Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

Functionalized silica nanoparticles coupled with nanoporous membrane for efficient ionic current rectification.

In the last few decades, tremendous effort has been dedicated to mimicking the efficient ionic current rectification (ICR) of biological nanopores. Nanoporous membranes and singular nanopores with ICR functionality have been fabricated using advanced, yet costly technologies. We herein demonstrate that a simple, novel, and robust ICR platform can be constructed using 80 nm silica nanoparticles and a piece of 15 nm track-etched polycarbonate membrane. Efficient ICR can be obtained when voltages of different polarities are applied across the membrane, due to the asymmetric electrophoretic migration of silica nanoparticles whose surfaces are modified with different functional groups. The effect of pore size, ionic strength, pH, voltage magnitude, and density of silica nanoparticles on the efficiency of the ICR system has been systematically investigated in this report. Our results clearly show that smaller pore, lower ionic strength, appropriate pH value, higher electrical field strength, lower density of silica nanoparticles can generally enhance the efficiency of the ICR system. The principles of this new ICR system may find many potential applications in controllable drug delivery, energy storage and water purification.

Hypertriglyceridemia in Apoa5-/- mice results from reduced amounts of lipoprotein lipase in the capillary lumen.

Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we have suspected that the underlying cause is reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5-/- mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific mAbs, the binding of LPL Abs to heart and BAT capillaries (relative to CD31 or GPIHBP1 Abs) was reduced in Apoa5-/- mice. LPL levels in the postheparin plasma were also lower in Apoa5-/- mice. We suspected that a recent biochemical observation - that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity - could be related to the low intracapillary LPL levels in Apoa5-/- mice. We showed that an ANGPTL3/8-specific mAb (IBA490) and APOA5 normalized plasma triglyceride (TG) levels and intracapillary LPL levels in Apoa5-/- mice. We also showed that ANGPTL3/8 detached LPL from heparan sulfate proteoglycans and GPIHBP1 on the surface of cells and that the LPL detachment was blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5-/- mice and further illuminate the molecular mechanisms that regulate plasma TG metabolism.

Engineered Production of Strictosidine and Analogues in Yeast.

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fed-batch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

The potential of circulating cell free RNA as a biomarker in cancer.

Introduction: It is now clear that circulating cell-free ribonucleic acids (ccfRNAs), including messenger RNA (mRNA) and miRNA, are potential cancer biomarkers. As ccfmiRNA is relatively more stable than ccfmRNA, research should concentrate on developing novel methods to preserve the stability of ccfmRNA and standardization of the protocol which includes extraction, detection, and multicenter validation. Areas covered: This literature review concentrates on the potential of ccfRNA being used as a biomarker in cancer, with special focus on mRNAs and microRNAs (miRNAs). Expert opinion: With the advancement of high-throughput technologies such as RNA sequencing, a panel of biomarkers will be used for the diagnosis, prognosis and therapeutic monitoring of cancer patients. In order to achieve this important target, bioinformatics education to pathologists, scientists, and technologists in molecular diagnostic laboratories is essential. Moreover, the panel of these new ccfRNAs biomarkers has to obtain approval or clearance from an authority such as the US Food and Drug Administration (FDA), and the standard of utilizing these new protocols has to be recognized via accreditation exercise. Therefore, there is still a long way to go before an extensively use of ccfRNA biomarkers in cancer patients can be realized.

Electrical stimulation of acupuncture points and blood pressure responses to postural changes: a pilot study.

BACKGROUND: Application of transcutaneous electrical stimulation over acupuncture points (Acu-TENS) facilitates heart rate recovery after exercise and restores hemodynamic stability after open heart surgery. The role of Acu-TENS on cardiovascular parameters in response to postural changes has not been reported. OBJECTIVE: To investigate (1) the effect of Acu-TENS on blood pressure responses to -10º head-down postural change and (2) whether such effects were associated with modulation by the autonomic nervous system. METHOD: Sixteen healthy volunteers, mean age 22.8 (SD, 3.1) years, were subjected to a -10º head-down tilt from the supine position on 3 separate occasions and received in random order the following 3 intervention protocols for 40 minutes before the postural change: Acu-TENS (over bilateral acupuncture points, PC6), sham-TENS (TENS applied to the skin over the patellae), and control (no electrical output from the TENS device applied at PC6). Mean arterial pressure, large artery elasticity index, cardiac output, and heart rate were recorded and compared at different stimulation protocols in the supine and -10º head-down tilt positions. Spectral analysis of heart rate variability was used to determine any modulation by the autonomic nervous system. RESULTS: Change in large artery elasticity index was observed only in the Acu-TENS group (P < .05) and mean arterial pressure appeared most stable during Acu-TENS. Autonomic nervous system modulation was not apparent with spectral analysis, irrespective of intervention. Sympathetic activity predominated in all positions. CONCLUSION: Acu-TENS seems to reduce blood pressure changes with -10º head-down tilt with concomitant changes in arterial vessel tone.