Disulfide bond formation is not crucial for the heat-induced interaction between ß-lactoglobulin and milk fat globule membrane proteins.

During heat treatment of milk, ß-lactoglobulin (ß-LG) associates with the milk fat globule membrane (MFGM). The objective of this study was to examine different binding types that could be involved in this process. First, we tested the thiol-disulfide bond interchange between ß-LG and MFGM by heating raw milk (87°C, 8 min) in the presence of different reagents capable of preventing this interaction, and then evaluated the presence of ß-LG in resulting MFGM preparations by sodium dodecyl sulfate-PAGE. Contrary to commonly accepted theory, ß-LG still associated with MFGM when milk was heated in the presence of 10 mM N-ethylmaleimide, dithiobis-nitrobenzoic acid, or dithioerythritol. This finding indicated that noncovalent binding could be involved in the interaction, and therefore these were studied next. Preventing noncovalent interactions by heating milk in the presence of 8 M urea (to inhibit formation of hydrogen bonds) or 2 M NaCl (to inhibit electrostatic and hydrophobic interactions) reduced the association of ß-LG and MFGM. Inhibiting both hydrogen and disulfide bond formation by addition of 8 M urea and 10 mM dithioerythritol or inhibiting hydrophobic interactions with 0.2% sodium dodecyl sulfate completely prevented the association. In contrast to the simple thiol-disulfide interaction model, the results suggest a more complex understanding of the interactions between ß-LG and MFGM during heating of milk. This indicates that disulfide formation between ß-LG and proteins in the MFGM is not required for the association, but that hydrophobic interactions and hydrogen bonding may be crucial. This novel insight into ß-LG and MFGM association is in contrast to the current literature and requires further study.

Impact of industrial cream heat treatments on the protein composition of the milk fat globule membrane.

The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and ß-lactoglobulin as well as αs1- and ß-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.

Thermal effects on IgM-milk fat globule-mediated agglutination.

The process of agglutination causes firm cream layers in bovine milk, and a functioning agglutination mechanism is paramount to the quality of non-homogenized milks. The phenomenon is not well-described, but it is believed to occur due to interactions between immunoglobulins (Ig) and milk fat globules. For the first time, this paper demonstrates how the process of agglutination can be visualized using confocal laser scanning microscopy, rhodamine red and a fluoresceinisothiocynat-conjugated immunoglobulin M antibody. The method was used to illustrate the effect on agglutination of storage temperature and pasteurization temperature. Storage at 5 °C resulted in clearly visible agglutination which, however, was markedly reduced at 15 °C. Increasing storage temperature to 20 or 37 °C cancelled any detectable interaction between IgM and milk fat globules, whereby the occurrence of cold agglutination was documented. Increasing 20 s pasteurization temperatures from 69 °C to 71 °C and further to 73 °C lead to progressively higher inactivation of IgM and, hence, reduction of agglutination. Furthermore, 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that changes in storage temperature caused a redistribution of Ig-related proteins in milk fat globule membrane isolates. Poly-immunoglobulin G receptor was present in milk fat globule preparations stored at cold (4 °C) conditions, but absent at storage at higher temperature (25 °C). The findings provide valuable knowledge to dairy producers of non-homogenized milk in deciding the right pasteurization temperature to retain the crucial agglutination mechanism.

Neuronal ceroid lipofuscinosis (NCL) is caused by the entire deletion of CLN8 in the Alpenländische Dachsbracke dog.

Neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage diseases that have been described in a variety of dog breeds, where they are caused by different mutations in different genes. However, the causative gene defect in the breed Alpenländische Dachsbracke remained unknown so far. Here we present two confirmed cases of NCL in Alpenländische Dachsbracke dogs from different litters of the same sire with a different dam harboring the same underlying novel mutation in the CLN8 gene. Case 1, a 2-year-old male Alpenländische Dachsbracke was presented with neurological signs including disorientation, character changes including anxiety states and aggressiveness, sudden blindness and reduction of food intake. Magnetic resonance imaging (MRI) scans showed cerebral atrophy with dilation of all cerebral ventricles, thinning of the intermediate mass of the thalamus and widening of the cerebral sulci. Postmortem examination of the central nervous system (CNS) showed neuronal loss in the cerebral cortex, cerebellum and spinal cord with massive intracellular deposits of ceroid pigment. Additional ceroid-lipofuscin deposits were observed in the enteric nervous system and in macrophages within spleen, lymph nodes and lung. Ultrastructural analyses confirmed NCL with the presence of osmiophilic membrane bounded lamellar-like structures. Case 2, a 1,5-year old female Alpenländische Dachsbracke was presented with progressive generalized forebrain disease including mental changes such as fearful reactions to various kinds of external stimuli and disorientation. The dog also displayed seizures, absence of menace reactions and negative cotton-ball test with normal pupillary light reactions. The clinical and post mortem examination yielded similar results in the brain as in Case 1. Whole genome sequencing of Case 1 and PCR results of both cases revealed a homozygous deletion encompassing the entire CLN8 gene as the most likely causative mutation for the NCL form observed in both cases. The deletion follows recessive inheritance since the dam and a healthy male littermate of Case 1 were tested as heterozygous carriers. This is the first detailed description of CLN8 gene associated NCL in Alpenländische Dachsbracke dogs and thus provides a novel canine CLN8 model for this lysosomal storage disease. The presence of ceroid lipofuscin in extracerebral tissues may help to confirm the diagnosis of NCL in vivo, especially in new dog breeds where the underlying mutation is not known.

Injectable microcarriers as human mesenchymal stem cell support and their application for cartilage and degenerated intervertebral disc repair.

Degeneration of the intervertebral disc (IVD) is a progressive and chronic process, and the high incidence of discogenic disorders calls for new therapeutic approaches, such as cell-based therapies using three dimensional cultures and mesenchymal stem cells (MSC), which can differentiate to chondrogenic- and IVD-lineages. Here, we investigated the growth and differentiation of human MSC culture on biodegradable collagen scaffolds in order to obtain an injectable suspension. Commercially available wound dressings were downsized to dimensions between 100 and 1500 µm and seeded with freshly isolated or early passages MSC. Proliferation rate and chondrogenic differentiation potential was tested at oxygenation levels of 2%, 5%, 10% and 21% in static and dynamic cultures. Evaluation methods included cell viability test, disc marker genes expression (aggrecan, collagen type I and type II), histological detection of proteoglycans and immunohistochemical analysis. On microcarriers, freshly isolated MSC had lower proliferation rate and chondrogenic differentiation potential compared with early passages MSC. Proliferation of MSC was significantly increased 1.7-fold at 5% oxygen level and in combination with dynamic culture was further increased to 2.3-fold, with respect to normoxia. Chondrogenesis was positively affected by 2% and 5% hypoxia, as shown by increased transcription levels and protein expression of collagen type II and proteoglycan accumulation in static cultures, while it was inhibited in dynamic cultures. Collagen type I and aggrecan expression were not affected by hypoxia. In conclusion, collagen based microcarriers are a suitable support for in vitro MSC growth and chondrogenesis especially when cultured at 5% oxygen level.

Impact of the use of bowel for urinary diversion on perioperative complications and 90-day mortality in patients aged 75 years or older.

OBJECTIVE: A potential strategy to decrease the high complication rate of radical cystectomy (RC) in the elderly is to avoid the use of bowel for urinary diversion. The aim of this study was to address this issue in a multicentre study of patients aged ≥ 75 years. PATIENTS AND METHODS: We performed a retrospective, multicentre study of a consecutive series of patients aged ≥ 75 years who underwent RC for muscle-invasive bladder cancer between 2006 and 2010. Medical, surgical and wound complications were graded according to the modified Clavien-Dindo classification. RESULTS: A total of 256 patients (68% men, mean age 79.6 years) were analysed. 204 (80%) patients received a urinary diversion with use of bowel and 52 (20%) a ureterocutaneostomy (UC). Patients with UC were older (82.0 vs. 78.9 years, p < 0.001) and had a higher ASA score (2.6 vs. 2.3, p = 0.007), while the mean Charlson score was lower (4.2 vs. 5.6, p < 0.001). Patients with UC had a shorter operating time (279 vs. 311 min, p = 0.002) and a shorter period in the intensive care unit (0.9 vs. 2.2 days). The overall rate of severe complications graded as Clavien III-V was significantly lower in the UC group (11.5%) as compared to patients receiving bowel for urinary diversion (25.0%) (p = 0.003). Severe (Clavien grade III-V) medical (3.9 vs. 10.3%) and surgical (2.1 vs. 14.1%) complications were all less frequent in the UC group. Inpatient, 30- and 90-day mortality was 5.8, 7.7 and 17.3% in the UC group as compared to 3.9, 5.9 and 6.9% in the bowel cohort, respectively. CONCLUSION: UC following RC is associated with a lower complication rate in geriatric patients. The constantly increasing cohort of geriatric, multimorbid patients requiring cystectomy might justify reconsideration of this form of diversion.

Patiromer to Reduce Albuminuria Through Increased Renin Angiotensin Aldosterone System Inhibition in Patients With CKD-A Feasibility Trial.

Introduction: We tested the feasibility of adding a potassium binder to enable increased renin angiotensin aldosterone system inhibition (RAASi) and reduce albuminuria in patients with chronic kidney disease (CKD). In a controlled trial design, a potassium binder was introduced exclusively in patients developing hyperkalemia after intensified RAASi, thereby mirroring clinical decision-making. Methods: We planned to include 140 patients aged 18 to 80 years with estimated glomerular filtration rate (eGFR) 25 to 60 ml/min per 1.73 m2, albuminuria, and a history of hyperkalemia to an open-label, randomized trial comparing treatment with or without patiromer alongside maximally tolerated RAASi. Patients were randomized only if developing a documented P-potassium >5.5 mmol/l during run-in with intensified RAASi (losartan/spironolactone). The primary end point was change in urine albumin-creatinine ratio (UACR). Results: Screening among 800,000 individuals with available laboratory results yielded just 317 candidates meeting major selection criteria during 18⅔ months, with 75 ultimately included. Among them, only 23 developed P-potassium >5.5 mmol/l, qualifying for randomization. Consequently, only 20 participants completed the study, falling short of the planned 98, precluding a significant effect on the primary outcome. Inclusion and randomization challenges stemmed from a limited pool of eligible patients for intensified RAASi at risk of hyperkalemia, along with a lower than expected incidence of hyperkalemia during run-in. Conclusion: Despite extensive screening efforts, few eligible patients were identified, and fewer developed hyperkalemia during run-in. Hence, a trial design limited to CKD patients at high hyperkalemia risk and including a run-in phase appears unlikely to provide evidence for a potential renal benefit from additional use of potassium binders.

Bladder cancer--the neglected tumor: a descriptive analysis of publications referenced in MEDLINE and data from the register ClinicalTrials.gov.

BACKGROUND: Uro-oncological neoplasms have both a high incidence and mortality rate and are therefore a major public health problem. The aim of this study was to evaluate research activity in uro-oncology over the last decade. METHODS: We searched MEDLINE and ClinicalTrials.gov systematically for studies on prostatic, urinary bladder, kidney, and testicular neoplasms. The increase in newly published reports per year was analyzed using linear regression. The results are presented with 95% confidence intervals, and a p value <0.05 was considered statistically significant. RESULTS: The number of new publications per year increased significantly for prostatic, kidney and urinary bladder neoplasms (all <0.0001). We identified 1,885 randomized controlled trials (RCTs); also for RCTs, the number of newly published reports increased significantly for prostatic (p = 0.001) and kidney cancer (p = 0.005), but not for bladder (p = 0.09) or testicular (p = 0.44) neoplasms. We identified 3,114 registered uro-oncological studies in ClinicalTrials.gov. However, 85% of these studies are focusing on prostatic (45%) and kidney neoplasms (40%), whereas only 11% were registered for bladder cancers. CONCLUSIONS: While the number of publications on uro-oncologic research rises yearly for prostatic and kidney neoplasms, urothelial carcinomas of the bladder seem to be neglected despite their important clinical role. Clinical research on neoplasms of the urothelial bladder must be explicitly addressed and supported.

Sometimes, often, and always: exploring the vague meanings of frequency expressions.

The article describes a general two-step procedure for the numerical translation of vague linguistic terms (LTs). The suggested procedure consists of empirical and model components, including (1) participants' estimates of numerical values corresponding to verbal terms and (2) modeling of the empirical data using fuzzy membership functions (MFs), respectively. The procedure is outlined in two studies for data from N = 89 and N = 109 participants, who were asked to estimate numbers corresponding to 11 verbal frequency expressions (e.g., sometimes). Positions and shapes of the resulting MFs varied considerably in symmetry, vagueness, and overlap and are indicative of the different meanings of the vague frequency expressions. Words were not distributed equidistantly across the numerical scale. This has important implications for the many questionnaires that use verbal rating scales, which consist of frequency expressions and operate on the premise of equidistance. These results are discussed for an exemplar questionnaire (COPSOQ). Furthermore, the variation of the number of prompted LTs (5 vs. 11) showed no influence on the words' interpretations.

The molecular basis of white pericarps in African domesticated rice: novel mutations at the Rc gene.

Repeated phenotypic evolution can occur at both the inter- and intraspecific level and is especially prominent in domesticated plants, where artificial selection has favoured the same traits in many different species and varieties. The question of whether repeated evolution reflects changes at the same or different genes in each lineage can now be addressed using the domestication and improvement genes that have been identified in a variety of crops. Here, we document the genetic basis of nonpigmented ('white') pericarps in domesticated African rice (Oryza glaberrima) and compare it with the known genetic basis of the same trait in domesticated Asian rice (Oryza sativa). In some cases, white pericarps in African rice are apparently caused by unique mutations at the Rc gene, which also controls pericarp colour variation in Asian rice. In one case, white pericarps appear to reflect changes at a different gene or potentially a cis-regulatory region.