Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids.

The high-value carotenoid astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type-like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.

Expression of a carotenogenic gene allows faster biomass production by redesigning plant architecture and improving photosynthetic efficiency in tobacco.

Because carotenoids act as accessory pigments in photosynthesis, play a key photoprotective role and are of major nutritional importance, carotenogenesis has been a target for crop improvement. Although carotenoids are important precursors of phytohormones, previous genetic manipulations reported little if any effects on biomass production and plant development, but resulted in specific modifications in carotenoid content. Unexpectedly, the expression of the carrot lycopene ß-cyclase (DcLCYB1) in Nicotiana tabacum cv. Xanthi not only resulted in increased carotenoid accumulation, but also in altered plant architecture characterized by longer internodes, faster plant growth, early flowering and increased biomass. Here, we have challenged these transformants with a range of growth conditions to determine the robustness of their phenotype and analyze the underlying mechanisms. Transgenic DcLCYB1 lines showed increased transcript levels of key genes involved in carotenoid, chlorophyll, gibberellin (GA) and abscisic acid (ABA) biosynthesis, but also in photosynthesis-related genes. Accordingly, their carotenoid, chlorophyll, ABA and GA contents were increased. Hormone application and inhibitor experiments confirmed the key role of altered GA/ABA contents in the growth phenotype. Because the longer internodes reduce shading of mature leaves, induction of leaf senescence was delayed, and mature leaves maintained a high photosynthetic capacity. This increased total plant assimilation, as reflected in higher plant yields under both fully controlled constant and fluctuating light, and in non-controlled conditions. Furthermore, our data are a warning that engineering of isoprenoid metabolism can cause complex changes in phytohormone homeostasis and therefore plant development, which have not been sufficiently considered in previous studies.

The Functions of Chloroplast Glutamyl-tRNA in Translation and Tetrapyrrole Biosynthesis.

The chloroplast glutamyl-tRNA (tRNAGlu) is unique in that it has two entirely different functions. In addition to acting in translation, it serves as the substrate of glutamyl-tRNA reductase (GluTR), the enzyme catalyzing the committed step in the tetrapyrrole biosynthetic pathway. How the tRNAGlu pool is distributed between the two pathways and whether tRNAGlu allocation limits tetrapyrrole biosynthesis and/or protein biosynthesis remains poorly understood. We generated a series of transplastomic tobacco (Nicotiana tabacum) plants to alter tRNAGlu expression levels and introduced a point mutation into the plastid trnE gene, which has been reported to uncouple protein biosynthesis from tetrapyrrole biosynthesis in chloroplasts of the protist Euglena gracilis We show that, rather than comparable uncoupling of the two pathways, the trnE mutation is lethal in tobacco because it inhibits tRNA processing, thus preventing translation of Glu codons. Ectopic expression of the mutated trnE gene uncovered an unexpected inhibition of glutamyl-tRNA reductase by immature tRNAGlu We further demonstrate that whereas overexpression of tRNAGlu does not affect tetrapyrrole biosynthesis, reduction of GluTR activity through inhibition by tRNAGlu precursors causes tetrapyrrole synthesis to become limiting in early plant development when active photosystem biogenesis provokes a high demand for de novo chlorophyll biosynthesis. Taken together, our findings provide insight into the roles of tRNAGlu at the intersection of protein biosynthesis and tetrapyrrole biosynthesis.

Lycopene ß-cyclase expression influences plant physiology, development, and metabolism in tobacco plants.

Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. ß-Carotene is generated from lycopene by lycopene ß-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.

The plastid-encoded PsaI subunit stabilizes photosystem I during leaf senescence in tobacco.

PsaI is the only subunit of PSI whose precise physiological function has not yet been elucidated in higher plants. While PsaI is involved in PSI trimerization in cyanobacteria, trimerization was lost during the evolution of the eukaryotic PSI, and the entire PsaI side of PSI underwent major structural remodelling to allow for binding of light harvesting complex II antenna proteins during state transitions. Here, we have generated a tobacco (Nicotiana tabacum) knockout mutant of the plastid-encoded psaI gene. We show that PsaI is not required for the redox reactions of PSI. Neither plastocyanin oxidation nor the processes at the PSI acceptor side are impaired in the mutant, and both linear and cyclic electron flux rates are unaltered. The PSI antenna cross section is unaffected, state transitions function normally, and binding of other PSI subunits to the reaction centre is not compromised. Under a wide range of growth conditions, the mutants are phenotypically and physiologically indistinguishable from wild-type tobacco. However, in response to high-light and chilling stress, and especially during leaf senescence, PSI content is reduced in the mutants, indicating that the I-subunit plays a role in stabilizing PSI complexes.

Koro-Like Symptoms in an Adolescent Female With Schizophrenia.

