The role of anti-VEGF treatment for ROP is still being evaluated. Although the shorter half-life of ranibizumab makes it an attractive option, reactivation of ROP is possible. Physicians and families should be aware of this to follow infants closely for an extended period of time.

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Culturing rat islets in high glucose (HG) increased 1-(14)C-alpha-ketoisocaproate (KIC) oxidation compared with culturing them in low glucose. Leucine caused insulin secretion (IS) in low glucose but not in HG rat islets, whereas KIC did so in both. Pretreatment with HG for 40 min abolished leucine stimulation of IS by mouse islets and prevented the cytosolic Ca(2+) rise without inhibiting IS and Ca(2+) increments caused by KIC. When islets were pretreated without glucose and glutamine, aminooxyacetic acid (AOA) markedly decreased KIC effects. When islets were pretreated without glucose and with glutamine, AOA potentiated leucine effects but attenuated KIC effects. AOA stimulated glutamine oxidation in the presence but not the absence of +/-2-amino-2-norbornane-carboxylic acid, a nonmetabolized leucine analog. Pretreatment with HG and glutamine partially reversed AOA inhibition of KIC effects. Glucose increased intracellular ATP and GTP, whereas it decreased ADP and GDP in beta HC9 cells. Glutamate dehydrogenase activity of beta HC9 cell extracts was increased by leucine and attenuated by GTP, but it was potentiated by ADP. In conclusion, leucine and KIC stimulated beta-cells via distinct mechanisms. Glutamate dehydrogenase is the sensor of leucine, whereas transamination plays an important role in KIC stimulation of pancreatic beta-cells.

Background Circulating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues. Results Different RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2–3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation. Conclusions Small RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification. Electronic supplementary material The online version of this article (10.1186/s12864-019-5826-7) contains supplementary material, which is available to authorized users.

Alzheimer's disease (AD) is the fourth leading cause of death in adults, characterized by hallmark neuritic plaques and neurofibrillary tangles. Current treatments focus only on symptom relief. As a possible new treatment option for AD, huperzine A's chemistry, pharmacology, and clinical effectiveness are assessed. The chemical synthesis of huperzine A has been optimized, while an in vitro technique has provided a renewable plant source. Pharmacological studies showed that the drug inhibits the enzyme acetylcholinesterase reversibly and selectively. Huperzine A also displayed good pharmacokinetics with a rapid absorption and a wide distribution in the body at a low to moderate rate of elimination. Presently, inadequate toxicity data in human have been reported, yet animal studies demonstrated mild to moderate cholinergic side effects at therapeutic doses. Previous clinical trials have shown improvement in memory function using MMSE, MQ, ADAS-COG, and ADL tests. In an unpublished phase II clinical trial, the ADAS-COG and MMSE tests indicated cognitive enhancement at a dose of 0.4 mg, yet no improvement was observed at a dose of 0.2 mg. The MMSE scores indicated cognitive enhancement at 0.4 mg. Promising data suggested that huperzine A is well tolerated at doses up to 0.4 mg for 24 weeks. Therefore, huperzine A seems to be a potential treatment option for AD.

We have recently shown that leucine culture upregulates ATP synthase ␤-subunit (ATPS␤) and increases ATP level, cytosolic Ca 2؉ , and glucose-induced insulin secretion in rat islets. The aim is to test whether glucokinase expression is also affected in rat islets and its role in glucose sensitization during leucine culture. Leucine culture increased glucose-induced NAD(P)H level at 1 and 2 days but not at 1 week. The half-maximal effective concentration of the glucose response curve for NAD(P)H was left-shifted from 5-7 to 2-3 mmol/l. The effect was dose dependent and rapamycin insensitive. Leucine culture did not affect glyceraldehyde effects on NAD(P)H. Leucine pretreatment for 30 min had no effects on NAD(P)H levels. Leucine culture for 2 days also increased glucose-induced cytosolic Ca 2؉ elevation, ATP level, and insulin secretion. Leucine increase of glucokinase mRNA levels occurred as early as day 1 and lasted through 1 week. That of ATPS␤ did not occur until day 2 and lasted through 1 week. Leucine effects on both mRNAs were dose dependent. The upregulation of both genes was confirmed by Western blotting. Leucine culture also increased glucose-induced insulin secretion, ATP level, glucokinase, and ATPS␤ levels of type 2 diabetic human islets. In conclusion, leucine culture upregulates glucokinase, which increases NAD(P)H level, and ATPS␤, which increases oxidation of NADH and production of ATP. The combined upregulation of both genes increases glucoseinduced cytosolic Ca 2؉ and insulin secretion. ] (6 -8). As the rate-limiting enzyme of glycolysis, it is believed that glucokinase sets a strict control on glucose metabolism in pancreatic ␤-cells. However, overexpression of glucokinase or hexokinase I fails to increase the maximal insulin output induced by glucose, although it decreases the threshold for glucose-induced insulin secretion in pancreatic ␤-cells (9 -12). In pancreatic ␤-cells, most of the intracellular ATP comes from the oxidation of glucosederived pyruvate and oxidation of NADH in the mitochondria via the electron transport chain. Alternations in ATP synthesis results in changes of ␤-cell function and glucosestimulated insulin secretion (13,14). Mitochondrial metabolism may set the maximal limit of fuel-stimulated insulin secretion in ␤-cells by controlling the rate of ATP production (10). Inhibition of mitochondrial gene transcription, and presumably oxidation of glucose-derived pyruvate in the mitochondria, is found to suppress glucose-induced insulin secretion in ␤HC9 cells (15,16). Thus, glucokinase may not be the only rate-limiting step in glucose-induced insulin secretion of pancreatic ␤-cells. Other factor(s) may also be rate-limiting and contribute to the tight control of glucose-induced insulin secretion. As the key enzyme catalyzing the conversion of ADP and inorganic phosphate (Pi) to ATP by proton gradient in the electron transport chain, ATP synthase (complex V) may play an important role in ATP synthesis and hence glucose-induced insulin secretion. It had been established that ...

