Systemic Obstacles to Addressing Research Misconduct in Higher Education: A Case Study.

Several widely publicized incidents of academic research misconduct, combined with the politicization of the role of science in public health and policy discourse (e.g., COVID, immunizations) threaten to undermine faith in the integrity of empirical research. Researchers often maintain that peer-review and study replication allow the field to self-police and self-correct; however, stark disparities between official reports of academic research misconduct and self-reports of academic researchers, specifically with regard to data fabrication, belie this argument. Further, systemic imperatives in academic settings often incentivize institutional responses that focus on minimizing reputational harm rather than the impact of fabricated data on the integrity of extant and future research.

Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges.

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Development of a cyanobacterial heterologous polyketide production platform.

The development of new heterologous hosts for polyketides production represents an excellent opportunity to expand the genomic, physiological, and biochemical backgrounds that better fit the sustainable production of these valuable molecules. Cyanobacteria are particularly attractive for the production of natural compounds because they have minimal nutritional demands and several strains have well established genetic tools. Using the model strain Synechococcus elongatus, a generic platform was developed for the heterologous production of polyketide synthase (PKS)-derived compounds. The versatility of this system is based on interchangeable modules harboring promiscuous enzymes for PKS activation and the production of PKS extender units, as well as inducible circuits for a regulated expression of the PKS biosynthetic gene cluster. To assess the capability of this platform, we expressed the mycobacterial PKS-based mycocerosic biosynthetic pathway to produce multimethyl-branched esters (MBE). This work is a foundational step forward for the production of high value polyketides in a photosynthetic microorganism.

Enhanced biomass and oxidative stress tolerance of Synechococcus elongatus PCC 7942 overexpressing the DHAR gene from Brassica juncea.

OBJECTIVES: To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H2O2, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR). RESULTS: Under H2O2 stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H2O2 level decreased. In addition, under H2O2 stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains. CONCLUSION: By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H2O2 stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942.

To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the Synechococcus elongatus strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin-antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection. The pANS-based shuttle vectors were shown to be able to carry a large 20 kb DNA fragment containing a gene cluster for biosynthesis of the omega-3 fatty acid eicosapentaenoic acid. Based on quantitative PCR analysis, there are about 10 copies of pANS and 3 copies of the large native plasmid pANL per chromosome in S. elongatus. Fluorescence levels of GFP reporter genes in a pANS-based vector were about 2.5-fold higher than when in pANL or integrated into the chromosome. In addition to its native host, pANS-based shuttle vectors were also found to replicate stably in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. There were about 27 copies of a pANS-based shuttle vector, 9 copies of a pDU1-based shuttle vector and 3 copies of an RSF1010-based shuttle vector per genome when these three plasmids co-existed in Anabaena cells. The endogenous pANS from our S. elongatus laboratory strain was cloned in Escherichia coli, re-sequenced and re-annotated to update previously published sequencing data.

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria.

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

Regulation of gene expression in diverse cyanobacterial species by using theophylline-responsive riboswitches.

Cyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942, Leptolyngbya sp. strain BL0902, Anabaena sp. strain PCC 7120, and Synechocystis sp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-ß-d-thiogalactopyranoside induction of a lacI(q)-Ptrc promoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaena sp. strain PCC7120.

BACKGROUND: Anabaena (also Nostoc) sp. strain PCC7120, hereafter Anabaena, is a cyanobacterium that fixes atmospheric N2 in specialized cells called heterocysts. Heterocyst differentiation is regulated by a homodimeric transcription factor, HetR. HetR is expressed at a basal level in all cells but its expression increases in differentiating cells early after nitrogen deprivation. HetR is required for heterocyst development, and therefore nitrogen fixation and diazotrophic growth. Overexpression of HetR leads to multiple contiguous heterocysts (Mch phenotype). HetR binds in vitro to DNA fragments upstream of several genes upregulated in heterocysts, including hetZ, hetP, hepA, patS, pknE, and hetR itself. HetR binds an inverted repeat sequence upstream of a few of these genes; however, HetR binds to promoters that do not contain this sequence, such as the promoter regions for patS and pknE. RESULTS: We employed chromatin pull-down and deep sequencing (ChIP-seq) to globally identify HetR DNA targets in vivo at six hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Promoter-gfp reporter fusions were used to determine the spatiotemporal expression of four potential HetR-target genes. The promoter region for asr1469 was expressed transiently in differentiating heterocysts, alr3758 was upregulated in heterocysts, asl2028 was expressed in vegetative cells, and alr2242 was derepressed in vegetative cells of a hetR mutant strain. CONCLUSIONS: In addition to identifying known HetR target genes hetR and hetP, the ChIP-seq data were used to identify new potential HetR targets and to define a consensus HetR-binding site. The in vivo ChIP-seq analysis of HetR's regulon suggests a possible role for HetR in vegetative cells in addition to its role in heterocyst development. The potential HetR target genes identified in this study provide new subjects for future work on the role of HetR in gene regulation.

Local edge statistics provide information regarding occlusion and nonocclusion edges in natural scenes.

Edges in natural scenes can result from a number of different causes. In this study, we investigated the statistical differences between edges arising from occlusions and nonocclusions (reflectance differences, surface change, and cast shadows). In the first experiment, edges in natural scenes were identified using the Canny edge detection algorithm. Observers then classified these edges as either an occlusion edge (one region of an image occluding another) or a nonocclusion edge. The nonocclusion edges were further subclassified as due to a reflectance difference, a surface change, or a cast shadow. We found that edges were equally likely to be classified as occlusion or nonocclusion edges. Of the nonocclusion edges, approximately 33% were classified as reflectance changes, 9% as cast shadows, and 58% as surface changes. We also analyzed local statistical properties like contrast, average edge profile, and slope of the edges. We found significant differences between the contrast values for each category. Based on the local contrast statistics, we developed a maximum likelihood classifier to label occlusion and nonocclusion edges. An 80%-20% cross validation demonstrated that the human classification could be predicted with 83% accuracy. Overall, our results suggest that for many edges in natural scenes, there exists local statistical information regarding the cause of the edge. We believe that this information can potentially be used by the early visual system to begin the process of segregating objects from their backgrounds.

Glycogen metabolism is required for optimal cyanobacterial growth in the rapid light-dark cycle of low-Earth orbit.

Some designs for bioregenerative life support systems to enable human space missions incorporate cyanobacteria for removal of carbon dioxide, generation of oxygen, and treatment of wastewater, as well as providing a source of nutrition. In this study, we examined the effects of the short light-dark (LD) cycle of low-Earth orbit on algal and cyanobacterial growth, approximating conditions on the International Space Station, which orbits Earth roughly every 90 min. We found that growth of green algae was similar in both normal 12 h light:12 h dark (12 h:12 h LD) and 45':45' LD cycles. Three diverse strains of cyanobacteria were not only capable of growth in short 45':45' LD cycles, but actually grew better than in 12 h:12 h LD cycles. We showed that 45':45' LD cycles do not affect the endogenous 24 h circadian rhythms of Synechococcus elongatus. Using a dense library of randomly barcoded transposon mutants, we identified genes whose loss is detrimental for the growth of S. elongatus under 45':45' LD cycles. These include several genes involved in glycogen metabolism and the oxidative pentose phosphate pathway. Notably, 45':45' LD cycles did not affect the fitness of strains that carry mutations in the biological circadian oscillator or the clock input and output regulatory pathways. Overall, this study shows that cultures of cyanobacteria could be grown under natural sunlight of low-Earth orbit and highlights the utility of a functional genomic study in a model organism to better understand key biological processes in conditions that are relevant to space travel.

Synechococcus elongatus Argonaute reduces natural transformation efficiency and provides immunity against exogenous plasmids.

IMPORTANCE: S. elongatus is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used for genetic engineering to produce renewable biochemicals. Our findings reveal an SeAgo-based defense mechanism in S. elongatus against the horizontal transfer of genetic material. We demonstrate that deletion of the ago gene facilitates genetic studies and genetic engineering of S. elongatus.

