Optimization of ribosome profiling in plants including structural analysis of rRNA fragments.

BACKGROUND: Ribosome profiling (or Ribo-seq) is a technique that provides genome-wide information on the translational landscape (translatome). Across different plant studies, variable methodological setups have been described which raises questions about the general comparability of data that were generated from diverging methodologies. Furthermore, a common problem when performing Ribo-seq are abundant rRNA fragments that are wastefully incorporated into the libraries and dramatically reduce sequencing depth. To remove these rRNA contaminants, it is common to perform preliminary trials to identify these fragments because they are thought to vary depending on nuclease treatment, tissue source, and plant species. RESULTS: Here, we compile valuable insights gathered over years of generating Ribo-seq datasets from different species and experimental setups. We highlight which technical steps are important for maintaining cross experiment comparability and describe a highly efficient approach for rRNA removal. Furthermore, we provide evidence that many rRNA fragments are structurally preserved over diverse nuclease regimes, as well as across plant species. Using a recently published cryo-electron microscopy (cryo-EM) structure of the tobacco 80S ribosome, we show that the most abundant rRNA fragments are spatially derived from the solvent-exposed surface of the ribosome. CONCLUSION: The guidelines presented here shall aid newcomers in establishing ribosome profiling in new plant species and provide insights that will help in customizing the methodology for individual research goals.

Multiple evanescent white dot syndrome associated with COVID-19 infection: a case report.

BACKGROUND: To report a case of Multiple Evanescent White Dot Syndrome (MEWDS) one month after a COVID-19 infection in a female patient at an age unusual for the occurrence of this disease. CASE PRESENTATION: A 69-year-old Caucasian female reported the presence of floaters, photopsia, and enlarging vision loss in her left eye following the COVID-19 infection. Clinical and multimodal imaging was consistent with the MEWDS diagnosis. Fluorescein angiography examination revealed characteristic hyperfluorescent spots around the fovea in a wreath-like pattern. An extensive lab workup to rule out other autoimmune and infectious etiologies was inconclusive. Visual acuity and white dots resolved after a course of corticosteroids, which was confirmed on follow-up dilated fundus exam and multimodal imaging. CONCLUSIONS: MEWDS is a rare white dot syndrome that may occur following COVID-19 infection in addition to other reported ophthalmic disorders following this infection.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

Utilizing high-resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas.

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5' and 3' sections of transcripts and characteristics of eukaryotic translation termination, which are fundamentally distinct for chloroplast translation. In chloroplasts, a heterogeneous RPF size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulatory step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light-harvesting complex proteins, LHCs. By providing high-quality datasets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim to make ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation output levels of specific genes and their splice variants and for monitoring genome annotation.

Agrobacterium-Mediated Seedling Transformation to Measure Circadian Rhythms in Arabidopsis.

Circadian clocks are endogenous timing mechanisms that allow an organism to adapt cellular processes in anticipation of predictable changes in the environment. Luciferase reporters are well utilized as an effective, nondestructive method to measure circadian rhythms of promoter activity in Arabidopsis. Obtaining stable transgenic reporter lines can be laborious. Here, we report a protocol for Agrobacterium-mediated seedling transformation tailored for plant circadian studies. We show that period estimates generated from wild-type and clock-mutant seedlings transformed with circadian luciferase reporters are similar to rhythms obtained from equivalent stable transgenic seedlings. These experiments demonstrate the versatility and robustness of the protocol for testing new constructs or quickly assessing circadian effects in any genotype of interest.

Assessing sensitivity to change of the genomics outcome scale (GOS).

The Genetic Counseling Outcome Scale (GCOS-24) is a 24-item patient-reported outcome measure (PROM) that was developed to evaluate genetic counseling and testing services by measuring the construct of empowerment. The Genomics Outcome Scale (GOS) is a 6-item version of GCOS-24 that was designed to provide a PROM for use both within and outside clinical genetics services and reduce respondent burden. However, unlike GCOS-24, the sensitivity to change of the GOS has not yet been assessed in appropriate clinical settings. We carried out pre- and post-clinic surveys using the GOS to assess sensitivity to change of the GOS and produce before-and-after GOS data as part of a service evaluation. The survey was sent to patients attending the genetic counseling clinic for a first appointment at the All Wales Medical Genetic Service from 8 April 2019 to 18 September 2019. Patients attending disease management clinics, where genetic issues were not the primary concern, were excluded from this study. A total of 138 respondents were included in the final analysis. The result shows that empowerment scores, measured using the GOS, were significantly higher (p<0.05) after clinic attendance. The GOS shows good sensitivity to change, with a medium-to-large effect size (Cohen's d = 0.73). The result also shows that the service is delivering measurable benefits for its service users.

