Early diagnosis and treatment of Alzheimer's disease by targeting toxic soluble Aß oligomers.

Transient soluble oligomers of amyloid-ß (Aß) are toxic and accumulate early prior to insoluble plaque formation and cognitive impairment in Alzheimer's disease (AD). Synthetic cyclic D,L-α-peptides (e.g., 1) self-assemble into cross ß-sheet nanotubes, react with early Aß species (1-3 mers), and inhibit Aß aggregation and toxicity in stoichiometric concentrations, in vitro. Employing a semicarbazide as an aza-glycine residue with an extra hydrogen-bond donor to tune nanotube assembly and amyloid engagement, [azaGly6]-1 inhibited Aß aggregation and toxicity at substoichiometric concentrations. High-resolution NMR studies revealed dynamic interactions between [azaGly6]-1 and Aß42 residues F19 and F20, which are pivotal for early dimerization and aggregation. In an AD mouse model, brain positron emission tomography (PET) imaging using stable 64Cu-labeled (aza)peptide tracers gave unprecedented early amyloid detection in 44-d presymptomatic animals. No tracer accumulation was detected in the cortex and hippocampus of 44-d-old 5xFAD mice; instead, intense PET signal was observed in the thalamus, from where Aß oligomers may spread to other brain parts with disease progression. Compared with standard 11C-labeled Pittsburgh compound-B (11C-PIB), which binds specifically fibrillar Aß plaques, 64Cu-labeled (aza)peptide gave superior contrast and uptake in young mouse brain correlating with Aß oligomer levels. Effectively crossing the blood-brain barrier (BBB), peptide 1 and [azaGly6]-1 reduced Aß oligomer levels, prolonged lifespan of AD transgenic Caenorhabditis elegans, and abated memory and behavioral deficits in nematode and murine AD models. Cyclic (aza)peptides offer novel promise for early AD diagnosis and therapy.

Candida krusei is the major contaminant of ultrafiltration and reverse osmosis membranes used for cranberry juice production.

Ultrafiltration (UF) and reverse osmosis (RO) are commonly used for the clarification and concentration of fruit juices. However, one of the main limitations of filtration membranes is biofouling, which reduces membrane efficiency and can contaminate the filtered product and lead to spoilage. In this study, the microbial fouling layers of UF and RO membranes from a Canadian cranberry juice processing plant were characterized. Unlike the microbiota found in cranberry juice, which is dominated by Bacillus sp. and other bacteria, both UF and RO membranes were mainly colonized by several strains of the yeast Candida krusei. A variation in bacterial and yeasts count was observed between tubular UF and spiral-wound RO membranes, and the analysis of the spatial distribution highlighted the homogeneity of the contamination across each membrane. Surprisingly, RO membranes had a higher level of contamination when compared to UF membranes. Furthermore, six strains of C. krusei were further characterized through multilocus sequence typing analysis, five of which exhibited unique allelic profiles and two of which were found to contain a new TRP1 allele.

Occurrence of Peptide-Peptide Interactions during the Purification of Self-Assembling Peptide f1-8 from a ß-Lactoglobulin Tryptic Hydrolysate.

Self-assembling peptides have gained attention because of their nanotechnological applications. Previous work demonstrated that the self-assembling peptide f1-8 (Pf1-8) that is generated from the tryptic hydrolysis of ß-lactoglobulin can form a hydrogel after several purification steps, including membrane filtration and consecutive washes. This study evaluates the impact of each processing step on peptide profile, purity, and gelation capacity of each fraction to understand the purification process of Pf1-8 and the peptide-peptide interactions involved. We showed that peptide-peptide interactions mainly occurred through electrostatic and hydrophobic interactions, influencing the fraction compositions. Indeed, the purity of Pf1-8 did not correlate with the number of wash steps. In addition to Pf1-8, two other hydrophobic peptides were identified, peptide f15-20, and peptide f41-60. The gelation observed could be induced either through peptide-peptide interactions or through self-assembling, both being driven by non-covalent bond and more specifically hydrophobic interactions.

High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis Affect Mealworm Allergenic Proteins.

