiologic, anatomic, metabolic, neurodevelopmental, pharmacologic and MRI studies. In reviewing and analyzing evidence, we focus on data supporting interaction of the theorized harmful glycine mimetic at one or more of the following calcium inflow regulatory factors for neurons: the N-methyl D-aspartate (NMDA) receptor, the glycine receptor (GlyR) and/or the glycine transporter protein 1 (GlyT1). We postulate this harmful glycine mimetic to act by exerting a direct molecular disruption to calcium regulatory factors for neurons. This disruption appears to occur in a time sensitive, rather than a strictly dose-dependent manner, leading to haphazard disorganizations of the normally carefully choreographed steps of early neuronal migration. Within this analysis, we find support for the contention that a strong candidate for the putative harmful glycine mimetic is glyphosate, the active ingredient in the pervasive herbicide Roundup®. In addition to glyphosate’s molecular similarity to glycine, glyphosate is known to have a propensity to avidly bind minerals such as manganese and magnesium, which minerals are implicated in the normal functioning of several neuronal calcium inflow regulatory factors. Our theory highlights areas deserving of further study.

Comunicación corta

Imaging of High and Low Resolution Ebola Envelope GP Structures Composited with in silico Models of Difficult-to-Resolve Sections

Garry W Lynch, Stefanie S Portelli, Siu Wai Wong, Maria Rocha Costa, Ana Paula Mello Lemgruber, Peter Williamson, John S Sullivan, Robert Booy and Bret Church W

The Ebola surface glycoprotein, GP, facilitates receptor binding, cell infection and cytopathology, and is a principal target for immune protection. The structure of the GP central core structure and antibody target for the 1976 Ebola Zaire Mayinga strain of Ebola has been resolved by X-ray crystallography (e.g., pdb-ID: 3CSY). But other important GP regions have defied crystal structure determination. These include the apical mucin-like domain (MLD) that contains immuno-protective sites and is most distal to the membrane, and the membrane proximal C terminus region (meD) where GP is tethered to the Ebola surface. A molecular structure-based strategy in vaccine design necessitates detailed fine-structure knowledge of exposed sites for immuno-targeting. To address the lack of MLD and meD structures we have performed computational modeling of those regions using the programs iTasser, Phyre2 and CABS-flex. Candidate MLD model structures for the 1976 Mayinga strain were screened for continuity and fit with the 3CSY core and tested further for 3D compliance for its fit within the spatial boundaries of a lowresolution GP trimer cryoelectron tomograph (EMDB-ID: EMD-6003). Under these constraints only 1 of 6 initial MLD models generated by iTasser or Phyre2 met the required structural criteria of continuity, orientation and spatial fit. That best-fit structure (MLDm1p1) was additionally evaluated using the MolProbity geometric analysis, as well by the generation of highly similar and compatible structures (e.g., MLDm2c2) by the CABS-flex program. Notably, documented protective antibody sites of the MLD mapped to the MLDm1p1 apex surface, as anticipated for domain functionality or exposure to antibody attack. These studies describe best fit proximate in silico models of the MLD and meD regions of GP to for improved molecular understanding and therapy design. Biochemical and immunologic validation and refinements of the models are continuing, to establish to their biostructural compatibilities and value to inform on GP structure-function and immuno-target potential.

Artículo de investigación

Effects of BRCA1 Knockdown on CYP19a1/Aromatase and Steroid Receptor Expression in Ovarian and Tubal Cells

Jean S Fleming, Igor Ruza, Bryony A Thompson, Kai-Fai Lee and Adele G Woolley

BRCA1 mutation carriers have an increased lifetime risk of serous epithelial ovarian cancer (EOC), as well as breast cancer, but reasons for this increased risk remain elusive. We hypothesized that the reported relationship between cytochrome P450 aromatase (CYP19a1) and BRCA1 expression might be used to elucidate this pathway to EOC in women with BRCA1 mutations. Expression of BRCA1, CYP19a1 and steroid receptors was measured by qRT-PCR and immunoblotting in immortalized cell lines from the ovarian surface epithelium (hOSE17-1 and hOSE11-12), tubal epithelium (OE-E6/E7) and granulosa cell tumor (KGN), as well as MCF-7 mammary carcinoma cells, before and after BRCA1 knockdown with 2 nM siRNA, or stimulation of CYP19a1 expression with forskolin or phorbol-12-myristate-13-acetate (PMA). Low or no CYP19a1 expression was observed in all cell lines, except KGN cells. BRCA1 knockdown with 2 nM siRNA did not stimulate CYP19a1 expression in any cell line. Forskolin treatment increased CYP19a1 expression only in KGN cells and this resulted in a decrease in BRCA1 expression equivalent to 2 nM siRNA knockdown. We conclude that lowering BRCA1 expression in OSE and tubal epithelia cell lines does not stimulate CYP19a1 expression or change steroid receptor expression significantly. The mechanism by which BRCA1 mutation increases risk of serous EOC remains to be elucidated.

