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PubMed2005

New generation live vaccines against human respiratory syncytial virus designed by reverse genetics.

Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non-temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN alpha/beta antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use.

Animals↗
PubMed1998

Activity of glutamate dehydrogenase is increased in ammonia-stressed hybridoma cells.

The effect of added ammonia on the intracellular fluxes in hybridoma cells was investigated by metabolic-flux balancing techniques. It was found that, in ammonia-stressed hybridoma cells, the glutamate-dehydrogenase flux is in the reverse direction compared to control cells. This demonstrates that hybridoma cells are able to prevent the accumulation of ammonia by converting ammonia and alpha-ketoglutarate into glutamate. The additional glutamate that is produced by this flux, as compared to the control culture, is converted by the reactions catalyzed by alanine aminotransferase (45% of the extra glutamate) and aspartate aminotransferase (37%), and a small amount is used for the biosynthesis of proline (6%). The remaining 12% of the extra glutamate is secreted into the culture medium. The data suggest that glutamate dehydrogenase is a potential target for metabolic engineering to prevent ammonia accumulation in high-cell-density culture.

Amino Acids↗
PubMed2004

Chemoinducible gene therapy: a strategy to enhance doxorubicin antitumor activity.

A replication-defective adenoviral vector, Ad.Egr-TNF.11D, was engineered by ligating the CArG (CC(A/T)6GG) elements of the Egr-1 gene promoter upstream to a cDNA encoding human tumor necrosis factor-alpha. We report here that Ad.Egr-TNF.11D is activated by the clinically important anticancer agents cisplatin, cyclophosphamide, doxorubicin, 5-fluorouracil, gemcitabine, and paclitaxel. N-acetylcysteine, a free radical scavenger, blocked induction of tumor necrosis factor-alpha by anticancer agents, supporting a role for reactive oxygen intermediates in activation of the CArG sequences. Importantly, resistance of PC-3 human prostate carcinoma and PROb rat colon carcinoma tumors to doxorubicin in vivo was reversed by combining doxorubicin with Ad.Egr-TNF and resulted in significant antitumor effects. Treatment with Ad.Egr-TNF.11D has been associated with inhibition of tumor angiogenesis. In this context, a significant decrease in tumor microvessel density was observed following combined treatment with doxorubicin and Ad.Egr-TNF.11D as compared with either agent alone. These data show that Ad.Egr-TNF.11D is activated by diverse anticancer drugs.

Acetylcysteine↗
PubMed1996

Synthesis of alpha-helix-forming peptides by gene engineering methods and their characterization by circular dichroism spectra measurements.

Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.

Amino Acid Sequence↗
PubMed2001

Transposon tagging in maize.

Through recent government- and industry-sponsored efforts, several forward and reverse genetic screening programs have emerged over the past few years to aid in the genetic dissection of gene function in maize. Despite a US maize crop valued at $18.4 billion last year (http://www.ncga.com/03world/main/US_crop_value_2000.html) and rich genetic history, maize has taken a back seat to Arabidopsis thaliana as the model genetic system for plants over the past decade. With a fully sequenced genome, short generation time and small size, studies of Arabidopsis have provided plant scientists with a molecular framework for hormonal, developmental and environmental signaling pathways in plants. As investigations into Arabidopsis continue, our capacity to engineer biochemical pathways and alter plant physiological responses will become increasingly sophisticated. Nevertheless, approximately 130 million years have passed since monocot and higher eudicot lineages diverged. Thus, our ability to engineer agronomically important monocot grasses such as maize, rice and wheat will become increasingly limited by our lack of understanding of the physiological and morphological differences that have evolved in the monocots and higher eudicots. The sophisticated transposon collections now being generated for maize are but one of several recent projects (http://www.nsf.gov/bio/pubs/awards/genome01.htm) to provide grass researchers with essential tools for genome analysis. Because grain crops are such a closely related group, it is hoped that many of the findings made in one grass will be directly applicable to understanding the biology of another. The goal of this review is to highlight the recent developments in maize transposon-based gene characterization programs and provide a critical examination of the advantages and disadvantages each system offers.

DNA Transposable Elements↗
PubMed2005

Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo.