Edible insects have garnered increased interest as alternative protein sources due to the world's growing population. However, the allergenicity of specific insect proteins is a major concern for both industry and consumers. This preliminary study investigated the capacity of high hydrostatic pressure (HHP) coupled to enzymatic hydrolysis by Alcalase® or pepsin in order to improve the in vitro digestion of mealworm proteins, specifically allergenic proteins. Pressurization was applied as pretreatment before in vitro digestion or, simultaneously, during hydrolysis. The degree of hydrolysis was compared between the different treatments and a mass spectrometry-based proteomic method was used to determine the efficiency of allergenic protein hydrolysis. Only the Alcalase® hydrolysis under pressure improved the degree of hydrolysis of mealworm proteins. Moreover, the in vitro digestion of the main allergenic proteins was increased by pressurization conditions that were specifically coupled to pepsin hydrolysis. Consequently, HHP-assisted enzymatic hydrolysis represents an alternative strategy to conventional hydrolysis for generating a large amount of peptide originating from allergenic mealworm proteins, and for lowering their immunoreactivity, for food, nutraceutical, and pharmaceutical applications.

Evolution of cranberry juice compounds during in vitro digestion and identification of the organic acid responsible for the disruption of in vitro intestinal cell barrier integrity.

Cranberry juice is increasingly consumed for its richness in polyphenols having a positive impact on human health. Unfortunately, when regularly consumed, its high concentration in organic acids may cause some intestinal discomforts. In the present study, its organic acid content was reduced of 41% by electrodialysis with bipolar membrane (EDBM), and the resulted deacidified juice was divided in five different juices readjusted or not with different concentrations of citric and/or malic acid(s) corresponding to the concentration of this/these acid(s) recovered during EDBM or at the titratable acidity (TA) of the non-deacidified cranberry juice. The evolution of the cranberry juice main interesting compounds (organic acids and polyphenols), according to the concentration and nature of the organic acids present, was studied for the first time at each specific stages of the digestion. After digestion, Caco-2 cells were exposed to all digested juices to identify the organic acid(s) responsible for the loss of integrity of the epithelial barrier. It appeared that organic acid contents did not change during the different steps of the digestion while polyphenolic compounds decreased starting from the gastric phase. Whatever the organic acid concentration or nature, the concentration of PACs significantly decreased between the salivary and the gastric steps but was different according to their structure when the concentration of most of anthocyanins significantly decreased at the gastric step. Also, to the best of our knowledge, it was the first time that citric acid was demonstrated as the organic acid responsible for the loss of integrity of Caco-2 cell monolayers.

Effect of denatured whey protein concentrate and its fractions on cheese composition and rheological properties.

The objectives of this study were (1) to assess the effect of a denatured whey protein concentrate (DWPC) and its fractions on cheese yield, composition, and rheological properties, and (2) to separate the direct effect of the DWPC or its fractions on cheese rheological properties from the effect of a concomitant increase in cheese moisture. Semihard cheeses were produced at a laboratory scale, and mechanical properties were characterized by dynamic rheometry. Centrifugation was used to induce a moisture gradient in cheese to separate the direct contribution of the DWPC from the contribution of moisture to cheese mechanical properties. Cheese yield increased and complex modulus (G*) decreased when the DWPC was substituted for milk proteins in milk. For cheeses with the same moisture content, the substitution of denatured whey proteins for milk proteins had no direct effect on rheological parameters. The DWPC was fractionated to evaluate the contribution of its different components (sedimentable aggregates, soluble component, and diffusible component) to cheese yield, composition, and rheological properties. The sedimentable aggregates were primarily responsible for the increase in cheese yield when DWPC was added. Overall, moisture content explained to a large extent the variation in cheese rheological properties depending on the DWPC fraction. However, when the effect of moisture was removed, the addition of the DWPC sedimentable fraction to milk increased cheese complex modulus. Whey protein aggregates were hypothesized to act as active fillers that physically interact with the casein matrix and confer rigidity after pressing.

Impact of dianionic and dicationic linkers on tumor uptake and biodistribution of [64 Cu]Cu/NOTA peptide-based gastrin-releasing peptide receptors antagonists.

