Molecular biology

Cryptic species remain poorly studied in octopuses. Following the reinstatement of the cryptic species, Octopus americanus, suggestions that it should supplant two taxa, Octopus vulgaris types I & II, in the western central and southwestern Atlantic Ocean, respectively, had yet been investigated in southeast Florida. To delineate this species, an integrative approach including morphological assessments and phylogenetic analyses of mitochondrial (16S & COI) and nuclear (rhodopsin) genes was employed. The utility of swabbing as a minimally invasive alternative DNA sampling method to tissue was also investigated. This taxon displays similar morphological traits to O. americanus. Maximum Likelihood phylogenetic inference placed this taxon in a highly supported monophyletic group with O. americanus. Both sampling methods delineated this taxon, though tissue was more successful. These results indicate that southeast Florida’s O. vulgaris-like species represents O. americanus. Thorough species delineation approaches advance our understanding of biodiversity, evolution, and ecology, and inform management practices.

Beta-Amyloid (1-40) (Aβ40) is an aggregation prone protein, which undergoes a nucleation-dependent aggregation process causing the pathological neurodegeneration by amyloid plaque formation implicated in Alzheimer’s disease. In this thesis, we investigated the effects of small molecule modulators extracted from the marine invertebrate Pseudopterogorgia elisabethae on the Aβ40 amyloidogenic process using in- vitro ThT fluorescence assay and atomic force microscopy. We also investigated the effects of neutral and anionic phospholipid liposomes on Aβ40 aggregation. Our results show that a marine natural product Pseudopterosin-A and its derivatives can suppress and modulate the Aβ40 aggregation process. Furthermore, our results demonstrate that a neutral phospholipid liposome inhibits Aβ40 fibril formation, whereas the anionic liposomes promote it.

There is compelling evidence that smokers are less responsive to vaccination. We reported that both therapeutic and prophylactic vaccines fail to protect and cure animals from disease due to negative effects of nicotine on DCs’ ability to generate effector T cells. We have been investigating whether vaccine formulated with TLR agonist(s) could potentially overcome the immunosuppressive effects of nicotine on human DC-NK cross-talk essential for effector T cell generation. Monocyte-derived DCs and nicDCs were stimulated with individual and combined TLR agonists prior to co-culture with purified T cells. The phenotypes and cytokine profiles of T cell were assessed using Flow Cytometry and ELISA, respectively. We found nicDCs cultured with TLR-8/7 alone or in combination with TLR-3 produce quantitatively and qualitatively similar IFN-γ producing effector T cells when compared to control DCs. Our data suggest that the addition of appropriate TLR agonist to vaccine formulation could potentially overcome the immunosuppression seen in smokers, thereby containing the spread of infectious disease to vulnerable population

The research efforts refer to tracking homologus loci in the chromosomes of a pair of a species. The purpose is to infer the extent of maximum syntenic correlation when an exhaustive set of orthologs of the species are searched. Relevant bioinformatic analyses use comparative mapping of conserved synteny via Oxford grid. In medical diagnostic efforts, deducing such synteny correlation can help screening chromosomal aberration in genetic disorder pathology. Objectively, the present study addresses: (i) Cytogenetic framework of syntenic correlation and, (ii) applying information-theoretics to determine entropy-dictated synteny across an exhaustive set of orthologs of the test pairs of species.

Restrictive cardiomyopathy (RCM) is represented in part by left ventricular stiffness
and diastolic dysfunction. Missense mutations of the cardiac troponin I (cTnI) gene cause
idiopathic RCM. These mutations are located in the C-terminus of cTnI and affect cardiac
relaxation. Transgenic mouse models presenting the pathology observed in clinical
patients with RCM have been generated previously and express the mutant cTnI in their
hearts. RCM-linked mutations increase cardiac myofilament Ca2+ sensitivity and promote
diastolic dysfunction in the heart. Previous studies using double transgenic mice
(cTnI/R193H/ND) showed that ventricular relaxation is enhanced in the cTnI/R193H
transgenic mice. In this study, another double transgenic mouse model,
(cTnI/R193H/ND/KO), provides an avenue to investigate its rescuing effects on RCMlinked
mutations in the cTnI /R193H/KO mouse. Use of molecular biological techniques,
transgenic animal developments and murine echocardiography in this study has
culminated into a greater understanding of RCM and diastolic dysfunction.

