Marine actinomycete diversity and natural product discovery

Microbial natural products remain an important resource for drug discovery yet the microorganisms inhabiting the worlds oceans have largely been overlooked in this regard. The recent discovery of novel secondary metabolites from taxonomically unique populations of marine actinomycetes suggests that these bacteria add an important new dimension to microbial natural product research. Continued efforts to characterize marine actinomycete diversity and how adaptations to the marine environment affect secondary metabolite production will create a better understanding of the potential utility of these bacteria as a source of useful products for biotechnology.     

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO⁻) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO⁻ and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.     

Live attenuated lentivirus infection elicits polyfunctional simian immunodeficiency virus Gag-specific CD8+ T cells with reduced apoptotic susceptibility in rhesus macaques that control virus replication after challenge with pathogenic SIVmac239

HIV-specific CD8+ T cells that secrete multiple cytokines in response to Ag stimulation are associated with the control of virus replication during chronic HIV infection. To determine whether the presence of polyfunctional CD8+ T cell responses distinguishes protected and unprotected monkeys in a live attenuated lentivirus model, SIV Gag peptide-specific CD8+ T cell responses of simian HIV (SHIV) 89.6-vaccinated, SIVmac239-challenged rhesus macaques were compared in two monkeys that controlled challenge virus replication and two that did not. The ratio of Bcl-2+ Gag-specific CD8+ T cells to caspase-3+ Gag-specific CD8+ T cells was higher in the vaccinated-protected animals compared with unprotected monkeys. In addition, polyfunctional SIV-specific CD8+ T cells were consistently detected through 12 wk postchallenge in the protected animals but not in the unprotected animals. In the unprotected monkeys, there was an increased frequency of CD8+ T cells expressing markers associated with effector memory T cells. Further, there was increased annexin V expression in central memory T cells of the unprotected animals before challenge. Thus, monkeys that control viral replication after live attenuated SHIV infection have polyfunctional SIV-specific CD8+ T cells with an increased survival potential. Importantly, the differences in the nature of the SIV-specific CD8+ T cell response in the protected and unprotected animals are present during acute stages postchallenge, before different antigenic levels are established. Thus, the polyfunctional capacity and increased survival potential of CD8+ SIV-specific T cells may account for live attenuated, SHIV89.6-mediated protection from uncontrolled SIV replication.     

Autonomic Recovery Is Delayed in Chinese Compared with Caucasian following Treadmill Exercise

Caucasian populations have a higher prevalence of cardiovascular disease (CVD) when compared with their Chinese counterparts and CVD is associated with autonomic function. It is unknown whether autonomic function during exercise recovery differs between Caucasians and Chinese. The present study investigated autonomic recovery following an acute bout of treadmill exercise in healthy Caucasians and Chinese. Sixty-two participants (30 Caucasian and 32 Chinese, 50% male) performed an acute bout of treadmill exercise at 70% of heart rate reserve. Heart rate variability (HRV) and baroreflex sensitivity (BRS) were obtained during 5-min epochs at pre-exercise, 30-min, and 60-min post-exercise. HRV was assessed using frequency [natural logarithm of high (LnHF) and low frequency (LnLF) powers, normalized high (nHF) and low frequency (nLF) powers, and LF/HF ratio] and time domains [Root mean square of successive differences (RMSSD), natural logarithm of RMSSD (LnRMSSD) and R–R interval (RRI)]. Spontaneous BRS included both up-up and down-down sequences. At pre-exercise, no group differences were observed for any HR, HRV and BRS parameters. During exercise recovery, significant race-by-time interactions were observed for LnHF, nHF, nLF, LF/HF, LnRMSSD, RRI, HR, and BRS (up-up). The declines in LnHF, nHF, RMSSD, RRI and BRS (up-up) and the increases in LF/HF, nLF and HR were blunted in Chinese when compared to Caucasians from pre-exercise to 30-min to 60-min post-exercise. Chinese exhibited delayed autonomic recovery following an acute bout of treadmill exercise. This delayed autonomic recovery may result from greater sympathetic dominance and extended vagal withdrawal in Chinese.Trial Registration: Chinese Clinical Trial Register ChiCTR-IPR-15006684     

Characterization and manipulation of embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L.)

