Development of a Novel Perfusable Solution for ex vivo Preservation: Towards Photosynthetic Oxygenation for Organ Transplantation

Oxygen is the key molecule for aerobic metabolism, but no animal cells can produce it, creating an extreme dependency on external supply. In contrast, microalgae are photosynthetic microorganisms, therefore, they are able to produce oxygen as plant cells do. As hypoxia is one of the main issues in organ transplantation, especially during preservation, the main goal of this work was to develop the first generation of perfusable photosynthetic solutions, exploring its feasibility for ex vivo organ preservation. Here, the microalgae Chlamydomonas reinhardtii was incorporated in a standard preservation solution, and key aspects such as alterations in cell size, oxygen production and survival were studied. Osmolarity and rheological features of the photosynthetic solution were comparable to human blood. In terms of functionality, the photosynthetic solution proved to be not harmful and to provide sufficient oxygen to support the metabolic requirement of zebrafish larvae and rat kidney slices. Thereafter, isolated porcine kidneys were perfused, and microalgae reached all renal vasculature, without inducing damage. After perfusion and flushing, no signs of tissue damage were detected, and recovered microalgae survived the process. Altogether, this work proposes the use of photosynthetic microorganisms as vascular oxygen factories to generate and deliver oxygen in isolated organs, representing a novel and promising strategy for organ preservation.

Challenges in using CellTracker Green on foraminifers that host algal endosymbionts

The uses of fluorescent microscopy and fluorescent probes, such as the metabolically activated probe CellTracker™ Green CMFDA (CTG), have become common in studies of living Foraminifera. This metabolic requirement, as well as the relatively quick production of the fluorescent reaction products, makes CTG a prime candidate for determining mortality in bioassay and other laboratory experiments. Previous work with the foraminiferAmphistegina gibbosa, which hosts diatom endosymbionts, has shown that the species is capable of surviving both acute chemical exposure and extended periods of total darkness by entering a low-activity dormant state. This paper explores the use of CTG and fluorescent microscopy to determine mortality in such experiments, as well as to explore the physiology of dormant foraminifers. The application of CTG was found to be complicated by the autofluorescence of the diatom symbionts, which masks the signal of the CTG, as well as by interactions between CTG and propylene glycol, a chemical of interest known to cause dormancy. These complications necessitated adapting methods from earlier studies using CTG. Here we present observations on CTG fluorescence and autofluorescence inA. gibbosafollowing both chemical exposure and periods of total darkness. While CTG can indicate vital activity in dormant foraminifers, complications include underestimates of total survival and recovery, and falsely indicating dead individuals as live due to rapid microbial colonization. Nonetheless, the brightness of the CTG signal in dormant individuals exposed to propylene glycol supports previously published results of survival patterns inA. gibbosa. Observations of CTG fluorescence in individuals kept for extended periods in aphotic conditions indicate uptake of CTG may begin within 30 min of exposure to light, suggesting darkness-induced dormancy and subsequent recovery can occur on short time scales. These results suggest that CTG accurately reflects changes associated with dormancy, and can be useful in laboratory experiments utilizing symbiont-bearing foraminifers.

Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni

ABSTRACT Copper is both a required micronutrient and a source of toxicity in most organisms, including Campylobacter jejuni. Two proteins expressed in C. jejuni (termed CopA and CueO) have been shown to be a copper transporter and multicopper oxidase, respectively. We have isolated strains with mutations in these genes, and here we report that they were more susceptible to both the addition of copper in the growth media and to induced oxidative stress. Both mutant strains were defective in colonization of an avian host, and copper in the feed exacerbated the colonization deficiency. Overexpression of a cytoplasmic peptide derived from the normally periplasmic copper-binding region of CueO also caused copper intolerance compared to nonexpressing strains or strains expressing the non-copper-binding versions of the peptide. Taken together, the results indicate that copper toxicity in C. jejuni is due to a failure to effectively sequester cytoplasmic copper, resulting in an increase in copper-mediated oxidative damage. IMPORTANCE Copper is a required micronutrient for most aerobic organisms, but it is universally toxic at elevated levels. These organisms use homeostatic mechanisms that allow for cells to acquire enough of the element to sustain metabolic requirements while ensuring that lethal levels cannot build up in the cell. Campylobacter jejuni is an important foodborne pathogen that typically makes its way into the food chain through contaminated poultry. C. jejuni has a metabolic requirement for copper and encodes a copper detoxification system. In the course of studying this system, we have learned that it is important for avian colonization. We have also gained insight into how copper exerts its toxic effects in C. jejuni by promoting oxidative stress.

