Bovine CD14 gene characterization and relationship between polymorphisms and surface expression on monocytes and polymorphonuclear neutrophils

<p>Abstract</p> <p>Background</p> <p>CD14 is an important player in host innate immunity in that it confers lipopolysaccharide sensitivity to cell types like neutrophils, monocytes and macrophages. The study was aimed at characterizing the CD14 gene of cattle for sequence variations and to determine the effect of variations on the expression of the protein on the surfaces of monocytes and neutrophils in healthy dairy cows.</p> <p>Results</p> <p>Five SNPs were identified: two within the coding regions (g.A1908G and g.A2318G, numbering is according to GenBank No. <ext-link ext-link-type="gen" ext-link-id="EU148609">EU148609</ext-link>), one in the 5' (g.C1291T) and two in the 3' (g.A2601G and g.G2621T) untranslated regions. SNP 1908 changes amino acid 175 of the protein (p.Asn175Asp, numbering is according to GenBank No. <ext-link ext-link-type="gen" ext-link-id="ABV68569">ABV68569</ext-link>), while SNP 2318 involves a synonymous codon change. Coding region SNPs characterized three gene alleles <it>A </it>(GenBank No. <ext-link ext-link-type="gen" ext-link-id="EU148609">EU148609</ext-link>), <it>A</it>₁(GenBank No. <ext-link ext-link-type="gen" ext-link-id="EU148610">EU148610</ext-link>) and <it>B </it>(GenBank No. <ext-link ext-link-type="gen" ext-link-id="EU148611">EU148611</ext-link>) and two deduced protein variants A (<ext-link ext-link-type="gen" ext-link-id="ABV68569">ABV68569</ext-link> and <ext-link ext-link-type="gen" ext-link-id="ABV68570">ABV68570</ext-link>) and B (<ext-link ext-link-type="gen" ext-link-id="ABV68571">ABV68571</ext-link>). Protein variant A is more common in the breeds analyzed. All SNPs gave rise to 3 haplotypes for the breeds. SNP genotype 1908AG was significantly (P < 0.01) associated with a higher percentage of neutrophils expressing more CD14 molecules on their surfaces. The promoter region contains several transcription factor binding sites, including multiple AP-1 and SP1 sites and there is a high conservation of amino acid residues between the proteins of closely related species.</p> <p>Conclusion</p> <p>The study has provided information on sequence variations within the CD14 gene and proteins of cattle. The SNP responsible for an amino acid exchange may play an important role in the expression of CD14 on the surfaces of neutrophils. Further observations involving a larger sample size are required to validate our findings. Our SNP and association analyses have provided baseline information that may be used at defining the role of CD14 in mediating bacterial infections. The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.</p

The influence of subclinical mastitis on the protein composition and protease activities of raw milk from lactating Thai-crossbred dairy cows

Background and Aim: Mastitis in dairy cattle is associated with a high rate of morbidity and death, which has major implications for milk production and quality. This study aimed to investigate the protein component and the activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in raw milk samples with different testing scores determined using the California mastitis test (CMT). Materials and Methods: Thirty cows were employed in the study, and milk from each quarter was tested for subclinical mastitis (SCM). According to the results of CMT, raw milk samples were classified into five categories: Healthy (score 0), trace (score T), weakly positive (score 1), distinctly positive (score 2), and strongly positive (score 3) for somatic cell count (SCC). The total milk protein was analyzed using the Bio-Rad protein assay, and the milk protein composition was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In addition, gelatin zymography was used to evaluate changes in proteolytic abilities. Results: Milk samples with CMT scores of 1 and 3 had the highest total milk protein levels (32.25 ± 12.60 g/L and 32.50 ± 7.67 g/L, respectively), while the samples from healthy cows (CMT score 0) were only 6.75 ± 1.64 g/L. Globulin and lactoferrin were significantly increased in samples with a CMT score of 3 compared with those with other CMT scores. The bovine serum albumin level in samples with a CMT score of 2 was significantly (p < 0.05) higher than those with other CMT scores. No significant differences in casein abundance were found among samples with different CMT scores. Results from analysis of proteolytic activities demonstrated that the level of MMP-9 in samples with a CMT score of 3 was significantly (p < 0.05) higher than those with other CMT scores. Conclusion: The protein content and gelatinolytic activity of milk were drastically altered by the number of SCC, mainly due to SCM

Neutrophils as one of the major haptoglobin sources in mastitis affected milk

The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to define the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100–200 (n = 10); C: 201–500 (n = 10); and D: > 500 × 103 (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were ~10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immunofluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation

Caffeic acid phenethyl amide ameliorates ischemia/reperfusion injury and cardiac dysfunction in streptozotocin-induced diabetic rats

BACKGROUND: Caffeic acid phenethyl ester (CAPE) has been shown to protect the heart against ischemia/reperfusion (I/R) injury by various mechanisms including its antioxidant effect. In this study, we evaluated the protective effects of a CAPE analog with more structural stability in plasma, caffeic acid phenethyl amide (CAPA), on I/R injury in streptozotocin (STZ)-induced type 1 diabetic rats. METHODS: Type 1 diabetes mellitus was induced in Sprague–Dawley rats by a single intravenous injection of 60 mg/kg STZ. To produce the I/R injury, the left anterior descending coronary artery was occluded for 45 minutes, followed by 2 hours of reperfusion. CAPA was pretreated intraperitoneally 30 minutes before reperfusion. An analog devoid of the antioxidant property of CAPA, dimethoxyl CAPA (dmCAPA), and a nitric oxide synthase (NOS) inhibitor (Nω-nitro-l-arginine methyl ester [l-NAME]) were used to evaluate the mechanism involved in the reduction of the infarct size following CAPA-treatment. Finally, the cardioprotective effect of chronic treatment of CAPA was analyzed in diabetic rats. RESULTS: Compared to the control group, CAPA administration (3 and 15 mg/kg) significantly reduced the myocardial infarct size after I/R, while dmCAPA (15 mg/kg) had no cardioprotective effect. Interestingly, pretreatment with a NOS inhibitor, (l-NAME, 3 mg/kg) eliminated the effect of CAPA on myocardial infarction. Additionally, a 4-week CAPA treatment (1 mg/kg, orally, once daily) started 4 weeks after STZ-induction could effectively decrease the infarct size and ameliorate the cardiac dysfunction by pressure-volume loop analysis in STZ-induced diabetic animals. CONCLUSIONS: CAPA, which is structurally similar to CAPE, exerts cardioprotective activity in I/R injury through its antioxidant property and by preserving nitric oxide levels. On the other hand, chronic CAPA treatment could also ameliorate cardiac dysfunction in diabetic animals

Effect of bis(hydroxymethyl) alkanoate curcuminoid derivative MTH-3 on cell cycle arrest, apoptotic and autophagic pathway in triple-negative breast adenocarcinoma MDA-MB-231 cells: An in vitro study

Curcumin has been shown to exert potential antitumor activity in vitro and in vivo involved in multiple signaling pathways. However, the application of curcumin is still limited because of its poor hydrophilicity and low bio-availability. In the present study, we investigated the therapeutic effects of a novel and water soluble bis(hydroxymethyl) alkanoate curcuminoid derivative, MTH-3, on human breast adenocarcinoma MDA-MB-231 cells. This study investigated the effect of MTH-3 on cell viability, cell cycle and induction of autophagy and apoptosis in MDA-MB-231 cells. After 24-h treatment with MTH-3, a concentration-dependent decrease in MDA-MB-231 cell viability was observed, and the IC50 value was 5.37±1.22 μM. MTH-3 significantly triggered G2/M phase arrest and apoptosis in MDA-MB-231 cells. Within a 24-h treatment, MTH-3 decreased the CDK1 activity by decreasing CDK1 and cyclin B1 protein levels. MTH-3-induced apoptosis was further confirmed by morphological assessment and Annexin V/PI staining assay. Induction of apoptosis caused by MTH-3 was accompanied by an apparent increase of DR3, DR5 and FADD and, as well as a marked decrease of Bcl-2 and Bcl-xL protein expression. MTH-3 also decreased the protein levels of Ero1, PDI, PERK and calnexin, as well as increased the expression of IRE1α, CHOP and Bip that consequently led to ER stress and MDA-MB-231 cell apoptosis. In addition, MTH-3-treated cells were involved in the autophagic process and cleavage of LC3B was observed. MTH-3 enhanced the protein levels of LC3B, Atg5, Atg7, Atg12, p62 and Beclin-1 in MDA-MB-231 cells. Finally, DNA microarray was carried out to investigate the level changes of gene expression modulated by MTH-3 in MDA-MB-231 cells. Taken together, our results suggest that MTH-3 might be a novel therapeutic agent for the treatment of triple-negative breast cancer in the near future

The different catalytic roles of the metal- binding ligands in human 4-hydroxyphenylpyruvate dioxygenase