Mini reseña

Hypothesis: Spirulina may Slow the Growth and Spread of Ovarian Cancer by Interfering with Growth Factor Activity of Lysophophatidic Acid

Mark F McCarty

Lysophosphatidic acid (LPA) has emerged as a key autocrine growth factor for most ovarian cancers, promoting their proliferation, survival, invasiveness, dissemination within the peritoneal cavity, and angiogenic capacity. Effective LPA signaling requires activation of endosomal NADPH oxidase activity. Free bilirubin is now known to function intracellularly as a potent inhibitor of NADPH oxidase complexes. The cyanobacterial chromophore phycocyanobilin (PhyCB), via intracellular conversion to the bilirubin homolog phycocyanorubin, can likewise inhibit NADPH oxidase activity, and is orally active in this regard. The cell wall polysaccharides of cyanobacteria may also aid cancer control by activating innate immunity and inhibiting angiogenesis. Hence, consumption of edible cyanobacteria such as spirulina may have potential for slowing the growth and spread of ovarian cancer – as it has recently been shown to do with a human pancreatic adenocarcinoma.

Artículo de investigación

Whole-Exome Sequencing Identifies Novel Somatic Mutations in Chinese Breast Cancer Patients

Yanfeng Zhang, Qiuyin Cai, Xiao-Ou Shu, Yu-Tang Gao, Chun Li, Wei Zheng and Jirong Long

Most breast cancer genomes harbor complex mutational landscapes. Somatic alterations have been predominantly discovered in breast cancer patients of European ancestry; however, little is known about somatic aberration in patients of other ethnic groups including Asians. In the present study, whole-exome sequencing (WES) was conducted in DNA extracted from tumor and matched adjacent normal tissue samples from eleven early onset breast cancer patients who were included in the Shanghai Breast Cancer Study. We discovered 159 somatic missense and ten nonsense mutations distributed among 167 genes. The most frequent 50 somatic mutations identified by WES were selected for validation using Sequenom MassARRAY system in the eleven breast cancer patients and an additional 433 tumor and 921 normal tissue/blood samples from the Shanghai Breast Cancer Study. Among these 50 mutations selected for validation, 32 were technically validated. Within the validated mutations, somatic mutations in the TRPM6, HYDIN, ENTHD1, and NDUFB10 genes were found in two or more tumor samples in the replication stage. Mutations in the ADRA1B, CBFB, KIAA2022, and RBM25 genes were observed once in the replication stage. To summarize, this study identified some novel somatic mutations for breast cancer. Future studies will need to be conducted to determine the function of these mutations/genes in the breast carcinogenesis.

Artículo de investigación

Influence of a Regular, Standardized Meal on Lipid Profile of People with Diabetes

Susana Garrido, Fernando Pichel, Helena Neto, Helena Ramos, José Carlos Oliveira and Isabel Palma

Aims: The need to fast before lipid screening has been questioned in the past years. The aim of this study was to determine the influence of a light meal on the lipid profile in people with diabetes.
Methods: 115 participants with type 2 diabetes were recruited between April/2013-August/2014 from our Outpatient Diabetes Education Clinic. Clinical and analytical evaluation took place in 2 moments (8-hour fasting=t0; 2h after a light standardized meal=t1), with measurements of total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides.
Results: Triglycerides concentration increased between the 2 moments (median difference t1-t0=0.07 mmol/L, p=0.002) but the total cholesterol, LDL-cholesterol, HDL-cholesterol and non HDL- cholesterol did not change significantly. Performing an analysis according to the LDL-cholesterol therapeutic goals proposed by Adult Treatment Panel III, we found an agreement between fasting and postprandial assessments of 91.1% for the goal of 2.6 mmol/L (102/112), and of 97.3% for the goal of 1.8 mmol/L (109/112). The same analysis was performed for the secondary goal, non HDL-cholesterol.
Conclusion: The data presented suggest that the nonfasting lipid profile can be an alternative to the fasting lipid profile in selected patients. Larger studies are needed to confirm these results and demonstrate an association of nonfasting lipemia and cardiovascular risk in individuals with diabetes.

