Risk assessment for biopharmaceutical single-use manufacturing: A case study of upstream continuous processing.

In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.

Novel Peritoneal Sclerosis Rat Model Developed by Administration of Bleomycin and Lansoprazole.

In our preliminary experiment, peritoneal sclerosis likely induced by peritoneal dialysis was unexpectedly observed in the livers of rats given bleomycin and lansoprazole. We examined whether this peritoneal thickening around the liver was time-dependently induced by administration of both drugs. Male Wistar rats were injected with bleomycin and/or lansoprazole for 2 or 4 weeks. The 3YB-1 cell line derived from rat fibroblasts was treated by bleomycin and/or lansoprazole for 24 h. The administration of both drugs together, but not individually, thickened the peritoneal tissue around the liver. There was accumulation of collagen fibers, macrophages, and eosinophils under mesothelial cells. Expressions of Col1a1, Mcp1 and Mcp3 genes were increased in the peritoneal tissue around the liver and in 3YB-1 cells by the administration of both drugs together, and Opn genes had increased expressions in this tissue and 3YB-1 cells. Mesothelial cells indicated immunoreactivity against both cytokeratin, a mesothelial cell marker, and αSMA, a fibroblast marker, around the livers of rats given both drugs. Administration of both drugs induced the migration of macrophages and eosinophils and induced fibrosis associated with the possible activation of fibroblasts and the possible promotion of the mesothelial-mesenchymal transition. This might become a novel model of peritoneal sclerosis for peritoneal dialysis.

Complete genome sequence of a novel putative polerovirus detected in grapevine.

Next-generation sequencing detected a novel virus from grapevine cultivar 'Kishmish Chjornyj' from Russia. Its complete genome sequence of 5625 nucleotides includes seven open reading frames encoding seven putative proteins similar to those of members of the genus Polerovirus in the family Luteoviridae. The novel virus showed graft-transmissibility and was tentatively named "grapevine polerovirus 1" (GPoV-1). Phylogenetic analysis using complete genome sequences of GPoV-1 and members of the family Luteoviridae indicated that although GPoV-1 is a member of the genus Polerovirus, it is unique within its clade. GPoV-1 is the first polerovirus detected in grapevine.

Genetic variability of grapevine fabavirus variants and development of a broad-spectrum assay for their detection.

Complete RNA1 and RNA2 sequences of two and nearly complete genome sequences of six new variants of grapevine fabavirus found in Japan were compared to those of previously reported variants. Negative selection pressure was suggested, and no recombination events were detected in either RNA1 or RNA2. The first 18 nucleotides in both RNAs were predicted to form a stem-loop structure. The variants could be genetically divided into four groups based on RNA1 and two based on RNA2. A broad-spectrum reverse transcription polymerase chain reaction assay using a primer pair designed based on an RNA2 consensus sequence was able to detect all of the known variants.

Characterization of a distinct variant of hop stunt viroid and a new apscaviroid detected in grapevines.

Using next-generation sequencing, we detected a novel variant of hop stunt viroid (HSVd) in grapevine 'Chenin blanc' (Vitis vinifera L.) and a new viroid species in 'Nachubearmarie' (Vitis labrusca L. × V. vinifera). The HSVd variant termed HSVd-CB has 296 nucleotides with up to 82% sequence identity with other HSVd variants such as HSVd-AP1 (Genbank accession EF523826). Many nucleotide changes, deletions, and insertions were sporadically found in HSVd-CB relative to HSVd-AP1 in the pathogenic and variable domains. Although several variations were also found in the central domain, few variations were found in the terminal left and right domains, including no variations in the terminal conserved hairpin. The new viroid, tentatively termed Japanese grapevine viroid (JGVd), has 367 nucleotides and has genetic features characteristic of the genus Apscaviroid. JGVd shared the highest nucleotide sequence identity (68%) with a persimmon latent viroid (PLVd) in its overall genome. However, the JGVd sequence shows chimerism with the partial genomes of other apscaviroids from apple, grapevine, and citrus. Phylogenetic analyses showed that only HSVd-CB formed a distinct branch from the cluster of the other HSVd variants and JGVd and PLVd formed a distinct branch from all other grapevine-infecting apscaviroids.

Molecular characterization of a novel putative ampelovirus tentatively named grapevine leafroll-associated virus 13.

A novel putative ampelovirus was detected in grapevines that showed typical leafroll symptoms and was tentatively named grapevine leafroll-associated virus (GLRaV)-13 following the series of numbering of other GLRaVs. The complete genome of GLRaV-13 comprised 17,608 nt and contained eleven putative open reading frames, showing genetic features similar to those of viruses belonging to subgroup I of genus Ampelovirus. Phylogenetic trees based on the RNA-dependent RNA polymerase, heat shock protein 70 homolog, and coat protein showed that GLRaV-13 had the closest, but still distant, relationship to GLRaV-1 in the subgroup I cluster.

