Ml Of PBS Research Articles

Abstract 16849: Extracellular Vesicles Derived From Mesenchymal Stem Cells Protects Mice From Monocrotaline-Induced Pulmonary Hypertension and Improve Echocardiographic Parameters

Introduction: Pulmonary hypertension (PH) is a currently incurable disease with high morbidity and mortality. Treatment with extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) appeared to be effective and promising. Objective: We aimed at evaluating whether MSC-EVs could improve right ventricle (RV) and pulmonary artery (PA) functions in mice with PAH induced by monocrotaline (MCT). Methods: FVB mice (M/F – 6 weeks) received MCT injections 1x/week for 4 weeks (60mg/kg sc.) and were treated with MSCs-EVs 24 hours after each MCT injection (3x10 6 cells in 100μL PBS 1x, iv.). The experimental groups were: Control (vehicle-vehicle; n=9); MCT (MCT-vehicle; n=10); Control EVs (vehicle-EVs; n=8); MCT EVs (MCT-EVs; n=10); PA acceleration time (PAT), PA ejection time (PET) and tricuspid annular plane systolic excursion (TAPSE) were assessed by echocardiography 28 days after first MCT injection; Fulton index was used to calculate RV hypertrophy, and pulmonary vascular remodeling (WT/D) was assessed by histology. Results: PAT and PAT/PET were decreased by 22.6±5.7% and 15.6±4.9%, respectively, in MCT group compared to Control (p<0.01 and p<0.02). PAT was increased in Control EVs and MCT EVs groups when compared with MCT group (by 28.2±7.6%, p<0.01 and 20±6.9%, p<0.04, respectively). TAPSE (figure 1) was decreased in the MCT group compared with Control (42.9±6.7%, p<0.0001) and increased in both Control EVs (62.8±12.2%, p<0.0001) and MCT EVs (52.7±11.1%, p<0.0005) groups compared with MCT group. In addition, MCT group presented RV hypertrophy 49.4±16.5% higher than Control (p<0.03), and 29.1±10.4% higher than MCT EVs group (p<0.05). The WT/D was increased in MCT compared to Control (63.7±7.6%, p<0.0001), but decreased in Control EVs (44.9±4.6%, p<0.0001) and MCT EVs (44.3±5.4%, p<0.0001) in relation to MCT group. Conclusion: Treatment with MSC-EVs protects against the development of PH and improves PA and the RV function in MCT-treated mice.

Quantitative detection of ricin in beverages using trypsin/Glu-C tandem digestion coupled with ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS.The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion.Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1ng/mL in PBS and 0.5ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5-300ng/mL in PBS, 1.0-400ng/mL in milk, and 1.0-250ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9-105.2% for milk, and 95.3-118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3-9.4%, 0.7-8.9%, and 0.2-6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.

Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay.

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.

L-GLUTAMINE SUPPLEMENTATION ASSOCIATED WITH MODERATE AEROBIC TRAINING IMPROVES BIOMETRIC, GLYCEMIC PROFILE AND THE ANTIOXIDANT DEFENSE

Introduction: L-glutamine is a non-essential amino acid, whose intrinsic pool of appears to be depleted during catabolic conditions, such as intense or high duration exercise, and to avoid the exercise- related benefits. Therefore, its supplementation could provide an additional source of L-glutamine and prevent these effects. However, the oral intake of its free form has been discouraged, despites of some evidences reporting positive effects. Objective: to verify whether the L-glutamine supplementation (in its free form) could provide an additional improvement in biometric, glycemic and redox parameters, in animals undergoing moderate aerobic training (MAT). Methods: 28 Swiss male mice were divided into four groups: Cont (n=7), Ex (n=7), Glut (n=8), and Ex+Glut (n=6). Glut and Ex+Glut received gastric gavage of L-glutamine (1g/kg), while Cont and Ex groups received 100 µL of PBS one hour before exercising, five days/week, six weeks. Ex and Ex+Glut underwent moderate swimming, while Cont and Glut remained sedentary, for the same period. Mice started swimming with 2% of body weight attached to the tail during 20 min, and ended the experiment with 4% during 60 min. Results: L-glutamine supplementation increased the gastrocnemius mass and improved the glucose tolerance in animals submitted to MAT. It improved the antioxidant status in gastrocnemius, liver and pancreas, and declined it in adipose tissue in animals undergoing MAT. The drop of adipose antioxidant defense was associated with adiposity, while pancreas antioxidant activity was inversely associated with the glucose intolerance. Conclusion: L-glutamine (free form) improves biometric and glucose parameters, and enhances antioxidant activities.

