Leucine improves thiram-induced tibial dyschondroplasia and gut microbiota dysbiosis in broilers.

Thiram, a commonly used agricultural insecticide and fungicide, has been found to cause tibial dyschondroplasia (TD) in broilers, leading to substantial economic losses in the poultry industry. In this study, we aimed to investigate the mechanism of action of leucine in mitigating thiram-induced TD and leucine effects on gut microbial diversity. Broiler chickens were randomly divided into five equal groups: control group (standard diet), thiram-induced group (thiram 80 mg/kg from day 3 to day 7), and different concentrations of leucine groups (0.3%, 0.6%, 0.9% leucine from day 8 to day 18). Performance indicator analysis and tibial parameter analysis showed that leucine positively affected thiram-induced TD broilers. Additionally, mRNA expressions and protein levels of HIF-1α/VEGFA and Ihh/PTHrP genes were determined via quantitative real-time polymerase chain reaction and western blot. The results showed that leucine recovered lameness disorder by downregulating the expression of HIF-1α, VEGFA, and PTHrP while upregulating the expression of Ihh. Moreover, the 16 S rRNA sequencing revealed that the leucine group demonstrated a decrease in the abundance of harmful bacteria compared to the TD group, with an enrichment of beneficial bacteria responsible for producing short-chain fatty acids, including Alistipes, Paludicola, CHKCI002, Lactobacillus, and Erysipelatoclostridium. In summary, the current study suggests that leucine could improve the symptoms of thiram-induced TD and maintain gut microbiota homeostasis.

The autophagy-mediated mechanism via TSC1/mTOR signaling pathway in thiram-induced tibial dyschondroplasia of broilers.

Thiram is a member of the dithiocarbamate family and is widely used in agriculture, especially in low-income countries. Its residues lead to various diseases, among which tibial dyschondroplasia (TD) in broiler chickens is the most common. Recent studies have also demonstrated that thiram residues may harm human health. Our previous study showed that the activity of the mTOR (mammalian target of rapamycin) signaling pathway has changed after thiram exposure. In the current study, we investigated the effect of autophagy via the mTOR signaling pathway after thiram exposure in vitro and in vivo. Our results showed that thiram inhibited the protein expression of mTOR signaling pathway-related genes such as p-4EBP1 and p-S6K1. The analysis showed a significant increase in the expression of key autophagy-related proteins, including LC3, ULK1, ATG5, and Beclin1. Further investigation proved that the effects of thiram were mediated through the downregulation of mTOR. The mTOR agonist MHY-1485 reverse the upregulation of autophagy caused by thiram in vitro. Moreover, our experiment using knockdown of TSC1 resulted in chondrocytes expressing lower levels of autophagy. In conclusion, our results demonstrate that thiram promotes autophagy via the mTOR signaling pathway in chondrogenesis, providing a potential pharmacological target for the prevention of TD.

miR-199a/b-3p inhibits HCC cell proliferation and invasion through a novel compensatory signaling pathway DJ-1\Ras\PI3K/AKT.

Several studies have reported the effects of DJ-1 gene and miR-199a/b-3p on HCC development. However, whether miR-199a/b-3p regulates HCC progression through a novel compensatory signaling pathway involving DJ-1, Ras, and PI3K/AKT remains unknown. We used (TCGA, HPA, miRWalk and Target scan) databases, cancer and para-tissue HCC patients, dual-luciferase reporter gene analysis, proteomic imprinting, qPCR, cell proliferation, scratch, transport, and flow cytometry to detect the molecular mechanism of DJ-1 and miR-199a/b-3p co-expression in HCC cell lines. Bioinformatics analysis showed that DJ-1 was highly expressed in HCC ((P < 0.001) were closely associated with tumor stage (T), portal vein vascular invasion, OS, DSS, and PFI (P < 0.05); miR-199a/b-3p was lowly expressed in HCC (P < 0.001), which was the upstream regulator of DJ-1. Spearman coefficient r = -0.113, P = 0.031; Dual luciferase gene report verified the negative targeting relationship between them P< 0.001; Western blotting demonstrated that miR-199a/b-3p could inhibit the protein expression of DJ-1, Ras and AKT(P < 0.05); The results of CCK8, cell scratch, Transwell migration and flow cytometry showed that OE + DJ-1 increased the proliferation, migration and invasion ability of HepG2 cells, and decreased the apoptosis process, and the differences were statistically significant (P < 0.05), while miR-199a/b-3p had the opposite effect (P < 0.05).

