A non-invasive mouse model that recapitulates disuse-induced muscle atrophy in immobilized patients.

Disuse muscle atrophy occurs consequent to prolonged limb immobility or bed rest, which represents an unmet medical need. As existing animal models of limb immobilization often cause skin erosion, edema, and other untoward effects, we here report an alternative method via thermoplastic immobilization of hindlimbs in mice. While significant decreases in the weight and fiber size were noted after 7 days of immobilization, no apparent skin erosion or edema was found. To shed light onto the molecular mechanism underlying this muscle wasting, we performed the next-generation sequencing analysis of gastrocnemius muscles from immobilized versus non-mobilized legs. Among a total of 55,487 genes analyzed, 787 genes were differentially expressed (> fourfold; 454 and 333 genes up- and down-regulated, respectively), which included genes associated with muscle tissue development, muscle system process, protein digestion and absorption, and inflammation-related signaling. From a clinical perspective, this model may help understand the molecular/cellular mechanism that drives muscle disuse and identify therapeutic strategies for this debilitating disease.

Physical therapy in the intensive care unit: A cross-sectional study of three Asian countries.

BACKGROUND: Physical therapy (PT) is beneficial for critically ill patients, but the extent of its application in the intensive care unit (ICU) differs between countries. Here, we compared the extent of PT intervention in the ICU in Japan, the Philippines, and Taiwan by evaluating the sociodemographic and ICU-related profiles of ICU physical therapists. MATERIALS AND METHODS: In this cross-sectional study, a semistructured nationwide online survey was distributed to ICU physical therapists in the three countries. RESULTS: We analyzed the responses of 164 physical therapists from Japan, Philippines, and Taiwan. Significant differences were observed between the countries in all sociodemographic variables and the following ICU-related profiles of physical therapists: ICU work experience, duration of the ICU posting, number of hours per day spent in the ICU, on-call ICU PT service engagement, source of ICU patient referral, therapist-patient ratio, and ICU-related PT training participation (p < 0.05). Medical, surgical, and neurologic ICUs were the most common ICU workplaces of the ICU physical therapists, but only surgical and neurologic ICUs exhibited significant differences between the countries (p < 0.05). Standard PT techniques in the ICU were passive and active-assisted range of motion, positioning, and breathing exercises but were implemented with significantly different frequencies between the countries (p < 0.05). The most common challenge faced in ICU PT service delivery by respondents from all three countries was lack of training prior to ICU duty, and lack of training was even bigger challenge in Japan than in other two countries after adjustment of age, highest educational attainment, and work experience. CONCLUSION: The differences in the health-care system between Japan, the Philippines, and Taiwan were related to differences in the compliance with internationally recommended PT practice standards in the ICU, differences in the type of PT intervention prioritized, and the challenges encountered in ICU PT service delivery.

Transcriptomic Analyses of Exercise Training in Alzheimer's Disease Cerebral Cortex.

BACKGROUND: Research reported exercise could reduce Alzheimer's disease (AD) symptoms in human and animals. However, the molecular mechanism of exercise training via transcriptomic analysis was unclear especially in AD in the cortex area. OBJECTIVE: Investigate potential significant pathways in the cortex area that were affected by exercise during AD. METHODS: RNA-seq analysis, differential expressed genes, functional enrichment analysis, and GSOAP clustering analysis were performed in the isolated cerebral cortex from eight 3xTg AD mice (12 weeks old) randomly and equally divided into control (AD) and exercise training (AD-EX) group. Swimming exercise training in AD-EX group was conducted 30 min/day for 1 month. RESULTS: There were 412 genes significant differentially expressed in AD-EX group compared to AD group. Top 10 upregulated genes in AD-EX group against AD group mostly correlated with neuroinflammation, while top 10 downregulated genes mostly had connection with vascularization, membrane transport, learning memory, and chemokine signal. Pathway analysis revealed the upregulated interferon alpha beta signaling in AD-EX had association with cytokines delivery in microglia cells compared to AD and top 10 upregulated genes involved in interferon alpha beta were Usp18, Isg15, Mx1, Mx2, Stat1, Oas1a, and Irf9; The downregulated extracellular matrix organization in AD-EX had correlation with Aß and neuron cells interaction and Vtn was one of the top 10 downregulated genes involved in this pathway. CONCLUSION: Exercise training influenced 3xTg mice cortex through interferon alpha beta signaling upregulation and extracellular matrix organization downregulation based on transcriptomics analysis.

