bluuebunny/crossref_metadata_2025 huggingface dataset:
From Fast Oscillations to Circadian Rhythms: Coupling at Multiscale Frequency Bands in the Rodent Subcortical Visual System
Authors: Chrobok Lukasz , Belle Mino D. C. , Myung Jihwan
Published: December 2021

The subcortical visual system (SVS) is a unique collection of brain structures localised in the thalamus, hypothalamus and midbrain. The SVS receives ambient light inputs from retinal ganglion cells and integrates this signal with internal homeostatic demands to influence physiology. During this processing, a multitude of oscillatory frequency bands coalesces, with some originating from the retinas, while others are intrinsically generated in the SVS. Collectively, these rhythms are further modulated by the day and night cycle. The multiplexing of these diverse frequency bands (from circadian to infra-slow and gamma oscillations) makes the SVS an interesting system to study coupling at multiscale frequencies. We review the functional organisation of the SVS, and the various frequencies generated and processed by its neurons. We propose a perspective on how these different frequency bands couple with one another to synchronise the activity of the SVS to control physiology and behaviour.
Isolation of Mitochondria From Fresh Mice Lung Tissue
Authors: Caldeira Dayene de Assis Fernandes , Oliveira Dahienne Ferreira de , Cavalcanti-de-Albuquerque João Paulo , Nascimento Jose Hamilton Matheus , Zin Walter Araujo , Maciel Leonardo
Published: December 2021

Direct analysis of isolated mitochondria enables a better understanding of lung dysfunction. Despite well-defined mitochondrial isolation protocols applicable to other tissues, such as the brain, kidney, heart, and liver, a robust and reproductive protocol has not yet been advanced for the lung. We describe a protocol for the isolation of mitochondria from lung tissue aiming for functional analyses of mitochondrial O2 consumption, transmembrane potential, reactive oxygen species (ROS) formation, ATP production, and swelling. We compared our protocol to that used for heart mitochondrial function that is well-established in the literature, and achieved similar results.
Test–Retest Reliability of Physiological Variables During Submaximal Seated Upper-Body Poling in Able-Bodied Participants
Authors: Ettema Marlou , Brurok Berit , Baumgart Julia Kathrin
Published: December 2021

Purpose: To investigate the test–retest reliability of physiological variables across four different test days and four different submaximal exercise intensities during seated upper-body poling (UBP).

Methods: Thirteen abled-bodied, upper-body trained men (age 29±3years; body mass 84±12kg; height 183±5cm) performed four submaximal 4-min stages of seated UBP on four separate test days. The four submaximal stages were set at individual power outputs corresponding to a rating of perceived exertion of 9, 11, 13, and 15. The absolute reliability for pairwise test-day comparisons of the physiological variables was investigated with the smallest detectable change percentage (%SDC) and the relative reliability with the interclass correlation coefficient (ICC).

Results: Absolute and relative reliability across test-day comparisons and submaximal stages were moderate to excellent for all variables investigated (V̇O2 – %SDC range: 5–13%, ICC range: 0.93–0.99; HR – %SDC range: 6–9%, ICC range: 0.91–0.97) other than blood lactate, for which absolute reliability was poor and relative reliability highly variable (%SDC range: 26–69%, ICC range: 0.44–0.92). Furthermore, absolute and relative reliability were consistent across the low-to-moderate exercise intensity spectrum and across test days.

Conclusion: Absolute and relative test–retest reliability were acceptable for all investigated physiological variables but blood lactate. The consistent test–retest reliability across the exercise intensity spectrum and across test days indicates that a familiarization period to the specific exercise modality may not be necessary. For generalizability, these findings need to be confirmed in athletes with a disability by future large-scale studies.
Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm
Authors: Anjos Catarina , Santos Ana Luísa , Duarte Daniel , Matias Domitília , Cabrita Elsa
Published: December 2021

Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.
Changes in Diaphragmatic Function Induced by an Increased Inspiratory Load Experienced by Military Divers: An Ultrasound Study
Authors: Rives Sarah , Schmid Bruno , Chaumet Guillaume , Brégeon Fabienne , Boussuges Alain
Published: December 2021

Background: Inspiratory loading is experienced by military divers when they use rebreather device. Our objective was to assess the changes in diaphragm function induced by an increase in inspiratory load at values similar to those experienced by divers in real life.

Methods: We recorded the excursion and the thickness of the right hemidiaphragm in 22 healthy male volunteers under inspiratory load conditions, using ultrasound in B- and M-mode. The measurements were performed at tidal volume and during breathing at 50% of inspiratory capacity. The breathing rate was regulated and similar in the various sessions with and without load.

