Short-Term Storability of Alginate-Encapsulated Persian Violet Microshoots for Germplasm Exchange

Microshoots have been widely used for micropropagation. It may be necessary to store microshoots for a short period of time, for example in germplasm exchange needing transport to other research groups. Here, we investigated the short-term storability of alginate-encapsulated Persian violet (Exacum affine Balf. f. ex Regel) microshoots at 4 °C and 25 °C. After storage, the encapsulated microshoots were sown on basal Murashige and Skoog medium for germination and viability determination using tetrazolium chloride staining. The results showed that one or five microshoots encapsulated with a single alginate layer could be stored at 4 °C for up to 30 days, while the percentages of germination and viability of the microshoots encapsulated with two layers of alginate were greatly reduced upon storage. This is the first report on the storability of alginate-encapsulated multiple microshoots, which could be a more efficient way to encapsulate microshoots used for short-term cold storage.

Preconditioning With Intermittent Hypobaric Hypoxia Attenuates Stroke Damage and Modulates Endocytosis in Residual Neurons

Background: Moderate hypobaric hypoxia induces cerebral ischemic tolerance. We investigated the optimal method for applying hypobaric hypoxia preconditioning at 5,000 m to ischemic brain tissue and combined it with proteomics to determine the mechanisms underlying this effect.Methods: Male SD rats were randomly grouped as S (sham, n = 20), M (middle cerebral artery occlusion [MCAO], n = 28), H2M (intermittent hypobaric hypoxia preconditioned MCAO group, 2 h/day, 10 days, n = 20), H6M (intermittent hypobaric hypoxia preconditioned MCAO group, 6 h/day, 10 days, n = 28), and HpM (persistent hypobaric hypoxia preconditioned MCAO group, 10 days, n = 28). The permanent MCAO model was established based on the Zea Longa method. Infarction was assessed with the modified neurological severity score (mNSS) and 2,3,5-triphenyl tetrazolium chloride staining. The total protein expression of the neuron-specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3), cleaved-caspase-3, and interleukin 6 (IL-6) was determined using western blotting. We assessed the peri-infarct cortex's ultrastructural changes. A label-free proteomic study and western blot verification were performed on the most effective preconditioned group.Results: The H6M group showed a lower infarct volume (p = 0.0005), lower mNSS score (p = 0.0009) than the M group. The H2M showed a lower level of IL-6 (p = 0.0213) than the M group. The caspase-3 level decreased in the H2M (p = 0.0002), H6M (p = 0.0025), and HpM groups (p = 0.0054) compared with that in the M group. Cleaved-caspase-3 expression decreased in the H2M (p = 0.0011), H6M (p < 0.0001), and HpM groups (p < 0.0001) compared with that in the M group. The neurons' ultrastructure and the blood-brain barrier in the peri-infarct tissue improved in the H2M and H6M groups. Immunofluorescence revealed increased NeuN-positive cells in the peri-infarct tissue in the H6M group (p = 0.0003, H6M vs. M). Protein expression of Chmp1a, Arpc5, and Hspa2 factors related to endocytosis were upregulated in the H6M compared with those of the M group (p < 0.05 for all) on western blot verification of label-free proteomics.Conclusions: Intermittent hypobaric hypoxia preconditioning exerts a neuroprotective effect in a rat stroke model. Persistent hypobaric hypoxia stimulation exhibited no significant neuroprotective effect. Intermittent hypoxic preconditioning for 6 h/day for 10 days upregulates key proteins in clathrin-dependent endocytosis of neurons in the cortex.

Root K Affinity Drivers and Photosynthetic Characteristics in Response to Low Potassium Stress in K High-Efficiency Vegetable Soybean

Significant variations of potassium absorption and utilization exist in vegetable soybean. Pot and hydroponic experiments were carried out to examine the characteristics of root potassium (K) affinity-associated drivers and photosynthesis in vegetable soybean (edamame) [Glycine max (L.) Merr.] with different K efficiency. Two K high-efficiency vegetable soybean genotypes (Line 19 and Line 20) and two K low-efficiency genotypes (Line 7 and Line 36) were investigated in low K and normal K conditions. The root of K high-efficiency genotypes had a higher K+ affinity associated with a higher maximum K+ uptake rate (Imax), but lower Michaelis constant for K+ absorption (Km) and lower compensation concentration for K+ uptake (Cmin). Seedlings of K high-efficiency genotypes also had higher root vigor [triphenyl tetrazolium chloride (TTC) reduction method] and greater absorbing activity (methylene blue method), especially in the low K condition. Furthermore, the root bleeding-sap rate of K high-efficiency genotypes in low K stress was 9.9–24.3% greater than that of normal K conditions, which was accompanied by a relatively higher K concentration of root bleeding-sap in contributing to K+ upward flux. The root of K high-efficiency vegetable soybean genotypes exhibited K+ high-affinity and driving advantages. Photosynthetic parameters of K high-efficiency vegetable soybean genotypes were less affected by low K stress. Low K stress decreased the net photosynthetic rate of K high-efficiency genotypes by 6.1–6.9%, while that of K low-efficiency genotypes decreased by 10.9–15.7%. The higher chlorophyll (Chl) a/b ratio with enhanced relative content of Chl a in response to low K stress might be an adapted mechanism for K high-efficiency genotypes to maintain photosynthetic capacity. Stronger root K affinity drivers associated with photosynthetic adaptability to low K stress are the key factors in determining the K high-efficiency of vegetable soybeans.

