The Effect of Botulinum Toxin on Flap Viability of the Posterior Thigh Perforator Flap in Rats.

BACKGROUND: The use of perforator propeller flaps in lower limb reconstruction has increased recently. Many pharmacological agents are used to increase flap viability. Botulinum toxin has been used in various types of flaps in the literature. However, there is no study regarding the use of botulinum toxin in the lower limb propeller flaps. This study investigates the effect of botulinum toxin administration on flap survival for lower limb propeller flap in rats. MATERIALS AND METHODS: The study included 20 male Wistar albino rats, divided into two groups with a flap rotation of 90° in group 1 and 180° in group 2. In both groups, botulinum toxin was administered to the right thigh and a physiological saline solution was applied to the left thigh. Five days later, flaps were elevated over the posterior aspect of the right and left thighs and inset after 90° and 180° rotation was performed. Histopathological, immunohistochemical, and necrosis area analyses were performed. RESULTS: Necrosis area, edema, polymorphonuclear leukocyte infiltration, and necrosis were found to be higher on the left side of the groups, whereas epidermal thickness, collagen density, vascularization, and hair root density were found to be higher on the right side of the groups. No significant difference was found between the right posterior thighs in either group on any parameter other than vascularization. Histopathologically and immunochemically statistically significant differences were found between the two groups. CONCLUSIONS: The present study found that botulinum toxin increases flap viability in lower limb perforator-based propeller flaps.

Ameliorating Effects of ß-Glucan on Epigastric Artery Island Flap Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury has been one of the culprits of tissue injury and flap loss after island flap transpositions. Thus, significant research has been undertaken to study how to prevent or decrease the spread of ischemia-reperfusion injury. Preventive effects of ß-glucan on ischemia-reperfusion injury in the kidney, lung, and small intestine have previously been reported. In this study, we present the ameliorating effects of ß-glucan on ischemia-reperfusion injury using the epigastric artery island-flap in rats. MATERIALS AND METHODS: Thirty Wistar-Albino rats were equally divided into three groups: sham, experimental model, and treatment groups. In the sham group, an island flap was elevated and sutured back to the original position without any ischemia. In the experimental model group, the same-sized flap was elevated and sutured back with 8 h of ischemia and consequent 12 h of reperfusion. In the treatment group, 50 mg per kilogram ß-glucan was administered to the rats using an orogastric tube for 10 d before the experiment. The same-sized flap is elevated and sutured back to its original position with 8 h of ischemia and 12 h of consequent reperfusion in the treatment group. Tissue biopsies were taken on the first day of the experimental surgery. Tissue neutrophil aggregation and vascular responses were evaluated by histological examinations. Tissue oxidant and antioxidant enzyme levels are evaluated biochemically after tissue homogenization. Topographic follow-up and evaluation of the flaps were maintained, and photographs were taken on the first and seventh day of the experimental surgery. RESULTS: Topographic flap survival was significantly better in the ß-glucan administered group. The neutrophil number, malondialdehyde, and myeloperoxidase levels were significantly lower while glutathione peroxidase and superoxide dismutase levels were significantly higher in the ß-glucan administered group respective to the experimental model group. CONCLUSIONS: Based on the results of our study, we can conclude that ß-glucan is protective against ischemia-reperfusion injury. Our study presents the first experimental evidence of such an effect on skin island flaps.

Light promoted brown staining of protoplasm by Ag+ is ideal to test wheat pollen viability rapidly.

Pollen viability is crucial for wheat breeding programs. The unique potential of the protoplasm of live cells to turn brown due to the synthesis of silver nanoparticles (AgNPs) through rapid photoreduction of Ag+, was exploited for testing wheat pollen viability. Ag+-viability test medium (consisting of 0.5 mM AgNO3 and 300 mM KNO3) incubated with wheat pollen turned brown within 2 min under intense light (~600 µmol photon flux density m-2s-1), but not in dark. The brown medium displayed AgNPs-specific surface plasmon resonance band in its absorption spectrum. Light microscopic studies showed the presence of uniformly stained brown protoplasm in viable pollen incubated with Ag+-viability medium in the presence of light. Investigations with transmission electron microscope coupled with energy dispersive X-ray established the presence of distinct 5-35 nm NPs composed of Ag. Powder X-ray diffraction analysis revealed that AgNPs were crystalline and biphasic composed of Ag0 and Ag2O. Conversely, non-viable pollen and heat-killed pollen did not turn brown on incubation with Ag+-medium in light. We believe that the viable wheat pollen turn brown rapidly by bio-transforming Ag+ to AgNPs through photoreduction. Our findings furnish a novel simplest and rapid method for testing wheat pollen viability.