Results: The rebreather device used by French military divers leads to an increase in inspiratory load of close to 30 cmH2O. Consequently, the session under load was performed using a device set to this threshold. Significant increases in the excursion and the thickening fraction of the diaphragm were observed between the sessions at tidal volume and at high volume. With addition of the inspiratory load, the excursion of the right hemidiaphragm increased significantly from 2.3 to 3.4cm at tidal volume and from 3.9 to 4.7cm at high volume. The thickening fraction increased significantly from 30.4 to 76.6% at tidal volume and from 70 to 123% at high volume. The statistical analysis demonstrated that assessment of the changes of the thickening fraction during breathing at tidal volume was the most relevant marker to assess the impact of the inspiratory load on the diaphragm.

Conclusion: Diaphragm ultrasound can be used to assess the changes in the diaphragm contraction pattern secondary to an increase in the respiratory load that can be generated by use a diving apparatus. The recording of the changes of the motion, and more importantly of the thickness of the diaphragm, during the breathing cycle is able to provide relevant information regarding the inspiratory load.
Risk of New-Onset Atrial Fibrillation Post-cavotricuspid Isthmus Ablation in Typical Atrial Flutter Without History of Atrial Fibrillation
Authors: Li Jia-hui , Xie Hai-yang , Chen Yan-qiao , Cao Zhong-jing , Tang Qing-hui , Guo Xiao-gang , Sun Qi , Ma Jian
Published: December 2021

Aims: The aim was to describe the incidence of atrial fibrillation (AF) after cavotricuspid isthmus (CTI) ablation in patients with typical atrial flutter (AFL) without history of AF and to identify risk factors for new-onset AF after the procedure.

Methods: A total of 191 patients with typical AFL undergoing successful CTI ablation were enrolled. Patients who had history of AF, structural heart disease, cardiac surgery, or ablation or who received antiarrhythmic drug after procedure were excluded. Clinical and electrophysiological data were collected.

Results: There were 47 patients (24.6%) developing new AF during a follow-up of 3.3 ± 1.9 years after CTI ablation. Receiver operating characteristic (ROC) curves indicated that the cut-off values of left atrial diameter (LAD) and CHA2DS2-VASc score were 42 mm and 2, with area under the curve of 0.781 and 0.550, respectively. The multivariable Cox regression analysis revealed that obstructive sleep apnea (OSA) [hazard ratio (HR) 3.734, 95% confidence interval (CI) 1.470–9.484, P = 0.006], advanced interatrial block (aIAB) (HR 2.034, 95% CI 1.017–4.067, P = 0.045), LAD > 42 mm (HR 2.710, 95% CI 1.478–4.969, P = 0.001), and CHA2DS2-VASc score > 2 (HR 2.123, 95% CI 1.118–4.034, P = 0.021) were independent risk factors of new-onset AF.

Conclusion: A combination of OSA, aIAB, LAD > 42 mm, and CHA2DS2-VASc > 2 was a strongly high risk for new-onset AF after ablation for typical AFL, and it had significance in postablation management in clinical practice.
Unsuccessful and Successful Clinical Trials in Acute Respiratory Distress Syndrome: Addressing Physiology-Based Gaps
Authors: Villar Jesús , Ferrando Carlos , Tusman Gerardo , Berra Lorenzo , Rodríguez-Suárez Pedro , Suárez-Sipmann Fernando
Published: December 2021

