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The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analysing the genotypes and phenotypes of the resulting transformed lines.

Background: Jehovah's Witnesses pose a clinical challenge in the setting of critical anemia. Most do not accept transfusions, but some accept hemoglobin-based oxygen carriers on a compassionate-use basis. PEGylated carboxyhemoglobin bovine (PCHB) is an acellular dual-action carbon monoxide (CO)-releasing and oxygen transfer agent currently being investigated in Phase II clinical trials., Case Report: We present the case of a 42-year-old Jehovah's Witness with an acute upper gastrointestinal bleed and hemorrhagic shock who required emergent PCHB for stabilization during lifesaving interventions. After PCHB infusion, the patient's shock and encephalopathy improved with decreased vasopressor requirement. Through gastroenterology and interventional radiology procedures, the patient's bleeding stabilized. While receiving five additional doses of PCHB and other supportive therapies (iron, folate, vitamin B12, darbepoetin alfa), the patient was extubated and weaned off vasopressors., Conclusions: PCHB was used to stabilize (bridge) a critically ill anemic patient for lifesaving interventions without adverse effects. Additional studies are warranted to explore the drug's safety profile and efficacy in patients declining blood products., (© 2018 AABB.)

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.

The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analyzing the genotypes and phenotypes of the resulting transformed lines.

An impurity produced in the synthesis of compound I is separated and identified as its enantiomer II using normal-phase chiral high-performance liquid chromatography (HPLC) with UV absorbance, optical rotation (OR) and mass spectrometric (MS) detection. The results show that the impurity II and compound I have equal and opposite specific rotations, identical MS spectra and the same MS-MS fragmentation pattern, as required for enantiomers. The procedures presented demonstrate a novel combination of methods for enantiomer identification and characterization that do not require the preparation of individual enantiomer markers or even the racemic mixture, thus reducing the need for additional synthetic work.

A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S-enantiomer was less active. We show that enantiomeric selectivity of antiviral for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoselective inhibition of DNA polymerase from human cytomegalovirus.