Preparation and characteristics of W/O microemulsion stabilized with polyglycerylpolyricinoleate as a potential system for oral insulin delivery

Aim. The aim of the present work was to develop the composition and study the characteristics of water-in-oil microemulsion stabilized with polyglycerylpolyricinoleate — Tween 80 — ethanol mixture as a potential system for oral insulin delivery. Materials and methods. To determine the boundaries of the regions of existence of water-in-oil microemulsion in the pseudo-three component systems water — polyglycerylpolyricinoleate (PG-3-PR, Gobiotics BV, Netherlands)/ Tween 80/ethanol — paraffin oil, mixtures of paraffin oil and surfactants with oil — surfactant ratios from 9.5:0.5 to 0.5: 9.5 (wt.) were thoroughly mixed and titrated with an aqueous phase (distilled water).Compositions with the value of hydrophilic-lipophilic balance of the PG-3-PR — Tween 80 mixture equal to 6.15 were studied. Among several types of formed systems, a single-phase region corresponding to a homogeneous, optically transparent, liquid water-in-oil microemulsion was determined. The kinetic and thermodynamic stability of a number of compositions, including those containing insulin (Actrapid HM, Novo Nordisk А/С, Denmark), was studied. The values of the effective viscosity of microemulsions at different ratios of surfactant — oil and surfactant — co-surfactant were determined using a vibration viscometer. Based on the results obtained, a composition was selected to study the kinetics of insulin release into a model environment that simulated the environment of the small intestine. Insulin solution (the control sample) and the insulin-containing microemulsion were placed in the dialysis bags and immersed in 50 mL of PBS (pH 7.4) in a shaking incubator at 180 rpm and 37 ° С. At predetermined intervals, the aliquots of dissolution media were withdrawn, and the concentration of the released peptide was determined by the Bradford assay using a UV spectrophotometer at 595 nm. Results. The composition with 9:1 surfactant — co-surfactant ratio, containing 10 % of the aqueous phase (an insulin solution with a concentration of 100 IU / ml), which remained stable both during three cycles of freezing/thawing and heating/cooling, and after long-term storage at room temperature, was selected to study the kinetics of in vitro release of the peptide into the model medium. The effective viscosity of the sample was 2.4±0.04 Pa.s. The microemulsion sample demonstrated a prolonged release of insulin within 48 hours of the experiment (43 %). Conclusions. As a result, the boundaries of the existence of microemulsion regions in pseudo-three — component systems water — polyglycerylpolyricinoleate / Tween 80 / ethanol — paraffin oil were established, as well as the values of the effective viscosity of a number of compositions were determined. The study of the kinetic and thermodynamic stability of the obtained systems, including those containing insulin, as well as the study of the kinetics of the release of biologically active substance from the microemulsion into the model medium, allowed us to determine the optimal composition for further development of nanoscale dosage forms intended for prolonged delivery of insulin to the gastrointestinal tract.

Intratunical injection of human urine-derived stem cells derived exosomes prevents fibrosis and improves erectile function in a rat model of Peyronie's disease.

We aimed to evaluate the effects of intratunical injection of exosomes derived from human urine-derived stem cells (USC-exo) on plaque formation and erectile function in a transforming growth factor-β1 (TGF-β1) induced Peyronie's disease (PD) rat model. Twenty-four SD rats were randomly assigned equally to three groups: (I) Sham group (50μl phosphate-buffered saline [PBS] injected into the tunica albuginea [TA]), (II) PD group (0.5μg TGF-β1 in 50μl PBS injected into the TA) and (III) USC-exo group (0.5 ug TGF-β1 plus 100μg USC-exo injected into the TA at the same day). The maximum intracavernous pressure (ICPmax ) and mean arterial pressure (MAP) of each group were evaluated 4weeks after injection. The plaque formation, fibrosis, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) in the TA were evaluated. Four weeks after injection, USC-exo group showed more significantly enhanced ICPmax and ICPmax /MAP than PD group (p<.05). USC-exo could significantly ameliorate the TA fibrosis that could be associated with the inhibition of transdifferentiation of fibroblasts into myofibroblasts, decreased expression of TIMPs (TIMP-1, 2, 3) and increased activity of MMPs (MMP-1, 3, 9) in the TA. According to these findings, USC-exo can be a new candidate for the prevention of PD.