Mesenchymal stem cells (MSCs) have generated a great deal of interest in clinical application because of their potential use in regenerative medicine and tissue engineering. However, the therapeutic application of MSCs still remain limited unless the favorable effect of MSCs for tumor growth in vivo and the long-term safety of the clinical applications of MSCs are better understood. In this study, MSCs derived from fetal bone marrow (FMSCs) and adult MSCs (AMSCs) alone or FMSCs and AMSCs with tumor cell line (F6 or SW480) together were transplanted subcutaneously into BALB/c-nu/nu mice to observe the outgrowth of tumor, and the characteristics of tumor cells were investigated by pathological and immunohistochemical methods, flow cytometry and real-time quantitative PCR. The results showed that both FMSCs and AMSCs could favor tumor growth in vivo. The pathologic examination revealed that tumor tissues had rich vessel distribution, extensive necrosis and invasion surrounding normal tissues, such as muscular tissue and subcutaneous tissue. In the immunohistochemical examination, tumor cells mixed with MSCs transplanted subcutaneously exhibited elevated capability of proliferation, rich angiogenesis in tumor tissues and highly metastatic ability. To understand whether MSCs affected the general properties of the tumor cells in vivo, the expression of some surface antigens and Bmi-1 gene of tumor tissue cells was detected in this study. The results indicated that these parameters were not affected after the interaction of MSCs with tumor cells in vivo. These findings suggested that MSCs could favor tumor growth in vivo. It is necessary to carry out a study for assurance of the long-term safety before MSCs were used as a therapy tools in regenerative medicine and tissue engineering.

Adult↗
PubMedDate not supplied

Characterization of antitumor immunization to a defined melanoma antigen using genetically engineered murine dendritic cells.

A murine model of dendritic cell (DC)-based genetic immunization to a defined human melanoma antigen (Ag), MART-1/Melan-A (MART-1), was developed. The MART-1 gene was stably transfected into the nonimmunogenic mouse fibrosarcoma cell line NFSA that is syngeneic in C3Hf/Sem/Kam (C3H, H-2k) mice to generate the NFSA(MART1) cell line. In vivo protection from a lethal NFSA(MART1) tumor challenge could be generated by DCs transduced with a recombinant adenovirus (AdV) vector expressing MART-1 (AdVMART1). This model has the following characteristics: (a) immunological specificity and memory, (b) comparable protection for varying transduction multiplicities of infection, cell doses, and sites of DC inoculation but, interestingly, worse protection with increasing numbers of vaccinations, (c) the ability to treat small established tumors, (d) an absolute requirement for CD8 and CD4 T cells, (e) generation of MART-1-specific splenic cytotoxic T lymphocytes, and (f) up-regulation of both T helper type 1 and T helper type 2 cytokines. Genetically engineered DCs presenting defined tumor Ags represent an attractive method to generate effective immune responses.

Animals↗
PubMed2002

Derivation of type II alveolar epithelial cells from murine embryonic stem cells.

Embryonic stem (ES) cell pluripotency is being investigated increasingly to obtain specific cell lineages for tissue engineering. However, the possibility that ES cells can give rise to lung tissue has not been tested. We hypothesized that lung epithelial cells (type II pneumocytes) can be derived in vitro from murine ES cells. After withdrawal of leukemia inhibitory factor (LIF) and formation of embryoid bodies in maintenance medium for 10, 20, and 30 days, differentiating ES cells were kept in the same medium or transferred to serum-free small airway growth medium (SAGM) for a further 3 or 14 days of culture. The presence of type II pneumocytes in the resulting mixed cultures was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) of surfactant protein C (SPC) mRNA, immunostaining of SPC, and electron microscopy of osmiophilic lamellar bodies only at 30 days sampling time. SAGM appeared to be more favorable for type II cell formation than ES medium. No SPC transcripts were found in differentiating cells grown under the same conditions without formation of embryoid bodies. These findings could form the basis for the enrichment of ES cell-derived cultures with type II pneumocytes, and provide an in vitro system for investigating mechanisms of lung repair and regeneration.

Animals↗
PubMed1996

Molecular therapies for vascular diseases.

Vascular disease is the most common cause of death in the industrialized world. Although significant progress has been made in treating these disorders, more therapeutic agents must be developed that effectively prevent, arrest, or reverse this disease. Recent insights into the pathogenesis of vascular disease have opened up a new frontier of molecular therapies that target molecules as diverse as adhesion molecules and transcription factors. The biological rationale for these new therapies and their prospects for success are discussed.

Animals↗
PubMed2004

Transcriptional responses of intestinal epithelial cells to infection with Vibrio cholerae.