Low-Level Laser Therapy Induces Melanoma Tumor Growth by Promoting Angiogenesis.

The effects of low-level laser therapy (LLLT) on tumor growth are inconsistent. In this study, we investigated the effects of LLLT on melanoma tumor growth and angiogenesis. C57/BL6 mice were challenged with B16F10 melanoma cells and treated with LLLT for 5 consecutive days; untreated mice were used as controls. Tumor weight, angiogenesis, immunohistochemistry, and protein levels were compared between the treated and untreated mice. In an in vitro experiment, B16F10 cells were treated with LLLT. Proteins were extracted and subjected to Western blot analysis for analyzing signaling pathways. Compared with the findings in the untreated mice, tumor weight substantially increased in the treated mice. Both immunohistochemical and Western blot analyses revealed markedly increased levels of CD31, a biomarker of vascular differentiation, in the LLLT group. In B16F10 cells, LLLT considerably induced the phosphorylation of extracellular signal-regulated kinase (ERK), which, in turn, phosphorylated p38 mitogen-activated protein kinase (MAPK). Furthermore, LLLT induced the expression of vascular endothelial growth factor, but not hypoxia-inducible factor-1α, through the ERK/p38 MAKP signaling pathways. Our findings indicate that LLLT induces melanoma tumor growth by promoting angiogenesis. Therefore, it should be avoided in patients with melanoma.

Ketamine, benzoate, and sarcosine for treating depression.

Studies have demonstrated the beneficial therapeutic effects of sarcosine, benzoate, and ketamine (including esketamine and arketamine) on depression. These drugs mainly act by modulating N-methyl-d-aspartate glutamate receptors (NMDARs) and reducing inflammation in the brain. Although ketamine, benzoate, and sarcosine act differently as the antagonists or coagonists of NMDARs, they all have demonstrated efficacy in animal models or human trials. In vitro and in vivo studies have indicated that sarcosine, benzoate, and ketamine exert their anti-inflammatory effects by inhibiting microglial activity. This review summarizes and compares the efficacy of the possible therapeutic mechanisms of sarcosine, benzoate, ketamine, esketamine, and arketamine. These compounds act as both NMDAR modulators and anti-inflammatory drugs and thus can be effective in the treatment of depression.

Effects of Different Pedaling Positions on Muscle Usage and Energy Expenditure in Amateur Cyclists.

BACKGROUND: Inappropriate cycling positions may affect muscle usage strategy and raise the level of fatigue or risk of sport injury. Dynamic bike fitting is a growing trend meant to help cyclists select proper bikes and adjust them to fit their ergometry. The purpose of this study is to investigate how the "knee forward of foot" (KFOF) distance, an important dynamic bike fitting variable, influences the muscle activation, muscle usage strategy, and rate of energy expenditure during cycling. METHODS: Six amateur cyclists were recruited to perform the short-distance ride test (SRT) and the graded exercise tests (GXT) with pedaling positions at four different KFOF distances (+20, 0, -20, and -40 mm). The surface electromyographic (EMG) and portable energy metabolism systems were used to monitor the muscle activation and energy expenditure. The outcome measures included the EMG root-mean-square (RMS) amplitudes of eight muscles in the lower extremity during the SRT, the regression line of the changes in the EMG RMS amplitude and median frequency (MF), and the heart rate and oxygen consumption during the GXT. RESULTS: Our results revealed significant differences in the muscle activation of vastus lateralis, vastus medialis, and semitendinosus among four different pedaling positions during the SRT. During GXT, no statistically significant differences in muscle usage strategy and energy expenditure were found among different KFOF. However, most cyclists had the highest rate of energy expenditure with either KFOF at -40 mm or 20 mm. CONCLUSIONS: The KFOF distance altered muscle activation in the SRT; however, no significant influence on the muscle usage strategy was found in the GXT. A higher rate of energy expenditure in the extreme pedaling positions of KFOF was observed in most amateur cyclists, so professional assistance for proper bike fitting was recommended.

