Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)₆ small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)₆ tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni²⁺-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)₆ tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5′-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)₆ tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.     

vanI: a novel d-Ala-d-Lac vancomycin resistance gene cluster found in Desulfitobacterium hafniense

The glycopeptide vancomycin was until recently considered a drug of last resort against Gram-positive bacteria. Increasing numbers of bacteria, however, are found to carry genes that confer resistance to this antibiotic. So far, 10 different vancomycin resistance clusters have been described. A chromosomal vancomycin resistance gene cluster was previously described for the anaerobic Desulfitobacterium hafniense Y51. We demonstrate that this gene cluster, characterized by its d-Ala-d-Lac ligase-encoding vanI gene, is present in all strains of D. hafniense, D. chlororespirans and some strains of Desulfosporosinus spp. This gene cluster was not found in vancomycin-sensitive Desulfitobacterium or Desulfosporosinus spp., and we show that this antibiotic resistance can be exploited as an intrinsic selection marker for Desulfitobacterium hafniense and D. chlororespirans. The gene cluster containing vanI is phylogenetically only distantly related with those described from soil and gut bacteria, but clusters instead with vancomycin resistance genes found within the phylum Actinobacteria that include several vancomycin-producing bacteria. It lacks a vanH homologue, encoding a D-lactate dehydrogenase, previously thought to always be present within vancomycin resistance gene clusters. The location of vanH outside the resistance gene cluster likely hinders horizontal gene transfer. Hence, the vancomycin resistance cluster in D. hafniense should be regarded a novel one that we here designated vanI after its unique d-Ala-d-Lac ligase.     

Influence of GLP-1 on Myocardial Glucose Metabolism in Healthy Men during Normo- or Hypoglycemia

Background and Aims

Glucagon-like peptide-1 (GLP-1) may provide beneficial cardiovascular effects, possibly due to enhanced myocardial energetic efficiency by increasing myocardial glucose uptake (MGU). We assessed the effects of GLP-1 on MGU in healthy subjects during normo- and hypoglycemia.

Materials and Methods

We included eighteen healthy men in two randomized, double-blinded, placebo-controlled cross-over studies. MGU was assessed with GLP-1 or saline infusion during pituitary-pancreatic normo- (plasma glucose (PG): 4.5 mM, n = 10) and hypoglycemic clamps (PG: 3.0 mM, n = 8) by positron emission tomography with ¹⁸fluoro-deoxy-glucose (¹⁸F-FDG) as tracer.

Results

In the normoglycemia study mean (± SD) age was 25±3 years, and BMI was 22.6±0.6 kg/m² and in the hypoglycemia study the mean age was 23±2 years with a mean body mass index of 23±2 kg/m². GLP-1 did not change MGU during normoglycemia (mean (+/− SD) 0.15+/−0.04 and 0.16+/−0.03 µmol/g/min, P = 0.46) or during hypoglycemia (0.16+/−0.03 and 0.13+/−0.04 µmol/g/min, P = 0.14). However, the effect of GLP-1 on MGU was negatively correlated to baseline MGU both during normo- and hypoglycemia, (P = 0.006, r² = 0.64 and P = 0.018, r² = 0.64, respectively) and changes in MGU correlated positively with the level of insulin resistance (HOMA 2IR) during hypoglycemia, P = 0.04, r² = 0.54. GLP-1 mediated an increase in circulating glucagon levels at PG levels below 3.5 mM and increased glucose infusion rates during the hypoglycemia study. No differences in other circulating hormones or metabolites were found.

Conclusions

While GLP-1 does not affect overall MGU, GLP-1 induces changes in MGU dependent on baseline MGU such that GLP-1 increases MGU in subjects with low baseline MGU and decreases MGU in subjects with high baseline MGU. GLP-1 preserves MGU during hypoglycemia in insulin resistant subjects.ClinicalTrials.gov registration numbers: {"type":"clinical-trial","attrs":{"text":"NCT00418288","term_id":"NCT00418288"}}NCT00418288: (hypoglycemia) and {"type":"clinical-trial","attrs":{"text":"NCT00256256","term_id":"NCT00256256"}}NCT00256256: (normoglycemia).     

Velocity and Directionality of the Electrohysterographic Signal Propagation

Objective

The initiation of treatment for women with threatening preterm labor requires effective distinction between true and false labor. The electrohysterogram (EHG) has shown great promise in estimating and classifying uterine activity. However, key issues remain unresolved and no clinically usable method has yet been presented using EHG. Recent studies have focused on the propagation velocity of the EHG signals as a potential discriminator between true and false labor. These studies have estimated the propagation velocity of individual spikes of the EHG signals. We therefore focus on estimating the propagation velocity of the entire EHG burst recorded during a contraction in two dimensions.

Study Design

EHG measurements were performed on six women in active labor at term, and a total of 35 contractions were used for the estimation of propagation velocity. The measurements were performed using a 16-channel two-dimensional electrode grid. The estimates were calculated with a maximum-likelihood approach.

Results

The estimated average propagation velocity was 2.18 (±0.68) cm/s. No single preferred direction of propagation was found.

Conclusion

The propagation velocities estimated in this study are similar to those reported in other studies but with a smaller intra- and inter-patient variation. Thus a potential tool has been established for further studies on true and false labor contractions.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

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Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.