High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells.

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Role of HDAC6-STAT3 in immunomodulatory pathways in Colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common malignant neoplasms and the second leading cause of death from tumors worldwide. Therefore, there is a great need to study new therapeutical strategies, such as effective immunotherapies against these malignancies. Unfortunately, many CRC patients do not respond to current standard immunotherapies, making it necessary to search for adjuvant treatments. Histone deacetylase 6 (HDAC6) is involved in several processes, including immune response and tumor progression. Specifically, it has been observed that HDAC6 is required to activate the Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor involved in immunogenicity, by activating different genes in these pathways, such as PD-L1. Over-expression of immunosuppressive pathways in cancer cells deregulates T-cell activation. Therefore, we focused on the pharmacological inhibition of HDAC6 in CRC cells because of its potential as an adjuvant to avoid immunotolerance in immunotherapy. We investigated whether HDAC6 inhibitors (HDAC6is), such as Nexturastat A (NextA), affected STAT3 activation in CRC cells. First, we found that NextA is less cytotoxic than the non-selective HDACis panobinostat. Then, NextA modified STAT3 and decreased the mRNA and protein expression levels of PD-L1. Importantly, transcriptomic analysis showed that NextA treatment affected the expression of critical genes involved in immunomodulatory pathways in CRC malignancies. These results suggest that treatments with NextA reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulatory genes involved in the inflammatory and immune responses. Therefore, targeting HDAC6 may represent an interesting adjuvant strategy in combination with immunotherapy.

Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin.

The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

Randomised, crossover comparison of the single-use SoftSeal and the LMA Unique laryngeal mask airways.

We performed a randomised, crossover, single-blind trial among 168 patients, to compare the single-use SoftSeal and LMA Unique laryngeal mask airways in spontaneously breathing adults. Size-3 and -4 laryngeal mask airways, inserted by experienced anaesthetists, performed equivalently for successful first-time placement (148 (91%) vs 155 (96%) for the SoftSeal and LMA Unique, respectively). The SoftSeal was more often rated as difficult to insert (27 (17%) vs 4 (3%); p < 0.001) and was more likely to show evidence of mucosal trauma after the first insertion (14 (10%) vs 5 (4%); OR 1.3 (95% CI 1.3-11.3); p < 0.05). The fibreoptic view of the larynx was better through the SoftSeal (vocal cords not visible in 27 (17%) vs 44 (27%); p < 0.05) and it more frequently provided a ventilation seal at 20 cmH(2)O (93 (59%) vs 62 (39%); OR 2.15 (CI 1.44-3.21); p < 0.001). In contrast to the LMA Unique(trade mark), its cuff pressure did not increase during nitrous oxide anaesthesia (median (IQR [range]) decrease 3 (- 20-23 [-40-94]) cmH(2)O vs increase of 16 (-2-39 [-54-112]) cmH(2)O; p < 0.01). Both devices were equivalent for the success of first-time insertion and performed satisfactorily clinically. There were some performance differences, but either appears suitable for airway management in spontaneously breathing patients.

[Comparison of value of four immunological methods (AST, ADNase B, AHy, ANADase) by A-streptococcal infections and their significance in the routine diagnostic (author's transl)]. / Vergleich der Aussagekraft von vier immunologischen Nachweismethoden (AST, ADNase B, AHy, ANADase) bei A-Streptokokken-Infektionen und ihre Bedeutung für die Routinediagnostik

In the sera of approximately 600 patients of all age groups, who were on clinical suspicion of an A-streptococcal infection, AST, ADNase B, AHy and ANADase were determined. The ANADase alone detects 90% of the acute A-streptococcal infections, the AHy and the ADNase B 80% and the ASO 50%. In skin infections superinfected by A-streptococci, the AHy is positive in 85%, the ANADase in 80%, the ADNase B in 75% and the AST in 40% of the cases. The most frequent A-streptococcal infection, Angina tonsillaris, is determined in the best manner possible through the combination ADNase B + ANADase + AHy. According to the statistical evaluation, the ADNase B and the ANADase are the most reliable tests. The age group of the 4 to 15 year-olds reaches for ASO, ADNase B and AHy a percentage of highly increased titers twice as high in comparison with the adult group. In no case does one test determine 100% of the collective; therefore, a combination of several antibody reactions is desirable for the diagnosis. Increased titers should be checked every four weeks, also normal titers with patients, who continue on the clinical suspicion of having an A-streptococcal infection, because their blood sample may have been taken before the immune reaction occurred.

NMR parameter contrast in abundant-spin images of solids.

Solid state nuclear magnetic resonance imaging (NMRI) techniques have been steadily improving over the years. Today high-resolution images of rigid solids are now accomplished by many different means. For abundant nuclei, the combination of multiple-pulse line narrowing and pulsed field gradients have greatly improved both the resolution and sensitivity of the imaging experiment, but often at the expense of the chemical information in the material. In this paper we discuss means of incorporating NMR parameters in the imaging experiment to generate image contrast which provides information about local variations in the chemistry of the material.

Weak Combination Bands in the 3-µm Region of Ethane.

