Recovery from organ-specific autoimmune diseases largely relies on the mobilization of endogenous repair mechanisms and local factors that control them. Natural killer (NK) cells are swiftly mobilized to organs targeted by autoimmunity and typically undergo numerical contraction when inflammation wanes. We report the unexpected finding that NK cells are retained in the brain subventricular zone (SVZ) during the chronic phase of multiple sclerosis in humans and its animal model in mice. These NK cells were found preferentially in close proximity to SVZ neural stem cells (NSCs) that produce interleukin-15 and sustain functionally competent NK cells. Moreover, NK cells limited the reparative capacity of NSCs following brain inflammation. These findings reveal that reciprocal interactions between NSCs and NK cells regulate neurorepair.

The current study investigates the transient human physiological and comfort responses during sedentary activity following a period of elevated activity in a hot condition. Such metabolic and thermal down-steps are common in buildings as occupants arrive after commuting in summer. It creates a serious problem for thermostatic control, since arriving occupants find their transition uncomfortably warm at temperatures that resident occupants find comfortable. Fifty-nine participants (29 men, 30 women) dressed in 0.6 clo were tested while sedentary for 60 min in 26 °C, after having been exposed to 30 °C for 15min, during which they performed activities metabolically simulating commuting: sitting (SE- 1.2 met), or doing three levels of stair-step exercises: low (LEx- 2.2 met), medium (MEx - 3.0 met), and high (HEx - 4.4 met). Subjective comfort and physiological responses (metabolic rate, skin temperature, skin blood flow rate, heart rate, core temperature, and skin wettedness) were collected. Results show that sedentary conditions at 26°C became comfortable and acceptable within 2 min, but thermal sensation required much longer to change from ‘warm’ or ‘hot’ to ‘neutral’: 0, 8, 17, 30 min after SE, LEx, MEx, HEx respectively. Skin wettedness and core temperature did not recover within the60 min. The delays are mainly due to body heat stored during the exercise. A room temperature of 26°C may not provide sufficient cooling after summer commuting. Localized convective cooling of transitional spaces and work areas by ceiling or desk fans represent a way to enhance comfort recovery.

The current study investigates the transient human physiological and comfort responses during sedentary activity following a period of elevated activity in a hot condition. Such metabolic and thermal down-steps are common in buildings as occupants arrive after commuting in summer. It creates a serious problem for thermostatic control, since arriving occupants find their transition uncomfortably warm at temperatures that resident occupants find comfortable. Fifty-nine participants (29 men, 30 women) dressed in 0.6 clo were tested while sedentary for 60 min in 26 °C, after having been exposed to 30 °C for 15min, during which they performed activities metabolically simulating commuting: sitting (SE- 1.2 met), or doing three levels of stair-step exercises: low (LEx- 2.2 met), medium (MEx - 3.0 met), and high (HEx - 4.4 met). Subjective comfort and physiological responses (metabolic rate, skin temperature, skin blood flow rate, heart rate, core temperature, and skin wettedness) were collected. Results show that sedentary conditions at 26°C became comfortable and acceptable within 2 min, but thermal sensation required much longer to change from ‘warm’ or ‘hot’ to ‘neutral’: 0, 8, 17, 30 min after SE, LEx, MEx, HEx respectively. Skin wettedness and core temperature did not recover within the60 min. The delays are mainly due to body heat stored during the exercise. A room temperature of 26°C may not provide sufficient cooling after summer commuting. Localized convective cooling of transitional spaces and work areas by ceiling or desk fans represent a way to enhance comfort recovery.

A biotinylated heparosan hexasaccharide was synthesized using a one-pot multi-enzyme strategy, in situ activation and transfer of N-trifluoroacetylglucosamine (GlcNTFA) to a heparin backbone significantly improved the synthetic efficiency. The biotinylated hexasaccharide could serve as a flexible core to diversify its conversion into heparan sulfate isoforms with potential biological applications and therapeutics.

Unlabelled

Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering.

Importance

In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

Background and purpose

Autoimmune responses can occur when antigens from the central nervous system are presented to lymphocytes in the periphery or central nervous system in several neurological diseases. However, whether autoimmune responses emerge after brain ischemia and their impact on clinical outcomes remains controversial. We hypothesized that brain ischemia facilitates the genesis of autoimmunity and aggravates ischemic brain injury.

Methods

Using a mouse strain that harbors a transgenic T-cell receptor to a central nervous system antigen, MOG_35-55 (myelin oligodendrocyte glycoprotein) epitope (2D2), we determined the anatomic location and involvement of antigen-presenting cells in the development of T-cell reactivity after brain ischemia and how T-cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion and photothrombotic stroke models were used in this study. We also quantified the presence and status of T cells from brain slices of ischemic patients.

Results

By coupling transfer of labeled MOG_35-55-specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG_91-108 and MOG_103-125 in transient middle cerebral artery occlusion and photothrombotic stroke models. This reactivity and T-cell activation first occur locally in the brain after ischemia. Also, microglia act as antigen-presenting cells that effectively present MOG antigens, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T-cell activation and MOG-specific T cells are present in the brain of patients with ischemic stroke.

Conclusions

Our findings suggest that brain ischemia activates and diversifies T-cell responses locally, which exacerbates ischemic brain injury.

Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.

Quantification, characterization and biofunctional studies of N-glycans on proteins remain challenging tasks due to complexity, diversity and low abundance of these glycans. The availability of structurally defined N-glycans (especially isomers) libraries is essential to help on solving these tasks. We reported herein an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycans. Starting with 5 chemically prepared building blocks, 8 N-glycan core structures containing one or two terminal N-acetyl-D-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in the convergent fragment coupling strategy. Each of these core structures was then extended to 5 to 15 N-glycan sequences by enzymatic reactions catalyzed by 4 robust glycosyltransferases. Success in synthesizing N-glycans with Neu5Gc and core-fucosylation further expanded the ability of enzymatic extension. High performance liquid chromatography with an amide column enabled rapid and efficient purification (>98% purity) of N-glycans in milligram scales. A total of 73 N-glycans (63 isomers) were successfully prepared and characterized by MS² and NMR. The CSEE strategy provides a practical approach for "mass production" of structurally defined N-glycans, which are important standards and probes for Glycoscience.