Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.     

Host-Seeking Behavior and Dispersal of Triatoma infestans,a Vector of Chagas Disease,under Semi-field Conditions

Chagas disease affects millions of people in Latin America. The control of this vector-borne disease focuses on halting transmission by reducing or eliminating insect vector populations. Most transmission of Trypanosoma cruzi, the causative agent of Chagas disease, involves insects living within or very close to households and feeding mostly on domestic animals. As animal hosts can be intermittently present it is important to understand how host availability can modify transmission risk to humans and to characterize the host-seeking dispersal of triatomine vectors on a very fine scale. We used a semi-field system with motion-detection cameras to characterize the dispersal of Triatoma infestans, and compare the behavior of vector populations in the constant presence of hosts (guinea pigs), and after the removal of the hosts. The emigration rate – net insect population decline in original refuge – following host removal was on average 19.7% of insects per 10 days compared to 10.2% in constant host populations (p = 0.029). However, dispersal of T. infestans occurred in both directions, towards and away from the initial location of the hosts. The majority of insects that moved towards the original location of guinea pigs remained there for 4 weeks. Oviposition and mortality were observed and analyzed in the context of insect dispersal, but only mortality was higher in the group where animal hosts were removed (p-value <0.01). We discuss different survival strategies associated with the observed behavior and its implications for vector control. Removing domestic animals in infested areas increases vector dispersal from the first day of host removal. The implications of these patterns of vector dispersal in a field setting are not yet known but could result in movement towards human rooms.     

Development of advanced host cell protein enrichment and detection strategies to enable process relevant spike challenge studies

An orthogonal chromatography methodology for the enrichment of host cell protein (HCP) species relative to monoclonal antibody (mAb) products was developed and applied for the successful enrichment of HCP from post‐Protein A process pools for seven different mAb products. An advanced two‐dimensional liquid chromatography/mass spectrometry platform (2D‐LC/MS^E) was utilized to demonstrate that the HCP enriched material was representative, in terms of species content, to pre‐enriched process pools. The HCP enrichment methodology was scaled up for two different mAb products, and this process relevant enriched HCP material was used to conduct advanced spike challenge studies to demonstrate the utility of the approach for the understanding of (1) quantitative HCP clearance, (2) individual species clearance, and (3) species clearance redundancy across polishing chromatography steps. The combined ability to enrich process relevant HCP, detect individual HCP species with 2D‐LC/MS^E technology, and conduct advanced challenge studies with process relevant material surmounts prior limitations to high integrity process challenge study implementation, and facilitates significant process understanding for development of risk‐based control strategies and strategic process design. This also demonstrates implementation of a foundational strategy for conducting spike‐challenge studies using process‐relevant impurities isolated from processes of interest using orthogonal approaches. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:983–989, 2015     

THE EVOLUTION OF PHENOTYPIC CORRELATIONS AND “DEVELOPMENTAL MEMORY”

Development introduces structured correlations among traits that may constrain or bias the distribution of phenotypes produced. Moreover, when suitable heritable variation exists, natural selection may alter such constraints and correlations, affecting the phenotypic variation available to subsequent selection. However, exactly how the distribution of phenotypes produced by complex developmental systems can be shaped by past selective environments is poorly understood. Here we investigate the evolution of a network of recurrent nonlinear ontogenetic interactions, such as a gene regulation network, in various selective scenarios. We find that evolved networks of this type can exhibit several phenomena that are familiar in cognitive learning systems. These include formation of a distributed associative memory that can “store” and “recall” multiple phenotypes that have been selected in the past, recreate complete adult phenotypic patterns accurately from partial or corrupted embryonic phenotypes, and “generalize” (by exploiting evolved developmental modules) to produce new combinations of phenotypic features. We show that these surprising behaviors follow from an equivalence between the action of natural selection on phenotypic correlations and associative learning, well‐understood in the context of neural networks. This helps to explain how development facilitates the evolution of high‐fitness phenotypes and how this ability changes over evolutionary time.     

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.     

TELOCYTES IN ENDOCARDIUM: Electron Microscope Evidence

The term TELOCYTES was very recently introduced, for replacing the name Interstitial Cajal‐Like Cells (ICLC). In fact, telocytes are not really Cajal‐like cells, they being different from all other interstitial cells by the presence of telopodes, which are cell‐body prolongations, very thin (under the resolving power of light microscopy), extremely long (tens up to hundreds of micrometers), with a moniliform aspect (many dilations along), and having caveolae. The presence of telocytes in epicardium and myocardium was previously documented. We present here electron microscope images showing the existence of telocytes, with telopodes, at the level of mouse endocardium. Telocytes are located in the subendothelial layer of endocardium, and their telopodes are interposed in between the endocardial endothelium and the cardiomyocytes bundles. Some telopodes penetrate from the endocardium among the cardiomyocytes and surround them, eventually. Telopodes frequently establish close spatial relationships with myocardial blood capillaries and nerve endings. Because we may consider endocardium as a ‘blood–heart barrier’, or more exactly as a ‘blood–myocardium barrier’, telocytes might have an important role in such a barrier being the dominant cell population in subendothelial layer of endocardium.     

