Bacteriophage-mediated decolonization of Klebsiella pneumoniae in a novel Galleria mellonella gut colonization model with Enterobacteriaceae.

Galleria mellonella larvae have emerged as an invertebrate model for investigating bacterial pathogenesis and potential therapies, addressing ethical concerns related to mammalian models. This model has the advantage of having a simple gut microbiome, which is suitable for gut colonization studies. Intestinal colonization by Enterobacteriaceae significantly contributes to the spread of antibiotic resistance. This study aimed to establish a novel Enterobacteriaceae gut colonization larval model and assess its suitability for evaluating distinct antimicrobial efficacies. Larvae were force-fed sequentially with bacterial doses of K. pneumoniae and E. coli at 0, 24, and 48 h, with survival monitoring at 24 h intervals. Bacterial counts were assessed after 48 h and 120 h of force-feeding. Successfully colonized larvae were subjected to one-time force feeding of a bacteriophage cocktail (107 PFU/larvae) or MIC-based meropenem and ciprofloxacin. The colonized bacterial load was quantified by CFU count. Three doses of 106 CFU/larvae resulted in stable gut colonization, independent of the K. pneumoniae or E. coli strain. Compared with the control, force-feeding of the bacteriophage reduced the colonization of the strain Kp 419614 by 5 log10 CFU/larvae, while antibiotic treatment led to a 3 log10 CFU/larval reduction. This novel G. mellonella model provides a valuable alternative for gut colonization studies, facilitating proof-of-concept investigations and potentially reducing or replacing follow-up experiments in vertebrate models.

In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes.

EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 µM) or curcumin (10 µg ml(-1)) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 µg ml(-1)). In response to TNF-α (25 ng ml(-1))-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 µm for TNF-α (23.5%) and 15 µm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

Comparison of the anticatabolic effects of leucine and Ca-ß-hydroxy-ß-methylbutyrate in experimental models of cancer cachexia.

OBJECTIVE: Loss of skeletal muscle is the most debilitating feature of cancer cachexia, and there are few treatments available. The aim of this study was to compare the anticatabolic efficacy of L-leucine and the leucine metabolite ß-hydroxy-ß-methylbutyrate (Ca-HMB) on muscle protein metabolism, both in vitro and in vivo. METHODS: Studies were conducted in mice bearing the cachexia-inducing murine adenocarcinoma 16 tumor, and in murine C2 C12 myotubes exposed to proteolysis-inducing factor, lipopolysaccharide, and angiotensin II. RESULTS: Both leucine and HMB were found to attenuate the increase in protein degradation and the decrease in protein synthesis in murine myotubes induced by proteolysis-inducing factor, lipopolysaccharide, and angiotensin II. However, HMB was more potent than leucine, because HMB at 50 µM produced essentially the same effect as leucine at 1 mM. Both leucine and HMB reduced the activity of the ubiquitin-proteasome pathway as measured by the functional (chymotrypsin-like) enzyme activity of the proteasome in muscle lysates, as well as Western blot quantitation of protein levels of the structural/enzymatic proteasome subunits (20 S and 19 S) and the ubiquitin ligases (MuRF1 and MAFbx). In vivo studies in mice bearing the murine adenocarcinoma 16 tumor showed a low dose of Ca-HMB (0.25 g/kg) to be 60% more effective than leucine (1 g/kg) in attenuating loss of body weight over a 4-d period. CONCLUSION: These results favor the clinical feasibility of using Ca-HMB over high doses of leucine for the treatment of cancer cachexia.

Attenuation of muscle wasting in murine C2C 12 myotubes by epigallocatechin-3-gallate.

BACKGROUND: Loss of muscle protein is a common feature of wasting diseases where currently treatment is limited. This study investigates the potential of epigallocatechin-3-gallate (EGCg), the most abundant catechin in green tea, to reverse the increased protein degradation and rescue the decreased protein synthesis which leads to muscle atrophy. METHODS: Studies were conducted in vitro using murine C2C12 myotubes. Increased protein degradation and reduced rates of protein synthesis were induced by serum starvation and tumour necrosis factor-α (TNF-α). RESULTS: EGCg effectively attenuated the depression of protein synthesis and increase in protein degradation in murine myotubes at concentrations as low as 10 µM. Serum starvation increased expression of the proteasome 20S and 19S subunits, as well as the proteasome 'chymotrypsin-like' enzyme activity, and these were all attenuated down to basal values in the presence of EGCg. Serum starvation did not increase expression of the ubiquitin ligases MuRF1 and MAFbx, but EGCg reduced their expression below basal levels, possibly due to an increased expression of phospho Akt (pAkt) and phospho forkhead box O3a (pFoxO3a). Attenuation of protein degradation by EGCg was increased in the presence of ZnSO4, suggesting an EGCg-Zn(2+) complex may be the active species. CONCLUSION: The ability of EGCg to attenuate depressed protein synthesis and increase protein degradation in the myotubule model system suggests that it may be effective in preserving skeletal muscle mass in catabolic conditions.