Use Of Sequences Research Articles

Genome Sequence of Cupriavidus metallidurans Strain H1130, Isolated from an Invasive Human Infection

Cupriavidus metallidurans H1130 was repeatedly isolated from different blood culture sets taken from a patient suffering from significant nosocomial septicemia. Here, we announce the H1130 genome sequence for use in comparative analyses and for exploring the adaptation and pathogenic potential of this bacterium.

An autonomously replicating sequence for use in a wide range of budding yeasts

The initiation of DNA replication at replication origins is essential for the duplication of genomes. In yeast, the autonomously replicating sequence (ARS) property of replication origins is necessary for the stable maintenance of episomal plasmids. However, because the sequence determinants of ARS function differ among yeast species, current ARS modules are limited for use to a subset of yeasts. Here, we describe a short ARS sequence that functions in at least 10 diverse species of budding yeast. These include, but are not limited to members of the Saccharomyces, Lachancea, Kluyveromyces, and Pichia (Komagataella) genera spanning over 500million years of evolution. In addition to its wide species range, this ARS and an optimized derivative confer improved plasmid stability relative to other currently used ARS modules.

Near full-length genome analysis of HCV genotype 5 strains from South Africa

Hepatitis C virus (HCV) genotype 5 is the predominant genotype in South Africa. However, to date, only 2 full-length genotype 5 genomes have been sequenced and only one is from South Africa. This study characterized HCV genotype 5 sequences from South Africa, including six near full-length genomes, as well as the E1 region from an additional 12 genotype 5 samples. Phylogenetic analysis of these near full-length genome sequences revealed that all genotype 5 sequences formed a close cluster with high bootstrap support. Bayesian analysis of the E1 region was used to estimate the time of the most recent common ancestor (tMRCA). The tMRCA for HCV genotype 5a was estimated at 114–134years before the last sampling date. In conclusion, this study provides six near full-length genotype 5 nucleotide sequences for use as references to design efficient vaccines and for the development of new antiviral agents, and provides further insight into the diversity of HCV genotypes circulating in South Africa.

<I>Mycobacterium tuberculosis</I> detection in blood using multiplex nested polymerase chain reaction

Polymerase chain reaction (PCR) offers great promise for the rapid, sensitive and specific diagnosis of tuberculosis (TB). However, blood TB PCR is still not standardised. Evaluation of the Blacklight® Card to standardise TB PCR in blood samples using a multiplex nested PCR method targeting insertion sequence 6110 and MPB64 sequence for use as an auxiliary tool in the diagnosis of TB. The study focused on the standardisation of the Blacklight Card as a new extraction tool and its evaluation using 130 blood samples randomly collected from patients with clinical symptoms of TB. Of 130 cases, PCR was positive in 22 samples (17%). The overall sensitivity and specificity of the PCR assay was respectively 95.7% and 100%; the positive and negative predictive values were respectively 85.1% and 99.2%. These results suggest that for the detection of Mycobacterium tuberculosis DNA in peripheral blood mononuclear cells, the Blacklight Card may be a useful tool for the rapid diagnosis of disseminated and extra-pulmonary forms of TB as well as in difficult-to-diagnose cases in which a diagnosis is urgently needed due to the severity of the patient's status.

Analysis of mitochondrial DNA HVR1 haplotype of pure-bred domestic dogs in Japan

To develop DNA markers for forensic analysis, we examined the hypervariable region 1 (HVR1) sequences of 447 pure-bred domestic dogs (Canis lupus familiaris) that had been bred and raised in Japan. HVR1 is a 660-bp stretch of mitochondrial (mt) DNA. Among the 447 HVR1 sequences examined, we identified 58 haplotypes from 47 single nucleotide polymorphisms (SNPs) and two insertion-deletion (InDel) polymorphisms. The haplotype diversity inferred from inter-breed analysis (N=154, 88 breeds) was 0.929±0.011. Intra-breed analysis showed that the haplotype diversity of Golden Retrievers (N=53), Labrador Retrievers (N=67), Miniature Dachshunds (N=61), Toy Poodles (N=62), and Welsh Corgis (N=50) was 0.624±0.052, 0.722±0.029, 0.922±0.010, 0.877±0.020, and 0.443±0.084, respectively. The results of this genotype analysis were used to construct a dataset consisting of dog mtDNA HVR1 sequences for use in forensic applications in Japan.