Effect of exercise training on cardiac mitochondrial respiration, biogenesis, dynamics, and mitophagy in ischemic heart disease.

Objective: Cardiac mitochondrial dysfunction was found in ischemic heart disease (IHD). Hence, this study determined the effects of exercise training (ET) on cardiac mitochondrial respiration and cardiac mitochondrial quality control in IHD. Methods: A narrative synthesis was conducted after searching animal studies written in English in three databases (PubMed, Web of Science, and EMBASE) until December 2020. Studies that used aerobic exercise as an intervention for at least 3 weeks and had at least normal, negative (sedentary IHD), and positive (exercise-trained IHD) groups were included. The CAMARADES checklist was used to check the quality of the included studies. Results: The 10 included studies (CAMARADES score: 6-7/10) used swimming or treadmill exercise for 3-8 weeks. Seven studies showed that ET ameliorated cardiac mitochondrial respiratory function as manifested by decreased reactive oxygen species (ROS) production and increased complexes I-V activity, superoxide dismutase 2 (SOD2), respiratory control ratio (RCR), NADH dehydrogenase subunits 1 and 6 (ND1/6), Cytochrome B (CytB), and adenosine triphosphate (ATP) production. Ten studies showed that ET improved cardiac mitochondrial quality control in IHD as manifested by enhanced and/or controlled mitochondrial biogenesis, dynamics, and mitophagy. Four other studies showed that ET resulted in better cardiac mitochondrial physiological characteristics. Conclusion: Exercise training could improve cardiac mitochondrial functions, including respiration, biogenesis, dynamics, and mitophagy in IHD. Systematic review registration: https://www.crd.york.ac.uk/prospero/ display_record.php?RecordID=226817, identifier: CRD42021226817.

Impact of Storage Condition on Chemical Composition and Antifungal Activity of Pomelo Extract against Colletotrichum gloeosporioides and Anthracnose in Post-harvest Mango.

Anthracnose caused by Colletotrichum leads to a tremendous post-harvest mango loss. While chemical fungicides are applied to control anthracnose, natural alternatives are preferred due to food safety and environmental concerns. Pomelo extract (PE) exhibits a broad spectrum of antimicrobial activities; however, its effect against anthracnose is unknown. Here we investigated the chemical profile of PE using GC-MS and the anti-anthracnose activity of PE using in vitro and in vivo assays. We also evaluated the impact of storage temperature (0°, 5°, 10°, 20°, -20°, and -80 °C) and light conditions on the composition and antifungal activity of PE. We found that PE inhibited C. gloeosporioides in vitro with an IC50 of 3.2 mL L-1. Applying chitosan-based coating incorporated with 20 mL L-1 PE significantly suppressed anthracnose in post-harvest 'Keitt' mango. A storage temperature below 5 °C substantially preserved major compounds and the antifungal activity of PE after 6 m of storage. Finally, we showed that applying d-limonene, the key constituent of PE, inhibited C. gloeosporioides in vitro (IC50: 10.9 mM) and suppressed anthracnose in vivo. In conclusion, we demonstrated that the application of PE and d-limonene are sustainable methods for anthracnose control in post-harvest crops and established the preservation protocol for PE.

Development of a traditional Chinese medicine-based agent for the treatment of cancer cachexia.

