The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

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Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The microbiome, of which the bacterial component alone (microbiota), is estimated to include 10 times more cells than human cells of the body, blooms immediately after birth and evolves in composition and complexity throughout childhood. The gut microbiome has a profound impact on gastrointestinal tract development, maintenance of mucosal surface integrity, and contributes to the nutritional status of the host and thus plays a pivotal role in health and disease. New technologies have enabled the detailed characterization of normal microbial symbionts and dysbiosis-disease associations. This review summarizes the stepwise establishment of the intestinal microbiota, influential environmental factors, and how this may be perturbed in preterm very-low-birth-weight infants. The contribution of the microbiota to provision of energy and nutrients for intestinal development and the nutritional status of the host are reviewed. In addition, the crucial role of the gut microbiota in maintaining mucosal integrity is explored along with how its breakdown can lead to sepsis, necrotizing enterocolitis, and systemic inflammatory response syndrome. Finally, the role of enteral feeding type (human milk, formula, and nutrient fortification) in mediating these processes is discussed, and guidance is provided for nutritional strategies to promote health in these fragile infants.

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Gut microbiota of the very-low-birth-weight infant

Frozen storage is often used by milk banks to preserve expressed human milk for later use. Optimal storage and handling conditions which ensure minimum alteration of lipid composition have not been well defined. Therefore we investigated the effect of rapid freeze−thawing and storage conditions (−20 and −70° C) on the free fatty acid (FFA) levels and on the activities of lipoprotein lipase (LPL) and bile salt−stimulated lipase (BSSL) in human milk. Since during mechanical expression leakage of serum components into milk may occur, we also investigated the effect of the presence of serum on human milk LPL during storage. Lipase activity levels were unaffected by rapid freeze−thawing (×3) followed by storage for 1 month at −20 or −70° C. LPL activity (nmol FFA released/ml milk/min) was 414 ± 128, 451 ± 37, and 351 ± 20 and BSSL activity (μmol FFA/ml milk/min) was 5.7 ± 0.7, 5.5 ± 0.8, and 5.7 ± 0.2 in fresh, freeze−thawed, and stored milk, respectively. FFA levels (% of total lipid) were 3.01 ± 1.05 and 10.3 ± 1.6 in fresh−frozen milk stored at −7 0 and −20° C for 5 months, and 3.78 ± 1.08 and 13.60 ± 1.25 in specimens of freeze−thawed (x3) before storage at −70 or −20° C. Addition of serum had no effect on milk LPL at either temperature. We conclude that LPL and BSSL remain fully active during frozen storage of human milk and that milk fat is hydrolyzed at −20° C but not at −70° C. We suggest that banked human milk be stored routinely at −70° C.

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Lipases and lipids in human milk: effect of freeze-thawing and storage.

Human milk is seen not only as a food, but as a functional and dynamic biologic system. It provides nutrients, bioactive components, and immune factors, promoting adequate and healthy growth of newborn infants. When mothers cannot supply their children, donated breast milk is the nutrition recommended by the World Health Organization, as it is a better alternative than infant formula. However, because of the manner in which donor milk is handled in human milk banks (HMB) many of the properties ascribed to mother's own milk are diminished or destroyed. The major process responsible for these losses is Holder pasteurization. High-pressure processing (HPP) is a novel nonthermal pasteurization technology that is being increasingly applied in food industries worldwide, primarily as an alternative to thermal treatment. This is due to its capacity to inactivate microorganisms while preserving both nutritional and bioactive components of foods. This review describes human milk composition and preservation, and critically discusses HMB importance and practices, highlighting HPP as a potential nonthermal pasteurization technology for human milk preservation. HPP technology is described and the few currently existing studies of its effects in human milk are presented.

Human Milk Composition and Preservation: Evaluation of High-pressure Processing as a Nonthermal Pasteurization Technology

Background: Provision of pasteurized donor human milk, as a bridge to mother’s own milk, is the standard of care for very low-birth-weight infants in hospital. The aim of this research was to confirm that Holder pasteurization (62.5°C for 30 min) would be sufficient to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in donated human milk samples. Methods: We spiked frozen milk samples from 10 donors to the Rogers Hixon Ontario Human Milk Bank with SARSCoV-2 to achieve a final concentration of 1 × 107 TCID50/mL (50% of the tissue culture infectivity dose per mL). We pasteurized samples using the Holder method or held them at room temperature for 30 minutes and plated serial dilutions on Vero E6 cells for 5 days. We included comparative controls in the study using milk samples from the same donors without addition of virus (pasteurized and unpasteurized) as well as replicates of Vero E6 cells directly inoculated with SARS-CoV-2. We reported cytopathic effects as TCID50/mL. Results: We detected no cytopathic activity in any of the SARS-CoV-2–spiked milk samples that had been pasteurized using the Holder method. In the SARSCoV-2–spiked milk samples that were not pasteurized but were kept at room temperature for 30 minutes, we observed a reduction in infectious viral titre of about 1 log. Interpretation: Pasteurization of human milk by the Holder method (62.5°C for 30 min) inactivates SARS-CoV-2. Thus, in the event that donated human milk contains SARS-CoV-2 by transmission through the mammary gland or by contamination, this method of pasteurization renders milk safe for consumption and handling by care providers. 	Trial registration: Chinese Clinical Trial Registry, No. ChiCTR2000031301.

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Holder pasteurization of donated human milk is effective in inactivating SARS-CoV-2.

enormous number of references, which make up between a fourth and a third of the entire book (eg, chap¬ ters 5, 6, and 7 have 27, 48, and 28 pages of text and 17,13, and 10 pages of reference citations, respectively). Quite appropriately, a chapter is included on leukemia as a second malignancy, but it is hard to under¬ stand why this chapter follows chap¬ ters on infectious agents and immu¬ nologie factors instead of the chapter

D G Day

Papers

Human milk banking.