In the clinical laboratory, flow cytometry assays are critical to providing diagnostic and prognostic information to the treating clinicians. A validation or verification provides confidence that the ...assay will yield reliable results that can be trusted to make critical medical decisions. The following performance specifications should be included in a validation for laboratory developed tests as needed: accuracy (or trueness), precision (reproducibility and repeatability), detection capability, selectivity, reference range, and sample and reagent stability. We define these terms and present our approach to validation of several common flow cytometry assays, including examples of a leukemia/lymphoma assay and a paroxysmal nocturnal hemoglobinuria (PNH) assay.
Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk after lactose and lipids. Preclinical research has demonstrated that HMOs and specifically ...2'-fucosyllactose (2'-FL) are more than a prebiotic and have multiple functions, including immune, gut, and cognition benefits. Previously, human milk has been the only source for significant levels of HMOs. The most abundant HMO in most mothers' breast milk is 2'-FL. Recently, 2'-FL has been synthesized and shown to be structurally identical to the 2'-FL found in human milk. 2'-FL HMO is now available in some commercial infant formulas. The purpose of this narrative review was to summarize the clinical experiences of feeding infant formula supplemented with the HMO, 2'-FL. Most of these studies investigated standard intact milk protein-based infant formulas containing 2'-FL, and one evaluated a partially hydrolyzed whey-based formula. Collectively, these clinical experiences demonstrated that 2'-FL being added to infant formula was safe, well-tolerated, and absorbed and excreted with similar efficiency to 2'-FL in human milk. Further, infants that were fed formula with 2'-FL had immune benefits, fewer parent-reported respiratory infections, and improved symptoms of formula intolerance. Ultimately, infant formula with 2'-FL supports immune and gut health and is closer compositionally and functionally to human milk.
Alzheimer's disease (AD) is a progressive, neurodegenerative disease, characterised by decline of memory, cognitive function and changes in behaviour. Generic markers of lipid peroxidation are ...increased in AD and reactive oxygen species have been suggested to be involved in the aetiology of cognitive decline. Carotenoids are depleted in AD serum, therefore we have compared serum lipid oxidation between AD and age-matched control subjects before and after carotenoid supplementation. The novel oxidised phospholipid biomarker 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) was analysed using electrospray ionisation tandem mass spectrometry (MS) with multiple reaction monitoring (MRM), 8-isoprostane (IsoP) was measured by ELISA and ferric reducing antioxidant potential (FRAP) was measured by a colorimetric assay.
AD patients (n=21) and healthy age-matched control subjects (n=16) were supplemented with either Macushield™ (10mg meso-zeaxanthin, 10mg lutein, 2mg zeaxanthin) or placebo (sunflower oil) for six months.
The MRM-MS method determined serum POVPC sensitively (from 10µl serum) and reproducibly (CV=7.9%). At baseline, AD subjects had higher serum POVPC compared to age-matched controls, (p=0.017) and cognitive function was correlated inversely with POVPC (r=−0.37; p=0.04). After six months of carotenoid intervention, serum POVPC was not different in AD patients compared to healthy controls. However, POVPC was significantly higher in control subjects after six months of carotenoid intervention compared to their baseline (p=0.03). Serum IsoP concentration was unrelated to disease or supplementation. Serum FRAP was significantly lower in AD than healthy controls but was unchanged by carotenoid intervention (p=0.003).
In conclusion, serum POVPC is higher in AD patients compared to control subjects, is not reduced by carotenoid supplementation and correlates with cognitive function.
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•POVPC can be determined in serum using a robust and sensitive MS-MRM method.•POVPC is higher in AD patient serum compared to age-matched controls.•POVPC correlates inversely with mini-mental state examination.•POVPC is increased in control sera after carotenoid supplementation.