In this study, we investigated for the first time the influence of 2-aminoethyl-piperazine-1-carboxylic acid (APCA) and amino-hexanedioic-1-acid (AHDA) on tumor uptake and elimination kinetics of [64 Cu]-radiolabeled gastrin releasing peptide receptors (GRPR) antagonists. Three GRPR antagonists containing the RM26 sequence were synthesized and conjugated with NOTA via different linkers (LK): polyethylene glycol (PEG-neutral), APCA (dicationic) or AHDA (dianionic). The NOTA-LK-RM26 peptides were radiolabeled with 64 Cu to assess their pharmacokinetic and positron emission tomography (PET) imaging properties using PC3 tumor-bearing athymic nude mice. The inhibition constants (Ki ) of the 3 nat Cu/NOTA-LK-RM26 peptides bearing PEG, dicationic and dianionic linkers were 0.98 ± 0.48 nM, 0.95 ± 0.21 nM, and 17.97 ± 2.79 nM, respectively. The [64 Cu] NOTA-LK-RM26 conjugates were prepared with labeling yields superior to 95% and specific activities of 67 to 77 TBq/mmol. The 3 radiopeptides were stable in vivo and showed GRPR-specific uptake in pancreas with a very fast washout of this tissue observed for [64 Cu]-NOTA-AHDA-RM26 peptide. Results from imaging studies displayed specific PC3 tumor uptake for both [64 Cu]-NOTA-APCA- and AHDA-RM26, similar kidney elimination and fast liver washout. Considering their adequate imaging characteristics, [64 Cu]-NOTA-LK-RM26 bearing APCA- and AHDA-linkers are promising candidates for GRPR-targeted PET imaging prostate cancer.

Eco-efficiency as a prioritization tool in the reduction of food waste in restaurants.

Striving towards eco-efficiency means creating more value while generating a product or service with a reduced environmental impact. This quest makes it possible to link objectives associated with both the environmental and the economic pillars of sustainable development. Eco-efficiency could therefore be an interesting tool to evaluate the impacts of food waste (FW) and the potential of various food waste reduction strategies (FWRSs). However, the use of eco-efficiency to assess the interest in implementing FWRSs has never been explored in the foodservice sector. This work firstly aims to carry out an in-depth analysis of the costs of the FW generation of an independent restaurant. Secondly, based on these costs data and on some previously documented environmental impact data with a life cycle assessment according to ISO 14045:2012, this work also aims to model and evaluate the performance of FWRSs from a perspective of improving a restaurant eco-efficiency. The impact of each FWRS on the eco-efficiency of the restaurant under study was measured by modeling their economic and environmental net benefits over three implementation periods (one week, one month and six months) and under scenarios of strong and weak adherence. The study identified the most eco-efficient FWRSs to be implemented to reduce FW in the studied restaurant. In addition, key factors affecting eco-efficiency were raised, namely the period following the implementation of FWRSs, the FW reduction rate between FWRSs affecting the same type of FW, the specificity of the FWRSs and their ability to limit the waste of vegetables, meat, sea products and food requiring significant processing time by the cooks. Thus, these elements will guide foodservice managers in adopting FWRSs aimed at reducing FW generated in their restaurant and at improving its eco-efficiency. In addition, this work proposes a new methodology intended for the scientific community to identify FWRSs that have a strong impact on a restaurant eco-efficiency.

Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of ß-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

Nisin Purification from a Cell-Free Supernatant by Electrodialysis in a Circular Economy Framework.

Nisin, an antimicrobial peptide produced by Lactococcus lactis strains, is a promising natural preservative for the food industry and an alternative to antibiotics for the pharmaceutical industry against Gram-positive bacteria. Nisin purification is commonly performed using salting out and chromatographic techniques, which are characterized by their low yields, the use of solvents and the production of large volumes of effluents. In the present work, the purification of nisin from a cell-free supernatant (CFS), after the production of nisin by fermentation on a whey permeate medium, was studied using ammonium sulfate precipitation and electrodialysis (ED) as a promising eco-friendly process for nisin purification. Results showed an increase in nisin precipitation using a 40% ammonium sulfate saturation (ASS) level with a purification fold of 73.8 compared with 34.5 and no purification fold for a 60% and 20% ASS level, respectively. The results regarding nisin purification using ED showed an increase in nisin purification and concentration fold, respectively, of 21.8 and 156 when comparing the final product to the initial CFS. Nisin-specific activity increased from 75.9 ± 4.4 to 1652.7 ± 236.8 AU/mg of protein. These results demonstrated the effectiveness of ED coupled with salting out for nisin purification compared with common techniques. Furthermore, the process was noteworthy for its relevance in a circular economy scheme, as it does not require any solvents and avoids generating polluting effluents. It can be employed for the purification of nisin and the recovery of salts from salting out, facilitating their reuse in a circular economy.

