Current Status and Challenges in Rotigotine Delivery.

Rotigotine is a non-ergoline, high lipophilic dopamine agonist. It is indicated as the first-line therapy for Parkinson's disease (PD) and Restless Leg Syndrome (RLS). However, the precise mechanism of rotigotine is yet to be known. Rotigotine has similar safety and tolerability to the other oral non-ergolinic dopamine antagonists in clinical trials, which include nausea, dizziness and somnolence. Neupro® was the first marketed transdermal patch formulation having rotigotine. The transdermal delivery system is advantageous as it enables continuous administration of the drug, thus providing steady-state plasma drug concentration for 24-hours. Intranasal administration of rotigotine allows the drug to bypass the blood-brain barrier enabling it to reach the central nervous system within minutes. Rotigotine can also be formulated as an extended-release microsphere for injection. Some challenges remain in other routes of rotigotine administration such as oral, parenteral and pulmonary, whereby resolving these challenges will be beneficial to patients as they are less invasive and comfortable in terms of administration. This review compiles recent work on rotigotine delivery, challenges and its future perspective.

Evaporation and dispersion of respiratory droplets from coughing.

Understanding how respiratory droplets become droplet nuclei and their dispersion is essential for understanding the mechanisms and control of disease transmission via droplet-borne and airborne routes. A theoretical model was developed to estimate the size of droplet nuclei and their dispersion as a function of the ambient humidity and droplet composition. The model-predicted dried droplet nuclei size was 32% of the original diameter, which agrees with the maximum residue size in the classic study by Duguid, 1946, Edinburg Med. J., 52, 335 and the validation experiment in this study, but is smaller than the 50% size predicted by Nicas et al., 2005, J. Occup. Environ. Hyg., 2, 143. The droplet nuclei size at a relative humidity of 90% (25°C) could be 30% larger than the size of the same droplet at a relative humidity of less than 67.3% (25°C). The trajectories of respiratory droplets in a cough jet are significantly affected by turbulence, which promotes the wide dispersion of droplets. We found that medium-sized droplets (e.g., 60 µm) are more influenced by humidity than are smaller and larger droplets, while large droplets (≥100 µm), whose travel is less influenced by humidity, quickly settle out of the jet.

Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR inhibitor. These data reveal that biomarker-directed trials for FGFR1-amplified SCC require assessment of FGFR1 protein expression and uncover novel therapeutic strategies for FGFR1-amplified SCC with low FGFR1 protein expression.

Functional status and health-related quality of life in patients with primary intracranial tumour.

This study aimed to evaluate the functional status and HRQoL in patients with primary intracranial tumours in Malaysia. Karnofsky Performance Scale (KPS) and Modified Barthel Index (MBI) were used to assess the functional status whereas EORTC core Quality of Life Questionnaire (QLQ-C30) and Brain Cancer Module (BN-20) questionnaires were used to assess the HRQoL. Thirty-eight patients with primary intracranial tumours admitted for surgery in University Malaya Medical Center were recruited. These assessments were administered before surgery (baseline) and six months after surgery (follow-up). All patients received some form of rehabilitation interventions after surgery. The global HRQoL and functional status of these patients showed improvement at six months after surgery. Emotional Functioning score showed the greatest improvement among the functional domains (63 vs 86, p=0.003). Reduction in symptom burden such as fatigue, nausea, vomiting, pain and headache were also noted at follow-up together with less future uncertainty (p<0.05). Pearson correlation revealed statistically significant positive correlation between functional status and HRQoL at baseline and follow-up, in particular, global health status (r=0.50 and r=0.67), physical functioning (r=0.53 and r=0.90) and role functioning (r=0.34 and r=0.77). Thus, from the correlation found, improving a patient's function and independence level throughout all stages of care, even before any surgical intervention is offered would improve the HRQoL concurrently.

Use of adjunctive treatments in improving patient outcome in Fournier's gangrene.