BACKGROUND: Despite recent advances in understanding the pathophysiology of cancer cachexia, prevention/treatment of this debilitating disease remains an unmet medical need. METHODS: We developed an integrated, multi-tiered strategy involving both in vitro and in vivo muscle atrophy platforms to identify traditional Chinese medicine (TCM)-based anti-cachectic agents. In the initial screening, we used inflammatory cytokine-induced atrophy of C2C12 myotubes as a phenotypic screening platform to assess the protective effects of TCMs. The selected TCMs were then evaluated for their abilities to protect Caenorhabditis elegans from age-related reduction of mobility and contractility, followed by the C-26 colon adenocarcinoma mouse model of cachexia to confirm the anti-muscle atrophy effects (body/skeletal muscle weights, fibre size distribution, grip strengths, and serum IL-6). Transcriptome analysis, quantitative real-time polymerase chain reaction, and immunoblotting were performed to gain understanding of the potential mechanism(s) by which effective TCM protected against C26 tumour-induced muscle atrophy. RESULTS: Of 29 widely used TCMs, Dioscorea radix (DR) and Mu Dan Pi (MDP) showed a complete protection (all P values, 0.0002) vis-à-vis C26 conditioned medium control in the myotube atrophy platform. MDP exhibited a unique ability to ameliorate age-associated decreases in worm mobility, accompanied by improved total body contractions, relative to control (P < 0.0001 and <0.01, respectively), which, however, was not noted with DR. This differential in vivo protective effect between MDP and DR was also confirmed in the C-26 mouse model. MDP at 1000 mg/kg (MDP-H) was effective in protecting body weight loss (P < 0.05) in C-26 tumour-bearing mice without changing food or water intake, accompanied by the restoration of the fibre size distribution of hindleg skeletal muscles (P < 0.0001) and the forelimb grip strength (P < 0.05). MDP-treated C-26-tumour-bearing mice were alert, showed normal posture and better body conditions, and exhibited lower serum IL-6 levels (P = 0.06) relative to vehicle control. This decreased serum IL-6 was associated with the in vitro suppressive effect of MDP (25 and 50 µg/mL) on IL-6 secretion into culture medium by C26 cells. RNA-seq analysis, followed by quantitative real-time polymerase chain reaction and/or immunoblotting, shows that MDP's anti-cachectic effect was attributable to its ability to reverse the C-26 tumour-induced re-programming of muscle homoeostasis-associated gene expression, including that of two cachexia drivers (MuRF1 and Atrogin-1), in skeletal muscles. CONCLUSIONS: All these findings suggest the translational potential of MDP to foster new strategies for the prevention and/or treatment of cachexia. The protective effect of MDP on other types of muscle atrophy such as sarcopenia might warrant investigations.

Detection and genetic characterization of the novel torque teno virus group 6 in Taiwanese general population.

Torque teno virus (TTV) is one of the most common human viruses and can infect an individual with multiple genotypes chronically and persistently. TTV group 6 is a recently discovered phylogenetic group first isolated from eastern Taiwan indigenes, but whether the TTV group 6 was also prevalent in the general population still unknown. One hundred and three randomly collected blood samples from general population and 66 TTV positive DNA samples extracted from Taiwan indigenes were included. A group-6-specific PCR was developed for re-screen over TTV positive samples. Two TTV group 6 positive samples from general population were cloned and sequenced for identifying mix-infected TTVs and confirming their classification by maximum-likelihood and Bayesian inference phylogeny. TTV group 6 can be detected in 4.5% (4/89) and 7.6% (5/66) of TTV positive samples from Taiwanese general population and eastern Taiwan indigenes, respectively. Sample VC09 was mix-infected with TTV groups 3 and 6. Sample VC99 was mix-infected with TTV groups 3, 4 and 6. A highly diverse triple overlapping region was observed, which may represent a unique phenomenon of TTV. The group-6-specific PCR can successfully detect TTV group 6. TTV group 6 may be prevalent worldwide regardless of the geographic region and/or ethnic groups.

d-Amino Acids and pLG72 in Alzheimer's Disease and Schizophrenia.

Numerous studies over the last several years have shown that d-amino acids, especially d-serine, have been related to brain and neurological disorders. Acknowledged neurological functions of d-amino acids include neurotransmission and learning and memory functions through modulating N-methyl-d-aspartate type glutamate receptors (NMDARs). Aberrant d-amino acids level and polymorphisms of genes related to d-amino acids metabolism are associated with neurodegenerative brain conditions. This review summarizes the roles of d-amino acids and pLG72, also known as d-amino acid oxidase activator, on two neurodegenerative disorders, schizophrenia and Alzheimer's disease (AD). The scope includes the changes in d-amino acids levels, gene polymorphisms of G72 genomics, and the role of pLG72 on NMDARs and mitochondria in schizophrenia and AD. The clinical diagnostic value of d-amino acids and pLG72 and the therapeutic importance are also reviewed.