Gestational Diabetes Mellitus, Human Milk Composition, and Infant Growth.

Background: Gestational diabetes mellitus (GDM) is known to affect human milk composition. Aims of this study were to compare macronutrient and energy content of human milk of women with (GDM+) and without GDM (GDM-), to assess the association between maternal health and human milk macronutrient and energy content and association between human milk macronutrient and energy content and infant growth. Study Design and Methods: Two months after delivery, hindmilk samples were collected. Triglyceride (TG), lactose, and protein content of human milk were measured. An oral glucose tolerance test was performed. Infant weight and length at birth and 2 months were collected. Weight-for-age (WAZ) and weight-for-length z-scores were calculated. Results: Twenty-four GDM+ and 29 GDM- women were included. Protein, lactose, and energy content of human milk were similar between groups. TG concentration was higher in GDM+ than in GDM- women (6.3 ± 2.0 versus 5.3 ± 1.2, p = 0.04). This difference was no longer significant after adjustment for maternal age and infant sex (p = 0.23). Maternal age was associated with TG (r = 0.28, p = 0.04) and lactose (r = -0.30, p = 0.03), while fasting glucose was associated with proteins (r = 0.30, p = 0.03) and tended to be associated with TG (r = 0.27, p = 0.05) and energy (r = 0.24, p = 0.08). TG levels in human milk were associated with weight (ß: 0.26, 95% confidence interval [CI]: 0.02 to 0.50) and WAZ (ß: 0.40, 95% CI: 0.05 to 0.75) at 2 months among children unexposed (GDM-) to GDM, but not among children exposed (GDM+) Conclusions: In conclusion, GDM status, maternal age, and fasting glucose level were associated with human milk composition. Finally, TG in human milk was associated with infant growth among GDM- children but not among GDM+ children. ClinicalTrials.gov NCT02872402.

68Ga-DOTATATE Prepared from Cyclotron-Produced 68Ga: An Integrated Solution from Cyclotron Vault to Safety Assessment and Diagnostic Efficacy in Neuroendocrine Cancer Patients.

Cyclotron production of 68Ga is a promising approach to supply 68Ga radiopharmaceuticals. To validate this capability, an integrated solution for a robust synthesis of 68Ga-DOTATATE prepared from cyclotron-produced 68Ga was achieved. A retrospective comparison analysis was performed on patients who underwent PET/CT imaging after injection of DOTATATE labeled with 68Ga produced by a cyclotron or eluted from a generator to demonstrate the clinical safety and diagnostic efficacy of the radiopharmaceutical as a routine standard-of-care diagnostic tool in the clinic. Methods: An enriched pressed 68Zn target was irradiated by a cyclotron with a proton beam set at 12.7 MeV for 100 min. The fully automated process uses an in-vault dissolution system in which a liquid distribution system transfers the dissolved target to a dedicated hot cell for the purification of 68GaCl3 and radiolabeling of DOTATATE using a cassette-based automated module. Quality control tests were performed on the resulting tracer solution. The internal radiation dose for 68Ga-DOTATATE was based on extrapolation from rat biodistribution experiments. A retrospective comparison analysis was performed on patients who underwent PET/CT imaging after injection of DOTATATE labeled with cyclotron- or generator-produced 68Ga. Results: The synthesis of 68Ga-DOTATATE (20.7 ± 1.3 GBq) with high apparent molar activity (518 ± 32 GBq/µmol at the end of synthesis) was completed in 65 min, and the radiopharmaceutical met the requirements specified in the European Pharmacopoeia monograph on 68Ga-chloride (accelerator-produced) solution for radiolabeling. 68Ga-DOTATATE was stable for at least 5 h after formulation. The dosimetry calculated with OLINDA for cyclotron- and generator-produced 68Ga-DOTATATE was roughly equivalent. The SUVmean or SUVmax of tumoral lesions with cyclotron-produced 68Ga-DOTATATE was equivalent to that with generator-produced 68Ga. Among physiologic uptake levels, a significant difference was found in kidneys, spleen, and stomach wall, with lower values in cyclotron-produced 68Ga-DOTATATE in all cases. Conclusion: Integrated cyclotron production achieves reliable high yields of clinical-grade 68Ga-DOTATATE. The clinical safety and imaging efficacy of cyclotron-produced 68Ga-DOTATATE in humans provide supporting evidence for its use in routine clinical practice.