Rapid sensing response for phenol with CuO nanoparticles

The human beings are directly or indirectly affected with the environmental pollution, excreted from the industries in the form of liquids such as inorganic and organic hazardous material. As compared to the inorganic chemicals, the organic aromatic compounds are more toxic for the nature. The phenol (PhOH), which is one the largest applied organic compounds in industries and hose hold chemical, which affect to the human health. Due to the wide applicability of copper oxide (CuO) nanomaterial in numerous areas such as optoelectronics, photocatalysis, solar cells, and energy evolution etc; easy to process and provide enhanced and rapid detectible responses against phenol is utilized in this study. The CuO-NPs were synthesized at low refluxing (at 65°C, 90 min) temperature and were well characterize via XRD (X-ray diffraction pattern), FESEM (Field emission scanning electron microscopy), TEM (Transmission electron microscopy) and FTIR (Fourier transform infrared spectroscopy), revels the spherical shaped morphology (∼10 nm). The NPs were pasted on a glassy carbon electrode (GCE) to ascertain their sensing efficiency against PhOH via three electrode system. The efficiency of modified electrode was evaluated under different parameters,concentration effect of PhOH (very low, mid and high) (1, 10 and 31 μL/100 mL of PBS) tested and discloses that the sensor efficiency is very high and concentration dependent at all detectable limits. The formed sensor was also tested with different potentials from 1, 5, 10, 20, 50 and 100 mV/s and it’s sequential to the potential. The chronoamperometry (CuO-NPs/GCE) was investigated in absence and presence and of PhOH at four different voltages +0.05 to +1.2 V from 0 to 1500s to observe the effect of time, which is time reliant. The reproducibility and stability were also tested in terms of cyclic response for seven days (RSD 2.13%), which further displayed that the processed sensor is much reliable for a longer period. To understand well, furthermore, a possible mechanism was also presented, based on obtained results.

Neurotoxicity of HIV-1 Tat is attributed to its penetrating property

We have recently engineered an exosomal Tat (Exo-Tat) which can activate latent HIV-1 in resting CD4+ T lymphocytes from antiretroviral treated HIV-1 infected patients. HIV-1 Tat protein can penetrate cell membrane freely and secrete into extracellular medium. Exo-Tat loses this penetrating property. HIV-1 Tat protein can damage the synaptic membranes contributing to the development of dementia in HIV-1 infected patients. To investigate whether the penetrating property attributes to synaptic damage in vivo, we have generated adeno-associated viruses AAV-Tat and AAV-Exo-Tat viruses. Vehicle control or AAV viruses (1 × 1012 GC/mouse in 200 μl PBS) were injected into Balb/cj mice via tail veins. The mRNA and protein expression levels in blood, brain, heart, intestine, kidney, liver, lung, muscle and spleen were determined on day 21. Intravenously injected AAV-Tat or AAV-Exo-Tat mainly infects liver and heart. Short-term expression of Tat or Exo-Tat doesn’t change the expression levels of neuronal cytoskeletal marker β3-tubulin and synaptic marker postsynaptic density 95 protein (PSD-95). Wild-type Tat, but not Exo-Tat, reduces the expression level of synaptic marker synaptophysin significantly in mice, indicating that penetrating property of HIV-1 Tat protein attributes to synaptic damage.

