Development of the Low-Temperature Inducible System for Recombinant Protein Production in Escherichia coli Nissle 1917.

The pET system is commonly used for producing foreign proteins in Escherichia coli, but its reliance on the costly and metabolically demanding inducer IPTG limits its industrial use. This study engineered a low-temperature inducible system (LTIS) in E. coli Nissle 1917 (EcN) by combining the T7 expression system with the thermal inducible mechanism CI857-λPRPL to generate the new LTIS strain, ENL7P. The strain ENL7P-sfGFP-Km underwent overnight culture at 37 °C for 14-16 h, followed by subculturing at 30 °C for 24 h. This resulted in a notable 5.53-fold increase in the sfGFP induction rate when the strain was cultivated under 37-30 °C conditions. Moreover, gene expression was induced using a two-stage strategy. Initially, the strain was cultured overnight at 39 °C for 14-16 h, followed by a subculture at 30 °C for 6 h, and finally, another subculture at 30 °C for 24 h. This cultivation strategy led to an impressive 158.37-fold induction rate for sfGFP. Similar effects could be achieved through utilization of the LTIS system for inducing the production of thermophilic trehalose synthase from Thermus antranikianii (TaTS). The results of this study proved that the LTIS system has the potential for industrial applications.

Integration of Multiple Phage Attachment Sites System to Create the Chromosomal T7 System for Protein Production in Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic used to treat gastrointestinal diseases. The probiotic and endotoxin-free characteristics of EcN support its potential to be developed into a microbial expression system. With this aim, in this study, the powerful T7 expression system was constructed in the cryptic plasmid-free EcN (EcNP) to generate the T7 expression host ENL6P. The concept of multiple copies of gene expression cassettes regulated by the chromosomal T7 promoter was promoted due to plasmid instability issues with protein production in ENL6P. The integration of multiple phage attachment sites (IMPACT) system, which combined Cre-lox72, CRIM, and lambda red recombinase systems, was designed to simplify the manipulation and achieve the multiple φ80 bacterial attachment sites (attB) in ENL6P to generate the new strain ENL6PP4 with four φ80 attB sites. The strain can simultaneously integrate four copies of gene expression cassettes in the chromosome to produce recombinant proteins. The IMPACT systems incorporated several tools in gene editing to rapidly achieve more robust and stable microbial strains for research and various industrial applications.

Leptin protects brain from ischemia/reperfusion-induced infarction by stabilizing the blood-brain barrier to block brain infiltration by the blood-borne neutrophils.

The cellular and molecular mechanisms underlying leptin-mediated brain protection against cerebral ischemia were investigated at the blood-brain barrier (BBB) and neutrophil level. Through the ischemia/reperfusion (I/R) animal model, we found that leptin expression level was significantly decreased in ischemic hemisphere. Brain injection with leptin (15 µg/kg, intracisternally) could block the I/R-increased BBB permeability, activation of matrix metallopeptidase 9 (MMP-9) and brain infiltration of blood-borne neutrophils to reduce the infarct volume of ischemic brain. The brain expression level of tight junction protein ZO-1 as well as number and motility of neutrophils in blood was all increased by the same injection, indicating BBB stability (rather than reduction in neutrophils) played a major role in the leptin-inhibited brain infiltration of neutrophils. Leptin-mediated protection of BBB was further confirmed in vitro, through a BBB cellular model under the in vitro ischemic condition (G/R: glucose-oxygen-serum deprivation followed by GOS restoration). The results showed that leptin again could block the G/R-increased neutrophil adherence to EC layer as well as BBB permeability, likely by stimulating the endothelial expression of ZO-1 and VE-Cadherin. The study has demonstrated that leptin could protect ischemic brain via multiple ways (other than neuronal protection), by inhibiting the BBB permeability, brain infiltration of the blood-borne neutrophils and neutrophil adherence to vascular ECs. The role of leptin in vascular biology of stroke could further support its therapeutic potential in other neurodegenerative diseases, associated with BBB disorder.

