Polysaccharide Biocatalysis: From Synthesizing Carbohydrate Standards to Establishing Characterization Methods

Starch, glycogen, and cellulose are all around us. They are eaten and used on a daily basis but they are not understood completely. Even though these carbohydrates are simple, concerning their repeating unit, they are hard to characterize. In order to try to understand as much as possible about their structure and the relationship between their molecular structure and physical properties, it is very practical to create such polysaccharides, for instance enzymatically, characterize them, and use them as standards for the characterization of natural ones. Therefore, the main objective of this Trend article is to outline different enzymatic routes to such carbohydrates, possibilities for their characterization, and the characterization of natural ones.     

A pilot wastewater‐based epidemiology assessment of anabolic steroid use in Queensland,Australia

Anabolic‐androgenic steroids are synthetic compounds prohibited due to their performance‐enhancing characteristics. The use of these substances is known to cause health‐related issues, which highlights the importance of being able to evaluate the scale of consumption by the general population. However, most available research on the analysis of anabolic steroids is focused on animals and athletes in connection with doping. The potential of wastewater‐based epidemiology as an intelligence tool for the assessment of community level use of anabolic steroids is presented herein. A liquid chromatography tandem mass spectrometry method was developed for the analysis of 10 anabolic‐androgenic steroids and 14 endogenous hormones in influent wastewater. The validated method was applied to sixteen 24‐hour composite wastewater influent samples that were collected over a period of five years from two wastewater treatment plants in Queensland, Australia. Nine investigated compounds were found to be present at concentrations between 14 and 611 ng L^?1 which translated into 3–104 mg excreted per 1000 individuals per day. It was concluded that the developed analytical method is suitable for the analysis of AAS in wastewater matrix. Additionally, both the inclusion of metabolites and further investigation into deconjugation by enzymatic hydrolysis would aid in understanding and evaluating community anabolic steroid use. For the first time, this study presents the application of wastewater‐based epidemiology on anabolic‐androgenic steroids in Australia.     

Efficacy of SpyGlass~(TM)-directed biopsy compared to brush cytology in obtaining adequate tissue for diagnosis in patients with biliary strictures

AIM: To evaluate the diagnostic yield(inflammatory activity) and efficiency(size of the biopsy specimen) of SpyGlassTM-guided biopsy vs standard brush cytology in patients with and without primary sclerosing cholangitis(PSC).METHODS: At the University Medical Center Mainz, Germany, 35 consecutive patients with unclear biliarylesions(16 patients) or long-standing PSC(19 patients) were screened for the study. All patients underwent a physical examination, lab analyses, and abdominal ultrasound. Thirty-one patients with non-PSC strictures or with PSC were scheduled to undergo endoscopic retrograde cholangiography(ERC) and subsequent per-oral cholangioscopy(POC). Standard ERC was initially performed, and any lesions or strictures were localized. POC was performed later during the same session. The Boston Scientific SpyGlass SystemTM(Natick, MA, United States) was used for choledochoscopy. The biliary tree was visualized, and suspected lesions or strictures were biopsied, followed by brush cytology of the same area. The study endpoints(for both techniques) were the degree of inflammation, tissue specimen size, and the patient populations(PSC vs non-PSC). Inflammatory changes were divided into three categories: none, low activity, and high activity. The specimen quantity was rated as low, moderate, or sufficient.RESULTS: SpyGlassTM imaging and brush cytology with material retrieval were performed in 29 of 31(93.5%) patients(23 of the 29 patients were male). The median patient age was 45 years(min, 20 years; max, 76 years). Nineteen patients had known PSC, and 10 showed non-PSC strictures. No procedure-related complications were encountered. However, for both methods, tissues could only be retrieved from 29 pa-tients. In cases of inflammation of the biliary tract, the diagnostic yield of the SpyGlassTM-directed biopsies was greater than that using brush cytology. More tissue material was obtained for the biopsy method than for the brush cytology method(P = 0.021). The biopsies showed significantly more inflammatory characteristics and greater inflammatory activity compared to the cy-tological investigation(P = 0.014). The greater quantity of tissue samples proved useful for both PSC and non-PSC patients.CONCLUSION: SpyGlassTM imaging can be recom-mended for proper inflammatory diagnosis in PSC pa-tients. However, its value in diagnosing dysplasia wasnot addressed in this study and requires further investi-gation.     

Environmental enrichment extends ocular dominance plasticity into adulthood and protects from stroke-induced impairments of plasticity