Theranostic 64Cu-DOTHA2-PSMA allows low toxicity radioligand therapy in mice prostate cancer model.

Introduction: We have previously shown that copper-64 (64Cu)-DOTHA2-PSMA can be used for positron emission tomography (PET) imaging of prostate cancer. Owing to the long-lasting, high tumoral uptake of 64Cu-DOTHA2-PSMA, the objective of the current study was to evaluate the therapeutic potential of 64Cu-DOTHA2-PSMA in vivo. Methods: LNCaP tumor-bearing NOD-Rag1nullIL2rgnull (NRG) mice were treated with an intraveinous single-dose of 64Cu-DOTHA2-PSMA at maximal tolerated injected activity, natCu-DOTHA2-PSMA at equimolar amount (control) or lutetium-177 (177Lu)-PSMA-617 at 120 MBq to assess their impact on survival. Weight, well-being and tumor size were followed until mice reached 62 days post-injection or ethical limits. Toxicity was assessed through weight, red blood cells (RBCs) counts, pathology and dosimetry calculations. Results: Survival was longer with 64Cu-DOTHA2-PSMA than with natCu-DOTHA2-PSMA (p < 0.001). Likewise, survival was also longer when compared to 177Lu-PSMA-617, although it did not reach statistical significance (p = 0.09). RBCs counts remained within normal range for the 64Cu-DOTHA2-PSMA group. 64Cu-DOTHA2-PSMA treated mice showed non-pathological fibrosis and no other signs of radiation injury. Human extrapolation of dosimetry yielded an effective dose of 3.14 × 10-2 mSv/MBq, with highest organs doses to gastrointestinal tract and liver. Discussion: Collectively, our data showed that 64Cu-DOTHA2-PSMA-directed radioligand therapy was effective for the treatment of LNCaP tumor-bearing NRG mice with acceptable toxicity and dosimetry. The main potential challenge is the hepatic and gastrointestinal irradiation.

Novel radiolabeled peptides for breast and prostate tumor PET imaging: (64)Cu/and (68)Ga/NOTA-PEG-[D-Tyr(6),ßAla(11),Thi(13),Nle(14)]BBN(6-14).

Bombesin (BBN)-based radiolabeled peptides exhibit promising properties for targeted imaging of gastrin-releasing peptide receptors (GRPR)-positive tumors. The aim of this study was to evaluate with positron emission tomography (PET) the pharmacokinetic and imaging properties of two novel BBN-based radiolabeled peptides, (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14), for diagnosis of breast and prostate cancers using small animal models. Competitive binding assays on T47D breast and PC3 prostate cancer cells showed that the affinity for GRPR depends on the complexed metal and can vary up to a factor of about 3; (64)Cu/NOTA-PEG-BBN(6-14) was found to have the lowest inhibition constant (1.60 ± 0.59 nM). (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14) presented similar cell uptake on T47D and PC3 cells and were stable in vivo. Biodistribution studies of radiolabeled peptides carried out in Balb/c and tumor-bearing Balb/c nude mice showed that (64)Cu/NOTA-PEG-BBN(6-14) presented higher GRPR-mediated uptake in pancreas and adrenal glands, but comparable PC3 tumor uptake as (68)Ga/NOTA-PEG-BBN(6-14). Finally, receptor-dependent responses were observed during blocking studies with unlabeled peptide in both biodistribution and small-animal PET imaging studies. Our results confirmed the dependence of the affinity and pharmacokinetics of BBN-based radiopeptides on the complexed radiometal. Interspecies differences between mouse and human GRPR binding properties were also noted in these preclinical studies. Considering their good imaging characteristics, both (64)Cu/NOTA-PEG-BBN(6-14) and (68)Ga/NOTA-PEG-BBN(6-14) are promising candidates for GRPR-targeted PET imaging of breast and prostate cancers.