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a non-haem iron(II)-dependent oxygenase that catalyses the conversion of 4-hydroxyphenylpyruvate (HPP) to homogentisate (HG). In the active site, a strictly conserved 2-His-1-Glu facial triad co-ordinates the iron ready for catalysis. Substitution of these residues resulted in about a 10-fold decrease in the metal binding affinity, as measured by isothermal titration calorimetry, and a large reduction in enzyme catalytic efficiencies. The present study revealed the vital role of the ligand Glu349 in enzyme function. Replacing this residue with alanine resulted in loss of activity. The E349G variant retained 5% activity for the coupled reaction, suggesting that co-ordinating water may be able to support activation of the trans-bound dioxygen upon substrate binding. The reaction catalysed by the H183A variant was fully uncoupled. H183A variant catalytic activity resulted in protein cleavage between Ile267 and Ala268 and the production of an N-terminal fragment. The H266A variant was able to produce 4-hydroxyphenylacetate (HPA), demonstrating that decarboxylation had occurred but that there was no subsequent product formation. Structural modelling of the variant enzyme with bound dioxygen revealed the rearrangement of the co-ordination environment and the dynamic behaviour of bound dioxygen in the H266A and H183A variants respectively. These models suggest that the residues regulate the geometry of the reactive oxygen intermediate during the oxidation reaction. The mutagenesis and structural simulation studies demonstrate the critical and unique role of each ligand in the function of HPPD, and which correlates with their respective co-ordination position.</jats:p

Bowel Preparation for Capsule Endoscopy: A Prospective Randomized Multicenter Study

Background/Aims: The ability to visualize the small bowel mucosa by capsule endoscopy is limited. Moreover, studies involving small-bowel preparation with purgative drugs have failed to establish which preparations produce better images and higher diagnostic yields. The aim of this study was to evaluate the efficacies and diagnostic yields of different bowel preparations. Methods: A cohort of 134 patients with suspected small bowel disease was randomly assigned to 3 groups. Patients in group A (n=44) fasted for 12 h before being administered an M2A capsule (Given Imaging, Yoqneam, Israel). Patients in group B (n=45) were asked to drink two doses of 45 mL of sodium phosphate (NaP) with water during the afternoon and evening on the day before the procedure and to drink at least 2 L of water thereafter. Patients in group C (n=45) drank 2 L of a polyethylene glycol (PEG) lavage solution the evening before the procedure. Results: Overall cleansing of the small bowel was adequate in 43% of patients in group A, 77% of those in group B, and 56% of those in group C (group A vs; group B, p=0.001). Diagnoses for obscure gastrointestinal bleeding were established in 9 patients (39%) in group A, 16 patients (69%) in group B, and 14 patients (50%) in group C. No significant difference in diagnostic yield was observed between groups. Conclusions: Bowel preparation with NaP for capsule endoscopy improved small-bowel mucosal visualization when compared to 12-h overnight fasting. (Gut and Liver 2009;3:180-185)Wei W, 2008, AM J GASTROENTEROL, V103, P77, DOI 10.1111/j.1572-0241.2007.01633.xCheon JH, 2007, GUT LIVER, V1, P118van Tuyl SAC, 2007, ENDOSCOPY, V39, P1037, DOI 10.1055/s-2007-966988Ben-Soussan E, 2005, J CLIN GASTROENTEROL, V39, P381FIREMAN Z, 2005, WORLD J GASTROENTERO, V11, P5863DAI N, 2005, GASTROINTEST ENDOSC, V61, P28Viazis N, 2004, GASTROINTEST ENDOSC, V60, P534Niv Y, 2004, SCAND J GASTROENTERO, V39, P1005, DOI 10.1080/00365520410003209Fireman Z, 2004, ISRAEL MED ASSOC J, V6, P521Albert J, 2004, GASTROINTEST ENDOSC, V59, P487Pennazio M, 2004, GASTROENTEROLOGY, V126, P643, DOI 10.1053/j.gastro.2003.11.057Mylonaki M, 2003, GUT, V52, P1122Costamagna G, 2002, GASTROENTEROLOGY, V123, P999, DOI 10.1053/gast.2002.35988Lewis BS, 2002, GASTROINTEST ENDOSC, V56, P349, DOI 10.1067/mge.2002.126906Kastenberg D, 2001, GASTROINTEST ENDOSC, V54, P705Aronchick CA, 2000, GASTROINTEST ENDOSC, V52, P346