Fournier's gangrene is a polymicrobial necrotising soft tissue infection (NSTI) affecting the perineum and scrotum. It is rapidly progressive and destructive, and is associated with high morbidity and mortality. Management protocol includes prompt diagnosis, early institution of antibiotic therapy and adequate wound debridement, usually requiring multiple operations. The resultant defect can be left to heal by secondary intention, or surgical coverage can be undertaken. We report Fournier's gangrene in a 60-year-old diabetic man and his successful treatment with skin grafting, which utilised a multidisciplinary approach and adjuncts, including negative-pressure wound therapy and hyperbaric oxygen therapy. We also review the literature related to these adjuncts and discuss their usefulness in the management of NSTIs.

MicroRNA-125b expands hematopoietic stem cells and enriches for the lymphoid-balanced and lymphoid-biased subsets.

MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.

Niche recycling through division-independent egress of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

The adhesion molecule esam1 is a novel hematopoietic stem cell marker.

Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

A directed approach for engineering conditional protein stability using biologically silent small molecules.

The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (DeltaDeltaG(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.

Differential expression and pathological significance of autocrine motility factor/glucose-6-phosphate isomerase expression in human lung carcinomas.

To clarify the involvement of autocrine motility factor (AMF) in the phenotype and biological profiles of human lung carcinomas, we analysed protein and mRNA expression in a total of 180 cases. Immunohistochemistry revealed positive staining in 67.2%, with the highest frequency in squamous cell carcinoma (SCC; 90.8%) and the lowest in small cell carcinoma (SmCC; 27.8%). In SCC, the staining frequency and intensity correlated with the degree of morphological differentiation. Generally, the expression levels in immunoblotting analysis corresponded well with immunohistochemical positivity. However, there was less agreement between protein and mRNA levels: in SmCC and large cell carcinomas (LCCs), mRNA showed higher, but protein showed lower expression. Among non-small cell lung carcinomas (NSCLCs), AMF protein levels correlated inversely with tumour size, but tumours exhibiting lymph node metastasis showed higher mRNA expression. In cultured lung carcinoma cells which comprised all histological subtypes, AMF was detected in the lysates of all ten cell lines. Secreted AMF protein was detected in the conditioned media from six cell lines, most of which were SmCC or LCC. Thus, a particular subset of lung carcinomas secrete AMF, which may promote cell motility via autocrine stimulation through its cognate receptor and cause the biological aggressiveness seen in SmCC and LCC. Moreover, treatment by proteasome inhibitors resulted in increased cellular AMF in five cell lines, suggesting that intracellular AMF levels are regulated by both secretion and proteasome-dependent degradation. In conclusion, AMF was detected in a major proportion of lung carcinomas, and may play a part not only in proliferation and/or progression of the tumours, but also, possibly, in the differentiation of SCC. Furthermore, higher mRNA expression may be related to the high metastatic potential of NSCLC and increased protein secretion, leading to a more aggressive phenotype, such as the invasiveness of SmCC and LCC.

A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

Frequencies of HER-2/neu expression and gene amplification in patients with oesophageal squamous cell carcinoma.

The utilisation of antitumour T cells induced by cancer vaccination with HER-2 peptides or antibodies (Herceptin) against HER-2, as immunotherapy for oesophageal cancer, is a novel and attractive approach. It is important to clarify the frequencies of HER-2 expression and gene amplification in patients with oesophageal squamous cell carcinoma (SCC) and to evaluate the relationship between HER-2 status and HLA haplotype, since the candidates for HER-2 peptide-based vaccination are restricted to a certain HLA haplotype. We determined the frequency of HER-2 expression using the HercepTest for immunohistochemistry and HER-2 gene amplification by fluorescence in situ hybridisation (FISH) assay in oesophageal SCC (n=66). HER-2-positive tumours (1+/2+/3+) analysed by a HercepTest were observed in 30.3% of all the patients and HER-2 gene amplification evaluated by FISH was observed in 11.0% of all the patients, in which all HercepTest (3+) tumours were found to have gene amplification and three of six moderately positive (2+) tumours showed gene amplification. Furthermore, HER-2-positive cells were present more diffusely and were larger within each tumour in the patients who were HercepTest 3+ than those who were HercepTest 1+. Moreover, the survival rate in HER-2-positive group was significantly worse than that in HER-2-negative group. Also, the survival rate in the patients with HER-2 gene amplification was significantly worse than that without HER-2 gene amplification. In addition, oesophageal SCC patients with both HLA-A24-positive and HER-2-positive tumours (1+/2+/3+) accounted for 26% of these cases, and both HLA-A2- and HER-2-positive tumours accounted for 18% of them.