Impact of Ultra-High Pressure Homogenization on the Structural Properties of Egg Yolk Granule.

Ultra-high pressure homogenization (UHPH) is a promising method for destabilizing and potentially improving the techno-functionality of the egg yolk granule. This study's objectives were to determine the impact of pressure level (50, 175 and 300 MPa) and number of passes (1 and 4) on the physico-chemical and structural properties of egg yolk granule and its subsequent fractions. UHPH induced restructuration of the granule through the formation of a large protein network, without impacting the proximate composition and protein profile in a single pass of up to 300 MPa. In addition, UHPH reduced the particle size distribution up to 175 MPa, to eventually form larger particles through enhanced protein-protein interactions at 300 MPa. Phosvitin, apovitellenin and apolipoprotein-B were specifically involved in these interactions. Overall, egg yolk granule remains highly stable during UHPH treatment. However, more investigations are needed to characterize the resulting protein network and to evaluate the techno-functional properties of UHPH-treated granule.

Impacts of pH and Base Substitution during Deaerator Treatments of Herring Milt Hydrolysate on the Odorous Content and the Antioxidant Activity.

Despite the biological interest in herring milt hydrolysate (HMH), its valorization is limited by its unpleasant odor resulting from the presence of mainly amine and carbonyl compounds. Recently, a deaerator was demonstrated as an interesting avenue to reduce the odorous content of HMH. However, the removal rate of amine and carbonyl compounds was highly dependent on the operating conditions, and the impact of such a process on the biological potential of HMH was not considered. Therefore, this study aimed to optimize the deaerator process by assessing the impacts of the combination of deaerator treatments at neutral and basic pH, the increase in pH from 10 to 11, and the substitution of NaOH by KOH on the odorous content and the antioxidant activity of HMH. Results showed that the highest deodorization rate of HMH was obtained when a deaerator treatment at neutral pH was combined with another one at basic pH using KOH for alkalization. This condition resulted in a decrease in the dimethylamine and trimethylamine contents by 70%, while certain compounds such as 2,3-pentanedione, methional, (E,E)-2,4-heptadienal, or (E,Z)-2,6-nonadienal were almost completely removed. Removal mechanisms of the targeted compounds were totally identified, and the performance of the developed process was confirmed by sensory analysis. Lastly, it was shown that the antioxidant potential of HMH was not affected by the deodorization process. These results demonstrated the feasibility of deodorizing a complex matrix without affecting its biological potential.

Preservation of Anti-cytomegalovirus Activity in Human Milk Following High-Pressure Processing Compared to Holder Pasteurization.

Pasteurized donor human milk is recommended for hospitalized preterm infants when mother's own milk is unavailable. Our aim was to compare the antiviral activity of human milk processed by Holder pasteurization (HoP) or high-pressure processing (HPP) against representative enveloped and non-enveloped viruses including cytomegalovirus and hepatitis A virus. Expressed milk from 20 donors collected from the Ontario Milk Bank was combined into 10 pools, each from two unique donors. Each pool was processed by HoP (62.5°C, 30 min) or HPP (500 MPa, 8 min, 4°C) and subsequently inoculated with cytomegalovirus or hepatitis A virus to achieve a final concentration of 5-log plaque-forming units/mL. Plaque reduction assays were used to quantify detectable virus after 30 min incubation (room temperature). Post hoc experiments using a 4 h incubation time were conducted if reductions were detected at 30 min. Irrespective of processing, cytomegalovirus concentrations declined in all pools after 30 min incubation (P < 0.0001). Milk processed by HoP exhibited significantly less reduction compared to raw milk (P = 0.0069). In post hoc experiments, anti-cytomegalovirus activity was maintained at 4 h, with high inter-pool variability. Hepatitis A virus concentration remained unchanged after 30 min incubation in raw and processed milk. Anti-cytomegalovirus activity in human milk is preserved following HoP and HPP, persisting up to 4 h post-inoculation; anti-hepatitis A virus activity was not observed in raw or processed milk. Further research is needed to understand how HoP or promising alternative processing methods affect the antiviral activity of donated milk, given its potential importance to recipient infants.