Modulation of apoptosis and Inflammasome activation in chondrocytes: co-regulatory role of Chlorogenic acid.

BACKGROUND: The B-cell lymphoma 2 (Bcl-2) protein regulates programmed cell death throughout the disease conditions by upholding apoptotic pathways. However, the mechanism by which it's expressed in chondrocytes still needs to be studied in chondrocyte-related disorders. Additionally, exploring the potential therapeutic role of Chlorogenic acid (CGA) in confluence with Bcl-2 modulation is of significant interest. METHODS: In vivo and in vitro studies were performed according to our previous methodologies. The chondrocytes were cultured in specific growth media under standard conditions after expression verification of different microRNAs through high-throughput sequencing and verification of Bcl-2 involvement in tibial growth plates. The effect of Bcl-2 expression was investigated by transfecting chondrocytes with miR-460a, siRNA, and their negative controls alone or in combination with CGA. The RNA was extracted and subjected to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot analysis and immunofluorescence assays were performed to visualize the intracellular localization of Bcl-2 and associated proteins related to apoptotic and inflammasome pathways. Moreover, apoptosis through flow cytometry was also performed to understand the modulation of concerning pathways. RESULTS: The suppression of Bcl-2 induced higher apoptosis and mitochondrial dysfunction, leading to IL-1ß maturation and affecting the inflammasome during chondrocyte proliferation. Conversely, overexpression attenuated the activation, as evidenced by reduced caspase activity and IL-1ß maturation. In parallel, CGA successfully reduced siRNA-induced apoptosis by decreasing Cytochrome C (Cyto C) release from the mitochondria to the cytoplasm, which in turn decreased Caspase-3 and Caspase-7 cleavage with Bcl-2-associated X protein (Bax). Furthermore, siBcl-2 transfection and CGA therapy increased chondrocyte proliferation and survival. The CGA also showed a promising approach to maintaining chondrocyte viability by inhibiting siRNA-induced apoptosis. CONCLUSIONS: Targeting Bcl-2-mediated regulation might be a possible treatment for chondrocyte-related conditions. Moreover, these results add knowledge of the complicated processes underlying chondrocyte function and the pathophysiology of related diseases, highlighting the significance of target specific therapies. Video Abstract.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions.

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4-7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8-18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

Synthesis and characterization of piperic acid conjugates with homochiral and heterochiral dipeptides containing phenylalanine and their application in skin cancer.

The current work demonstrates the synthesis and characterization of piperic acid conjugates with homochiral/heterochiral dipeptides containing phenylalanine as anti-skin cancer agents. The conjugates PA-DPhe-LPhe-OH, FC-1; PA-LPhe-DPhe-OH, FC-2; PA-DPhe-DPhe-OH, FC-3; and PA-DPhe-DPhe-OH, FC-4 were synthesized, characterized and assessed for cytotoxicity against melanoma cell lines of human and murine origin. Among all, PA-DPhe-DPhe-OH (FC-3) conjugate was identified as a potential cytotoxic lead against melanoma cells by delineating the anti-proliferative and anti-migratory potential together with its anti-inflammatory potential against pro-inflammatory interleukins (IL-1ß, IL-6, and IL-8). Evidences from western blotting, fractionation, and immunocytochemistry experiments suggest that Stat-3 is a critical signaling molecule involved in the FC-3 mechanism of action. The results denote that FC-3 profoundly ablates Stat-3 expression, phosphorylation, and nuclear translocation. Stat-3 mRNA analysis revealed that FC-3 did not alter the transcription of Stat-3. However, in cells where proteasome mediated degradation was inhibited, FC-3 failed to check the Stat-3 expression implying that FC-3 augments the proteasomal degradation of Stat-3. Of note, FC-3 failed to reverse the IL-6 mediated hyperactivation of Stat-3 in A375 cells. Critically, in Stat-3 deficient cancer cells, the anti-clonogenic and anti-migratory potential of FC-3 was significantly subdued. Further, the in vivo efficacy of FC-3 was validated in the two-step (DMBA/TPA) chemically induced mouse skin cancer model. The FC-3-treated cohorts of mice unveiled a significant decrease in the cumulative number of tumors besides attenuation of tumor growth with respect to the vehicle-treated mice. Lastly, in corroboration with our in vitro findings, serum collected from mice groups at various intervals during the treatment regimen demonstrated decrement in IL-1ß and IL-6 levels in FC-3 treated groups compared to the vehicle-treated group.