Cerebral Cortex Apoptosis in Early Aged Hypertension: Effects of Epigallocatechin-3-Gallate.

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg-94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

Effects of Treadmill Exercise on Neural Mitochondrial Functions in Parkinson's Disease: A Systematic Review of Animal Studies.

This systematic review sought to determine the effects of treadmill exercise on the neural mitochondrial respiratory deficiency and neural mitochondrial quality-control dysregulation in Parkinson's disease. PubMed, Web of Science, and EMBASE databases were searched through March 2020. The English-published animal studies that mentioned the effects of treadmill exercise on neural mitochondria in Parkinson's disease were included. The CAMARADES checklist was used to assess the methodological quality of the studies. Ten controlled trials were included (median CAMARADES score = 5.7/10) with various treadmill exercise durations (1-18 weeks). Seven studies analyzed the neural mitochondrial respiration, showing that treadmill training attenuated complex I deficits, cytochrome c release, ATP depletion, and complexes II-V abnormalities in Parkinson's disease. Nine studies analyzed the neural mitochondrial quality-control, reporting that treadmill exercise improved mitochondrial biogenesis, mitochondrial fusion, and mitophagy in Parkinson's disease. The review findings supported the hypothesis that treadmill training could attenuate both neural mitochondrial respiratory deficiency and neural mitochondrial quality-control dysregulation in Parkinson's disease, suggesting that treadmill training might slow down the progression of Parkinson's disease.

From Menopause to Neurodegeneration-Molecular Basis and Potential Therapy.

The impacts of menopause on neurodegenerative diseases, especially the changes in steroid hormones, have been well described in cell models, animal models, and humans. However, the therapeutic effects of hormone replacement therapy on postmenopausal women with neurodegenerative diseases remain controversial. The steroid hormones, steroid hormone receptors, and downstream signal pathways in the brain change with aging and contribute to disease progression. Estrogen and progesterone are two steroid hormones which decline in circulation and the brain during menopause. Insulin-like growth factor 1 (IGF-1), which plays an import role in neuroprotection, is rapidly decreased in serum after menopause. Here, we summarize the actions of estrogen, progesterone, and IGF-1 and their signaling pathways in the brain. Since the incidence of Alzheimer's disease (AD) is higher in women than in men, the associations of steroid hormone changes and AD are emphasized. The signaling pathways and cellular mechanisms for how steroid hormones and IGF-1 provide neuroprotection are also addressed. Finally, the molecular mechanisms of potential estrogen modulation on N-methyl-d-aspartic acid receptors (NMDARs) are also addressed. We provide the viewpoint of why hormone therapy has inconclusive results based on signaling pathways considering their complex response to aging and hormone treatments. Nonetheless, while diagnosable AD may not be treatable by hormone therapy, its preceding stage of mild cognitive impairment may very well be treatable by hormone therapy.

Photobiomodulation Therapy to Promote Angiogenesis in Diabetic Mice with Hindlimb Ischemia.

Objective: To assess whether photobiomodulation therapy (PBMT) induces angiogenesis in diabetic mice with hindlimb ischemia (HLI). Background: Patients with diabetes mellitus (DM) are at high risk of developing peripheral arterial disease (PAD) in the lower extremities. PBMT has been shown to promote angiogenesis both in vitro and in vivo and could be a treatment for DM patients with PAD. Methods: Femoral artery ligation/excision in mice was performed to induce HLI as an animal model of PAD. PBMT at a dose of 660 nm and 1.91 J/cm2 was delivered for 10 min on 5 consecutive days after the HLI surgery. Control mice received HLI only. Mice in the DM group were injected with streptozocin to induce diabetes before HLI surgery. Mice in the laser and DM+ laser groups received both HLI and PBMT, and the latter group had induced DM. After the laser treatment, lower limb blood flow was evaluated by laser Doppler. The capillary density and CD31 were analyzed by immunofluorescence staining, and protein levels of vascular endothelial growth factor (VEGF)-A, hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinases (ERK) were measured by Western blotting of tissue samples. Results: Compared with the control and DM mice, the laser and DM+ laser groups had more than double the capillary density and blood perfusion rate. Levels of CD31 and VEGF-A proteins in groups that received laser were increased by 1.9- to 3.2-fold compared with groups that did not undergo laser treatment. Animals treated with PBMT exhibited significantly increased HIF-1α expression and ERK phosphorylation compared with animals that did not receive this treatment, and the amount of phospho-eNOS and iNOS increased and decreased, respectively. Conclusions: PBMT can induce therapeutic angiogenesis, indicating that low intensity laser could be a novel treatment for PAD patients.