Intervertebral disc (IVD) degeneration causes low-back pain through disc compression, prolapse and herniation. Inflammation of the IVD and subsequent degeneration produce altered glycosylation ...profiles in several animal models of IVD injury and ageing, although the function of this altered glycosylation pattern in a human is unknown. Altered N-glycome, specifically sialylated and fucosylated N-glycosylation motif expression, might play a role in inflammation and disease progression. Healthy (foetal and adolescent idiopathic scoliosis) and degenerated (lumbar degeneration) human IVD glycosylation patterns were studied using lectin histochemistry. Small-molecule fluorinated sugar analogues (3Fax-Peracetyl Neu5Ac; 2F-Peracetyl-Fucose) were used to inhibit sialylation and fucosylation in an in vitro model of inflammation, to investigate their effects on the glycosignature, cell metabolism, extracellular matrix synthesis and cell migration. The effects of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 on glycosylation in human nucleus pulposus cells were investigated by lectin histochemistry, PCR and enzyme-linked immunosorbent assay (ELISA). In the in vitro model of IVD degeneration, cytokine-induced inflammation-induced hypersialylation was observed, as indicated by Sambucus nigra I binding. However, this modification was inhibited by the sialyltransferase inhibitor. Inhibition of sialylation and fucosylation modulates cell migration and protein translation of catabolic enzymes in response to inflammation. The altered patterns of glycosylation in human tissue in degeneration was consistent with previous IVD studies in murine, bovine and ovine models. The present study was the first functional investigation of glycosylation in human degenerated IVD, elucidating the role of the glycome in disease progression and identified potential therapeutic targets for future regenerative therapies.
The installation of solar facilities is increasing rapidly in the Mojave Desert USA, with the largest facility in North America (3227 ha) currently being built 30 km north of Las Vegas, NV. At the ...state level, Nevada (USA) has developed an energy plan to diversify its energy portfolio by 2030 with green energy representing 50% of the energy produced. Although solar is considered a clean energy, it does require significant amounts of land and as such may have negative consequences at the habitat and ecosystem levels. A multi-year study was conducted to assess the impact a photovoltaic facility in the Mojave Desert had on the growth and physiological response of two native shrubs (Ambrosia dumosa and Larrea tridentata) growing inside and outside the facility. These species were selected because they were the dominant species at the site and are representative of desert scrub communities throughout the Mojave Desert. At the time of construction, native plants and washes were left intact inside the solar facility. The solar panel arrays were separated at either 8 m or 10 m. Plants were selected for monitoring on the basis of location: at the panel drip line, below the panels, or midway between panel rows. Abiotic factors, including PAR, reference evapotranspiration, precipitation, soil water in storage, and infiltration, were monitored bi-monthly. The growth and physiological status of the plants were assessed by monitoring leaf water potential, chlorophyll index, canopy temperatures, non-structural carbohydrates in the roots and stems, leaf tissue ion concentrations, stem elongation, and seed production. Plants at the bottom edges of the panels received more precipitation due to runoff from the panels, which led to increased soil moisture in the long spacing but not the short spacing. The lower soil water in storage in the short spacing was related to greater growth and higher soil water extraction. Although the area under the panels provided shade in the summer and warmer temperatures in the winter, the incoming PAR was reduced by as much as 85%, causing plants growing under the panels to be spindly with lower canopy volume (L. tridentata, p = 0.03) and seed yield (A. dumosa, p = 0.05). Ambrosia plants remained green in color year-round (not going into winter dormancy) inside the facility and had elevated levels of starch in their roots and stems compared with plants growing at the outside control sites (p < 0.001). Larrea growing outside the facility had lower xylem water potentials compared with those inside the facility (p < 0.001), lower chlorophyll index (p < 0.001, Ambrosia as well), and lower stem elongation (p < 0.001), supporting the conclusion that both Larrea and Ambrosia performed better inside the facility. Shifts in δ13 C suggested greater water-use efficiency at the locations with the least amount of soil water in storage. Our results support the installation of solar facilities that minimize the impact on native plants and wash connectivity (ecovoltaics), which should translate into a reduced negative impact at the habitat and ecosystem levels. Basedon our results, energy companies that embrace ecovoltaic systems that take an engineering and biological approach should provide acceptable environments for desert fauna. However, corridors (buffers) will need to be maintained between solar facilities, and fences will need to have openings that allow for the continuous flow of animals and resources.