Application of Generic System Design Process for Various Design -2 Creation of Bicycle Design with Observations of Bicycle User's Activities, Supposing Sequences of Use and Situations

Application of Generic System Design Process for Various Design -2 Creation of Bicycle Design with Observations of Bicycle User's Activities, Supposing Sequences of Use and Situations

Castration-resistant Prostate Cancer: Novel Therapeutics Pre- or Post- Taxane Administration

Until 2010 docetaxel was the only agent with a proven survival benefit in the treatment of castration-resistant prostate cancer (CRPC). Recent evidence suggests that CRPC is caused by augmented androgen/androgen receptor (AR) signaling involving AR hypersensitivity, promiscuous activation of the AR, de novo production of androgens and activation of the AR by cytokines and growth factors; these findings have led to the development of novel agents targeting AR signaling. Several novel drugs targeting the AR axis, including the cytochrome P17 inhibitor abiraterone acetate and anti-androgen enzalutamide (MDV3100), have shown promising results in prolonging survival in clinical trials in a postchemotherapy setting. Because of these encouraging findings, these drugs have also been evaluated in a pre-chemotherapy setting. In addition, several novel drugs targeting non-AR signaling, including the novel taxoid compound cabazitaxel, antisense oligonucleotide OGX-011 (custirsen), sipuleucel-T immunotherapy and Alpharadin-based radiotherapy, have also been demonstrated to improve overall survival in CRPC. However, there is no consensus with regard to the sequence in which these novel drugs and taxanes should be used in the treatment of CRPC. In this review, we summarize recently developed and developing novel agents for use against CRPC. We also discuss the sequence of use of these agents and taxanes, mainly from the standpoint of factors related to drug resistance.

Representing Objects, Relations, and Sequences

Vector symbolic architectures (VSAs) are high-dimensional vector representations of objects (e.g., words, image parts), relations (e.g., sentence structures), and sequences for use with machine learning algorithms. They consist of a vector addition operator for representing a collection of unordered objects, a binding operator for associating groups of objects, and a methodology for encoding complex structures. We first develop constraints that machine learning imposes on VSAs; for example, similar structures must be represented by similar vectors. The constraints suggest that current VSAs should represent phrases ("The smart Brazilian girl") by binding sums of terms, in addition to simply binding the terms directly. We show that matrix multiplication can be used as the binding operator for a VSA, and that matrix elements can be chosen at random. A consequence for living systems is that binding is mathematically possible without the need to specify, in advance, precise neuron-to-neuron connection properties for large numbers of synapses. A VSA that incorporates these ideas, Matrix Binding of Additive Terms (MBAT), is described that satisfies all constraints. With respect to machine learning, for some types of problems appropriate VSA representations permit us to prove learnability rather than relying on simulations. We also propose dividing machine (and neural) learning and representation into three stages, with differing roles for learning in each stage. For neural modeling, we give representational reasons for nervous systems to have many recurrent connections, as well as for the importance of phrases in language processing. Sizing simulations and analyses suggest that VSAs in general, and MBAT in particular, are ready for real-world applications.

Unmet Needs in the Treatment of Rheumatoid Arthritis

Biologics have greatly improved the management of rheumatoid arthritis (RA), demonstrating efficacy and safety in alleviating symptoms, inhibiting bone erosion, and preventing loss of function. Unmet therapeutic needs in RA remain; however, further advances require an understanding of issues left unaddressed under the current treatment paradigm. Most biologic-na?ve and biologic-pretreated patients who initiate a biologic therapy, for example, do not reach American College of Rheumatology 50% (ACR50) response, and few achieve remission. Responses are often not durable, prompting frequent treatment switching. Predictive markers are unavailable to guide therapy selection, and clinical trial data are lacking on whether a tumor necrosis factor inhibitor (TNFi) is the best first-line biologic and on the optimal sequence of use for the different biologics. Risk of serious infection is the major safety concern. Translating preclinical and clinical findings into new therapeutics may help address unmet needs. An increasing body of evidence indicates that the cytokine interleukin (IL)-17A represents an important therapeutic target; ongoing trials with IL-17A inhibitors will determine whether these agents can address some of the unmet needs associated with current biologics.

