A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

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Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.

Reaction of magnesium amidinate complexes of the form [{MesC(NR) 2 }MgBr(OEt 2)] 2 (R = i Pr, Dipp, Mes) with the potassium salts of transition metal anions K[CpFe(CO) 2 ] (K[Fp]) and K[Co(CO) 3 (PCy 3)](THF) 2 gave the complexes {MesC(NR) 2 }MgFp(THF) and {MesC(NR) 2 }Mg{Co(CO) 3 (PCy 3)}(THF). Single crystal X-ray diffraction studies of {MesC(NR) 2 }Mg{Co(CO) 3 (PCy 3)}(THF) for R = i Pr and Dipp confirm these to have Mg-Co bonds in the solid state. Reaction of the structurally similar magnesium guanidinate complex {Me 2 NC(NDipp) 2 }MgI(OEt 2) with the aforementioned transition metal anions and additional K[Co(CO) 3 (PPh 3)](THF) gave the series of complexes [{Me 2 NC(NDipp) 2 }MgFp] 2 , {Me 2 NC(NDipp) 2 }Mg{Co(CO) 3 (PCy 3)}(OEt 2) and {Me 2 NC(NDipp) 2 }Mg{Co(CO) 3 (PPh 3)}(OEt 2). Structural authentication by X-ray crystallography showed [{Me 2 NC(NDipp) 2 }MgFp] 2 to be a very rare example of a base-free alkaline earth-transition bonded complex, having two Mg-Fe bonds. IR and diffusion NMR spectroscopy were carried out to gain further insight into the solid state and solution phase structures.