The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Retracted: Intra‐arterial transplantation of human bone marrow mesenchymal stem cells (hBMMSCs) improves behavioral deficits and alters gene expression in rodent stroke model

Stroke is a multi‐factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 10⁶ human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q‐PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation‐specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper‐methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo‐methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.

     

Senescence‐associated superoxide dismutase influences mitochondrial gene expression in budding tunicates

A recent study has shown that in the budding tunicate Polyandrocarpa misakiensis, the mitochondrial respiratory chain (MRC) dramatically attenuates the gene activity during senescence. In this study, we examined the possible involvement of superoxide dismutase (SOD) in the attenuation of gene expression of cytochrome c oxidase subunit 1 (COX1) in aged zooids. By RT‐PCR and in situ hybridization, Cu/Zn‐SOD (SOD1) was found to be expressed in most cells and tissues of buds and juvenile zooids but showed a conspicuous decline in senescent adult zooids, except in the gonad tissue in which the cytoplasm of juvenile oocytes was stained heavily. This expression pattern of SOD1 was similar to that of COX1. In contrast to SOD1, Mn‐SOD (SOD2) was expressed constitutively in both somatic and germline tissues of buds, juvenile zooids, and senescent adult zooids. Knockdown of SOD1 by RNAi diminished the gene activity of not only SOD1 but also of COX1. The resultant zooids had transient deficiencies in growth and budding, and they recovered from these deficiencies approximately 1 month later. Our results indicate that in P. misakiensis, SOD1 is a senescence‐associated nuclear gene and that the experimental decline in SOD1 gene expression accompanies the attenuation of MRC gene activity. Although it is uncertain how SOD1 is downregulated during tunicate senescence, the decreased SOD1 activity could be one of the main causes of MRC gene attenuation during normal senescence.     

ICAM‐1 and MKL‐1 polymorphisms impose considerable impacts on coronary heart disease occurrence

This study was aimed to explore the correlation of intercellular adhesion molecule‐1 (ICAM‐1) K469E and megakaryoblastic leukaemia factor‐1 (MKL‐1) ?184C/T polymorphisms with the susceptibility to coronary heart disease (CHD) in the Chinese Han population. 100 CHD patients and 91 healthy people that had no blood connection with each other were enrolled in this case‐control study. ICAM‐1 and MKL‐1 polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) approach. Multiple logistic regression was used to analyse the correlation between polymorphisms of ICAM‐1 and MKL‐1 and CHD susceptibility. Differences of genotype and allele frequencies of the two SNPs between case and control groups were analysed by chi‐square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were indicated relative susceptibility of CHD. The distributions of ICAM‐1 and MKL‐1 polymorphisms in each group conformed to Hardy‐Weinberg equilibrium (HWE). After adjusting for traditional risk factors, the TT genotype frequency of MKL‐1 ?184C/T polymorphism was found significantly higher in case group than in control group (P < .05). Meanwhile, T allele frequency increased in case group compared with control group, and the differences had statistical significance (P = .04, OR = 2.34, 95% CI = 1.34‐5.26). Logistic regression analysis in this study proved that smoking, hypertension, diabetes and triglyceride (TG) were all risk factors for CHD ICAM‐1 K469E polymorphism has no association with the onset of CHD. But MKL‐1 ?184C/T polymorphism is associated with the risk of CHD and T allele might be a susceptibility factor for CHD.     

