Preclinical Proficiency-Based Model of Ultrasound Training.

BACKGROUND: Graduate medical education is being transformed from a time-based training model to a competency-based training model. While the application of ultrasound in the perioperative arena has become an expected skill set for anesthesiologists, clinical exposure during training is intermittent and nongraduated without a structured program. We developed a formal structured perioperative ultrasound program to efficiently train first-year clinical anesthesia (CA-1) residents and evaluated its effectiveness quantitatively in the form of a proficiency index. METHODS: In this prospective study, a multimodal perioperative ultrasound training program spread over 3 months was designed by experts at an accredited anesthesiology residency program to train the CA-1 residents. The training model was based on self-learning through web-based modules and instructor-based learning by performing perioperative ultrasound techniques on simulators and live models. The effectiveness of the program was evaluated by comparing the CA-1 residents who completed the training to graduating third-year clinical anesthesia (CA-3) residents who underwent the traditional ultrasound training in the residency program using a designed index called a "proficiency index." The proficiency index was composed of scores on a cognitive knowledge test (20%) and scores on an objective structured clinical examination (OSCE) to evaluate the workflow understanding (40%) and psychomotor skills (40%). RESULTS: Sixteen CA-1 residents successfully completed the perioperative ultrasound training program and the subsequent evaluation with the proficiency index. The total duration of training was 60 hours of self-based learning and instructor-based learning. There was a significant improvement observed in the cognitive knowledge test scores for the CA-1 residents after the training program (pretest: 71% [0.141 ± 0.019]; posttest: 83% [0.165 ± 0.041]; P < .001). At the end of the program, the CA-1 residents achieved an average proficiency index that was not significantly different from the average proficiency index of graduating CA-3 residents who underwent traditional ultrasound training (CA-1: 0.803 ± 0.049; CA-3: 0.823 ± 0.063, P = .307). CONCLUSIONS: Our results suggest that the implementation of a formal, structured curriculum allows CA-1 residents to achieve a level of proficiency in perioperative ultrasound applications before clinical exposure.

Faculty-Focused Perioperative Ultrasound Training Program: A Single-Center Experience.

OBJECTIVE: Comprehensive educational initiatives in ultrasound for practicing physicians are lacking. We developed a perioperative ultrasound training program for faculty to offer a broad orientation to the principles and clinical applications of ultrasound. DESIGN AND SETTING: The program consisted of two phases. Phase one, which introduced ultrasound via didactic and hands-on components, had six live sessions. At the end of each, participants completed a knowledge quiz. During the sixth session, faculty participated in an objective structured clinical examination (OSCE). For phase two, faculty attended two to three sessions (8-10 hours each) of in-depth individualized training and demonstrated supervised performance of ultrasound-related procedures of their choice. PARTICIPANTS: Participants included Anesthesia faculty at Beth Israel Deaconess Medical Center. MEASUREMENTS AND MAIN RESULTS: On average 30 faculty members attended each live session for phase one; 12 completed phase two. There was a significant difference in quiz scores across the six sessions (p < 0.001) with scores for Session 6 being significantly higher than for Session 1 (p < 0.001). The average mean and median scores on the three OSCE stations were 95.63% and 98.33%, respectively. For phase two, the 8 participants who received training in regional anesthesia each performed > 10 blocks on patients over two days; 5 of the 7 participants who received training in transthoracic echocardiography each completed more than 15 examinations on simulators and 10 examinations on patients. CONCLUSION: It is possible to implement a departmental educational program for ultrasound to improve ultrasound knowledge and skills in practicing anesthesiologists.

Summative Objective Structured Clinical Examination Assessment at the End of Anesthesia Residency for Perioperative Ultrasound.

While standardized examinations and data from simulators and phantom models can assess knowledge and manual skills for ultrasound, an Objective Structured Clinical Examination (OSCE) could assess workflow understanding. We recruited 8 experts to develop an OSCE to assess workflow understanding in perioperative ultrasound. The experts used a binary grading system to score 19 graduating anesthesia residents at 6 stations. Overall average performance was 86.2%, and 3 stations had an acceptable internal reliability (Kuder-Richardson formula 20 coefficient >0.5). After refinement, this OSCE can be combined with standardized examinations and data from simulators and phantom models to assess proficiency in ultrasound.

