Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB₅ toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB₅ toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB₅ toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA’s active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin’s mechanism of action.     

Towards sustainable electrochemical energy storage: solution-based processing of polyquinone composites

Continuous adoption of renewable energy sources and the proliferation of electric transportation technologies push towards sustainable energy storage solutions. Consequently, a solution-based up-scalable synthesis approach is developed for polymeric quinone composites with graphene. Cellulose nanocrystals play a vital role in achieving greener processing and improving the composite electrochemical energy storage performance. The synthesis method emphasizes using aqueous reaction media, incorporates low-cost and biomass-derived feedstocks, avoids critical or scarce materials, and maintains temperatures below 200 °C. Stable aqueous graphene dispersions were obtained by hydrothermal reduction of electrochemically exfoliated graphene oxide in the presence of cellulose nanocrystals. Dispersions served as a reaction medium for quinone cationic polymerization, leading to core–shell type structures of polymer-covered mono-to-few layer graphene, thanks to the nanosheet restacking prevention effect provided by cellulose nanocrystal dispersions. A sample consisting of 5 wt% cellulose nanocrystals and 5 wt% graphene achieved storage metrics of 720.5 F g⁻¹ and 129.6 mA h g⁻¹ at 1 A g⁻¹, retaining over 70% of the performance after 1000 charge/discharge cycles.

A valid one-pot, low temperature and readily scalable aqueous processing route towards sustainable production of organic electrode-based battery/capacitive systems.     

Acute renal infarction: A diagnostic challenge

Acute renal infarction is a relatively rare and scarcely reported condition. Contrast enhanced CT scan is essential for diagnosing this condition. The most common etiology of this condition is cardioembolic, however up to 59% of cases could be classified as idiopathic acute renal infarction.Here we present a case of a 41?year-old male who was admitted for acute onset right flank pain with fevers and rigors. A CT abdomen with contrast showed findings concerning for right pyelonephritis or renal infarction. Urinalysis was negative for infection. He did not respond to treatment with intravenous antibiotics and was thus diagnosed with acute renal infarction. Work-up for common etiologies was negative and the renal infarction was presumed to be idiopathic. He was discharged home on Enoxaparin.In conclusion, acute renal infarction is a rare condition which should be suspected in patients presenting with acute flank/abdominal pain in whom the more common etiologies have been ruled out.     

Five myofibrillar lesion types in eccentrically challenged,unloaded rat adductor longus muscle—a test model

Sarcomere disruptions are observed in the adductor longus (AL) muscles following voluntary reloading of spaceflown and hindlimb suspension unloaded (HSU) rat, which resemble lesions in eccentrically challenged muscle. We devised and tested an eccentric contraction (ECCON) test system for the 14‐day HSU rat AL. Six to 7 hours following ECCON, ALs were fixed to allow immunostaining and electron microscopy (EM). Toluidine blue‐stained histology semithin sections were screened for lesion density (#/mm²). Serial semithin sections from the ECCON group were characterized for myosin immunointensity of lesions. Five myofibrillar lesion types were identified in histological semithin sections: focal contractions; wide A‐bands; opaque areas; missing A‐bands; and hyperstretched sarcomeres. Lesion density by type was greater for ECCON than NonECCON ALs (P ≤ 0.05; focal contractions and opaque regions). Lesion density (#‐of‐all‐five‐types/mm²) was significantly different (ECCON: 23.91 ± 10.58 vs. NonECCON: 5.48 ± 1.28, P ≤ 0.05; ECCON vs. SHAM: 0.00 ± 0.00; P ≤ 0.025). PostECCON optimal tension decreased (Poi‐drop, 17.84 ± 4.22%) and was correlated to lesion density (R² = 0.596), but prestretch tension demonstrated the highest correlation with lesion density (R² = 0.994). In lesions, the darkly staining A‐band lost the normally organized thick filament alignment to differing degrees across the different lesion types. Ranking the five lesion types by a measure of lesion length deformation (hypercontracted to hyperstretched) at the light microscopy level, related to the severity of thick filament registry loss across the lesion types at the electron microscopic level. This ranking suggested that the five lesion types seen in semithin sections at the light level represented a lesion progression sequence and paralleled myosin immunostaining loss as the distorted A‐band filaments spread across the hyperlengthening lesion types. Lesion ultrastructure indicated damage involved calcium homeostasis loss (focal contraction lesions) and “thick‐filamentcentering” failure of titin (wide A‐band lesions) in the early stages of lesion development. Anat Rec 254:39–52, 1999. © 1999 Wiley‐Liss, Inc.     