Quantitative methods in microscopy to assess pollen viability in different plant taxa.

High-quality pollen is a prerequisite for plant reproductive success. Pollen viability and sterility can be routinely assessed using common stains and manual microscope examination, but with low overall statistical power. Current automated methods are primarily directed towards the analysis of pollen sterility, and high throughput solutions for both pollen viability and sterility evaluation are needed that will be consistent with emerging biotechnological strategies for crop improvement. Our goal is to refine established labelling procedures for pollen, based on the combination of fluorescein (FDA) and propidium iodide (PI), and to develop automated solutions for accurately assessing pollen grain images and classifying them for quality. We used open-source software programs (CellProfiler, CellProfiler Analyst, Fiji and R) for analysis of images collected from 10 pollen taxa labelled using FDA/PI. After correcting for image background noise, pollen grain images were examined for quality employing thresholding and segmentation. Supervised and unsupervised classification of per-object features was employed for the identification of viable, dead and sterile pollen. The combination of FDA and PI dyes was able to differentiate between viable, dead and sterile pollen in all the analysed taxa. Automated image analysis and classification significantly increased the statistical power of the pollen viability assay, identifying more than 75,000 pollen grains with high accuracy (R2 = 0.99) when compared to classical manual counting. Overall, we provide a comprehensive set of methodologies as baseline for the automated assessment of pollen viability using fluorescence microscopy, which can be combined with manual and mechanized imaging systems in fundamental and applied research on plant biology. We also supply the complete set of pollen images (the FDA/PI pollen dataset) to the scientific community for future research.

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.

The ROS-associated programmed cell death causes the decline of pollen viability recovered from cryopreservation in Paeonia lactiflora.

KEY MESSAGE: After cryopreservation, the occurrence of apoptosis-like programmed cell death events induced by the accumulation of ROS reduces pollen viability. Cryopreservation, as a biotechnological means for long-term preservation of pollen, has been applied to many species. However, after cryopreservation, the viability of pollen significantly decreases via a mechanism that is not completely clear. In this study, the pollen of Paeonia lactiflora 'Zi Feng Chao Yang', which exhibits significantly reduced viability after liquid nitrogen (LN2) storage, was used to study the relationship among pollen viability, programmed cell death (PCD) and reactive oxygen species (ROS). The apoptosis rate was increased significantly in pollen with decreased viability after cryopreservation, and the changes in ROS generation and hydrogen peroxide (H2O2) were consistent with the apoptosis rate. Correlation analysis results showed that the apoptosis rate is positively correlated with ROS generation and H2O2 content. In addition, ascorbic acid (AsA), glutathione (GSH) and ascorbic acid reductase (APX) levels were significantly correlated with ROS and H2O2. After LN2 preservation for 8 months, the exogenous antioxidants AsA and GSH at appropriate concentrations significantly decreased H2O2 content, inhibited PCD indicator levels, and increased cryopreserved pollen viability. These observations suggest that PCD occurred in pollen during LN2 preservation for 1-8 months and was induced by the accumulation of ROS in pollen after cryopreservation, thus explaining the main reasons for the reduction in pollen viability after cryopreservation in LN2.

The Tapetal Major Facilitator NPF2.8 Is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana.

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Although the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that a member of the nitrate/peptide transporter family is required for the accumulation and transport of pollen-specific flavonol 3-o-sophorosides, characterized by a glycosidic ß-1,2-linkage, to the pollen surface of Arabidopsis (Arabidopsis thaliana). Ectopic, transient expression in Nicotiana benthamiana epidermal leaf cells demonstrated localization of this flavonol sophoroside transporter (FST1) at the plasmalemma when fused to green fluorescent protein (GFP). We also confirmed the tapetum-specific expression of FST1 by GFP reporter lines driven by the FST1 promoter. In vitro characterization of FST1 activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1 insertion mutant by complementation with an FST1 genomic fragment restored the accumulation of flavonol glycosides in pollen grains to wild-type levels, corroborating the requirement of FST1 for transport of flavonol-3-o-sophorosides from the tapetum to the pollen surface.

