Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.     

Genomic Characterization of Listeria monocytogenes Strains Involved in a Multistate Listeriosis Outbreak Associated with Cantaloupe in US

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.     

A common genetic network underlies substance use disorders and disruptive or externalizing disorders

Here we summarize evidence obtained by our group during the last two decades, and contrasted it with a review of related data from the available literature to show that behavioral syndromes involving attention deficit/hyperactivity disorder (ADHD), externalizing disorders, and substance-use disorder (SUD) share similar signs and symptoms (i.e., have a biological basis as common syndromes), physiopathological and psychopathological mechanisms, and genetic factors. Furthermore, we will show that the same genetic variants harbored in different genes are associated with different syndromes and that non-linear interactions between genetic variants (epistasis) best explain phenotype severity, long-term outcome, and response to treatment. These data have been depicted in our studies by extended pedigrees, where ADHD, externalizing symptoms, and SUD segregate and co-segregate. Finally, we applied here a new formal network analysis using the set of significantly replicated genes that have been shown to be either associated and/or linked to ADHD, disruptive behaviors, and SUD in order to detect significantly enriched gene categories for protein and genetic interactions, pathways, co-expression, co-localization, and protein domain similarity. We found that networks related to pathways involved in axon guidance, regulation of synaptic transmission, and regulation of transmission of nerve impulse are overrepresented. In summary, we provide compiled evidence of complex networks of genotypes underlying a wide phenotype that involves SUD and externalizing disorders.     

Isolation and characterization of polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum)

We report on the development of nine polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum) using a combination of enriched and unenriched subgenomic libraries. Based on the small percentage of positive clones in the unenriched library (0.4%) it appears that microsatellites are very scarce in nurse shark genomes. Numbers of alleles at polymorphic loci ranged from two to 15; observed heterozygosity ranged from 0.17 to 0.90. We expect these loci to be useful for studies of breeding structure and paternity.     

Evidence for progressive phosphorus limitation over long-term ecosystem development: Examination of a biogeochemical paradigm

Aims

To test predictions of ecosystem theory for changes in P cycling over primary succession, we determined soil phosphorus (P) in labile, primary mineral, organic, and occluded forms along a chronosequence of five wave cut terraces known as the “Ecological Staircase”. The Ecological Staircase terraces (T1-T5) transition naturally from fertile native coastal forests in California, USA, to diminutive pygmy vegetation over the span of?>?500,000 years of pedogenesis.

Methods

Soil P fractions were quantified to a depth of 40 cm on T1-T5 using a modified Hedley P fractionation procedure.

Results

Overall results confirmed the Walker and Syers Model of Phosphorus Transformations During Pedogenesis: total P declined from youngest (194 mg/kg P) to oldest (127 mg/kg P) sites; primary-mineral P decreased sharply from T1 to older sites; and occluded P dominated P pools at the oldest pygmy sites (T3-T5). In addition, foliar P concentrations declined markedly in the pygmy forest, and N/P of vegetation (T1: 6.03, T5: 14.4) and N/P_organic of mineral soils (T1: 6.10, T5: 25.3) increased significantly over time.

Conclusions

Results point to P as the primary limiting nutrient in the pygmy forest, exemplifying the terminal steady-state of ecosystem retrogression that underlies the persistence of this unique ecosystem.     

Elabela/toddler: New peptide with a promising future in cancer diagnostic and therapy

Elabela/toddler is the second endogenous ligand recently identified after Apelin, that binds to the G protein-coupled receptor APJ. Elabela is a 54-amino acid peptide initially identified in fish and human genomes and classified as noncoding. This precursor can be cleaved to shorter sequences (32, 21, and 11 amino acids), which bind and activate APJ, and can be blocked by APJ antagonists. Contrary to Apelin and APJ, widely distributed in organs and tissues, Elabela expression is more restricted, and different studies have revealed the potential role of Elabela in cancers. This review summarizes the current studies focusing on the role of Elabela in different cancers.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Investigating substrate promiscuity in cyclooxygenase-2: the role of Arg-120 and residues lining the hydrophobic groove

The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H(2) from arachidonic acid (AA). In its catalytically productive conformation, AA binds within the cyclooxygenase channel with its carboxylate near Arg-120 and Tyr-355 and ω-end located within a hydrophobic groove above Ser-530. Although AA is the preferred substrate for both isoforms, COX-2 can oxygenate a broad spectrum of substrates. Mutational analyses have established that an interaction of the carboxylate of AA with Arg-120 is required for high affinity binding by COX-1 but not COX-2, suggesting that hydrophobic interactions between the ω-end of substrates and cyclooxygenase channel residues play a significant role in COX-2-mediated oxygenation. We used structure-function analyses to investigate the role that Arg-120 and residues lining the hydrophobic groove play in the binding and oxygenation of substrates by murine (mu) COX-2. Mutations to individual amino acids within the hydrophobic groove exhibited decreased rates of oxygenation toward AA with little effect on binding. R120A muCOX-2 oxygenated 18-carbon ω-6 and ω-3 substrates albeit at reduced rates, indicating that an interaction with Arg-120 is not required for catalysis. Structural determinations of Co(3+)-protoporphyrin IX-reconstituted muCOX-2 with α-linolenic acid and G533V muCOX-2 with AA indicate that proper bisallylic carbon alignment is the major determinant for efficient substrate oxygenation by COX-2. Overall, these findings implicate Arg-120 and hydrophobic groove residues as determinants that govern proper alignment of the bisallylic carbon below Tyr-385 for catalysis in COX-2 and confirm nuances between COX isoforms that explain substrate promiscuity.