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.

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.

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.

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.

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.

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.

Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice.

Blocking monoclonal antibodies (mAbs) specific to mouse interleukin-1 receptor antagonist (IL-1ra) were prepared by immunizing Armenian hamsters with recombinant mouse IL-1ra. A sensitive and specific ELISA against mouse IL-1ra was also established. In Propionibacterium acnes-induced liver injury, P. acnes induced transient increase of serum tumor necrosis factor-alpha levels but not those of IL-1ra, IL-1, and IL-6. However, subsequent lipopolysaccharide (LPS) challenge induced the increase of serum levels of all these cytokines and the peak serum IL-1ra level was more than 20 times as high as serum IL-1 levels. Immunohistochemical analysis demonstrated that IL-1ra was predominantly produced by hepatocytes during the course of the priming phase by P. acnes and eliciting phase by LPS challenge. Furthermore, the administration of a mAb to mouse IL-1ra exacerbates the liver injury induced by P. acnes and sublethal dose of LPS, suggesting a protective role of endogenous IL-1ra in this liver injury model.

erbB-2 overexpression but no activation of beta-Catenin gene in extramammary Paget's disease.

Our previous study in extramammary Paget's disease showed neither p53 mutations nor allelic loss at selected loci implicated in other cancers, suggesting a pathogenesis of this skin cancer different from other common epithelial malignancies. To examine further the genetic defects in extramammary Paget's disease, we carried out molecular genetic analyses in 31 tumor samples obtained from 27 cases of extramammary Paget's disease without underlying malignancies. Immunohistochemistry using CB-11 monoclonal antibody revealed either membrane or cytoplasmic erbB-2 oncoprotein overexpression in none of the 13 primary in situ tumors, but in one recurrent in situ tumor, 10 of 13 invasive primary tumors and two of four lymph node metastases. Sensitive dual color fluorescence in situ hybridization analysis using probes for erbB-2 gene locus and chromosome 17 pericentromere, however, revealed different erbB-2 gene status in the erbB-2 overexpressing tumors. One recurrent in situ tumor and one lymph node metastasis showed definite gene amplification characterized by multiple scattered signals or a few large clustered erbB-2 signals, whereas four tumors with predominantly cytoplasmic erbB-2 overexpression were thought to have low-grade gene amplification. The remaining six tumors overexpressing erbB-2 showed no increase of erbB-2 copy numbers. No evidence of abnormal activation of the beta-catenin gene, a critical mediator of Wnt signaling pathway, in any tumor by immunohistochemical staining and by direct sequencing and reverse transcription-polymerase chain reaction analysis was found. Frequent overexpression of erbB-2 by either gene amplification or possible transcriptional activation in invasive primary tumors and metastases suggests an important part for this oncogene in the progression of extramammary Paget's disease.

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.

Expression of glial fibrillary acidic protein (GFAP) in peripheral nerve sheath tumors. A comparative study of immunoreactivity of GFAP, vimentin, S-100 protein, and neurofilament in 38 schwannomas and 18 neurofibromas.

Immunoreactivity of glial fibrillary acidic protein (GFAP) in 38 schwannomas and 18 neurofibromas was evaluated and compared with the reactivity of vimentin, S-100 protein, and neurofilament protein. All cases were positive for vimentin and S-100 protein. GFAP was positively stained in the neoplastic cells of 15 of 38 schwannomas (38%) and in two of 18 neurofibromas (11%). The extensively stained GFAP-positive tumors tended to be deeply situated in the body. The GFAP-positive cells were usually spindle-shaped and appeared preferentially in the perivascular region of hyalinized, thick blood vessels.

Ha-ras codon 12 mutation in papillary tumors of the urinary-bladder - a retrospective study.

This report concerns the retrospective examination of low grade, low stage (Ta/T1) bladder tumors for the Ha-ras codon 12 G-->T mutation. The patients studied had a minimum of 5 years follow-up and were grouped into 3 categories: (i) patients with no recurring tumors, (ii) patients with recurring Ta/T1 tumors and (iii) patients who subsequently developed high grade, high stage tumors. A heminested, non-isotopic, allele-specific polymerase chain reaction (PCR) amplification assay was used. The codon 12 G-->T mutation was found in 10/27 specimens from patients with non-recurring Ta/T1 tumors; in 11/27 initial and 12/23 recurring Ta/T1 tumors, and in 5/8 initial Ta/T1 lesions and 8/12 subsequently developed high grade/stage tumors. Although there was no correlation between disease recurrence and mutation, these results indicate that a relatively large proportion of patients with Ta/T1 tumors of the bladder have cells with the Ha-ras codon 12 G-->T substitution.

Gastric carcinoma cells with endocrine differentiation show no evidence of proliferation.

The proliferative activity of gastric cancer cells with endocrine features was evaluated in five cases by means of a double-immunostaining procedure. The endocrine cells were recognized by a monoclonal antibody to chromogranin A (CGA) and the proliferative activity by a monoclonal antibody to proliferating cell nuclear antigen (PCNA). With the use of two different chromogens it was easy to determine whether CGA was located in the cytoplasm and whether PCNA was located in the nucleus of the same section. The CGA-positive endocrine cells of the normal gastric antral mucosa could be readily distinguished from the PCNA-positive cells scattered in the mucosal neck zones. Over 1,000 CGA-positive cancer cells were counted per case. A few cells (average, less than 1.0%) exhibited faint nuclear staining with anti-PCNA; in no instance was unequivocal PCNA reactivity demonstrable in the gastric cancer cells with endocrine differentiation. By contrast, the PCNA reaction was positive in one fourth to one third of the other cancer cells. These observations suggest that gastric cancer cells with endocrine features are differentiated and do not participate in the cell cycle.