Hospital acquired viral respiratory tract infections: An underrecognized nosocomial infection.

BACKGROUND: Our study aimed to describe the incidence, epidemiology of respiratory viruses and outcomes in hospital acquired viral respiratory infections (HAVRI). METHODS: We conducted a retrospective observational study on all adults and children with hospital acquired viral respiratory infections between July 2012 and April 2019. Clinical and microbiological data were collected in a major tertiary level hospital in North Queensland. Morbidity indicators were the length of stay, need for intensive care and mechanical ventilation. Length of stay was analyzed with the Kruskal-Wallis test and mortality with the Chi-Square test. RESULTS: A total of 283 patients tested positive for a respiratory virus and fulfilled the criteria for a hospital acquired infection. Individuals in the younger age group were more likely to be admitted to intensive care and need mechanical ventilation. A higher mortality was found with individuals in the older age category. The morbidity and mortality did not differ based on the virus type. Influenza A was the most common respiratory virus associated with hospital acquired viral respiratory infections. CONCLUSION: Hospital acquired viral respiratory infections contribute significantly to morbidity and mortality regardless of the virus species.

The calmodulin-like protein, CML39, is involved in regulating seed development, germination, and fruit development in Arabidopsis.

KEY MESSAGE: We show that the calcium sensor, CML39, is important in various developmental processes from seeds to mature plants. This study bridges previous work on CML39 as a stress-induced gene and highlights the importance of calcium signalling in plant development. In addition to the evolutionarily-conserved Ca2+ sensor, calmodulin (CaM), plants possess a large family of CaM-related proteins (CMLs). Using a cml39 loss-of-function mutant, we investigated the roles of CML39 in Arabidopsis and discovered a range of phenotypes across developmental stages and in different tissues. In mature plants, loss of CML39 results in shorter siliques, reduced seed number per silique, and reduced number of ovules per pistil. We also observed changes in seed development, germination, and seed coat properties in cml39 mutants in comparison to wild-type plants. Using radicle emergence as a measure of germination, cml39 mutants showed more rapid germination than wild-type plants. In marked contrast to wild-type seeds, the germination of developing, immature cml39 seeds was not sensitive to cold-stratification. In addition, germination of cml39 seeds was less sensitive than wild-type to inhibition by ABA or by treatments that impaired gibberellic acid biosynthesis. Tetrazolium red staining indicated that the seed-coat permeability of cml39 seeds is greater than that of wild-type seeds. RNA sequencing analysis of cml39 seedlings suggests that changes in chromatin modification may underlie some of the phenotypes associated with cml39 mutants, consistent with previous reports that orthologs of CML39 participate in gene silencing. Aberrant ectopic expression of transcripts for seed storage proteins in 7-day old cml39 seedlings was observed, suggesting mis-regulation of early developmental programs. Collectively, our data support a model where CML39 serves as an important Ca2+ sensor during ovule and seed development, as well as during germination and seedling establishment.

Transcript profiling indicates a widespread role for bacterial-type phosphoenolpyruvate carboxylase in malate-accumulating sink tissues.