Future Directions in Metastatic Castration Resistant Prostate Cancer (mCRPC): Clinical Rationale and Use of New-Generation Hormonal Therapies

Several recent studies in mCRPC have identified the mechanisms of tumoral growth after the disease becomes unresponsive to standard hormonal therapy. These studies have highlighted the importance of residual intratumoral androgens in maintaining androgen receptor (AR) transcriptional activity in mCRPC [1]. Based on this findings, it has been possible to develop drugs, blocking the key enzyme in the biosynthesis of androgens through the inhibition of cytochrome p450 17 (CYP17) such as Abiraterone Acetate (AA) and drugs which directly target the AR including Enzalutamide (E) and Orteronel. Before this new knowledge, mCRPC treatment benefited from chemotherapy with taxanes. Recently a new taxane, Cabazitaxel (C), was approved in second line setting in association with prednisone. Retrospective analyses have tried to clarify the current role of chemotherapy in mCRPC patients and the right chemotherapy sequence of use of chemotherapy compared to new hormonal agents. Moreover, it would be important to address changes in the endpoints used in clinical trials, based on the stage of disease including the presence tumor-related symptoms, in order to identify the right therapeutic strategy.

Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna?

BackgroundThe megadiverse Neotropical freshwater ichthyofauna is the richest in the world with approximately 6,000 recognized species. Interestingly, they are distributed among only 17 orders, and almost 80% of them belong to only three orders: Characiformes, Siluriformes and Perciformes. Moreover, evidence based on molecular data has shown that most of the diversification of the Neotropical ichthyofauna occurred recently. These characteristics make the taxonomy and identification of this fauna a great challenge, even when using molecular approaches. In this context, the present study aimed to test the effectiveness of the barcoding methodology (COI gene) to identify the mega diverse freshwater fish fauna from the Neotropical region. For this purpose, 254 species of fishes were analyzed from the Upper Parana River basin, an area representative of the larger Neotropical region.ResultsOf the 254 species analyzed, 252 were correctly identified by their barcode sequences (99.2%). The main K2P intra- and inter-specific genetic divergence values (0.3% and 6.8%, respectively) were relatively low compared with similar values reported in the literature, reflecting the higher number of closely related species belonging to a few higher taxa and their recent radiation. Moreover, for 84 pairs of species that showed low levels of genetic divergence (<2%), application of a complementary character-based nucleotide diagnostic approach proved useful in discriminating them. Additionally, 14 species displayed high intra-specific genetic divergence (>2%), pointing to at least 23 strong candidates for new species.ConclusionsOur study is the first to examine a large number of freshwater fish species from the Neotropical area, including a large number of closely related species. The results confirmed the efficacy of the barcoding methodology to identify a recently radiated, megadiverse fauna, discriminating 99.2% of the analyzed species. The power of the barcode sequences to identify species, even with low interspecific divergence, gives us an idea of the distribution of inter-specific genetic divergence in these megadiverse fauna. The results also revealed hidden genetic divergences suggestive of reproductive isolation and putative cryptic speciation in some species (23 candidates for new species). Finally, our study constituted an important contribution to the international Barcoding of Life (iBOL.org) project, providing barcode sequences for use in identification of these species by experts and non-experts, and allowing them to be available for use in other applications.

A Characteristic Analysis of Ergonomic Console Layout Studies Using Optimization Techniques

Objective: The present study systematically analyzed the characteristics of ergonomic layout optimization methods by a comprehensive literature survey. Background: Although layout design methods for ergonomic placement of controls and displays on a console have been developed, understanding of their characteristics is lacking. Method: The present study analyzed layout optimization papers published past 20 years from the following four aspects: optimization model, optimization algorithm, design principle, and constraint/assumption. Results: The existing layout optimization methods based on various optimization techniques consider only a partial set of four layout principles(importance, frequency of use, sequence of use, and functional grouping) and two ergonomic criteria(visibility and reach). In addition, the existing methods oversimplify components in various sizes, shapes, and angles by assuming the equality of the components in size and shape. Conclusion: A more effective layout optimization method is needed which considers the layout principles and ergonomic criteria in a comprehensive manner and reflect the diversity of components in size and shape. Application: The identified characteristics on the existing layout optimization methods can be applicable to development of a better ergonomic console layout design method.

Abstract P4-16-04: Outcome of stage II/III breast cancer treated with neoadjuvant versus adjuvant radiotherapy in British Columbia.

