High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method

Feasibility and safety of setting up a donor breastmilk bank in a neonatal prem unit in a resource limited setting: An observational, longitudinal cohort study

<p>Abstract</p> <p>Background</p> <p>The beneficial effects of human milk on decreasing rates of paediatric infections such as necrotizing enterocolitis (NEC) and sepsis have been clearly demonstrated. Donor breastmilk has been encouraged as the milk of choice when a mother's own breastmilk is not available. The objectives of this study were to assess feasibility of providing donor breastmilk to infants in a resource limited Neonatal Prem Unit (NPU). In addition we sought to determine whether donor breastmilk could be safely pasteurized and administered to infants without any adverse events.</p> <p>Methods</p> <p>Low birth weight infants < 1800 g and under 32 weeks gestational age were followed up in the NPU over a 3 week period; feeding data and morbidity data was collected in order to determine if there were any adverse events associated with donor breastmilk. Samples of pasteurized breastmilk were cultured to check for any bacterial contamination.</p> <p>Results</p> <p>191 infants met the inclusion criteria of whom 96 received their mother's own breastmilk. Of the 95 infants who were potentially eligible to receive donor milk, only 40 did in fact receive donor milk. There was no evidence of bacterial contamination in the samples analyzed, and no evidence of adverse events from feeding with donor breastmilk.</p> <p>Conclusion</p> <p>It is feasible to supply donor breastmilk to infants in an NPU in a resource limited setting, however staff needs to be sensitized to the importance of donor breastmilk to improve uptake rates. Secondly we showed that it is possible to supply donor breastmilk according to established guidelines with no adverse events therefore making it possible to prevent NEC and other side effects often associated with formula feeding of premature infants.</p

Infant feeding counselling in Uganda in a changing environment with focus on the general population and HIV-positive mothers - a mixed method approach

<p>Abstract</p> <p>Background</p> <p>Health workers' counselling practices are essential to improve infant feeding practices. This paper will assess how infant feeding counselling was done and experienced by counsellors and mothers in Eastern Uganda in the context of previous guidelines. This has implications for implementation of the new infant feeding guidelines from 2009.</p> <p>Methods</p> <p>This paper combines qualitative and quantitative data from Mbale District in Eastern Uganda. Data was collected from 2003 to 2005 in a mixed methods approach. This includes: key-informant interviews among eighteen health workers in the public hospital, health clinics and non-governmental organisations working with people living with HIV, fifteen focus group discussions in the general population and among clients from an HIV clinic, two cross-sectional surveys including 727 mothers from the general population and 235 HIV-positive mothers.</p> <p>Results</p> <p>The counselling sessions were often improvised. Health workers frequently had pragmatic approaches to infant feeding as many clients struggled with poverty, stigma and non-disclosure of HIV. The feasibility of the infant feeding recommendations was perceived as challenging among health workers, both for HIV-positive mothers and in the general population. Group counselling with large groups was common in the public health service. Some extra infant feeding teaching capacities were mobilised for care-takers of undernourished children. A tendency to simplify messages giving one-sided information was seen. Different health workers presented contradicting simplified perspectives in some cases. Outdated training was a common concern with many health workers not being given courses or seminars on infant feeding since professional graduation. Other problems were minimal staffing, lack of resources, and programs being started and subsequently stopped abruptly. Many of the HIV-counsellors in the non-governmental organisations got extended training in counselling which seemed to be beneficial.</p> <p>Conclusions</p> <p>Health workers were faced with challenges related to workload, resources, scientific updating, and also a need to adjust to frequent changes in programs, recommendations and guidelines. The clients were faced with difficult choices, poverty, lack of education and stigma. Feasibility of the recommendations was a major concern. Systematic approaches to update health workers should be a priority.</p

Sleep: A Novel Mechanistic Pathway, Biomarker, and Treatment Target in the Pathology of Alzheimer's Disease?

Sleep disruption appears to be a core component of Alzheimer's disease (AD) and its pathophysiology. Signature abnormalities of sleep emerge before clinical onset of AD. Moreover, insufficient sleep facilitates accumulation of amyloid-β (Aβ), potentially triggering earlier cognitive decline and conversion to AD. Building on such findings, this review has four goals, evaluating: (i) associations and plausible mechanisms linking NREM sleep disruption, Aβ, and AD, (ii) a role for NREM sleep disruption as a novel factor linking cortical Aβ to impaired hippocampus-dependent memory consolidation, (iii) the potential diagnostic utility of NREM sleep disruption as a new biomarker of AD, and (iv) the possibility of sleep as a new treatment target in aging, affording preventative and therapeutic benefits