Models of Somatic Hypermutation Targeting and Substitution Based on Synonymous Mutations from High-Throughput Immunoglobulin Sequencing Data

Analyses of somatic hypermutation (SHM) patterns in B cell immunoglobulin (Ig) sequences contribute to our basic understanding of adaptive immunity, and have broad applications not only for understanding the immune response to pathogens, but also to determining the role of SHM in autoimmunity and B cell cancers. Although stochastic, SHM displays intrinsic biases that can confound statistical analysis, especially when combined with the particular codon usage and base composition in Ig sequences. Analysis of B cell clonal expansion, diversification, and selection processes thus critically depends on an accurate background model for SHM micro-sequence targeting (i.e., hot/cold-spots) and nucleotide substitution. Existing models are based on small numbers of sequences/mutations, in part because they depend on data from non-coding regions or non-functional sequences to remove the confounding influences of selection. Here, we combine high-throughput Ig sequencing with new computational analysis methods to produce improved models of SHM targeting and substitution that are based only on synonymous mutations, and are thus independent of selection. The resulting “S5F” models are based on 806,860 Synonymous mutations in 5-mer motifs from 1,145,182 Functional sequences and account for dependencies on the adjacent four nucleotides (two bases upstream and downstream of the mutation). The estimated profiles can explain almost half of the variance in observed mutation patterns, and clearly show that both mutation targeting and substitution are significantly influenced by neighboring bases. While mutability and substitution profiles were highly conserved across individuals, the variability across motifs was found to be much larger than previously estimated. The model and method source code are made available at http://clip.med.yale.edu/SH

Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes

Gene Expression Analysis of Forskolin Treated Basilar Papillae Identifies MicroRNA181a as a Mediator of Proliferation

Auditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients.Gene expression was profiled in forskolin treated (i.e., proliferating) and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6), 48 (n = 6), and 72 (n = 12) hours in culture. In the forskolin-treated epithelia there was significant (p<0.05; >two-fold change) upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a), which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells.These studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells

Use of systemic steroids in management of a pregnant patient with ARDS.

Learning Objectives: Steroids have been used in the management of ARDS. All studies which have shown the benefit of using steroids in ARDS excluded pregnant patients and there is no data to support the use of steroids in pregnant patients with ARDS. Methods: 16 y/o female with a past medical history of childhood asthma, pregnant in her 33rd week, who failed outpatient mild UTI treatment with Nitrofurantoin presented to the ER with fever, chills and worsening back pain. She was found to be febrile, tachycardic with leukocytosis. She was admitted for sepsis secondary to pyelonephritis due to pan-susceptible E Coli and was started on Ceftriaxone. The following day, she had worsening respiratory distress with increasing oxygen requirements. Imaging showed complete consolidation of the almost entire right lung. She required 12L HFNC. Antibiotics were escalated and she was transferred to a tertiary care center for escalation of care. Blood cultures and tests for respiratory pathogens remained negative. She was intubated after 2 days for worsening respiratory distress and hypoxic respiratory failure. Despite 5 days of broad spectrum antibiotics, her respiratory status was unchanged. Repeat imaging now showed diffuse airspace opacities in the left lung. She had no evidence of cardiogenic pulmonary edema and a diagnosis of ARDS was made. Her ventilator settings were then managed per ARDSNet protocol. 2 days later she was started on Methylprednisolone per Meduri protocol for ARDS. After 7 days of broad spectrum antibiotics, she was continued on Ceftriaxone to complete total 14 days of antibiotics for pyelonephritis. 3 days after starting corticosteroid, she underwent cesarean section due to pre-eclampsia. She required high FiO2 and PEEP to maintain oxygenation. Weaning her ventilator settings was a gradual process. She was extubated to HFNC after 17 days of intubation. Hospital course complicated by pneumomediastinum and pneumothorax requiring drainage catheter and mood disorder. Clinical status then improved and the patient was transferred to a general medical floor. After 1 month of hospitalization, she was discharged home on 2L NC and a tapering dose of prednisone. PFTs done a week after her discharge showed severe restriction with severely reduced DLCO at 24% with 92%oxygen saturation on room air. Results: This case demonstrated successful use of steroids in a pregnant patient with ARDS. Adverse effects like pregnancy induced hypertension, muscle weakness, mood disorder should be kept in mind

Marine microalgae flocculation and focused beam reflectance measurement

The production of biodiesel from fast growing and lipid containing marine microalgal species is sustainably and economically more promising than fresh water microalgae. However, like fresh water microalgae, the process is limited by numerous factors such as the generally dilute nature of microalgal cultures, and the small size of microalgae cells, except for multi-cellular species such as Spirulina. Current dewatering technologies are sufficiently able to separate microalgae from the culture media; however the economics of the process makes these technologies ineffective as they are all too expensive when compared with the low cost of biodiesel from other sources. Centrifugation, which is one of the current technologies, is highly energy intensive especially in a large-scale setting. Filtration techniques such as tangential flow filtration has the potential to be a low cost dewatering technique, however there is a cost issue associated with the replacement of clogged membranes. Flocculation is another commonly used dewatering technique that has the advantage of using less energy under optimum conditions. Thus process development for marine microalgae flocculation could be an essential step to revolutionize biodiesel production from microalgae.In this work, mixed cultures of marine microalgal species were obtained from semi-continuous laboratory reactors and flocculation was investigated using polyelectrolyte (polymer) flocculants. Cationic, anionic and non-ionic polyelectrolyte flocculants were tested using the standard jar stirrer test at varying pH and temperature. All three flocculant types displayed suitability for microalgae flocculation with the cationic polymer obtaining the highest flocculation efficiency of 89.9% at an optimum concentration of 4. mg/L. Focused beam reflectance measurements (FBRM) showed real time changes in microalgal flocs size during the flocculation process. This data is essential to understand the kinetics of microalgal flocs formation, to ensure the stability of the floc formation process, and to monitor and evaluate the performance of the flocculation process. © 2010 Elsevier B.V