Abstract Backgroud: Neoadjuvant radiotherapy (NRT) is generally recommended after systemic therapy for inoperable stage III breast cancer. At the BCCA, neoadjuvant radiation is also frequently offered for patients with operable node positive disease after neoadjuvant chemotherapy. There is a lack of randomised trial data comparing outcomes in stage II/III breast cancer when radiation therapy is delivered neoadjuvantly versus adjuvantly. Aim: The primary objective of this study is to assess the clinical outcomes, as measured by relapse-free survival (RFS), overall survival (OS), and breast cancer-specific survival (BCSS), of women with stage II/III breast cancer treated with neoadjuvant chemotherapy and either neoadjuvant or adjuvant radiotherapy. Methods: Patients were identified by linking the Breast Cancer Outcomes Unit (BCOU) with the British Columbia Cancer Agency Pharmacy data repository. Inclusion criteria included: Female, referred to BCCA with newly diagnosed disease, clinical stage II or III breast cancer, neoadjuvant chemotherapy, breast surgery performed as part of the initial treatment plan, RT (given adjuvantly or neo-adjuvantly to the breast/chest wall +/− regional nodes). Patients were excluded if they had a previous or synchronous in situ or invasive breast cancer. Demographic data, treatment characteristics, ER, PR and HER-2 status (when available) were extracted. Data was analyzed using descriptive statistics and Kaplan Méier curves survival analyses were produced using SPSS, V. 14. Results: Between Jan 1, 1995 and Dec 31, 2008, 687 patients with stage II/III disease were identified. 394 patients received neo-adjuvant and 293 patients received adjuvant radiation. Patients treated with neoadjuvant vs. adjuvant RT differed in age (median: 51yrs vs. 49yrs, p &amp;lt; 0.001), margin status (86.5% negative/5.1% positive/ 8.4% unknown vs. 94.2% negative/1.7% positive/4.1% unknown, p = 0.02), surgery (94.2% vs. 87.7% mastectomy, p = 0.003), and type of chemotherapy (50.3% vs. 24.6% anthracycline only, p &amp;lt; 0.001) respectively. No differences in 5-year RFS (60.0 vs. 64.8 %, respectively, p = 0.07), 5 year BCSS (67.9 vs. 73.1 %, respectively p = 0.17) and 5-year OS (66.2 vs. 72.5 %, respectively, p = 0.06) were seen in the neoadjuvant vs. adjuvant population. Conclusion: Within the limitations of this retrospective study, the sequence of use of radiation pre-operative compared to post-operative following neoadjuvant chemotherapy appears to have similar clinical outcomes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-16-04.

Bevacizumab-containing regimens after cetuximab failure in Kras wild-type metastatic colorectal carcinoma

Bevacizumab and cetuximab both improve treatment efficacy when administered with chemotherapy for metastatic colorectal carcinoma (mCRC). Cetuximab has enhanced efficacy in Kras wild-type tumors. However, inferior outcomes have been demonstrated concerning the concurrent use of bevacizumab and cetuximab with chemotherapy. There is an urgent need to define the optimal sequence of use of these two agents. With regard to the pre-clinical data that increased VEGF expression is associated with acquired resistance to anti-EGFR antibody, we performed a retrospective analysis on the outcomes of patients who received bevacizumab-containing regimens after cetuximab failure in Kras wild-type mCRC. From January 2006 to December 2011, patients who received bevacizumab-containing regimens for mCRC in our institution were reviewed. Patients were eligible for further analysis if the following criteria were met: i) Kras wild-type mCRC; ii) chemotherapy and cetuximab received as immediate prior treatment; iii) chemotherapy and bevacizumab received as the index line of treatment; and iv) imaging conducted for response evaluation. Outcome measures included median progression-free survival (mPFS) and objective response rate (ORR). Targeted adverse events were recorded in accordance with two prospective observational cohort studies; the BRiTE and BEAT studies. Fifty patients who received bevacizumab-containing regimens were reviewed and 18 of them met the criteria for further analysis. After a median follow-up of 12.1 months, the mPFS for the total group of patients was 26.3 weeks (95% CI, 19.5–33.0 weeks) with an ORR of 38.9%. Two patients (11.1%) had hypertension that required additional anti-hypertensive drugs and one patient did not survive due to a bowel perforation. No arterial thromboembolic events (ATEs), post-operative wound-healing complications (POWHCs) or grade III/IV bleeding were observed. In patients with Kras wild-type mCRC, bevacizumab-containing regimens following cetuximab failure have modest activity and manageable toxicity.