The acute respiratory distress syndrome (ARDS) is a severe form of acute hypoxemic respiratory failure caused by an insult to the alveolar-capillary membrane, resulting in a marked reduction of aerated alveoli, increased vascular permeability and subsequent interstitial and alveolar pulmonary edema, reduced lung compliance, increase of physiological dead space, and hypoxemia. Most ARDS patients improve their systemic oxygenation, as assessed by the ratio between arterial partial pressure of oxygen and inspired oxygen fraction, with conventional intensive care and the application of moderate-to-high levels of positive end-expiratory pressure. However, in some patients hypoxemia persisted because the lungs are markedly injured, remaining unresponsive to increasing the inspiratory fraction of oxygen and positive end-expiratory pressure. For decades, mechanical ventilation was the only standard support technique to provide acceptable oxygenation and carbon dioxide removal. Mechanical ventilation provides time for the specific therapy to reverse the disease-causing lung injury and for the recovery of the respiratory function. The adverse effects of mechanical ventilation are direct consequences of the changes in pulmonary airway pressures and intrathoracic volume changes induced by the repetitive mechanical cycles in a diseased lung. In this article, we review 14 major successful and unsuccessful randomized controlled trials conducted in patients with ARDS on a series of techniques to improve oxygenation and ventilation published since 2010. Those trials tested the effects of adjunctive therapies (neuromuscular blocking agents, prone positioning), methods for selecting the optimum positive end-expiratory pressure (after recruitment maneuvers, or guided by esophageal pressure), high-frequency oscillatory ventilation, extracorporeal oxygenation, and pharmacologic immune modulators of the pulmonary and systemic inflammatory responses in patients affected by ARDS. We will briefly comment physiology-based gaps of negative trials and highlight the possible needs to address in future clinical trials in ARDS.
Cardiac Effects of Treadmill Running at Different Intensities in a Rat Model
Authors: Yan Zhipeng , Zeng Ni , Li Jieting , Liao Tao , Ni Guoxin
Published: December 2021

Purpose: In this study, we investigated the effect of treadmill exercise training on cardiac hypertrophy, collagen deposition, echo parameters and serum levels of cardiac troponin I (cTnI) in rats, and how they differ with various exercise intensities, hence exploring potential signal transduction.

Methods: Male Sprague-Dawley rats were randomly divided into sedentary (SED), low-intensity running (LIR), medium-intensity running (MIR), and high-intensity running (HIR) groups. Each exercise group had 3 subgroups that were sacrificed for cardiac tissue analyses at 1, 4, and 8 weeks, respectively, and all rats participated in a daily 1 h treadmill routine 5 days per week. Echocardiographic measurements were performed 24 h after the last exercise session. Additionally, myocardium samples and blood were collected for histological and biochemical examinations. Changes in the extracellular signal-regulated kinases 1/2 (ERK1/2) signal pathway were detected by Western blotting.

Results: After a week of running, ventricular myocyte size and the phosphorylation of ERK1/2 increased in the HIR group, while left ventricular (LV) diastolic diameter values and LV relative wall thickness increased in the LIR and MIR groups. In addition, we observed heart enlargement, cTnI decrease, and ERK1/2 signal activation in each of the exercise groups after 4 weeks of running. However, the HIR group displayed substantial rupture and increased fibrosis in myocardial tissue. In addition, compared with the LIR and MIR groups, 8 weeks of HIR resulted in structural damage, fiber deposition, and increased cTnI. However, there was no difference in the activation of ERK1/2 signaling between the exercise and SED groups.

Conclusion: The effect of running on cardiac hypertrophy was intensity dependent. In contrast to LIR and MIR, the cardiac hypertrophy induced by 8 weeks of HIR was characterized by potential cardiomyocyte injury, which increased the risk of pathological development. Furthermore, the ERK signaling pathway was mainly involved in the compensatory hypertrophy process of the myocardium in the early stage of exercise and was positively correlated with exercise load. However, long-term exercise may attenuate ERK signaling activation.
Comparison of V̇O2-Kinetic Parameters for the Management of Heart Failure
Authors: Wagner Jonathan , Niemeyer Max , Infanger Denis , Pfister Otmar , Myers Jonathan , Schmidt-Trucksäss Arno , Knaier Raphael
Published: December 2021

Objective: The aim of this study was to analyze whether V̇O2-kinetics during cardiopulmonary exercise testing (CPET) is a useful marker for the diagnosis of heart failure (HF) and to determine which V̇O2-kinetic parameter distinguishes healthy participants and patients with HF.

Methods: A total of 526 healthy participants and 79 patients with HF between 20 and 90 years of age performed a CPET. The CPET was preceded by a 3-min low-intensity warm-up and followed by a 3-min recovery bout. V̇O2-kinetics was calculated from the rest to exercise transition of the warm-up bout (on-kinetics), from the exercise to recovery transition following ramp test termination (off-kinetics) and from the initial delay of V̇O2 during the warm-up to ramp test transition (ramp-kinetics).

Results: V̇O2 off-kinetics showed the highest z-score differences between healthy participants and patients with HF. Furthermore, off-kinetics was strongly associated with V̇O2peak. In contrast, ramp-kinetics and on-kinetics showed only minimal z-score differences between healthy participants and patients with HF. The best on- and off-kinetic parameters significantly improved a model to predict the disease severity. However, there was no relevant additional value of V̇O2-kinetics when V̇O2peak was part of the model.