Koro is a disorder characterized by the fear of genitals shrinking and retracting into the body.1 While it is not included in the cultural concepts of distress in DSM-5, DSM-IV-TR described koro as "an episode of sudden and intense anxiety that the penis (or, in female patients, the vulva and nipples) will recede into the body and possibly cause death." The term koro originates from the Malay language, referring to the retracting motion of the head of turtles or tortoises into their shells. In the United States and Europe, the term genital retraction syndrome is commonly used instead of koro. In classical koro, cultural belief plays a role in its origin and spread, often in epidemics in Asian countries.2 On the other hand, Koro-like syndrome (KLS) has been reported worldwide secondary to medical and psychiatric disorders.2 Similar to koro, KLS is more prevalent in males, with only 3 documented cases in female patients worldwide (Table 1). We present the first case to our knowledge of KLS in an adolescent female patient with schizophrenia. The patient's parents gave consent for the publication of this case report.

Risk factors for injuries in landslide- and flood-affected populations in Uganda.

INTRODUCTION: The frequency of occurrence of natural disasters has increased over the past several decades, which necessitates a better understanding of human vulnerability, particularly in low-resource settings. This paper assesses risk factors for injury in the March 2010 floods and landslides in Eastern Uganda, and compares the effects of location, injury type, and severity. METHODS: A stratified cluster survey of the disaster-affected populations was conducted five months after onset of the disasters. Probability proportional to size sampling was used to sample 800 households, including 400 affected by floods in Butaleja District and 400 affected by landslides in Bududa District. RESULTS: Flood- and landslide-affected populations were surveyed in July 2010 using a stratified cluster design. The odds of injury were 65% higher in the flood-affected groups than the landslide-affected groups in a logistic regression (OR = 0.35; 95% CI, 0.24-0.52; P < .001). The injury rate was greater in individuals under 42 years of age, and location of injury was a contributing factor. More people were injured in the flood-affected population as compared with the landslide-affected population, and injuries were more severe. CONCLUSIONS: This study illustrates differences between populations injured by flood and landslide disasters that occurred simultaneously in Eastern Uganda in 2010. In areas where landslides are prone to occur due to massive rainfalls or floods, preventative measures, such as early warning systems and evacuation, are more likely to increase the likelihood of people surviving, while for areas with massive floods, immediate and effective medical attention can save lives and improve injury outcomes.

Postabortal Bleeding: A Diagnostic Dilemma Resolved As Arteriovenous Malformation and Successfully Managed With Uterine Artery Embolization.

Uterine arteriovenous malformations (AVM) are rare and may be missed in routine clinical practice, often concealing their existence until dire consequences emerge. This potentially lethal condition can manifest abruptly, with torrential postabortal bleeding, as a grim reminder of its risky nature. Here, we present a rare case of para 1, living 1, abortion 2, initially subjected to dilatation and evacuation due to a missed abortion at a peripheral healthcare facility, subsequently developing torrential bleeding despite all the conservative measures. So she was referred to our hospital in view of heavy vaginal bleeding following the earlier instrumentation; the differential diagnosis of molar pregnancy and AVM was made clinically. As per speculum examination, the presence of remnants of abortion was seen, and the possibility of an invasive mole was suspected. Ultrasonography with color Doppler showed uterine AVM, which was further confirmed by magnetic resonance imaging (MRI). In order to save her life while preserving the uterus, a multidisciplinary approach was involved in managing this patient, consisting of interventional radiologists to perform uterine artery embolization (UAE).

Family experiences of antenatal counselling of spina bifida: a systematic review.

OBJECTIVE: To describe families' experiences of antenatal counselling of spina bifida. DESIGN: Systematic review. METHODS: MEDLINE, CINAHL, PsycINFO and Embase databases were searched using a combination of Medical Subject Headings and text/abstract terms. Case reports, survey results and qualitative interview data were included. The quality of research was evaluated using the Critical Appraisal Skills Programme checklist. RESULTS: 8 papers were included. Families described shock and grief at diagnosis, with some immediately offered termination of pregnancy (TOP) even though they knew little about the condition. Positive and negative aspects of care were found. Teams that were gentle, kind and empathetic, who did not use jargon, and highlighted positive and negative aspects of the baby's life were seen favourably. Callous language, and overly negative or incorrect counselling was not, particularly if there was pressure to agree to TOP. Families based their decisions on how they would cope, the effect on siblings and the baby's likely quality of life. Prenatal surgery was viewed positively. The views of families who chose TOP, were happy with their care, partners, families, and the LGBTQ+ community were under-represented in the literature. CONCLUSIONS: Unlike other conditions where limited data on outcome exist or the spectrum is genuinely broad, the outcomes of children with spina bifida is well described. Poor aspects of antenatal counselling were described frequently by families, and further work is needed to capture the full spectrum of views on antenatal counselling, how it can be improved, and what training and resources healthcare professionals need to perform it better.