Evaluation of the condition for storing pollen grains of japanese melon \(\textit{Cucumis melo}\) L. (Cucurbitaceae)

In recent years, studies have been carried on the selection and breeding of melon to reduce its production cost. The F1 seeds of this species are usually created by parental lines. Notably, the parental lines are separated from each other to secure the breeding materials. However, there are no studies on establishing suitable conditions for storing pollen grains of Japanese melon Cucumis melo L. In this study, pollen grains of C. melo were dried at 38 oC temperature, for 1.5 hours, which served as the raw materials. After storage in acetone at 5 oC for 3, 5, 7 days, or in liquid nitrogen for 5 days, pollen grains were stained with triphenyl tetrazolium chloride (TTC) to evaluate their viability and pollination ability. Then, the following parameters including pollen grains’ survival rate, morphology analysis of pollen grains, pollen grains’ fruiting rate, fruit weight, firm seed rate, and germination of melon seeds were analyzed. The study results show that the grains’ survival rates were 86.58%, 88.38%, 87.28% or 88.58% in the group stored in acetone for 3, 5, 7 days or liquid nitrogen for 5 days, respectively. The fertilized fruit rate was 100% in the group stored in liquid nitrogen for 5 days. But the rate of germination from firm seeds was highest, with 94.00% in the group stored in acetone for 3 days. The best method is storing the pollen grains in acetone for 3 days.

Midazolam suppresses ischemia/reperfusion-induced cardiomyocyte apoptosis by inhibiting the JNK/p38 MAPK signaling pathway

Myocardial ischemia/reperfusion (I/R) injury causes irreversible injury to the heart, causing mainly acute myocardial infarction. Midazolam is a benzodiazepine commonly utilized in anesthesia and intensive care. Research has indicated midazolam plays a critical role in many diseases. However, the function of midazolam in myocardial injury induced by I/R warrants further investigation. The infarct size was examined through 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The CK-MB, LDH, and AST levels were tested using commercial kits. Cell apoptosis was determined through TUNEL or flow cytometry assays. Bax, Bcl-2, cleaved caspase-3, p-p38, p38, p-JNK, JNK, p-ERK, and ERK expression was examined through western blot. In our study, midazolam was shown to suppress the infarct size and myocardial enzyme leakage in I/R rats. Additionally, midazolam was found to retard cardiomyocyte apoptosis. The JNK/p38 MAPK signaling pathway in I/R rats was inhibited by midazolam. Our findings demonstrated that in H/R-mediated H9C2 cells, anisomycin abolished the suppressive effects of midazolam on the JNK/p38 MAPK signaling pathway. Next, exploration discovered that anisomycin abolished the cytoprotective effects of midazolam on H/R-treated H9C2 cell apoptosis. Midazolam retarded I/R-induced cardiomyocyte apoptosis by inhibiting the JNK/p38 MAPK signaling pathway. These results may provide new insight into the treatment of myocardial I/R injury.

First Report of Xanthomonas campestris pv. campestris Causing Marginal Leaf Necrosis of Arugula (Eruca vesicaria subsp. sativa) in Serbia