Conclusion: V̇O2 off-kinetics appears to be superior for distinguishing patients with HF and healthy participants compared with V̇O2 on-kinetics and ramp-kinetics. If V̇O2peak cannot be determined, V̇O2 off-kinetics provides an acceptable substitute. However, the additional value beyond that of V̇O2peak cannot be provided by V̇O2-kinetics.
Importance of Windows of Exposure to Maternal High-Fat Diet and Feto-Placental Effects: Discrimination Between Pre-conception and Gestational Periods in a Rabbit Model
Authors: Rousseau-Ralliard Delphine , Aubrière Marie-Christine , Daniel Nathalie , Dahirel Michèle , Morin Gwendoline , Prézelin Audrey , Bertrand Jérémy , Rey Catherine , Chavatte-Palmer Pascale , Couturier-Tarrade Anne
Published: December 2021

Context and Aim: Lipid overnutrition in female rabbits, from prepuberty, leads to impaired metabolism (dyslipidemia and increased adiposity) and follicular atresia, and, when continued during gestation, affects offspring phenotype with intrauterine growth retardation (IUGR) and leads to placental and lipid metabolism abnormalities. Growth retardation is already observed in embryo stage, indicating a possible implication of periconceptional exposure. The objective of this study was to discriminate the effects of preconception and gestational exposures on feto-placental development.

Materials and Methods: Rabbit 1-day zygotes were collected from female donors under control (CD) or high-fat-high-cholesterol (HD) diet and surgically transferred to the left and right uterus, respectively, of each H (n = 6) or C (n = 7) synchronized recipients. Close to term, four combinations, CC (n = 10), CH (n = 13), HC (n = 13), and HH (n = 6), of feto-placental units were collected, for biometry analyses. Fatty acid (FA) profiles were determined in placental labyrinth, decidua, fetal plasma, and fetal liver by gas chromatography and explored further by principal component analysis (PCA). Candidate gene expression was also analyzed by RT-qPCR in the placenta and fetal liver. Data were analyzed by Kruskal–Wallis followed by Dunn’s pairwise comparison test. Combinations of different data sets were combined and explored by multifactorial analysis (MFA).

Results: Compared to controls, HH fetuses were hypotrophic with reduced placental efficiency and altered organogenesis, CH presented heavier placenta but less efficient, whereas HC presented a normal biometry. However, the MFA resulted in a good separation of the four groups, discriminating the effects of each period of exposure. HD during gestation led to reduced gene expression (nutrient transport and metabolism) and big changes in FA profiles in both tissues with increased membrane linoleic acid, lipid storage, and polyunsaturated-to-saturated FA ratios. Pre-conception exposure had a major effect on fetal biometry and organogenesis in HH, with specific changes in FA profiles (increased MUFAs and decreased LCPUFAs).

Conclusion: Embryo origin left traces in end-gestation feto-placental unit; however, maternal diet during gestation played a major role, either negative (HD) or positive (control). Thus, an H embryo developed favorably when transferred to a C recipient (HC) with normal biometry at term, despite disturbed and altered FA profiles.
Cannabinoid Therapeutics in Chronic Neuropathic Pain: From Animal Research to Human Treatment
Authors: Campos Raquel Maria P. , Aguiar Andrey F. L. , Paes-Colli Yolanda , Trindade Priscila Martins Pinheiro , Ferreira Bruna K. , de Melo Reis Ricardo A. , Sampaio Luzia S.
Published: December 2021

Despite the importance of pain as a warning physiological system, chronic neuropathic pain is frequently caused by damage in the nervous system, followed by persistence over a long period, even in the absence of dangerous stimuli or after healing of injuries. Chronic neuropathic pain affects hundreds of millions of adults worldwide, creating a direct impact on quality of life. This pathology has been extensively characterized concerning its cellular and molecular mechanisms, and the endocannabinoid system (eCS) is widely recognized as pivotal in the development of chronic neuropathic pain. Scientific evidence has supported that phyto-, synthetic and endocannabinoids are efficient for pain management, while strong data arise from the therapeutic use of Cannabis-derived products. The use of medicinal Cannabis products is directed toward not only relieving symptoms of chronic pain, but also improving several aspects of patients’ welfare. Here, we review the involvement of eCS, along with other cellular and molecular elements, in chronic neuropathic pain pathology and how this system can be targeted for pain management.
Using Salivary Biomarkers for Stress Assessment in Offshore Saturation Diving: A Pilot Study
Authors: Monnoyer Roxane , Lautridou Jacky , Deb Sanjoy , Hjelde Astrid , Eftedal Ingrid
Published: December 2021