The Effect of miR-140-5p with HDAC4 towards Growth and Differentiation Signaling of Chondrocytes in Thiram-Induced Tibial Dyschondroplasia.

There is evidence to suggest that microRNA-140-5p (miR-140), which acts as a suppressor, is often elevated and has a role in various malignancies. Nevertheless, neither the function nor the mechanisms in chondrocytes linked with bone disorders, e.g., tibial dyschondroplasia (TD), have been satisfactorily established. The purpose of this study was to look into the role of microRNA-140-5p (miR-140) and its interaction with HDAC4 in chondrocytes, as well as the implications for tibial dyschondroplasia (TD), with a particular focus on the relationship between low miR-140 expression and poor pathologic characteristics, as well as its physiological effects on chondrocyte growth, differentiation, and chondrodysplasia. In this investigation, we discovered that TD had a reduced expression level of the miR-140. There was a correlation between low miR-140 expression, poor pathologic characteristics, and the short overall survival of chondrocytes. Our findings show an aberrant reduction in miR-140 expression, and HDAC4 overexpression caused disengagement in resting and proliferation zones. This further resulted in uncontrolled cell proliferation, differentiation, and chondrodysplasia. Mechanistically, HDAC4 inhibited the downstream transcription factors MEF2C and Runx2 and interacted with Col-Ⅱ, Col-X, and COMP. However, miR-140 binding to the 3'-UTR of HDAC4 resulted in the growth and differentiation of chondrocytes. Moreover, the expression of HDAC4 through LMK-235 was significantly decreased, and the expression was significantly increased under ITSA-1, referring to a positive feedback circuit of miR-140 and HDAC4 for endochondral bone ossification. Furthermore, as a prospective treatment, the flavonoids of Rhizoma drynariae (TFRD) therapy increased the expression of miR-140. Compared to the TD group, TFRD treatment increased the expression of growth-promoting and chondrocyte differentiation markers, implying that TFRD can promote chondrocyte proliferation and differentiation in the tibial growth plate. Hence, directing this circuit may represent a promising target for chondrocyte-related bone disorders and all associated pathological bone conditions.

Socioeconomic disparities in surgery for carotid artery disease in England.

BACKGROUND: Carotid artery disease and stroke are more prevalent in socioeconomically deprived areas. The aim was to investigate socioeconomic disparities in carotid artery disease surgery rates and in outcomes following surgery. METHODS: The study used population-based ecological and cohort study designs, 31 672 census areas in England, hospital admissions from April 2006 to March 2018, the Index of Multiple Deprivation 2010 as the area-level deprivation indicator, and Poisson, logistic, and Cox regression. RESULTS: A total of 54 377 patients (67 per cent men) from a population aged 55 years and older of 14.7 million had carotid artery disease procedures (95 per cent carotid endarterectomy). Carotid endarterectomy rates were 116 per cent (95% c.i. 101 to 132) higher in men and 180 per cent (95% c.i. 155 to 207) higher in women aged 55-64 years in the most compared with the least socioeconomically deprived areas by quintile. However, this difference diminished and appeared to reverse with increasing age, with 24 per cent (95% c.i. 14 to 33) and 12 per cent (95% c.i. -3 to 24) lower carotid endarterectomy rates respectively in men and women aged 85 years and older in the most deprived areas. Patients in deprived areas having carotid endarterectomy were more likely to have been admitted as symptomatic emergency carotid artery disease admissions. Mortality, and a combined outcome of mortality or stroke-related re-admission, were both worse in patients living in more deprived areas and were only partially accounted for by the higher prevalence of co-morbidities. There was, however, no clear pattern of association between deprivation and elective waiting time for carotid endarterectomy. CONCLUSIONS: These results provide evidence of socioeconomic disparities in surgery for carotid artery disease. Clear policies are needed to address these disparities.