Arugula (Eruca vesicaria subsp. sativa (Miller) Thell., syn. Brassica eruca L.), is an annual cruciferous crop that is increasingly grown for fresh consumption in Serbia. In November 2018, a few detached leaves of cultivated arugula originating from a local producer, showing necrotic lesions, were observed in a fresh vegetable market in Belgrade, Serbia. Information about the disease incidence and severity was not available. Intensity of the observed symptoms was low, but it could be a consequence of the produce quality selection for the market. The leaves developed irregular chlorotic lesions starting from the leaf edge, and tissue within some of them turned dark brown and necrotic (Fig. 1a). From the lesions on different leaves, smooth, bright yellow pigmented, round and opalescent bacterial colonies were isolated on nutrient agar (NA) medium after 72 h of incubation at 26°C. Six bacterial isolates, obtained from three leaf subsamples which induced hypersensitive reaction in tobacco leaves (Nicotiana tabacum L. cv. Samsun), were selected for further studies. On yeast - dextrose – CaCO3 medium, the strains formed characteristic creamy yellow, mucoid, opaque and convex colonies. All isolates were Gram-negative, strictly aerobic, non-fluorescent and catalase positive, did not produce oxidase nor arginine dehydrolase, and did not show pectynolitic activity on potato tuber slices. They hydrolyzed starch, gelatine and esculin, used glucose and sucrose, but not arabinose as a carbon source, and did not reduce nitrates. They grew at 36°C, and tolerated 5% NaCl and 0.02% triphenyl-tetrazolium chloride (Lelliott and Stead, 1987). These growth characteristics were similar as for the reference Xanthomonas campestris pv. campestris (Xcc) strain KFB 105, used in all tests as a positive control (Obradović et al., 2000). The isolates were further characterized by polymerase chain reaction (PCR) using primers DLH120/DLH125, specific for the hrpF gene region of X. campestris according to Berg et al. (2005). Specific DNA fragment of 619 bp was amplified for all tested isolates. Amplification and partial sequencing of the gyrB gene of four isolates was performed using set of primers described by Parkinson et al. (2007). All obtained partial gyrB sequences were identical to each other. According to BLAST analysis (GenBank Acc. Nos. MW508894 - MW508897) they shared 100% of sequence identity with different Xcc strains and 99.5 % with the X.c. pv. raphani pathotype strain, deposited in the NCBI GenBank database. Pathogenicity of the isolates was tested by spraying leaves of 3-week old E. sativa seedlings grown in a commercial potting mix in a greenhouse, with a 24 h-old bacterial culture suspended in sterile distilled water (107 CFU/ml). Xcc strain KFB 105 was used as positive and sterile distilled water as negative control. Inoculated plants were incubated under plastic bags for 48 h and further maintained in a greenhouse at approx. 28°C. On inoculated plants, chlorotic lesions, spreading from the leaf margins, further coalescing into irregular, V-shaped tissue necrosis associated with blackening of veins, developed up to two weeks after inoculation (Fig. 1b, c). The colonies reisolated from symptomatic leaves were identified using PCR, as described above. Based on studied characteristics, all six isolates associated with arugula leaf lesions in Serbia belong to a clonal population. They were identified as X. campestris pv. campestris, the causal agent of black rot, a major disease affecting crucifers, including arugula worldwide (Romero et al., 2008; Rosenthal, et al., 2018). So far, it has been described on Brassica oleracea and B. napus in Serbia (Obradović et al., 2001; Popović et al., 2019). This is the first report of Xcc infecting arugula in this country. The severity of the symptoms developed on artificially inoculated plants indicated significant potential of the pathogen to affect arugula crop in conditions favoring infection. Being a minor crop, accurate information about severity of arugula diseases in Serbia is not available. Lack of crop rotation and close proximity of other Xcc host species on a farm could contribute to further spreading of this problem. Follow up of this arugula disease should reveal the distribution, population structure and genetic diversity of Xcc strains affecting this crop in Serbia.

Upregulation of FoxM1 protects against ischemia/reperfusion-induced myocardial injury

Background. Ischemia/reperfusion (I/R) induced lethal tissue injury in myocardium. FoxM1 (Forkhead Box M1), expressed in proliferating cardiac progenitor cells, could regulate myocardial development. However, the role of FoxM1 in I/R-induced myocardial injury has not been reported yet. Methods. Rats were conducted with regional ischemia followed by reperfusion in myocardium through ligation of the left anterior descending coronary artery. Triphenyl-tetrazolium chloride staining was utilized to assess the infarct size. ELISA was performed to detect activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). Protein expression of FoxM1 in heart tissues and H9c2 were determined by western blot. H9c2 cells were used to establish a hypoxia/reoxygenation cell model, and the cell viability, proliferation and apoptosis were evaluated by MTT, EdU (5-ethynyl-2’-deoxyuridine) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, respectively. Adenovirus (Ad)-mediated over-expression of FoxM1 was injected into the anterior wall of the left ventricle of rats to evaluate the role of FoxM1 on in vivo I/R-induced myocardial injury. Results. FoxM1 was reduced in heart tissues isolated from rats post myocardial I/R injury. Forced FoxM1 expression increased cell viability and proliferation of hypoxia/reoxygenation-induced H9c2, while repressed the cell apoptosis with increased Bcl-2 and decreased Bax and cleaved caspase-3. Injection of Ad-FoxM1 suppressed infarct size of the heart and decreased activities of CK-MB and LDH. Conclusion. FoxM1 attenuated I/R-induced myocardial injury, providing potential therapeutic target for the disease.