Downregulation of growth hormone 1 gene in the cerebellum and prefrontal cortex of rats with depressive-like behavior.

Depressive-like behaviors in animals are usually assessed by standardized behavioral tests such as the forced swimming test (FST). However, individual variation in test performance may obscure group differences and thereby hinder the discovery of genes responsible for depression. Few reports have shown the influence of individual variability in identifying the genes associated with depressive-like behaviors. In this study, we conducted microarray analysis to identify genes differentially expressed in the prefrontal cortex (PFC) and cerebellum of rats stratified by FST immobility ratio (% immobility in 5 min) into a control group [immobility ratio: -1 to +1 standard deviation (SD) from the mean] and a depressive group (immobility ratio: +1 to +2 SDs above the mean). Genes differentially expressed in both the cerebellum and PFC of the depressive group were Alas2, Gh1, Hba-a2, Hbb, Hbb-b1, Hbe2, LOC689064, Mrps10, Mybpc, Olf6415, and Pfkb1. Ingenuity Pathway Analysis identified Gh1 as a hub gene in the networks of differentially expressed genes in both brain regions. This study indicates that the depressive-like behavior may be related to the decrease of Gh1 expression in the cerebellum and PFC.

An assemblage of divergent variants of a novel putative closterovirus from American persimmon.

Deep-sequencing analysis of nucleic acids extracted from leaf tissue of an American persimmon (Diospyros virginiana L.) and subsequent-sequencing analyses uncovered at least four distinct closterovirus-like molecules. Two complete genomes of 18,569 and 18,030 nucleotides (nt) and partial genomes of 4,899 and 9,019 nt were determined. The two complete genomes encoded 11 potential open reading frames and the characteristic organization of closteroviruses. Among the four genomes, the putative heat shock protein 70 homolog (HSP70h), RNA-dependent RNA polymerase, and coat protein showed 82-85, 72-91, and 84-87 % amino acid sequence identities, respectively. These results suggested that the four identified viruses could be divergent variants in a single host plant. The phylogenetic tree based on HSP70h showed that their closest relative, although distant, is Olive leaf yellowing-associated virus, a putative unassigned member of the family Closteroviridae. The name Persimmon virus B was proposed for this new virus, representing another unassigned member of the family.

Host cell proteins in monoclonal antibody processing: Control, detection, and removal.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Genetic characterization of novel putative rhabdovirus and dsRNA virus from Japanese persimmon.

Deep-sequencing analysis of nucleic acids from leaf tissue of Japanese persimmon trees exhibiting fruit apex disorder in some fruits detected two molecules that were graft transmitted to healthy seedlings. One of the complete genomes consisted of 13 467 nt and encoded six genes similar to those of plant rhabdoviruses. The virus formed a distinct cluster in the genus Cytorhabdovirus with lettuce necrotic yellows virus, lettuce yellow mottle virus and strawberry crinkle virus in a phylogenetic tree based on the L protein (RNA-dependent RNA polymerase, RdRp). The other consisted of 7475 nt and shared a genome organization similar to those of some insect and fungal viruses having dsRNA genomes. In a phylogenetic tree using the RdRp sequence of several unassigned dsRNA viruses, the virus formed a possible new genus cluster with two insect viruses, Circulifer tenellus virus 1 and Spissistilus festinus virus 1, and one plant virus, cucurbit yellows-associated virus.

Characterization of a new apscaviroid from American persimmon.

A unique circular molecule of 358 nucleotides was detected in American persimmon (Diospyros virginiana L.). The molecule was graft-transmissible and had genetic characteristics of members of the genus Apscaviroid. It had the highest sequence similarity (72-73 %) to citrus viroid VI (CVd-VI) and formed a clade with CVd-VI, citrus dwarfing viroid, and apple dimple fruit viroid in a phylogenetic tree. The molecule was not detected in citrus, unlike CVd-VI, which infects citrus and persimmon, and it was genetically distant from persimmon latent viroid, which infects persimmon only. The genetic and biological features indicated that the molecule may be a member of a new apscaviroid species.

RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana.

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5'-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3' half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway.

Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. An in vitro experimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver.

Oral administration of linoleic acid immediately before glucose load ameliorates postprandial hyperglycemia.