Therapeutic Effect of Repurposed Temsirolimus in Lung Adenocarcinoma Model.

Lung cancer is one of the major cause of cancer-related deaths worldwide. The poor prognosis and resistance to both radiation and chemotherapy urged the development of potential targets for lung cancer treatment. In this study, using a network-based cellular signature bioinformatics approach, we repurposed a clinically approved mTOR inhibitor for renal cell carcinomans, temsirolimus, as the potential therapeutic candidate for lung adenocarcinoma. The PI3K-AKT-mTOR pathway is known as one of the most frequently dysregulated pathway in cancers, including non-small-cell lung cancer. By using a well-documented lung adenocarcinoma mouse model of human pathophysiology, we examined the effect of temsirolimus on the growth of lung adenocarcinoma in vitro and in vivo. In addition, temsirolimus combined with reduced doses of cisplatin and gemcitabine significantly inhibited the lung tumor growth in the lung adenocarcinoma mouse model compared with the temsirolimus alone or the conventional cisplatin-gemcitabine combination. Functional imaging techniques and microscopic analyses were used to reveal the response mechanisms. Extensive immunohistochemical analyses were used to demonstrate the apparent effects of combined treatments on tumor architecture, vasculature, apoptosis, and the mTOR-pathway. The present findings urge the further exploration of temsirolimus in combination with chemotherapy for treating lung adenocarcinoma.

The genome and transcriptome of Phalaenopsis yield insights into floral organ development and flowering regulation.

The Phalaenopsis orchid is an important potted flower of high economic value around the world. We report the 3.1 Gb draft genome assembly of an important winter flowering Phalaenopsis 'KHM190' cultivar. We generated 89.5 Gb RNA-seq and 113 million sRNA-seq reads to use these data to identify 41,153 protein-coding genes and 188 miRNA families. We also generated a draft genome for Phalaenopsis pulcherrima 'B8802,' a summer flowering species, via resequencing. Comparison of genome data between the two Phalaenopsis cultivars allowed the identification of 691,532 single-nucleotide polymorphisms. In this study, we reveal that the key role of PhAGL6b in the regulation of labellum organ development involves alternative splicing in the big lip mutant. Petal or sepal overexpressing PhAGL6b leads to the conversion into a lip-like structure. We also discovered that the gibberellin pathway that regulates the expression of flowering time genes during the reproductive phase change is induced by cool temperature. Our work thus depicted a valuable resource for the flowering control, flower architecture development, and breeding of the Phalaenopsis orchids.

Regulation of Intermittent Hypoxia on Brain Dopamine in Amphetaminized Rats.

We investigated intermittent hypoxia (IH) on dopamine (DA) release in rat brain treated with or without amphetamine (AMPH). Rats were divided into four groups including normoxia, IH, AMPH, and AMPH + IH treatments. The cerebrospinal fluid (CSF) was collected and the DA levels were detected by high performance liquid chromatography (HPLC). The plasma prolactin (PRL) concentration was measured by radioimmunoassay (RIA). We found that IH reduced basal DA concentration in media prefrontal cortex (mPFC), but increased that in striatum, where DA level was also increased in rats treated with AMPH or AMPH + IH. Angiotensin II (Ang II) increased the DA release in mPFC and striatum and this effect was enhanced in AMPH + IH group. The stimulatory effect of IH on plasma PRL was attenuated in presence of AMPH. Tyrosine hydroxylase (TH) expression was decreased by IH, but increased by AMPH + IH in mPFC. IH or AMPH treatment decreased the expression of vesicular monoamine transporter-2 (VMAT-2) in rat brain. These data suggested that IH altered the DA release and changed the protein expression levels in different parts of rat brain treated with AMPH. IH may play a role in regulating DA metabolism in AMPH addiction.

Risk factors correlated with risk of insulin resistance using homeostasis model assessment in adolescents in Taiwan.

