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Article ; Online: GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model.

Dufu, Kobina / Alt, Carsten / Strutt, Steven / Partridge, James / Tang, Tzechiang / Siu, Vincent / Liao-Zou, Hilary / Rademacher, Peter / Williams, Alexander T / Muller, Cynthia R / Geng, Xin / Pochron, Mira Patel / Dang, Annie Nguyen / Cabrales, Pedro / Li, Zhe / Oksenberg, Donna / Cathers, Brian E

British journal of haematology

2023 Volume 202, Issue 1, Page(s) 173–183

Abstract: The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream ... ...

Abstract	The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.
MeSH term(s)	Humans ; Animals ; Mice ; Hemolysis ; Disease Models, Animal ; Oxygen ; Anemia, Sickle Cell/drug therapy ; Erythrocytes ; Hemoglobins ; Hemoglobin, Sickle
Chemical Substances	Oxygen (S88TT14065) ; Hemoglobins ; Hemoglobin, Sickle
Language	English
Publishing date	2023-03-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80077-6
ISSN	1365-2141 ; 0007-1048
ISSN (online)	1365-2141
ISSN	0007-1048
DOI	10.1111/bjh.18771
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Article ; Online: RNA-dependent RNA targeting by CRISPR-Cas9.

Strutt, Steven C / Torrez, Rachel M / Kaya, Emine / Negrete, Oscar A / Doudna, Jennifer A

eLife

2018 Volume 7

Abstract: Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and ... ...

Abstract	Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.
MeSH term(s)	CRISPR-Associated Protein 9/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Gene Targeting/methods ; Hydrolysis ; RNA/genetics ; RNA/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Recombination, Genetic
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; RNA (63231-63-0) ; CRISPR-Associated Protein 9 (EC 3.1.-)
Language	English
Publishing date	2018-01-05
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.32724
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Article ; Online: Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor.

Bagchi, Samik / Lamendella, Regina / Strutt, Steven / Van Loosdrecht, Mark C M / Saikaly, Pascal E

Scientific reports

2016 Volume 6, Page(s) 28327

Abstract: Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S ...

Abstract	Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S rRNA gene sequencing to study the changes in community composition, metabolic gene content and gene expression in a granular anammox reactor with the objective of understanding the molecular mechanism of anammox growth and multiplication that led to formation of large granules. Size distribution analysis revealed the spatial distribution of granules in which large granules having higher abundance of anammox bacteria (genus Brocadia) dominated the bottom biomass. Metatranscriptomics analysis detected all the essential transcripts for anammox metabolism. During the later stage of reactor operation, higher expression of ammonia and nitrite transport proteins and key metabolic enzymes mainly in the bottom large granules facilitated anammox bacteria activity. The high activity resulted in higher growth and multiplication of anammox bacteria and expanded the size of the granules. This conceptual model for large granule formation proposed here may assist in the future design of anammox processes for mainstream wastewater treatment.
Language	English
Publishing date	2016-06-20
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep28327
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Article ; Online: New CRISPR-Cas systems from uncultivated microbes.

Burstein, David / Harrington, Lucas B / Strutt, Steven C / Probst, Alexander J / Anantharaman, Karthik / Thomas, Brian C / Doudna, Jennifer A / Banfield, Jillian F

Nature

2016 Volume 542, Issue 7640, Page(s) 237–241

Abstract: CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein ... ...

Abstract	CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.
MeSH term(s)	Amino Acid Sequence ; Archaea/genetics ; Bacteria/genetics ; Base Sequence ; Biotechnology/trends ; CRISPR-Associated Proteins/genetics ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/physiology ; DNA/genetics ; DNA/metabolism ; Escherichia coli/genetics ; Gene Expression Profiling ; Genome/genetics ; Metagenomics ; RNA, Bacterial/genetics ; RNA, Bacterial/metabolism ; Reproducibility of Results
Chemical Substances	CRISPR-Associated Proteins ; RNA, Bacterial ; DNA (9007-49-2)
Language	English
Publishing date	2016-12-22
Publishing country	England
Document type	Journal Article
ZDB-ID	120714-3
ISSN	1476-4687 ; 0028-0836
ISSN (online)	1476-4687
ISSN	0028-0836
DOI	10.1038/nature21059
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Article ; Online: A bacterial Argonaute with noncanonical guide RNA specificity.

Kaya, Emine / Doxzen, Kevin W / Knoll, Kilian R / Wilson, Ross C / Strutt, Steven C / Kranzusch, Philip J / Doudna, Jennifer A

Proceedings of the National Academy of Sciences of the United States of America

2016 Volume 113, Issue 15, Page(s) 4057–4062

Abstract: Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and ... ...

Abstract	Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5'-hydroxylated guide RNAs rather than the 5'-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5' phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5'-hydroxylated guide.
MeSH term(s)	Argonaute Proteins/chemistry ; Argonaute Proteins/genetics ; Argonaute Proteins/metabolism ; Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Bacterial ; Models, Molecular ; RNA, Guide, CRISPR-Cas Systems/metabolism
Chemical Substances	Argonaute Proteins ; Bacterial Proteins ; RNA, Guide, CRISPR-Cas Systems
Language	English
Publishing date	2016-03-30
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1524385113
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Article ; Online: Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences.

Damerla, Rama Rao / Knickelbein, Kelly E / Strutt, Steven / Liu, Fu-Jun / Wang, Hong / Opresko, Patricia L

Cell cycle (Georgetown, Tex.)

2012 Volume 11, Issue 16, Page(s) 3036–3044

Abstract: Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular ... ...