Analysis of ROH patterns in the Noriker horse breed reveals signatures of selection for coat color and body size

Overlapping runs of homozygosity (ROH islands) shared by the majority of a population are hypothesized to be the result of selection around a target locus. In this study we investigated the impact of selection for coat color within the Noriker horse on autozygosity and ROH patterns. We analyzed overlapping homozygous regions (ROH islands) for gene content in fragments shared by more than 50% of horses. Long‐term assortative mating of chestnut horses and the small effective population size of leopard spotted and tobiano horses resulted in higher mean genome‐wide ROH coverage (S_ROH) within the range of 237.4–284.2 Mb, whereas for bay, black and roan horses, where rotation mating is commonly applied, lower autozygosity (S_ROH from 176.4–180.0 Mb) was determined. We identified seven common ROH islands considering all Noriker horses from our dataset. Specific islands were documented for chestnut, leopard spotted, roan and bay horses. The ROH islands contained, among others, genes associated with body size (ZFAT, LASP1 and LCORL/NCAPG), coat color (MC1R in chestnut and the factor PATN1 in leopard spotted horses) and morphogenesis (HOXB cluster in all color strains except leopard spotted horses). This study demonstrates that within a closed population sharing the same founders and ancestors, selection on a single phenotypic trait, in this case coat color, can result in genetic fragmentation affecting levels of autozygosity and distribution of ROH islands and enclosed gene content.     

Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation‐mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N‐terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1‐PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1‐PP1 protects the origin‐binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1‐depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1‐targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.     

A metabolic signature for long life in the Caenorhabditis elegans Mit mutants

Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial‐derived α‐ketoacids and α‐hydroxyacids that are produced by long‐lived Mit mutants but not by other long‐lived mutants or by short‐lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α‐ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild‐type animals was reduced using RNA interference we observed an unprecedented effect on lifespan – as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype.     

Development of the pancreas in medaka,Oryzias latipes,from embryo to adult

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1‐ and ptf1a‐transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior–posterior axis.     

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down‐regulated by alcohol in cell lines and animal models. This down‐regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real‐time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down‐regulation of hepcidin expression leading to up‐regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.     

Novel three‐way complex rearrangement of TRPM1‐PUM1‐LCK in a case of agminated Spitz nevi arising in a giant congenital hyperpigmented macule

The genetic anomalies associated with the agminated variant of Spitz nevus have so far been limited to HRAS G13R mutations, especially when arising within a nevus spilus. A previous report exposed the case of a man with a giant pigmented macule involving his upper right limb and trunk. Since childhood, Spitz nevi have been periodically arising, within the pigmented area. The histopathology of several lesions displayed the usual criteria of junctional, compound, or intradermal Spitz nevi with a diversity of cytomorphological and architectural features. Some lesions spontaneously regressed. Genetic studies confirmed in three lesions an identical translocation involving TRPM1, PUM1, and LCK. No mutations in HRAS, NRAS, BRAF, or other known fusion genes linked to Spitz nevus were detected. LCK break‐apart fluorescence in situ hybridization confirmed the rearrangement was present not only in the melanocytic proliferation but also in the surrounding non‐spitzoid melanocytes. This report expands the list of genetic alterations involved both in giant congenital macules and in agminated Spitz nevi, and also extends the concept of mosaicism in melanocytes to gene translocations.     

INDUCER OF CBF EXPRESSION 1 integrates cold signals into FLOWERING LOCUS C‐mediated flowering pathways in Arabidopsis

Plants constantly monitor changes in photoperiod and temperature throughout the year to synchronize flowering with optimal environmental conditions. In the temperate zones, both photoperiod and temperature fluctuate in a somewhat predictable manner through the seasons, although a transient shift to low temperature is also encountered during changing seasons, such as early spring. Although low temperatures are known to delay flowering by inducing the floral repressor FLOWERING LOCUS C (FLC), it is not fully understood how temperature signals are coordinated with photoperiodic signals in the timing of seasonal flowering. Here, we show that the cold signaling activator INDUCER OF CBF EXPRESSION 1 (ICE1), FLC and the floral promoter SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) constitute an elaborate signaling network that integrates cold signals into flowering pathways. The cold‐activated ICE1 directly induces the gene encoding FLC, which represses SOC1 expression, resulting in delayed flowering. In contrast, under floral promotive conditions, SOC1 inhibits the binding of ICE1 to the promoters of the FLC gene, inducing flowering with a reduction of freezing tolerance. These observations indicate that the ICE1‐FLC‐SOC1 signaling network contributes to the fine‐tuning of flowering during changing seasons.     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