Ultrasound as a Screening Tool for Central Venous Catheter Positioning and Exclusion of Pneumothorax.

OBJECTIVES: Although real-time ultrasound guidance during central venous catheter insertion has become a standard of care, postinsertion chest radiograph remains the gold standard to confirm central venous catheter tip position and rule out associated lung complications like pneumothorax. We hypothesize that a combination of transthoracic echocardiography and lung ultrasound is noninferior to chest radiograph when used to accurately assess central venous catheter positioning and screen for pneumothorax. SETTING: All operating rooms and surgical and trauma ICUs at the institution. DESIGN: Single-center, prospective noninferiority study. PATIENTS: Patients receiving ultrasound-guided subclavian or internal jugular central venous catheters. INTERVENTIONS: During ultrasound-guided central venous catheter placement, correct positioning of central venous catheter was accomplished by real-time visualization of the guide wire and positive right atrial swirl sign using the subcostal four-chamber view. After insertion, pneumothorax was ruled out by the presence of lung sliding and seashore sign on M-mode. MEASUREMENTS AND MAIN RESULTS: Data analysis was done for 137 patients. Chest radiograph ruled out pneumothorax in 137 of 137 patients (100%). Lung ultrasound was performed in 123 of 137 patients and successfully screened for pneumothorax in 123 of 123 (100%). Chest radiograph approximated accurate catheter tip position in 136 of 137 patients (99.3%). Adequate subcostal four-chamber views could not be obtained in 13 patients. Accurate positioning of central venous catheter with ultrasound was then confirmed in 121 of 124 patients (97.6%) as described previously. CONCLUSIONS: Transthoracic echocardiography and lung ultrasound are noninferior to chest x-ray for screening of pneumothorax and accurate central venous catheter positioning. Thus, the point of care use of ultrasound can reduce central venous catheter insertion to use time, exposure to radiation, and improve patient safety.

Genome-wide analysis of heavy metal ATPases (HMAs) in Poaceae species and their potential role against copper stress in Triticum aestivum.

Plants require copper for normal growth and development and have evolved an efficient system for copper management based on transport proteins such as P1B-ATPases, also known as heavy metal ATPases (HMAs). Here, we report HMAs in eleven different Poaceae species, including wheat. Furthermore, the possible role of wheat HMAs in copper stress was investigated. BlastP searches identified 27 HMAs in wheat, and phylogenetic analysis based on the Maximum Likelihood method demonstrated a separation into four distinct clades. Conserved motif analysis, domain identification, gene structure, and transmembrane helices number were also identified for wheat HMAs using computational tools. Wheat seedlings grown hydroponically were subjected to elevated copper and demonstrated toxicity symptoms with effects on fresh weight and changes in expression of selected HMAs TaHMA7, TaHMA8, and TaHMA9 were upregulated in response to elevated copper, suggesting a role in wheat copper homeostasis. Further investigations on these heavy metal pumps can provide insight into strategies for enhancing crop heavy metal tolerance in the face of heavy metal pollution.

An integrated remediation approach using combinations of biochar, Rhizobium leguminosarum, and Vigna radiata for immobilizing and dissipating cadmium contaminants from the soil-mustard plant system.

Cadmium (Cd) contamination of soils is an environmental concern, as cadmium harms food crops and can therefore impact human health. The use of combinations of biochar (seeded with Rhizobium leguminosarum) and Vigna radiata (as an intercrop) has the potential to reduce the mobilization of Cd from soil via mustard plants (Brassica juncea). Mustard plants are grown as a food and oil production crop that is consumed worldwide. However, this plant has the property of hyperaccumulation; thus, it bioaccumulates Cd in its tissues, which in turn, if eaten, can become part of the human food chain. Hence, reducing Cd bioaccumulation in mustard plants is crucial to making these plants a reliable and safe source of food for consumption. To improve soil sorption capacity and immobilization efficiency, biochar (in the form of wheat husk) was mixed with R. leguminosarum and intercropped (using V. radiata) with mustard plants for further investigation. Sampling was performed at an early growth stage (i.e., at 30 days) and at maturity (i.e., at 60 days) to determine the impact of Cd on a plant's morphophysiological attributes. Data were analyzed in two ways: first by analysis of variance (ANOVA) and then by the post hoc Tukey's honestly significant difference (HSD) test. The statistical analysis concluded that combinations effectively improved plant traits by 65%-90% in the early growth stage and by 70%-90% in the maturity stage. The T6 treatment combination [i.e., biochar + R. leguminosarum + V. radiata (BC + RL + VR)] provided the most effective results in terms of growth, biomass, pod yield, and pigmentation content. In addition, this combination reduced the translocation of Cd in mustard plants by 70%-95%. The combination of BC + RL + VR effectively reduced Cd contamination of mustard tissue and provided a suitable growing environment for the plants. A post-harvesting soil analysis using X-ray diffraction (XRD) found that Cd was undetectable in soil. This provides clear confirmation that these approaches can lead to Cd soil remediation. Moreover, this study provided insight into the responses of different morphophysiological attributes of mustard plants to Cd stress and could aid in developing Cd stress tolerance in mustard plants.