Variation in dietary cation-anion differences (DCAD) of feed ingredients in relation to milk fever disease in dairy cattle

<p>Milk fever is an important disease that affect lactating cow due to the shortage of calcium circulation after parturition. Incidence of milk fever can be minimized by changing diet acidity/alkalinity before parturition to enhance Ca release of bone, and minimizing it excretion through several regulatory mechanisms. However, cow’s regulatory mechanisms are inadequate in its ability to satisfy the increased metabolic requirement of calcium. Many formulas have been suggested in literature for calculating Dietary Cation-Anion Differences (DCAD) in attempts to acidify diets to minimize the incidence of milk fever. Thus, selection of feed ingredients, and used formula (DCAD below 0 mEq/kg) are important when formulating diet to reach appropriate acidification of the cows’ blood. The aim of current study is to characterize and to measure DCAD of different feed ingredients (Listed in: National Research Council (NRC, 2001)) using the most used equations reported in the literature which are highly correlated with the incidence of milk fever. Tabulated DCAD values showed that the ability of most forages to cause acidification of the cow is not possible and few feed ingredients possessed mild-strong acidic effect. However, using ingredients with acidic effect have nutritional and economic limitations especially in dairy diets. This screening study showed that mostly used feed ingredients in Jordan possess alkaline effect. The magnitude of DCAD¹ ((Na⁺+ K⁺) + (Cl^-)), DCAD² ((Na⁺+ K⁺) + (Cl^-+ S^-2)) and DCAD³ ((Na⁺+ K⁺) + (Cl^-+ 0.6S^-2)) of different feed ingredients mainly used in Jordan ranged from 93.5 - 592.7 mEq/kg, 31.2 - 349.5 mEq/kg, and 56.1 - 446.8 mEq/kg, respectively. Thus, incorporation of acidifying ingredients is necessary when feeding dry cows without compromising feed intake when cows fed under Jordanian conditions. Several nutritional strategies have been suggested to acidify complete diet, and positively enhance Ca releasing from bones to decrease the possibility occurring milk fever in dairy cows.</p>

The Effect of Processing and Ingredient Interactions on Thiamine Degradation in Canned Cat Food: A Modern Nutrition - Health Dilemma

Water soluble vitamins play an integral role in normal metabolic function in cats. Thiamine deficiencies are a common issue in the pet food industry because thiamine degrades easily during processing. Specifically, when cats are fed a diet low in thiamine they may devel op life threatening health issues including anorexia, ventroflexion, neurological impairment, and possibly death within a few weeks if not treated. However, little research has been published using a pet food matrix regarding what specific factors in pet f ood processing result in the most losses and whether these can be controlled. Thiamine can be degraded in a canned food due to heat, moisture, long - term storage, sulfites, pH, and thiaminase enzyme activity. Thermal processes used to produce wet pet foods sold in cans, pouches, and trays are required to be heat treated for extended periods of time. This is detrimental to thiamine retention. Because the cat, like other carnivores, has a very high metabolic requirement for thiamine, they are susceptible to th ese losses. For this reason, supplementation is often a logical step. However, survival of more than 10% of the thiamine may not be assured. This review summarizes the prevailing literature on the topic with application to pet food. Further, suggestions re garding potential investigations to remedy the issue are discussed. Finding an optimal time x temperature x pH x ingredient combination is a real possibility that has the potential to save many cats in the future.