tRNAs Are Stable After All: Pitfalls in Quantification of tRNA from Starved Escherichia coli Cultures Exposed by Validation of RNA Purification Methods.

tRNAs and ribosomal RNAs are often considered stable RNAs. In contrast to this view, we recently proposed that tRNAs are degraded during amino acid starvation and drug-induced transcription inhibition. However, reevaluation of our experimental approach revealed that common RNA extraction methods suffer from alarming extraction and size biases that can lead to gross underestimation of RNA levels in starved Escherichia coli populations. Quantification of tRNAs suffers additional biases due to differing fractions of tRNAs with base modifications in growing versus starved bacteria. Applying an improved methodology, we measured tRNA levels after starvation for amino acids, glucose, phosphate, or ammonium and transcription inhibition by rifampicin. We report that tRNA levels remain largely unaffected in all tested conditions, including several days of starvation. This confirms that tRNAs are remarkably stable RNAs and serves as a cautionary tale about quantification of RNA from cells cultured outside the steady-state growth regime. rRNA, conversely, is extensively degraded during starvation. Thus, E. coli downregulates the translation machinery in response to starvation by reducing the ribosome pool through rRNA degradation, while a high concentration of tRNAs available to supply amino acids to the remaining ribosomes is maintained. IMPORTANCE We show that E. coli tRNAs are remarkably stable during several days of nutrient starvation, although rRNA is degraded extensively under these conditions. The levels of these two major RNA classes are considered to be strongly coregulated at the level of transcription. We demonstrate that E. coli can control the ratio of tRNAs per ribosome under starvation by means of differential degradation rates. The question of tRNA stability in stressed E. coli cells has become subject to debate. Our in-depth analysis of RNA quantification methods reveals hidden technical pitfalls at every step of the analysis, from RNA extraction to target detection and normalization. Most importantly, starved E. coli populations were more resilient to RNA extraction than unstarved populations. The current results underscore that the seemingly trivial task of quantifying an abundant RNA species is not straightforward for cells cultured outside the exponential growth regime.

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids.

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

Deep learning for twelve hour precipitation forecasts.

Existing weather forecasting models are based on physics and use supercomputers to evolve the atmosphere into the future. Better physics-based forecasts require improved atmospheric models, which can be difficult to discover and develop, or increasing the resolution underlying the simulation, which can be computationally prohibitive. An emerging class of weather models based on neural networks overcome these limitations by learning the required transformations from data instead of relying on hand-coded physics and by running efficiently in parallel. Here we present a neural network capable of predicting precipitation at a high resolution up to 12 h ahead. The model predicts raw precipitation targets and outperforms for up to 12 h of lead time state-of-the-art physics-based models currently operating in the Continental United States. The results represent a substantial step towards validating the new class of neural weather models.

TOR kinase activity in Chlamydomonas reinhardtii is modulated by cellular metabolic states.

Target of rapamycin (TOR) kinase is a sensor and a central integrator of internal and external metabolic cues. However, in algae and in higher plants, the components of TOR kinase signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in Chlamydomonas reinhardtii using the phosphorylation status of its putative downstream target, CrS6K. Using this assay, we probe the modulation of cellular TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy, and nitrogen (N) starvation. Importantly, we uncover that excess acetate in the medium leads to high cellular reactive oxygen species levels, triggering autophagy and a concomitant drop in TOR kinase activity in a dose-dependent manner, thus leading to a N-starvation-like cellular phenotype, even when nitrogen is present.

Effect of enhanced detailing and mass media on community use of oral rehydration salts and zinc during a scale-up program in Gujarat and Uttar Pradesh.

