Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties.

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needs.

Transcriptome Analysis and Intraspecific Variation in Spanish Fir (Abies pinsapo Boiss.).

Spanish fir (Abies pinsapo Boiss.) is an endemic, endangered tree that has been scarcely investigated at the molecular level. In this work, the transcriptome of Spanish fir was assembled, providing a large catalog of expressed genes (22,769), within which a high proportion were full-length transcripts (12,545). This resource is valuable for functional genomics studies and genome annotation in this relict conifer species. Two intraspecific variations of A. pinsapo can be found within its largest population at the Sierra de las Nieves National Park: one with standard green needles and another with bluish-green needles. To elucidate the causes of both phenotypes, we studied different physiological and molecular markers and transcriptome profiles in the needles. "Green" trees showed higher electron transport efficiency and enhanced levels of chlorophyll, protein, and total nitrogen in the needles. In contrast, needles from "bluish" trees exhibited higher contents of carotenoids and cellulose. These results agreed with the differential transcriptomic profiles, suggesting an imbalance in the nitrogen status of "bluish" trees. Additionally, gene expression analyses suggested that these differences could be associated with different epigenomic profiles. Taken together, the reported data provide new transcriptome resources and a better understanding of the natural variation in this tree species, which can help improve guidelines for its conservation and the implementation of adaptive management strategies under climatic change.

Is plastidic glutamine synthetase essential for C3 plants? A tale of photorespiratory mutants, ammonium tolerance and conifers.

Agriculture faces the considerable challenge of having to adapt to a progressively changing climate (including the increase in CO2 levels and temperatures); environmental impact must be reduced while at the same time crop yields need to be maintained or increased to ensure food security. Under this scenario, increasing plants' nitrogen (N) use efficiency and minimizing the energy losses associated with photorespiration are two goals of crop breeding that are long sought after. The plastidic glutamine synthetase (GS2) enzyme stands at the crossroads of N assimilation and photorespiration, and is therefore a key candidate for the improvement of crop performance. The GS2 enzyme has long been considered essential for angiosperm survival under photorespiratory conditions. Surprisingly, in Arabidopsis GS2 is not essential for plant survival, and its absence confers tolerance towards ammonium stress, which is in conflict with the idea that NH4+ accumulation is one of the main causes of ammonium stress. Altogether, it appears that the 'textbook' view of this enzyme must be revisited, especially regarding the degree to which it is essential for plant growth under photorespiratory conditions, and the role of NH4+ assimilation during ammonium stress. In this article we open the debate on whether more or less GS2 is a desirable trait for plant productivity.

Periprosthetic joint infections in femoral neck fracture patients treated with hemiarthroplasty - should we use antibiotic-loaded bone cement?

BACKGROUND: Hemiarthroplasty is the most common treatment in elderly patients with displaced intra-capsular femoral neck fracture (FNF). Prosthetic joint infection (PJI) is one of the most feared and frequent complications post-surgery because of the frail health status of these patients and the need for fast track surgery. Therefore, priorities should lie in effective preventive strategies to mitigate this burden. AIM: To determine how much the implementation of the routine use of antibiotic-loaded bone cement (ALBC) as a relatively easy-to-apply amendment to the surgical practice reduces the infection rate in our hemiarthroplasty cohort. METHODS: We retrospectively assessed all demographic, health status and treatment-related data of our FNF patients undergoing cemented hemiarthroplasty in the period from 2011 to 2017; 241 patients were further analyzed after exclusion of patients with cancer-related sequelae and those who died before the end of the 1-year observation period. The PJI rate as diagnosed on basis of the Musculoskeletal Infection Society (MSIS) criteria 2011 was determined for each included patient and compared in function of the bone cement used for hip stem fixation. Patients were split into a group receiving a plain bone cement in the period from January 2011 to June 2013 (non-ALBC group) and into a group receiving an ALBC in the period July 2013 to December 2017 (ALBC group). Data analysis was performed with statistical software. We further calculated the cost-efficacy of the implementation of routine use of ALBC in the second group balancing the in-hospital infection related treatment costs with the extra costs of use of ALBC. RESULTS: In total 241 FNF patients who received cemented hemiarthroplasty in the period from January 2011 to January 2017 were eligible for inclusion in this retrospective study. There were 8 PJI cases identified in the ALBC group among n = 94 patients, whereas 28 PJI cases were observed in the non-ALBC group among n = 147 patients. The statistical analysis showed an infection risk reduction of 55.3% (in particular due to the avoidance of chronic delayed infections) in the ALBC group (95%CI: 6.2%-78.7%; P = 0.0025). The cost-evaluation analysis demonstrated a considerable cost saving of 3.500 € per patient, related to the implementation of routine use of ALBC in this group. CONCLUSION: Use of ALBC is a potent infection preventive factor in FNF patients receiving cemented hemiarthroplasties. It was further found to be highly cost-effective.

