Fluorogenic RNA-based biomaterials for imaging and tracking the cargo of extracellular vesicles.

Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.

Biomolecular Condensates Can Enhance Pathological RNA Clustering.

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.

RNAs undergo phase transitions with lower critical solution temperatures.

Co-phase separation of RNAs and RNA-binding proteins drives the biogenesis of ribonucleoprotein granules. RNAs can also undergo phase transitions in the absence of proteins. However, the physicochemical driving forces of protein-free, RNA-driven phase transitions remain unclear. Here we report that various types of RNA undergo phase separation with system-specific lower critical solution temperatures. This entropically driven phase separation is an intrinsic feature of the phosphate backbone that requires Mg2+ ions and is modulated by RNA bases. RNA-only condensates can additionally undergo enthalpically favourable percolation transitions within dense phases. This is enabled by a combination of Mg2+-dependent bridging interactions between phosphate groups and RNA-specific base stacking and base pairing. Phase separation coupled to percolation can cause dynamic arrest of RNAs within condensates and suppress the catalytic activity of an RNase P ribozyme. Our work highlights the need to incorporate RNA-driven phase transitions into models for ribonucleoprotein granule biogenesis.

The impact of kangaroo mother care on work of breathing and oxygen saturation in very low birth weight infants with respiratory insufficiency.

BACKGROUND: Kangaroo mother care (KMC) is defined as prolonged skin to skin care between a mother and infant with the infant lying in prone position on mom's chest. KMC decreases morbidity and mortality and promotes physiologic stability. The aim of this study is to measure work of breathing (WOB) during KMC in very low birth weight (VLBW) infants on non-invasive respiratory support. METHODS: A prospective observational pilot study was conducted comparing WOB indices during standard care (SC) and KMC. Respiratory inductive plethysmography (RIP) measured WOB indices non-invasively: phase angle and labored breathing index. VLBW infants who were stable on non-invasive respiratory support were randomized to receive RIP measurements during KMC or during SC first. Summary statistics and mixed linear models were used to compare WOB and vital signs. RESULTS: A total of 32 infants were consented for the study, data collection and analysis was completed on 28 infants. There were no significant differences in mean phase angle during KMC or SC (73.5±4.6 SE deg vs 66.8±3.9 SE deg, p = 0.25). No differences in WOB and vital signs were detected. Controlling for respiratory support or randomization/first location did not change the results. CONCLUSION: In this pilot cohort, infants demonstrated no differences in work of breathing indices or oxygen saturation during KMC or SC while receiving non-invasive respiratory support. KMC appears to be safe and well tolerated with no worsened WOB. Larger studies should be performed to confirm our findings.

FISHing on a Budget.

Sensitive quantification of RNA transcripts via fluorescence in situ hybridization (FISH) is a ubiquitous part of understanding quantitative gene expression in single cells. Many techniques exist to identify and localize transcripts inside the cell, but often they are costly and labor intensive. Here we present a method to use a singly labeled short DNA oligo probe to perform FISH in yeast cells. This method is effective for highly constrained FISH applications where the target length is limited (<200 nucleotides). This method can quantify different RNA isoforms or enable the use of fluorescence resonance energy transfer (FRET) to detect co-transcription of neighboring sequence blocks. Since this method relies on a single probe, it is also more cost-effective than a multiple probe labeling strategy.

Stability and nuclear localization of yeast telomerase depend on protein components of RNase P/MRP.

RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways.

Dual-probe RNA FRET-FISH in Yeast.

mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure processes such as alternate initiation or splicing variation of the transcript. Unlike in a conventional FISH method using multiple probes to target a single transcript, FRET is limited to the use of two probes labeled with matched dyes and requires the use of sensitized emission. Any widefield microscope capable of sensitive single molecule detection of Cy3 and Cy5 should be able to measure FRET in yeast cells. Alternatively, a FRET-FISH method can be used to unambiguously ascertain identity of the transcript without the use of a guide probe set used in other FISH techniques.

Single-probe RNA FISH in Yeast.

Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.

mRNA detection in budding yeast with single fluorophores.

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

Anti-NMDA receptor encephalitis: report of ten cases and comparison with viral encephalitis.

The California Encephalitis Project (CEP), established in 1998 to explore encephalitic etiologies, has identified patients with N-methyl-D-aspartate receptor (NMDAR) antibodies, the likely etiology of their encephalitis. This study compares the presentation of such patients to those with viral encephalitis, so that infectious disease clinicians may identify individuals with this treatable disorder. Patients were physician-referred, and standardized forms were used to gather demographic, clinical, and laboratory data. Features of anti-NMDAR+ patients were compared with the viral encephalitides of enteroviral (EV), rabies, and herpes simplex-1 (HSV-1) origins. Sixteen cases with confirmed viral etiologies were all negative on NMDAR antibody testing. Ten anti-NMDAR+ patients were profiled with a median age of 18.5 years (range 11-31 years). None were Caucasian. They had a characteristic progression with prominent psychiatric symptoms, autonomic instability, significant neurologic abnormalities, and seizures. Two had a teratoma, and, of the remaining eight, four had serologic evidence of acute Mycoplasma infection. The clinical and imaging features of anti-NMDAR+ patients served to differentiate this autoimmune disorder from HSV-1, EV, and rabies. Unlike classic paraneoplastic encephalitis, anti-NMDAR encephalitis affects younger patients and is often treatable. The association of NMDAR antibodies in patients with possible Mycoplasma pneumoniae infection warrants further study.

The subspecialty training, practice type, and geographical distribution of recently trained ophthalmologists: a study of male and female physicians.

OBJECTIVE: To characterize the distribution of male and female ophthalmologists with regard to practice type, subspecialty training, rural-urban distribution, and regional distribution. METHODS: Ophthalmology Matching Program files containing the records of residents who began their second year at accredited programs between 1986 and 1990 (inclusive), were compared to membership files of the American Academy of Ophthalmology. Practice locations for each individual were classified according to region, stage, and Rural-Urban Continuum County Code, as defined by the US Department of Agriculture. RESULTS: This cohort comprised 2,494 individuals, 77.1% (1922) of whom were male and 22.9% (572) of whom were female. Group practice was most common (55.9% for women and 61.3% for men). More women were in salaried positions associated with health maintenance organizations (p = 0.006) and academic settings (p < 0.001) than were men. Notable differences in subspecialty choice were restricted to pediatric ophthalmology, chosen three times more frequently by women, and vitreoretinal diseases/surgery, chosen twice as often by men. Only 5.6% of women selected nonmetropolitan practice locales compared to approximately twice that percentage of men. The Middle Atlantic and New England regions attracted more women, while the South Atlantic attracted more men.

Locus of control, machiavellianism, and managerial job performance.

In this study, we examined the moderating effect of locus of control on the relationship between Machiavellianism and job performance of store managers in a retail setting. Our results indicated a significant moderating effect for managers who perceive that they have an external control orientation but not for managers with an internal control orientation.

Relating locus of control to Machiavellianism and managerial achievement.

Rotter's Locus of Control Scale, Christie and Geis' Mach IV Scale, and Fineman's Work Preference Questionnaire were administered to a sample of 60 retail specialty store managers. While there were no significant correlations between locus of control and managerial achievement, there were significant correlations between locus of control and Machiavellianism for the over-all sample and for men but not for women. In addition, men possessed higher mean internal-control orientation than women.