The aging biological clock in Neurospora crassa

The biological clock affects aging through ras‐1 (bd) and lag‐1, and these two longevity genes together affect a clock phenotype and the clock oscillator in Neurospora crassa. Using an automated cell‐counting technique for measuring conidial longevity, we show that the clock‐associated genes lag‐1 and ras‐1 (bd) are true chronological longevity genes. For example, wild type (WT) has an estimated median life span of 24 days, while the double mutant lag‐1, ras‐1 (bd) has an estimated median life span of 120 days for macroconidia. We establish the biochemical function of lag‐1 by complementing LAG1 and LAC1 in Saccharomyces cerevisiae with lag‐1 in N. crassa. Longevity genes can affect the clock as well in that, the double mutant lag‐1, ras‐1 (bd) can stop the circadian rhythm in asexual reproduction (i.e., banding in race tubes) and lengthen the period of the frequency oscillator to 41 h. In contrast to the ras‐1 (bd), lag‐1 effects on chronological longevity, we find that this double mutant undergoes replicative senescence (i.e., the loss of replication function with time), unlike WT or the single mutants, lag‐1 and ras‐1 (bd). These results support the hypothesis that sphingolipid metabolism links aging and the biological clock through a common stress response     

Bacillus subtilis QST 713 Confers Protection to Tomato Plants Against Pseudomonas syringae pv tomato and Induces Plant Defence‐related Genes

Bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is an economically important disease of tomato, resulting in yield loss of marketable fruit. Management of bacterial speck is a challenge in commercial production fields due to the limited efficacy of current disease management strategies, as the pathogen acquires resistance to antibiotics and fixed copper bactericides and host resistance has not proven durable. Therefore, it is essential to develop alternative disease management strategies, like biological control. In this study, the efficacy of the commercially available biocontrol agent Bacillus subtilis QST 713 along with copper hydroxide was tested against Pst under greenhouse conditions. QST 713 reduced significantly disease severity and incidence compared to control and the copper hydroxide treatment; subsequently, the Pst population was lower in the QST 713‐treated plants compared to control. In parallel, QST 713 and copper hydroxide increased plant height compared to control and mock plants. Furthermore, the quantitative PCR analysis of PR1a, PR1b and Pin2 expression suggests a positive role for Pin2 in the plant protective activity of QST 713, as Pin2 expression was significantly higher in the QST 713‐treated plants challenged with Pst compared to the control Pst‐inoculated plants.     

Clinical and pathological associations of the activating RAC1 P29S mutation in primary cutaneous melanoma

Activating mutations in the GTPase RAC1 are a recurrent event in cutaneous melanoma. We investigated the clinical and pathological associations of RAC1^P29S in a cohort of 814 primary cutaneous melanomas with known BRAF and NRAS mutation status. The RAC1^P29S mutation had a prevalence of 3.3% and was associated with increased thickness (OR=1.6 P = 0.001), increased mitotic rate (OR=1.3 P = 0.03), ulceration (OR=2.4 P = 0.04), nodular subtype (OR=3.4 P = 0.004), and nodal disease at diagnosis (OR=3.3 P = 0.006). BRAF mutant tumors were also associated with nodal metastases (OR=1.9 P = 0.004), despite being thinner at diagnosis than BRAF WT (median 1.2 mm versus 1.6 mm, P < 0.001). Immunohistochemical analysis of 51 melanomas revealed that 47% were immunoreactive for RAC1. Melanomas were more likely to show RAC1 immunoreactivity if they were BRAF mutant (OR=5.2 P = 0.01). RAC1 may therefore be important in regulating the early migration of BRAF mutant tumors. RAC1 mutations are infrequent in primary melanomas but may accelerate disease progression.