158 research outputs found
Photo‐biocatalytic Cascades:Combining Chemical and Enzymatic Transformations Fueled by Light
In the field of green chemistry, light – an attractive natural agent – has received particular attention for driving biocatalytic reactions. Moreover, the implementation of light to drive (chemo)enzymatic cascade reactions opens up a golden window of opportunities. However, there are limitations to many current examples, mostly associated with incompatibility between the enzyme and the photocatalyst. Additionally, the formation of reactive radicals upon illumination and the loss of catalytic activities in the presence of required additives are common observations. As outlined in this review, the main question is how to overcome current challenges to the exploitation of light to drive (chemo)enzymatic transformations. First, we highlight general concepts in photo-biocatalysis, then give various examples of photo-chemoenzymatic (PCE) cascades, further summarize current synthetic examples of PCE cascades and discuss strategies to address the limitations
Towards a representative reference for MRI-based human axon radius assessment using light microscopy
Non-invasive assessment of axon radii via MRI bears great potential for clinical and neuroscience research as it is a main determinant of the neuronal conduction velocity. However, there is a lack of representative histological reference data at the scale of the cross-section of MRI voxels for validating the MRI-visible, effective radius (reff). Because the current gold standard stems from neuroanatomical studies designed to estimate the bulk-determined arithmetic mean radius (rarith) on small ensembles of axons, it is unsuited to estimate the tail-weighted reff. We propose CNN-based segmentation on high-resolution, large-scale light microscopy (lsLM) data to generate a representative reference for reff. In a human corpus callosum, we assessed estimation accuracy and bias of rarith and reff. Furthermore, we investigated whether mapping anatomy-related variation of rarith and reff is confounded by low-frequency variation of the image intensity, e.g., due to staining heterogeneity. Finally, we analyzed the error due to outstandingly large axons in reff. Compared to rarith, reff was estimated with higher accuracy (maximum normalized-root-mean-square-error of reff: 8.5 %; rarith: 19.5 %) and lower bias (maximum absolute normalized-mean-bias-error of reff: 4.8 %; rarith: 13.4 %). While rarith was confounded by variation of the image intensity, variation of reff seemed anatomy-related. The largest axons contributed between 0.8 % and 2.9 % to reff. In conclusion, the proposed method is a step towards representatively estimating reff at MRI voxel resolution. Further investigations are required to assess generalization to other brains and brain areas with different axon radii distributions
Pharmacologic treatment with CPI-613 and PS48 decreases mitochondrial membrane potential and increases quantity of autolysosomes in porcine fibroblasts
A metabolic phenomenon known as the Warburg effect has been characterized in certain cancerous cells, embryonic stem cells, and other rapidly proliferative cell types. Previously, our attempts to induce a Warburg-like state pharmaceutically via CPI-613 and PS48 treatment did augment metabolite production and gene expression; however, this treatment demonstrated a Reverse Warburg effect phenotype observed in cancer-associated stroma. In the current study, we inquired whether the mitochondria were affected by the aforementioned pharmaceutical treatment as observed in cancerous stromal fibroblasts. While the pharmaceutical agents decreased mitochondrial membrane potential in porcine fetal fibroblasts, the number and size of mitochondria were similar, as was the overall cell size. Moreover, the fibroblasts that were treated with CPI-613 and PS48 for a week had increased numbers of large autolysosome vesicles. This coincided with increased intensity of LysoTracker staining in treated cells as observed by flow cytometry. Treated fibroblasts thus may utilize changes in metabolism and autophagy to mitigate the damage of treatment with pharmaceutical agents. These findings shed light on how these pharmaceutical agents interact and how treated cells augment metabolism to sustain viability. c2019, The Author(s).Includes bibliographical references
The role of outer membrane proteins and lipopolysaccharides for the sensitivity of escherichia coli to antimicrobial peptides