Swertia bimaculata moderated liver damage in mice by regulating intestine microbiota.

Swertia bimaculata (SB) is a medicinal herb in China having an array of therapeutic and biological properties. This study aimed to explore the attenuating effect of SB on carbon tetrachloride (CCl4) induced hepato-toxicity by regulation of gut microbiome in ICR mice. For this purpose, CCl4 was injected intraperitoneally in different mice groups (B, C, D and E) every 4th day for a period of 47 days. Additionally, C, D, and E groups received a daily dose (50 mg/kg, 100 mg/kg, and 200 mg/kg respectively) of Ether extract of SB via gavage for the whole study period. The results of serum biochemistry analysis, ELISA, H&E staining, and sequencing of the gut microbiome, indicated that SB significantly alleviates the CCl4-induced liver damage and hepatocyte degeneration. The serum levels of alanine transaminase, aspartate aminotransferase, malondialdehyde, interleukin 1 beta and tumor necrosis factor-alpha were significantly lower in SB treated groups compared to control while levels of glutathione peroxidase were raised. Also, the sequencing data indicate that supplementation with SB could restore the microbiome and its function in CCl4-induced variations in intestinal microbiome of mice by significantly downregulating the abundances of pathogenic intestinal bacteria species including Bacteroides, Enterococcus, Eubacterium, Bifidobacterium while upregulating the levels of beneficial bacteria like Christensenella in the gut. In conclusion, we revealed that SB depicts a beneficial effect against hepatotoxicity induced by CCl4 in mice through the remission of hepatic inflammation and injury, through regulation of oxidative stress, and by restoring gut microbiota dysbiosis.

Amino acid modified OCMC-g-Suc-ß-CD nanohydrogels carrying lapatinib and ginsenoside Rg1 exhibit high anticancer activity in a zebrafish model.

Nanohydrogels show great potential as efficient drug carriers due to their biocompatibility, low toxicity, and high water absorbability. In this paper, we prepared two O-carboxymethylated chitosan (OCMC)-based polymers functionalized with ß-cyclodextrin (ß-CD) and amino acid. The structures of the polymers were characterized by Fourier Transform Infrared (FTIR) Spectroscopy. Morphological study was carried out on a Transmission Electron Microscope (TEM), and the results indicated that the two polymers had irregular spheroidal structure with some pores distributed on their surface. The average particle diameter was below 500 nm, and the zeta potential was above +30 mV. The two polymers were further used for preparing nanohydrogels loaded with anticancer drugs lapatinib and ginsenoside Rg1, and the resulting nanohydrogels showed high drug loading efficiency and pH-sensitive (pH = 4.5) drug release behavior. In vitro cytotoxicity investigation revealed that the nanohydrogels exhibited high cytotoxicity against lung cancer (A549) cells. In vivo anticancer investigation was performed in a transgenic Tg(fabp10:rtTA2s-M2; TRE2:EGFP-kras V12) zebrafish model. The results showed that the synthesized nanohydrogels significantly inhibited the expression of EGFP-kras v12 oncogene in zebrafish liver, and the L-arginine modified OCMC-g-Suc-ß-CD nanohydrogels loading lapatinib and ginsenoside Rg1 showed the best results.

Whether the Golgi protein 73 could be a diagnostic serological marker in hepatocellular carcinoma: a meta analysis.