Co‐ordinated autophagy with resveratrol and γ‐tocotrienol confers synergetic cardioprotection

This study compared two dietary phytochemicals, grape‐derived resveratrol and palm oil‐derived γ‐tocotrienol, either alone or in combination, on the contribution of autophagy in cardioprotection during ischaemia and reperfusion. Sprague‐Dawley rats weighing between 250 and 300 g were randomly assigned to one of the following groups: vehicle, ischaemia/reperfusion (I/R), resveratrol + I/R, γ‐tocotrienol + I/R, resveratrol +γ‐tocotrienol + I/R. For resveratrol treatments, the rats were gavaged with resveratrol (2.5 mg/kg) for 15 days while for γ‐tocotrienol experiments the rats were gavaged with γ‐tocotrienol (0.3 mg/kg) for 30 days. For the combined resveratrol +γ‐tocotrienol experiments, the rats were gavaged with γ‐tocotrienol for 15 days, and then gavaging continued with resveratrol along with γ‐tocotrienol for a further period of 15 days. After 30 days, isolated perfused hearts were subjected to 30 min. of global ischaemia followed by 2 hrs of reperfusion. Our results showed for the first time that at least in part, the cardioprotection (evidenced from the ventricular performance, myocardial infarct size and cardiomyocyte apoptosis) with resveratrol and γ‐toctrienol was achieved by their abilities to induce autophagy. Most importantly, resveratrol and γ‐tocotrienol acted synergistically providing greater degree of cardioprotection simultaneously generating greater amount of survival signal through the activation of Akt‐Bcl‐2 survival pathway. Autophagy was accompanied by the activation of Beclin and LC3‐II as well as mTOR signalling, which were inhibited by either 3‐methyl adenine (3‐MA) or Wortmannin. The autophagy was confirmed from the results of transmission electron microscopy and light microscopy as well as with confocal microscopy. It is tempting to speculate that during ischaemia and reperfusion autophagy along with enhanced survival signals helps to recover the cells from injury.     

Marek's disease virus type 2 (MDV-2)-encoded microRNAs show no sequence conservation with those encoded by MDV-1

MicroRNAs (miRNAs) are increasingly being recognized as major regulators of gene expression in many organisms, including viruses. Among viruses, members of the family Herpesviridae account for the majority of the currently known virus-encoded miRNAs. The highly oncogenic Marek's disease virus type 1 (MDV-1), an avian herpesvirus, has recently been shown to encode eight miRNAs clustered in the MEQ and LAT regions of the viral genome. The genus Mardivirus, to which MDV-1 belongs, also includes the nononcogenic but antigenically related MDV-2. As MDV-1 and MDV-2 are evolutionarily very close, we sought to determine if MDV-2 also encodes miRNAs. For this, we cloned, sequenced, and analyzed a library of small RNAs from the lymphoblastoid cell line MSB-1, previously shown to be coinfected with both MDV-1 and MDV-2. Among the 5,099 small RNA sequences determined from the library, we identified 17 novel MDV-2-specific miRNAs. Out of these, 16 were clustered in a 4.2-kb long repeat region that encodes R-LORF2 to R-LORF5. The single miRNA outside the cluster was located in the short repeat region, within the C-terminal region of the ICP4 homolog. The expression of these miRNAs in MSB-1 cells and infected chicken embryo fibroblasts was further confirmed by Northern blotting analysis. The identification of miRNA clusters within the repeat regions of MDV-2 demonstrates conservation of the relative genomic positions of miRNA clusters in MDV-1 and MDV-2, despite the lack of sequence homology among the miRNAs of the two viruses. The identification of these novel miRNAs adds to the growing list of virus-encoded miRNAs.     

Retrospective study of the prevalence of high-risk human papillomaviruses among Croatian women

The infection with Human papillomavirus (HPV) is the necessary cause for cervical cancer. There are at least 15 High-Risk (HR) HPV types that are significantly associated with progression of cervical intraepithelial neoplasia to cervical cancer. Since previous studies showed that the prevalence of HPV in cervical cancers varies among different geographic regions, we wanted to investigate the prevalence of HPV types in Croatia, especially low abundant HR HPV types. By means of consensus primers directed polymerase chain reaction (PCR), we analysed cervical DNA samples of 2,136 Croatian women, mostly with abnormal cervical smears, in order to detect the presence of HPV Type-specific primers were then used to determine Low-Risk (LR) HPV types 6/11 and HR HPV types 16, 18, 31, 33, 45, 52 and 58. Out of 2,136 specimens, 1,255 (58.8%) were positive for HPV More than half of positive samples were typed (64.5%) and 35.5% still remained untyped. Multiple HPV infections were found in 10.3% of the cases. The most prevalent type, including both single and multiple infections, was HPV16 with the prevalence of 15.9%, followed by HPV types 31, 6/11, 33, 18, 52, 45 and 58 with 8.7%, 7.1%, 4.5%, 3.8%, 2.3%, 1.2% and 1.1%, respectively. The significant increase of frequency from Low-grade Squamous Intraepithelial Lesions (LSIL) to High-grade Squamous Intraepithelial Lesions (HSIL) was observed for HR HPV types 16, 18, 31 and 33 but not 45, 52 and 58. The frequency of unknown HPV types was almost the same in cervical specimens of women with LSIL and those with HSIL, 19.8% and 21.1%, respectively. The prevalence of HPV infection rate decreased significantly with patient age from 68.5% (age group 12 to 24 years) to 38.8% (age group 45 to 54 years). But, in women aged 55 or older the overall prevalence increased to 56.6%. Our results indicate that prevalence of HR HPV types in Croatia is similar to other countries. We suggest that HPV positive women in Croatia should be closely monitored by typing for HR HPV types: 16, 18, 31, 33, 45, 52 and 58.     

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.