A revised view on the evolution of glutamine synthetase isoenzymes in plants.

Glutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS genes of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g. monocots and eudicots) as a result of the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity. This new view of GS evolution in plants, including a new cytosolic GS group (GS1a), has important functional implications in the context of plant metabolism adaptation to global changes.

An Innovative Technique for Intraoperative Radiological Assessment of Pelvic and Spine Fractures in Austere Environment.

INTRODUCTION: Managing pelvic and spine fractures in the austere environment can be challenging even for skilled orthopedic surgeons, largely due to the difficulty of radiological visualization of bone structures. We present a simple alternative to the metallic operating table by placing the patient on a spinal board that will allow for a better radiological assessment of these fractures. MATERIAL AND METHODS: A cross-sectional, descriptive, and retrospective study was carried out in the period between 2015 and 2020. The study population was all patients with pelvic o spine fractures, managed surgically using a spinal board in the Spanish Role 2 or in the Spanish Role 4. RESULTS: Seven patients underwent surgery in total using a spinal board, pelvic fracture being the the main diagnosis (n = 6; 85.71%). The distribution of surgical procedures was as follows: percutaneous pedicle screw fixation (one case), external fixation of the pelvis (two cases), percutaneous screw fixation of the pelvis (two cases), and open reduction and internal fixation of the pelvis (two cases). The mean duration of surgical interventions was 52 minutes, and a successful reduction and synthesis of the fracture was obtained in all the patients, with no complications reported. CONCLUSION: A spinal board can be a useful, simple, and effective device for the orthopedic surgeon in the deployed setting. The described technique is simple, fast, and efficient in getting a complete radiological assessment of complex regions such as the pelvis and the spine, facilitating the surgical management of these injuries.

A Comparative Study Between Two Combat Injury Severity Scores.

INTRODUCTION: In recent years, specific trauma scoring systems have been developed for military casualties. The objective of this study was to examine the discrepancies in severity scores of combat casualties between the Abbreviated Injury Scale 2005-Military (mAIS) and the Military Combat Injury Scale (MCIS) and a review of the current literature on the application of trauma scoring systems in the military setting. METHODS: A cross-sectional, descriptive, and retrospective study was conducted between May 1, 2005, and December 31, 2014. The study population consisted of all combat casualties attended in the Spanish Role 2 deployed in Herat (Afghanistan). We used the New Injury Severity Score (NISS) as reference score. Severity of each injury was calculated according to mAIS and MCIS, respectively. The severity of each casualty was calculated according to the NISS based on the mAIS (Military New Injury Severity Score-mNISS) and MCIS (Military Combat Injury Scale-New Injury Severity Score-MCIS-NISS). Casualty severity were grouped by severity levels (mild-scores: 1-8, moderate-scores: 9-15, severe-scores: 16-24, and critical-scores: 25-75). RESULTS: Nine hundred and eleven casualties were analyzed. Most were male (96.37%) with a median age of 27 years. Afghan patients comprised 71.13%. Air medevac was the main casualty transportation method (80.13). Explosion (64.76%) and gunshot wound (34.68%) mechanisms predominated. Overall mortality was 3.51%. Median mNISS and MCIS-NISS were similar in nonsurvivors (36 [IQR, 25-49] vs. [IQR, 25-48], respectively) but different in survivors, 9 (IQR, 4-17) vs. 5 (IQR, 2-13), respectively (P < .0001). The mNISS and MCIS-NISS were discordant in 34.35% (n = 313). Among cases with discordant severity scores, the median difference between mNISS and MCIS-NISS was 9 (IQR, 4-16); range, 1 to 57. CONCLUSION: Our study findings suggest that discrepancies in injury severity levels may be observed in one in three of the casualties when using mNISS and MCIS-NISS.

Ammonium regulates the development of pine roots through hormonal crosstalk and differential expression of transcription factors in the apex.

