Continuous removal of endocrine disruptors by versatile peroxidase using a two‐stage system

The oxidant Mn³⁺‐malonate, generated by the ligninolytic enzyme versatile peroxidase in a two‐stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol‐A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17β‐estradiol, and 17α‐ethinylestradiol) were removed from wastewater at degradation rates in the range of 28–58 µg/L·min, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn³⁺‐malonate (hydraulic retention time as well as Na‐malonate and H₂O₂ feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3–8.8 mg/L) and environmental (1.2–6.1 µg/L) concentrations. Finally, when the two‐stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14‐fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (～8 h). This study demonstrates the feasibility of the two‐stage enzymatic system for removing EDCs both at high and environmental concentrations. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:908–916, 2015     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

The identification of a series of novel, soluble non-peptidic neuropeptide Y Y2 receptor antagonists

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.     

Promoter diversity in multigene transformation

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression.     

Thermal clamping of temperature-regulating flowers reveals the precision and limits of the biochemical regulatory mechanism

The flowers of several families of seed plants warm themselves when they bloom. In some species, thermogenesis is regulated, increasing the rate of respiration at lower ambient temperature (T _a) to maintain a somewhat stable floral temperature (T _f). The precision of this regulation is usually measured by plotting T _f over T _a. However, such measurements are influenced by environmental conditions, including wind speed, humidity, radiation, etc. This study eliminates environmental effects by experimentally ‘clamping’ T _f at constant, selected levels and then measuring stabilized respiration rate. Regulating flowers show decreasing respiration with rising T _f (Q ₁₀ < 1). Q ₁₀ therefore becomes a measure of the biochemical ‘precision’ of temperature regulation: lower Q ₁₀ values indicate greater sensitivity of respiration to T _f and a narrower range of regulated temperatures. At the lower end of the regulated range, respiration is maximal, and further decreases in floral temperature cause heat production to diminish. Below a certain tissue temperature (‘switching temperature’), heat loss always exceeds heat production, so thermoregulation becomes impossible. This study compared three species of thermoregulatory flowers with distinct values of precision and switching temperature. Precision was highest in Nelumbo nucifera (Q ₁₀ = 0.16) moderate in Symplocarpus renifolius (Q ₁₀ = 0.48) and low in Dracunculus vulgaris (Q ₁₀ = 0.74). Switching temperatures were approximately 30, 15 and 20°C, respectively. There were no relationships between precision, switching temperature or maximum respiration rate. High precision reveals a powerful inhibitory mechanism that overwhelms the tendency of temperature to increase respiration. Variability in the shape and position of the respiration–temperature curves must be accounted for in any explanation of the control of respiration in thermoregulatory flowers.     

HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts,and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1α-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1α-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1α in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1α showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1α also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1α increased tumor mass by ∼2-fold and tumor volume by ∼3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1α also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1α is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1α expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1α in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1α in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a three-fold reduction in tumor volume. We conclude that HIF1α activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly “feed” cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their “self-digestion.” Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other “classic” oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed “autophagic tumor stroma model of cancer.”Key words: caveolin-1, autophagy, mitophagy, the Warburg effect, tumor stroma, hypoxia, HIF1A, NFκB, compartment-specific oncogenesis, cancer-associated fibroblasts     

Preharvest application of exogenous abscisic acid (ABA) or an ABA analogue does not affect endogenous ABA concentration of onion bulbs

Bulb abscisic acid (ABA) concentration has been shown to decrease in stored onions, and onset of sprouting to occur at minimal ABA concentration. It was postulated that increasing prestorage bulb ABA concentration could increase storage life. Analogues of ABA that enhance biological activity and resist degradation are available and are becoming commercially viable. Exogenous ABA and an ABA analogue (8′-methylene ABA methyl ester; PBI-365) were applied separately as preharvest foliar sprays to six onion cultivars with varying storage potential. Quality indicators including pyruvate, total soluble solids and firmness were determined at regular intervals during storage. Neither ABA treatment increased endogenous bulb ABA concentration. Bulb ABA concentration decreased during storage and the onset of sprouting occurred at a minimal ABA concentration (ca. 50–120 ng g⁻¹ DW). This was followed by an increase in ABA concentration as sprout growth continued. No straightforward relationship between ABA and carbohydrate metabolism could be determined.     

Knocking down the diencephalic thyrotropin-releasing hormone precursor gene normalizes obesity-induced hypertension in the rat

We recently showed that diencephalic TRH may mediate the central leptin-induced pressor effect. Here, to study the role of TRH in obesity-induced hypertension (OIH), we used a model of OIH produced by a high-fat diet (HFD, 45 days) in male Wistar rats. After 4 wk, body weight and systolic arterial blood pressure (SABP) increased in HFD animals. Plasma leptin was correlated with peritoneal adipose tissue. Then, we treated OIH animals with an antisense oligodeoxynucleotide and small interfering (si)RNA against the prepro-TRH. Antisense significantly decreased diencephalic TRH content and SABP at 24 and 48 h posttreatment. Similar effects were observed with siRNA against prepro-TRH but for up to 4 wk. Conversely, vehicle, an inverted antisense sequence and siRNA against green fluorescence protein, produced no changes. SABP decrease seems to be owing to an inhibition of the obesity-enhanced sympathetic outflow but not to an alteration in thyroid status. Using a simple OIH model we demonstrated, for the first time, that central TRH participates in the hypertension induced by body weight gain probably through its well-known action on sympathetic activity. Thus the TRH-leptin interaction may contribute to the strong association between hypertension and obesity.