Sample-to-answer COVID-19 nucleic acid testing using a low-cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out.

With its origin estimated around December 2019 in Wuhan, China, the ongoing SARS-CoV-2 pandemic is a major global health challenge. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used to selectively remove primer dimers, inactivate the reaction post-amplification and allowing enhanced fluorescence detection via a smartphone camera. Sample-to-answer analysis within 1 hour from sample collection and a detection limit of approximately 100 RNA copies in 10 µL reaction volume were achieved. The platform was validated with a panel of 162 nasopharyngeal swab samples collected from patients with COVID-19 symptoms, providing a sensitivity of 96.6% (82.2-99.9%, 95% CI) for samples with Ct values below 26 and a specificity of 100% (90-100%, 95% CI), thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics to resource-limited settings.

Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples.

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

USA300 methicillin-resistant Staphylococcus aureus in Stockholm, Sweden, from 2008 to 2016.

Methicillin-resistant Staphylococcus aureus (MRSA) USA300 isolates have been recognized globally, not only in community but also in healthcare settings. USA300 isolates were initially resistant only to methicillin, but resistance to non-ß-lactams has emerged with time. To evaluate the prevalence and antimicrobial susceptibility of USA300 isolates in Stockholm, we conducted a nine-year retrospective study. Of 5359 consecutive MRSA cases in Stockholm, isolates from 285 cases were USA300 strains according to the pulsed-field gel electrophoresis pattern. Of these cases, repeated isolates with altered antibiotic resistance patterns were observed in six individuals. Therefore, antimicrobial susceptibility testing was performed on totally 291 isolates. To study the phylogenetic relatedness of isolates in transmission events and genomic resistance traits, 35 isolates were further studied by whole genome sequencing (WGS). The incidence of MRSA was increased from 17.6 per 100,000 inhabitants in 2008 to 37.3 per 100,000 inhabitants in 2016, while the proportion of USA300 cases declined from 6.6% in 2008 to 2.6% in 2016. Among the USA300 isolates, 73.5% were community-associated, 21.3% healthcare-associated, and 5.2% had unknown acquisition. The highest resistance rate among non-ß-lactams was found in erythromycin (86%), followed by fluoroquinolones (68-69%). 57% of the isolates were resistant to both erythromycin and fluoroquinolone. Simultaneous resistance to four non-ß-lactam antibiotic classes was found in six isolates. Four isolates were susceptible to all non-ß-lactam antibiotics. Ceftaroline, daptomycin, linezolid, mupirocin, rifampicin, teicoplanin, telavancin, trimethoprim-sulfamethoxazole and vancomycin retained full activity in the study. WGS analysis indicated that isolates from an outbreak were phylogenetically closely related. In conclusion, USA300 MRSA isolates in Stockholm have neither been limited to the community setting, nor remained susceptible to non-ß-lactam agents. WGS is becoming a useful tool in tracing transmission events. The results herein provide the most up-to-date and comprehensive information regarding status of USA300 strains in this geographic area.

The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme.

Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.

Differential transcriptional regulation of orthologous dps genes from two closely related heterocyst-forming cyanobacteria.

In cyanobacteria, DNA-binding proteins from starved cells (Dps) play an important role in the cellular response to oxidative and nutritional stresses. In this study, we have characterized the cell-type specificity and the promoter regions of two orthologous dps genes, Npun_R5799 in Nostoc punctiforme and alr3808 in Anabaena sp. PCC 7120. A transcriptional start site (TSS), identical in location to the previously identified proximal TSS of alr3808, was identified for Npun_R5799 under both combined nitrogen supplemented and N2-fixing growth conditions. However, only alr3808 was also transcribed from a second distal TSS. Sequence homologies suggest that the promoter region containing the distal TSS is not conserved upstream of orthologous genes among heterocyst-forming cyanobacteria. The analysis of promoter GFP-reporter strains showed a different role in governing cell-type specificity between the proximal and distal promoter of alr3808. We here confirmed the heterocyst specificity of the distal promoter of alr3808 and described a very early induction of its expression during proheterocyst differentiation. In contrast, the complete promoters of both genes were active in all cells. Even though Npun_R5799 and alr3808 are orthologs, the regulation of their respective expression differs, indicating distinctions in the function of these cyanobacterial Dps proteins depending on the strain and cell type.

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

BACKGROUND: In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. RESULTS: Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. CONCLUSIONS: The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.

Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium.

Trichodesmium--a widespread marine cyanobacterium with unusual nitrogen fixation properties.

The last several decades have witnessed dramatic advances in unfolding the diversity and commonality of oceanic diazotrophs and their N2 -fixing potential. More recently, substantial progress in diazotrophic cell biology has provided a wealth of information on processes and mechanisms involved. The substantial contribution by the diazotrophic cyanobacterial genus Trichodesmium to the nitrogen influx of the global marine ecosystem is by now undisputable and of paramount ecological importance, while the underlying cellular and molecular regulatory physiology has only recently started to unfold. Here, we explore and summarize current knowledge, related to the optimization of its diazotrophic capacity, from genomics to ecophysiological processes, via, for example, cellular differentiation (diazocytes) and temporal regulations, and suggest cellular research avenues that now ought to be explored.

Diazocyte development in the marine diazotrophic cyanobacterium Trichodesmium.

The establishment of non-diazotrophic cultures of the filamentous marine cyanobacterium Trichodesmium erythraeum IMS101 enabled the first detailed investigation of the process leading to the development of its unique nitrogen-fixing cell type, the diazocyte. Trichome heterogeneity was apparent already within 3-8 h, while the differentiation of mature diazocytes, containing the nitrogenase enzyme, required 27 h after the removal of combined nitrogen. The distribution of 'pro-diazocytes' within the trichomes correlates with the localization of mature diazocytes, which suggests that pattern regulation is an early event during diazocyte development. The development was initially identified as changes in the subcellular ultrastructure, most notably the degradation of glycogen granules and gas vacuoles. These changes were preceded by the induced expression of the global nitrogen regulator ntcA at an early stage of combined nitrogen deprivation, followed by elevated expression of genes related to nitrogen metabolism and their corresponding proteins. The strongest induction (10-fold) was related to the transcription of the respiratory gene coxB2, apparent already at an early stage, which suggests an important role for respiration and the subsequent energy generation in the subcellular changes found, and in the creation of the reducing environment required for nitrogen fixation in diazocytes.

Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes.

Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and sub-tropical oceans. It is unique in that it exclusively fixes N(2) at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N(2)-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N(2) fixation activity. The diazotrophic regime (N(2) versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N(2) fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N(2)-fixing physiology.

Temporal separation of cell division and diazotrophy in the marine diazotrophic cyanobacterium Trichodesmium erythraeum IMS101.

Examination of the diurnal patterns of basic cellular processes in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium revealed that the division of cells occurred throughout the diurnal cycle, but that it oscillated and peaked at an early stage in the dark period. Transcription of the early cell division gene ftsZ and the occurrence of the FtsZ protein showed a similar diurnal rhythmicity that preceded the division of cells. DNA replication (dnaA gene transcription) occurred before the transcription of ftsZ and hetR, the latter encoding the key heterocyst differentiation protein. Transcription of ftsZ and hetR in turn preceded the development of the nitrogen-fixing diazocytes and nifH transcription, and were at the minimum when diazotrophy was at the maximum. The nifH gene transcription showed a negative correlation to the circadian clock gene kaiC. Together, the data show a temporal separation between cell division and diazotrophy on a diurnal basis.