Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli

Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3' terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.     

Synthesis and evaluation of in vitro antiviral activity of novel phenoxy acetic acid derivatives

Several substituted phenoxy acetic acid derived pyrazolines were synthesized by the reaction between 2-{4-[3-(2,4-dihydroxyphenyl)-3-oxo-1-propenyl]-2-methoxyphenoxy} acetic acid and substituted acid hydrazides and were tested for their in vitro cytotoxicity and antiviral activity. None of the compounds showed any specific antiviral activity [50% antivirally effective concentration (EC(50)) > or = 5-fold lower than minimum cytotoxic concentration]. The most cytotoxic of the series was 2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-hydroxybenzoyl-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3(j)), with a minimum cytotoxic concentration of 0.16 microg/mL in human embryonic lung (HEL) cells.     

Bacterial programmed cell death and multicellular behavior in bacteria

Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin–antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.     

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-β and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated β-strand sheets forming a cross-β configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-β amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.