Traditional sparkling wines are produced by the refermentation of a base wine with yeast in the bottle followed by a critical period of aging. During the often lengthy aging process, yeast undergoes cell death and autolysis to release cellular compounds that over time ultimately contribute to the flavor and appearance of the product. While accelerating yeast autolysis for sparkling wine production has been the focus of several studies, employing overexpressed native yeast alleles for this purpose remains poorly explored. Here, we show that the overexpression of native yeast genes, specifically selected autophagic genes, results in accelerated cell death in nitrogen starvation and base wine refermentation. We show ATG3 or ATG4 overexpression has pleiotropic intracellular ramifications including reduced turnover of autophagic cargo, vacuolar fragmentation, abnormal accumulation of lipids, and accelerated accumulation of reactive oxygen species (ROS), all of which precede accelerated cell death. Our findings suggest that the increased expression of autophagy-related genes, such as ATG3 and ATG4, in industrial wine yeast can serve as a suitable marker or breeding strategy to accelerate the cell death and autolysis of wine yeast during sparkling wine production.

The discovery that repeat expansions in the C9orf72 gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has revolutionized our understanding of these diseases. Substantial headway has been made in characterizing C9orf72-mediated disease and unravelling its underlying aetiopathogenesis. Three main disease mechanisms have been proposed: loss of function of the C9orf72 protein and toxic gain of function from C9orf72 repeat RNA or from dipeptide repeat proteins produced by repeat-associated non-ATG translation. Several downstream processes across a range of cellular functions have also been implicated. In this article, we review the pathological and mechanistic features of C9orf72-associated FTD and ALS (collectively termed C9FTD/ALS), the model systems used to study these conditions, and the probable initiators of downstream disease mechanisms. We suggest that a combination of upstream mechanisms involving both loss and gain of function and downstream cellular pathways involving both cell-autonomous and non-cell-autonomous effects contributes to disease progression.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript variants contain upstream open reading frames (uORFs) in their 5'untranslated region (5'TR). To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγ gene, we constructed wild-type (psiCHECK2-cPPARγ3-5'UTR-WT) and a uORF mutant (the upstream ATG (uATG) was mutated to stop codon TGA) 5'UTR reporters (psiCHECK2-cPPARγ3- 5'UTR-Mut) of chicken PPARγ transcript variant 3 (cPPARγ3). These two reporters were individually transfected into immortalized chicken pre-adipocytes (ICPA) and DF1 cells, and the renilla luciferase (hRluc) activity and mRNA expression level were detected by reporter assay and qRT-PCR. The results showed that the hRluc activity of the mutated 5'UTR was significantly higher than that of the wild-type 5'UTR in ICPA cells (P<0.01), and the hRluc activity of the mutated 5'UTR tended to be higher than that of the wild-type 5'UTR in DF1 cells, but this difference did not reach statistical significance (P>0.05). The qRT-PCR analysis showed, in ICPA cells, the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct (P<0.01). In DF1 cells, the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct, but this difference did not reach statistical significance (P>0.05). To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF, we constructed the expression plasmids bearing the full-length coding region of chicken PPARγ gene plus either wild-type or mutant uORF 5'UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5'UTR of the cPPARγ3 inhibits its translation.

This prospective study explored an optimal conditioning regimen to ensure engraftment with minimal toxicity in adult patients with severe aplastic anemia (SAA) who received haplo-identical stem cell transplantation from a related mismatched donor (Haplo-SCT). We explored a safe and sufficient dose of rabbit ATG (Thymoglobulin®) in combination with 800 cGy total body irradiation (TBI) and fludarabine (Flu, 30 mg/m2 /day) for 5 days using step-by-step dose de-escalation. The dose of ATG was de-escalated from 10 mg/kg (group 1), to 7.5 mg/kg (group 2), to 5 mg/kg (group 3), and the TBI dose was reduced to 600 cGy (group 4) beginning in October 2014. If one patient developed transplant-related mortality (TRM) with engraftment in a group, we moved to the next lower dose group. Thirty-four patients were enrolled in groups 1-3 (n=10) and 4 (n=24). All patients achieved primary engraftment. The incidence of acute GVHD (grade ≥2) and chronic GVHD (≥ moderate) was 29.4% and 14.7%, respectively. With a median follow-up of 56.6 and 21.8 months in groups 1-3 and group 4, respectively, the 2-year probability of overall survival (91.7% in group 4 vs. 70% in groups 1-3, P = 0.155) and GVHD-free survival (78.4% in group 4 vs. 50% in groups 1-3, P = 0.115) was shown tended to be better in group 4. This study explored an optimal conditioning with step-by-step de-escalation dosage of ATG and TBI to reduce TRM with sustained graft function. TBI-600 cGy/Flu/intermediate-dose ATG resulted in feasible outcomes of Haplo-SCT for adult patients with SAA. This article is protected by copyright. All rights reserved.