Health monitoring during offshore saturation diving is complicated due to restricted access to the divers, the desire to keep invasive procedures to a minimum, and limited opportunity for laboratory work onboard dive support vessels (DSV). In this pilot study, we examined whether measuring salivary biomarkrers in samples collected by the divers themselves might be a feasible approach to environmental stress assessment. Nine saturation divers were trained in the passive drool method for saliva collection and proceeded to collect samples at nine time points before, during, and after an offshore commercial saturation diving campaign. Samples collected within the hyperbaric living chambers were decompressed and stored frozen at −20°C onboard the DSV until they were shipped to land for analysis. Passive drool samples were collected without loss and assayed for a selection of salivary biomarkers: secretory immunoglobulin A (SIgA), C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukins IL-6, IL-8, IL-1β, as well as cortisol and alpha-amylase. During the bottom phase of the hyperbaric saturation, SIgA, CRP, TNF-α, IL-8 and IL-1β increased significantly, whereas IL-6, cortisol and alpha-amylase were unchanged. All markers returned to pre-dive levels after the divers were decompressed back to surface pressure. We conclude that salivary biomarker analysis may be a feasible approach to stress assessment in offshore saturation diving. The results of our pilot test are consonant with an activation of the sympathetic nervous system related to systemic inflammation during hyperbaric and hyperoxic saturation.
Unmanned Aerial System-Based Weed Mapping in Sod Production Using a Convolutional Neural Network
Authors: Zhang Jing , Maleski Jerome , Jespersen David , Waltz F. C. , Rains Glen , Schwartz Brian
Published: December 2021

Weeds are a persistent problem on sod farms, and herbicides to control different weed species are one of the largest chemical inputs. Recent advances in unmanned aerial systems (UAS) and artificial intelligence provide opportunities for weed mapping on sod farms. This study investigates the weed type composition and area through both ground and UAS-based weed surveys and trains a convolutional neural network (CNN) for identifying and mapping weeds in sod fields using UAS-based imagery and a high-level application programming interface (API) implementation (Fastai) of the PyTorch deep learning library. The performance of the CNN was overall similar to, and in some classes (broadleaf and spurge) better than, human eyes indicated by the metric recall. In general, the CNN detected broadleaf, grass weeds, spurge, sedge, and no weeds at a precision between 0.68 and 0.87, 0.57 and 0.82, 0.68 and 0.83, 0.66 and 0.90, and 0.80 and 0.88, respectively, when using UAS images at 0.57 cm–1.28 cm pixel–1 resolution. Recall ranges for the five classes were 0.78–0.93, 0.65–0.87, 0.82–0.93, 0.52–0.79, and 0.94–0.99. Additionally, this study demonstrates that a CNN can achieve precision and recall above 0.9 at detecting different types of weeds during turf establishment when the weeds are mature. The CNN is limited by the image resolution, and more than one model may be needed in practice to improve the overall performance of weed mapping.
Regulatory Network of Cotton Genes in Response to Salt, Drought and Wilt Diseases (Verticillium and Fusarium): Progress and Perspective
Authors: Billah Masum , Li Fuguang , Yang Zhaoen
Published: December 2021

In environmental conditions, crop plants are extremely affected by multiple abiotic stresses including salinity, drought, heat, and cold, as well as several biotic stresses such as pests and pathogens. However, salinity, drought, and wilt diseases (e.g., Fusarium and Verticillium) are considered the most destructive environmental stresses to cotton plants. These cause severe growth interruption and yield loss of cotton. Since cotton crops are central contributors to total worldwide fiber production, and also important for oilseed crops, it is essential to improve stress tolerant cultivars to secure future sustainable crop production under adverse environments. Plants have evolved complex mechanisms to respond and acclimate to adverse stress conditions at both physiological and molecular levels. Recent progresses in molecular genetics have delivered new insights into the regulatory network system of plant genes, which generally includes defense of cell membranes and proteins, signaling cascades and transcriptional control, and ion uptake and transport and their relevant biochemical pathways and signal factors. In this review, we mainly summarize recent progress concerning several resistance-related genes of cotton plants in response to abiotic (salt and drought) and biotic (Fusarium and Verticillium wilt) stresses and classify them according to their molecular functions to better understand the genetic network. Moreover, this review proposes that studies of stress related genes will advance the security of cotton yield and production under a changing climate and that these genes should be incorporated in the development of cotton tolerant to salt, drought, and fungal wilt diseases (Verticillium and Fusarium).
New Technologies and Strategies for Grapevine Breeding Through Genetic Transformation
Authors: Campos Gabriela , Chialva Constanza , Miras Silvana , Lijavetzky Diego
Published: December 2021

