Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Varying the length of dim nocturnal illumination differentially affects the pacemaker controlling the locomotor activity rhythm of Drosophila jambulina

Photic entrainment of animals in the field is basically attributed to their exposure to the dimly lit nights flanked by the dawn and dusk twilight transitions. This implicates the functional significance of the dimly lit nights as that of the twilight transitions. Recently, the authors have demonstrated that the dimly lit night at 0.0006 lux altered the attributes of the circadian rhythm of locomotor activity of Drosophila jambulina. The present study examined whether the durations of such dimly lit nights affect the entrainment and free-running rhythmicity of D. jambulina. Flies were subjected for 10 days to two types of 24-h lighting regimes in which the photophase (L) was at 10 lux for all flies but the scotophase, which varied in duration from 9 to 15 h, was either at 0 lux (D phase) for control flies or 0.0006 lux (the artificial starlight or S phase) for experimental flies. Thereafter, they were transferred to constant darkness (DD) to compare the after-effects of the dimly lit nights on the period (τ) of free-running rhythm in DD with that of the completely dark nights. Control flies were entrained by all LD cycles, but the experimental flies were entrained only by five LS cycles in which the duration of the S phases ranged from 10 to 14 h. The two LS cycles with very short (9 h) and long (15 h) S phases rendered the flies completely arrhythmic. Control flies started activity shortly before lights-on and continued well after lights-off. The experimental flies, however, commenced activity several hours prior to lights-on but ended activity abruptly at lights-off as the result of a negative masking effect of nocturnal illumination. Length of the midday rest was considerably shorter in the control than in the experimental flies in each lighting regime. The active phase in the control flies was predictably shortened; nonetheless, it was invariable in the experimental flies as the nights lengthened. Transfer from lighting regimes to DD initiated robust free-running rhythmicity in all flies including the arrhythmic ones subjected to LS cycles with 9 and 15 h of scotophases. The τ was profoundly affected by the nocturnal irradiance of the prior entraining lighting regime, as it was always shorter in the experimental than in the control flies. Thus, these results indisputably demonstrate the changes in fundamental properties of the circadian pacemaker of D. jambulina were solely attributed to the extremely dim nocturnal irradiance. This strain of D. jambulina is entrained essentially by the dimly lit natural nights, since it is never exposed to the prevailing photic cues such as the twilight transitions or bright photoperiod, owing to the dense vegetation of its habitat.     

Prognostic significance of cyclooxygenase-2 and response to chemotherapy in invasive ductal breast carcinoma patients by real time surface plasmon resonance analysis

Cyclooxygenase-2 (COX-2), an inducible enzyme, has been implicated in the progression and angiogenesis of breast cancer. The aim of the study is to quantify the concentration of COX-2 and its association with clinico-pathological parameters and response to treatment in patients with invasive ductal carcinoma receiving both neo-adjuvant and adjuvant chemotherapy. The level of COX-2 was estimated using a novel biosensor-based surface plasmon resonance technique in serum of 84 patients with breast cancer (48 patients of neo-adjuvant chemotherapy and 36 patients of adjuvant chemotherapy) and 40 age- and gender-matched normal individuals. A significant increase in COX-2 level was observed in patients compared with normal individuals (p>0.0001). The COX-2 level in serum was found to be significantly higher in patients with lymph node involvement (p<0.0061). 68% (33/48) of the patients receiving neo-adjuvant chemotherapy showed significantly (p<0.0025) reduced COX-2 levels. This study shows significant decrease of COX-2 level in patients with breast cancer treated with both neo-adjuvant and adjuvant chemotherapy. Estimation of COX-2 level in serum may serve as a tumor biomarker in patients with breast cancer.     

Co-administration of IL-1+IL-6+TNF-α with Mycobacterium tuberculosis infected macrophages vaccine induces better protective T cell memory than BCG

BCG has been administered globally for more than 75 years, yet tuberculosis (TB) continues to kill more than 2 million people annually. Further, BCG protects childhood TB but is quite inefficient in adults. This indicates that BCG fails to induce long-term protection. Hence there is a need to explore alternative vaccination strategies that can stimulate enduring T cell memory response. Dendritic cell based vaccination has attained extensive popularity following their success in various malignancies. In our previous study, we have established a novel and unique vaccination strategy against Mycobacterium tuberculosis (M. tb) and Salmonella typhimurium by utilizing infected macrophages (IM). In short-term experiments (30 days), substantial degree of protection was observed. However, remarkable difference was not observed in long-term studies (240 days) due to failure of the vaccine to generate long-lasting memory T cells. Hence, in the present study we employed T cell memory augmenting cytokines IL-1+IL-6+TNF-α and IL-7+IL-15 for the induction of the enhancement of long-term protection by the vaccine. We co-administered the M. tb infected macrophages vaccine with IL-1+IL-6+TNF-α (IM-1.6.α) and IL-7+IL-15 (IM-7.15). The mice were then rested for a reasonably large period (240 days) to study the bona fide T cell memory response before exposing them to aerosolized M. tb. IM-1.6.α but not IM-7.15 significantly improved memory T cell response against M. tb, as evidenced by recall responses of memory T cells, expansion of both central as well as effector memory CD4 and CD8 T cell pools, elicitation of mainly Th1 memory response, reduction in the mycobacterial load and alleviated lung pathology. Importantly, the protection induced by IM-1.6.α was significantly better than BCG. Thus, this study demonstrates that not only antigen-pulsed DCs can be successfully employed as vaccines against cancer and infectious diseases but also macrophages infected with M. tb can be utilized with great efficacy especially in protection against TB.     

Complete genome sequence of Coraliomargarita akajimensis type strain (04OKA010-24)

Coraliomargarita akajimensis Yoon et al. 2007 is the type species of the genus Coraliomargarita. C. akajimensis is an obligately aerobic, Gram-negative, non-spore-forming, non-motile, spherical bacterium that was isolated from seawater surrounding the hard coral Galaxea fascicularis. C. akajimensis is of special interest because of its phylogenetic position in a genomically under-studied area of the bacterial diversity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Puniceicoccaceae. The 3,750,771 bp long genome with its 3,137 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

The determination of spatial pattern inDictyostelium discoideum

Free-living amoebae of the cellular slime mouldDictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case ofDictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) ‘coin tossing’, whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) ‘chemical kinetics’, whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other; and (iii) ‘positional information’, in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arisesin situ     

Unravelling the emerging threats of microplastics to agroecosystems

Reviews in Environmental Science and Bio/Technology - In the past few decades, pollution from microplastics has emerged as an important issue on a global scale. These plastic particles are mainly...