Bacterial resistance to classical antibiotics is emerging worldwide. The number of infections caused by multidrug resistant bacteria is increasing and becoming a serious threat for human health globally. In particular, Gram-negative pathogens including multidrug resistant Escherichia coli are of serious concern being resistant to the currently available antibiotics. All Gram-negative bacteria are enclosed by an outer membrane which acts as an additional protection barrier preventing the entry of toxic compounds including antibiotics and antimicrobial peptides (AMPs). In this study we report that the outer membrane component lipopolysaccharide (LPS) plays a crucial role for the antimicrobial susceptibility of E. coli BW25113 against the cationic AMPs Cap18, Cap11, Cap11-1-18m2, melittin, indolicidin, cecropin P1, cecropin B, and the polypeptide antibiotic colistin, whereas the outer membrane protease OmpT and the lipoprotein Lpp only play a minor role for the susceptibility against cationic AMPs. Increased susceptibility toward cationic AMPs was found for LPS deficient mutants of E. coli BW25113 harboring deletions in any of the genes required for the inner part of core-oligosaccharide of the LPS, waaC, waaE, waaF, waaG, and gmhA. In addition, our study demonstrates that the antimicrobial activity of Cap18, Cap11, Cap11-1-18m2, cecropin B, and cecropin P1 is not only dependent on the inner part of the core oligosaccharide, but also on the outer part and its sugar composition. Finally, we demonstrated that the antimicrobial activity of selected Cap18 derivatives harboring amino acid substitutions in the hydrophobic interface, are non-active against wild-type E. coli ATCC29522. By deleting waaC, waaE, waaF, or waaG the antimicrobial activity of the non-active derivatives can be partially or fully restored, suggesting a very close interplay between the LPS core oligosaccharide and the specific Cap18 derivative. Summarizing, this study implicates that the nature of the outer membrane component LPS has a big impact on the antimicrobial activity of cationic AMPs against E. coli. In particular, the inner as well as the outer part of the core oligosaccharide are important elements determining the antimicrobial susceptibility of E. coli against cationic AMPs
Electron Bio-Imaging Centre (eBIC): the UK national research facility for biological electron microscopy
The recent resolution revolution in cryo-EM has led to a massive increase in demand for both time on high-end cryo-electron microscopes and access to cryo-electron microscopy expertise. In anticipation of this demand, eBIC was set up at Diamond Light Source in collaboration with Birkbeck College London and the University of Oxford, and funded by the Wellcome Trust, the UK Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) to provide access to high-end equipment through peer review. eBIC is currently in its start-up phase and began by offering time on a single FEI Titan Krios microscope equipped with the latest generation of direct electron detectors from two manufacturers. Here, the current status and modes of access for potential users of eBIC are outlined. In the first year of operation, 222 d of microscope time were delivered to external research groups, with 95 visits in total, of which 53 were from unique groups. The data collected have generated multiple high- to intermediate-resolution structures (2.8–8 Å), ten of which have been published. A second Krios microscope is now in operation, with two more due to come online in 2017. In the next phase of growth of eBIC, in addition to more microscope time, new data-collection strategies and sample-preparation techniques will be made available to external user groups. Finally, all raw data are archived, and a metadata catalogue and automated pipelines for data analysis are being developed
Comparison of insulin detemir and insulin glargine in subjects with type 1 diabetes using intensive insulin therapy
WSTĘP. Celem niniejszej pracy było porównanie kontroli
glikemii oraz ryzyka hipoglikemii podczas stosowania
insuliny detemir 2 × dziennie lub glarginy
raz dziennie u chorych na cukrzycę typu 1.
MATERIAŁ I METODY. Podczas trwającego 26 tygodni
wieloośrodkowego, otwartego badania z grupami
równoległymi 320 chorym na cukrzycę typu 1
podawano albo insulinę detemir 2 × dziennie albo
glarginę raz dziennie, każdorazowo w połączeniu
z przedposiłkowymi iniekcjami insuliny aspart.
WYNIKI. Po 26 tygodniach wartość HbA1c zmniejszyła
się z 8,8% do 8,2% w grupie przyjmującej insulinę
detemir i z 8,7% do 8,2% w grupie leczonej glarginą.
Wartość stężenia glukozy w osoczu (PG) mierzonego
na czczo w domu była niższa podczas stosowania
glarginy niż w czasie podawania insuliny detemir (7,0 mmol/l vs. 7,7 mmol/l; p < 0,001). Ogólny
9-punktowy profil mierzonych w domu wartości glikemii
był porównywalny między grupami (p = 0,125).
Nie stwierdzono istotnej różnicy w zmienności pomiarów
PG u 1 pacjenta (p = 0,437). Zróżnicowanie
pomiarów przedposiłkowego stężenia PG u 1 badanego
było niższe podczas stosowania insuliny detemir
niż glarginy (p < 0,05). Ogólne ryzyko hipoglikemii
było podobne, nie stwierdzono różnic w występowaniu
potwierdzonych epizodów hipoglikemii. Ryzyko
silnej, nocnej hipoglikemii wynosiło odpowiednio:
72% i 32% i było niższe w grupie stosującej insulinę
detemir (p < 0,05). Zwiększenie masy ciała
nie różniło się znacząco między grupami przyjmującymi
insulinę detemir i glarginę (0,52 kg vs. 0,96 kg;
p = 0,193).