Ammonium is a prominent source of inorganic nitrogen for plant nutrition, but excessive amounts can be toxic for many species. However, most conifers are tolerant to ammonium, a relevant physiological feature of this ancient evolutionary lineage. For a better understanding of the molecular basis of this trait, ammonium-induced changes in the transcriptome of maritime pine (Pinus pinaster Ait.) root apex have been determined by laser capture microdissection and RNA sequencing. Ammonium promoted changes in the transcriptional profiles of multiple transcription factors, such as SHORT-ROOT, and phytohormone-related transcripts, such as ACO, involved in the development of the root meristem. Nano-PALDI-MSI and transcriptomic analyses showed that the distributions of IAA and CKs were altered in the root apex in response to ammonium nutrition. Taken together, the data suggest that this early response is involved in the increased lateral root branching and principal root growth, which characterize the long-term response to ammonium supply in pine. All these results suggest that ammonium induces changes in the root system architecture through the IAA-CK-ET phytohormone crosstalk and transcriptional regulation.

Epitranscriptome changes triggered by ammonium nutrition regulate the proteome response of maritime pine roots.

Epitranscriptome constitutes a gene expression checkpoint in all living organisms. Nitrogen is an essential element for plant growth and development that influences gene expression at different levels such as epigenome, transcriptome, proteome, and metabolome. Therefore, our hypothesis is that changes in the epitranscriptome may regulate nitrogen metabolism. In this study, epitranscriptomic modifications caused by ammonium nutrition were monitored in maritime pine roots using Oxford Nanopore Technology. Transcriptomic responses mainly affected transcripts involved in nitrogen and carbon metabolism, defense, hormone synthesis/signaling, and translation. Global detection of epitranscriptomic marks was performed to evaluate this posttranscriptional mechanism in un/treated seedlings. Increased N6-methyladenosine (m6A) deposition in the 3'-UTR was observed in response to ammonium, which seems to be correlated with poly(A) lengths and changes in the relative abundance of the corresponding proteins. The results showed that m6A deposition and its dynamics seem to be important regulators of translation under ammonium nutrition. These findings suggest that protein translation is finely regulated through epitranscriptomic marks likely by changes in mRNA poly(A) length, transcript abundance and ribosome protein composition. An integration of multiomics data suggests that the epitranscriptome modulates responses to nutritional, developmental and environmental changes through buffering, filtering, and focusing the final products of gene expression.

Inorganic Nitrogen Form Determines Nutrient Allocation and Metabolic Responses in Maritime Pine Seedlings.

Nitrate and ammonium are the main forms of inorganic nitrogen available to plants. The present study aimed to investigate the metabolic changes caused by ammonium and nitrate nutrition in maritime pine (Pinus pinaster Ait.). Seedlings were grown with five solutions containing different proportions of nitrate and ammonium. Their nitrogen status was characterized through analyses of their biomass, different biochemical and molecular markers as well as a metabolite profile using 1H-NMR. Ammonium-fed seedlings exhibited higher biomass than nitrate-fed-seedlings. Nitrate mainly accumulated in the stem and ammonium in the roots. Needles of ammonium-fed seedlings had higher nitrogen and amino acid contents but lower levels of enzyme activities related to nitrogen metabolism. Higher amounts of soluble sugars and L-arginine were found in the roots of ammonium-fed seedlings. In contrast, L-asparagine accumulated in the roots of nitrate-fed seedlings. The differences in the allocation of nitrate and ammonium may function as metabolic buffers to prevent interference with the metabolism of photosynthetic organs. The metabolite profiles observed in the roots suggest problems with carbon and nitrogen assimilation in nitrate-supplied seedlings. Taken together, this new knowledge contributes not only to a better understanding of nitrogen metabolism but also to improving aspects of applied mineral nutrition for conifers.

NADH-GOGAT Overexpression Does Not Improve Maize (Zea mays L.) Performance Even When Pyramiding with NAD-IDH, GDH and GS.

Maize plants overexpressing NADH-GOGAT were produced in order to determine if boosting 2-Oxoglurate production used as a carbon skeleton for the biosynthesis of amino acids will improve plant biomass and kernel production. The NADH-GOGAT enzyme recycles glutamate and incorporates carbon skeletons into the ammonium assimilation pathway using the organic acid 2-Oxoglutarate as a substrate. Gene pyramiding was then conducted with NAD-IDH and NADH-GDH, two enzymes also involved in the synthesis of 2-Oxoglurate. NADH-GOGAT overexpression was detrimental for shoot biomass production but did not markedly affect kernel yield. Additional NAD-IDH and NADH-GDH activity did not improve plant performance. A decrease in kernel production was observed when NADH-GDH was pyramided to NADH-GOGAT and NAD-IDH. This decrease could not be restored even when additional cytosolic GS activity was present in the plants overexpressing the three enzymes producing 2-Oxoglutarate. Detailed leaf metabolic profiling of the different transgenic plants revealed that the NADH-GOGAT over-expressors were characterized by an accumulation of amino acids derived from glutamate and a decrease in the amount of carbohydrates further used to provide carbon skeletons for its synthesis. The study suggests that 2-Oxoglutarate synthesis is a key element acting at the interface of carbohydrate and amino acid metabolism and that its accumulation induces an imbalance of primary carbon and nitrogen metabolism that is detrimental for maize productivity.