The Effect of Leonurine on Multiterritory Perforator Flap Survival in Rats.

BACKGROUND: Leonurine (Leo), a natural active compound of Leonurus cardiaca, has been shown to possess various biological activities. However, it is not known whether Leo promotes perforator flap survival. METHODS: In this study, a perforator flap was outlined in the rat dorsum. The rats that survived surgery were divided randomly to control and Leo groups (n = 36 per group). Flap viability, flap perfusion, and level of protein linked with oxidative stress, cell apoptosis, and angiogenesis were evaluated. RESULTS: Relative to control group, the Leo group showed significantly higher the flap survival percentage (70.5% versus 90.2%, P < 0.05) and blood perfusion (197.1 versus 286.3, P < 0.05). Leo also increased 1.8-fold mean vessel density and upregulated 2.1-fold vascular endothelial growth factor protein expression compared with the control group, both of which indicate increased angiogenesis. Moreover, it significantly inhibited apoptosis by lowering caspase-3 activity. Superoxide dismutase expression was remarkably elevated in Leo group compared with the control group (56.0 versus 43.2 U/mg/protein, P < 0.01), but malondialdehyde quantities were significantly lower in the Leo group compared with control group (41.9 versus 57.5 nmol/mg/protein, P < 0.05). CONCLUSIONS: Leo may serve as an effective drug for improving perforator flap survival in rats via antioxidant and antiapoptotic mechanisms and promotion of angiogenesis.

Protective effects of icariin on dorsal random skin flap survival: An experimental study.

This study was performed to examine the protective effects of icariin (ICA) on ischemic random skin flaps. A rat random-pattern skin flap model was established, and animals in the low-dose and high-dose experimental groups were administered ICA intraperitoneally at doses of 40 and 80 mg/kg, respectively, once daily for 7 days after the initial surgery. Control rats received vehicle according to the same schedule. Survival rates were observed and recorded using transparent graph paper, and flaps were obtained and stained with hematoxylin and eosin (H&E). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the flap tissue were assessed. The blood flow volume was determined by the laser Doppler method, and tissue expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-1ß, and phosphodiesterase 5 (PDE5) were scored immunohistochemically. The levels of proinflammatory cytokines, including tumor necrosis factor-α and IL-6, were determined by enzyme-linked immunosorbent assay. The main flap survival area was significantly larger in rats treated with ICA than in vehicle-treated controls. H&E staining showed an inhibitory effect of ICA on inflammation, especially at the high dose. In addition, ICA treatment was associated with decreases in the tissue MDA level, proinflammatory cytokine production, and the level of PDE5, but increases in SOD activity, blood flow volume, and the level of VEGF expression. The findings of the present study suggest that ICA is a potential therapeutic agent for random-pattern skin flap necrosis in the clinical setting.

Manganese-enhanced MRI of the myocardium.

Gadolinium-based contrast media are widely used in cardiovascular MRI to identify and to highlight the intravascular and extracellular space. After gadolinium, manganese has the second highest paramagnetic moment and was one of the first MRI contrast agents assessed in humans. Over the last 50 years, manganese-enhanced MRI (MEMRI) has emerged as a complementary approach enabling intracellular myocardial contrast imaging that can identify functional myocardium through its ability to act as a calcium analogue. Early progress was limited by its potential to cause myocardial depression. To overcome this problem, two clinical formulations of manganese were developed using either chelation (manganese dipyridoxyl diphosphate) or coadministration with a calcium compound (EVP1001-1, Eagle Vision Pharmaceuticals). Preclinical studies have demonstrated the efficacy of MEMRI in quantifying myocardial infarction and detecting myocardial viability as well as tracking altered contractility and calcium handling in cardiomyopathy. Recent clinical data suggest that MEMRI has exciting potential in the quantification of myocardial viability in ischaemic cardiomyopathy, the early detection of abnormalities in myocardial calcium handling, and ultimately, in the development of novel therapies for myocardial infarction or heart failure by actively quantifying viable myocardium. The stage is now set for wider clinical translational study of this novel and promising non-invasive imaging modality.

3D Myocardial Scar Prediction Model Derived from Multimodality Analysis of Electromechanical Mapping and Magnetic Resonance Imaging.