Vibrio cholerae is a noninvasive enteric bacterium that causes the severe diarrheal disease cholera. Candidate cholera vaccines have been engineered by deleting genes encoding known virulence factors in V. cholerae; however, many of these attenuated strains were still reactogenic in human volunteers. In this study, DNA arrays were utilized to monitor the transcriptional responses of human intestinal epithelial cells (T84) to eight strains of V. cholerae, including attenuated, toxigenic, and environmental isolates. cDNA probes generated from host RNA samples were hybridized against low- and high-density gene arrays. V. cholerae induced the transcription of a variety of host genes and repressed the expression of a lower number of genes. Expression patterns were confirmed for certain genes by reverse transcriptase PCR and enzyme-linked immunosorbent assays. A core subset of genes was found to be differentially regulated in all experiments. These genes included genes involved in innate mucosal immunity, intracellular signaling, and cellular proliferation. Reactogenic vaccine strains induced greater expression of genes for certain proinflammatory cytokines than nonreactogenic strains. Wild-type and attenuated derivatives induced and repressed many genes in common, although there were differences in the transcription profiles. These results indicate that the types of host genes modulated by attenuated V. cholerae, and the extent of their induction, may mediate the symptoms seen with reactogenic cholera vaccine strains.

Chemokines↗
PubMedDate not supplied

Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain.

The use of cell lines utilized as biologic "minipumps" to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme for GABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17 cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33 degrees C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39 degrees C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vector alone, contained no GAD67 or GABA at either temperature. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hind paw. When 33G10.17 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7-week period after grafts of 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 2-3 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers a novel approach to pain management.

Animals↗
PubMed2006

Lentivirus-like particles without reverse transcriptase elicit efficient immune responses.

Following infection by HIV or SIV, reverse transcriptase (RT) directs the conversion of the single-stranded RNA genome into a double-stranded DNA molecule that integrates into the host cell genome. RT encodes for several immunogenic epitopes that are desirable for inclusion in a human vaccine for HIV infection, however, issues of safety have dampened enthusiasm for inclusion of an enzymatically-active RT molecule into an AIDS vaccine. In this study, virally-regulated, replication-incompetent lentiviral particles were expressed from DNA plasmids. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted and mutations were engineered into capsid to decreases RNA packaging. Virus-like particles incorporated no RT (HIV-VLP DeltaRT or SHIV-VLP DeltaRT) or contained a full-length enzymatically-inactivated RT molecule (HIV-VLP or SHIV-VLP). Each secreted VLP was enveloped with a lipid bilayer derived from primate cells with embedded, native viral envelopes in similar concentrations as infectious virions. BALB/c mice were vaccinated (weeks 0, 3, and 6) with purified VLPs via intranasal inoculation in the presence of cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs). All VLPs, with or without RT, elicited both robust humoral and cellular immune responses to Gag, Pol, and Env antigens. Therefore, the lack of RT enhances the safety of these VLPs for use in future human clinical trials without a significant reduction in the overall immunogenicity of these VLP immunogens.

AIDS Vaccines↗
PubMed2003

Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK).

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.

Animals↗
PubMed1999

Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers.

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, the M246Ala --> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.

Alanine↗
PubMed2004

Dietary carotenoids, connexins and cancer: what is the connection?

Carotenoids and retinoids are chemically related; indeed a major source of vitamin A in humans occurs through enzymic cleavage of beta-carotene. However, most dietary carotenoids cannot be converted into retinoids. Retinoids have demonstrated cancer-preventive activities in humans and experimental models; however, their toxicity has precluded wide-scale clinical use. In contrast, carotenoids are essentially non-toxic and their cancer-preventive activities, although strongly supported by epidemiological studies, have only been satisfactorily demonstrated in experimental systems. We have shown that in an experimental cell culture system consisting of carcinogen-treated 10T1/2 cells, both retinoids and all dietary carotenoids examined can reversibly inhibit neoplastic transformation in the post-initiation phase of carcinogenesis. This activity strongly correlates with their ability to increase gap junctional intercellular communication by up-regulating the expression of the gene CX43 (connexin43). Connexins comprise the structural unit of gap junctions, organelles which allow direct transfer of signals, nutrients and waste products between contacting cells. CX43 is the most widely expressed member of the gap junction family of genes, and we have demonstrated that its expression is strongly down-regulated in human cancers and in several premalignant conditions. When several human tumour cell lines were genetically engineered to conditionally express CX43 under the influence of a tetracycline promoter, their neoplastic phenotype was strongly attenuated. Specifically, induced cells were inhibited from growing in an anchorage-independent manner and, additionally, growth as xenografts in immunocompromised animals was also strongly attenuated. Growth inhibition in suspension was associated both with increased G(1) cell-cycle arrest and with increased apoptosis. We propose a model whereby junctional communication allows the transfer of growth inhibitory signals from normal to neoplastic cells and that retinoids and carotenoids, by increasing signal transfer, act to prevent cancer.

Anticarcinogenic Agents↗
PubMed2006

An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae.

The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora. The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-resistant plants by using a suite of genes.

Ascomycota↗
PubMed2006

Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension.

The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.

Animals↗
PubMed2026

OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.

Sequence Analysis, DNA↗