Plant natural products: history, limitations and the potential of cambial meristematic cells.

Humans have utilised plant derived natural products as medicines for millenia. Moreover, many contemporary pharmaceuticals are also natural products or derivatives thereof. However, the full potential of these compounds remains to be exploited because often they are: complex and difficult to synthesise; found in low quantities; produced by undomesticated and sometimes rare plants; and, their synthesis is routinely influenced by weather conditions. Potentially, the in vitro culture of cells from the corresponding plant species could circumvent some of these problems but the growth of plant cells on an industrial scale is also problematic. The recent isolation and culture of cambial meristematic cells (CMCs), stem cells which ordinarily generate the plant vasculature, may now provide a key platform technology to help realise the full potential of plant natural products.

Genome-wide identification, comprehensive characterization of transcription factors, cis-regulatory elements, protein homology, and protein interaction network of DREB gene family in Solanum lycopersicum.

Tomato is a drought-sensitive crop which has high susceptibility to adverse climatic changes. Dehydration-responsive element-binding (DREB) are significant plant transcription factors that have a vital role in regulating plant abiotic stress tolerance by networking with DRE/CRT cis-regulatory elements in response to stresses. In this study, bioinformatics analysis was performed to conduct the genome-wide identification and characterization of DREB genes and promoter elements in Solanum lycopersicum. In genome-wide coverage, 58 SlDREB genes were discovered on 12 chromosomes that justified the criteria of the presence of AP2 domain as conserved motifs. Intron-exon organization and motif analysis showed consistency with phylogenetic analysis and confirmed the absence of the A3 class, thus dividing the SlDREB genes into five categories. Gene expansion was observed through tandem duplication and segmental duplication gene events in SlDREB genes. Ka/Ks values were calculated in ortholog pairs that indicated divergence time and occurrence of purification selection during the evolutionary period. Synteny analysis demonstrated that 32 out of 58 and 47 out of 58 SlDREB genes were orthologs to Arabidopsis and Solanum tuberosum, respectively. Subcellular localization predicted that SlDREB genes were present in the nucleus and performed primary functions in DNA binding to regulate the transcriptional processes according to gene ontology. Cis-acting regulatory element analysis revealed the presence of 103 motifs in 2.5-kbp upstream promoter sequences of 58 SlDREB genes. Five representative SlDREB proteins were selected from the resultant DREB subgroups for 3D protein modeling through the Phyre2 server. All models confirmed about 90% residues in the favorable region through Ramachandran plot analysis. Moreover, active catalytic sites and occurrence in disorder regions indicated the structural and functional flexibility of SlDREB proteins. Protein association networks through STRING software suggested the potential interactors that belong to different gene families and are involved in regulating similar functional and biological processes. Transcriptome data analysis has revealed that the SlDREB gene family is engaged in defense response against drought and heat stress conditions in tomato. Overall, this comprehensive research reveals the identification and characterization of SlDREB genes that provide potential knowledge for improving abiotic stress tolerance in tomato.

Identification and Expression Analysis of Stilbene Synthase Genes in Arachis hypogaea in Response to Methyl Jasmonate and Salicylic Acid Induction.