BACKGROUND: The Value of Golgi protein 73 (GP73) in the diagnosis of Hepatocellular carcinoma (HCC) remains controversial, especially in its differentiation between HCC and cirrhosis. Besides, some papers showed that GP73 levels are correlated with liver fibrosis. This study conducts a meta-analysis to evaluate the value of GP73 in diagnosing HCC and differential diagnosing HCC from liver cirrhosis. METHODS: 36 studies with a sample size of 8314 cases concerning the accuracy of GP73 in the diagnosis of HCC were selected through a systematic review. Seven of these studies included a total of 438 HCC samples and 426 cirrhosis samples and calculated the sensitivity and specificity of GP73 for differential diagnosing HCC from cirrhosis. QUADAS (quality assessment of diagnostic accuracy studies) was used to evaluate the quality of literature. Statistical analyses were performed using StataSE16 software. RESULTS: The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio and the area under the curve were 0.79(95%CI 0.74-0.83),0.85(95%CI 0.80-0.89),5.4(95%CI 3.8-7.5), 0.25(95%CI 0.20-0.31), 22(95%CI 13-35), and 0.88 for GP73 diagnosing HCC;0.74(95%CI 0.64-0.81),0.70(95%CI 0.49-0.85),2.40(95%CI 1.3-4.7),0.38(95%CI 0.23-0.61),6(95%CI 2-19), and 0.78 for GP73 differential diagnosing HCC from liver cirrhosis. CONCLUSION: The results suggest that GP73 has a high diagnostic value for HCC and a moderate value for differential diagnosis of HCC from liver cirrhosis.

Comparison of proliferation and osteogenic differentiation potential of bovine adipose tissue and bone marrow derived stem cells.

Bone marrow derived stem cells (BMSC) are the most utilized cell type in the field of bone regeneration. Although BMSC are both safe and efficacious, the search for alternative sources for stem cells continues. We investigated bovine BMSC and adipose tissue derived mesenchymal stem cells (ATSC) using immunofluorescence and PCR. We further compared the osteogenic differentiation potentials of both sources of stem cells. We assessed alkaline phosphatase (ALP) enzyme levels and calcium deposition in differentiating cells at days 7, 14 and 21 to compare the osteogenic differentiation capability of both cell types. We found that ATSC expressed significantly higher ALP levels compared to BMSC throughout osteogenic differentiation. Calcium deposition was greater in ATSC than BMSC at days 7 and 14. By the end of day 21, BMSC produced greater calcium deposition. We found that ATSC undergo osteogenic differentiation more rapidly than BMSC, but BMSC provide greater mineralization over longer periods.

Socioeconomic disparities in abdominal aortic aneurysm repair rates and survival.

BACKGROUND: Abdominal aortic aneurysm (AAA) is more prevalent in socioeconomically disadvantaged areas. This study investigated socioeconomic disparities in AAA repair rates and survival. METHODS: The study used ecological and cohort study designs, from 31 672 census areas in England (April 2006 to March 2018), the Index of Multiple Deprivation 2010 as the area-level deprivation indicator, and Poisson, logistic and Cox regression. RESULTS: Some 77 606 patients (83.4 per cent men) in four age categories (55-64, 65-74, 75-84, 85 or more years) were admitted with AAA from a population aged at least 55 years of 14.7 million. Elective open and endovascular repair rates were 41 (95 per cent c.i. 23 to 61) and 60 (36 to 89) per cent higher respectively among men aged 55-64 years in the most versus least deprived areas by quintile. This differences diminished and appeared to reverse with increasing age, with 26 (-1 to 45) and 25 (13 to 35) per cent lower rates respectively in men aged 85 years or more in the most deprived areas. Men admitted from more deprived areas were more likely to die in hospital without aneurysm repair. Among those who had aneurysm repair, this was more likely to be for a ruptured aneurysm than among men from less deprived areas. For intact aneurysm repair, they were relatively more likely to have this during an emergency admission. The mortality rate after repair was higher for men from more deprived areas, although the hazard diminished with age. Patterns were unclear for women. CONCLUSION: There were clear socioeconomic disparities in operation rates, mode of presentation, and outcome for AAA surgery. Policies are needed to address these disparities.

Enhanced Healing Activity of Manuka Honey and Nitrofurazone Composite in Full-Thickness Burn Wounds in the Rabbit Model.

