The ability to track labeled cancer cells in vivo would allow researchers to study their distribution, growth, and metastatic potential within the intact organism. Magnetic resonance (MR) imaging is invaluable for tracking cancer cells in vivo as it benefits from high spatial resolution and the absence of ionizing radiation. However, many MR contrast agents (CAs) required to label cells either do not significantly accumulate in cells or are not biologically compatible for translational studies. We have developed carbon-based nanodiamond–gadolinium(III) aggregates (NDG) for MR imaging that demonstrated remarkable properties for cell tracking in vivo. First, NDG had high relaxivity independent of field strength, a finding unprecedented for gadolinium(III) [Gd(III)]–nanoparticle conjugates. Second, NDG demonstrated a 300-fold increase in the cellular delivery of Gd(III) compared to that of clinical Gd(III) chelates without sacrificing biocompatibility. Further, we were able to monitor the tumor growth of NDG-labeled flank tumors by T1- and T2-weighted MR imaging for 26 days in vivo, longer than was reported for other MR CAs or nuclear agents. Finally, by utilizing quantitative maps of relaxation times, we were able to describe tumor morphology and heterogeneity (corroborated by histological analysis), which would not be possible with competing molecular imaging modalities.

Pancreatic adenocarcinoma has a 5 year survival of approximately 3% and median survival of 6 months and is among the most dismal of prognoses in all of medicine. This poor prognosis is largely due to delayed diagnosis where patients remain asymptomatic until advanced disease is present. Therefore, techniques to allow early detection of pancreatic adenocarcinoma are desperately needed. Imaging of pancreatic tissue is notoriously difficult, and the development of new imaging techniques would impact our understanding of organ physiology and pathology with applications in disease diagnosis, staging, and longitudinal response to therapy in vivo. Magnetic resonance imaging (MRI) provides numerous advantages for these types of investigations; however, it is unable to delineate the pancreas due to low inherent contrast within this tissue type. To overcome this limitation, we have prepared a new Gd(III) contrast agent that accumulates in the pancreas and provides significant contrast enhancement by MR imaging. We describe the synthesis and characterization of a new dithiolane-Gd(III) complex and a straightforward and scalable approach for conjugation to a gold nanoparticle. We present data that show the nanoconjugates exhibit very high per particle values of r1 relaxivity at both low and high magnetic field strengths due to the high Gd(III) payload. We provide evidence of pancreatic tissue labeling that includes MR images, post-mortem biodistribution analysis, and pancreatic tissue evaluation of particle localization. Significant contrast enhancement was observed allowing clear identification of the pancreas with contrast-to-noise ratios exceeding 35:1.

Efficient and highly organized regulation of transcription is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.

Biogeography of microbial populations remains to be poorly understood, and a novel technique of single cell sorting promises a new level of resolution for microbial diversity studies. Using single cell sorting, we compared saturated NaCl brine environments (32-35 %) of the South Bay Salt Works in Chula Vista in California (USA) and Santa Pola saltern near Alicante (Spain). Although some overlap in community composition was detected, both samples were significantly different and included previously undiscovered 16S rRNA sequences. The community from Chula Vista saltern had a large bacterial fraction, which consisted of diverse Bacteroidetes and Proteobacteria. In contrast, Archaea dominated Santa Pola's community and its bacterial fraction consisted of the previously known Salinibacter lineages. The recently reported group of halophilic Archaea, Nanohaloarchaea, was detected at both sites. We demonstrate that cell sorting is a useful technique for analysis of halophilic microbial communities, and is capable of identifying yet unknown or divergent lineages. Furthermore, we argue that observed differences in community composition reflect restricted dispersal between sites, a likely mechanism for diversification of halophilic microorganisms.

Background The determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterized relationships between SARS-CoV-2 RNAemia and disease severity, clinical deterioration, and specific EPCs. Methods We used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterized the role of RNAemia in predicting clinical severity and EPCs using elastic net regression. Results 23.0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1.4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAemia within 10 days of symptom onset, reached maximum clinical severity within 16 days, and symptom resolution within 33 days. Initially RNAaemic patients were more likely to manifest severe disease (OR 6.72 [95% CI, 2.45 – 19.79]), worsening of disease severity (OR 2.43 [95% CI, 1.07 – 5.38]), and EPCs (OR 2.81 [95% CI, 1.26 – 6.36]). RNA load correlated with maximum severity (r = 0.47 [95% CI, 0.20 – 0.67]). Conclusions dPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.

