Esophageal wall blood perfusion during contraction and transient lower esophageal sphincter relaxation in humans

We recently reported that esophageal contraction reduces esophageal wall perfusion in an animal study. Our aim was to determine esophageal wall blood perfusion (EWBP) during esophageal contraction and transient lower esophageal sphincter relaxations (TLESRs) in humans. We studied 12 healthy volunteers. A custom-designed laser Doppler probe was anchored to the esophageal wall, 4-6 cm above the LES, by use of the Bravo pH system so that the laser light beam stay directed toward the esophageal mucosa. A high-resolution manometry equipped with impedance electrodes recorded esophageal pressures and reflux events. Synchronized pressure, impedance, pH, and EWBP recordings were obtained during dry and wet swallows and following a meal. Stable recordings of laser Doppler EWBP were only recorded when the laser Doppler probe was firmly anchored to the esophageal wall. Esophageal contractions induced by dry and wet swallows resulted in 46 ± 9% and 60 ± 10% reduction in the EWBP, respectively (compared to baseline). Reduction in EWBP was directly related to the amplitude (curvilinear fit) and duration of esophageal contraction. Atropine reduced the esophageal contraction amplitude and decreased the EWBP reduction associated with esophageal contraction. TLESRs were also associated with reduction in the EWBP, albeit of smaller amplitude (29 ± 3%) but longer duration (19 ± 2 s) compared with swallow-induced esophageal contractions. We report 1) an innovative technique to record EWBP for extended time periods in humans and 2) contraction of circular and longitudinal muscle during peristalsis and selective longitudinal muscle contraction during TLESR causes reduction in the EWBP; 3) using our innovative technique, future studies may determine whether esophageal wall ischemia is the cause of esophageal pain/heartburn.     

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.     

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.     

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) for Monitoring of Drug Response in Primary Cancer Spheroids

Malignant ascites is a fluid, which builds up in the abdomen and contains cancer cells in the form of single cells or multicellular clusters called spheroids. Malignant ascites has been observed in patients suffering from ovarian, cervical, gastric, colorectal, pancreatic, endometrial, or primary liver cancer. The spheroids are believed to play a major role in chemo resistance and metastasis of the cancer. To ease the discomfort of patients, malignant ascites (MA) is often drained from the abdomen using a procedure called paracentesis. MA retrieved via this minimal invasive procedure is a great source for cancer spheroids, which can be used for testing chemotherapeutic drugs and drug combinations. Herein, the existing workflow is adapted to make concurrent monitoring of drug accumulation, drug response, and drug metabolites feasible using primary spheroids or spheroids grown without a scaffolding matrix. To achieve this, those spheroids are embedded in matrigel, before fixing them with formalin. This makes it possible to process, store, and ship samples at room temperature. This new approach might be used to choose the best targeted therapy for each patient and thereby facilitate personalized medicine.     

Habitat shift and time budget of the Tibetan argali: the influence of livestock grazing

Livestock production is the primary source of livelihood and income in most of the high steppe and alpine regions of the Indian Trans-Himalaya. In some areas, especially those established or proposed for biodiversity conservation, recent increases in populations of domestic livestock, primarily sheep and goats, have raised concern about domestic animals competitively excluding wild herbivores from the rangelands. We evaluated the influence of domestic sheep and goat grazing on the habitat use and time budget of the endangered Tibetan argali Ovis ammon hodgsoni in the proposed Gya-Miru Wildlife Sanctuary, Ladakh, India. We asked if the domestic sheep and goat grazing and collateral human activities relegate the argali to sub-optimal habitats, and alter their foraging time budgets. Data were collected on habitat use and time budget of a population of c. 50 argalis before and after c. 2,000 sheep and goats moved onto their winter pasture in the Tsabra catchment of the aforementioned reserve. Following the introduction of domestic sheep and goats, argalis continued to use the same catchment but shifted to steeper habitats, closer to cliffs, with lower vegetation cover, thus abandoning previously used plant communities with denser cover. Argalis’ active time spent foraging also decreased by 10% in response to the presence of livestock. These results suggest a clear disturbance effect of livestock on argalis, and indicate a potential for competition, conceivably a significant disadvantage for argalis in winter when forage availability is minimal.     

Application of the LUminometric Methylation Assay to ecological species: tissue quality requirements and a survey of DNA methylation levels in animals

The LUminometric Methylation Assay (LUMA) measures global DNA methylation. LUMA depends on digestion of DNA with methyl‐sensitive and methyl‐insensitive restriction enzymes, followed by pyrosequencing. Until recently, LUMA has been principally used for biomedical research. Here, we use chickens as a model to investigate sample quality issues relating to LUMA and then apply the method to ecological species. First, we assessed the effect of tissue storage conditions on DNA methylation values. This is an important consideration for ecological species because samples are not always ideally preserved and LUMA is sensitive to poor DNA quality. We found that good quality LUMA data could be obtained from chicken liver and brain tissues stored at 21 °C for at least 2 and 12 h, respectively. Longer storage times introduced nonspecific peaks to pyrograms which were associated with reduced DNA methylation. Repeatedly, freezing and thawing the tissues did not affect LUMA data. Second, we measured DNA methylation in 12 species representing five animal classes: amphibians (African and Western clawed frog), reptiles (green anole lizard), fish (yellow perch, goldfish, lake trout), mammals (American mink, polar bear, short‐beaked common dolphin, Atlantic white‐sided dolphin) and birds (chicken, Japanese quail). We saw a pattern of high DNA methylation in fish (84–87%), and intermediate levels in mammals (68–72%) and birds (52–71%). This pattern corresponds well with previous measures of DNA methylation generated by HPLC. Our data represent the first CpG methylation values to be reported in several species and provide a basis for studying patterns of epigenetic inheritance in an ecological context.     

Phenotypic T Cell Exhaustion in a Murine Model of Bacterial Infection in the Setting of Pre-Existing Malignancy

While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8⁺ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8⁺ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8⁺ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8⁺ T cells and total CD4⁺ and CD8⁺ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8⁺ T cells in the cancer group. Taken together, these data suggest that cancer''s immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8⁺ T cell functionality and differentiation.