Burns cause many significant changes in metabolism and inflammatory reactions, leading to poor regeneration in animals and humans. A list of medicines to treat burns is available in the market. But due to the high cost of these medicines, these are unaffordable, especially for farmers of middle-class families of Africa and Asia. Therefore, a low-cost complementary treatment has always been a topic of many researchers, and there is a dire need of time for the welfare of animals to save them. The current study was planned to scrutinize the therapeutic effects of Manuka honey and Nitrofurazone ointments on full-thickness burn wounds in the rabbit model. The healing efficacy was performed through wound contraction rate, hematological analysis, the thickness of dermis and epidermis, and collagen content percentage. Histopathology was performed after taking biopsy samples at the end of the research. Based on statistical analysis using wound healing time (days, D), the combination (MO + NT) resulted in a shorter period (27 D ± 1) than the average healing time of controlled (36 ± 2), Manuka ointment (31.33 D ± 1.52), and Nitrofurazone ointment (32 ± 1). A significant decrease in the count of red blood cell (RBC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in all treatments was noticed mainly in MO + NT. Furthermore, burns induced a significant difference (p < 0.05) in the white blood cells (WBCs) count levels in the MO-treated group. While the level of platelets (PLTs) was not significantly different from the healthy control group. Histopathological assessment (epithelialization, fibrosis, and angiogenesis) of skin showed burn healing to be better in MO and MO + NT groups. In conclusion, the composite of Manuka honey with Nitrofurazone led to the faster recovery than other treatments.

The Patterns of Acquired Upper and Lower Extremity Amputation at a Tertiary Centre in Saudi Arabia.

INTRODUCTION: Upper and lower extremity amputations are associated with variable degrees of physical disability. In Saudi Arabia, disability still represents a major challenge for healthcare systems. There are insufficient data to describe the incidence and prevalence of impairment and disability. This study attempts to identify the patterns of limb amputations at a tertiary centre. METHODS: A retrospective chart review of the data of patients who received integrated tertiary healthcare in an amputation rehabilitation program (ARP) from 2013 to 2018 at King Fahad Medical City, Riyadh, Saudi Arabia was conducted. Data were collected using the demographic data and clinical history of amputees. RESULTS: A total of 412 patients were included in the study. Transtibial amputation (70%) and partial hand amputation (48%) were the most common levels for lower and upper limb amputations, respectively. There was a significantly higher rate of lower limb amputations secondary to vascular causes than that of upper limb amputations, which were more related to traumatic causes. Most patients, 213 (52%), were enrolled in an amputation rehabilitation program over a year after their amputation. CONCLUSION: Vascular amputation is the most common cause of amputation. Most patients entered the rehabilitation program over a year after amputation. National guidelines for the prevention and management of the risk factors for vascular amputations should be developed.

Response of Olive Shoots to Salinity Stress Suggests the Involvement of Sulfur Metabolism.

Global warming has two dangerous global consequences for agriculture: drought, due to water scarcity, and salinization, due to the prolonged use of water containing high concentrations of salts. Since the global climate is projected to continue to change over this century and beyond, choosing salt-tolerant plants could represent a potential paramount last resort for exploiting the secondary saline soils. Olive is considered moderately resistant to soil salinity as compared to other fruit trees, and in the present study, we investigated the influence of NaCl solutions (ranging from 0 to 200 mM) in a salt-tolerant (cv Canino) and two of its transgenic lines (Canino AT17-1 and Canino AT17-2), overexpressing tobacco osmotin gene, and in a salt-sensitive (Sirole) olive cultivar. After four weeks, most of the shoots of both Canino and Sirole plants showed stunted growth and ultimate leaf drop by exposure to salt-enriched media, contrary to transgenic lines, that did not show injuries and exhibited a normal growth rate. Malondialdehyde (MDA) content was also measured as an indicator of the lipid peroxidation level. To evaluate the role of the S assimilatory pathway in alleviating the adverse effects of salt stress, thiols levels as well as extractable activities of ATP sulfurylase (ATPS) and O-acetyl serine(thiol)lyase (OASTL), the first and the last enzyme of the S assimilation pathway, respectively, have been estimated. The results have clearly depicted that both transgenic lines overexpressing osmotin gene coped with increasing levels of NaCl by the induction of S metabolism, and particularly increase in OASTL activity closely paralleled changes of NaCl concentration. Linear correlation between salt stress and OASTL activity provides evidence that the S assimilation pathway plays a key role in adaptive response of olive plants under salt stress conditions.