64Cu-DOTHA2-PSMA, a Novel PSMA PET Radiotracer for Prostate Cancer with a Long Imaging Time Window.

Prostate cancer imaging and late-stage management can be improved with prostate-specific membrane antigen (PSMA)-targeting radiotracers. We developed a PSMA positron emission tomography (PET) radiotracer, DOTHA2-PSMA radiolabeled with 64Cu (T1/2: 12.7 h), to leverage its large imaging time window. This preclinical study aimed to evaluate the biological and imaging properties of 64Cu-DOTHA2-PSMA. Its stability was assessed in plasma ex vivo and in mice. Cellular behavior was studied for up to 48 h in LNCaP cells. Biodistribution studies were performed in balb/c mice for up to 48 h. Dynamic (1 h) and static (4 h and 24 h) PET imaging was completed in LNCaP tumor-bearing mice. 64Cu-DOTHA2-PSMA was stable ex vivo in plasma and reached cellular internalization up to 34.1 ± 4.9% injected activity (IA)/106 cells at 48 h post-injection (p.i.). Biodistribution results showed significantly lower uptake in kidneys than 68Ga-PSMA-617, our reference PET tracer (p < 0.001), but higher liver uptake at 2 h p.i. (p < 0.001). PET images showed 64Cu-DOTHA2-PSMA's highest tumoral uptake at 4 h p.i., with a significant difference between blocked and non-blocked groups from the time of injection to 24 h p.i. The high stability and tumor uptake with a long tumor imaging time window of 64Cu-DOTHA2-PSMA potentially contribute to the prostate cancer theranostic approach and its local recurrence detection.

Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates.

Several bifunctional chelates (BFCs) were investigated as carriers of (64)Cu for PET imaging. The most widely used chelator for (64)Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N'''-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with (64)Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H(2)N-Aoc-[d-Tyr(6),ßAla(11),Thi(13),Nle(14)]bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the (64)Cu/Oxo-DO3A complex remained intact after 20 h while the (64)Cu/DOTA-BBN complex was completely demetalated. In contrast, both (64)Cu/NOTA- and (64)Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that (64)Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.

Scale-Up and Long-Term Study of Electrodialysis with Ultrafiltration Membrane for the Separation of a Herring Milt Hydrolysate.

Electrodialysis with ultrafiltration membrane (EDUF) was selected to separate a herring milt hydrolysate (HMH) in a scale-up and long-term study for the recovery of bioactive peptides. The scale-up was performed to maximise peptide recovery by placing a total membrane area of 0.08 m2 for each anionic and cationic compartment. Twelve consecutive runs were carried out, for a total of 69 h, with minimal salt solution cleaning in between experiments. The final peptide migration rate showed that cationic peptides had a higher average migration rate (5.2 ± 0.8 g/m2·h), compared to anionic peptides (4.7 ± 1.1 g/m2·h). Migration was also selective according to peptide identifications and molecular mass distribution where only small molecular weights were found (<1000 Da) in both recovery compartments. The areal system resistance slightly decreased during each run and the averaged values were stable in between experiments since they were all found in the 95% confidence interval. In addition, total relative energy consumption was quite consistent with an average value of 39.95 ± 6.47 Wh/g all along the 12 consecutive runs. Finally, according to membrane characterization, there was no visual fouling on the different membranes present in the EDUF cell after 69 h of treatment. This may be due to the salt cleaning in between experiments which allowed removal of peptides from the membranes, thus allowing recovering initial system working parameters at the beginning of each run. The entire process was revealed to be very consistent and repeatable in terms of peptide migration, global system resistance, and energy consumption. To the best of our knowledge, this is the first time such EDUF conditions (membrane surface, duration, and minimal salt cleaning between experiments) are being tested on a complex hydrolysate.