Grapevine, as other woody perennials, has been considered a recalcitrant crop to produce transgenic plants. Since the production of transgenic and/or edited plants requires the ability to regenerate plants from transformed tissues, this step is often the biggest bottleneck in the process. The objective of this work is to review the state of the art technologies and strategies for the improvement of grapevine transformation and regeneration, focusing on three aspects: (i) problems associated with grapevine transformation; (ii) genes that promote grapevine regeneration; and (iii) vehicles for gene delivery. Concerning the first aspect, it is well documented that one of the main factors explaining the low success rate in obtaining transgenic plants is the regeneration process. After transgenic integration into receptor cells, tissue culture is required to regenerate transgenic seedlings from transformed cells. This process is time consuming and often requires the addition of environmentally damaging reagents (antibiotics and herbicides) to the culture medium to select transgenic plants. On the other hand, the expression of genes such as the so-called developmental regulators (DR), which induce specific development programs, can be used to avoid traditional tissue culture methods. The ectopic expression of specific combinations of DR in somatic cells has the potential to induce de novo meristems in diverse crops, including grapevine. Successful genome editing by de novo reprogramming of plant meristems in somatic tissues has been reported. Moreover, it has been shown that the expression of certain transcription factors can increase the regeneration efficiency in wheat, citrus, and rice. Finally, recent reports showed the use of nanoparticles, such as carbon dots (CDs), as an attractive alternative to Agrobacterium- and biolistic-mediated plant genetic transformation. In this way, the use of antibiotics in culture media is avoided, overcoming the loss of viability of plant tissues and accelerating the regeneration processes. It has been shown that CDs can act as a vehicle to transport plasmids to plant cells in transient transformation in several crops without negative impacts on photosynthesis or growth. Based on these advances, it is possible to combine these new available strategies and technologies to overcome the regeneration problems of species such as grapevine and other crops considered as recalcitrant.
Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies
Authors: Conti Gabriela , Xoconostle-Cázares Beatriz , Marcelino-Pérez Gabriel , Hopp Horacio Esteban , Reyes Carina A.
Published: December 2021

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation proceduresvia Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving thein vitroregeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.
Combined Stress Conditions in Melon Induce Non-additive Effects in the Core miRNA Regulatory Network
Authors: Villalba-Bermell Pascual , Marquez-Molins Joan , Marques María-Carmen , Hernandez-Azurdia Andrea G. , Corell-Sierra Julia , Picó Belén , Monforte Antonio J. , Elena Santiago F. , Gomez Gustavo G.
Published: December 2021

Climate change has been associated with a higher incidence of combined adverse environmental conditions that can promote a significant decrease in crop productivity. However, knowledge on how a combination of stresses might affect plant development is still scarce. MicroRNAs (miRNAs) have been proposed as potential targets for improving crop productivity. Here, we have combined deep-sequencing, computational characterization of responsive miRNAs and validation of their regulatory role in a comprehensive analysis of response of melon to several combinations of four stresses (cold, salinity, short day, and infection with a fungus). Twenty-two miRNA families responding to double and/or triple stresses were identified. The regulatory role of the differentially expressed miRNAs was validated by quantitative measurements of the expression of the corresponding target genes. A high proportion (ca. 60%) of these families (mainly highly conserved miRNAs targeting transcription factors) showed a non-additive response to multiple stresses in comparison with that observed under each one of the stresses individually. Among those miRNAs showing non-additive response to stress combinations, most interactions were negative, suggesting the existence of functional convergence in the miRNA-mediated response to combined stresses. Taken together, our results provide compelling pieces of evidence that the response to combined stresses cannot be easily predicted from the study individual stresses.
Integrated Transcriptome Analysis and Single-Base Resolution Methylomes of Watermelon (Citrullus lanatus) Reveal Epigenome Modifications in Response to Osmotic Stress
Authors: Zhu Fangming , Li Mingyan , Yan Manwen , Qiao Fei , Jiang Xuefei
Published: December 2021