Abstract PR11: Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube*

Abstract Background: High demand for liquid biopsy tests in cancer research increases the need for combined approaches that allow multimodal testing from a limited blood volume. Although crosslinking-based fixatives allow for efficient preservation of the circulating cell-free DNA (ccfDNA) profile in whole blood, analysis of mRNA from circulating tumor cells (CTCs) is hampered. Currently, there are no blood collection tubes available that allow RNA analysis from CTCs after prolonged (&amp;gt;48 hours) storage. In this research study, we present a newly developed combined workflow for blood stabilization and subsequent analysis of CTC RNA, ccfDNA, and gDNA after prolonged (≥72 hours) storage. Methods: To establish the multimodal workflow, blood was collected from healthy donors into PAXgene Blood ccfDNA Tubes*, aliquoted and manually spiked with 20 individual LNCaP95 cells/5 mL blood as a CTC model or 20 µL PBS as a control. Blood samples were spiked and stored at 2–8°C for 3, 24, 48, and 72 hours before processing. CTCs were enriched and detected using the AdnaTest ProstateCancerPanel AR-V7† (QIAGEN). The plasma and cellular fraction of the CTC-depleted blood was then used to extract ccfDNA and gDNA, respectively. As a control for ccfDNA and gDNA yield measurements, plasma and the cellular fraction were also generated from whole samples (not CTC-depleted) spiked with 20 LNCaP95 cells/5 mL. The resulting multimodal workflow was tested: 1) in comparison to Streck Cell-Free DNA BCT; 2) to assess the impact of room temperature. Results: Tumor cells spiked into PAXgene Blood ccfDNA Tubes could be enriched and detected at experimental time points 3, 24, 48, and 72 hours after spiking with 100% test sensitivity within 24 hours storage and 91% test sensitivity after 72 hours storage at 2–8°C. In unspiked samples, the PAXgene stabilization solution did not cause false positive signals at any experimental time point. Yields of ccfDNA and gDNA were not statistically significantly affected by CTC capture (p&amp;gt;0.05). RNA from spiked tumor cells was detected in blood samples collected and stored in Streck Cell-Free DNA BCTs for 3 hours, but not at later time points. Storage of PAXgene stabilization solution-treated samples at room temperature is feasible, with 100% test sensitivity for CTC detection within 24 hours storage (similar to storage at 2–8°C) and 80% after 72 hours storage. Conclusions: Blood collected into PAXgene Blood ccfDNA Tubes and stored for up to 72 hours at 2–8°C or at room temperature can be used for multimodal analysis of CTC RNA, ccfDNA, and gDNA from a single blood sample. *For research use only. Not for use in diagnostic procedures. †For molecular biology applications. Not intended for the diagnosis, prevention, or treatment of a disease. This abstract is also being presented as Poster B18. Citation Format: Eric Provencher, Maike Remmel, Siegfried Hauch, Michael Otte, Andrea Ullius, Daniel Groelz, Anna Babayan. Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube* [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR11.

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

Anti-endometriotic effect of &lt;i&gt;Angelica sinensis&lt;/i&gt; (Oliv.) Diels extract in human endometriotic cells and rats

Purpose: To study the anti-endometriotic effect of Angelica sinensis (Oliv.) Diels extract (ASDE) in human endometriotic cells and rats.Method: Forty female rats were randomly divided into four groups (10 rats/group): control, endometriosis+danazol, endometriosis+high dose of ASDE and low dose of ASDE. The rats were orally administered either vehicle (200 μL of PBS) alone or ASDE (140, 280 and 560 mg/kg/day) for 5 weeks. Danazol was used as the control drug. After induction of endometriosis for 4 weeks, the rats were sacrificed by cervical dislocation and the peritoneum and visceral organs examined visually to measure the number of endometriotic lesions. Serum levels of cancer antigen 125 (CA-125) and interleukin 13 (IL-13), interleukin 18 (IL-18) and tumor necrosis factor-alpha (TNF-α) of peritoneal fluids of rats were measured using ELISA kits. Western blot assay was performed to measure the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions after 24 h of treatment with ASDE (30, 60, and 120 μg/mL).Results: ASDE-treated rats displayed reduced numbers of total endometriotic lesions when compared with vehicle-treated controls (p &lt; 0.01). When the rats were treated with high dose of ASDE, serum CA-125 level, as well as IL-18 and TNF-α levels in peritoneal fluids were significantly lower than that of the control group (p &lt; 0.01); however, IL-13 level in peritoneal fluids was significantly higher than that of the control group (p &lt; 0.01). ASDE treatment significantly suppressed the levels of MMP-2 and MMP-9 protein in 11Z cell (p &lt; 0.01).Conclusion: The results reveal that ASDE exhibits significant anti-endometriotic effect by inhibiting inflammatory factors in rats. Thus, the plant extract can potentially be developed for the clinical management of endometriosis.&#x0D; Keywords: Angelica sinensis, Endometriosis, Cancer antigen, Endometriotic lesions, Matrix metalloproteinase

In-vivo Assessment of Neurotoxicological Effect of Mugwort Leaves on Induced Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating ailment in which the loss of myelin destructs conduction along the affected axons typically resulting in the conduction block. The motive of this protocol is to carry out the neuroprotective activity of Artemisia vulgaris (Mugwort) by induced demyelination of ethidium Bromide in wistar rats. The leaf methanolic extract was treated at 100mg/kg and 200mg/kg weight administered through oral route and continued for 28 days in demyelinated rats. Demyelination was affected by administering intracranial injection of toxin (ethidium Bromide) at the dose of 1µg/0.03ml of PBS per kg body weight. The potency of the extract was analysed in the terms of their behavioural study on the first, second and fourth week. The animals were immolate after 28 days and subjected to histopathological assessment. The unearthing from behavioural histopathological and biochemical studies evince that the methanol extract of Artemisia vulgaris have potential protective effect on the ethidium bromide induced demyelinated rats.