The study aims to discover risk factors significantly correlated with insulin resistance among adolescents in Taiwan. A total of 339 study subjects were recruited in this cross-sectional study. A self-administered questionnaire and physical examinations including anthropometrics and biochemistry profiles were collected. Insulin resistance was assessed using homeostasis model assessment for insulin resistance (HOMA-IR). Study subjects had a significantly increased risk of IR for those with abnormal level of body mass index (odds ratio [OR] = 3.54; 95% confidence interval [CI] = 1.81-6.91), body fat (OR = 2.71; 95% CI = 1.25-5.88), and waist circumference (OR = 25.04; 95% CI = 2.93-214.14) when compared with those who have normal values. Furthermore, a significantly joint effect of 10.86-fold risk for HOMA-IR abnormality among body fat, body mass index, and systolic blood pressure was observed. The identification of risk factors significantly correlated with IR will be important to prevent metabolic syndrome-related diseases and complications for adolescents in their future life.

Leptin and interleukin-1beta modulate neuronal glutamate release and protect against glucose-oxygen-serum deprivation.

Molecular mechanism underlying leptin-mediated neuronal protection against glucose-oxygen-serum deprivation (GOSD) insult was investigated by focusing on the interactions among leptin, Interleukin-1beta (IL-1beta) and glutamate and their impacts on the growth of neurons under GOSD. The trypan blue dye exclusion assay, 4', 6-diamidino-2-phenylindole (DAPI) assay, cytokine antibody array assay, immunocytochemical staining assay, glutamate determination kit, immunoblocking and chemical blocking strategies were applied to serve the study goal. Results showed that in response to 6 h of GOSD, cortical neurons can secrete significant amounts of leptin and IL-1beta to protect neurons from GOSD-induced cell damage. Serine/threonine kinase Akt (Akt) and extracellular signal-related kinase (ERK) inhibitors significantly reversed leptin-mediated neuroprotection. GOSD-induced IL-1beta was further enhanced by leptin in Akt/ERK-dependent manner. Blockade of endogenous leptin with specific antibodies significantly inhibited GOSD-induced IL-1beta expression and increased glutamate release from GOSD neurons. IL-1 blockade with IL-1 receptor antagonist (IL-1ra) on the other hand, inhibited leptin-mediated neuroprotection and suppression of glutamate release from GOSD neurons. Pre-treating GOSD neurons with leptin and IL-1beta in combined significantly increased their survival but decreased their releases of glutamate. The results indicate that leptin may act through Akt and ERK signaling pathways to protect neurons from GOSD insult; the protection was in part IL-1beta dependent and through which the glutamate release from GOSD neurons was inhibited. Therapeutic values of leptin and IL-1beta were suggested in the treatment of cerebral ischemia at early stage.

Molecules involve in the self-protection of neurons against glucose-oxygen-serum deprivation (GOSD)-induced cell damage.

Molecules involved in self-protection of neurons against glucose/oxygen/serum deprivation (GOSD) were investigated. Trypan blue dye exclusion assay, Western blotting, ELISA, cytokine antibody array and chemical blocking assay were applied in the study. Results showed that early induction (at 6h of GOSD) of cyclooxygenase-2 (COX-2), leptin, transforming growth factor-beta1 (TGF-beta1), glial-cell-line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) all played a compensatory role in the protection of neurons against GOSD. Decline of these molecules and peroxisome proliferators-activated receptor (PPAR)-gamma and -alpha since 12h of GOSD may lead to an irreversible neuronal death. Nitric oxide (NO) and superoxide at low concentrations were neuroprotective whereas at high concentrations were detrimental to neurons. Accumulation of NO and superoxide at late stage of GOSD should therefore be prevented. The study provided a useful platform for screening of potential anti-ischemic drugs and also explained why GOSD neuron derived conditioned medium (NCM) only exerted a time-restricted neuroprotection.

Auditory and visual toxicity during deferoxamine therapy in transfusion-dependent patients.