Strategic Orthopedic Evacuations to the Spanish Role 4 During a Decade (2009-2018).

INTRODUCTION: Casualty evacuation is a key point in medical support to military operations, sometimes being necessary to transfer them to National Territory for a definitive diagnosis and treatment. The aim of this work is to analyze the patients evacuated from Areas of Operations to the Orthopedic Surgery and Traumatology Unit of the Spanish Role 4 Medical Treatment Facility in the last 10 years. MATERIAL AND METHODS: A cross-sectional, descriptive, and retrospective study carried out in the period between January 1, 2009 and December 31, 2018. The study population was all personnel evacuated from the Area of Operations to Spanish Role 4. For categorical variables, absolute and relative percent frequencies were used. Spanish military authorization was obtained to perform this study. This study has been approved by the Ethics and Clinical Research Committee of the Defense Central Hospital "Gómez Ulla" (code 12/17). RESULTS: A total of 520 medical evacuations have been performed on Role 4, of which 227 were on the Orthopedic Surgery and Traumatology Unit. Seven percent of the evacuees were categorized as "combat" casualties. The areas of operations from which more patients have been evacuated were Afghanistan and Lebanon, 30.39% and 19.38%, respectively. The most frequent lesion pattern was the fracture affecting the extremities. Accidents (n = 98, 43.17%) and sport (n = 57; 25.3%) were the main causes of injury. Sixty-seven (29.51%) patients were treated surgically in Role 4. No deaths were recorded among patients in this series. CONCLUSION: Evacuations because of trauma cause more than half of the medical repatriations carried out on the National Territory, of which the majority are due to fractures affecting the limbs, especially the bones of the hand. Sport was the first preventable cause of injury among evacuated patients. Our results are similar to the experience obtained by other allied armed forces. It is a moral imperative and a fundamental necessity for the Spanish military medical services to promote and maintain the Spanish Role 4 Medical Treatment Facility as an indispensable element in medical support for international missions.

Multi-stage surgery for a multiple-level spondylodiscitis caused by multidrug-resistant Mycobacterium avium complex.

The objective of this article is to report a rare case of atypical mycobacterial spinal spondylodiscitis with multiple-level involvement and the successful treatment by multi-stage surgical intervention. Reports on the surgical management of atypical mycobacterial spondylodiscitis are lacking. A 71-year-old woman with a confirmed diagnosis of multiple-level spondylodiscitis of L2-L3 and L5-S1 caused by Mycobacterium avium complex (MAC). The patient underwent a two-stage surgical treatment (first: posterior instrumentation; second: anterior debridement with anterior lumbar interbody fusion). At 1 year after surgery, the patient suffered a proximal junctional failure secondary to a vertebral fracture that was solved with a proximal extension of the fusion using a percutaneous technique. The patient was successfully discharged with good pain control, satisfactory correction, no neurologic complications and an overall satisfactory outcome. A rare case of antibiotic-resistant multi-level spondylodiscitis due to MAC was treated successfully with multi-stage surgical treatment. Surgery in this patient group remains challenging due to the technical complexities and the difficulty of choosing the instrumentation levels.

Understanding plant nitrogen nutrition through a laboratory experiment.

Plant nitrogen nutrition is an essential topic in biology that should be included in scientific education. Nitrogen availability is one of the primary limiting factors for plant growth, and these organisms are the primary support for the pluricellular terrestrial life, since they are the fundamental group of autotrophs. For this reason, the use of nitrogen fertilizers is in the basis of the Green Revolution that led to an extraordinary increase in the food production during the twentieth century. To illustrate the importance of plant nitrogen nutrition, a new laboratory experience for students is presented in this manuscript. The aim of the following laboratory teaching activity is training of students through the evaluation of metabolic, biochemical, and molecular markers that are related to the nitrogen nutritional status of plants. For this purpose, cherry tomato plants (Solanum lycopersicum var. cerasiforme) were used, since they exhibit rapid growth and good differential biomass accumulation in response to changes in the nitrogen supply. The proposed laboratory experiment enables the development of the entire protocol for postgraduate students or in a reduced version for students from lower educational levels. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):450-458, 2019.

Use of Oxygen Tissue Monitoring in Patients With Compartment Syndrome: Two Clinical Cases and Literature Review.