Machine Learning Sequence Classification Techniques: Application to Cysteine Protease Cleavage Prediction

Sequence classification is one of the most fundamental machine learning tasks in computational biology nowadays. With the wide availability of large corpora of annotated sequences, the use of supervised learning techniques can greatly speed up the process of identifying new sequences sharing certain function or properties. Many methods have been proposed over the years and we hope to provide an introduction to some of the more prominent ones by focussing on protease cleavage prediction: a typical representative of this class of problem. The variety of proteolytic action modes between cysteine-proteases covers a broad range of complexity level and feature specificity, illustrating the strengths and limitations of the different machine learning techniques used on them. This review briefly introduces the particulars of predicting cleavage by calpains and caspases. We then offer some general practical considerations on treating sequences for use with machine learning algorithms, before covering specific methods. The methods presented range from basic position-based statistical models to more technically advanced methods such as Markov models or kernel-based algorithms, as well as methods with more restricted goals such as decision trees. With each family of algorithms, examples of implementations are introduced and their performances compared, along with particular strengths and weaknesses. With this review, we aim to provide useful elements of decision toward choosing an existing method or developing a new one, based on the complexity and specific needs of a given sequence classification problem. Keywords: Calpain, caspase, cleavage prediction, cysteine proteases, machine learning, protease, proteolysis, sequence classification, Vector Encoding, Markov Models

HIV-1 diversity in Cameroon: new insights on the evolution of the virus

Background Given the central role of HIV-1 diversity in the HIV pandemic and its impact on vaccine development, it is imperative that global molecular epidemiology surveillance continues. Constantly improving phylogeneticsbased analytical techniques and rapidly expanding HIV sequence datasets promise to yield important insights into the origin, evolution and spread of HIV-1. Methods In an effort to study the phylogeography and phylodynamics of the HIV-1 M epidemic in the early 20th century, we analysed 50 plasma samples from HIV-infected blood donors from Cameroon. Full length gag sequences were generated and aligned using MUSCLE along with a representative selection of HIV sequences from the rest of the world and all published gag sequences from Cameroon and other West African countries. A maximum likelihood phylogenetic tree was constructed from these sequences following removal of recombinant sequence fragments by a blinded fully exploratory screen for recombination using RDP3. Results All the Cameroonian sequences were derived from HIV-1 M viruses. The phylogenetic tree indicated that at least one of the newly-sequenced CRF02_AG viruses is an outlier of the CRF02_AG clade and may help resolve the controversy surrounding the origins of this clade. Furthermore, isolates from Cameroon were spread throughout the phylogenetic tree clustering with different subtypes and circulating recombinant forms, a finding consistent with West Africa being the geographic origin of the global HIV epidemic. Importunately, our blind recombination screen suggested that many divergent Cameroonian viruses previously identified as being unique recombinant forms, may be divergent, relatively non-recombinant, but under-sampled subtype-level lineages. Conclusion Lineages diverging early after the origin of HIV-1 M are likely still circulating in Cameroon and could be suitable for retracing the movement and evolutionary dynamics of HIV-1 during the earliest stages of the pandemic. These lineages will be useful for reconstructing ancestral HIV-1M sequences for use as vaccine immunogens.

P-0248 Bevacizumab after Cetuximab Failure in Kras Wild-Type Metastatic Colorectal Carcinoma