Modulation of angiogenesis by topical application of leptin and high and low molecular heparin using the Japanese quail chorioallantoic membrane model.

Pathological angiogenesis characterized by uncontrollable vessel growth is an accompanying feature of many diseases. The avian embryo chorioallantoic membrane (CAM) is an excellent model for angiogenesis research. In our study we used a less common Japanese quail CAM model for the testing of angiogenic potential of leptin, high-molecular (heparin sodium) andlow-molecular (nadroparin calcium) heparins. Heparins play a significant role in vascular endothelial cell function, and they are able to modulate the activities of angiogenic growth factors. On embryonic day 7 leptin (5 μg per CAM), heparin sodium (75 IU per CAM) and nadroparin calcium (47.5 IU per CAM) in 500 μl PBS were applied on the CAM surface. After 24 h the fractal dimension (Df) of the vasculature was evaluated. Samples from each group were histologically analyzed and VEGF-A and Quek1 expression were detected by qPCR. Df was significantly increased in the leptin group. A moderate stimulatory effect of heparin sodium and an inhibitory effect of nadroparin calcium were observed. Both leptin and heparin sodium caused a noticeable increase in the CAM thickness compared to the control and nadroparin calcium groups. We observed an increased number of blood vessels and accumulation of fibroblasts. There was no significant impact on gene expression of VEGF-A and Quek1 24 h after treatment, however, trends similar to the changes in Df and CAM thickness were present. The resulting effect of nadroparin administration on Quek1 levels was exactly the opposite to that of leptin (p < 0.05).

CD44‐Targeted Monoclonal Antibody is Anti‐Inflammatory in an In‐Vivo Model of Monosodium Urate Crystal Induced Peritonitis

CD44‐Targeted Monoclonal Antibody is Anti‐Inflammatory in an <i>In‐Vivo</i> Model of Monosodium Urate Crystal Induced Peritonitis

YAP Involves in Reduced Anabolic Response to Functional Overload in Lewis Lung Carcinoma‐bearing Mice

Cancer‐associated weight loss, also known as cachexia, is connected to poor prognosis, reduced response to chemotherapy, and impaired patient quality of life. While resistance exercise/muscle contraction is a promising therapy, anabolic resistance, the suppressed ability to induce protein synthesis, is present and can’t be solely elucidated by the change of mTOR signaling. Yes‐associated protein (YAP) is a downstream effector of Hippo signaling and is known to control skeletal muscle size in response to mechanical stimulus. However, the role of YAP on anabolic resistance in cancer cachexia needs to be clarified. The purpose of this study was to determine whether the state of YAP activity after functional overload is altered in cachectic mice. Approximately three‐month‐old male C57BL6 mice were divided into two groups: Control group with PBS (100 μl) injection (Con, n=5) and tumor‐implantation group with Lewis lung Carcinoma (LLC) cells injection (2×105 cells in 100 μl PBS, LLC, n=5). Injection was made at the right flank under anesthesia. Four weeks after injection tumor growth was visible, synergist ablation surgery was performed on the left leg (Overload) and the right leg served as an internal control with sham surgery (Sham). Puromycin (0.04 μmol/g body weight, BW) was injected 30 minutes prior to sacrifice and plantaris muscles (PLAN) were removed and snap‐frozen at day 7 following the surgery. After total protein was extracted, routine Western blotting was performed using 60~120 μg of the total protein. Paired t‐test (Sham vs. Overload) and unpaired t‐test (Con vs. LLC) were used for statistical analysis and the levels of significance was set at 0.05. PLAN weights were comparable between Con and LLC with sham surgery (19.8±1.2 mg vs. 17.3±0.5 mg for Con and LLC, respectively, p=0.077). Seven‐day functional overload increased PLAN weights regardless of tumor inoculation, but the degree of hypertrophy was different (42.7±4.0% vs. 27.6±4.6% for Con and LLC, respectively, p&lt;0.05). LLC mice showed reduced levels of phosphorylated p70S6K (p‐p70) with sham surgery (1.0±0.3 vs. 0.4±0.1, for Con and LLC, respectively, p&lt;0.05) and those with SA surgery (4.4±0.7 vs. 1.6±0.7, for Con and LLC, p&lt;0.05). In harmony with these results, the basal levels of muscle protein synthesis in LLC mice were decreased by 51% compared to Con mice and the levels remained lower after functional overload (2.1±0.1 vs. 1.1±0.1 for Con and LLC, respectively, p&lt;0.05). Interestingly, the basal phosphorylated levels of YAP (p‐YAP) were elevated in LLC mice (1.0±0.1 vs. 2.2±0.1, for Con and LLC, respectively, p&lt;0.05). However, functional overload did not alter the levels of p‐YAP in LLC mice while Con mice significantly increased the levels by 55% (p&lt;0.05). In conclusion, the data suggest that YAP plays a role, at least in part, in decreased anabolic response to functional overload in LLC‐bearing mice.Support or Funding InformationSupported by Louisiana Board of Regents Support Fund (LEQSF(2017‐20)‐RD‐A‐22) to SS.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