Phenotypically complex living materials containing engineered cyanobacteria.

The field of engineered living materials lies at the intersection of materials science and synthetic biology with the aim of developing materials that can sense and respond to the environment. In this study, we use 3D printing to fabricate a cyanobacterial biocomposite material capable of producing multiple functional outputs in response to an external chemical stimulus and demonstrate the advantages of utilizing additive manufacturing techniques in controlling the shape of the fabricated photosynthetic material. As an initial proof-of-concept, a synthetic riboswitch is used to regulate the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942 within a hydrogel matrix. Subsequently, a strain of S. elongatus is engineered to produce an oxidative laccase enzyme; when printed within a hydrogel matrix the responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a tool in environmental bioremediation. Finally, cells are engineered for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in volumetric 3D-printed designs, we demonstrate programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

Altered Carbon Partitioning Enhances CO2 to Terpene Conversion in Cyanobacteria.

Photosynthetic terpene production represents one of the most carbon and energy-efficient routes for converting CO2 into hydrocarbon. In photosynthetic organisms, metabolic engineering has led to limited success in enhancing terpene productivity, partially due to the low carbon partitioning. In this study, we employed systems biology analysis to reveal the strong competition for carbon substrates between primary metabolism (e.g., sucrose, glycogen, and protein synthesis) and terpene biosynthesis in Synechococcus elongatus PCC 7942. We then engineered key "source" and "sink" enzymes. The "source" limitation was overcome by knocking out either sucrose or glycogen biosynthesis to significantly enhance limonene production via altered carbon partitioning. Moreover, a fusion enzyme complex with geranyl diphosphate synthase (GPPS) and limonene synthase (LS) was designed to further improve pathway kinetics and substrate channeling. The synergy between "source" and "sink" achieved a limonene titer of 21.0 mg/L. Overall, the study demonstrates that balancing carbon flux between primary and secondary metabolism can be an effective approach to enhance terpene bioproduction in cyanobacteria. The design of "source" and "sink" synergy has significant potential in improving natural product yield in photosynthetic species.

Heterologous Expression in Anabaena of the Columbamide Pathway from the Cyanobacterium Moorena bouillonii and Production of New Analogs.

Columbamides are chlorinated acyl amide natural products, several of which exhibit cannabinomimetic activity. These compounds were originally discovered from a culture of the filamentous marine cyanobacterium Moorena bouillonii PNG5-198 collected from the coastal waters of Papua New Guinea. The columbamide biosynthetic gene cluster (BGC) had been identified using bioinformatics, but not confirmed by experimental evidence. Here, we report the heterologous expression in Anabaena (Nostoc) PCC 7120 of the 28.5 kb BGC that encodes for columbamide biosynthesis. The production of columbamides in Anabaena is investigated under several different culture conditions, and several new columbamide analogs are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition to previously characterized columbamides A, B, and C, new columbamides I-M are produced in these experiments, and the structure of the most abundant monochlorinated analog, columbamide K (11), is fully characterized. The other new columbamide analogs are produced in only small quantities, and structures are proposed based on high-resolution-MS, MS/MS, and 1H NMR data. Overexpression of the pathway's predicted halogenases resulted in increased productions of di- and trichlorinated compounds. The most significant change in production of columbamides in Anabaena is correlated with the concentration of NaCl in the medium.

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120.

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.