ABSTRACT Introduction Bevacizumab and cetuximab both improve treatment efficacy when given with chemotherapy for metastatic colorectal carcinoma (mCRC) in both first- and second-line setting. Cetuximab has enhanced efficacy in Kras wild-type tumor. However, inferior outcomes were reported in two phase III randomised controlled trials for the concurrent use of bevacizumab and cetuximab with chemotherapy. There is an urgent need to define the optimal sequence of use of these two agents. With regard to the pre-clinical data that increased VEGF expression is associated with acquired resistance to anti-EGFR antibody, we performed a retrospective analysis on the outcomes of patients who received bevacizumab-containing regimens after cetuximab failure in Kras wild-type mCRC. Methods From January 2006 to December 2011, patients who received bevacizumab-containing regimens for mCRC in our institution were reviewed. Patients were eligible for further analysis if all the following criteria were met: [1] Kras wild-type mCRC, [2] received chemotherapy and cetuximab as immediate prior treatment, [3] received chemotherapy and bevacizumab as the index line of treatment and [4] had imaging for response evaluation. Outcome measures included median progression-free survival (mPFS) and objective response rate (ORR). Targeted adverse events were recorded in accordance with the two prospective observational cohort studies, the BRiTE and BEAT study. Adverse events include gastrointestinal perforation [perforation, intra-abdominal abscess, and fistula], arterial thromboembolic events (ATE) [myocardial infarction, cerebrovascular accident, transient ischemic attack, and unstable angina], postoperative bleeding or wound-healing complications (POWHC), grade 3/4 bleeding, and hypertension requiring additional anti-hypertensive. Results Fifty patients received bevacizumab-containing regimens were reviewed and 18 of them met all the criteria for further analysis. The median age was 56.5 and 10 patients had metastasis in multiple organs. After a median follow up of 12.1 months, the mPFS for the whole group of patients was 26.3 weeks (95% CI 19.5 – 33.0 weeks) with an ORR of 38.9%. For the 8 patients who received bevacizumab-containing regimens at second line, 1 complete response and 3 partial responses were observed, giving an ORR of 50%. The mPFS was 27.4 weeks (95% CI 2.0-52.8 weeks). For the 10 patients treated at third line, 3 PRs were observed, giving an ORR of 30%. The mPFS was 23.9 weeks (95% CI 19.7 – 28.1 weeks). Regarding previous exposure to oxaliplatin, irinotecan and 5FU, patients who exposed to only two of these agents, compared with those who exposed to all three agents, were more likely to respond to bevacizumab-containing regimen after cetuximab failure (Odds ratio 5.0, 95% CI 0.44-56.6). 2 patients (11.1%) had hypertension that required additional anti-hypertensive and 1 patient died of bowel perforation. The event of bowel perforation was unlikely attributed to bevacizumab since the event occurred 78 days after the last dose of bevacizumab while the patient was receiving hypofractionated palliative radiotherapy to the pelvis. There was no case of ATE, POWHC and grade 3/4 bleeding. Conclusion In patients with Kras wild-type mCRC, bevacizumab-containing regimens after cetuximab failure have promising activity and manageable toxicity. Further study is needed to compare switching to bevacizumab versus cetuximab beyond progression in this specific cohort of patients.

Shakespeare's Perversion: A Reading of Sonnet 20

This article reviews the critical debate about the sexual politics of Shakespeare's Sonnet 20, the “Master-Mistress” sonnet. It argues that, while the sonnet is available to queer readings, it draws its power from the way it queers desire while perverting gender. That is, Sonnet 20 does not offer a queer sexuality, one which refuses gender identifications, but a reading of the relationship between desire and gender which demonstrates the way gender binds desire. This is why the homoeroticism in the sonnet cannot be repressed, even as its queerness cannot be celebrated. Sonnet 20 speaks to the sequence's use of Petrarchism, and allows us to see beyond the rules which make its speaking possible, at the same time as it offers a focus on the constitutive power of structure. The sonnet's sexual meanings are irreducible even as its gendered meanings are conventional. In its use of gender, the article argues, Sonnet 20 makes perverts of us all.

CHLOROPLAST GENETIC TOOL FOR THE GREEN MICROALGAE HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

At present, there is strong commercial demand for recombinant proteins, such as antigens, antibodies, biopharmaceuticals, and industrial enzymes, which cannot be fulfilled by existing procedures. Thus, an intensive search for alternative models that may provide efficiency, safety, and quality control is being undertaken by a number of laboratories around the world. The chloroplast of the eukaryotic microalgae Haematococcus pluvialis Flotow has arisen as a candidate for a novel expression platform for recombinant protein production. However, there are important drawbacks that need to be resolved before it can become such a system. The most significant of these are chloroplast genome characterizations, and the development of chloroplast transformation vectors based upon specific endogenous promoters and on homologous targeting regions. In this study, we report the identification and characterization of endogenous chloroplast sequences for use as genetic tools for the construction of H. pluvialis specific expression vectors to efficiently transform the chloroplast of this microalga via microprojectile bombardment. As a consequence, H. pluvialis shows promise as a platform for expressing recombinant proteins for biotechnological applications, for instance, the development of oral vaccines for aquaculture.

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation.