Early data on long-term efficacy and safety of inotersen in patients with hereditary transthyretin amyloidosis: a 2-year update from the open-label extension of the NEURO-TTR trial.

BACKGROUND AND PURPOSE: Hereditary transthyretin (hATTR) amyloidosis causes progressive polyneuropathy resulting from transthyretin (TTR) amyloid deposition throughout the body, including the peripheral nerves. The efficacy and safety of inotersen, an antisense oligonucleotide inhibitor of TTR protein production, were demonstrated in the pivotal NEURO-TTR study in patients with hATTR polyneuropathy. Here, the long-term efficacy and safety of inotersen are assessed in an ongoing open-label extension (OLE) study. METHODS: Patients who completed NEURO-TTR were eligible to enroll in the OLE (NCT02175004). Efficacy assessments included the modified Neuropathy Impairment Score plus seven neurophysiological tests composite score (mNIS + 7), the Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) questionnaire total score and the Short-Form 36 Health Survey (SF-36) Physical Component Summary (PCS) score. Safety and tolerability were also assessed. RESULTS: Overall, 97% (135/139) of patients who completed NEURO-TTR enrolled in the OLE. Patients who received inotersen for 39 cumulative months in NEURO-TTR and the OLE continued to show benefit; patients who switched from placebo to inotersen in the OLE demonstrated improvement or stabilization of neurological disease progression by mNIS + 7, Norfolk QOL-DN and SF-36 PCS. No new safety concerns were identified. There was no evidence of increased risk for grade 4 thrombocytopenia or severe renal events with increased duration of inotersen exposure. CONCLUSION: Inotersen slowed disease progression and reduced deterioration of quality of life in patients with hATTR polyneuropathy. Early treatment with inotersen resulted in greater long-term disease stabilization than delayed initiation. Routine platelet and renal safety monitoring were effective; no new safety signals were observed.

Evolving projection analysis of multicomponent mixtures.

The application of evolving projection analysis (EPA) to second-order bilinear data sets consisting of more than two components is described. EPA is a method for rank analysis of ordered data matrices where the components appear sequentially as a function of time or some other ordinal variable. It was found that extension of the method to mixtures of more than two components was best accomplished using principal components analysis to preprocess the data. The algorithm is demonstrated using simulated four-component chromatographic data, and experimental data from liquid chromatography (three- and four-component mixtures) and a spectrophotometric titration (four components), both employing UV-visible diode array detection.

Unusual complications following laparoscopic Nissen fundoplication.

Laparoscopic Nissen fundoplication is commonly used to treat medically refractory gastroesophageal reflux disease. The most frequent severe complications following laparoscopic Nissen fundoplication are pneumothorax, gastroesophageal leak, and splenic injury. Prompt recognition and treatment of complications are important in reducing subsequent morbidity and mortality. This report describes two postoperative complications: (1) delayed diagnosis of a postoperative gastric perforation despite an unrevealing work-up for postoperative fever and abdominal pain; and (2) postoperative pancreatitis following revision of a fundoplication.

A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking.

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in CSF are being used in clinical practice. One of the most critical factors in CSF biomarker research is the inadequate powering of studies because of the lack of sufficient samples that can be obtained in single-center studies. Therefore, collaboration between investigators is needed to establish large biobanks of well-defined samples. Standardized protocols for biobanking are a prerequisite to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by preanalytical factors. Here, a consensus report on recommendations for CSF collection and biobanking is presented, formed by the BioMS-eu network for CSF biomarker research in multiple sclerosis. We focus on CSF collection procedures, preanalytical factors, and high-quality clinical and paraclinical information. The biobanking protocols are applicable for CSF biobanks for research targeting any neurologic disease.

Evaluation of glutamic acid and glycine as sources of nonessential amino acids for lake trout (Salvelinus namaycush) and rainbow trout (Salmo gairdnerii).

A semi-purified test diet which contained either glutamic acid or glycine as the major source of nonessential amino acids (NEAA) was fed to lake and rainbow trout. Trout fed the diet containing glutamic acid consistently showed better growth and feed conversion efficiencies than those fed the diets containing glycine. The data indicate that these trout utilize glutamic acid more efficiently than glycine when no other major sources of NEAA are present.

Effect of excess dietary riboflavin on growth of rainbow trout.

Fingerling rainbow trout were fed semipurified diets containing graded levels of supplemental riboflavin (3, 35, 42, 49, 56, 63, 70, 100 and 600 mg/kg of diet) to determine if dietary riboflavin in excess of requirements decreases growth. In three trials with three sizes of fish (mean initial weights, 0.5, 3.2 and 4.3 g) and two water temperatures (8.3 degrees and 15 degrees C), no significant diet-related growth inhibition was detected. The results suggest that rainbow trout, like other animals, are insensitive to excesses of dietary riboflavin and that the growth depression reported by other workers was the result of some other dietary influence or of faulty experimental design.

A map of the cleavage sites for endonuclease AvaI in the chromosome of bacteriophage lambda.

The linear order of nine fragments generated by the action of endonuclease AvaI on the DNA of bacteriophage lambda was determined from the altered fragmentation patterns of bacteriophages containing known deletions and of hybrids of bacteriophages lambda and phi80. Digestion of 5'-terminally 32P-labelled bacteriophage-lambda DNA was used to identify the terminal fragments. Measurement of relative fragment lengths permitted rough mapping of the endonuclease-AvaI cleavage sites relative to the ends of the bacteriophage-lambda chromosome. The fragment order was confirmed and the map refined by analysis of the fragmentation of derivative phages containing single cleavage sites for endonuclease EcoRI.

The nucleotide sequences recognized by endonucleases AvaI and AvaII from Anabaena variabilis.

Determination of the 5'-terminal sequences flanking all the individual cleavage sites for endonuclease AvaI in bacteriophage-lambda DNA has shown that this enzyme recognizes the hexanucleotide sequences: (Formula: see text), This sequence is cut as shown by the arrows to give single-stranded 5'-tetranucleotide protrusions (cohesive ends). Endonucleases SmaI, XhoI and XmaI recognize different symmetrical subsets of this sequence and provide independent evidence for the occurrence of these subsets at particular endonuclease-AvaI cleavage sites in the bacteriophage-lambda genome. Further evidence for this structure came from the demonstration that DNA fragments generated by endonuclease AvaI can be ligated to form a discrete set of larger molecules and from nearest-neighbor analysis which showed that cytosine residues occurred at the 3'-side of cleavage points. The observation that endonuclease AvaII recognized a subset of the sites recognized by AsuI [Hughes, Bruce & Murray (1979) Biochem. J. 185, 59-63[ led to the deduction that AvaII recognize the pentanucleotide sequence: (Formula: see text), and breaks internucleotide bonds at the positions indicated by the arrows.

Cloning an Aspergillus nidulans developmental gene by transformation.

We have developed a transformation system for Aspergillus nidulans giving a frequency of transformation high enough to screen a gene bank from which we were able to isolate and clone the A. nidulans developmental gene brlA by visual selection. The vector contains the selective marker argB+, and with it a frequency of transformation of 500 stable transformants/micrograms plasmid DNA can regularly be achieved. The evidence suggests that transformation is by integration but spontaneous excision of integrated plasmids is apparently frequent enough to allow the recovery of transforming plasmids in Escherichia coli.

Effects of feeding a high level of D-glucose on liver function in juvenile white sturgeon (Acipenser transmontanus).

Juvenile white sturgeon (Acipenser transmontanus) were fed three isonitrogenous and isoenergetic diets containing either 35% D-glucose (HC), a mixture of 20% dextrin and 10% cellulose (MC), or 23% cellulose (LC), to investigate the effects of dietary carbohydrate on liver function. After 8-week feeding, body weight gain of fish fed the HC diet was consistently higher than that of fish fed the MC and LC diets, but was not significantly different from the MC-fed fish. Fish fed the HC diet had significantly (p < 0.05) higher feed efficiencies and liver glycogen concentrations than fish fed the MC and LC diets. Sturgeon were injected intravenously with 10 mg kg(-1) body weight of sulfobromophthalein (BSP) and post-injection blood taken from the caudal vein at 15, 30, 60, and 120 min. No significant differences in plasma BSP concentrations were found among the treatments at these times. Plasma hemoglobin and activities of aspartate and alanine aminotransferase were not affected by the diets. This study suggests that the HC diet does not adversely affect liver function or weight gain. Inclusion of high dietary levels of digestible and inexpensive carbohydrates in commercial sturgeon feeds seems promising, but long-term feeding trials should be conducted to confirm this assertion.

The isolation and characterization of a sequence-specific endonuclease from Anabaena subcylindrica.

An endonuclease, AsuI, was isolated from extracts of Anabaena subcylindrica on the basis of gel-electrophoretic analysis of digests of bacteriophage-lambda DNA with the paritally purified extracts. The enzyme requires Mg2+, but no other cofactors. Endonuclease AsuI recognizes the interrupted tetranucleotide sequence: (Formula: see tex), and breaks the phosphodiester bonds indicated by the arrows to leave single-stranded trinucleotide projections at the 5'-termini of the DNA fragments.

Branched-chain amino acid aminotransferase activity in the tissues of lake trout.

The enzyme branched-chain amino acid aminotransferase (BCAT) was found in five tissues of fingerling lake trout, Salvelinus namaycush, (listed in order of decreasing tissue specific activity): posterior kidney, skeletal muscle, gill, liver, and anterior kidney. This pattern is consistent with that found in other animals. The results of this study seem to indicate that BCAT in the liver of lake trout has a higher specific activity than that of the rat and that the specific activity is higher in both the liver and skeletal muscle than it is in these organs of the chick.

Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis.

Two endonucleases, AvaI and AvaII, were isolated from Anabaena variabilis on the basis of their ability to make a limited number of breaks at specific points in bacteriophage lambda DNA. Neither enzyme has cofactor requirements beyond Mg2+. Endonuclease AvaI makes eight breaks in the phage lambda chromosome at which the 5'-terminal sequence is pPy-C-G-N. AvaII endonuclease cuts phage lambda DNA more extensively, yielding fragments with the 5'-terminal sequence G-T-C-N or G-A-C-N. Neither enzyme generates cohesive ends.

Messenger RNA from isolated aleurone cells directs the synthesis of an alpha-galactosidase found in the endosperm during germination of guar (Cyamopsis tetragonaloba) seed.

In cereals and some legumes the aleurone layer is a site of synthesis of enzymes which mobilize endosperm reserves. It has been established whether or not the aleurone cells of the seed endosperm of Cyamopsis tetragonaloba are a site of synthesis of α-galactosidase. The isolation and cultivation of aleurone cells demonstrated that they contain mRNA which directs the synthesis and secretion of α-galactosidase into the endosperm where along with a ß-mannanase it is responsible for the degradation of the galactomannan storage polymer. A method was developed to purify the mRNA from the aleurone cells of germinating seeds. This mRNA was analysed by: i) Northern blot hybridization using oligo-nucleotide mixed probes derived from the protein's NH2-terminal amino acid sequence and ii) in vitro translation in a wheat germ system and detection of the α-galactosidase protein using antibodies. The molecular mass of the protein synthesized in vitro is slightly larger (44 kDa) than that of the mature α-galactosidase (40.5 kDa) which is as expected for the precursor of a secreted protein.

Biomicroscopic and histologic pathology of the eye in riboflavin deficient rainbow trout (Salmogairdneri).

Quadruplicate lots of 35 fingerling rainbow trout (mean body weight 11.2 g) were fed for 20 weeks a complete diet or one deficient in riboflavin. Five fish from each low were examined for ocular lesions each week. At the end of the experiment eyes of three fish form each lot were examined histologically. Beginning with the 14th week, epithelial changes were present in the cornea; they appeared as thickening of the stroma and endothelium and resulted in clouding. Opacity of the lens originated in the posterior subcapsular cortex and progressed to lens fiber autolysis and liquefaction.

The isolation and sequence analysis of two seed-expressed acyl carrier protein genes from Brassica napus.

Genomic Southern blot analysis of Brassica napus DNA indicates that seed-expressed acyl carrier protein (ACP) is encoded by a multigene family of some 35 genes/haploid genome. Two genomic clones encoding B. napus ACP have been isolated and sequenced. The coding sequences of the 2 respective genes were found to be perfectly homologous to 2 distinct B. napus seed-expressed cDNAs and therefore represent seed-expressed forms of ACP. The 2 genomic ACP sequences share 94% homology within their coding sequences. Both genes are interrupted by 3 intervening sequences whose position within the 2 coding sequences is conserved. RNase protection studies were used to map the transcription start site of one of the genes and to provide further evidence that the gene is seed-expressed. The expression of a sub-group of the ACP gene family was found to be developmentally regulated in concert with the storage lipid synthetic phase of seed development. The coding sequence of both B. napus genes are highly homologous (96% and 93% respectively) to a Brassica campestris ACP cDNA sequence, suggesting that they may have evolved from this ancestral gene.

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.

Plastid-localised seed acyl-carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family.

Acyl-carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N-terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 51-amino-acid N-terminal extension. Immunocytochemical studies showed ACP to be localised solely within the plastids of B. napus seed tissue and it would therefore appear that the N-terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.