The sigE gene is required for normal expression of heterocyst-specific genes in Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 produces specialized cells for nitrogen fixation called heterocysts. Previous work showed that the group 2 sigma factor sigE (alr4249; previously called sigF) is upregulated in differentiating heterocysts 16 h after nitrogen step-down. We now show that the sigE gene is required for normal heterocyst development and normal expression levels of several heterocyst-specific genes. Mobility shift assays showed that the transcription factor NtcA binds to sites in the upstream region of sigE and that this binding is enhanced by 2-oxoglutarate (2-OG). Deletions of the region containing the NtcA binding sites in P(sigE)-gfp reporter plasmids showed that the sites contribute to normal developmental regulation but are not essential for upregulation in heterocysts. Northern RNA blot analysis of nifH mRNA revealed delayed and reduced transcript levels during heterocyst differentiation in a sigE mutant background. Quantitative reverse transcription-PCR (qRT-PCR) analyses of the sigE mutant showed lower levels of transcripts for nifH, fdxH, and hglE2 but normal levels for hupL. We developed a P(nifHD)-gfp reporter construct that showed strong heterocyst-specific expression. Time-lapse microscopy of the P(nifHD)-gfp reporter in a sigE mutant background showed delayed development and undetectable green fluorescent protein (GFP) fluorescence. Overexpression of sigE caused accelerated heterocyst development, an increased heterocyst frequency, and premature expression of GFP fluorescence from the P(nifHD)-gfp reporter.

Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation.

BACKGROUND: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth. RESULTS: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism. CONCLUSIONS: Directional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation. We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development. These data provide detailed information on the Anabaena transcriptome as filaments undergo heterocyst development and begin nitrogen fixation.

Anabaena sp. strain PCC 7120 conR contains a LytR-CpsA-Psr domain, is developmentally regulated, and is essential for diazotrophic growth and heterocyst morphogenesis.

The conR (all0187) gene of the filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 is predicted to be part of a family of proteins that contain the LytR-CpsA-Psr domain associated with septum formation and cell wall maintenance. The conR gene was originally misannotated as a transcription regulator. Northern RNA blot analysis showed that conR expression was upregulated 8 h after nitrogen step-down. Fluorescence microscopy of a P(conR)-gfp reporter strain revealed increased GFP fluorescence in proheterocysts and heterocysts beginning 9 h after nitrogen step-down. Insertional inactivation of conR caused a septum-formation defect of vegetative cells grown in nitrate-containing medium. In nitrate-free medium, mutant filaments formed abnormally long heterocysts and were defective for diazotrophic growth. Septum formation between heterocysts and adjacent vegetative cells was abnormal, often with one or both poles of the heterocysts appearing partially open. In a conR mutant, expression of nifH was delayed after nitrogen step-down and nitrogenase activity was approximately 70 % of wild-type activity, indicating that heterocysts of the conR mutant strain are partially functional. We hypothesize that the diazotrophic growth defect is caused by an inability of the heterocysts to transport fixed nitrogen to the neighbouring vegetative cells.

Novel Development of Predictive Feature Fingerprints to Identify Chemistry-Based Features for the Effective Drug Design of SARS-CoV-2 Target Antagonists and Inhibitors Using Machine Learning.

A unique approach to bioactivity and chemical data curation coupled with random forest analyses has led to a series of target-specific and cross-validated predictive feature fingerprints (PFF) that have high predictability across multiple therapeutic targets and disease stages involved in the severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2)-induced COVID-19 pandemic, which include plasma kallikrein, human immunodeficiency virus (HIV)-protease, nonstructural protein (NSP)5, NSP12, Janus kinase (JAK) family, and AT-1. The approach was highly accurate in determining the matched target for the different compound sets and suggests that the models could be used for virtual screening of target-specific compound libraries. The curation-modeling process was successfully applied to a SARS-CoV-2 phenotypic screen and could be used for predictive bioactivity estimation and prioritization for clinical trial selection; virtual screening of drug libraries for the repurposing of drug molecules; and analysis and direction of proprietary data sets.

The circadian clock and darkness control natural competence in cyanobacteria.

The cyanobacterium Synechococcus elongatus is a model organism for the study of circadian rhythms. It is naturally competent for transformation-that is, it takes up DNA from the environment, but the underlying mechanisms are unclear. Here, we use a genome-wide screen to identify genes required for natural transformation in S. elongatus, including genes encoding a conserved Type IV pilus, genes known to be associated with competence in other bacteria, and others. Pilus biogenesis occurs daily in the morning, while natural transformation is maximal when the onset of darkness coincides with the dusk circadian peak. Thus, the competence state in cyanobacteria is regulated by the circadian clock and can adapt to seasonal changes of day length.