So far, the investigation in cancer cell lines of the modulation of cancer growth and progression by oxysterols, in particular 27-hydroxycholesterol (27HC), has yielded controversial results. The ...primary aim of this study was the quantitative evaluation of possible changes in 27HC levels during the different steps of colorectal cancer (CRC) progression in humans. A consistent increase in this oxysterol in CRC mass compared to the tumor-adjacent tissue was indeed observed, but only in advanced stages of progression (TNM stage III), a phase in which cancer has spread to nearby sites. To investigate possible pro-tumor properties of 27HC, its effects were studied in vitro in differentiated CaCo-2 cells. Relatively high concentrations of this oxysterol markedly increased the release of pro-inflammatory interleukins 6 and 8, monocyte chemoattractant protein-1, vascular endothelial growth factor, as well as matrix metalloproteinases 2 and 9. The up-regulation of all these molecules, which are potentially able to favor cancer progression, appeared to be dependent upon a net stimulation of Akt signaling exerted by supra-physiological amounts of 27HC.
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•27-hydroxycholesterol (27HC) is involved in colorectal cancer (CRC) progression.•27HC reaches high pathological concentrations in CRC advanced stage in humans.•27HC in vitro increases cell release of pro-tumor inflammatory mediators.•27HC favors survival and invasiveness-related cell signals in intestinal cells.
A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that ...result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes.
BACKGROUND: Hospital malnutrition is a significant problem that still remains under‐recognised and under‐treated in India. The present study assessed the effects of oral nutritional supplementation ...(ONS) in conjunction with dietary counselling versus dietary counselling (control) alone in malnourished patients when given in hospital and post‐hospital discharge. METHODS: The present study was conducted in nine private and four public hospitals. Patients from various medical wards were screened for malnutrition using modified Subjective Global Assessment (mSGA) and randomised to control (n = 106) or ONS (n = 106) for 12 weeks. Two servings (460 mL) of ONS were prescribed daily, providing 432 kcal, 16 g of protein and 28 micronutrients. The primary outcome was weight gain over 12 weeks. Other outcomes included change in body mass index (BMI), serum pre‐albumin, albumin and C‐reactive protein levels, energy and nutrient intakes, and handgrip strength at weeks 4, 8 and 12, as well as mSGA score at week 12. RESULTS: The mean age of patients was 39 years. Fifty‐five percent were males and 90.3% were moderately malnourished (mSGA score B) at baseline. At week 12, ONS significantly improved certain parameters compared to control: weight (2.0 versus 0.9 kg; P < 0.001), BMI (0.76 versus 0.37 kg m–²; P < 0.001) and energy intake per day (560 versus 230 kcal; P < 0.05). There were no differences in biochemical parameters and mSGA score between groups. Additionally, patients on ONS who were more functionally impaired at baseline had significantly greater weight gain and improved handgrip strength scores than controls. CONCLUSIONS: ONS use throughout hospital stay and post‐hospital discharge significantly improved energy intake and weight in malnourished Indian patients. Those patients with poorer functional status at baseline demonstrated the most benefit.
A field study was conducted in the Mojave Desert (USA) to assess the influence of a large photo voltaic facility on heat and water transport into an adjacent creosote (Larrea tridentata) bursage ...(Ambrosia dumosa) plant community. Air temperature, plant physiological status, soil water in storage and precipitation were monitored over a two to four year period. A service road built 27 years before the construction of the PV facility decoupled the wash system at the site leading to a significant decline in soil moisture, canopy level NDVI values and mid-day leaf xylem water potentials (p < 0.001) down gradient from the PV facility. Measurements along a 900 m gradient suggested that plants closer to where the wash was decoupled were placed under significantly greater stress during the higher environmental demand summer months. Air temperatures measured at three 10 m meteorological towers revealed warmer night time temperatures at the two towers located in close association with the solar facility (Inside Facility—IF and Adjacent to facility—AF), compared to the Down Gradient Control tower (DGC). As the warmer air was displaced down gradient, the temperature front advanced into the creosote—bursage plant community with values 5 to 8 °C warmer along an east west front just north of tower AF. Based on our research in Eldorado Valley, NV, USA, a down gradient zone of about 300 m was impacted to the greatest extent (water and heat), suggesting that the spacing between solar facilities will be a critical factor in terms of preserving high quality habitat for the desert tortoise and other species of concern. Greater research is needed to identify habitat zones acceptable for animal populations (especially the desert tortoise) within areas of high solar energy development and this should be done prior to any fragmentation of the ecosystem.
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