Abstract	Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]6 sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.
MeSH term(s)	Base Sequence ; Cell Line, Tumor ; DNA Replication ; Exodeoxyribonucleases/genetics ; Exodeoxyribonucleases/metabolism ; G-Quadruplexes ; Gene Rearrangement ; Genes, Reporter ; Genes, Suppressor ; Genetic Vectors/genetics ; Genetic Vectors/metabolism ; Humans ; Molecular Sequence Data ; Mutagenesis ; RNA, Transfer/genetics ; RNA, Transfer/metabolism ; RecQ Helicases/genetics ; RecQ Helicases/metabolism ; Repetitive Sequences, Nucleic Acid ; Sequence Deletion ; Telomere/genetics ; Telomere/metabolism ; Werner Syndrome/enzymology ; Werner Syndrome/genetics ; Werner Syndrome Helicase
Chemical Substances	supF tRNA ; RNA, Transfer (9014-25-9) ; Exodeoxyribonucleases (EC 3.1.-) ; RecQ Helicases (EC 3.6.4.12) ; WRN protein, human (EC 3.6.4.12) ; Werner Syndrome Helicase (EC 3.6.4.12)
Language	English
Publishing date	2012-08-08
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2146183-1
ISSN	1551-4005 ; 1538-4101 ; 1554-8627
ISSN (online)	1551-4005
ISSN	1538-4101 ; 1554-8627
DOI	10.4161/cc.21399
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Article ; Online: Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy.

Ulrich, Nikea / Rosenberger, Abigail / Brislawn, Colin / Wright, Justin / Kessler, Collin / Toole, David / Solomon, Caroline / Strutt, Steven / McClure, Erin / Lamendella, Regina

Applied and environmental microbiology

2016 Volume 82, Issue 12, Page(s) 3525–3536

Abstract: Unlabelled: Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a ... ...

Abstract	Unlabelled: Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in which Betaproteobacteria and Gammaproteobacteria decreased in 16S rRNA gene relative abundance, while the relative abundance of members of the Firmicutes increased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains of Legionella, Campylobacter, Arcobacter, and Helicobacter, as well as bacteria of fecal origin (e.g., Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event. Importance: In order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study investigated the bacterial communities during and after Superstorm Sandy to provide fine time point resolution of dynamic changes in bacterial composition. This study adds to the current literature by revealing the variation in bacterial community structure during the course of a storm. This study employed high-throughput DNA sequencing, which generated a deep analysis of inter- and intracommunity responses during a significant storm event. This study has highlighted the utility of applying high-throughput sequencing for water quality monitoring purposes, as this approach enabled a more comprehensive investigation of the bacterial community structure. Altogether, these data suggest a drastic restructuring of the stream bacterial community during a storm event and highlight the potential of high-throughput sequencing approaches for assessing the microbiological quality of our environment.
MeSH term(s)	Bacteria/classification ; Bacteria/genetics ; Biota ; Cluster Analysis ; Computational Biology ; Cyclonic Storms ; DNA, Bacterial/chemistry ; DNA, Bacterial/genetics ; DNA, Ribosomal/chemistry ; DNA, Ribosomal/genetics ; High-Throughput Nucleotide Sequencing ; Longitudinal Studies ; Pennsylvania ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; Rivers/microbiology ; Sequence Analysis, DNA
Chemical Substances	DNA, Bacterial ; DNA, Ribosomal ; RNA, Ribosomal, 16S
Language	English
Publishing date	2016-06-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/AEM.00520-16
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Article: Assessment of the Deepwater Horizon oil spill impact on Gulf coast microbial communities.

Lamendella, Regina / Strutt, Steven / Borglin, Sharon / Chakraborty, Romy / Tas, Neslihan / Mason, Olivia U / Hultman, Jenni / Prestat, Emmanuel / Hazen, Terry C / Jansson, Janet K

Frontiers in microbiology

2014 Volume 5, Page(s) 130

Abstract: One of the major environmental concerns of the Deepwater Horizon oil spill in the Gulf of Mexico was the ecological impact of the oil that reached shorelines of the Gulf Coast. Here we investigated the impact of the oil on the microbial composition in ... ...

Abstract	One of the major environmental concerns of the Deepwater Horizon oil spill in the Gulf of Mexico was the ecological impact of the oil that reached shorelines of the Gulf Coast. Here we investigated the impact of the oil on the microbial composition in beach samples collected in June 2010 along a heavily impacted shoreline near Grand Isle, Louisiana. Successional changes in the microbial community structure due to the oil contamination were determined by deep sequencing of 16S rRNA genes. Metatranscriptomics was used to determine expression of functional genes involved in hydrocarbon degradation processes. In addition, potential hydrocarbon-degrading Bacteria were obtained in culture. The 16S data revealed that highly contaminated samples had higher abundances of Alpha- and Gammaproteobacteria sequences. Successional changes in these classes were observed over time, during which the oil was partially degraded. The metatranscriptome data revealed that PAH, n-alkane, and toluene degradation genes were expressed in the contaminated samples, with high homology to genes from Alteromonadales, Rhodobacterales, and Pseudomonales. Notably, Marinobacter (Gammaproteobacteria) had the highest representation of expressed genes in the samples. A Marinobacter isolated from this beach was shown to have potential for transformation of hydrocarbons in incubation experiments with oil obtained from the Mississippi Canyon Block 252 (MC252) well; collected during the Deepwater Horizon spill. The combined data revealed a response of the beach microbial community to oil contaminants, including prevalence of Bacteria endowed with the functional capacity to degrade oil.
Language	English
Publishing date	2014-04-03
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2014.00130
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