Characterization and optimization of the embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L. sub-species indica and japonica) are described. Histological and morphological analyses revealed that the parenchymatous ground tissue present in the region between the second whorl of sterile bracts and the base of the fertile bracts, the embryogenically competent region (ECR), was involved in the embryogenic response. Initial cell divisions within the ECR occurred in the vicinity of the pro-vascular regions of the spikelet. Continued cell divisions resulted in groups of proliferating units and each single proliferating unit was the product of a coordinated behavior of neighboring cells functioning as a morphogenic group. Further proliferation of this embryogenic tissue was due to the development of cambium-like tissue(s) often forming an embryogenic stratum which under optimal culture conditions produced plants at a high frequency. The morphogenic pathways governing plant regeneration from spikelets of the immature rice inflorescence were dependent upon the growth-regulator composition of the culture medium. Three different modes of plant regeneration were observed: (i) direct plant regeneration, (ii) plant regeneration with an intervening callus phase (prolific non-embryogenic growth associated with unorganized, loose and mucilaginous tissue), and (iii) plant regeneration without an intervening callus phase (compact embryogenie tissue with highly organized growth). The efficiency of plant regeneration, via somatic embryogenesis without an intervening callus phase, was increased by optimizing the culture conditions. In a two-step procedure, immature inflorescences of rice were first cultured on a conditioning medium supplemented with 2.0 mg · 1^–1 2,4-dichlorophenoxyacetic acid + 1.5 mg · 1^–1 kinetin + 0.75 mg · 1^–1 -naphthaleneacetic acid for a period of two weeks. The conditioning medium, with the appropriate culture conditions, allowed redirection of partially differentiated cells of the ECR into embryogenically competent pro-embryogenic groups. Maturation of these pro-embryogenic groups was achieved by transferring them to an embryo proliferation medium, and plants could then be regenerated at a high frequency upon their transfer to the regeneration medium.Abbreviations CM conditioning medium - 2,4-D 2,4-dichlorophenoxyacetic acid - ECR embryogenically competent region - NAA -naphthaleneacetic acid - SEM scanning electron micrograph Thanks are extended to Stephanie Lara, Barbara Bricks, Kay Robbinson-Beers, James Haudenshield, Dave Bayer and Gene Nester, for their invaluable help during the course of this research. The research was partly supported by the Rockefeller Foundation through the Rice Biotechnology program as a grant to W.J.L. and a Postdoctoral Fellowship to J.R.R.     

Biodegradation of the Alkaline Cellulose Degradation Products Generated during Radioactive Waste Disposal

The anoxic, alkaline hydrolysis of cellulosic materials generates a range of cellulose degradation products (CDP) including α and β forms of isosaccharinic acid (ISA) and is expected to occur in radioactive waste disposal sites receiving intermediate level radioactive wastes. The generation of ISA''s is of particular relevance to the disposal of these wastes since they are able to form complexes with radioelements such as Pu enhancing their migration. This study demonstrates that microbial communities present in near-surface anoxic sediments are able to degrade CDP including both forms of ISA via iron reduction, sulphate reduction and methanogenesis, without any prior exposure to these substrates. No significant difference (n = 6, p = 0.118) in α and β ISA degradation rates were seen under either iron reducing, sulphate reducing or methanogenic conditions, giving an overall mean degradation rate of 4.7×10⁻² hr⁻¹ (SE±2.9×10⁻³). These results suggest that a radioactive waste disposal site is likely to be colonised by organisms able to degrade CDP and associated ISA''s during the construction and operational phase of the facility.     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

Use of signal quality measurements to gain efficiency in the analysis of cDNA microarray data

This research provides a new way to measure error in microarray data in order to improve gene expression analysis.Microarray data contains many sources of error.In order to glean information about mRNA expression levels,the true signal must first be segregated from noise.This research focuses on the variation that can be captured at the spot level in cDNA microarray images.Variation at other levels,due to differences at the array,dye,and block levels,can be corrected for by a variety of existing normalization procedures.Two signal quality estimates that capture the reliability of each spot printed on a microarray are described.A parametric estimate of within-spot vari ance,referred to here as σ s2pot,assumes that pixels follow a normal distribution and are spatially correlated.A non-parametric estimate of error,called the mean square prediction error(MSPE),assumes that spots of high quality possess pixels that are similar to their neighbors.This paper will provide a framework to use either spot quality measure in downstream analysis,specifically as weights in regression models.Using these spot quality estimates as weights can result in greater efficiency,in a statistical sense,when modeling microarray data.     

Characterisation of Tat protein transport complexes carrying inactivating mutations

The Tat system functions to transport folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Tat transport involves a high molecular weight TatBC-containing complex that transiently associates with TatA during protein translocation. Sedimentation equilibrium experiments were used to determine a protein-only molecular mass for the TatBC complex of 630+/-30kDa, suggesting that it contains approximately 13 copies of the TatB and TatC protomers. Point mutations that inactivate Tat transport have previously been identified in each of TatA, TatB, and TatC. Analysis of the TatBC complexes formed by these inactive variants demonstrates that the amino acid substitutions neither affect the composition of the TatBC complex nor cause accumulation of the assembled TatABC translocation site. In addition, the TatA protein is shown not to be required for the assembly or stability of the TatBC complex.