Combining C-H functionalisation and flow photochemical heterocyclic metamorphosis (FP-HM) for the synthesis of benzo[1,3]oxazepines

C-H Activation/functionalisation and Flow Photochemical Heterocyclic Metamorphosis (FP-HM) have been combined to synthesize a library of benzo[1,3]oxazepines, a rarely described heterocyclic family. This combined protocol allows a range of arylated products to be made from simple starting materials, and the cheap flow photochemical system has proven effective for rapid synthesis of gram-quantities of benzo[1,3]oxazepines

The Homeodomain Protein Defective Proventriculus Is Essential for Male Accessory Gland Development to Enhance Fecundity in Drosophila

The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. We provide the first evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity

Starvation Induced Cell Death in Autophagy-Defective Yeast Mutants Is Caused by Mitochondria Dysfunction

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants

Regulation of the autophagy protein LC3 by phosphorylation

PKA puts the brakes on autophagy by inhibiting LC3 recruitment to autophagosomes

Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels

Reoperations after first lumbar disc herniation surgery; a special interest on residives during a 5-year follow-up

BACKGROUND: The overall rate of operations after recurrent lumbar disc herniation has been shown to be 3–11%. However, little is known about the rate of residives. Thus the aim of this study was to explore the cumulative rates of re-operations and especially residive disc herniations at the same side and level as the primary disc herniation after first lumbar disc herniation surgery and the factors that influence the risk of re-operations over a five year follow-up study. METHODS: 166 virgin lumbar disc herniation patients (mean age 42 years, 57% males) were studied. Data on patients' initial disc operations and type and timing of re-operations during the follow-up were collected from patient files. Back and leg pain on visual analog scale and employment status were collected by questionnaires. RESULTS: The cumulative rate of re-operations for lumbar disc herniation was 10.2% (95% Cl 6.0 to 15.1). The rate of residives at initial site was 7.4% (95% Cl 3.7 to 11.3) and rate of lumbar disc herniations at other sites was 3.1% (95% Cl 0.6 to 6.2). The occurrence of residive lumbar disc herniations was evenly distributed across the 5 years. Neither age, gender, preoperative symptoms, physical activity nor employment had effect on the probability of re-operation. CONCLUSION: Seven percent of the lumbar disc patients had a residive lumbar disc operation within five years of their first operation. No specific factors influencing the risk for re-operation were found

Computed tomographic analysis of the quality of trunk muscles in asymptomatic and symptomatic lumbar discectomy patients

Background: No consensus exists on how rehabilitation programs for lumbar discectomy patients with persistent complaints after surgery should be composed. A better understanding of normal and abnormal postoperative trunk muscle condition might help direct the treatment goals. Methods: A three-dimensional CT scan of the lumbar spine was obtained in 18 symptomatic and 18 asymptomatic patients who had undergone a lumbar discectomy 42 months to 83 months (median 63 months) previously. The psoas muscle (PS), the paraspinal muscle mass (PA) and the multifidus muscle (MF) were outlined at the L3, L4 and L5 level. Of these muscles, fat free Cross Sectional Area (CSA) and fat CSA were determined. CSA of the lumbar erector spinae (LES = longissimus thoracis + iliocostalis lumborum) was calculated by subtracting MF CSA from PA CSA. Mean muscle CSA of the left and right sides was calculated at each level. To normalize the data for interpersonal comparison, the mean CSA was divided by the CSA of the L3 vertebral body (mCSA = normalized fat-free muscle CSA; fCSA = normalized fat CSA). Differences in CSA between the pain group and the pain free group were examined using a General Linear Model (GLM). Three levels were examined to investigate the possible role of the level of operation. Results: In lumbar discectomy patients with pain, the mCSA of the MF was significantly smaller than in pain-free subjects (p = 0.009) independently of the level. The mCSA of the LES was significantly smaller in pain patients, but only on the L3 slice (p = 0.018). No significant difference in mCSA of the PS was found between pain patients and pain-free patients (p = 0.462). The fCSA of the MF (p = 0.186) and of the LES (p = 0.256) were not significantly different between both populations. However, the fCSA of the PS was significantly larger in pain patients than in pain-free patients. (p = 0.012). The level of operation was never a significant factor. Conclusions: CT comparison of MF, LES and PS muscle condition between lumbar discectomy patients without pain and patients with protracted postoperative pain showed a smaller fat-free muscle CSA of the MF at all levels examined, a smaller fat-free muscle CSA of the LES at the L3 level, and more fat in the PS in patients with pain. The level of operation was not found to be of importance. The present results suggest a general lumbar muscle dysfunction in the pain group, in particular of the deep stabilizing muscle system

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general

Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation

Arabidopsis thaliana phosphatidylinositol 3-kinase (AtVPS34) functions in the development and germination of pollen by catalyzing the biosynthesis of phosphatidylinositol 3-phosphate (PI3P). In yeast, Vps15p is required for the membrane targeting and activity of Vps34. The expression of Arabidopsis thaliana VPS15 (AtVPS15), an ortholog of yeast Vps15, is mainly detected in pollen grains and pollen tubes. To determine its role in pollen development and pollen tube growth, we attempted to isolate the T-DNA insertion mutants of AtVPS15; however, homozygous lines of atvps15 were not obtained from the progeny of atvps15/+ heterozygotes. Genetic analysis revealed that the abnormal segregation is due to the failure of transmission of the atvps15 allele through pollen. Most pollen grains from the atvps15/+ genotype are viable, with normal exine structure and nuclei, but some mature pollen grains are characterized with unusual large vacuoles that are not observed in pollen grains from the wild AtVPS15 genotype. The germination ratio of pollen from the atvps15/+ genotype is about half when compared to that from the wild AtVPS15 genotype. When supplied with PI3P, in vitro pollen germination of the atvps15/+ genotype is greatly improved. Presumably, AtVPS15 functions in pollen development and germination by regulating PI3P biosynthesis in Arabidopsis

Heterologous Expression of ATG8c from Soybean Confers Tolerance to Nitrogen Deficiency and Increases Yield in Arabidopsis

Nitrogen is an essential element for plant growth and yield. Improving Nitrogen Use Efficiency (NUE) of crops could potentially reduce the application of chemical fertilizer and alleviate environmental damage. To identify new NUE genes is therefore an important task in molecular breeding. Macroautophagy (autophagy) is an intracellular process in which damaged or obsolete cytoplasmic components are encapsulated in double membraned vesicles termed autophagosomes, then delivered to the vacuole for degradation and nutrient recycling. One of the core components of autophagosome formation, ATG8, has been shown to directly mediate autophagosome expansion, and the transcript of which is highly inducible upon starvation. Therefore, we postulated that certain homologs of Saccharomyces cerevisiae ATG8 (ScATG8) from crop species could have potential for NUE crop breeding. A soybean (Glycine max, cv. Zhonghuang-13) ATG8, GmATG8c, was selected from the 11 family members based on transcript analysis upon nitrogen deprivation. GmATG8c could partially complement the yeast atg8 mutant. Constitutive expression of GmATG8c in soybean callus cells not only enhanced nitrogen starvation tolerance of the cells but accelerated the growth of the calli. Transgenic Arabidopsis over-expressing GmATG8c performed better under extended nitrogen and carbon starvation conditions. Meanwhile, under optimum growth conditions, the transgenic plants grew faster, bolted earlier, produced larger primary and axillary inflorescences, eventually produced more seeds than the wild-type. In average, the yield was improved by 12.9%. We conclude that GmATG8c may serve as an excellent candidate for breeding crops with enhanced NUE and better yield