Gene shuttling: moving of cloned DNA into and out of eukaryotic cells.

Successful shuttling of cloned DNA in eukaryotic cells should allow isolation of expressed genes. We tested the utility of cosmids for moving DNA into and out of eukaryotic cells. The unique cleavage of DNA at the cos site by the terminase function of lambda was exploited to maintain the linkage between the vector and inserted gene sequences, a prerequisite for successful rescue of the transforming DNA from high molecular weight DNA of the eukaryotic transformant. A cosmid recombinant containing the HSV thymidine kinase gene and a lambda recombinant containing the chicken thymidine kinase gene were used to test the feasability of this method. It was found that these recombinants can be rescued with high efficiency from DNA of HAT-resistant cells.     

Effects of the surfactant tween 80 on enzymatic hydrolysis of newspaper

The effects of Tween 80 on the enzymatic hydrolysis of newspaper were tested. By monitoring sugar production it was found that the surfactant (0.1%) increased the rate and extent of cellulose saccharification. Consequently, the rate of enzyme usage in the hydrolysis reactor was improved by 33%. In addition, in the presence of surfactant the recovery of enzymes was higher. Analysis of the enzyme solution showed that with Tween 80 present larger fractions of enzyme remained in solution throughout hydrolysis. Thus, it appears that the surfactant hindered the immobilization of the enzymes on the substrates by reducing the strength of adsorption.     

Studies on (pro)insulin biosynthesis and secretion of pancreatic islets of sand rats (Psammomys obesus) after different feeding conditions.

It was possible to induce different metabolic states in sand rats of our breeding colony or in newly caught Egyptian sand rats, respectively, by feeding a pellet diet or vegetable diet (green cabbage). Newly captured sand rats fed only on native food were used as reference group (group C). Plasma IRI-level and glucose in vivo and [3H]-leucine incorporation into proinsulin and insulin, insulin secretion and insulin content in vitro were investigated. Sand rats fed on pellet chow and ad libitum (group B) developed a hyperinsulinism and showed higher sensitivity of [3H]-leucine incorporation into proinsulin and insulin to glucose (maximal stimulation at 3 mM) and increased incorporation rates in vitro. Restriction of pellet food to 35-40 kcal/animal/day (group A) lead to changes of all parameters, which were investigated in the same direction as in group B, but to a much smaller extent. Newly captured sand rats, which were fed green cabbage for 4 to 6 weeks divided into two groups: One group (group D1) was comparable to the normal group (C) in IRI levels, glucose levels, glucose sensitivity and amount of [3H]-leucine incorporation. The other group (group D2) tended to group A. Marked changes in insulin content and insulin secretion of isolated pancreatic islets could not be found in any group.     

Utilization of cellulosic materials through enzymatic hydrolysis. I. Fermentation of hydrolysate to ethanol and single-cell protein

Ethanol fermentation studies were conducted with Saccharomyces cerevisiae ATCC #4126, to determine the optimal conditions of oxygen tension and feed sugar concentration. In long-term continuous culture maximum ethanol production was found to occur at 0.07 mmHg oxygen tension and 10% glucose feed concentration. Preliminary process design and cost studies are developed for industrial scale fermentations to produce ethanol and torula yeast from sugars obtained by enzymatic hydrolysis of newsprint.     

Utilization of cellulosic materials through enzymatic hydrolysis. II. Preliminary assessment of an integrated processing scheme

An integrated processing scheme is described for the conversion of a cellulosic waste (newsprint) to sugars by enzymatic hydrolysis and then to ethanol and yeast by fermentation. The unconverted solids are burned to produce process energy requirements and surplus electrical power. Preliminary designs and cost studies are developed to provide a rough perspective on the potential economic feasibility of this method of cellulose utilization.     

Phosphoproteomics identifies microglial Siglec‐F inflammatory response during neurodegeneration

Alzheimer’s disease (AD) is characterized by the appearance of amyloid‐β plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK‐p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec‐F which was upregulated on a subset of reactive microglia. The human paralog Siglec‐8 was also upregulated on microglia in AD. Siglec‐F and Siglec‐8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV‐2 cell line and human stem cell‐derived microglia models. Siglec‐F overexpression activates an endocytic and pyroptotic inflammatory response in BV‐2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine‐based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV‐2 cells. Collectively, our results point to an important role for mouse Siglec‐F and human Siglec‐8 in regulating microglial activation during neurodegeneration.     

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.