Leucine-Rich Repeat Kinase 1 Regulates Autophagy through Turning On TBC1D2-Dependent Rab7 Inactivation

Autophagy is a conserved process that enables catabolic and degradative pathways. Rab family proteins, which are active in the GTP-bound form, regulate the transport and fusion of autophagosomes. However, it remains unclear how each cycle of Rab activation and inactivation is precisely regulated. Here, we show that leucine-rich repeat kinase 1 (LRRK1) regulates autophagic flux by controlling Rab7 activity in autolysosome formation. Upon induction of autophagy, LRRK1 was recruited via an association with VAMP7 to the autolysosome, where it activated the Rab7 GTPase-activating protein (GAP) TBC1D2, thereby switching off Rab7 signaling. Consistent with this model, LRRK1 deletion caused mice to be vulnerable to starvation and disrupted autolysosome formation, as evidenced by the accumulation of enlarged autolysosomes with undegraded LC3-II and persistently high levels of Rab7-GTP. This defect in autophagic flux was partially rescued by a mutant form of TBC1D2 with elevated Rab7-GAP activity. Thus, the spatiotemporal regulation of Rab7 activity during tunicamycin-induced autophagy is regulated by LRRK1.     

Genomic analysis identifies candidate pathogenic variants in 9 of 18 patients with unexplained West syndrome

West syndrome, which is narrowly defined as infantile spasms that occur in clusters and hypsarrhythmia on EEG, is the most common early-onset epileptic encephalopathy (EOEE). Patients with West syndrome may have clear etiologies, including perinatal events, infections, gross chromosomal abnormalities, or cases followed by other EOEEs. However, the genetic etiology of most cases of West syndrome remains unexplained. DNA from 18 patients with unexplained West syndrome was subjected to microarray-based comparative genomic hybridization (array CGH), followed by trio-based whole-exome sequencing in 14 unsolved families. We identified candidate pathogenic variants in 50 % of the patients (n = 9/18). The array CGH revealed candidate pathogenic copy number variations in four cases (22 %, 4/18), including an Xq28 duplication, a 16p11.2 deletion, a 16p13.1 deletion and a 19p13.2 deletion disrupting CACNA1A. Whole-exome sequencing identified candidate mutations in known epilepsy genes in five cases (36 %, 5/14). Three candidate de novo mutations were identified in three cases, with two mutations occurring in two new candidate genes (NR2F1 and CACNA2D1) (21 %, 3/14). Hemizygous candidate mutations in ALG13 and BRWD3 were identified in the other two cases (14 %, 2/14). Evaluating a panel of 67 known EOEE genes failed to identify significant mutations. Despite the heterogeneity of unexplained West syndrome, the combination of array CGH and whole-exome sequencing is an effective means of evaluating the genetic background in unexplained West syndrome. We provide additional evidence for NR2F1 as a causative gene and for CACNA2D1 and BRWD3 as candidate genes for West syndrome.     

Xmrk‐induced melanoma progression is affected by Sdf1 signals through Cxcr7

Chemokine signals mediated by Sdf1/Cxcl12 through the chemokine receptor Cxcr4 are thought to play an instructive role in tumor migration and organ‐specific metastasis. We have used a small aquarium fish model to contribute to a better understanding of how the course of melanoma development is influenced by Sdf1 signals in vivo. We studied oncogene‐induced skin tumor appearance and progression in the transgenic medaka (Oryzias latipes) melanoma model. Similar to humans, invasive medaka melanomas show increased levels of sdf1, cxcr4, and cxcr7 gene expression. Stable transgenic fish lines overexpressing sdf1 exclusively in pigment cells showed a reduction in melanoma appearance and progression. Remarkably, diminished levels of functional Cxcr7, but not of Cxcr4b, resulted in strongly reduced melanoma invasiveness and a repression of melanoma. Our results thereby indicate that Sdf1 signals via Cxcr7 are able to constrain melanoma growth in vivo and that these signals influence tumor outcome.     

Worldwide core collections of tea (Camellia sinensis) based on SSR markers

Tea (Camellia sinensis (L.) O. Kuntze) is the world’s most popular beverage crop. However, to date, no core collection has been selected from worldwide germplasm resources on the basis of genotype data. In this study, we analyzed 788 tea germplasm accessions using 23 simple sequence repeat (SSR) markers. Our population structure analysis divided the germplasms into a Japanese group and an exotic group. The latter could be divided into var. sinensis and var. assamica. The genetic diversity was higher in germplasms from China, Taiwan, India, and Sri Lanka than in those from other countries, and low in germplasms from Japan. Using the number of SSR alleles as a measure of genetic diversity, we developed a core collection consisting of 192 accessions and three subcore collections with 96, 48, and 24 accessions. Although the results might be affected by marker-selection bias, the core 192 collection adequately covered the range of variation of the 788 accessions in floral morphology, and the chemical composition of first-flush leaves. These collections will be powerful tools for breeding and genetic research in tea.     