A naturally-occurring recessive lethal mutation in axolotls, Ambystoma mexicanum, is an intriguing model for studying tropomyosin expression regulation. Homozygous embryos(c/c) form hearts that are deficient in tropomyosin, lack organized myofibrils and fail to beat. Previous studies have shown that a non-coding RNA gene, MIR (Myofibril Inducing RNA), is sufficient to rescue the non-beating homozygous recessive mutant hearts by promoting sarcomeric tropomyosin expression that leads to formation of organized myofibrils and beating hearts. Real time RT-PCR reveals that mutant hearts express the same level mRNA of the alpha-tropomyosin and TM4 type tropomyosin (ATmC-3) gene as normal embryonic hearts. These genes show no differences with regard to the splicing patterns of normal and mutant. Using protease inhibitor LLnL and E-64d treatments and two-dimensional Western blots of normal and mutant hearts, it is found that mutant hearts express all tropomyosin protein isoforms as normal hearts but protein expression are at low levels. These studies suggest that there is a failure in the translational or posttranslational control mechanisms for tropomyosin protein synthesis in cardiac mutant axolotl hearts during development.

Restrictive cardiomyopathy (RCM) is a cardiac muscle disorder characterized by increased ventricular stiffness and diastolic dysfunction. Patients with RCM often present severe cardiac problems which usually lead to heart failure and sudden death. No effective treatment is available for RCM which makes the finding of novel efficient therapies an urgent necessity. Great progress in molecular biology techniques and advances in transgenic animal development provide great opportunities for the study of RCM and other cardiovascular diseases encountered in clinical patients.... Our laboratory is among the first to generate transgenic mouse models of RCM based on cardiac troponin I (cTnI) missense mutations. In this study, transgenic mice that suffer from RCM have been generated to understand the factors behind the diastolic dysfunction associated with that myocardial disease.... The information obtained from this study allows a better understanding of the role of troponin in RCM and the factors behind the physiopathology of the disease. It will also offer a therapeutic strategy taking into account the physiological characteristic of RCM.

We have studies oxidative damage of RNA, a major type of cellular macromolecules. RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic acid damages in Escherichia coli (E.coli) are present in RNA as shown by high levels of 8-oxo-G, an oxidized form of guanine. Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither RNA structure nor association with proteins protects RNA from oxidation... Our results demonstrate a major role for RNA degradation in controlling oxidized RNA. We have identified activities that may work in specific pathways for selectively degrading damaged RNA. These activities may play pivotal rold in controlling oxidized RNA and protecting cells under oxidative stress.

Carbohydrate Active Enzyme family 6 (CA6) glycosyltransferases (GTs) are type II transmembrane proteins localized in the Golgi apparatus. CA6 GTs have a GT-A fold, a type of structure that resembles the Rossman fold and catalyze the transfer either galactose (Gal) or N-acetylgalactosamine (GalNAc) from the UDP nucleotide sugar to an non-reducing terminal Gal or GalNAc on an acceptor via an a-1,3 linkage. In this reaction, the anomeric configuration of the sugar moiety of the donor is retained in the product. CA6 GTs includes the histo-blood group A and B GTs, a-galactosyltransferase (a3GT), Forssman glycolipid synthase (FS), isogloboside 3 synthase (iGb3) in mammals. a3GT and its products (a-Gal epitode) are present in most mammals but are absent in humans and old world primates because of inactivating mutations. The absence of a3GT and its products results in the production of anti-a-Gal epitope natural antibodies in these species.