Effect of (in)organic additives on microbially induced calcium carbonate precipitation

\ua9 The Author(s) 2023. Aim: This study aimed to understand the morphological effects of (in)organic additives on microbially induced calcium carbonate precipitation (MICP). Methods and results: MICP was monitored in real time in the presence of (in)organic additives: bovine serum albumin (BSA), biofilm surface layer protein A (BslA), magnesium chloride (MgCl2), and poly-L-lysine. This monitoring was carried out using confocal microscopy to observe the formation of CaCO3 from the point of nucleation, in comparison to conditions without additives. Complementary methodologies, namely scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction, were employed to assess the visual morphology, elemental composition, and crystalline structures of CaCO3, respectively, following the crystals’ formation. The results demonstrated that in the presence of additives, more CaCO3 crystals were produced at 100 min compared to the reaction without additives. The inclusion of BslA resulted in larger crystals than reactions containing other additives, including MgCl2. BSA induced a significant number of crystals from the early stages of the reaction (20 min) but did not have a substantial impact on crystal size compared to conditions without additives. All additives led to a higher content of calcite compared to vaterite after a 24-h reaction, with the exception of MgCl2, which produced a substantial quantity of magnesium calcite. Conclusions: The work demonstrates the effect of several (in)organic additives on MICP and sets the stage for further research to understand additive effects on MICP to achieve controlled CaCO3 precipitation

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ 1, γ 2 or γ 3 isoform. When assayed at a physiological ATP concentration (5 mM), γ 1- and γ 2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ 3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ 1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ 2 and γ 3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation; with γ 2 complexes, ADP had similar potency to AMP, but with γ 1 and γ 3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles

Efficient detection of RNA–protein interactions using tethered RNAs

The diverse localization of transcripts in cells suggests that there are many specific RNA–protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by ∼10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3′-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3′-UTR. Our tRSA technique should find wide application in mapping the RNA–protein interactome

An Unbiased Evaluation of CK2 Inhibitors by Chemoproteomics: Characterization of Inhibitor Effects on CK2 and Identification of Novel Inhibitor Targets

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-

Predicting the response to sorafenib in hepatocellular carcinoma: where is the evidence for phosphorylated extracellular signaling-regulated kinase (pERK)?

The approval of sorafenib and active development of many other molecularly targeted agents in hepatocellular carcinoma (HCC) have presented a challenge to understand the mechanism of action of sorafenib and identify predictive biomarkers to select patients more likely to benefit from sorafenib. The preclinical study by Zhang and celleagues published this month in BMC Medicine provides preliminary evidence that baseline phosphorylated extracellular signaling-regulated kinase (pERK) may be a relevant marker to reflect the level of constitutive activation of the RAF/mitogen-activated protein kinase kinase (MEK)/ERK signaling pathway and has the potential value in predicting response to sorafenib. The clinical data from the initial single arm phase II study and preliminary report from the randomized phase III study also suggest the correlation of baseline archived tumor pERK levels and time to tumor progression in HCC patients. Whether baseline pERK will prove to be a useful predictive biomarker of response and clinical benefits for sorafenib in HCC will need to be validated in future large prospective studies