The infrared spectrum of ethane in the region of the CH-stretching fundamental vibrations, between 2600 and 3300 cm(-1), was recorded with a Bruker IFS120HR Fourier transform spectrometer. Jet-cooled and room-temperature conditions were used at 0.01 and 0.005 cm(-1) instrumental resolution, respectively. Ten weak combination bands were observed and vibrationally assigned, in addition to the three principal bands in the region (nu(5), nu(7), and nu(8) + nu(11) ( parallel component)). A stack-by-stack analysis was performed for five of the combination bands. Their origins were determined as follows: nu(8) + nu(12) ( parallel component) at 2665.1512(30) cm(-1), nu(2) + nu(6) at 2753.326(32) cm(-1), nu(6) + nu(11) at 2844.13069(75) cm(-1), nu(8) + nu(11) ( perpendicular component) at 2930.705(90) cm(-1), and nu(4) + nu(10) at 3255.568(50) cm(-1). Effective rovibrational parameters were obtained for the corresponding upper states. New information was also obtained on the five remaining combination bands. Their approximate origins are: nu(8) + nu(12) ( perpendicular component) at 2660 cm(-1), nu(2) + nu(4) + nu(12) at 2860 cm(-1), nu(4) + 2nu(9) + nu(12) at 3090 cm(-1), 3nu(4) + nu(8) + nu(9) at 3157 cm(-1), and nu(4) + 2nu(8) at 3225 cm(-1). Copyright 1999 Academic Press.

Effective Rotation-Vibration Parameters for the nu8 and nu4 + nu12 Bands of Ethane.

The jet-cooled spectrum of ethane in the region of the nu8 perpendicular fundamental vibration at 1472 cm-1 was recorded with a Fourier transform spectrometer at 0.01 cm-1 instrumental resolution. About 1150 lines were assigned to 18 subbands of nu8 ranging from K"DeltaK = -9 to +8. About 160 lines were assigned to three subbands (K"DeltaK = 0 to +2) of the nu4 + nu12 combination band near 1481 cm-1. Torsional splittings were observed for the majority of the transitions. A stack-by-stack analysis was performed, resulting in effective rovibrational parameters for the upper states nu8 and nu4 + nu12. A number of perturbations was observed and is discussed. Copyright 1999 Academic Press.

Determination of 207Pb2+ chemical shift tensors from precise powder lineshape analysis.

207Pb solid state NMR powder spectra at 296 K are presented for PbSO4, PbMoO4, PbCrO4, PbCO3, PbTiO3, PbZrO3, Pb(NO3)2, Pb(SCN)2, and PbS. Analysis for principal values of the anisotropic chemical shift tensors of the generally very broad spectra included the frequency dependent excitation of the pulse sequence used. Commonly used solid and liquid secondary shift standards for lead were studied with high precision as a function of temperature between 295 K and 315 K to establish a clean 207Pb shift scale. Errors in the existing literature are discussed.

Exploration of xenon as a potential cardiostable sedative: a comparison with propofol after cardiac surgery.

Xenon anaesthesia is thought to have minimal haemodynamic side-effects. It is, however, expensive and requires special delivery systems for economic use. In this randomised cross-over study, we: (i) investigated the haemodynamic profile and recovery characteristics of xenon compared with propofol sedation in postoperative cardiac surgery patients, and (ii) evaluated a fully closed breathing system to minimise xenon consumption. We demonstrated a significantly faster recovery from xenon (3 min 11 s) than propofol sedation (25 min 23 s). Relative to propofol, xenon sedation produced no change in heart rate or mean arterial pressure and there were significantly higher mean values for central venous pressure (10.6 vs. 8.9 mmHg), pulmonary artery occlusion pressure (11.2 vs. 9.5 mmHg), mean pulmonary artery pressure (20.1 vs. 18.3 mmHg) and systemic vascular resistance index (2170 vs. 1896 dyn.s.cm-5.m-2). The haemodynamic profile seen with propofol reflected its known vasodilator effects. This was supported by the almost identical left ventricular stroke work indexes seen with both methods of sedation.

Submillimeter-Wave Absorption Spectroscopy of the Ar-CO Complex: Detection of the van der Waals Bending Vibration

Van der Waals bending rovibrational transitions of the Ar-CO complex were observed between 308 and 383 GHz using submillimeter-wave absorption spectroscopy in a pulsed supersonic jet. The submillimeter radiation was generated by a phase-stabilized backward wave oscillator source. Fourteen transitions of the P and R branch of the bending vibration were measured with an accuracy of about 100 kHz. The frequencies were fitted using effective molecular parameters including the band origin and the rotational constants for the ground state and for the excited bending vibrational state.

High-Resolution Jet-Cooled Spectroscopy of SF6: The nu2 + nu6 Combination Band of 32SF6 and the nu3 Band of the Rare Isotopomers.

The Fourier transform infrared spectrum of SF6 was recorded in a supersonic expansion jet of an SF6/argon mixture. The SF6:Ar seeding ratio was 2:3. The instrumental bandwidth was 0.005 cm-1. A globar source and an MCT detector were used. A rotational temperature of approximately 30 K was achieved. The nu2 + nu6 combination band of 32SF6 was analyzed using a modified version of the spherical top data system (STDS) programs developed in Dijon. A very good fit was obtained for this band with an rms of 0.0036 cm-1. The effective Hamiltonian was developed up to fourth order for the nu2 + nu6 part, to second order for the nu2 and ground state parts, and to first order for the nu6 part. Five hundred twenty-one transitions were assigned, 40 of them reaching the F2u forbidden sublevel. The positions of the two F1u and F2u sublevels are found to be 991.276 and 989.487 cm-1, respectively. The nu3 band of the 33SF6 and 34SF6 isotopomers were also analyzed. Parameters and simulations are presented. A first detection of the nu3 Q branch of 36SF6 (0.02% natural abundance) is reported. Copyright 1998 Academic Press.