Phosphoenolpyruvate carboxylase (PEPC) is an important regulatory enzyme situated at a key branch point of central plant metabolism. Plant genomes encode several plant-type PEPC (PTPC) isozymes, along with a distantly related bacterial-type PEPC (BTPC). BTPC is expressed at high levels in developing castor oil seeds where it tightly interacts with co-expressed PTPC polypeptides to form unusual hetero-octameric Class-2 PEPC complexes that are desensitized to allosteric inhibition by L-malate. Analysis of RNA-Seq and microarray transcriptome datasets revealed two distinct patterns of tissue-specific BTPC expression in vascular plants. Species such as Arabidopsis thaliana, strawberry, rice, maize, and poplar mainly exhibited pollen- or floral-specific BTPC expression. By contrast, BTPC transcripts were relatively abundant in developing castor, cotton, and soybean seeds, cassava tubers, as well as immature tomato, cucumber, grape, and avocado fruit. Immunoreactive 118 kDa BTPC polypeptides were detected on immunoblots of cucumber and tomato fruit extracts. Co-immunoprecipitation established that as in castor, BTPCs physically interact with endogenous PTPCs to form Class-2 PEPC complexes in tomato and cucumber fruit. We hypothesize that Class-2 PEPCs simultaneously maintain rapid anaplerotic PEP carboxylation and respiratory CO2 refixation in diverse, biosynthetically active sinks that accumulate high malate levels.

The Deltoid Lift: A Comparison Study of Exposure Area in Proximal Humeral Approaches.

BACKGROUND: Adequate exposure of the proximal humerus is necessary to address atypical or complex proximal humerus fractures and orthopedic tumors. Surgical management may be difficult through existing approaches due to their limited nature and the delicate neurovascular anatomy of the shoulder. The deltoid lift, a previously described extensile approach, can be incorporated into the surgeon's armamentarium as an alternative exposure to the proximal humerus. The objective of this study was to quantify and compare the humeral exposure achieved through the deltoid lift with the standard direct lateral deltoid-splitting, anterolateral acromial, and deltopectoral approaches in terms of surface area and exposure of key anatomic landmarks. METHODS: Each approach was performed a minimum of 8 times on 18 cadaveric specimens. After identifying landmarks, exposure area of exposed humerus was quantified using digital images and ImageJ software. RESULTS: The deltoid lift yielded an average exposure area of 46 cm2. Comparison of the exposure area for the deltoid lift against each of the other approaches yielded statistical significance ( P < .01). The exposure provided was 2-folds greater than that of the next most extensive approach. All anatomic landmarks were directly visible through the deltoid lift as compared with the remaining approaches, through which only 1 landmark was directly visualized and only 2 of the 3 remaining were palpable through the approach. CONCLUSIONS: The deltoid lift extensile surgical exposure to the proximal humerus provides the largest humeral exposure with the greatest visibility of landmarks relative to the 3 most widely utilized standard approaches.

EPHA4-FC TREATMENT REDUCES ISCHEMIA/REPERFUSION-INDUCED INTESTINAL INJURY BY INHIBITING VASCULAR PERMEABILITY.

The inflammatory response is characterized by increased endothelial permeability, which permits the passage of fluid and inflammatory cells into interstitial spaces. The Eph/ephrin receptor ligand system plays a role in inflammation through a signaling cascade, which modifies Rho-GTPase activity. We hypothesized that blocking Eph/ephrin signaling using an EphA4-Fc would result in decreased inflammation and tissue injury in a model of ischemia/reperfusion (I/R) injury. Mice undergoing intestinal I/R pretreated with the EphA4-Fc had significantly reduced intestinal injury compared to mice injected with the control Fc. This reduction in I/R injury was accompanied by significantly reduced neutrophil infiltration, but did not affect intestinal inflammatory cytokine generation. Using microdialysis, we identified that intestinal I/R induced a marked increase in systemic vascular leakage, which was completely abrogated in EphA4-Fc-treated mice. Finally, we confirmed the direct role of Eph/ephrin signaling in endothelial leakage by demonstrating that EphA4-Fc inhibited tumor necrosis factor-α-induced vascular permeability in human umbilical vein endothelial cells. This study identifies that Eph/ephrin interaction induces proinflammatory signaling in vivo by inducing vascular leak and neutrophil infiltration, which results in tissue injury in intestinal I/R. Therefore, therapeutic targeting of Eph/ephrin interaction using inhibitors, such as EphA4-Fc, may be a novel method to prevent tissue injury in acute inflammation by influencing endothelial integrity and by controlling vascular leak.

The Deltoid Lift: A Cadaveric Analysis and the Literature Review of a Novel Surgical Approach to the Proximal Humerus.

