Modern approaches to therapeutic oligonucleotide manufacturing.

Therapeutic oligonucleotides are a powerful drug modality with the potential to treat many diseases. The rapidly growing number of therapies that have been approved and that are in advanced clinical trials will place unprecedented demands on our capacity to manufacture oligonucleotides at scale. Existing methods based on solid-phase phosphoramidite chemistry are limited by their scalability and sustainability, and new approaches are urgently needed to deliver the multiton quantities of oligonucleotides that are required for therapeutic applications. The chemistry community has risen to the challenge by rethinking strategies for oligonucleotide production. Advances in chemical synthesis, biocatalysis, and process engineering technologies are leading to increasingly efficient and selective routes to oligonucleotide sequences. We review these developments, along with remaining challenges and opportunities for innovations that will allow the sustainable manufacture of diverse oligonucleotide products.

An enzyme cascade enables production of therapeutic oligonucleotides in a single operation.

Therapeutic oligonucleotides have emerged as a powerful drug modality with the potential to treat a wide range of diseases; however, the rising number of therapies poses a manufacturing challenge. Existing synthetic methods use stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. We report a biocatalytic approach to efficiently produce oligonucleotides in a single operation where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks and aqueous conditions. We demonstrate the versatility of this methodology through the synthesis of clinically relevant oligonucleotide sequences containing diverse modifications.

Predation drives morphological convergence in the Gambusia panuco species group among lotic and lentic habitats.

Fish morphology is often constrained by a trade-off between optimizing steady vs. unsteady swimming performance due to opposing effects of caudal peduncle size. Lotic environments tend to select for steady swimming performance, leading to smaller caudal peduncles, whereas predators tend to select for unsteady swimming performance, leading to larger caudal peduncles. However, it is unclear which aspect of performance should be optimized across heterogeneous flow and predation environments and how this heterogeneity may affect parallel phenotypic evolution. We investigated this question among four Gambusia species in north-eastern Mexico, specifically the riverine G. panuco, the spring endemics G. alvarezi and G. hurtadoi, and a fourth species, G. marshi, found in a variety of habitats with varying predation pressure in the Cuatro Ciénegas Basin and Río Salado de Nadadores. We employed a geometric morphometric analysis to examine how body shapes of both male and female fish differ among species and habitats and with piscivore presence. We found that high-predation and low-predation species diverged morphologically, with G. marshi exhibiting a variable, intermediate body shape. Within G. marshi, body morphology converged in high-predation environments regardless of flow velocity, and fish from high-predation sites had larger relative caudal peduncle areas. However, we found that G. marshi from low-predation environments diverged in morphology between sub-basins of Cuatro Ciénegas, indicating other differences among these basins that merit further study. Our results suggest that a morphological trade-off promotes parallel evolution of body shape in fishes colonizing high-predation environments and that changing predation pressure can strongly impact morphological evolution in these species.

Estimating the risks of ovarian hyperstimulation syndrome (OHSS): implications for egg donation for research.

This study aims to provide an estimate of women's risk of ovarian hyperstimulation syndrome (OHSS) when undergoing superovulation to donate eggs for research. This is an essential prerequisite for appropriate informed consent. In the absence of sufficiently large numbers of egg donors to assess the risk, comparative data was obtained from women undergoing the same superovulation protocol for in vitro fertilization (IVF) treatment. In this prospective study 339 women, who developed >/=20 follicles after superovulation in their first treatment cycle (total number of treatment cycles during the same period - 2417), were intensively monitored on five occasions, between human chorionic gonadotrophin and pregnancy test, according to our routine clinical protocol. Hospital admission was needed for 49 (14.5%) women, 13 (3.8%) needed intravenous fluids and 9 (2.7%) needed paracentesis. The admission rates were similar in pregnant and non-pregnant women (13.5% vs. 15%); the need for intravenous fluids and paracentesis were 3.2% vs. 2.3% and 6.3% vs. 2.3%, respectively. The peak increase in haematocrit occurred on Day 4 after hCG, and the mean day of hospital admission was Day 5. If an egg donor develops <20 follicles, she can be reassured that the risk of OHSS is very small (<0.1%). If >/=20 follicles develop, her risk of hospital admission due to OHSS is <15%. The absence of pregnancy in egg donors does not eliminate the risk of OHSS. Given the timescale of development of the haematological and biochemical abnormalities, egg donors who develop >/=20 follicles should be actively monitored for the first week after egg collection.

Improving on-line information for potential living kidney donors.