Involvement of Cholinergic, Adrenergic, and Glutamatergic Network Modulation with Cognitive Dysfunction in Alzheimer's Disease.

Alzheimer's disease (AD), the most common cause of dementia, is a progressive neurodegenerative disease. The number of AD cases has been rapidly growing worldwide. Several the related etiological hypotheses include atypical amyloid ß (Aß) deposition, neurofibrillary tangles of tau proteins inside neurons, disturbed neurotransmission, inflammation, and oxidative stress. During AD progression, aberrations in neurotransmission cause cognitive decline-the main symptom of AD. Here, we review the aberrant neurotransmission systems, including cholinergic, adrenergic, and glutamatergic network, and the interactions among these systems as they pertain to AD. We also discuss the key role of N-methyl-d-aspartate receptor (NMDAR) dysfunction in AD-associated cognitive impairment. Furthermore, we summarize the results of recent studies indicating that increasing glutamatergic neurotransmission through the alteration of NMDARs shows potential for treating cognitive decline in mild cognitive impairment or early stage AD. Future studies on the long-term efficiency of NMDA-enhancing strategies in the treatment of AD are warranted.

Exercise training attenuates cardiac inflammation and fibrosis in hypertensive ovariectomized rats.

This study investigated the effects of exercise training on cardiac inflammatory and cardiac fibrotic pathways in female spontaneously hypertensive rats (SHR), which were divided into a sham-operated sedentary hypertensive group (SHR-S), a sedentary hypertensive ovariectomized group (SHR-O), or a hypertensive ovariectomized group with treadmill exercise training (SHR-OT; 60 min/day, 5 days/wk) for 8 wk. Normotensive female Wistar-Kyoto rats (WKY) served as controls. SOD and catalase (CAT) activities were significantly increased in the SHR-OT group, when compared with the SHR-S or SHR-O groups. The protein levels of estrogen receptor (ER)-α and ER-ß became decreased in the SHR-O group, when compared with the WKY or SHR-S groups, but were not changed in the SHR-OT group. The protein level of the angiotensin II type I receptor (AT1R) was increased in the SHR-S group but did not further change in the SHR-O group, whereas it was decreased in the SHR-OT group. The inflammatory-related protein levels of TNF-α, p-NF-κB, cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6, as well as the fibrotic-related protein levels of transforming growth factor-ß (TGF-ß), p-Smad2/3, connective tissue growth factor (CTGF), tissue-type plasminogen activator (tPA), matrix metalloproteinase (MMP)-9, and collagen I were increased in the SHR-S group and increased further in the SHR-O group, whereas they were decreased in the SHR-OT group. The coexistence of hypertension and ovariectomy additively increased cardiac inflammatory and fibrotic pathways partially through hypertension-enhanced AT1R and ovariectomy-depressed estrogen receptors. Exercise training appeared to suppress hypertensive ovariectomized heart-induced inflammatory and fibrotic pathways possibly through decreasing AT1R but not through estrogen receptors.NEW & NOTEWORTHY The coexistence of hypertension and ovariectomy appeared to increase cardiac inflammatory and fibrotic pathways likely through hypertension-enhanced angiotensin II type I receptor and ovariectomy-depressed estrogen receptors. Exercise training on a treadmill could prevent hypertensive ovariectomized heart-induced cardiac inflammation and fibrosis via an inflammatory pathway [TNF-α, p-IKK-α/ß, p-NF-κB, cyclooxygenase 2 (COX-2), iNOS, and IL-6] and fibrotic pathway [transforming growth factor-ß (TGF-ß), p-Smad2/3, connective tissue growth factor (CTGF), tissue-type plasminogen activator (tPA), matrix metalloproteinase (MMP)-9, and collagen I] possibly through decreasing angiotensin II type I receptor but not through estrogen receptors.