In vivo cell tracking is vital for understanding migrating cell populations, particularly cancer and immune cells. Magnetic resonance (MR) imaging for long-term tracking of transplanted cells in live organisms requires cells to effectively internalize Gd(III) contrast agents (CAs). Clinical Gd(III)-based CAs require high dosing concentrations and extended incubation times for cellular internalization. To combat this, we have devised a series of Gd(III)-gold nanoconjugates (Gd@AuNPs) with varied chelate structure and nanoparticle-chelate linker length, with the goal of labeling and imaging breast cancer cells. These new Gd@AuNPs demonstrate significantly enhanced labeling compared to previous Gd(III)-gold-DNA nanoconstructs. Variations in Gd(III) loading, surface packing, and cell uptake were observed among four different Gd@AuNP formulations suggesting that linker length and surface charge play an important role in cell labeling. The best performing Gd@AuNPs afforded 23.6 ± 3.6 fmol of Gd(III) per cell at an incubation concentration of 27.5 μM—this efficiency of Gd(III) payload delivery (Gd(III)/cell normalized to dose) exceeds that of previous Gd(III)-Au conjugates and most other Gd(III)-nanoparticle formulations. Further, Gd@AuNPs were well-tolerated in vivo in terms of biodistribution and clearance, and supports future cell tracking applications in whole-animal models.

Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well-documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500 bp) identified many closely and more distantly related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes indicates that accumulation of genomic variation is rapid: faster than the rate of third codon substitutions.

BackgroundThe determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterise the relationships between SARS-CoV-2 RNAaemia and disease severity, clinical deterioration, and specific EPCs.MethodsWe used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from nasopharyngeal swabs and plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterised the role of RNAaemia in predicting clinical severity and EPCs using elastic net regression.Findings23·0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1·4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAaemia 10 days after onset of symptoms, but took 16 days to reach maximum severity, and 33 days for symptoms to resolve. Initially RNAaemic patients were more likely to manifest severe disease (OR 6·72 [95% CI, 2·45 – 19·79]), worsening of disease severity (OR 2·43 [95% CI, 1·07 - 5·38]), and EPCs (OR 2·81 [95% CI, 1·26 – 6·36]). RNA load correlated with maximum severity (r = 0·47 [95% CI, 0·20 - 0·67]).InterpretationdPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAaemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAaemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.FundingNIH/NIAID (Grants R01A153133, R01AI137272, and 3U19AI057229 – 17W1 COVID SUPP #2) and a donation from Eva Grove.Research in contextEvidence before this studyThe varied clinical manifestations of COVID-19 have directed attention to the distribution of SARS-CoV-2 in the body. Although most concentrated and tested for in the nasopharynx, SARS-CoV-2 RNA has been found in blood, stool, and numerous tissues, raising questions about dissemination of viral RNA throughout the body, and the role of this process in disease severity and extrapulmonary complications. Recent studies have detected low levels of SARS-CoV-2 RNA in blood using either quantitative reverse transcriptase real-time PCR (qPCR) or droplet digital PCR (dPCR), and have associated RNAaemia with disease severity and biomarkers of dysregulated immune response.Added value of this studyWe quantified SARS-CoV-2 RNA in the nasopharynx and plasma of patients presenting to the Emergency Department with COVID-19, and found an array-based dPCR platform to be markedly more sensitive than qPCR for detection of SARS-CoV-2 RNA, with a simplified workflow well-suited to clinical adoption. We collected serial plasma samples during patients’ course of illness, and showed that SARS-CoV-2 RNAaemia peaks early, while clinical condition often continues to worsen. Our findings confirm the association between RNAaemia and disease severity, and additionally demonstrate a role for RNAaemia in predicting future deterioration and specific extrapulmonary complications.Implications of all the available evidenceVariation in SARS-CoV-2 RNAaemia may help explain disparities in disease severity and extrapulmonary complications from COVID-19. Testing for RNAaemia with dPCR early in the course of illness may help guide patient triage and management.