Soy and Gastrointestinal Health: A Review.

Soybean is the most economically important legume globally, providing a major source of plant protein for millions of people; it offers a high-quality, cost-competitive and versatile base-protein ingredient for plant-based meat alternatives. The health benefits of soybean and its constituents have largely been attributed to the actions of phytoestrogens, which are present at high levels. Additionally, consumption of soy-based foods may also modulate gastrointestinal (GI) health, in particular colorectal cancer risk, via effects on the composition and metabolic activity of the GI microbiome. The aim of this narrative review was to critically evaluate the emerging evidence from clinical trials, observational studies and animal trials relating to the effects of consuming soybeans, soy-based products and the key constituents of soybeans (isoflavones, soy proteins and oligosaccharides) on measures of GI health. Our review suggests that there are consistent favourable changes in measures of GI health for some soy foods, such as fermented rather than unfermented soy milk, and for those individuals with a microbiome that can metabolise equol. However, as consumption of foods containing soy protein isolates and textured soy proteins increases, further clinical evidence is needed to understand whether these foods elicit similar or additional functional effects on GI health.

How Healthy Are Non-Traditional Dietary Proteins? The Effect of Diverse Protein Foods on Biomarkers of Human Health.

Future food security for healthy populations requires the development of safe, sustainably-produced protein foods to complement traditional dietary protein sources. To meet this need, a broad range of non-traditional protein foods are under active investigation. The aim of this review was to evaluate their potential effects on human health and to identify knowledge gaps, potential risks, and research opportunities. Non-traditional protein sources included are algae, cereals/grains, fresh fruit and vegetables, insects, mycoprotein, nuts, oil seeds, and legumes. Human, animal, and in vitro data suggest that non-traditional protein foods have compelling beneficial effects on human health, complementing traditional proteins (meat/poultry, soy, eggs, dairy). Improvements in cardiovascular health, lipid metabolism, muscle synthesis, and glycaemic control were the most frequently reported improvements in health-related endpoints. The mechanisms of benefit may arise from their diverse range of minerals, macro- and micronutrients, dietary fibre, and bioactive factors. Many were also reported to have anti-inflammatory, antihypertensive, and antioxidant activity. Across all protein sources examined, there is a strong need for quality human data from randomized controlled intervention studies. Opportunity lies in further understanding the potential effects of non-traditional proteins on the gut microbiome, immunity, inflammatory conditions, DNA damage, cognition, and cellular ageing. Safety, sustainability, and evidence-based health research will be vital to the development of high-quality complementary protein foods that enhance human health at all life stages.

Vegetation expansion in the subnival Hindu Kush Himalaya.

The mountain systems of the Hindu Kush Himalaya (HKH) are changing rapidly due to climatic change, but an overlooked component is the subnival ecosystem (between the treeline and snow line), characterized by short-stature plants and seasonal snow. Basic information about subnival vegetation distribution and rates of ecosystem change are not known, yet such information is needed to understand relationships between subnival ecology and water/carbon cycles. We show that HKH subnival ecosystems cover five to 15 times the area of permanent glaciers and snow, highlighting their eco-hydrological importance. Using satellite data from the Landsat 5, 7 and 8 missions, we measured change in the spatial extent of subnival vegetation from 1993 to 2018. The Landsat surface reflectance-derived Normalized Difference Vegetation Index product was thresholded at 0.1 to indicate the presence/absence of vegetation. Using this product, the strength and direction of time-series trends in the green pixel fraction were measured within three regions of interest. We controlled for cloud cover, snow cover and evaluated the impact of sensor radiometric differences between Landsat 7 and Landsat 8. Using Google Earth Engine to expedite data processing tasks, we show that there has been a weakly positive increase in the extent of subnival vegetation since 1993. Strongest and most significant trends were found in the height region of 5,000-5,500 m a.s.l. across the HKH extent: R2  = .302, Kendall's τ = 0.424, p < .05, but this varied regionally, with height, and according to the sensors included in the time series. Positive trends at lower elevations occurred on steeper slopes whilst at higher elevations, flatter areas exhibited stronger trends. We validated our findings using online photographs. Subnival ecological changes have likely impacted HKH carbon and water cycles with impacts on millions of people living downstream, but the strength and direction of impacts of vegetation expansion remain unknown.