Screening for novel ENU-induced rhythm, entrainment and activity mutants.

Chemical mutagenesis has provided an opportunity to develop and expand the repertoire of behavioural mutants for gene function studies. With this in mind, we have established a screen in mice for mutations affecting circadian rhythms, entrainment to light and other wheel-running parameters. The screen consists of an assessment of mouse wheel-running activity in a 12:12 h light/dark cycle for 7-10 days followed by assessment in constant darkness for up to 20 days. Responses to light are assessed using two protocols; a 15 minute light pulse given at circadian time 16 on the tenth day in constant darkness and an additional 12 h of light upon transition from light/dark conditions to constant darkness. To date, approximately 1300 progeny of chemically mutagenised mice have been screened. Computer-aided assessment of wheel-running parameters has helped in identifying abnormal phenotypes in approximately 5% of all animals screened. Inheritance testing of mice with abnormal phenotypes has confirmed the number of robustly inherited mutant phenotypes to be 1% of the total screened. Confirmed mutants including those affecting free-running period, light-responsiveness and wheel-running endurance have been identified. Thus far, low-resolution map positions have been established for four mutants by completing genome scans in backcross progeny. Mutant loci do not correspond with those previously associated with wheel-running behaviour. This result confirms that phenotype-driven approaches such as this should continue to provide material for mammalian gene function studies.

Boxer's pericardium.

A 65-year-old retired professional boxer presented with progressively worsening shortness of breath, peripheral oedema and mild abdominal swelling over a period of 6 months. His only past medical history was hypertension. Subsequent investigations revealed chylous ascites, pericardial constriction and bilateral chylothorax. He had uneventful pericardectomy, and post-operatively the chylothorax resolved only after administration of octreotide for 10 days. The histopathological features of fibrosis, haemosiderin deposition in the pericardium and abundant haemosiderin-laden macrophages are consistent with chronic resolving haemopericardium. These findings suggested that the cause of pericardial constriction was repeated chest trauma from boxing.

Plasma, intestine and tumor levels of 5-fluorouracil in mice bearing L1210 ascites tumor following oral administration of 5-fluorouracil, UFT (mixed compound of tegafur and uracil), carmofur and 5'-deoxy-5-fluorouridine.

Several 5-fluorouracil (5-FU) derivatives, 1-hexylcarbamoyl-5-fluorouracil (HCFU), 5'-deoxy-5-fluorouridine (5'-DFUR) and UFT (mixed compound of tegafur and uracil), have been developed and clinically widely used. However, comparative pharmacokinetic studies of the parent compound and other fluorinated drivatives have not been precisely reported. The dosage of the oral clinical use for human cancer of 5-FU, HCFU, 5'-DFUR and UFT as tegafur (FT) is 200-300mg/d, 600mg/d, 800-1,200mg/d and 300-600mg/d respectively. These amounts of the drugs are almost equimolar. Previously, we reported the effect of oral equimolar administration of each four drugs on thymidilate synthase activity, deoxyribonucleotide metabolism and cell cycle progression in L1210 ascites tumor. (1,2) In this study, we examined the antitumor effect and 5-FU concentration in the plasma, intestine and tumor after oral equimolar administrations of each drug using BDF1 mice bearing L1210 ascites tumor. In our study, UFT showed the best life prolongation among these four drugs. The intestine 5-FU level was highest by treatment with 5-FU during the initial 4 h. The plasma 5-FU level was highest by treatment with HCFU for 4 h. But the tumor 5-FU level was highest by treatment with UFT over the 24 h. In spite of the high plasma 5-FU concentration after the treatment with HCFU, the 5-FU concentration in the tumor was below the detectable level until 24 h. These findings suggested that the highest specific accumulation of 5-FU in tumor cells may explain the best therapeutic results of UFT.