Par-4 activation restrains EMT-induced chemoresistance in PDAC by attenuating MDM-2.

BACKGROUND: We recently reported prostate apoptosis response 4 (Par-4), a potential tumor suppressor protein restrains epithelial-mesenchymal transition (EMT) properties and promotes mesenchymal-epithelial transition (MET) in invasive cancer cells by repressing Twist-1 promoter activity. Here, we demonstrate that genetic as well as pharmacological modulation of Par-4 by NGD16 (a small molecule antimetastatic agent), limits EMT-induced chemoresistance in aggressive cancer cells by suppressing MDM-2, a downstream effector of Twist-1. METHODS: Matrigel invasion assay, gelatin degradation assay, cell scattering assay, MTT assay and colony formation assay were used to study the proliferation and migration abilities of invasive cancer cells. Immunoblotting, immunocytochemistry, and immunoprecipitation analysis were utilized for determining protein expression and protein-protein interaction. 4T1 aggressive mouse carcinoma model was employed to evaluate tumor growth and lung metastasis. RESULTS: Treatment of gemcitabine (nucleoside analogue anticancer agent) to pancreatic cancer (Panc-1, MiaPaca-2) and breast cancer (MDA-MB-231) cells amplified MDM-2 expression along with increase in EMT properties. Conversely, NGD16 boosted expression of tumor suppressor Par-4 and inhibited invasion and migration abilities of these cells. Moreover, induction of Par-4 effectively diminished MDM-2 along with pro-EMT markers, whereas, augmented the expression of epithelial markers. Furthermore, siRNA-mediated silencing of Par-4 divulged that NGD16 exerts its EMT inhibitory effects in a Par-4-dependent manner. Mechanistically, Par-4 activation provokes p53 by disrupting MDM-2-p53 interaction, which restored epithelial characteristics in cancer cells. Additionally, partial knockdown of MDM-2 through siRNA pronounced the anti-proliferative and anti-invasive effects of NGD16. Finally, NGD16 efficiently inhibited tumor growth and lung metastasis in mouse mammary carcinoma model without showing any undesirable effects. CONCLUSION: Our findings unveil Par-4 as a key therapeutic target and NGD16 (the pharmacological modulator of Par-4) are potential tools to suppress EMT and associated chemoresistance, which could be exploited clinically for the treatment of aggressive cancers.

Mixed methods study to develop the content validity and the conceptual framework of the electronic patient-reported outcome measure for vascular conditions.

OBJECTIVE: The aim of this paper is to describe the stages undertaken to generate the items and conceptual framework of a new electronic personal assessment questionnaire for vascular conditions. DESIGN: A mixed methods study: First a survey of vascular clinicians was completed to identify the most common conditions treated in vascular clinics and wards. Quantitative systematic reviews were done to identify validated patient-reported outcome measures (PROMs) for direct inclsuion in the new instrument. However, due to scarcity of validated PROMs, the items of the new instrument were mainly based on a large qualitative study of patients and systematic reviews of the qualitative evidence . This was followed by a quantitative clinicians' consensus study and, finally, a qualitative face validity study with patients. PARTICIPANTS: Vascular patients participated in the primary qualitative study and the face validity study. In the qualitative study, 55 patients were interviewed, and for the face validity, 19 patients gave feedback. Twelve clinicians completed the survey and 13 completed two cycles of the clinicians' consensus study. RESULTS: The items and scales in the electronic personal assessment questionnaire for vascular conditions (ePAQ-VAS) were generated based on the results of five systematic reviews evaluating existing PROMs for possible inclusion in ePAQ-VAS, five systematic reviews of qualitative evidence, a primary qualitative study involving 55 patients and clinicians' input. One hundred and sixty-eight items were initially generated, of which 59 were eliminated by the expert panel due to repetition. The instrument was divided into one generic and three disease-specific sections (abdominal aortic aneurysm, carotid artery disease and lower limb vascular conditions). In each section, items were grouped together into putative scales. Fifty-five items were grouped across eight scales; the remaining items were kept as individual items, because of relevance to service users. CONCLUSIONS: This multidimensional electronic questionnaire covers the most common vascular conditions. This is particularly important for patients presenting with mixed symptoms or multiple conditions. This tool captures symptomatology, health related quality of life (HRQoL) and other clinically relevant data, such as experience with services and comorbidities.