Introduction: Fatty acids are a major nutrient in dietary fat, some of which are ligands of long-chain fatty acid receptors, including G-protein-coupled receptor (GPR) 40 and GPR120. Pretreatment with GPR40 agonists enhanced the secretion of insulin in response to elevating blood glucose levels after glucose load in a diabetes model, but pretreatment with GPR120 agonist did not ameliorate postprandial hyperglycemia. This study examined whether oral administration of linoleic acid (LA), a GPR40 and GPR120 agonist, immediately before glucose load would affect the elevation of postprandial blood glucose levels in rats. Methods: Male rats and rats with type 1 diabetes administered streptozocin were orally administered LA, trilinolein, α-linolenic acid (α-LA), oleic acid, TAK-875, or TUG-891 immediately before glucose load. Blood glucose levels were measured before, then 15, 30, 60 and 120 min after glucose load. CACO-2 cells were used to measure the uptake of [14C] α-MDG for 30 min with or without LA. Gastric content from rats administered LA was collected 15 and 30 min after glucose load, and blood samples were collected for measurement of glucagon-like peptide 1 (GLP-1) and cholecystokinin concentrations. Results: The elevation of postprandial blood glucose levels was slowed by LA but not by trilinolein in rats without promotion of insulin secretion, and this effect was also observed in rats with type 1 diabetes. The uptake of α-MDG, an SGLT-specific substrate, was, however, not inhibited by LA. Gastric emptying was slowed by LA 15 min after glucose load, and GLP-1, but not cholecystokinin, level was elevated by LA 15 min after glucose load. TUG-891, a GPR120 agonist, ameliorated postprandial hyperglycemia but TAK-875, a GPR40 agonist, did not. Pretreatment with AH7614, a GPR120 antagonist, partially canceled the improvement of postprandial hyperglycemia induced by LA. α-LA, which has high affinity with GPR120 as well as LA, slowed the elevation of postprandial blood glucose levels, but oleic acid, which has lower affinity with GPR120 than LA, did not. Conclusion: Oral administration of LA immediately after glucose load ameliorated postprandial hyperglycemia due to slowing of gastric emptying via promotion of GLP-1 secretion. The mechanisms may be associated with GPR120 pathway.

The characteristics of serious suicide attempters in Japanese adolescents--comparison study between adolescents and adults.

BACKGROUND: Suicide is the leading cause of death among Japanese adolescents, and they may commit suicide differently from adults. However, there are few studies in medical-based data concerning adolescent patients seriously attempting suicide. We aimed to explore the characteristics of serious suicide attempts in Japanese adolescents, comparing them with those in adults. METHODS: We investigated adolescents who seriously attempted suicide and were treated at the Critical Care Medical Center (CCMC) of Nippon Medical School Hospital between 2000 and 2010, and we compared them with adult suicide attempters treated during 2009. We retrospectively studied medical records and collected clinical data and socio-demographic factors, including age, sex, psychiatric symptoms or diagnosis, methods of suicide attempt, motives for suicide attempt, previous deliberate self-harm, previous psychiatric history, parent loss experience, and previous psychiatric history in the family. RESULTS: Adolescent attempters were 15 males and 44 females, 13 to 18 years old (mean 16.39). Adult attempters were 37 males and 65 females, 19 to 79 years old (mean 39.45). In comparison to adult attempters, adolescent attempters were more frequently diagnosed with Borderline Personality Disorder (BPD), had more school problems and parent loss experience, but they had less financial problems. Gender differences between adolescents and adults were examined, and male adolescent attempters were found to be more frequently diagnosed with schizophrenia and had less financial problems than their adult counterparts, while female adolescent attempters were more frequently diagnosed with BPD, had more school problems and parent loss, but they had less previous psychiatric history than their adult counterparts. CONCLUSIONS: Our findings indicated that adolescent attempters were more frequently diagnosed with BPD and had more school problems and parent loss experience but had less financial problems. Additionally, in male adolescent attempters, identifying patients with schizophrenia seemed important, as it was their most frequent psychiatric diagnosis. For female adolescents, adequately assessing family function and interpersonal conflicts seemed important, as they were more often diagnosed with BPD and had more school and family problems.

Yersinia enterocolitica bacteremia and enterocolitis in a previously healthy 20-month-old girl.

Yersinia enterocolitica is a gram-negative bacillus that can cause illness ranging from a self-limiting enterocolitis to life-threatening bacteremia. Y. enterocolitica biotype 1B, serotype O:8 (1B/O:8), is the most pathogenic of the Yersinia species because of the presence of the high-pathogenicity island and the Yersinia virulence plasmid (pYV). Here, we report a pediatric case of Y. enterocolitica 1B/O:8 bacteremia and enterocolitis. A 20-month-old girl was admitted to hospital with fever,pharyngitis, and abdominal pain on day 2. Blood culture on admission was positive for Y. enterocolitica 1B/O:8. Stool culture on day 5 after cefotaxime treatment was also positive for Y. enterocolitica 1B/O:8, but only after cold enrichment at 4°C for 3 weeks. PCR assays identified the pYV only in stool specimens, indicating that strains from routine blood culture at 37°C lacked the pYV. The present case showed the usefulness of stool culture with cold enrichment and agglutination test for the diagnosis of Y. enterocolitica infection. We would therefore like to emphasize the importance of collection and preservation of stool specimens for the identification of pYV. To our knowledge, this is the first reported pediatric case of Y. enterocolitica 1B/O:8 bacteremia.

[Delirium, insomnia in hospitalization and ramelteon].

Ramelteon acts on a highly selective melatonin (MT1/MT2) receptor agonist. During delirium in postoperative and critically ill patients, a disruption of the sleep-wake cycle is frequently observed. Melatonin has a key role in the regulation of the sleep-wake cycle, so this raised the hypothesis that alterations in the metabolism of melatonin might play an important role in the development of delirium. Ramelteon and exogenous melatonin may have a prophylactic treatment for frail older persons at high risk for delirium that is safe, effective, and easily implementable in daily practice. However, there is currently insufficient evidence that ramelteon and exogenous melatonin is effective in preventing or treating postoperative delirium and sleep disorders in the critically ill patient.

Cardiac and vascular gene profiles in an animal model of takotsubo cardiomyopathy.

We investigated cardiac and vascular gene profiles in response to immobilization stress (IMO) in rats, an animal model of emotional stress-induced takotsubo cardiomyopathy using microarray analysis, followed by re-confirmation with real-time reverse transcription-polymerase chain reaction. Expression levels of the identified genes were further estimated by pretreatment with an α1-adrenoceptor blocker and/or a ß1-adrenoceptor blocker. In response to IMO, expression of 46 genes was significantly altered in the heart and that of 49 genes was significantly altered in the aorta. Pathway analysis with DAVID Bioinformatics Resources indicated that regulation of transcription and response to endogenous stimulation were the top two scoring pathways. Altered expression of cardiac genes was blunted by pretreatment with a ß1-adrenoceptor blocker or α1 + ß1-adrenoceptor blockers. In contrast, that of aortic genes was blunted by pretreatment with an α1-adrenoceptor blocker or α1 + ß1-adrenoceptor blockers. Activation of α1-adrenoceptor in the blood vessels or activation of ß1-adrenoceptors in the heart were mainly responsible for emotional stress-induced alteration of cardiac and vascular gene profiles.

Meningitis and septicemia caused by nontypeable Haemophilus influenzae in a previously healthy 2-year-old girl.

Nontypeable Haemophilus influenzae (NTHi) commonly colonizes the upper respiratory tract of children and causes otitis media, sinusitis, and bronchitis. Invasive NTHi diseases such as meningitis and septicemia have rarely been reported, especially in children with underlying predisposing conditions such as head trauma and immune compromise. However, we report a previously healthy 2-year-old girl who developed meningitis and septicemia caused by NTHi biotype ΙΙΙ. She was treated with dexamethasone, meropenem, and ceftriaxone, and recovered uneventfully. We wish to emphasize that NTHi should be borne in mind as a potential pathogen that can cause meningitis and septicemia, even in previously healthy children.

Yorkie drives Ras-induced tumor progression by microRNA-mediated inhibition of cellular senescence.

The activation of Ras signaling is a major early event of oncogenesis in many contexts, yet paradoxically, Ras signaling induces cellular senescence, which prevents tumorigenesis. Thus, Ras-activated cells must overcome senescence to develop into cancer. Through a genetic screen in Drosophila melanogaster, we found that the ETS family transcriptional activator Pointed (Pnt) was necessary and sufficient to trigger cellular senescence upon Ras activation and blocked Ras-induced tumor growth in eye-antennal discs. Through analyses of mosaic discs using various genetic tools, we identified a mechanism of tumor progression in which loss of cell polarity, a common driver of epithelial oncogenesis, abrogated Ras-induced cellular senescence through microRNA-mediated inhibition of Pnt. Mechanistically, polarity defects in Ras-activated cells caused activation of the Hippo effector Yorkie (Yki), which induced the expression of the microRNA bantam bantam-mediated repression of the E3 ligase-associated protein Tribbles (Trbl) relieved Ras- and Akt-dependent inhibition of the transcription factor FoxO. The restoration of FoxO activity in Ras-activated cells induced the expression of the microRNAs miR-9c and miR-79, which led to reduced pnt expression, thereby abrogating cellular senescence and promoting tumor progression. Our findings provide a mechanistic explanation for how Ras-activated tumors progress toward malignancy by overcoming cellular senescence.