Combination therapy with astaxanthin and epidermal neural crest stem cells improves motor impairments and activates mitochondrial biogenesis in a rat model of spinal cord injury

Spinal cord injury (SCI), a multifactorial disease, can lead to irreversible motor and sensory disabilities. Cell therapy in combination with pharmacological agents can be a promising approach to attenuate SCI damages. Epidermal neural crest stem cells (EPI-NCSCs) extracted from bulge hair follicle in adults are attractive candidates due to the possibility of autologous transplantation. This study evaluated the effect of EPI-NCSCs combined with astaxanthin (Ast), a potent antioxidant, on damages induced by SCI.Male rats were treated with Ast (0.2 mM) and EPI-NCSCs (106/10 μl PBS) alone and combined together after SCI contusion. Motor function was assessed by Basso, Beattie and Bresnahan (BBB) test on days 1, 3, 7, 14, 21, 28, 35 and 42 post-injury. Motor neurons number and myelin level were evaluated on days 14 and 42 using Nissl and Luxol Fast Blue staining. The gene expression of mitochondrial biogenesis involved factors (PGC1α, NRF1 and TFAM) was measured by qPCR.All treatments improved motor function, with the highest BBB score in Ast + Cell compared to Ast and Cell. Decreased motor neurons number and myelin level following SCI, were increased by Ast, Cell and Ast + Cell, but combination therapy significantly had a better effect. We observed reduction in PGC1α, NRF1, and TFAM expression in spinal tissue after SCI, and treatment with Cell and Ast + Cell significantly restored NRF1 and TFAM mRNA levels.These results suggested that Ast in combination with EPI-NCSCs has better effects on behavioral dysfunction, motor neuron loss and demyelination after SCI. These protective effects may be attributed to mitochondrial biogenesis activation.

Pirfenidone ameliorates the formation of choroidal neovascularization in mice.

The formation and development of choroidal neovascularization (CNV) is accompanied by inflammation and fibrosis. Existing treatments are expensive and can cause irreversible complications. Pirfenidone (PFD) exerts anti-inflammatory and anti-fibrotic effects; however, its applications in the eye remain unclear. Male C57BL/6J mice (aged 6–8 weeks) were used to explore whether PFD can inhibit the formation of laser-induced CNV. The localization of transforming growth factor β2 (TGFβ2) was determined through immunofluorescent staining. After laser photocoagulation, the vehicle and PFD groups were intravitreally injected with 1 µl PBS and 1 µl 0.5% PFD, respectively. At day 7 after intravitreal injection, the expression of TGFβ2 and vascular endothelial growth factor (VEGF) was assessed. Fundus fluorescein angiography was performed to investigate the extent of fluorescence leakage, and the CNV areas were analyzed using a choroidal flat mount. The results demonstrated that, on day 7 after photocoagulation, the expression of TGFβ2 and VEGF was reduced in the experimental group. In addition, fluorescein angiography showed that the leakage area of CNV was significantly smaller in the PFD injection group than those observed in the control and vehicle groups. Moreover, the areas of CNV in the PFD injection group were smaller compared with those reported in the other two injection groups. Histopathological and TUNEL analyses performed on day 28 revealed that there were no notable abnormalities on the layers of the neural retina of PFD-treated mice. In conclusion, intravitreal injection of PFD inhibited the formation of CNV in mice, likely via the downregulation of VEGF and TGFβ2, which did not cause damage to the mouse retina after 28 days of treatment.

Deleterious Effect of NMDA Plus Kainate on the Inner Retinal Cells and Ganglion Cell Projection of the Mouse.

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal “b” wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the “a” wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber.

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa