Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying

The influence of low temperature vacuum drying process parameters on the survival, metabolic activity and residual water content of three different bacterial strains (Lactobacillus paracasei ssp. paracasei, Lactobacillus delbrueckii ssp. bulgaricus and Bifidobacterium lactis) was investigated. Shelf temperature and chamber pressure were varied and optimized by response surface methodology with regard to survival and residual water content. It is shown that the survival rate after low temperature vacuum drying is comparable to that of freeze drying. Based on the optimization experiments the combined influence of fermentation pH and drying process parameters was studied for the most detrimental and the best process condition, respectively. The results show that interactions between process and fermentation conditions have to be taken in account and that these influences are highly strain specific.     

Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus)

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.     

Mutations in Calmodulin Cause Ventricular Tachycardia and Sudden Cardiac Death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

Promotion of Human Early Embryonic Development and Blastocyst Outgrowth In Vitro Using Autocrine/Paracrine Growth Factors

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.     

Leaf breakdown in an Atlantic Rain Forest stream

The hypothesis of this study was that colonizers in decaying leaf litter prefer native species (Erythrina verna) to exotic ones (Eucalyptus camaldulensis and Protium heptaphyllum). Therefore, native species are expected to show higher breakdown rates, increased biomass, richness and density of invertebrate species, and increased biomass of decomposer fungi. Breakdown of leaf litter from these three species was assessed in an Atlantic Rain Forest stream. Four samples were collected during a period of 90 days and washed on a sieve to separate the invertebrates. Then, a series of leaf disks were cut to determine ash‐free dry mass and fungal biomass, and the remaining material was oven‐dried to determine the dry weight. Eucalyptus camaldulensis and E. verna showed higher breakdown rates than P. heptaphyllum, due to differences in leaf physical and chemical characteristics. The harder detritus (P. heptaphyllum) broke down more slowly than detritus with high concentrations of labile compounds (E. camaldulensis). The density of the invertebrates associated with detritus increased with time. There were no differences in density, taxonomic richness or biomass of invertebrates among the leaf types, which indicated that the invertebrates did not distinguish between exotic and native detritus. Fungal colonization varied among samples; E. camaldulensis showed the lowest ergosterol concentrations, mainly due to a high concentration of total phenolics. The detritus with the highest hardness value was colonized most slowly by fungi. These results showed that leaf breakdown in Atlantic Rain Forest streams could be affected either by changes in riparian vegetation, or by becoming more savanna‐like process due to climate change.     

Facts From Feces: Nitrogen Still Measures Up as a Nutritional Index for Mammalian Herbivores

Abstract: Fecal nitrogen (FN) has been applied widely as an index of dietary quality in studies of nutritional ecology of free-ranging and captive vertebrate herbivores, particularly ruminants. Three related articles in the Journal of Wildlife Management (JWM; Leslie and Starkey 1985, 1987; Hobbs 1987) have been cited (n = 150) in 87 publications and 39 peer-reviewed journals. The critique by Hobbs (1987) and the reply by Leslie and Starkey (1987) on limitations and appropriate applications of FN have been used to justify use of FN or negate its value as a nutritional proxy. We undertook a retrospective analysis of FN applications since 1985, largely because we sensed that methodological cautions noted in the 3 JWM publications were not being followed, leading to faulty conclusions and management applications, and that application protocols needed updating. From January 1986 through July 2007, the 107 species-by-continent applications of FN, citing the 3 JWM publications singly or in any combination, were diverse; FN was used in various ways on 5 continents and for 50 wild and domestic species. Cumulative rates of departure from recommended FN applications increased in recent years, largely in studies that compare different species while failing to fully acknowledge that differences likely reflected digestive capabilities rather than differences in some aspect of dietary intake. Post-1985 research on plant secondary compounds (e.g., tannins) has refined limitations to the application of FN, permitting more straightforward protocols than were possible in 1985. Although use does not necessarily reflect value, the number of published applications during the past 22 years indicates that peer reviewers from a variety of scientific disciplines view FN as a suitable proxy for nutritional status, which can be used to contrast study units when carefully defined by the study design. Any index can have shortcomings, and there are still circumstances when application of FN is problematic. Precise prediction of intake with FN under field conditions is still hampered by inherent variability, but contrasts of comparable study units and species can be appropriate. Published protocols for FN, as amended herein, should be adhered to, and conclusions are strengthened by the use of multiple nutritional indices.