BACKGROUND: The Clinton Health Access Initiative implemented a program from 2012-2016 to increase use of oral rehydration salts (ORS) and zinc to treat diarrhea in children under five in three states in India: Gujarat, Madhya Pradesh, and Uttar Pradesh. The program interventions included detailing and development of a rural supply chain to reach private rural health care providers, training of Accredited Social Health Activists (ASHAs), technical support to the state governments, and a mass media campaign targeted at caregivers. In Gujarat and Uttar Pradesh, some of the program activities, such as detailing and ASHA trainings, were targeted to high-burden focal districts, thus providing an opportunity to study their effect compared to statewide activities that covered all districts, such as the mass media campaign. Our study aimed to estimate the effect of activities on ORS and zinc use. METHODS: Household surveys were conducted at two points during the program and in both focal and non-focal districts. We used a difference-in-difference quasi-experimental approach to estimate the effect of the enhanced activities in focal districts and mass media campaign on the odds of a child being treated with ORS and zinc. FINDINGS: Focal district interventions were associated with a significant increase in the odds of a diarrhea episode receiving ORS in Gujarat and Uttar Pradesh. Living in focal districts increased the odds of receiving ORS in Gujarat and Uttar Pradesh by factors of 3.42 (95% CI = 1.39-8.33) and 2.29 (95% CI = 1.19-4.39), respectively. Focal district interventions were also associated with 15.02 (95% CI = 2.97-75.19) greater odds of receiving both ORS and zinc in Gujarat. In Uttar Pradesh, where the mass media campaign was focused, exposure to the campaign further modified the odds of receiving ORS and combined ORS and zinc by 1.38 (95% CI = 1.04-1.84) and 1.57 (95% CI = 1.01-2.46), respectively. CONCLUSION: Comprehensive public and private provider interventions combined with mass media are effective strategies for increasing ORS and zinc use.

Horizontal Transfer of a Synthetic Metabolic Pathway between Plant Species.

Transgene expression from the plastid (chloroplast) genome provides unique advantages, including high levels of foreign protein accumulation, convenient transgene stacking in operons, and increased biosafety due to exclusion of plastids from pollen transmission [1, 2]. However, applications in biotechnology and synthetic biology are severely restricted by the very small number of plant species whose plastid genomes currently can be transformed [3, 4]. Here we report a simple method for the introduction of useful plastid transgenes into non-transformable species. The transgenes tested comprised a synthetic operon encoding three components of a biosynthetic pathway for producing the high-value ketocarotenoid astaxanthin in the plastids of the cigarette tobacco, Nicotiana tabacum. Transplastomic N. tabacum plants accumulated astaxanthin to up to 1% of the plants' dry weight. We then used grafting, a procedure recently shown to facilitate horizontal genome transfer between plants [5-7], to let the transgenic chloroplast genome move across the graft junction from N. tabacum plants into plants of the nicotine-free tree species Nicotiana glauca. Transplastomic N. glauca trees expressing the synthetic pathway were recovered at high frequency, thus providing a straightforward method for extension of the transplastomic technology to new species.

The role of NGOs in child injury prevention: an organizational assessment of one network of NGOs.

Unintentional injuries are estimated to claim the lives of more than 875,000 children each year; millions more live with long-term consequences and permanent disabilities. The epidemiology of injuries has become clearer in the past decade. NGOs need to work in concert with each other to address the global burden of injuries by sharing information. Several NGOs have heeded this call, and the field has seen the emergence of global organizations aimed at highlighting the burden of injuries and streamlining injury prevention activities worldwide. Safe Kids Worldwide Inc. (SKWW) is a global network in 16 countries whose mission is to address the burden of injuries in children under 15 by harnessing the potential of local NGOs. An organizational assessment was conducted of SKWW which included structured organizational assessment, functional organizational mapping and contextual analysis that allowed for an in-depth examination of the strengths and challenges of SKWW's injury prevention approach. Over one year, primary and secondary data were collected and analyzed from headquarters and individual country offices. SKWW appears to be an effective model and has experienced a strong momentum and growth over the last two decades. Global NGOs that address the burden of injuries should start by defining a clear and universal strategic goal, build on local successes, maximize their strengths, and create avenues for stronger country engagement.

Risk factors for mortality in landslide- and flood-affected populations in Uganda.

OBJECTIVE: Assess mortality risk factors including age, sex, and disaster type, in the March 2010 floods and landslides in Eastern Uganda and to compare time period, cause, location, and receipt of medical care among landslide and flood fatalities. DESIGN: A stratified cluster survey was conducted of 802 affected households in community and camp locations. SETTING: Flood and landslide affected populations in the East Uganda the districts of Baduda and Butaleja. RESPONDENTS: Adult household members in 802 households were surveyed regarding household member deaths in the floods and landslides areas. MAIN OUTCOME MEASURES: The primary outcome measure was the odds of death associated with age, sex, and disaster type (flood or landslide). The secondary outcome measure was the odds of event (landslide or flood) among fatalities associated with sex, age, time period of death, and cause of death. RESULTS: The odds of death were significantly higher in landslide affected populations than in flood affected populations (OR 3.06, 95% CI 2.20-4.25, p < 0.001). Time period of death (p = 0.016), type of death (p < 0.001), death after seeking medical care (p = 0.033), month of death (p < 0.001), and cause of death (p < 0.001) were significantly associated with increased odds of death due to landslides as compared to floods. CONCLUSIONS: More deaths occurred due to landslides than floods, and landslide deaths were more immediate, with a majority occurring on the day of the event. Females and younger age groups faced a greater risk of death from the landslide than the flood.