Many cardiac catheter interventions require accurate discrimination between healthy and infarcted myocardia. The gold standard for infarct imaging is late gadolinium-enhanced MRI (LGE-MRI), but during cardiac procedures electroanatomical or electromechanical mapping (EAM or EMM, respectively) is usually employed. We aimed to improve the ability of EMM to identify myocardial infarction by combining multiple EMM parameters in a statistical model. From a porcine infarction model, 3D electromechanical maps were 3D registered to LGE-MRI. A multivariable mixed-effects logistic regression model was fitted to predict the presence of infarct based on EMM parameters. Furthermore, we correlated feature-tracking strain parameters to EMM measures of local mechanical deformation. We registered 787 EMM points from 13 animals to the corresponding MRI locations. The mean registration error was 2.5 ± 1.16 mm. Our model showed a strong ability to predict the presence of infarction (C-statistic = 0.85). Strain parameters were only weakly correlated to EMM measures. The model is accurate in discriminating infarcted from healthy myocardium. Unipolar and bipolar voltages were the strongest predictors.

Monophasic action potential amplitude for substrate mapping.

Although radiofrequency ablation has revolutionized the management of tachyarrhythmias, the rate of arrhythmia recurrence is a large drawback. Successful substrate identification is paramount to abolishing arrhythmia, and bipolar voltage electrogram's narrow field of view can be further reduced for increased sensitivity. In this report, we perform cardiac mapping with monophasic action potential (MAP) amplitude. We hypothesize that MAP amplitude (MAPA) will provide more accurate infarct sizes than other mapping modalities via increased sensitivity to distinguish healthy myocardium from scar tissue. Using the left coronary artery ligation Sprague-Dawley rat model of ischemic heart failure, we investigate the accuracy of in vivo ventricular epicardial maps derived from MAPA, MAP duration to 90% repolarization (MAPD90), unipolar voltage amplitude (UVA), and bipolar voltage amplitude (BVA) compared with gold standard histopathological measurement of infarct size. Numerical analysis reveals discrimination of healthy myocardium versus scar tissue using MAPD90 (P = 0.0158) and UVA (P < 0.001, n = 21). MAPA and BVA decreased between healthy and border tissue (P = 0.0218 and 0.0015, respectively) and border and scar tissue (P = 0.0037 and 0.0094, respectively). Contrary to our hypothesis, BVA mapping performed most accurately regarding quantifying infarct size. MAPA mapping may have high spatial resolution for myocardial tissue characterization but was quantitatively less accurate than other mapping methods at determining infarct size. BVA mapping's superior utility has been reinforced, supporting its use in translational research and clinical electrophysiology laboratories. MAPA may hold potential value for precisely distinguishing healthy myocardium, border zone, and scar tissue in diseases of disseminated fibrosis such as atrial fibrillation.NEW & NOTEWORTHY Monophasic action potential mapping in a clinically relevant model of heart failure with potential implications for atrial fibrillation management.

Reconstruction of the ovary microenvironment utilizing macroporous scaffold with affinity-bound growth factors.

Implementing ovarian tissue engineering for the maturation of primordial follicles, the most abundant follicle population in the ovary, holds great potential for women fertility preservation. Here, we evaluated whether macroporous alginate scaffolds with affinity-bound bone morphogenetic protein-4 (BMP-4) could mimic the ovary microenvironment and support the culture and growth of primordial follicles seeded with supporting ovarian cells. Porcine primordial follicles developed in the alginate scaffolds up to the pre-antral stage within 21 days. Affinity-bound BMP-4 significantly contributed to follicular maturation, as evident by the 5-fold increase in the number of developing follicles and enhanced estradiol secretion in these cultures compared to when BMP-4 was added to cultures with no affinity binding. After 21 days in culture, an increase in GDF-9/AMH gene expression, which is correlated with follicular development, was statistically significant when BMP-4 was affinity bound, compared to all other scaffold groups. When developed in-vivo, after xeno-transplantation of the follicle devices supplemented with additional angiogenic factors, the follicles reached antral size and secreted hormones at levels leading to restoration of ovarian function in ovariectomized severe combined immunodeficiency (SCID) mice. Altogether, our results provide first affirmation for the applicability of macroporous alginate scaffolds as a suitable platform for promoting follicle maturation in-vitro and in-vivo, and lay the foundations for the advantageous use of affinity binding presentation of growth factors to cultured follicles.

Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss.

Traumatic composite bone-muscle injuries, such as open fractures, often require multiple surgical interventions and still typically result in long-term disability. Clinically, a critical indicator of composite injury severity is vascular integrity; vascular damage alone is sufficient to assign an open fracture to the most severe category. Challenging bone injuries are often treated with bone morphogenetic protein 2 (BMP-2), an osteoinductive growth factor, delivered on collagen sponge. Previous studies in a composite defect model found that a minimally bridging dose in the segmental defect model was unable to overcome concomitant muscle damage, but the effect of BMP dose on composite injuries has not yet been studied. Here, we test the hypotheses that BMP-2-mediated functional regeneration of composite extremity injuries is dose dependent and can be further enhanced via co-delivery of adipose-derived microvascular fragments (MVF), which have been previously shown to increase tissue vascular volume. Although MVF did not improve healing outcomes, we observed a significant BMP-2 dose-dependent increase in regenerated bone volume and biomechanical properties. This is the first known report of an increased BMP-2 dose improving bone healing with concomitant muscle damage. While high dose BMP-2 delivery can induce heterotopic ossification (HO) and increased inflammation, the maximum 10 µg dose used in this study did not result in HO and was associated with a lower circulating inflammatory cytokine profile than the low dose (2.5 µg) group. These data support the potential benefits of an increased, though still moderate, BMP-2 dose for treatment of bone defects with concomitant muscle damage. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res.

Comparison of two different laser photobiomodulation protocols on the viability of random skin flap in rats.

To identify the best low level laser photobiomodulation application site at the same irradiation time to increase the viability of the skin flap in rats. Eighteen male rats (Rattus norvegicus: var. Albinus, Rodentia Mammalia) were randomly distributed into three groups (n = 6). Group I (GI) was submitted to simulated laser photobiomodulation; group II (GII) was submitted to laser photobiomodulation at three points in the flap cranial base, and group III (GIII) was submitted to laser photobiomodulation at 12 points distributed along the flap. All groups were irradiated with an Indium, Galium, Aluminum, and Phosphorus diode laser (InGaAlP), 660 nm, with 50 mW power, irradiated for a total time of 240 s in continuous emission mode. The treatment started immediately after performing the cranial base random skin flap (10 × 4 cm2 dimension) and reapplied every 24 h, with a total of five applications. The animals were euthanized after the evaluation of the percentage of necrosis area, and the material was collected for histological analysis on the seventh postoperative day. GII animals presented a statistically significant decrease for the necrosis area when compared to the other groups, and a statistically significant increase in the quantification of collagen when compared to the control. We did not observe a statistical difference between the TGFß and FGF expression in the different groups evaluated. The application of laser photobiomodulation at three points of the flap cranial base was more effective than at 12 points regarding the reduction of necrosis area.

Variable pollen viability and effects of pollen load size on components of seed set in cultivars and feral populations of oilseed rape.

Pollination success is important for crop yield, but may be cultivar dependent. Less is known about which floral traits influence pollination success. Floral traits, e.g. traits related to attraction and reward, can also contribute to gene flow via pollen, the latter being of particular importance in oilseed rape (Brassica napus) where gene flow occurs between plants of crop, volunteer and feral origin as well as related taxa. We investigated the relationship between pollen load size and seed set in winter oilseed rape. We compared variability in pollen-viability traits, flower production (flowers from the main raceme times number of branches) and seed number and weight per siliqua among cultivars and feral populations (growing outside of agricultural fields) under controlled conditions. Both seed number and weight were saturated at relatively low pollen loads in the tested cultivar. Pollen viability and estimated flower production differed among cultivars, indicating that these traits could contribute to yield variability. Seed weight per siliqua, but not pollen traits or flower production, was lower in ferals compared to cultivars. Thus, while the probability of establishment may be reduced in ferals (due to lower seed weight per siliqua) this will not necessarily impact their contribution to gene flow via pollen. In oilseed rape a relatively low pollen load may be sufficient for full seed set in some cultivars, suggesting less dependence on insect pollination for high yield than generally expected. Our results also showed that previously less investigated floral traits, such as pollen viability, pollen tube growth rate and flower number, can differ between cultivars. Studies of these traits may provide targets for increasing crop yield and provide general knowledge about gene flow between cultivated, feral and related wild populations.