Reporte de un caso

Gene Mutation in Tuberous Sclerosis Complex (TSC) and Reversal Treatment of the Mal-formation in Collagens Synthesis: Treatment of Angiofibromas via LLysine Therapy

Samira A Barghouthi and Abdalla MWalwil

Angiofibromas are malformation of skin caused by genetic disorders and gene mutation. The purpose of this case study is to find a natural and cost effective solution to overcome the formation and proliferation of angiofibroma structures. At the present time there no simple treatment is available for angiofaibromas and the only available techniques involve the use of carbon dioxide and laser. Besides being costly these techniques need repetitive treatments over a long time and no guarantee of permanent removal of these angiofaibromas. They seem to reform in the same location and in some cases may extend to the surrounding area. The manifestation of these, in the facial area, is in addition to the disfiguring of the face cause health problems as a result of bleeding and possible infection. These angiofaibromas with sizes ranging from granules on the micrometer size to larger ones with diameters on the millimeter scale. They are being reddish to brown, depending on the amount of blood vessels and vacuolated blood. Why are angiofibromas developed around the onset of puberty and accompanying hormonal change? What are the physiological changes that occur at this age? Are they associated with skin and protein synthesis and are conducive to the development of angiofibromas?

Because of the fact that these are considered to be genetics the only treatments proposed and performed by medical-doctors only symptomatic treatments. In this paper we propose a treatment of the actual protein synthesis and an enhancement of the production of the proper collagen protein to sharply minimize the formation or may be in some cases totally eradicating the angiofaibromas or in the worst scenario just decrease of blood vacuoles supplement and hence shrinking the fibromas. Results presented here are based on case studies and are very encouraging to perform clinical testing on a larger scale, i.e., a larger group of patients younger than 20 years of age. The limitation of such study is defined under testing patients who do not have any angiofibromas but who are likely to develop angiofibromas. We did not have this kind of sample in the group, which would strengthen the results by providing a baseline on the effectiveness of L-Lysin in inhibiting the initial development of the facial fibromas. In future study this could be compensated for by conducting some tissue culture and monitoring the effect of various amino acids on normal cells vis-à-vis mutated cell lines. This type of research is very crucial for both researchers and consumers as it emphasizes the concept of simplicity in prevention and treatment. Angiofibromas are also caused as side effects of some seizure medications such as phenytoin and the results are devastating for the patients. We hope that maybe some will benefit from this research by following healthy diets with regard to the inclusion of essential amino acids in a healthy diet especially that of L-Lysine.

Artículo de investigación

Cold Stress and Presence of Pseudomonas fluorescens Affect Listeria monocytogenes Biofilm Structure and Response to Chitosan

Puga CH, Orgaz B, Muñoz S and SanJose C

Life in biofilms (BF) provides microorganisms with protection against different adverse conditions and agents. In food industry, as they can host and transfer to food both pathogenic and spoilage microorganism, they have to be constantly kept under control. Many hygienic practices and disinfectants aim at preventing and/or destroying BF, and chitosan has a promising future in this respect. Listeria monocytogenes (Lm) is a dangerous foodborne pathogen that can live in BF and survive many restrictive conditions used to preserve foods, such as refrigeration. In this work, nine Lm strains, persistently or sporadically isolated from a meat processing plant, were cultured at 20°C and 4°C to obtain mature BF either in isolation or with Pseudomonas fluorescens (Pf), both species being simultaneously inoculated at similar low population levels. Pf was more compatible with the persistent Lm strains than with the rest, enhancing or maintaining their viable counts in the corresponding dual species BF. All dual species BF formed at 4°C were much thinner than those formed at 20°C, but contained more cells per cm3 of BF biomass. Chitosan damage was observed both as reduction of Lm viable cells and by confocal laser scanning microscopy (CLSM) with Live/Dead stains. In Lm monospecies BF, 1 h chitosan exposure reduced viable counts between 3 and 6 Log when cultured at 20°C and 2-4 Log when at 4°C. Both temperature of BF formation and Lm strain affected their susceptibility to chitosan in dual species BF. CLSM showed focalized chitosan injuries in binary BF, particularly in those with persistent Lm strains.

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