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Detailed, comprehensive, and timely reporting on population health by underlying causes of disability and premature death is crucial to understanding and responding to complex patterns of disease and injury burden over time and across age groups, sexes, and locations. The availability of disease burden estimates can promote evidence-based interventions that enable public health researchers, policy makers, and other professionals to implement strategies that can mitigate diseases. It can also facilitate more rigorous monitoring of progress towards national and international health targets, such as the Sustainable Development Goals. For three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has filled that need. A global network of collaborators contributed to the production of GBD 2021 by providing, reviewing, and analysing all available data. GBD estimates are updated routinely with additional data and refined analytical methods. GBD 2021 presents, for the first time, estimates of health loss due to the COVID-19 pandemic. Methods: The GBD 2021 disease and injury burden analysis estimated years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries using 100 983 data sources. Data were extracted from vital registration systems, verbal autopsies, censuses, household surveys, disease-specific registries, health service contact data, and other sources. YLDs were calculated by multiplying cause-age-sex-location-year-specific prevalence of sequelae by their respective disability weights, for each disease and injury. YLLs were calculated by multiplying cause-age-sex-location-year-specific deaths by the standard life expectancy at the age that death occurred. DALYs were calculated by summing YLDs and YLLs. HALE estimates were produced using YLDs per capita and age-specific mortality rates by location, age, sex, year, and cause. 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws. Uncertainty was propagated at each step of the estimation process. Counts and age-standardised rates were calculated globally, for seven super-regions, 21 regions, 204 countries and territories (including 21 countries with subnational locations), and 811 subnational locations, from 1990 to 2021. Here we report data for 2010 to 2021 to highlight trends in disease burden over the past decade and through the first 2 years of the COVID-19 pandemic. Findings: Global DALYs increased from 2·63 billion (95% UI 2·44–2·85) in 2010 to 2·88 billion (2·64–3·15) in 2021 for all causes combined. Much of this increase in the number of DALYs was due to population growth and ageing, as indicated by a decrease in global age-standardised all-cause DALY rates of 14·2% (95% UI 10·7–17·3) between 2010 and 2019. Notably, however, this decrease in rates reversed during the first 2 years of the COVID-19 pandemic, with increases in global age-standardised all-cause DALY rates since 2019 of 4·1% (1·8–6·3) in 2020 and 7·2% (4·7–10·0) in 2021. In 2021, COVID-19 was the leading cause of DALYs globally (212·0 million [198·0–234·5] DALYs), followed by ischaemic heart disease (188·3 million [176·7–198·3]), neonatal disorders (186·3 million [162·3–214·9]), and stroke (160·4 million [148·0–171·7]). However, notable health gains were seen among other leading communicable, maternal, neonatal, and nutritional (CMNN) diseases. Globally between 2010 and 2021, the age-standardised DALY rates for HIV/AIDS decreased by 47·8% (43·3–51·7) and for diarrhoeal diseases decreased by 47·0% (39·9–52·9). Non-communicable diseases contributed 1·73 billion (95% UI 1·54–1·94) DALYs in 2021, with a decrease in age-standardised DALY rates since 2010 of 6·4% (95% UI 3·5–9·5). Between 2010 and 2021, among the 25 leading Level 3 causes, age-standardised DALY rates increased most substantially for anxiety disorders (16·7% [14·0–19·8]), depressive disorders (16·4% [11·9–21·3]), and diabetes (14·0% [10·0–17·4]). Age-standardised DALY rates due to injuries decreased globally by 24·0% (20·7–27·2) between 2010 and 2021, although improvements were not uniform across locations, ages, and sexes. Globally, HALE at birth improved slightly, from 61·3 years (58·6–63·6) in 2010 to 62·2 years (59·4–64·7) in 2021. However, despite this overall increase, HALE decreased by 2·2% (1·6–2·9) between 2019 and 2021. Interpretation: Putting the COVID-19 pandemic in the context of a mutually exclusive and collectively exhaustive list of causes of health loss is crucial to understanding its impact and ensuring that health funding and policy address needs at both local and global levels through cost-effective and evidence-based interventions. A global epidemiological transition remains underway. Our findings suggest that prioritising non-communicable disease prevention and treatment policies, as well as strengthening health systems, continues to be crucially important. The progress on reducing the burden of CMNN diseases must not stall; although global trends are improving, the burden of CMNN diseases remains unacceptably high. Evidence-based interventions will help save the lives of young children and mothers and improve the overall health and economic conditions of societies across the world. Governments and multilateral organisations should prioritise pandemic preparedness planning alongside efforts to reduce the burden of diseases and injuries that will strain resources in the coming decades. Funding: Bill &amp; Melinda Gates Foundation