Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors

Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale ( 5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency - one verses four impellers. Results: In both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen ( DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space. Conclusion: Thus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.open1123sciescopu

Cationic Amino Acids Specific Biomimetic Silicification in Ionic Liquid: A Quest to Understand the Formation of 3-D Structures in Diatoms

The intricate, hierarchical, highly reproducible, and exquisite biosilica structures formed by diatoms have generated great interest to understand biosilicification processes in nature. This curiosity is driven by the quest of researchers to understand nature's complexity, which might enable reproducing these elegant natural diatomaceous structures in our laboratories via biomimetics, which is currently beyond the capabilities of material scientists. To this end, significant understanding of the biomolecules involved in biosilicification has been gained, wherein cationic peptides and proteins are found to play a key role in the formation of these exquisite structures. Although biochemical factors responsible for silica formation in diatoms have been studied for decades, the challenge to mimic biosilica structures similar to those synthesized by diatoms in their natural habitats has not hitherto been successful. This has led to an increasingly interesting debate that physico-chemical environment surrounding diatoms might play an additional critical role towards the control of diatom morphologies. The current study demonstrates this proof of concept by using cationic amino acids as catalyst/template/scaffold towards attaining diatom-like silica morphologies under biomimetic conditions in ionic liquids

A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial–mesenchymal transition

Expression of the essential EMT inducer Snail1 is inhibited by miR-34 through a p53-dependent regulatory pathway

Truly form-factor–free industrially scalable system integration for electronic textile architectures with multifunctional fiber devices

Funding Information: This work was supported by the European Commission (H2020, 1D-NEON, grant agreement ID: 685758). J.M.K. and L.G.O. acknowledge the support from the U.K. Research and Innovation (EPSRC, EP/P027628/1). We thank Y. Bernstein and J. Faulkner for helping with grammar check. Funding Information: Acknowledgments Funding:ThisworkwassupportedbytheEuropeanCommission(H2020,1D-NEON,grant agreementID:685758).J.M.K.andL.G.O.acknowledgethesupportfromtheU.K.Researchand Innovation(EPSRC,EP/P027628/1).W ethankY .BernsteinandJ.Faulknerforhelpingwith grammarcheck.Authorcontributions:S.L.andJ.M.K.conceivedtheproject.S.L.,L.G.O.,P .B., R.Martins,andJ.M.K.supervisedtheproject.S.L.andH.L.developedF-PD.S.L.,Y .-W .L., G.-H.A., D.-W .S., J.I.S.,andS.C.developedF-SC.C.L.F ., A.S.,R.I.,P .B., andR.Martinsdevelopedfiber transistor.S.L.,H.L.,andS.C.developedF-LED.ThefiberdeviceswereevaluatedbyS.L.,H.W .C., D.-W .S., H.L.,S.J.,S.D.H.,S.Y .B., S.Z.,W .H.-C., Y .-H.S., X.-B.F ., T .H.L., J.-W .J., andY .K. The developmentofweavingprocesswasconductedbyS.L.,H.W .C., F .M.M., P .J., andV .G.C. Thelaser interconnectionwasdevelopedbyS.L.,H.W .C., K.U.,M.E.,andM.S.Thetextiledemonstrations werecharacterizedbyS.L.,H.W .C., D.-W .S., J.Y ., S.S.,U.E.,S.N.,A.C.,A.M.,R.Momentè,J.G.,N.D., S.M.,C.-H.K.,M.L.,A.N.,D.J.,M.C.,andY .C. ThismanuscriptwaswrittenbyS.L.andJ.M.K.and reviewed by H.W .C., D.-W .S., M.C.,L.G.O., P .B., E.F ., and G.A.J.A. All authors discussed the results andcommentedonthemanuscript.Competinginterests:Theauthorsdeclarethattheyhave nocompetinginterests.Dataandmaterialsavailability:Alldataneededtoevaluatethe conclusionsinthepaperarepresentinthepaperand/ortheSupplementaryMaterials. Publisher Copyright: Copyright © 2023 The Authors, some rights reserved.An integrated textile electronic system is reported here, enabling a truly free form factor system via textile manufacturing integration of fiber-based electronic components. Intelligent and smart systems require freedom of form factor, unrestricted design, and unlimited scale. Initial attempts to develop conductive fibers and textile electronics failed to achieve reliable integration and performance required for industrial-scale manufacturing of technical textiles by standard weaving technologies. Here, we present a textile electronic system with functional one-dimensional devices, including fiber photodetectors (as an input device), fiber supercapacitors (as an energy storage device), fiber field-effect transistors (as an electronic driving device), and fiber quantum dot light-emitting diodes (as an output device). As a proof of concept applicable to smart homes, a textile electronic system composed of multiple functional fiber components is demonstrated, enabling luminance modulation and letter indication depending on sunlight intensity.publishersversionpublishe

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Crystal structures of penicillin-binding protein D2 from listeria monocytogenes and structural basis for antibiotic specificity

-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of -lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the -lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of -lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important -lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of -lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against -lactam-resistant PBPs. Copyright © 2018 American Society for Microbiology. All Rights Reserve

Recombinant Yeasts Using an SUC2 Promoter Production and Secretion Patterns of Cloned Glucoamylase in Plasmid-Harboring and Chromosome-Integrated Recombinant Yeasts Employing an SUC2 Promoter

Abstract To understand the differences in production and secretion patterns between plasmid-harboring and chromosome-integrated recombinant yeasts, the two recombinant Saccharomyces cerevisiae yeasts, containing the structural glucoamylase STA gene and the SUC2 promoter, were investigated. Both systems were regulated by glucose concentration in the culture broth. First, the glucoamylase activity per gene copy number of the chromosome-integrated recombinant yeast was 2.8-to 5.6-fold higher than that of the plasmid-harboring recombinant yeast. Overburdened owing to high copy number, the plasmid-harboring recombinant yeast gave lower glucoamylase activity per gene copy number. Second, the efficiency of signal sequence was compared; the secretion efficiency of glucoamylase in the plasmid-harboring recombinant yeast was higher than that in the chromosome-integrated recombinant yeast at 96 h of cultivation (74 vs 65%). We postulated that the higher level of secretion efficiency of the plasmid-harboring recombinant yeast resulted because the production level did not reach the capacity of the secretory apparatus of the host yeast. However, the specific 82 Cha et al. Applied Biochemistry and Biotechnology Vol. 87, 2000 secretion rate was much higher in the chromosome-integrated recombinant yeast even though the final secretion efficiency was lower. The lower secretion rate in the plasmid-harboring recombinant yeast could be explained by an adverse effect caused by higher production rate. Finally, the optimal glucose concentration for glucoamylase production in the chromosomeintegrated recombinant yeast culture was lower than that in the plasmidharboring recombinant yeast culture owing to gene dosage effect