Individuals who consider becoming living kidney donors often search the internet for reliable information before contacting the transplant center. The quality of such information requires due consideration. Using the search engines Google and Yahoo and the WebMD information portal, two reviewers independently abstracted data on the classification, readability, and general quality of websites. The coverage and accuracy of each site's discussion of the risks, benefits, and process of living donation was also assessed against a checklist of recommended information. Eighty-six unique websites on living kidney donation were found. Most were created by transplant programs and transplant organizations. Although the content of most sites was accurate, almost all (98%) were written above the recommended patient reading level (i.e., fifth grade). On average, each site covered 38% of the recommended information on living donation (range 8-76%). Educational topics of potential long-term medical risks, psychological risks, and expected benefits to the donor were often missing. The most visited websites were often not ranked among the best sites to provide information. By better understanding the nature of on-line information, transplant professionals can direct their patients to the best available websites. Local educational efforts, including the effective use of internet resources, will ensure living donation and complete understanding of the risks by potential donors and recipients.

Linkage between proton binding and folding in RNA: implications for RNA catalysis.

Small ribozymes use their nucleobases to catalyse phosphodiester bond cleavage. The hepatitis delta virus ribozyme employs C75 as a general acid to protonate the 5'-bridging oxygen leaving group, and to accomplish this task efficiently, it shifts its pKa towards neutrality. Simulations and thermodynamic experiments implicate linkage between folding and protonation in nucleobase pKa shifting. Even small oligonucleotides are shown to fold in a highly co-operative manner, although they do so in a context-specific fashion. Linkage between protonation and co-operativity of folding may drive pKa shifting and provide for enhanced function in RNA.

Internet use and its relationship to lonelines.

The association between Robert Weiss's bimodal theory of loneliness and Internet use was examined. The degree of social and emotional loneliness was assessed using the Social and Emotional Loneliness scale. This was compared with self-report measures of Internet use and the breadth of one's network of friends, both online and on a face-to-face basis. Low levels of social and emotional loneliness were both associated with high degrees of face-to-face networks of friends, while high levels of Internet use were associated with low levels of social loneliness and high levels of emotional loneliness. This supports recent research that has found that the Internet can decrease social well-being, even though it is often used as a communication tool.

Interpretations of retrograde amnesia: old problems redux.

Recent evidence indicates that an old memory reactivated by cueing becomes labile and vulnerable to an amnesic treatment. Although the 'reconsolidation' concept derived from these findings challenges the traditional consolidation theory, here we argue that the new concept suffers from some of the same limitations as the earlier model. We propose an alternative retrieval-based theory that accommodates the recent data, as well as other puzzling related observations.

SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells.

Neurotensin (NT) is an autocrine growth factor for some small cell lung cancer (SCLC) cells. In this communication, the effects of a non-peptide NT receptor antagonist, SR48692, were investigated using SCLC cells. (3)H-SR48692 bound with high affinity (IC(50) = 20 nM) to NCI-H209 cells. Also, NT and SR48692 inhibited specific (125)I-NT binding with high affinity (IC(50) values of 2 and 200 nM). In contrast, the NT(2) receptor agonist, levocabastine, had little effect on specific (125)I-NT binding, second messenger production and proliferation using NCI-H209 cells. SR48692 (5 microM) antagonized the ability of NT (10 nM) to cause elevated cytosolic Ca2+ in Fura-2 AM loaded NCI-H209 cells. SR48692 antagonized the ability of NT to cause elevation of c-fos mRNA in these cells. Using a MTT proliferation assay, SR48692 inhibited NCI-H209 and H345 proliferation in a concentration-dependent manner. Using a clonogenic assay, 1 microM SR48692, reduced NCI-H209 colony number. Also, SR48692 (0.4 mg/kg per day) inhibited NCI-H209 xenograft proliferation in nude mice. These results suggest that SR48692 is a NT(1) receptor antagonist which inhibits SCLC growth.

The metabolism of BW2258U89, a GRP receptor antagonist.

BW2258U89 is a gastrin releasing peptide (GRP) receptor antagonist which inhibits the proliferation of the neuroendocrine tumor small cell lung cancer (SCLC). Here the biological activity of BW2258U89 and its metabolite were investigated. Using mass spectroscopy (LC-ESI/MS) techniques, three major peaks for BW2258U89 were observed with mass/charge (m/z) ratios of 1081.6, 541.4 and 361.4. After metabolism by mouse plasma enzymes, the major product had a m/z ratio of 1082.5, 541.9 and 361.8 suggesting that BW2258U89 was deamidated. Deamidated (Da) BW2258U89 was synthesized and it inhibited ((125)I-Tyr(4)) BB binding to NCI-H345 SCLC cells with an IC(50)value of 450 nM; BW2258U89 had an IC(50)value of 17 nM. BW2258U89 (1 microM) antagonized the ability of 50 nM BB to elevate cytosolic Ca(2+)in NCI-H345 cells, whereas 1 microM (Da) BW2258U89 did not. One micromolar BW2258U89 antagonized the increase in NCI-H345 c-fos mRNA caused by 10 nM BB, whereas 1 microM (Da) BW2258U89 had little effect. One microM BW2258U89 inhibited NCI-H345 clonal growth significantly whereas 1 microM (Da) BW2258U89 did not. These data suggest that an amidated C-terminal is important for antagonism of SCLC GRP receptors by BW2258U89.