DNA methylation plays an important role against adverse environment by reshaping transcriptional profile in plants. To better understand the molecular mechanisms of watermelon response to osmotic stress, the suspension cultured watermelon cells were treated with 100mM mannitol, and then a methylated cytosines map was generated by whole genome bisulfite sequencing (WGBS). Combined with transcriptome sequencing, the effects of osmotic stress on differentially methylated expressed genes (DMEGs) were assessed. It was found that genes related to plant hormone synthesis, signal transduction, osmoregulatory substance-related and reactive oxygen species scavenging-related enzyme could rapidly respond to osmotic stress. The overall methylation level of watermelon decreased after osmotic stress treatment, and demethylation occurred in CG, CHG, and CHH contexts. Moreover, differentially methylated expressed genes (DMEGs) were significantly enriched in RNA transport, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of secondary metabolites, especially in biosynthesis of osmolytes synthase genes. Interestingly, demethylation of a key enzyme gene Cla014489 in biosynthesis of inositol upregulated its expression and promoted accumulation of inositol, which could alleviate the inhibition of cell growth caused by osmotic stress. Meanwhile, a recombinant plasmid pET28a-Cla014489 was constructed and transferred into Escherichia coli BL21 for prokaryotic expression and the expression of ClMIPS protein could improve the tolerance of E. coli to osmotic stress. The effect of methylation level on the expression properties of inositol and its related genes was further confirmed by application of DNA methylation inhibitor 5-azacytidine. These results provide a preliminary insight into the altered methylation levels of watermelon cells in response to osmotic stress and suggest a new mechanism that how watermelon cells adapt to osmotic stress.
The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets
Authors: do Nascimento Antonia Maiara Marques , Polesi Luiza Giacomolli , Back Franklin Panato , Steiner Neusa , Guerra Miguel Pedro , Castander-Olarieta Ander , Moncaleán Paloma , Montalbán Itziar Aurora
Published: December 2021

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL–1 of L-glutamine, 1,050 mgL–1 of L-asparagine, 350 mgL–1 of L-arginine, and 35 mgL–1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.
Longitudinal Pattern of Aerenchyma Formation Using the Ti-Gompertz Model in Rice Adventitious Roots
Authors: Chen Yun , Li Guoming , Zhao Buhong , Zhang Yajun , Liu Kun , Nadeeshika Samarawickrama Priyadarshani , Wu Xiaoxia , Lv Bing , Liu Lijun
Published: December 2021

The longitudinal pattern of root aerenchyma formation of its relationship with the function of adventitious roots in rice remains unclear. In this study, the percentage of the aerenchyma area to the cross-sectional area (i.e., aerenchyma percentage) was fit with four non-linear models, namely, W0-Gompertz, Ti-Gompertz, logistic, and von Bertalanffy. Goodness-of-fit criteria such as the R2, the Akaike information criterion (AIC), and the Bayesian information criterion (BIC) were used to select the model. The bias of the parameters was evaluated using the difference between the ordinary least squares-based parameter estimates and the mean of 1,000 bootstrap-based parameter estimates and the symmetry of the distributions of these parameters. The results showed that the Ti-Gompertz model, which had a high goodness-of-fit with an R2 close to 1, lower AIC and BIC values, parameter estimates close to being unbiased, and good linear approximation, provided the best fit for the longitude pattern of rice aerenchyma formation with different root lengths among the competing models. Using the second- and third-order derivatives according to the distance from the root apex, the critical points of Ti-Gompertz were calculated. The rapid stage for aerenchyma formation was from the maximum acceleration point (1.38–1.76 cm from the root apex) to the maximum deceleration point (3.13–4.19 cm from the root apex). In this stage, the aerenchyma percentage increased by 5.3–15.7% per cm, suggesting that the cortical cells tended to die rapidly for the aerenchyma formation rather than for the respiration cost during this stage. Meanwhile, the volume of the aerenchyma of the entire roots could be computed using the integral function of the Ti-Gompertz model. We proposed that the longitudinal pattern of root aerenchyma formation modeled by the Ti-Gompertz model helped to deeply understand the relationship between the anatomical traits and physiological function in rice adventitious roots.
Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis
Authors: Liu Chanchan , Gao Qiyu , Shang Zhuo , Liu Jian , Zhou Siwei , Dang Jingjie , Liu Licheng , Lange Iris , Srividya Narayanan , Lange B. Markus , Wu Qinan , Lin Wei
Published: December 2021

Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq., Nepeta tenuifolia Briq., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (−)-pulegone reductase from Nepeta tenuifolia (NtPR), which can catalyze (−)-pulegone to (+)-menthone and (−)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from M. piperita (MpPR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of MpPR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.
Reprogramming of Histone H3 Lysine Methylation During Plant Sexual Reproduction
Authors: Fang Huihui , Shao Yuke , Wu Gang
Published: December 2021

Plants undergo extensive reprogramming of chromatin status during sexual reproduction, a process vital to cell specification and pluri- or totipotency establishment. As a crucial way to regulate chromatin organization and transcriptional activity, histone modification can be reprogrammed during sporogenesis, gametogenesis, and embryogenesis in flowering plants. In this review, we first introduce enzymes required for writing, recognizing, and removing methylation marks on lysine residues in histone H3 tails, and describe their differential expression patterns in reproductive tissues, then we summarize their functions in the reprogramming of H3 lysine methylation and the corresponding chromatin re-organization during sexual reproduction inArabidopsis, and finally we discuss the molecular significance of histone reprogramming in maintaining the pluri- or totipotency of gametes and the zygote, and in establishing novel cell fates throughout the plant life cycle. Despite rapid achievements in understanding the molecular mechanism and function of the reprogramming of chromatin status in plant development, the research in this area still remains a challenge. Technological breakthroughs in cell-specific epigenomic profiling in the future will ultimately provide a solution for this challenge.
Acclimating Cucumber Plants to Blue Supplemental Light Promotes Growth in Full Sunlight
Authors: Kang Chenqian , Zhang Yuqi , Cheng Ruifeng , Kaiser Elias , Yang Qichang , Li Tao
Published: December 2021

Raising young plants is important for modern greenhouse production. Upon transfer from the raising to the production environment, young plants should maximize light use efficiency while minimizing deleterious effects associated with exposure to high light (HL) intensity. The light spectrum may be used to establish desired traits, but how plants acclimated to a given spectrum respond to HL intensity exposure is less well explored. Cucumber (Cucumis sativus) seedlings were grown in a greenhouse in low-intensity sunlight (control; ∼2.7 mol photons m–2 day–1) and were treated with white, red, blue, or green supplemental light (4.3 mol photons m–2 day–1) for 10 days. Photosynthetic capacity was highest in leaves treated with blue light, followed by white, red, and green, and was positively correlated with leaf thickness, nitrogen, and chlorophyll concentration. Acclimation to different spectra did not affect the rate of photosynthetic induction, but leaves grown under blue light showed faster induction and relaxation of non-photochemical quenching (NPQ) under alternating HL and LL intensity. Blue-light-acclimated leaves showed reduced photoinhibition after HL intensity exposure, as indicated by a high maximum quantum yield of photosystem II photochemistry (Fv/Fm). Although plants grown under different supplemental light spectra for 10 days had similar shoot biomass, blue-light-grown plants (B-grown plants) showed a more compact morphology with smaller leaf areas and shorter stems. However, after subsequent, week-long exposure to full sunlight (10.7 mol photons m–2 day–1), B-grown plants showed similar leaf area and 15% higher shoot biomass, compared to plants that had been acclimated to other spectra. The faster growth rate in blue-light-acclimated plants compared to other plants was mainly due to a higher photosynthetic capacity and highly regulated NPQ performance under intermittent high solar light. Acclimation to blue supplemental light can improve light use efficiency and diminish photoinhibition under high solar light exposure, which can benefit plant growth.
ATG8-Interacting Motif: Evolution and Function in Selective Autophagy of Targeting Biological Processes
Authors: Liu Wanqing , Liu Zinan , Mo Zulong , Guo Shaoying , Liu Yunfeng , Xie Qingjun
Published: December 2021

Autophagy is an evolutionarily conserved vacuolar process functioning in the degradation of cellular components for reuse. In plants, autophagy is generally activated upon stress and its regulation is executed by numbers of AuTophaGy-related genes (ATGs), of which the ATG8 plays a dual role in both biogenesis of autophagosomes and recruitment of ATG8-interacting motif (AIM) anchored selective autophagy receptors (SARs). Such motif is either termed as AIM or ubiquitin-interacting motif (UIM), corresponding to the LC3-interacting region (LIR)/AIM docking site (LDS) or the UIM docking site (UDS) of ATG8, respectively. To date, dozens of AIM or UIM containing SARs have been characterized. However, the knowledge of these motifs is still obscured. In this review, we intend to summarize the current understanding of SAR proteins and discuss the conservation and diversification of the AIMs/UIMs, expectantly providing new insights into the evolution of them in various biological processes in plants.