A generic approach for the development of short-term predictions of Escherichia coli and biotoxins in shellfish.

Microbiological contamination or elevated marine biotoxin concentrations within shellfish can result in temporary closure of shellfish aquaculture harvesting, leading to financial loss for the aquaculture business and a potential reduction in consumer confidence in shellfish products. We present a method for predicting short-term variations in shellfish concentrations of Escherichia coli and biotoxin (okadaic acid and its derivates dinophysistoxins and pectenotoxins). The approach was evaluated for 2 contrasting shellfish harvesting areas. Through a meta-data analysis and using environmental data (in situ, satellite observations and meteorological nowcasts and forecasts), key environmental drivers were identified and used to develop models to predict E. coli and biotoxin concentrations within shellfish. Models were trained and evaluated using independent datasets, and the best models were identified based on the model exhibiting the lowest root mean square error. The best biotoxin model was able to provide 1 wk forecasts with an accuracy of 86%, a 0% false positive rate and a 0% false discovery rate (n = 78 observations) when used to predict the closure of shellfish beds due to biotoxin. The best E. coli models were used to predict the European hygiene classification of the shellfish beds to an accuracy of 99% (n = 107 observations) and 98% (n = 63 observations) for a bay (St Austell Bay) and an estuary (Turnaware Bar), respectively. This generic approach enables high accuracy short-term farm-specific forecasts, based on readily accessible environmental data and observations.

Urine and serum midkine levels in an Australian chronic kidney disease clinic population: an observational study.

BACKGROUND AND OBJECTIVES: The cytokine midkine (MK) is pathologically implicated in progressive chronic kidney disease (CKD) and its systemic consequences and has potential as both a biomarker and therapeutic target. To date, there are no published data on MK levels in patients with different stages of CKD. This study aims to quantify MK levels in patients with CKD and to identify any correlation with CKD stage, cause, progression, comorbid disease or prescribed medication. METHODS: In this observational, single-centre study, demographic data were collected, and serum and urine assayed from 197 patients with CKD and 19 healthy volunteers in an outpatient setting. RESULTS: The median serum and urine MK level in volunteers was 754 pg/mL (IQR: 554-1025) and 239 pg/mL (IQR: 154-568), respectively. Compared with serum MK in stage 1 CKD (660 pg/mL, IQR: 417-893), serum MK increased in stage 3 (1878 pg/mL, IQR: 1188-2756; p<0.001), 4 (2768 pg/mL, IQR: 2065-4735; p<0.001) and 5 (4816 pg/mL, IQ: 37477807; p<0.001). Urine MK levels increased from stage 1 CKD (343 pg/mL, IQR: 147-437) to stage 3 (1007 pg/mL, IQR: 465-2766; p=0.07), 4 (2961 pg/mL, IQR: 1368-5686; p=0.005) and 5 (6722 pg/mL, IQR: 3796-10 060; p=0.001). Fractional MK excretion (FeMK) increased from stage 1 CKD (0.159, IQR: 0.145-0.299) to stage 3 (1.024, IQR: 0.451-1.886, p=0.047), 4 (3.39, IQR: 2.10-5.82, p=0.004) and 5 (11.95, IQR: 5.36-24.41, p<0.001). When adjusted for estimated glomerular filtration rate, neither serum nor urine MK correlated with primary CKD diagnosis or CKD progression (small sample). There was a positive correlation between protein:creatinine ratio and FeMK (p=0.003). Angiotensin blockade (adjusted for proteinuria) was associated with lower urine MK (p=0.018) and FeMK (p=0.025). CONCLUSION: MK levels sequentially rise with CKD stage beyond stage 2, and our data support existing animal evidence for an MK/renin angiotensin-system/proteinuria relationship. To what extent this is related to renal clearance versus pathology, or the consequences of chronically elevated MK levels requires further exploration.

Preclinical and clinical development of an anti-kappa free light chain mAb for multiple myeloma.

Monoclonal antibodies (mAb) have had tremendous success in treating a variety of cancers over the past twenty years. Yet despite their widespread clinical use, which includes treatments for haematological malignancies, there are still no approved mAb therapies for multiple myeloma (MM). This is likely to change within the next few years with a number of mAb therapies being assessed in late stage clinical trials, most notably, the anti-CS-1 mAb, elotuzumab, and the anti-CD38 mAb, daratumumab, which are currently being evaluated in Phase III clinical trials for MM. In this review, we will discuss the preclinical and clinical development of MDX-1097, a Phase II candidate which targets cell membrane-associated kappa immunoglobulin free light chains expressed on the surface of MM cells.

MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide.

MDX-1097 is an antibody specific for a unique B cell antigen called kappa myeloma antigen (KMA) that consists of cell membrane-associated free kappa light chain (κFLC). KMA was detected on kappa human multiple myeloma cell lines (κHMCLs), on plasma cells (PCs) from kappa multiple myeloma (κMM) patients and on κPC dyscrasia tissue cryosections. In primary κMM samples, KMA was present on CD38+ cells that were CD138 and CD45 positive and/or negative. MDX-1097 exhibited a higher affinity for KMA compared to κFLC and the latter did not abrogate binding to KMA. MDX-1097-mediated antibody-dependent cellular cytotoxicity (ADCC) and in vitro exposure of target cells to the immunomodulatory drug lenalidomide resulted in increased KMA expression and ADCC. Also, in vitro exposure of peripheral blood mononuclear cells (PBMCs) to lenalidomide enhanced MDX-1097-mediated ADCC. PBMCs obtained from myeloma patients after lenalidomide therapy elicited significantly higher levels of MDX-1097-mediated ADCC than cells obtained prior to lenalidomide treatment. These data establish KMA as a relevant cell surface antigen on MM cells that can be targeted by MDX-1097. The ADCC-inducing capacity of MDX-1097 and its potentiation by lenalidomide provide a powerful rationale for clinical evaluation of MDX-1097 alone and in combination with lenalidomide.

Cell membrane associated free kappa light chains are found on a subset of tonsil and in vitro-derived plasmablasts.

The monoclonal antibody, MDX-1097, is currently progressing through clinical trials as a possible therapy for multiple myeloma. MDX-1097 targets a cell membrane bound form of free immunoglobulin kappa light chain (FκLC), termed kappa myeloma antigen (KMA), which is found on the surface of malignant plasma cells. The clinical potential of MDX-1097 highlights the need to characterise the expression of its cognate antigen, KMA, in normal tissue. In this study, we have analysed the expression of KMA on B cell subsets found in tonsils, peripheral blood and bone marrow. We found KMA expression on a small population of tonsillar and in vitro derived plasmablasts. In contrast, no KMA expression was observed on peripheral blood or bone marrow resident B cell subsets. This study yields important insights into the possible subsets of B cells that might be depleted as a result of an immunotherapy targeting KMA.

Formation of assemblies on cell membranes by secreted proteins: molecular studies of free λ light chain aggregates found on the surface of myeloma cells.

We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

Optimisation is at the heart of the operation.

In our other article based around operating theatres in this issue of HEJ (see pages 64-72), we examine how some of the latest technology is benefiting users, but in this article--with all areas of the NHS charged with reducing energy consumption and cutting carbon emissions--Darren Jones, MD at carbon and energy management specialist, Low Carbon Europe, takes a detailed look, with the help of a 'real-life' case study based on recent experience at London's Heart Hospital, at operating theatre optimisation and HTM 03-01 audits.

The ability to interact with cell membranes suggests possible biological roles for free light chain.

During antibody synthesis, immunoglobulin light chains are produced in excess of heavy chains and, as a consequence, can be secreted by plasma cells as free light chains (FLC). Thus, FLC were considered to be a by-product of immunoglobulin synthesis, lacking any biological function or relevance. However, mounting evidence suggests that FLC are bioactive molecules. For example, FLC can induce antigen specific type I hypersensitivity and inhibit viral replication in encephalomyocarditis infected mice. We have recently shown that FLC can associate with the outer membrane of certain plasma cells via interaction with saturated phosphocholine lipids such as sphingomyelin. As these lipids are highly abundant in mammalian cell membranes, we set out to determine whether FLCs can bind to membranes from a variety of cell types. We found that FLCs bind to the plasma membrane of cells from a wide range of lineages. Interestingly, the highest level of binding was to monocytes. As these cells are professional antigen presenting cells, we postulate that membrane-associated FLCs may provide a novel mechanism of antigen uptake by these cells.

Characterization of a unique conformational epitope on free immunoglobulin kappa light chains that is recognized by an antibody with therapeutic potential.

