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Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. We also discuss the potential biotechnological applications of chloroplast genomes.

Summary Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

Summary Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4+ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4+CD25+FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.

We demonstrate the feasibility of a wearable, augmented reality–based nystagmus examination system, showing its preliminary diagnostic agreement with conventional video-oculography and its potential for portable vestibular assessment in patients with vertigo.

High mortality is common in mechanically ventilated patients with severe community-acquired pneumonia (CAP). This study sought to create prediction models and determine factors for the pneumonia patients under difficult ventilator weaning in a respiratory care center. In total, 353 CAP and hospital-acquired pneumonia (HAP) patients admitted to a respiratory care center (RCC) from January 1, 2015 to December 31, 2017 were included in this retrospective cohort study. Mortality and weaning risks factors were collected and analyzed for validating the prediction models. The study focuses primarily on CAP patients, with HAP data used to external validation. Among 270 CAP patients in model testing and validation, CURB-65 (Confusion, Uremia, Respiratory rate, Blood pressure, aged ≥65 years) and CUB-65 similarly predicted RCC survival (AUROCs ~65%). Three new RCC survival prediction models incorporated age ≥65, hypotension, BUN >19 mg/dl, ventilator type (replacing respiratory rate), and GCS ≤ 8 (replacing confusion), and either white blood cells (WBCs) or hemoglobin (Hb) were additionally included in model 1 or model 2. In CAP test samples, AUROCs with CAP test sample were 77.51% (model 1), 75.99% (model 2), and 77.97% (model 3). All three new models showed higher AUROCs than CURB-65, with significantly improved predictability in models 1 ( = 0.0354) and 3 ( = 0.0383). All three models demonstrated satisfactory performance (AUROC ≥ 80%) for predicting RCC survival in the CAP validation sample. These new models more accurately predicted survival during RCC admission. Furthermore, they offer clinicians a better predictive tool for survival in CAP patients facing difficult ventilator weaning.

Background Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants. Results Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant evolution, such as the transition to land and at the base of the angiosperm lineage. While detection of transcripts for a selection of non-NDH, photosynthesis related proteins was independent of the state of NDH, coordinate, lineage-specific loss of plastid NDH genes and expression of nuclear-encoded NDH subunits was documented in Pinaceae, gnetophytes, Orchidaceae and Geraniales confirming the independent and complete loss of NDH in these diverse seed plant taxa. Conclusion The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest that the NDH complex is of limited biological significance in contemporary plants. While NDH activity appears dispensable under favorable conditions, there were likely sufficiently frequent episodes of abiotic stress affecting terrestrial habitats to allow the retention of NDH activity. These findings reveal genetic factors influencing plant/environment interactions in a changing climate through 450 million years of land plant evolution.

Deep learning has become the most popular research subject in the fields of artificial intelligence (AI) and machine learning. In October 2013, MIT Technology Review commented that deep learning was a breakthrough technology. Deep learning has made progress in voice and image recognition, image classification, and natural language processing. Prior to deep learning, decision tree, linear discriminant analysis (LDA), support vector machines (SVM), k-nearest neighbors algorithm (K-NN), and ensemble learning were popular in solving classification problems. In this paper, we applied the previously mentioned and deep learning techniques to hairy scalp images. Hairy scalp problems are usually diagnosed by non-professionals in hair salons, and people with such problems may be advised by these non-professionals. Additionally, several common scalp problems are similar; therefore, non-experts may provide incorrect diagnoses. Hence, scalp problems have worsened. In this work, we implemented and compared the deep-learning method, the ImageNet-VGG-f model Bag of Words (BOW), with machine-learning classifiers, and histogram of oriented gradients (HOG)/pyramid histogram of oriented gradients (PHOG) with machine-learning classifiers. The tools from the classification learner apps were used for hairy scalp image classification. The results indicated that deep learning can achieve an accuracy of 89.77% when the learning rate is 1 × 10−4, and this accuracy is far higher than those achieved by BOW with SVM (80.50%) and PHOG with SVM (53.0%).

Orchids exhibit a range of unique flower shapes and are a valuable ornamental crop. MADS‐box transcription factors are key regulatory components in flower initiation and development. Changing the flower shape and flowering time can increase the value of the orchid in the ornamental horticulture industry. In this study, 28 MADS‐box genes were identified from the transcriptome database of the model orchid Erycina pusilla. The full‐length genomic sequences of these MADS‐box genes were obtained from BAC clones. Of these, 27 were MIKC‐type EpMADS (two truncated forms) and one was a type I EpMADS. Eleven EpMADS genes contained introns longer than 10 kb. Phylogenetic analysis classified the 24 MIKCᶜ genes into nine subfamilies. Three specific protein motifs, AG, FUL and SVP, were identified and used to classify three subfamilies. The expression profile of each EpMADS gene correlated with its putative function. The phylogenetic analysis was highly correlated with the protein domain identification and gene expression results. Spatial expression of EpMADS6, EpMADS12 and EpMADS15 was strongly detected in the inflorescence meristem, floral bud and seed via in situ hybridization. The subcellular localization of the 28 EpMADS proteins was also investigated. Although EpMADS27 lacks a complete MADS‐box domain, EpMADS27‐YFP was localized in the nucleus. This characterization of the orchid MADS‐box family genes provides useful information for both orchid breeding and studies of flowering and evolution.

The NAD(P)H dehydrogenase complex is encoded by 11 ndh genes in plant chloroplast (cp) genomes. However, ndh genes are truncated or deleted in some autotrophic Epidendroideae orchid cp genomes. To determine the evolutionary timing of the gene deletions and the genomic locations of the various ndh genes in orchids, the cp genomes of Vanilla planifolia , Paphiopedilum armeniacum , Paphiopedilum niveum , Cypripedium formosanum , Habenaria longidenticulata , Goodyera fumata and Masdevallia picturata were sequenced; these genomes represent Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae subfamilies. Four orchid cp genome sequences were found to contain a complete set of ndh genes. In other genomes, ndh deletions did not correlate to known taxonomic or evolutionary relationships and deletions occurred independently after the orchid family split into different subfamilies. In orchids lacking cp encoded ndh genes, non cp localized ndh sequences were identified. In Erycina pusilla , at least 10 truncated ndh gene fragments were found transferred to the mitochondrial (mt) genome. The phenomenon of orchid ndh transfer to the mt genome existed in ndh -deleted orchids and also in ndh containing species.