Compartment syndrome is defined as the pathology caused by the increase of pressure within a muscular compartment to the point where the vascular perfusion necessary for the viability of the tissues included therein is reduced. The diagnosis is established by clinical exam and pressure measurement. Measurement of intracompartmental pressure is an invasive method with no option of easy continuous monitoring. Continuous tissue oximetry, using near-infrared light spectroscopy, can estimate soft-tissue oxygenation several centimeters below the sensor placement. This method of monitoring has been used successfully in the diagnosis of compartment syndrome, presenting itself as a non-invasive method of continuous measurement that can be a very useful alternative in complex situations or doubtful cases. We present two clinical cases of patients with acute compartment syndrome, in which the use of near-infrared light spectroscopy was determinant, both for the diagnosis and to verify the surgical treatment performed.

Can we achieve universal health coverage without a focus on disability? Results from a national case-control study in Guatemala.

OBJECTIVE: To compare access to healthcare services for people with disabilities to those without disabilities, within a national case-control study in Guatemala. METHODS: We undertook a population-based case-control study, nested within a national survey in Guatemala. Cases with disabilities were people with self-reported difficulties in functioning. One control without disabilities was selected per case, matched by age, gender and cluster. Information was collected on: health status, access to health services and rehabilitation, and socioeconomic status. RESULTS: The study included 707 people with disabilities, and 465 controls. People with disabilities were more likely to report a serious health problem (aOR 2.8, 2.2-3.7) or doctor-diagnosis of one of 17 general health conditions (aOR 2.9, 2.2-3.8) as compared to controls without disabilities. People with disabilities were twice as likely as controls to have received treatment for a diagnosed condition (aOR 2.2, 1.7-2.8). Coverage of treatment for impairment-related health conditions was low, as was awareness and access to rehabilitation services. People with disabilities were more likely than controls to report being disrespected (aOR 1.9, 1.0-3.7) or finding it difficult to understand information given (aOR 1.6, 1.1-1.4). CONCLUSION: Efforts are needed to raise awareness about rehabilitation services and improve quality of health services for people with disabilities in Guatemala, to ensure that their rights are fulfilled and to assist in the achievement of Universal Health Coverage. Better tools are needed to measure healthcare access, including consideration of geographic access, quality and affordability, to allow the generation of comparable data on access to healthcare among people with disabilities.

Nitrogen Metabolism and Biomass Production in Forest Trees.

Low nitrogen (N) availability is a major limiting factor for tree growth and development. N uptake, assimilation, storage and remobilization are key processes in the economy of this essential nutrient, and its efficient metabolic use largely determines vascular development, tree productivity and biomass production. Recently, advances have been made that improve our knowledge about the molecular regulation of acquisition, assimilation and internal recycling of N in forest trees. In poplar, a model tree widely used for molecular and functional studies, the biosynthesis of glutamine plays a central role in N metabolism, influencing multiple pathways both in primary and secondary metabolism. Moreover, the molecular regulation of glutamine biosynthesis is particularly relevant for accumulation of N reserves during dormancy and in N remobilization that takes place at the onset of the next growing season. The characterization of transgenic poplars overexpressing structural and regulatory genes involved in glutamine biosynthesis has provided insights into how glutamine metabolism may influence the N economy and biomass production in forest trees. Here, a general overview of this research topic is outlined, recent progress are analyzed and challenges for future research are discussed.

Labeling Maize (Zea mays L.) Leaves with 15 NH4+ and Monitoring Nitrogen Incorporation into Amino Acids by GC/MS Analysis.

The human body contains approximately 3.2% nitrogen (N), mainly present as protein and amino acids. Although N exists at a high concentration (78%) in the air, it is not readily available to animals and most plants. Plants are however able to take up both nitrate (NO3- ) and ammonium (NH4+ ) ions from the soil and convert them to amino acids and proteins, which are excellent sources for all animals. Most N is available as the stable isotope 14 N, but a second form, 15 N, is present in very low concentrations. 15 N can be detected in extracts of plants by gas chromatography followed by mass spectrometry (GC/MS). In this protocol, the methods are described for tracing the pathway by which plants are able to take up 15 N-labeled nitrate and ammonium and convert them into amino acids and proteins. A protocol for extracting and quantifying amino acids and 15 N enrichment in maize (Zea mays L.) leaves labeled with 15 NH4+ is described. Following amino acid extraction, purification, and separation by GC/MS, a calculation of the 15 N enrichment of each amino acid is carried out on a relative basis to identify any differences in the dynamics of amino acid accumulation. This will allow a study of the impact of genetic modifications or mutations on key reactions involved in primary nitrogen and carbon metabolism. © 2018 by John Wiley & Sons, Inc.