Stilbene synthase is an important enzyme of the phenylpropanoid pathway, regulating the production of several biologically active stilbenoids. These compounds have antioxidant, anti-inflammatory, and anti-cancer properties. However, the detailed characterization of stilbene synthase genes in Arachis hypogaea has not yet been performed. In this study, the comprehensive characterization of stilbene synthase genes in A. hypogaea was conducted, commencing with identification, phylogenetic analysis, and study of their expression in response to exogenous hormonal treatment. We identified and isolated five AhSTSs genes and recorded their expression pattern in peanut (BARD-479) in response to methyl jasmonate (MeJA) and salicylic acid (SA) treatment. The presence of Chal_sti_synt, ACP_syn_III, and FAE1_CUT1_rppA domains in all AhSTSs indicated their role in the biosynthesis of stilbene and lipid metabolism. Cis-regulatory element analysis indicated their role in light responsiveness, defense responses, regulation of seed development, plant growth, and development. Despite close structural and functional similarities, expression and correlational analysis suggested that these genes may have a specific role in peanut, as individual AhSTS exhibited differential expression upon hormonal treatment in a genotype dependent manner. Further studies on functional characterization involving the transcriptional regulation of AhSTSs can clearly explain the differential expression of stilbene synthase genes to hormonal treatment.

Genome wide identification and characterization of nodulation related genes in Arachis hypogaea.

Nitrogen is an important plant nutrient that has a significant role in crop yield. Hence, to fulfill the needs of sustainable agriculture, it is necessary to improve biological nitrogen fixation in leguminous crops. Nod inducing gene families plays a crucial role in the interaction between rhizobia and legumes, leading to biological nitrogen fixation. However, nod inducing genes identification and characterization has not yet been performed in Arachis hypogaea. In this study, identification and genome-wide analysis of nod inducing genes are performed so that to explore their potential functions in the Arachis hypogaea for the first time. Nod genes were comprehensively analyzed by phylogenetic clustering analysis, gene structure determination, detection of conserved motifs, subcellular localization, conserved motifs, cis-acting elements and promoter region analysis. This study identified 42 Nod inducing genes in Arachis hypogaea, their sequences were submitted to NCBI and accession numbers were obtained. Potential involvement of these genes in biological nitrogen fixation has been unraveled, such as, phylogenetic analysis revealed that nod inducing genes evolved independently in Arachis hypogaea, the amino acid structures exhibited 20 highly conserved motifs, the proteins are present at different locations in cells and the gene structures revealed that all the genes are full-length genes with upstream intronic regions. Further, the promoter analysis determined a large number of cis-regulatory elements involved in nodulation. Moreover, this study not only provides identification and characterization of genes underlying developmental and functional stages of nodulation and biological nitrogen fixation but also lays the foundation for further revelation of nod inducing gene family. Besides, identification and structural analysis of these genes in Arachis hypogaea may provide a theoretical basis for the study of evolutionary relationships in future analysis.

Genome-wide analysis, identification, evolution and genomic organization of dehydration responsive element-binding (DREB) gene family in Solanum tuberosum.

BACKGROUND: The dehydration responsive element-binding (DREB) gene family plays a crucial role as transcription regulators and enhances plant tolerance to abiotic stresses. Although the DREB gene family has been identified and characterized in many plants, knowledge about it in Solanum tuberosum (Potato) is limited. RESULTS: In the present study, StDREB gene family was comprehensively analyzed using bioinformatics approaches. We identified 66 StDREB genes through genome wide screening of the Potato genome based on the AP2 domain architecture and amino acid conservation analysis (Valine at position 14th). Phylogenetic analysis divided them into six distinct subgroups (A1-A6). The categorization of StDREB genes into six subgroups was further supported by gene structure and conserved motif analysis. Potato DREB genes were found to be distributed unevenly across 12 chromosomes. Gene duplication proved that StDREB genes experienced tandem and segmental duplication events which led to the expansion of the gene family. The Ka/Ks ratios of the orthologous pairs also demonstrated the StDREB genes were under strong purification selection in the course of evolution. Interspecies synteny analysis revealed 45 and 36 StDREB genes were orthologous to Arabidopsis and Solanum lycopersicum, respectively. Moreover, subcellular localization indicated that StDREB genes were predominantly located within the nucleus and the StDREB family's major function was DNA binding according to gene ontology (GO) annotation. CONCLUSIONS: This study provides a comprehensive and systematic understanding of precise molecular mechanism and functional characterization of StDREB genes in abiotic stress responses and will lead to improvement in Solanum tuberosum.