Deferoxamine is a chelating agent that has extended the life expectancy of patients with thalassemia. In the 1980s, many investigators reported otologic and visual toxicity caused by deferoxamine. In July 1999 and 2 years later, the authors performed audiologic and ophthalmologic assessments in 30 transfusion-dependent patients receiving deferoxamine therapy (40-50 mg/kg per dose, subcutaneously for 8-10 hours, 4-7 days per week). In 1999, six patients (20%) had deferoxamine-related hearing impairment (>25 dB), all at high frequencies. Because the authors believed the benefits of chelation therapy outweighed the risk of ototoxicity, the dose of deferoxamine was not reduced. Two years later, the hearing impairment had not progressed in any of the patients. There was no association between ototoxicity and ferritin level. No patients had abnormalities of visual acuity or funduscopy in either 1999 or 2001. Based on this experience, deferoxamine at doses lower than 50 mg/kg/d was safe for the eyes and slightly toxic to the ears. The ototoxicity probably relates to individual susceptibility. Regular monitoring of auditory function and close follow-up of abnormal findings are recommended. According to this limited experience, reducing the dose or withdrawing deferoxamine might not be necessary if the hearing loss is stable in the face of ferritin levels above 2,000 ng/mL. Because of the relatively small patient numbers, more data are needed to confirm these conclusions.

Systemic infection of Kudoa lutjanus n. sp. (Myxozoa: Myxosporea) in red snapper Lutjanus erythropterus from Taiwan.

A new species of Kudoa lutjanus n. sp. (Myxosporea) is described from the brain and internal organs of cultured red snapper Lutjanus erythropterus from Taiwan. The fish, 260 to 390 g in weight, exhibited anorexia and poor appetite and swam in the surface water during outbreaks. Cumulative mortality was about 1% during a period of 3 wk. The red snapper exhibited numerous creamy-white pseudocysts, 0.003 to 0.65 cm (n = 100) in diameter, in the eye, swim bladder, muscle and other internal organs, but especially in the brain. The number of pseudocysts per infected fish was not correlated with fish size or condition. Mature spores were quadrate in apical view and suboval in side view, measuring 8.2 +/- 0.59 microm in width and 7.3 +/- 0.53 microm in length. The 4 valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 3.62 +/- 0.49 microm in length and 2.2 +/- 0.49 microm in width. Mild inflammatory responses or liquefaction of host tissue were associated with K. lutjanus n. sp. infection. The junction of shell valves appeared as overlapping, straight lines. The polar filament formed 2 to 3 coils. A general PCR (polymerase chain reaction) primer for Kudoa amplified the small subunit (SSU) rDNA sequences, and the amplified gene was sequenced. It was evident from the phylogenetic tree that the 3 strains tested, AOD93020M, AOD93028M and AOD93028B, were identical and belonged to the Kudoa SS rRNA subgroup. The evolutionary tree showed that these strains form a unique clade, at a distance from other Kudoa species and myxosporeans. The spore's morphological and ultrastructural characteristics, as well as the SS rDNA properties of the isolates, were also essentially identical and served to distinguish them from representative Kudoa. It is, therefore, proposed that the strains isolated from the diseased red snapper be assigned to a new species.

New analogues of AHMA as potential antitumor agents: synthesis and biological activity.

A series of new analogues of 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA, 1) and AHMA-ethylcarbamate (2) were synthesized by introducing an O-alkylcarboxylic acid esters to the CH(2)OH function, displacing the CH(2)OH function with a dimethylaminocarboxamido group or with a methyl function introduced at the meta-, para- or ortho-position to the NH(2) group to form 5-(9-acridinylamino)-m-toluidines (AMTs), 5-(9-acridinylamino)-p-toluidines (APTs) or 5-(9-acridinylamino)-o-toluidines (AOTs), respectively. The inhibitions of a variety of human tumor cell growth, interactions with DNA as well as inhibitory effect against topoisomerase II (Topo II) of these new agents were studied. Among AMT, APT and AOT derivatives with dimethylaminoethylcarboxamido and Me at C4 and C5 of acridine moiety (i.e., 21c, 23c and 26c) were more cytotoxic than AHMA (1) and AHMA-ethylcarbamate (2), depending upon the tumor cell line tested. Detailed structure-activity relationships of the new analogues were studied.