WNIOSKI. Leczenie insuliną detemir podawaną 2 × dziennie lub glarginą podawaną raz dziennie, każdorazowo
w połączeniu z insuliną aspart, powodowało
podobną kontrolę glikemii. Ogólne zagrożenie
hipoglikemią było porównywalne, natomiast
ryzyko nasilonej i nocnej hipoglikemii było istotnie
niższe podczas stosowania insuliny detemir.BACKGROUND. To compare glycaemic control and risk
of hypoglycaemia of twice-daily insulin detemir with
once-daily insulin glargine in subjects with type 1
diabetes.
MATERIAL AND METHODS. In this 26-week, multicentre,
open-label, parallel-group trial, 320 subjects
with type 1 diabetes received either insulin detemir
twice daily or insulin glargine once daily, each in
combination with premeal insulin aspart.
RESULTS. After 26 weeks, HbA1c had decreased from
8.8 to 8.2% in the insulin detemir group and from
8.7% to 8.2% in the insulin glargine group. Homemeasured
fasting plasma glucose (PG) was lower
with insulin glargine than with insulin detemir
(7.0 mmol/l vs. 7.7 mmol/l; p < 0.001). The overall
shape of the home-measured nine-point PG profiles
was comparable between treatments (p = 0.125).
Overall, there was no significant difference in
within-subject variation in PG (p = 0.437). Withinsubject
variation in predinner PG was lower with insulin
detemir than with insulin glargine (p < 0.05).
The overall risk of hypoglycaemia was similar with
no differences in confirmed hypoglycaemia. However,
the risk of severe and nocturnal hypoglycaemia
was 72% and 32%, respectively, lower with insulin detemir
than with insulin glargine (p < 0.05). Body weight
gain was not significantly different comparing
insulin detemir and insulin glargine (0.52 kg
vs. 0.96 kg, p = 0.193).
CONCLUSIONS. Treatment with twice-daily insulin
detemir or once-daily insulin glargine, each in combination
with insulin aspart, resulted in similar glycaemic
control. The overall risk of hypoglycaemia
was comparable, whereas the risks of both severe
and nocturnal hypoglycaemia were significantly lower
with insulin detemir
Phenylalanine hydroxylase contributes to serotonin synthesis in mice
This is the final version. Available from the Federation of American Society of Experimental Biology via the DOI in this record.Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.Saint Petersburg State University (SPbU)Bundesministerium für Bildung und Forschung (BMBF)Deutsches Zentrum für Herz-Kreislaufforschung (DZHK)Volkswagen Foundation (VolkswagenStiftung
Fiber-orientation independent component of R(2)* obtained from single-orientation MRI measurements in simulations and a post-mortem human optic chiasm
The effective transverse relaxation rate (R(2)*) is sensitive to the microstructure of the human brain like the g-ratio which characterises the relative myelination of axons. However, the fibre-orientation dependence of R(2)* degrades its reproducibility and any microstructural derivative measure. To estimate its orientation-independent part (R(2,iso)*) from single multi-echo gradient-recalled-echo (meGRE) measurements at arbitrary orientations, a second-order polynomial in time model (hereafter M2) can be used. Its linear time-dependent parameter, β(1), can be biophysically related to R(2,iso)* when neglecting the myelin water (MW) signal in the hollow cylinder fibre model (HCFM). Here, we examined the performance of M2 using experimental and simulated data with variable g-ratio and fibre dispersion. We found that the fitted β(1) can estimate R(2,iso)* using meGRE with long maximum-echo time (TE(max) ≈ 54 ms), but not accurately captures its microscopic dependence on the g-ratio (error 84%). We proposed a new heuristic expression for β(1) that reduced the error to 12% for ex vivo compartmental R(2) values. Using the new expression, we could estimate an MW fraction of 0.14 for fibres with negligible dispersion in a fixed human optic chiasm for the ex vivo compartmental R(2) values but not for the in vivo values. M2 and the HCFM-based simulations failed to explain the measured R(2)*-orientation-dependence around the magic angle for a typical in vivo meGRE protocol (with TE(max) ≈ 18 ms). In conclusion, further validation and the development of movement-robust in vivo meGRE protocols with TE(max) ≈ 54 ms are required before M2 can be used to estimate R(2,iso)* in subjects
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