Numerical modelling of flow and heat transfer in the rotating disc cavities of a turboprop engine.

A numerical analysis of the flow and heat transfer in the cavity between two co-rotating discs with axial inlet and radial outflow of fluid, a configuration common in gas turbine engines, is described. The results are compared with the experimental data of Northrop and Owen. The effectiveness of the k-epsilon turbulence model with the two-layer zonal model for near-wall treatment of Chen and Patel is tested for this type of flow. Using three-dimensional models it is shown that modelling discrete holes at the outlet as opposed to a continuous slot, which is the approximation inherent in the two-dimensional axisymmetric model, has little effect on the predicted Nusselt number distribution along the disc surface. Results of a conjugate heat transfer analysis of a spacer in the turbine section of a turboprop engine are then presented.

Interposed colon between remnants of the small intestine exhibits small bowel features in a patient with short bowel syndrome.

We describe herein the case of a 48-year-old man who underwent emergency massive resection of the small intestine due to a strangulated ileus, which led to short bowel syndrome (SBS), as he was left with only 7 cm of jejunum and 8 cm of ileum with ileocecal valve. He then received interposition of a colon segment between the jejunum and ileum remnants isoperistaltically. For 24 months after the operation, he has been able to tolerate oral intake, but still requires partial home parenteral nutritional support during the night on a bimonthly basis. Biochemical and nutritional parameters, including the analysis of minerals and trace elements, indicated that the patient was in relatively good health. Histological examination revealed that the mucosa of the interposed colon showed hypertrophy and hyperplasia of the crypt glands, and cells resembling Paneth cells which are usually seen in the small intestine, suggesting that the colon segment exhibits adaptive changes to the small intestine. Colon interposition may be a useful technique in patients with SBS when the small bowel is too short for the other surgical considerations.

Protein overexpression and gene amplification of c-erb B-2 in pulmonary carcinomas: a comparative immunohistochemical and fluorescence in situ hybridization study.

Amplification of the c-er bB-2 gene (located on 17q11.2--12) is accompanied by overexpression of its cell surface receptor product, p185(ERBB2). In pulmonary carcinomas, however, there has been disagreement between the reported frequencies of gene amplification and overexpression. To clarify their relationship, the correlation between the cellular expression of p185(ERBB2) and the level of c-erb B-2 gene amplification was studied. A total of 195 pulmonary carcinomas (182 primary and 13 metastatic) were examined immunohistochemically using a polyclonal antibody, which recognizes the internal domain of the human c-erb B-2 protein, and positive tumors were further examined for the gene amplification by dual-color fluorescence in situ hybridization using probes for centromere 17 and 17q11.2--12. By immunohistochemistry, distinct membrane staining was found in an adenocarcinoma, a large cell carcinoma and a metastatic carcinoma from the breast, and cytoplasmic and/or faint membranous staining was observed in 23 non-small cell lung carcinomas. It was in the two primaries and the metastatic carcinoma that more than 8-fold amplification of c-erb B-2 was found by fluorescence in situ hybridization. Especially, in the two primary carcinomas, tumor cells had amplified genes with the signals forming one or two clusters, indicating that the amplified gene was present in homogeneously staining regions. Among the 23 tumors, three tumors showed low-level amplification (less than 3-fold), which was differentiated from polysomy 17 found in the other two. In the 30 non-small cell lung carcinomas selected at random from 151 with negative immunostaining, there were five trisomy 17, but no tumors with the gene amplification. This suggests that although c-erb B-2 amplification in pulmonary carcinoma is rare, it occurs in the form of a homogeneously staining region and is thought to control the overexpression of the protein in the cell membrane. New adjuvant therapy using a humanized antibody to the oncoprotein may be beneficial to patients with these tumors.

Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450.

Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.