Desmoplastic reaction of gastric carcinoma: a light- and electron-microscopic immunohistochemical analysis using collagen type-specific antibodies.

The desmoplastic reaction in ten cases of gastric carcinoma was investigated light and electron immunohistochemically by using monospecific antibodies to collagen types. In addition to type I and III collagens, type V collagen was constantly recognized in the fibrous stroma, increasingly of the scirrhous carcinoma. Type IV collagen delineated the basement membranes of carcinoma nests linearly with occasional discontinuity, whereas in the scirrhous carcinoma, it was present along the thick bundles of collagenous fibers. Immunoelectron microscopic studies revealed that type I and III collagens were distributed on the collagen fibers, and type V collagen was stained in the margin of these fibers. These antibodies also reacted in the rough endoplasmic reticulum of fibroblasts or myofibroblasts in a few cases. Type IV collagen was localized in the periphery of smooth muscle cells, endothelial cells of collapsed capillaries, and myofibroblasts scattered in the stroma of scirrhous carcinoma. Carcinoma cells were not reactive with any antibodies examined. These findings suggest that type V collagen, as well as type I and III collagens, is involved in the formation of desmoplastic stroma, and that these collagens are reactively synthesized by fibroblasts and myofibroblasts in some interaction with invading carcinoma cells.

Detection of human cytomegalovirus, Epstein-Barr virus, and herpes simplex virus in diffuse interstitial pneumonia by polymerase chain reaction and immunohistochemistry.

Using formalin-fixed and paraffin-embedded tissues from autopsy, the authors examined infection by human cytomegalovirus, Epstein-Barr virus, and herpes simplex virus in 54 patients with primary or secondary diffuse interstitial pneumonia (DIP) by polymerase chain reaction and immunohistochemistry and compared it with that in 32 persons without lung complications. Polymerase chain reaction and immunohistochemistry demonstrated that approximately 40% and 30% of DIP were positive for human cytomegalovirus and Epstein-Barr virus, respectively, but none of 32 controls had evidence of infection by human cytomegalovirus and Epstein-Barr virus. The polymerase chain reaction was more sensitive than the immunohistochemical technique for detection of herpes simplex virus. The former technique revealed herpes simplex virus infection in approximately 90% of DIP and controls and the latter in approximately 50% of each group. However, immunohistochemistry had the advantage of demonstrating the morphologic location of infected cells and of allowing their semiquantitative evaluation. Herpes simplex virus was more extensively distributed in the lungs of several DIP cases than in those of controls, suggesting the reactivation of herpes simplex virus. Only DIP patients (31 cases [57.4%]) were infected by two or three kinds of herpesviruses. The combination of polymerase chain reaction and immunohistochemistry revealed that these herpesviruses proliferated in many cases of DIP.

Effects of subtilisin cleavage of monomeric actin on its nucleotide binding.

The kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1,N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 microM free Ca2+. Analysis of the dependence of k-ATP on free Ca2+ showed that the dissociation rate constant of tightly bound Ca2+ was not significantly changed by subtilisin cleavage. On the other hand, an equilibrium binding study using 8-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline N,N,N',N'-tetraacetic acid (Quin 2) showed that the affinity of tightly bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.

Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate.

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.

Numerical chromosome alterations in colorectal carcinomas detected by fluorescence in situ hybridization. Relationship to 17p and 18q allelic losses.

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific pericentromeric alpha-satellite DNA probes. Allelic losses were assessed by Southern blotting and by the polymerase chain reaction loss of heterozygosity method. Approximately 50% of the tumours were aneuploid. There was heterogeneity with respect to chromosome numbers, but gains and losses of chromosomes, or both, were detected in all carcinomas examined, including 10 that were nonaneuploid by image analysis. Trisomy 7 was found in 74% of the tumours, and monosomy of chromosome 18 in 32%. Allelic loss at chromosome 17p13.3 was evident in 13 of 26 informative cases, and only one case exhibited monosomy 17. In comparison monosomy 18 was found in 10 cases; 7 of them corresponded to approximately half of the cases with allelic loss within the DCC gene, and the other three were noninformative. These findings indicate that the loss of one chromosome 18 is an important mechanism producing allelic deletion of the DCC gene in colorectal carcinomas. Our data also suggest that monosomy 18 is a useful indicator for studying colorectal cancer progression on a cell by cell basis.

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.

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.

Ca2+-dependent regulation of the dynamic polarity of F-actin under the influence of tropomyosin and troponin.

A novel method that we have developed in the preceding paper to study the subunit exchange rates of F-actin (N. Suzuki and K. Mihashi, Biophys. Chem. 33 (1989) 177) was applied to regulated F-actin (a complex of F-actin, tropomyosin and troponin). We found that the dynamic polarity of regulated F-actin is modulated in a Ca2+-dependent manner, giving rise to strong suppression of the on/off rates of subunit exchange at the P-end. We interpreted this characteristic suppression as follows. Removal of Ca2+ from troponin C in regulated F-actin produces strong constraints on fluctuations in potential energy of an intermediate conformation of the terminal structure (P-end) which would be formed in the course of association and dissociation of the actin subunit.