Acetyl-CoA synthases are essential for maintaining histone acetylation under metabolic stress during zygotic genome activation in pigs.

ACSS1/2 converts acetate into acetyl-coenzyme A, which contributes to histone acetylation in the mitochondria and cytoplasm. Zygotic genome activation (ZGA) is critical for embryo development involving drastic histone modification. An efficient crRNAs-Cas13a targeting strategy was employed to investigate the ACSS1/2 function during ZGA. The results showed that nuclear accumulation of ACSS1 and ACSS2 occurs during ZGA. Knockdown of ACSS1/2 did not affect blastocyst formation when using a normal medium. On culturing embryos in a medium with acetate and no pyruvate (-P + Ace), knockdown of ACSS1 did not affect histone acetylation levels but significantly reduced ATP levels, whereas knockdown of ACSS2 significantly reduced histone acetylation levels in porcine embryos. Inhibition of fatty acid beta-oxidation by etomoxir significantly reduced ATP levels, which could be restored by acetate. The histone acetylation levels in the ACSS1 and ACSS2 knockdown groups both decreased considerably after etomoxir treatment. Moreover, acetate showed dose-dependent effects on SIRT1 and SIRT3 levels when under metabolic stress. The C-terminus of ACSS1 regulated the nuclear translocation. In conclusion, ACSS1/2 helps to maintain ATP and histone acetylation levels in porcine early embryos under metabolic stress during ZGA.

AGS3-dependent trans-Golgi network membrane trafficking is essential for compaction in mouse embryos.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.

Effects of omitting elective neck irradiation to nodal Level IB in nasopharyngeal carcinoma patients with negative Level IB lymph nodes treated by intensity-modulated radiotherapy: a Phase 2 study.

OBJECTIVE: To investigate the need for elective neck irradiation (ENI) to nodal Level IB in patients with nasopharyngeal carcinoma (NPC) with negative Level IB lymph nodes (IB-negative) treated by intensity-modulated radiotherapy (IMRT). METHODS: We conducted a Phase 2 prospective study in 123 newly diagnosed IB-negative patients with NPC treated by IMRT, who met at least 1 of the following criteria: (1) unilateral or bilateral Level II involvement with 1 of the following: Level IIA involvement or any Level II node ≥2 cm/with extracapsular spread; (2) ≥2 unilateral node-positive regions. Bilateral Level IB nodes were not contoured as part of the treatment target and treated electively. Level IB regional recurrence rate; pattern of treatment failure; 3-year overall survival (3y-OS), 3-year local control (3y-LC) and 3-year regional control (3y-RC) rates; toxicities; and dosimetric data for planning target volumes, organs at risk, Level IB and submandibular glands (SMGs) were evaluated. RESULTS: Two patients developed failures at Level IB (1.6%). The 3y-LC, 3y-RC and 3y-OS rates were 93.5%, 93.5% and 78.0%, respectively. Bilateral Level IB received unplanned high-dose irradiation with a mean dose (Dmean) ≥50 Gy in 60% of patients. The average Dmean of bilateral SMGs was approximately 53 Gy. CONCLUSION: ENI to Level IB may be unnecessary in IB-negative patients with NPC treated by IMRT. A further Phase 3 study is warranted. ADVANCES IN KNOWLEDGE: Based on the results of this first Phase 2 study, we suggest omitting ENI to Level IB in Ib-negative patients with NPC with extensive nodal disease treated by IMRT.

Target-oriented discovery of a new esterase-producing strain Enterobacter sp. ECU1107 for whole cell-catalyzed production of (2S,3R)-3-phenylglycidate as a chiral synthon of Taxol.

A new strain, Enterobacter sp. ECU1107, was identified among over 200 soil isolates using a two-step screening strategy for the enantioselective synthesis of (2S,3R)-3-phenylglycidate methyl ester (PGM), a key intermediate for production of a potent anticancer drug Taxol®. An organic-aqueous biphasic system was employed to reduce spontaneous hydrolysis of the substrate PGM and isooctane was found to be the most suitable organic solvent. The temperature and pH optima of the whole cell-mediated bioreaction were 40 °C and 6.0, respectively. Under these reaction conditions, the enantiomeric excess (ee(s)) of (2S,3R)-PGM recovered was greater than 99 % at approximately 50 % conversion. The total substrate loading in batch reaction could reach 600 mM. By using whole cells of Enterobacter sp. ECU1107, (2S,3R)-PGM was successfully prepared in decagram scale in a 1.0-l mechanically stirred reactor, affording the chiral epoxy ester in >99 % ee s and 43.5 % molar yield based on the initial load of racemic substrate.