Effects of Dietary Self-Monitoring,Physical Activity,Liraglutide 3.0 mg,and Placebo on Weight Loss in the SCALE IBT Trial

IntroductionIndividuals who enroll in intensive behavioral therapy (IBT) programs are asked to make several lifestyle changes simultaneously. However, few studies have examined the relative effects of adherence to different treatment components on weight loss.ObjectiveThis secondary analysis of the SCALE IBT trial assessed adherence to the medication regimen, dietary self-monitoring, and physical activity recommendations and their relative contributions to weight change in individuals with obesity who were provided with IBT combined with either liraglutide 3.0 mg or placebo.MethodsSCALE IBT was a double-blinded, multicenter, randomized controlled trial comparing 56-week weight losses in individuals with obesity who received liraglutide 3.0 mg (n = 142) or placebo (n = 140), as an adjunct to IBT. Adherence to dietary self-monitoring, physical activity, and medication usage (liraglutide or placebo) were measured during the 56-week treatment period. A regression model was used to estimate the relative contribution of adherence to each treatment component to weight loss at week 56.ResultsThe proportion of individuals who adhered to each intervention component decreased over time. Compared with non-adherence, complete adherence to dietary self-monitoring and physical activity recommendations were associated with estimated weight changes of −7.2% (95% CI −10.4 to −4.0; p < 0.0001) and −2.0% (95% CI −3.2 to −0.8; p = 0.0009), respectively. Complete adherence to liraglutide predicted an additional weight loss of −6.5% (95% CI −10.2 to −2.9; p = 0.0005) relative to individuals who did not adhere to the medication regimen, while adherence to placebo did not have a statistically significant effect on weight loss (p = 0.33).ConclusionsHigh adherence to dietary self-monitoring and use of liraglutide 3.0 mg was associated with clinically relevant weight loss with IBT and adjunctive pharmacotherapy. The effect of adherence to physical activity was significant but smaller.     

The postobstructive kidney. Observations on nephron function after the relief of 24 hr of ureteral ligation in the dog

After the relief of 24 hr of complete unilateral ureteral obstruction in the dog, the experimental kidney is characterized by a decrease in filtration rate and an increase in fractional and often absolute excretion of sodium before and after the administration of mannitol. In the hydrated state, the failure to conserve sodium is associated with increases in fractional free water clearance and fractional sodium supply to water-freeing sites signifying that the augmented sodium excretion is derived from a proximal source. In the hydropenic state there is decreased fractional free water reabsorption, and sometimes free water excretion, in the postobstructive kidney. An early plateau in free water reabsorption is associated with an increased fractional excretion of sodium. These findings are attributed to the early development of distal nephron impermeability to water as a result of enhanced distal tubular supply and transport of sodium. There is a decrease in maximal tubular reabsorptive capacity (Tm) of glucose in the post-obstructive kidney which is, however, less marked than the decrease in filtration rate. The fall in filtration rate is to some extent likely due to a dropping out of nephrons from the circulation while the remaining nephrons are hypoperfused. The magnitude of the sodium reabsorptive defect is markedly exaggerated as the concentration of nonreabsorbable solute (mannitol) in the glomerular perfusate is increased. It is concluded that the postobstructive increase in sodium excretion during mannitol administration is in part due to a limit in the capacity to reabsorb sodium against a concentration gradient in the proximal tubule.     

Specificity and mechanism of the histone methyltransferase Pr-Set7

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.