Photobiomodulation of a flowable matrix in a human skin ex vivo model demonstrates energy-based enhancement of engraftment integration and remodeling.

The use of dermal substitutes to treat skin defects such as ulcers has shown promising results, suggesting a potential role for skin substitutes for treating acute and chronic wounds. One of the main drawbacks with the use of dermal substitutes is the length of time from engraftment to graft take, plus the risk of contamination and failure due to this prolonged integration. Therefore, the use of adjuvant energy-based therapeutic modalities to augment and accelerate the rate of biointegration by dermal substitute engraftments is a desirable outcome. The photobiomodulation (PBM) therapy modulates the repair process, by stimulating cellular proliferation and angiogenesis. Here, we evaluated the effect of PBM on a collagen-glycosaminoglycan flowable wound matrix (FWM) in an ex vivo human skin wound model. PBM resulted in accelerated rate of re-epithelialization and organization of matrix as seen by structural arrangement of collagen fibers, and a subsequent increased expression of alpha-smooth muscle actin (α-SMA) and vascular endothelial growth factor A (VEGF-A) leading to an overall improved healing process. The use of PBM promoted a beneficial effect on the rate of integration and healing of FWM. We therefore propose that the adjuvant use of PBM may have utility in enhancing engraftment and tissue repair and be of value in clinical practice.

Preservation of rat limbs by hyperbaric carbon monoxide and oxygen.

Cold ischemia times ranging from <6 h to as long as 24 h are generally quoted as the limits for attempting the replantation of amputated extremities. In this study, we aimed to assess the effect of hyperbaric carbon monoxide (CO) and oxygen (O2) on rat limb preservation. Donor rat limbs were preserved in a chamber filled with hyperbaric CO and O2 for 3 days (CO + O2 3 days) or 7 days (CO + O2 7 days). Positive and negative control groups were created by using non-preserved limbs (NP) and limbs wrapped in saline-moistened gauze for 3 days (SMG 3 days), respectively. The survival rate of transplanted limbs at postoperative day 90 was 88% in the NP and 86% in the CO + O2 3 days. The corresponding survival rate was 50% in the CO + O2 7 days at postoperative day 90 but was 0% in the SMG 3 days at postoperative day 3. Muscle mass decreased in the CO + O2 3 days and CO + O2 7 days compared with the NP, but sciatic-tibial nerve conduction velocities did not differ. These results indicate that amputated extremities preservation with hyperbaric CO and O2 could extend the time limits of preservation, maintaining their viability for replantation.

Lack of the α1,3-Fucosyltransferase Gene (Osfuct) Affects Anther Development and Pollen Viability in Rice.

N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.

Involvement of CAT in the detoxification of HT-induced ROS burst in rice anther and its relation to pollen fertility.

KEY MESSAGE: HT-induced ROS burst in developing anther is closely related to the lowered CAT activity as the result of the markedly suppressed OsCATB transcript, thereby causing severe fertility injury for rice plants exposed to HT at meiosis stage. The reproductive stage of rice plants is highly sensitive to heat stress. In this paper, different rice cultivars were used to investigate the relationship of HT-induced floret sterility with reactive oxygen species (ROS) detoxification in rice anthers under well-controlled climatic conditions. Results showed that high temperature (HT) exposure significantly enhanced the ROS level and malondialdehyde (MDA) content in developing anther, and the increase in ROS amount in rice anther under HT exposure was closely associated with HT-induced decline in the activities of several antioxidant enzymes. For various antioxidant enzymes, SOD and CAT were more susceptible to the ROS burst in rice anther induced by HT exposure than APX and POD, in which SOD and CAT activity in developing anther decreased significantly by HT exposure, whereas APX activity was relatively stable among different temperature regimes. HT-induced decrease in CAT activity was attributable to the suppressed transcript of OsCATB. This occurrence was strongly responsible for HT-induced increase in ROS level and oxidative-damage in rice anther, thereby it finally caused significant reduction in pollen viability and floret fertility for the rice plants exposed to HT during meiosis. Exogenous application of 1000 µM salicylic acid (SA) may alleviate HT-induced reduction in pollen viability and floret fertility, concomitantly with the increased CAT activity and reduced ROS level in rice anther.