Ocular dominance (OD) plasticity in mouse primary visual cortex (V1) declines during postnatal development and is absent beyond postnatal day 110 if mice are raised in standard cages (SCs). An enriched environment (EE) promotes OD plasticity in adult rats. Here, we explored cellular mechanisms of EE in mouse V1 and the therapeutic potential of EE to prevent impairments of plasticity after a cortical stroke. Using in vivo optical imaging, we observed that monocular deprivation in adult EE mice (i) caused a very strong OD plasticity previously only observed in 4-wk-old animals, (ii) restored already lost OD plasticity in adult SC-raised mice, and (iii) preserved OD plasticity after a stroke in the primary somatosensory cortex. Using patch-clamp electrophysiology in vitro, we also show that (iv) local inhibition was significantly reduced in V1 slices of adult EE mice and (v) the GABA/AMPA ratio was like that in 4-wk-old SC-raised animals. These observations were corroborated by in vivo analyses showing that diazepam treatment significantly reduced the OD shift of EE mice after monocular deprivation. Taken together, EE extended the sensitive phase for OD plasticity into late adulthood, rejuvenated V1 after 4 mo of SC-rearing, and protected adult mice from stroke-induced impairments of cortical plasticity. The EE effect was mediated most likely by preserving low juvenile levels of inhibition into adulthood, which potentially promoted adaptive changes in cortical circuits.Ocular dominance (OD) plasticity induced by monocular deprivation (MD) is one of the best studied models of experience-dependent plasticity in the mammalian cortex (1, 2). OD plasticity in primary visual cortex (V1) of C57BL/6J mice is maximal at 4 wk of age, declines after 2–3 mo, and is absent beyond postnatal day 110 (PD110) if animals are raised in standard cages (SCs) (3–6). In 4-wk-old mice, 4 d of MD are sufficient to induce an OD shift to the open eye; therefore, neurons in the binocular V1, which are usually dominated by the contralateral eye in rodents (3, 7), become activated more equally by both eyes (5, 8). This juvenile OD shift is predominantly mediated by a decrease in the visual cortical responses to the deprived eye (1, 9–11), whereas significant OD shifts in older animals up to PD110 need 7 d of MD and are mediated primarily by increased open-eye responses in V1. Raising animals in an enriched environment (EE) gives them the opportunity of enhanced physical, social, and cognitive stimulation and influences brain physiology and behavior in many ways (12, 13). It has been shown previously that EE enhances visual system development in rats (14) and mice (15–17), increases levels of the brain-derived neurotrophic factor and serotonin (18), reduces both extracellular GABA levels (18, 19) and the density of ECM perineuronal nets (PNNs) (19), and promotes OD plasticity in adult and aging rats (18–21). Here, we explored cellular mechanisms of EE in V1 of mice and the therapeutic potential of EE to prevent impairments of plasticity after a cortical stroke. Furthermore, we studied whether EE would prolong the sensitive phase for OD plasticity into adulthood and also restore this form of plasticity in mice that were raised in SC until PD110 (i.e., in animals that were already beyond their sensitive phase for OD plasticity). Despite pharmacological detection of in vivo GABA levels, suggesting that EE reduces intracortical inhibition, direct electrophysiological evidence is still missing. We therefore recorded GABA, AMPA, and NMDA currents in slices from EE- and SC-raised mice and also tested the efficacy of diazepam injections to abolish OD plasticity of EE mice in vivo. Finally, we studied whether raising mice in EE would protect them from lesion-induced impairments of OD plasticity. Our results show that raising mice in EE preserved OD plasticity into late adulthood rejuvenated the brain after 3 mo of SC-rearing, and protected adult mice from stroke-induced impairments of cortical plasticity. Our electrophysiological measurements and diazepam treatment indicate that the plasticity-promoting effect of EE was primarily mediated by reduced intracortical inhibition compared with SC-raised mice. These results suggest EE as a preventive intervention to enhance and preserve plasticity in adulthood and after a cortical lesion.     

From the Cover: A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs

The recent success of antibody–drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on β-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubule-destabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.Microtubule-targeting agents such as the taxanes and the vinca alkaloids represent a successful class of anticancer drugs (1). Vinblastine, for example, is a microtubule-destabilizing agent (MDA) that is widely used in combination therapy for the treatment of childhood and adult malignancies (2). The broad clinical application of MDAs, however, is hampered by their severe adverse effects (3). This problem has been very recently addressed by the use of antibody–drug conjugate (ADC) approaches, which have revived interest in the development of highly potent MDAs for therapeutic use (4–6).For several important MDAs, the molecular mechanism of action on tubulin and microtubules has so far remained elusive. Rhizoxin, for example, is a potent MDA that has been investigated in phase 2 clinical trials, but for reasons poorly understood, it has demonstrated only very limited clinical efficacy (7). At the molecular level, it is well established that rhizoxin interferes with the binding of vinblastine to tubulin; however, the exact location of its binding site has been a matter of debate (8–10). Interestingly, biochemical and mutagenesis data suggest that the structurally unrelated MDA maytansine (9, 11), which is part of an ADC that was recently approved by the FDA for the treatment of advanced breast cancer (11, 12), and the phase 1 drug PM060184 (13, 14) (Fig. 1A) share a common tubulin-binding site with rhizoxin (9, 13, 14). These two latter drugs have also been reported to interfere with the binding of vinblastine; however, as for rhizoxin, the exact binding sites and modes of action of maytansine and PM060184 have not been elucidated (9, 14–16).Open in a separate windowFig. 1.Structure of the tubulin–rhizoxin F complex. (A) Chemical structures of rhizoxin F, maytansine, and PM060184. (B) Overall view of the T₂R-TTL–rhizoxin F complex. Tubulin (gray), RB3 (light green), and TTL (violet) are shown in ribbon representation; the MDA rhizoxin F (orange) and GDP (cyan) are depicted in spheres representation. As a reference, the vinblastine structure (yellow, PDB ID no. 1Z2B) is superimposed onto the T₂R complex. (C) Overall view of the tubulin–rhizoxin F interaction in two different orientations. The tubulin dimer with bound ligand (α-tubulin-2 and β-tubulin-2 of the T₂R-TTL–rhizoxin F complex) is shown in surface representation. The vinblastine structure is superimposed onto the β-tubulin chain to highlight the distinct binding site of rhizoxin F. All ligands are in sphere representation and are colored in orange (rhizoxin F), cyan (GDP), and yellow (vinblastine). (D) Close-up view of the interaction observed between rhizoxin F (orange sticks) and β-tubulin (gray ribbon). Interacting residues of β-tubulin are shown in stick representation and are labeled.To establish the exact tubulin-binding site of rhizoxin, maytansine, and PM060184 and to clarify their specific interactions with the protein, we have investigated the structures of the corresponding ligand–tubulin complexes by X-ray crystallography. Our data reveal a new tubulin-binding site and pharmacophore for small molecules, and binding to this site is associated with a distinct molecular mechanism for the inhibition of microtubule formation.     

Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.