PARK7/DJ-1 is a Parkinson disease- and cancer-associated protein that functions as a multifunctional protein involved in gene transcription regulation and anti-oxidative defense. Although PARK7 lacks the secretory signal sequence, it is secreted and plays important physiological and pathophysiological roles. Whereas secretory proteins that lack the endoplasmic reticulum-targeting signal sequence are secreted from cells by way of what is called the unconventional secretion mechanism, the specific processes responsible for causing PARK7 to be secreted across the plasma membrane have remained unclear. In the present study, we found that PARK7 secretion was increased by treatment with 6-OHDA via the unconventional secretory pathway in human neuroblastoma SH-SY5Y cells and MEF cells. We also found that 6-OHDA-induced PARK7 secretion was suppressed in Atg5-, Atg9-, or Atg16l1-deficient MEF cells or ATG16L1 knockdown SH-SY5Y cells, indicating that the autophagy-based unconventional secretory pathway is involved in PARK7 secretion. We moreover observed that 6-OHDA-derived electrophilic quinone induced oxidative stress as indicated by a decrease in glutathione levels, and that this was suppressed by pretreatment with antioxidant NAC. We further found that NAC treatment suppressed autophagy and PARK7 secretion. We also observed that 6-OHDA-induced autophagy was associated with activation of AMPK and ULK1 via a pathway which was independent of MTOR. Collectively these results suggest that electrophilic 6-OHDA quinone enhances oxidative stress, and that this is followed by AMPK-ULK1 pathway activation and induction of secretory autophagy to produce unconventional secretion of PARK7.

Rabbit anti-thymocyte globulin (ATG (Thymoglobulin)) kills T cells in vitro and probably also in vivo as it prevents graft-vs-host disease (GvHD) in patients. Recently we demonstrated that ATG at a clinically relevant concentration (10-50 mg/L) kills in vitro not only T cells but also leukemic blasts. In the present study, we investigated whether ATG kills not only leukemic blasts but also leukemic stem cells (LSCs). We used a flow cytometric assay of complement-mediated cytotoxicity (CDC). ATG-induced death of acute myeloid leukemia (AML) cells from patients newly diagnosed with AML was measured among blasts as well as LSCs. At 10 mg/L ATG, blasts but not LSCs were killed. At 50 mg/L ATG, both blasts and LSCs were killed. We also measured ATG-mediated killing of healthy individuals' hematopoietic stem cells (HSCs). Median 2% HSCs from blood and 15% HSCs from filgrastim-mobilized grafts were killed with 50 mg/L ATG, compared to 30% LSCs from the blood of AML patients (p = 0.001 and 0.022, respectively). In conclusion, LSCs are sensitive to ATG, however, only at a relatively high ATG concentration. At that concentration, LSCs are killed to a higher degree than HSCs.

Chronic subdural hematoma (CSDH) is fundamentally treatable, although it sometimes recurs. We observed, however, several cases of spontaneous resolution of CSDH outer membranes, even in a trabecular type of CSDH, after a trepanation surgical procedure. In this study, we examined the expression of molecules of the autophagy signaling pathway in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation were included in this study. By Western blot analysis, we examined the expression of mammalian target of rapamycin (mTOR); GβL; UNC-51-like kinase-1 (ULK1); Beclin-1; autophagy-related genes (Atg) 3, 5, 7, 12, 13, and 16L1β,α; the autophagy marker Light Chain3A/B (LC3A/B); and β-actin, which constitute the autophagy signaling pathway. The expression levels of Beclin-1, Atg12, and LC3A/B were also examined by immunohistochemistry. Almost all of these molecules could be detected in all samples. Beclin-1, Atg12, and LC3A/B were found to be localized in the endothelial cells of vessels and fibroblasts in CSDH. We detected molecules of the autophagy signaling pathway in CSDH outer membranes. Autophagy contributes to the tissue homeostatic process, maintaining cellular integrity by clearing debris. Our data suggest that autophagy might play an important role in the spontaneous resolution of CSDH. Therefore, these molecules may be novel therapeutic targets for the treatment of those with CSDH.

Simple sequence repeats (SSRs) or microsatellite markers derived from expressed sequence tags (ESTs) are routinely used for molecular assisted-selection breeding, comparative genomic analysis, and genetic diversity studies. In this study, we investigated 54,546 ESTs for the identification and development of SSR markers in Pogostemon cablin (Patchouli). In total, 1219 SSRs were identified from 1144 SSR-containing ESTs. Trinucleotides (80.8%) were the most abundant SSRs, followed by di- (10.8%), mono- (7.1%), and hexa-nucleotides (1.3%). The top six motifs were CCG/CGG (15.3%), AAG/CTT (15.0%), ACC/GGT (13.5%), AGG/CCT (12.4%), ATC/ATG (9.9%), and AG/CT (9.8%). On the basis of these SSR-containing ESTs, a total of 192 primer pairs were randomly designed and used for polymorphism analysis in 38 accessions collected from different geographical regions of Guangdong, China. Of the SSR markers, 45 were polymorphic and had allele variations from two to four. Furthermore, a transferability analysis of these primer pairs revealed a 10⁻40% cross-species transferability in 10 related species. This report is the first comprehensive study on the development and analysis of a large set of SSR markers in P. cablin. These markers have the potential to be used in quantitative trait loci mapping, genetic diversity studies, and the fingerprinting of cultivars of P. cablin.