Ghrelin Protects against Renal Damages Induced by Angiotensin-II via an Antioxidative Stress Mechanism in Mice

We explored the renal protective effects by a gut peptide, Ghrelin. Daily peritoneal injection with Ghrelin ameliorated renal damages in continuously angiotensin II (AngII)-infused C57BL/6 mice as assessed by urinary excretion of protein and renal tubular markers. AngII-induced increase in reactive oxygen species (ROS) levels and senescent changes were attenuated by Ghrelin. Ghrelin also inhibited AngII-induced upregulations of transforming growth factor-β (TGF-β) and plasminogen activator inhibitor-1 (PAI-1), ameliorating renal fibrotic changes. These effects were accompanied by concomitant increase in mitochondria uncoupling protein, UCP2 as well as in a key regulator of mitochondria biosynthesis, PGC1α. In renal proximal cell line, HK-2 cells, Ghrelin reduced mitochondria membrane potential and mitochondria-derived ROS. The transfection of UCP2 siRNA abolished the decrease in mitochondria-derived ROS by Ghrelin. Ghrelin ameliorated AngII-induced renal tubular cell senescent changes and AngII-induced TGF-β and PAI-1 expressions. Finally, Ghrelin receptor, growth hormone secretagogue receptor (GHSR)-null mice exhibited an increase in tubular damages, renal ROS levels, renal senescent changes and fibrosis complicated with renal dysfunction. GHSR-null mice harbored elongated mitochondria in the proximal tubules. In conclusion, Ghrelin suppressed AngII-induced renal damages through its UCP2 dependent anti-oxidative stress effect and mitochondria maintenance. Ghrelin/GHSR pathway played an important role in the maintenance of ROS levels in the kidney.     

System Vaccinology for the Evaluation of Influenza Vaccine Safety by Multiplex Gene Detection of Novel Biomarkers in a Preclinical Study and Batch Release Test

Vaccines are beneficial and universal tools to prevent infectious disease. Thus, safety of vaccines is strictly evaluated in the preclinical phase of trials and every vaccine batch must be tested by the National Control Laboratories according to the guidelines published by each country. Despite many vaccine production platforms and methods, animal testing for safety evaluation is unchanged thus far. We recently developed a systems biological approach to vaccine safety evaluation where identification of specific biomarkers in a rat pre-clinical study evaluated the safety of vaccines for pandemic H5N1 influenza including Irf7, Lgals9, Lgalsbp3, Cxcl11, Timp1, Tap2, Psmb9, Psme1, Tapbp, C2, Csf1, Mx2, Zbp1, Ifrd1, Trafd1, Cxcl9, β2m, Npc1, Ngfr and Ifi47. The current study evaluated whether these 20 biomarkers could evaluate the safety, batch-to-batch and manufacturer-to-manufacturer consistency of seasonal trivalent influenza vaccine using a multiplex gene detection system. When we evaluated the influenza HA vaccine (HAv) from four different manufactures, the biomarker analysis correlated to findings from conventional animal use tests, such as abnormal toxicity test. In addition, sensitivity of toxicity detection and differences in HAvs were higher and more accurate than with conventional methods. Despite a slight decrease in body weight caused by HAv from manufacturer B that was not statistically significant, our results suggest that HAv from manufacturer B is significantly different than the other HAvs tested with regard to Lgals3bp, Tapbp, Lgals9, Irf7 and C2 gene expression in rat lungs. Using the biomarkers confirmed in this study, we predicted batch-to-batch consistency and safety of influenza vaccines within 2 days compared with the conventional safety test, which takes longer. These biomarkers will facilitate the future development of new influenza vaccines and provide an opportunity to develop in vitro methods of evaluating batch-to-batch consistency and vaccine safety as an alternative to animal testing.     

Structural Property of DNA That Migrates Faster in Gel Electrophoresis,as Deduced by CD Spectroscopy

Bent DNAs are known to migrate slower than ordinary DNA in non-denaturing polyacrylamide gel electrophoresis. In contrast, several satellite DNAs have been shown to migrate fast. The structural property that causes the fast migration, however, is not clarified so far on molecular basis. We have investigated the structural property of a satellite DNA, which contains consecutive purine sequences and migrates faster in gel, by CD spectroscopy. Partial formation of an A-form–like structure has been suggested. Reduction in DNA length due to the formation of the A-form–like structure may be responsible for the fast migration. The pronounced rigidity of DNA may also contribute to the behavior.     

TRIM5 alpha Drives SIVsmm Evolution in Rhesus Macaques

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5^Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5^TFP/TFP or TRIM5^Cyp/TFP. Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5^TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5^Cyp. Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIM^TFP alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.     

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.