Potential oxygen consumption and community composition of sediment bacteria in a seasonally hypoxic enclosed bay

The dynamics of potential oxygen consumption at the sediment surface in a seasonally hypoxic bay were monitored monthly by applying a tetrazolium dye (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride [INT]) reduction assay to intact sediment core samples for two consecutive years (2012–2013). Based on the empirically determined correlation between INT reduction (INT-formazan formation) and actual oxygen consumption of sediment samples, we inferred the relative contribution of biological and non-biological (chemical) processes to the potential whole oxygen consumption in the collected sediment samples. It was demonstrated that both potentials consistently increased and reached a maximum during summer hypoxia in each year. For samples collected in 2012, amplicon sequence variants (ASVs) of the bacterial 16S rRNA genes derived from the sediment surface revealed a sharp increase in the relative abundance of sulfate reducing bacteria toward hypoxia. In addition, a notable shift in other bacterial compositions was observed before and after the INT assay incubation. It was Arcobacter (Arcobacteraceae, Campylobacteria), a putative sulfur-oxidizing bacterial genus, that increased markedly during the assay period in the summer samples. These findings have implications not only for members of Delta- and Gammaproteobacteria that are consistently responsible for the consumption of dissolved oxygen (DO) year-round in the sediment, but also for those that might grow rapidly in response to episodic DO supply on the sediment surface during midst of seasonal hypoxia.

Nobiletin Attenuates Pathological Cardiac Remodeling after Myocardial Infarction via Activating PPARγ and PGC1α

Rationale. Pathological cardiac remodeling serves as a vital mechanism during the progression from myocardial infarction (MI) to chronic heart failure (CHF). Nobiletin (NOB), an active monomer extracted from the peel of citrus fruit, has been reported to have beneficial effects in cardiovascular diseases. Our study was aimed at describing the specific mechanisms through which NOB protects against pathological cardiac remodeling after MI. Materials and Methods. C57BL/6 mice were treated with coronary artery ligation to generate an MI model, followed by treatment for 3 weeks with NOB (50 mg/kg/d) or vehicle (50 mg/kg/d), with or without the peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor T0070907 (1 mg/kg/d). Cardiac function (echocardiography, survival rate, Evans blue, and triphenyl tetrazolium chloride staining), fibrosis (Masson’s trichrome staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB)), hypertrophy (haematoxylin-eosin staining, wheat germ agglutinin staining, and qRT-PCR), and apoptosis (WB and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining) were evaluated. Hypoxia-induced apoptosis (TUNEL, WB) and phenylephrine- (PE-) induced pathological hypertrophy (immunofluorescence staining, qRT-PCR) models were established in primary neonatal rat ventricular myocytes (NRVMs). The effects of NOB with or without T0070907 were examined for the expression of PPARγ and PPARγ coactivator 1α (PGC1α) by WB in mice and NRVMs. The potential downstream effectors of PPARγ were further analyzed by WB in mice. Results. Following MI in mice, NOB intervention enhanced cardiac function across three predominant dimensions of pathological cardiac remodeling, which reflected in decreasing cardiac fibrosis, apoptosis, and hypertrophy decompensation. NOB intervention also alleviated apoptosis and hypertrophy in NRVMs. NOB intervention upregulated PPARγ and PGC1α in vivo and in vitro. Furthermore, the PPARγ inhibitor abolished the protective effects of NOB against pathological cardiac remodeling during the progression from MI to CHF. The potential downstream effectors of PPARγ were nuclear factor erythroid 2-related factor 2 (Nrf-2) and heme oxygenase 1 (HO-1). Conclusions. Our findings suggested that NOB alleviates pathological cardiac remodeling after MI via PPARγ and PGC1α upregulation.

A simple method for the extension of shelf life of cultures of Phytophthora species causing black pod disease of cacao (Theobroma cacao L.)

Black pod disease of cacao caused by Phytophthora palmivora and Phytophthora megakarya in Ghana take heavy toll of cacao production in the field. Intensive research has been carried out worldwide on these pathogens. However, viability of the cultures during prolonged storage has remained a major challenge in the research. This paper reports findings of assessment of six storage media viz sterilised distilled water (SDW), sterilised and unsterilized soil suspension (SSS and USS), vegetable 8 juice broth (V8JB), Oat Meal Agar slant under mineral oil (at 4°C) and empty tube. Viability of the cultures was assessed on V8JA and in tetrazolium chloride test. Ability of zoospores of the cultures to infect cacao leaf discs was used to assess growth vigour and pathogenicity. Phytophthora cultures stored in SDW (26 ± 2°C; alternating day light and night) were preserved for 60 days (5 years). Both P. palmivora and P. megakarya performed better on SDW and SSS than on USS due to removal of staling substances in the soil medium by the sterilisation. Vigour of growth and pathogenicity of the stored cultures required re-inoculation of host tissue (cacao pod) in order to maintain potency to continually infect host.