Genome-wide promoter analysis, homology modeling and protein interaction network of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum.

Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes, and hence plays a pivotal role in abiotic stress responses and tolerance in plants. The study aimed to develop a complete overview of the cis-acting regulatory elements (CAREs) present in S. tuberosum DREB gene promoters. A total of one hundred and four (104) cis-regulatory elements (CREs) were identified from 2.5kbp upstream of the start codon (ATG). The in-silico promoter analysis revealed variable sets of cis-elements and functional diversity with the predominance of light-responsive (30%), development-related (20%), abiotic stress-responsive (14%), and hormone-responsive (12%) elements in StDREBs. Among them, two light-responsive elements (Box-4 and G-box) were predicted in 64 and 61 StDREB genes, respectively. Two development-related motifs (AAGAA-motif and as-1) were abundant in StDREB gene promoters. Most of the DREB genes contained one or more Myeloblastosis (MYB) and Myelocytometosis (MYC) elements associated with abiotic stress responses. Hormone-responsive element i.e. ABRE was found in 59 out of 66 StDREB genes, which implied their role in dehydration and salinity stress. Moreover, six proteins were chosen corresponding to A1-A6 StDREB subgroups for secondary structure analysis and three-dimensional protein modeling followed by model validation through PROCHECK server by Ramachandran Plot. The predicted models demonstrated >90% of the residues in the favorable region, which further ensured their reliability. The present study also anticipated pocket binding sites and disordered regions (DRs) to gain insights into the structural flexibility and functional annotation of StDREB proteins. The protein association network determined the interaction of six selected StDREB proteins with potato proteins encoded by other gene families such as MYB and NAC, suggesting their similar functional roles in biological and molecular pathways. Overall, our results provide fundamental information for future functional analysis to understand the precise molecular mechanisms of the DREB gene family in S. tuberosum.

Expression Characterization of Flavonoid Biosynthetic Pathway Genes and Transcription Factors in Peanut Under Water Deficit Conditions.

Drought is one of the hostile environmental stresses that limit the yield production of crop plants by modulating their growth and development. Peanut (Arachis hypogaea) has a wide range of adaptations to arid and semi-arid climates, but its yield is prone to loss due to drought. Other than beneficial fatty acids and micronutrients, peanut harbors various bioactive compounds including flavonoids that hold a prominent position as antioxidants in plants and protect them from oxidative stress. In this study, understanding of the biosynthesis of flavonoids in peanut under water deficit conditions was developed through expression analysis and correlational analysis and determining the accumulation pattern of phenols, flavonols, and anthocyanins. Six peanut varieties (BARD479, BARI2011, BARI2000, GOLDEN, PG1102, and PG1265) having variable responses against drought stress have been selected. Higher water retention and flavonoid accumulation have been observed in BARI2011 but downregulation has been observed in the expression of genes and transcription factors (TFs) which indicated the maintenance of normal homeostasis. ANOVA revealed that the expression of flavonoid genes and TFs is highly dependent upon the genotype of peanut in a spatiotemporal manner. Correlation analysis between expression of flavonoid biosynthetic genes and TFs indicated the role of AhMYB111 and AhMYB7 as an inhibitor for AhF3H and AhFLS, respectively, and AhMYB7, AhTTG1, and AhCSU2 as a positive regulator for the expression of Ah4CL, AhCHS, and AhF3H, respectively. However, AhbHLH and AhGL3 revealed nil-to-little relation with the expression of flavonoid biosynthetic pathway genes. Correlational analysis between the expression of TFs related to the biosynthesis of flavonoids and the accumulation of phenolics, flavonols, and anthocyanins indicated coregulation of flavonoid synthesis by TFs under water deficit conditions in peanut. This study would provide insight into the role of flavonoid biosynthetic pathway in drought response in peanut and would aid to develop drought-tolerant varieties of peanut.

Molecular characterization of Leucoanthocyanidin reductase and Flavonol synthase gene in Arachis hypogaea.

