A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Crosslinker-Based Regulation of Swelling Behavior of Poly(N-isopropylacrylamide) Gels in a Post-Polymerization Crosslinking System.

A fundamental understanding of the effect of a crosslinker on gel properties is important for the design of novel soft materials because a crosslinking is a key component of polymer gels. We focused on post-polymerization crosslinking (PPC) system utilizing activated ester chemistry, which is a powerful tool due to structural diversity of diamine crosslinkers and less susceptibility to solvent effect compared to conventional divinyl crosslinking system, to systematically evaluate the crosslinker effect on the gel properties. A variety of alkyldiamine crosslinkers was employed for the synthesis of poly(N-isopropylacrylamide) (PNIPAAm) gels and it was clarified that the length of alkyl chains of diamine crosslinkers strongly affected the gelation reaction and the swelling behavior. The longer crosslinker induced faster gelation and decreased the swelling degree and the response temperature in water, while the crosslinking density did not significantly change. In addition, we were able to modify the polymer chains in parallel with crosslinking by using a monoamine modifier along with a diamine crosslinker. This simultaneous chain modification during crosslinking (SMC) was demonstrated to be useful for the regulation of the crosslinking density and the swelling behavior of PNIPAAm gels.

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization.

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-ß-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.