Our goal was to investigate whether leucine culture affects ␤-cell glucose sensing. One-day culture of rat islets with 10 mM leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mM glucose. Glucose-induced cytosolic free Ca 2؉ was increased at 1 week but not at 1 day of leucine culture. Glucose is the main secretagogue of insulin secretion from pancreatic ␤-cells. The mechanisms of glucose-induced insulin secretion have been studied extensively. Through glycolysis and oxidation, glucose increases pancreatic ␤-cell ATP/ADP ratio, which closes ATP-sensitive potassium (K ATP ) channels and depolarizes the cell membrane. This results in an influx of extracellular Ca 2ϩ and increase of free cytosolic [Ca 2ϩ ] that stimulates exocytosis of insulin granules (1-5). Another mechanism is K ATP channel-independent and involves increased effectiveness of [Ca 2ϩ ] (6, 7). In pancreatic ␤-cells, most of the intracellular ATP comes from the oxidation of glucose-derived pyruvate and oxidation of NADH in the mitochondria via the electron transport chain. Damage or inhibition of ATP synthesis results in ␤-cell dysfunction and impairs glucose-stimulated insulin secretion (8, 9). Some recent publications indicate that superoxide produced by hyperglycemia activates uncouplingprotein-2 (UCP2) and destroys the proton gradient between inner and outer mitochondrial membranes. This negatively affects the activity of ATP synthase, decreases ATP production, and impairs glucose-stimulated insulin secretion of pancreatic ␤-cells resulting in diabetes (8, 9). More recently, it is shown that mitochondrial metabolism sets the maximal limit of fuel stimulated-insulin secretion in ␤-cells (10). These findings imply that mitochondrial ATP synthesis may play a vital role in fuel-stimulated insulin secretion of pancreatic ␤-cells.Some amino acids, particularly leucine and its non-metabolizable analogue 2-amino-2-norbornanecarboxylic acid, have been known to stimulate insulin secretion from pancreatic ␤-cells by activation of glutamate dehydrogenase (11-15). More recently, the branched-chain amino acids, including leucine, isoleucine, and valine, have been reported to activate the mammalian target of rapamycin (mTOR) 1 signaling pathway (16 -19) in ␤-cells. Leucine stimulates protein synthesis and pancreatic ␤-cell proliferation via the mTOR signaling pathway at physiological concentrations (16). These studies indicate a new role of branched-chain amino acids in pancreatic ␤-cell biology in addition to serving as fuels or residues for protein synthesis.As the rate-limiting enzyme of glucose metabolism, it is believed that glucokinase sets a strict control on glucose metabolism in pancreatic ␤-cells. However, overexpression of glucokinase or hexokinase I fails to increase the maximal insulin output induced by glucose, although it decreases the threshold for glucose-induced insulin secretion in pancreatic ␤-cel...

The proteoglycan versican (VCAN) promotes tumor progression and enhances metastasis in several cancers; however, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Recent evidence suggests that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC. Thus, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC. VCAN expression was analyzed using three RCC and normal kidney cell lines as well as a clinical cohort of 84 matched ccRCC and normal renal tissues. Functional analyses on growth and progression properties were performed using VCAN depleted ccRCC cells. Microarray expression profiling was employed to investigate the target genes and biological pathways involved in VCAN-mediated ccRCC carcinogenesis. ccRCC had elevated VCAN expression in comparison with normal kidney in both cell lines and clinical specimens. The elevated expression of VCAN was significantly correlated with metastasis (P<0.001) and worse 5-year overall survival after radical nephrectomy (P=0.014). In vitro, VCAN knockdown significantly decreased cell proliferation and increased apoptosis in Caki-2 and 786-O cells, and this was associated with alteration of several TNF signaling-related genes such as TNF-α, BID, and BAK. Furthermore, VCAN depletion markedly decreased cell migration and invasion which correlated with reduction of MMP7 and CXCR4. These results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis and thus, is an attractive target for novel diagnostic, prognostic and therapeutic strategies.