Genome Editing and Protoplast Regeneration to Study Plant-Pathogen Interactions in the Model Plant Nicotiana benthamiana.

Biotic diseases cause substantial agricultural losses annually, spurring research into plant pathogens and strategies to mitigate them. Nicotiana benthamiana is a commonly used model plant for studying plant-pathogen interactions because it is host to numerous plant pathogens and because many research tools are available for this species. The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of several powerful tools available for targeted gene editing, a crucial strategy for analyzing gene function. Here, we demonstrate the use of various CRISPR-associated (Cas) proteins for gene editing of N. benthamiana protoplasts, including Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas12a (FnCas12a), and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID). We successfully mutated Phytoene Desaturase (PDS) and Ethylene Receptor 1 (ETR1) and the disease-associated genes RNA-Dependent RNA Polymerase 6 (RDR6), and Suppressor of Gene Silencing 3 (SGS3), and confirmed that the mutated alleles were transmitted to progeny. sgs3 mutants showed the expected phenotype, including absence of trans-acting siRNA3 (TAS3) siRNA and abundant expression of the GFP reporter. Progeny of both sgs3 and rdr6 null mutants were sterile. Our analysis of the phenotypes of the regenerated progeny indicated that except for the predicted phenotypes, they grew normally, with no unexpected traits. These results confirmed the utility of gene editing followed by protoplast regeneration in N. benthamiana. We also developed a method for in vitro flowering and seed production in N. benthamiana, allowing the regenerants to produce progeny in vitro without environmental constraints.

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.

KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.

Combined effects of 17ß-estradiol and exercise training on cardiac apoptosis in ovariectomized rats.

BACKGROUND: The purpose of this study was to investigate the combined 17ß-estradiol (E2) and exercise training on cardiac pro-survival and anti-apoptotic pathways in ovariectomized rats. METHODS: Fifty-six female Sprague-Dawley rats were divided into a sham-operated (Sham), a bilaterally ovariectomized (OVX), an OVX treated with E2 (OVX-E2; 10µg/kg/day), and an OVX with E2 and treadmill exercise training (OVX-E2-EX; 60 min/day, 5 days/week) for 10 weeks. Following 10 weeks of exercise training, rat hearts were isolated for the evaluation of Histopathological analysis, TUNEL assay, and Western blotting. RESULTS: The protein levels of estrogen receptor α (ERα), estrogen receptor ß (ERß), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-phosphatidylinositol 3-kinase (p-PI3K) (estrogen receptors/IGF-1-related survival pathway) were significantly increased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. The protein levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL) and phosphorylated-Bad (p-Bad) (Bcl-2 family survival pathway) were significantly increased in the OVX-E2-EX group when compared with the OVX group. Only the p-Bad was significantly increased in the OVX-E2 group when compared with the OVX group. The protein levels of truncation of Bid (t-Bid), Bcl-2-associated death promotor (Bad), Bcl-2-associated X protein (Bax), Cytochrome c, caspases-9, and caspases-3 (mitochondria-dependent apoptotic pathway), as well as the protein levels of tumor necrosis factor-α (TNF-α), Fas ligand, Fas receptors, Fas-associated death domain (FADD), activated caspase-8 and activated caspase-3 (Fas receptor-dependent apoptotic pathway) were significantly decreased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. Furthermore, when compared with the OVX-E2 group, the protein levels of ERß, IGF-1, IGF-1R, Bcl-2 and Bcl-xL were further enhanced in the OVX-E2-EX group as well as the protein levels of Cytochrome c, Fas receptors, FADD, activated caspase-8, activated caspase-9 and activated caspase-3 were further decreased in the OVX-EX-E2 group. CONCLUSIONS: Combined E2 and exercise training exhibited a positive effect of protection on ovariectomy-induced cardiac apoptosis by enhancing ERß-related survival pathways, which might provide a more effective therapeutic effect on cardiac protection in bilaterally oophorectomized or menopausal women than E2 treatment only.