Non-covalent labeling of human serum albumin with indocyanine green: a study by capillary electrophoresis with diode laser-induced fluorescence detection.

Indocyanine green (ICG) is a negatively charged, water-soluble, tricarbocyanine dye used primarily for medical imaging. ICG is only weakly fluorescent in the near-infrared region in its free (unbound) state in dilute aqueous solution. However, when non-covalently bound to protein, its fluorescence is greatly enhanced, making it a candidate for diode laser-induced fluorescence (diode-LIF) detection of proteins in capillary electrophoresis (CE). This paper investigates the suitability of ICG as a fluorescent label for the separation and detection of human serum albumin (HSA) by CE with diode-LIF detection. Specifically, we have considered the separation conditions necessary to resolve free ICG from ICG-HSA complexes; the limits of detection for free and HSA-bound ICG; the stability of aqueous ICG and ICG-HSA solutions over time; and the stoichiometry of the ICG-HSA complex.

Ticks and tick-borne diseases in Oklahoma.

Tick-borne diseases are common in Oklahoma, especially the eastern part of the state where tick prevalence is highest. Three species of hard ticks are present in Oklahoma that are known vectors of human disease--the American dog tick (Rocky Mountain spotted fever; RMSF), the lone star tick (ehrlichiosis) and the black-legged tick (Lyme disease). Oklahoma consistently ranks among the top states in numbers of reported RMSF cases, and Ehrlichiosis may be as prevalent as RMSF. Although Lyme disease is frequently reported in Oklahoma, over-diagnosing of this disease due to false-positive test results is common; positive or equivocal screening tests should be confirmed by Western immunoblot. At present, it is unclear whether the disease seen here is Lyme disease or another Lyme-like disease. If true Lyme disease is present in the state, it is probably rare. Physicians should be aware of the most recent recommendations for diagnosis, therapy and prevention of tick-borne diseases.

Stereoselective actions of halothane at GABA(A) receptors.

Isoflurane anesthesia exhibits stereoselectivity, and a corresponding stereoselectivity ((+)->(-)-isomer) has been reported at GABA(A) receptors in vitro. The objective of the present study was to determine if the positive modulatory actions of halothane at GABA(A) receptors exhibited a similar stereoselectivity. Both (R)- and (S)-halothane ((+)- and (-)- isomers, respectively) enhanced [3H]flunitrazepam binding to brain membranes in a concentration dependent manner without a significant difference in either potency (EC50) or efficacy (Emax). While both (R)- and (S)-halothane enhanced [3H]muscimol binding, the potency of the (+)-isomer was slightly greater than the corresponding (-)-isomer (0.91 +/- 0.17 versus 1.45 +/- 0.04% atmospheres, respectively (P < 0.02)). Thus, subtle structural differences between inhalational anesthetics can have a significant impact on the degree of stereoselectivity at the receptor level and may provide insights for the development of more specific drugs.

Distinct structural requirements for the direct and indirect actions of the anaesthetic etomidate at GABA(A) receptors.

1. The intravenous anaesthetic etomidate augments GABA-gated chloride currents (indirect action) and, at higher concentrations, evokes chloride currents in the absence of GABA (direct action). 2. In order to identify amino acid residues essential for these actions, site directed mutagenesis was performed on the beta3 subunit. 3. Mutation of an asparagine to a serine residue at position 290 dramatically reduced both etomidate-induced chloride currents and its ability to enhance [3H]flunitrazepam binding in HEK293 cells expressing alpha1beta3gamma2 recombinant GABA(A) receptors. 4. In contrast, the indirect effect of etomidate was retained, though its potency was reduced. 5. These findings indicate that there are distinct requirements for these dual actions of etomidate at GABA(A) receptors.

Distinct loci mediate the direct and indirect actions of the anesthetic etomidate at GABA(A) receptors.

Most general anesthetics produce two distinct actions at GABA(A) receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a beta subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABA(A) receptors, site-directed mutagenesis of the beta3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABA(A) receptor composed of alpha1beta3gamma2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate [3H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [3H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on beta3(Asn290) indicates that the dual actions of this intravenous anesthetic at GABA(A) receptors are mediated via distinct loci.