Molecular characterization of bovine amniotic fluid derived stem cells with an underlying focus on their comparative neuronal potential at different passages.

BACKGROUND: The excellence in the field of stem cell therapy demands alternative and more convenient stem cells for potential applications. Researchers have opted for least invasive and broadly multipotent cells with minimum ethical concerns. Bovine amniotic fluid derived mesenchymal stem cells (BAF-MSCs) due to their ease of collection and owing similar gestational length to that of human could be presumed as an attractive large animal model for biomedical and biotechnology research. METHODS: Bovine amniotic fluid derived stem cells were isolated from abattoir based samples and characterized for epithelial, neuronal, mesenchymal and pluripotent markers by qPCR and immunofluorescence studies at P1, P3, P5 and P7 alongside population doubling time, growth curve and multilineage differentiation studies. RESULTS: The cells were explored for unique expression of Sox2, which was observed to be up regulated with increase in passage number and Nestin was found to be downregulated during further passaging of mesenchymal cells in this study. The cells also co-expressed Oct ¾ at initial passages which diminished within further passages. Evidence regarding diversity and heterogeneity in different cell population in amniotic fluid was recorded by positive expression of epithelial cell markers like pan Cytokeratin and p63 during early passages. The study suggested that cells with higher expression of Sox2 generated comparatively larger neurospheres with comparative strong expression of Sox2 and Nestin by immunofluorescence staining and qPCR analysis. Besides BAF-MSCs derived neurospheres were also shown to express pro-neuronal markers like ß-III Tubulin, GAP43 and ASCL-1. CONCLUSIONS: This study explores and characterizes BAF-MSCs for their multipotent and neurogenic potentials and their use for clinical applications, though more detailed studies are needed to determine the exact pathways linked with neurogenic capacities of these cells and their morphological assessments at different gestational ages in bovines. The knowledge from the bovine model after detailed studies, proven safety and efficacy could also be used to understand substitutive strategies to investigate MSCs physiology at different trimesters and potential application of these cells for human and veterinary regenerative medicine provided the animal ethics are carefully monitored.

Outcomes of aortic aneurysm surgery in England: a nationwide cohort study using hospital admissions data from 2002 to 2015.

BACKGROUND: The United Kingdom aortic aneurysms (AA) services have undergone reconfiguration to improve outcomes. The National Health Service collects data on all hospital admissions in England. The complex administrative datasets generated have the potential to be used to monitor activity and outcomes, however, there are challenges in using these data as they are primarily collected for administrative purposes. The aim of this study was to develop standardised algorithms with the support of a clinical consensus group to identify all AA activity, classify the AA management into clinically meaningful case mix groups and define outcome measures that could be used to compare outcomes among AA service providers. METHODS: In-patient data about aortic aneurysm (AA) admissions from the 2002/03 to 2014/15 were acquired. A stepwise approach, with input from a clinical consensus group, was used to identify relevant cases. The data is primarily coded into episodes, these were amalgamated to identify admissions; admissions were linked to understand patient pathways and index admissions. Cases were then divided into case-mix groups based upon examination of individually sampled and aggregate data. Consistent measures of outcome were developed, including length of stay, complications within the index admission, post-operative mortality and re-admission. RESULTS: Several issues were identified in the dataset including potential conflict in identifying emergency and elective cases and potential confusion if an inappropriate admission definition is used. Ninety six thousand seven hundred thirty-five patients were identified using the algorithms developed in this study to extract AA cases from Hospital episode statistics. From 2002 to 2015, 83,968 patients (87% of all cases identified) underwent repair for AA and 12,767 patients (13% of all cases identified) died in hospital without any AA repair. Six thousand three hundred twenty-nine patients (7.5%) had repair for complex AA and 77,639 (92.5%) had repair for infra-renal AA. CONCLUSION: The proposed methods define homogeneous clinical groups and outcomes by combining administrative codes in the data. These methodologically robust methods can help examine outcomes associated with previous and current service provisions and aid future reconfiguration of aortic aneurysm surgery services.