The murine mAb, K-1-21, recognizes a conformational epitope expressed on free Ig kappa light chains (FκLCs) and also on cell membrane-associated FκLCs found on kappa myeloma cells. This has led to the development of a chimeric version of K-1-21, MDX-1097, which is being assessed in a Phase II clinical trial for the treatment of multiple myeloma. The epitope recognized by K-1-21 is of particular interest, especially in the context that it is not expressed on heavy chain-associated light chains such as in an intact Ig molecule. Using epitope excision techniques we have localized the K-1-21 epitope to a region spanning residues 104-110 of FκLC. This short strand of residues links the variable and constant domains, and is a flexible region that adopts different conformations in FκLC and heavy chain-associated light chain. We tested this region using site-directed mutations and found that the reactivity of K-1-21 for FκLC was markedly reduced. Finally, we applied in silico molecular docking to generate a model that satisfied the experimental data. Given the clinical potential of the Ag, this study may aid the development of next generation compounds that target the membrane form of FκLC expressed on the surface of myeloma plasma cells.

Free Ig light chains interact with sphingomyelin and are found on the surface of myeloma plasma cells in an aggregated form.

Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.

AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (approximately 1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 micromol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials.

Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.

Compound handling is a fundamental and critical step in compound screening throughout the drug discovery process. Although most compound-handling processes within compound management facilities use 100% DMSO solvent, conventional methods of manual or robotic liquid-handling systems in screening workflows often perform dilutions in aqueous solutions to maintain solvent tolerance of the biological assay. However, the use of aqueous media in these applications can lead to suboptimal data quality due to compound carryover or precipitation during the dilution steps. In cell-based assays, this effect is worsened by the unpredictable physical characteristics of compounds and the low DMSO tolerance within the assay. In some cases, the conventional approaches using manual or automated liquid handling resulted in variable IC(50) dose responses. This study examines the cause of this variability and evaluates the accuracy of screening data in these case studies. A number of liquid-handling options have been explored to address the issues and establish a generic compound-handling workflow to support cell-based screening across our screening functions. The authors discuss the validation of the Labcyte Echo reformatter as an effective noncontact solution for generic compound-handling applications against diverse compound classes using triple-quad liquid chromatography/mass spectrometry. The successful validation and implementation challenges of this technology for direct dosing onto cells in cell-based screening is discussed.

Controlled growth of triblock polyelectrolyte brushes.

We have achieved a significant breakthrough in the synthesis of polyelectrolyte brushes of controlled thickness and density, which has been demonstrated by the synthesis of triblock copolymer brushes composed of cationic, neutral, and anionic segments.

A 100-amino acid truncation in the cytoplasmic tail of glycoprotein 41 in the reference HIV type 1 strain RF.

Truncations of the cytoplasmic tail of the HIV-1 transmembrane (TM) protein are rare and almost always markedly reduce virus infectivity. We describe a truncation of the gp41 cytoplasmic tail in the commonly used early HIV-1 reference strain RF. This truncation apparently arose after continuous passage in H9 cells. We detected the truncation by Western blot as a size decrease in RF gp41 from 46 to approximately 34 kDa. The reduced size of RF gp41 observed was not due to differences in glycosylation. Viral DNA sequencing confirmed that a point mutation at Env residue 740 (Trp) introduced a premature stop codon, resulting in a 100-amino acid (13-kDa) truncation of the gp41 C terminus. This truncated RF species, termed RF(gp34), was characterized phenotypically by growth in Hut78 cells. Compared with other B clade HIV strains (IIIB, SF2, and NL4.3), RF(gp34) induced massive syncytia. Importantly, RF(gp34) also productively infected peripheral blood mononuclear cells in vitro.

Antibody-antigen kinetics following immunization of rainbow trout (Oncorhynchus mykiss) with a T-cell dependent antigen.

Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout (Oncorhynchus mykiss) by measuring antibody-antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants (k(diss.)). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k(diss.)) was observed over the ensuing course of the immune response. An average k(diss.) of 3.5 x 10(-4)+/-0.27 x 10(-4)sec(-1) was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly (p<0.05). Further reduction in k(diss.) was observed, with a low of 1.2 x 10(-4)+/-0.06 x 10(-4)sec(-1) being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k(diss.) as the immune response progressed. While these data clearly demonstrate a 2-3 fold increase in antibody-antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.