Arachis hypogaea (peanut) is a potential source of bioactive compounds including flavonols and proanthocyanidins, which have gained particular interest of metabolic engineering owing to their significance in the growth, development and defense responses in plants. To gain insight of proanthocyanidins and flavonols production in A. hypogaea, Leucoanthocyanidin reductase (AhLAR) and Flavonol synthase (AhFLS) enzymes responsible for their production, have been structurally, transcriptionally and functionally characterized. Structural and functional analysis of putative protein sequence of AhFLS indicated two functional motifs 2OG-FeII_Oxy and DIOX_N, while six functional motifs belonging to the families of NAD-dependent dehydratase, 3, ß hydroxysteroid dehydrogenase and NmrA-like family were observed in case of AhLAR. Promoter sequence analysis unraveled several promoter elements related to the development regulation, environmental stress responses and hormonal signaling. Furthermore, the expression analysis of AhFLS and AhLAR and accumulation pattern analysis of proanthocyanidins and flavonols in three selected cultivars of A. hypogaea under saline environment confirmed their role against salinity in genotype-dependent and stress level-dependent manner. Correlation studies revealed that AhFLS and AhLAR expression is not directly dependent on the antioxidant enzymes activity, biochemical and growth parameters but higher Pearson r value depicted some level of dependency. This detailed study of AhLAR and AhFLS can assist in the metabolic engineering of flavonoid biosynthetic pathway to produce stress tolerant varieties and production of proanthocyanidins and flavonols at an industrial scale.

Evaluating the cleavage efficacy of CRISPR-Cas9 sgRNAs targeting ineffective regions of Arabidopsis thaliana genome.

The CRISPR-Cas9 system has recently evolved as a powerful mutagenic tool for targeted genome editing. The impeccable functioning of the system depends on the optimal design of single guide RNAs (sgRNAs) that mainly involves sgRNA specificity and on-target cleavage efficacy. Several research groups have designed algorithms and models, trained on mammalian genomes, for predicting sgRNAs cleavage efficacy. These models are also implemented in most plant sgRNA design tools due to the lack of on-target cleavage efficacy studies in plants. However, one of the major drawbacks is that almost all of these models are biased for considering only coding regions of the DNA while excluding ineffective regions, which are of immense importance in functional genomics studies especially for plants, thus making prediction less reliable. In the present study, we evaluate the on-target cleavage efficacy of experimentally validated sgRNAs designed against diverse ineffective regions of Arabidopsis thaliana genome using various statistical tests. We show that nucleotide preference in protospacer adjacent motif (PAM) proximal region, GC content in the PAM proximal seed region, intact RAR and 3rd stem loop structures, and free accessibility of nucleotides in seed and tracrRNA regions of sgRNAs are important determinants associated with their high on-target cleavage efficacy. Thus, our study describes the features important for plant sgRNAs high on-target cleavage efficacy against ineffective genomic regions previously shown to give rise to ineffective sgRNAs. Moreover, it suggests the need of developing an elaborative plant-specific sgRNA design model considering the entire genomic landscape including ineffective regions for enabling highly efficient genome editing without wasting time and experimental resources.

Genome-wide analysis of bZIP, BBR, and BZR transcription factors in Triticum aestivum.

Transcription factors are regulatory proteins known to modulate gene expression. These are the critical component of signaling pathways and help in mitigating various developmental and stress responses. Among them, bZIP, BBR, and BZR transcription factor families are well known to play a crucial role in regulating growth, development, and defense responses. However, limited data is available on these transcription factors in Triticum aestivum. In this study, bZIP, BBR, and BZR sequences from Brachypodium distachyon, Oryza sativa, Oryza barthii, Oryza brachyantha, T. aestivum, Triticum urartu, Sorghum bicolor, Zea mays were retrieved, and dendrograms were constructed to analyze the evolutionary relatedness among them. The sequences clustered into one group indicated a degree of evolutionary correlation highlighting the common lineage of cereal grains. This analysis also exhibited that these genes were highly conserved among studied monocots emphasizing their common ancestry. Furthermore, these transcription factor genes were evaluated for envisaging conserved motifs, gene structure, and subcellular localization in T. aestivum. This comprehensive computational analysis has provided an insight into transcription factor evolution that can also be useful in developing approaches for future functional characterization of these genes in T. aestivum. Furthermore, the data generated can be beneficial in future for genetic manipulation of economically important plants.