Obtaining adequate exposure of the proximal humerus for anatomic reduction of complex intra-articular fractures or in the surgical treatment of tumor may be difficult. Here we describe a novel approach to the proximal humerus: the deltoid lift, and perform a cadaveric analysis objectively quantifying the exposure. The deltoid lift offers significantly greater exposure to the proximal humerus as compared with the deltopectoral approach.

Delayed presentation of nitroprusside-induced cyanide toxicity.

Cyanide toxicity is a rare complication of sodium nitroprusside that can be difficult to diagnose in critically ill patients. We describe a case of cyanide toxicity after cardiac surgery that presented as lactic acidosis after discontinuation of nitroprusside.

Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells.

BACKGROUND: Fetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function. RESULTS: We found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for 'sensing' migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties. CONCLUSIONS: We conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.

EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme.

Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.

Activation of ephrin A proteins influences hematopoietic stem cell adhesion and trafficking patterns.

OBJECTIVE: To determine if Eph receptors and ephrins can modulate the homing of hematopoietic cells in a murine bone marrow transplantation model. MATERIALS AND METHODS: EphA and ephrin A gene expression by mouse hematopoietic stem cells and the progenitor cell line FDCP-1 was determined by real-time reverse transcription polymerase chain reaction and flow cytometry. The effect of ephrin A activation on adhesion of hematopoietic progenitors was determined by in vitro adhesion assays in which cells were exposed to fibronectin or vascular cell adhesion molecule-1 (VCAM-1) and an increasing gradient of immobilized EphA3-Fc. Adhesion to fibronectin and VCAM-1 was further investigated using soluble preclustered EphA3-Fc. We used soluble unclustered EphA3-Fc as an antagonist to block endogenous EphA-ephrin A interactions in vivo. The effect of injecting soluble EphA3-Fc on the mobilization of hematopoietic progenitor cells was examined. We determined the effect on short-term homing by pretreating bone marrow cells with EphA3-Fc or the control IgG before infusion into lethally irradiated mice. RESULTS: Preclustered and immobilized EphA3-Fc increased adhesion of progenitor cells and FDCP-1 to fibronectin and VCAM-1 (1.6- to 2-fold higher adhesion; p < 0.05) relative to control (0 µ/cm(2) EphA3-Fc extracellular molecule alone). Injection of the antagonist soluble EphA3-Fc increased progenitor cell and colony-forming unit-spleen cells in the peripheral blood (42% greater colony-forming unit in culture; p < 0.05, 3.8-fold higher colony-forming unit-spleen) relative to control. CONCLUSION: Treating bone marrow cells with EphA3-Fc resulted in a reduction by 31% in donor stem cells homing to the bone marrow and accumulation of donor cells in recipient spleens (50% greater than control) and greater recovery of donor stem cells from the peripheral blood.

Sparse image reconstruction for molecular imaging.

The application that motivates this paper is molecular imaging at the atomic level. When discretized at subatomic distances, the volume is inherently sparse. Noiseless measurements from an imaging technology can be modeled by convolution of the image with the system point spread function (psf). Such is the case with magnetic resonance force microscopy (MRFM), an emerging technology where imaging of an individual tobacco mosaic virus was recently demonstrated with nanometer resolution. We also consider additive white Gaussian noise (AWGN) in the measurements. Many prior works of sparse estimators have focused on the case when H has low coherence; however, the system matrix H in our application is the convolution matrix for the system psf. A typical convolution matrix has high coherence. This paper, therefore, does not assume a low coherence H. A discrete-continuous form of the Laplacian and atom at zero (LAZE) p.d.f. used by Johnstone and Silverman is formulated, and two sparse estimators derived by maximizing the joint p.d.f. of the observation and image conditioned on the hyperparameters. A thresholding rule that generalizes the hard and soft thresholding rule appears in the course of the derivation. This so-called hybrid thresholding rule, when used in the iterative thresholding framework, gives rise to the hybrid estimator, a generalization of the lasso. Estimates of the hyperparameters for the lasso and hybrid estimator are obtained via Stein's unbiased risk estimate (SURE). A numerical study with a Gaussian psf and two sparse images shows that the hybrid estimator outperforms the lasso.