Application of the research electronic data capture (REDCap) system in a low- and middle income country- experiences, lessons, and challenges.

The challenges of reliably collecting, storing, organizing, and analyzing research data are critical in low- and middle-income countries (LMICs), particularly in Sub-Saharan Africa where several healthcare and biomedical research organizations have limited data infrastructure. The Research Electronic Data Capture (REDCap) System has been widely used by many institutions and hospitals in the USA for data collection, entry, and management and could help solve this problem. This study reports on the experiences, challenges, and lessons learned from establishing and applying REDCap for a large US-Nigeria research partnership that includes two sites in Nigeria, (the College of Medicine of the University of Lagos (CMUL) and Jos University Teaching Hospital (JUTH)) and Northwestern University (NU) in Chicago, Illinois in the United States. The largest challenges to this implementation were significant technical obstacles: the lack of REDCap-trained personnel, transient electrical power supply, and slow/intermittent internet connectivity. However, asynchronous communication and on-site hands-on collaboration between the Nigerian sites and NU led to the successful installation and configuration of REDCap to meet the needs of the Nigerian sites. An example of one lesson learned is the use of Virtual Private Network (VPN) as a solution to poor internet connectivity at one of the sites, and its adoption is underway at the other. Virtual Private Servers (VPS) or shared online hosting were also evaluated and offer alternative solutions. Installing and using REDCap in LMIC institutions for research data management is feasible; however, planning for trained personnel and addressing electrical and internet infrastructural requirements are essential to optimize its use. Building this fundamental research capacity within LMICs across Africa could substantially enhance the potential for more cross-institutional and cross-country collaboration in future research endeavors.

A combined prognostic factor for improved risk stratification of patients with oral cancer.

PURPOSE: We aimed to identify a combined prognostic factor for predicting better performance in risk stratification. MATERIALS AND METHODS: We reviewed the clinical and pathological variables of 316 patients with oral squamous cell carcinoma (OSCC) who underwent surgery. To identify a combined predictor, principal component analysis (PCA) was performed. RESULTS: Univariate analysis showed that the independent prognostic variables for overall survival (OS) were pathologic T stage (T1 vs T4, HR = 1.99, 95% CI: = 1.083-3.675, P = 0.026) and pathologic N stage (N0 vs N2, HR=1.90, 95% CI: = 1.17-3.08, P = 0.008). In the multivariate analysis, only pathologic T stage was significant (P = 0.006 and P = 0.007); however, the multivariate model was not significant (P = 0.191). The multivariate model became significant by including lymph node ratio (LNR) instead of pathologic N stage (P = 0.0025 in numeric LNR, P = 0.0007 in categorized LNR). Also, the performance of prediction model was improved by a combined prognostic factor (P = 0.0002). CONCLUSIONS: The newly identified combined prognostic factor included resection margin, differentiation, and LNR, and they were insignificant factors independently except for LNR. This combined prognostic factor showed a good performance although it did not include molecular markers; therefore, it may be used conveniently for risk stratification of patients with OSCC by combining only clinical information.

Maternal age and asthma in Latino populations.

BACKGROUND: Younger maternal age at birth is associated with increased risk of asthma in offspring in European descent populations, but has not been studied in Latino populations. OBJECTIVES: We sought to examine the relationship between maternal age at birth and prevalence of asthma in a nationwide study of Latino children. METHODS: We included 3473 Latino children aged 8-21 years (1696 subjects with physician-diagnosed asthma and 1777 healthy controls) from five US centres and Puerto Rico recruited from July 2008 through November 2011. We used multiple logistic regression models to examine the effect of maternal age at birth on asthma in offspring overall and in analyses stratified by ethnic subgroup (Mexican American, Puerto Rican and other Latino). Secondary analyses evaluated the effects of siblings, acculturation and income on this relationship. RESULTS: Maternal age < 20 years was significantly associated with decreased odds of asthma in offspring, independent of other risk factors (OR = 0.73, 95% CI: 0.57-0.93). In subgroup analyses, the protective effect of younger maternal age was observed only in Mexican Americans (OR = 0.53, 95% CI: 0.36, 0.79). In Puerto Ricans, older maternal age was associated with decreased odds of asthma (OR = 0.65, 95% CI: 0.44-0.97). In further stratified models, the protective effect of younger maternal age in Mexican Americans was seen only in children without older siblings (OR = 0.44, 95% CI: 0.23-0.81). CONCLUSION AND CLINICAL RELEVANCE: In contrast to European descent populations, younger maternal age was associated with decreased odds of asthma in offspring in Mexican American women. Asthma is common in urban minority populations but the factors underlying the varying prevalence among different Latino ethnicities in the United States is not well understood. Maternal age represents one factor that may help to explain this variability.

DRP1 inhibition rescues retinal ganglion cells and their axons by preserving mitochondrial integrity in a mouse model of glaucoma.

Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic neuropathies.

Racial/ethnic differences in self-reported and biologic measures of chronic stress in pregnancy.

OBJECTIVE: Racial differences in chronic maternal stress may contribute to disparities in pregnancy outcomes. The objective is to identify racial and ethnic differences in self-reported and biologic measures of stress between non-Hispanic black (NHB) and non-Hispanic white (NHW) pregnant women. STUDY DESIGN: NHB and NHW pregnant women were enrolled before 23 weeks of gestation in this prospective cohort study. Equal numbers of women were recruited with public vs private insurance in each racial group. Self-reported stress was measured and blood samples collected in the second and third trimesters were analyzed for serum Epstein-Barr virus antibody, C-reactive protein (CRP), corticotropin-releasing hormone (CRH) and adenocorticotropic hormone (ACTH). RESULTS: One hundred and twelve women were enrolled. NHW women reported more buffers against stress (P=0.04) and neighborhood satisfaction (P=0.02). NHB women reported more discrimination (P<0.001), food insecurity (P=0.04) and had significantly higher mean CRP levels and mean ACTH levels in the second and third trimesters. CONCLUSION: Significant differences in self-reported and biologic measures of chronic stress were identified between NHB and NHW pregnant women with similar economic characteristics. Future studies should investigate mechanisms underlying these differences and their relationship to pregnancy outcomes.

Inhibition of cyclophilin D by cyclosporin A promotes retinal ganglion cell survival by preventing mitochondrial alteration in ischemic injury.

Cyclosporin A (CsA) inhibits the opening of the mitochondrial permeability transition pore (MPTP) by interacting with cyclophilin D (CypD) and ameliorates neuronal cell death in the central nervous system against ischemic injury. However, the molecular mechanisms underlying CypD/MPTP opening-mediated cell death in ischemic retinal injury induced by acute intraocular pressure (IOP) elevation remain unknown. We observed the first direct evidence that acute IOP elevation significantly upregulated CypD protein expression in ischemic retina at 12 h. However, CsA prevented the upregulation of CypD protein expression and promoted retinal ganglion cell (RGC) survival against ischemic injury. Moreover, CsA blocked apoptotic cell death by decreasing cleaved caspase-3 protein expression in ischemic retina. Of interest, although the expression level of Bcl-xL protein did not show a significant change in ischemic retina treated with vehicle or CsA at 12 h, ischemic damage induced the reduction of Bcl-xL immunoreactivity in RGCs. More importantly, CsA preserved Bcl-xL immunoreactivity in RGCs of ischemic retina. In parallel, acute IOP elevation significantly increased phosphorylated Bad (pBad) at Ser112 protein expression in ischemic retina at 12 h. However, CsA significantly preserved pBad protein expression in ischemic retina. Finally, acute IOP elevation significantly increased mitochondrial transcription factor A (Tfam) protein expression in ischemic retina at 12 h. However, CsA significantly preserved Tfam protein expression in ischemic retina. Studies on mitochondrial DNA (mtDNA) content in ischemic retina showed that there were no statistically significant differences in mtDNA content among control and ischemic groups treated with vehicle or CsA. Therefore, these results provide evidence that the activation of CypD-mediated MPTP opening is associated with the apoptotic pathway and the mitochondrial alteration in RGC death of ischemic retinal injury. On the basis of these observations, our findings suggest that CsA-mediated CypD inhibition may provide a promising therapeutic potential for protecting RGCs against ischemic injury-mediated mitochondrial dysfunction.

CD84 is markedly up-regulated in Kawasaki disease arteriopathy.

The major goals of Kawasaki disease (KD) therapy are to reduce inflammation and prevent thrombosis in the coronary arteries (CA), but some children do not respond to currently available non-specific therapies. New treatments have been difficult to develop because the molecular pathogenesis is unknown. In order to identify dysregulated gene expression in KD CA, we performed high-throughput RNA sequencing on KD and control CA, validated potentially dysregulated genes by real-time reverse transcription-polymerase chain reaction (RT-PCR) and localized protein expression by immunohistochemistry. Signalling lymphocyte activation molecule CD84 was up-regulated 16-fold (P < 0·01) in acute KD CA (within 2 months of onset) and 32-fold (P < 0·01) in chronic CA (5 months to years after onset). CD84 was localized to inflammatory cells in KD tissues. Genes associated with cellular proliferation, motility and survival were also up-regulated in KD CA, and immune activation molecules MX2 and SP140 were up-regulated in chronic KD. CD84, which facilitates immune responses and stabilizes platelet aggregates, is markedly up-regulated in KD CA in patients with acute and chronic arterial disease. We provide the first molecular evidence of dysregulated inflammatory responses persisting for months to years in CA significantly damaged by KD.

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes.

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 µM H2O2)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ10 can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ10 not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ10 prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ10 preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ10 may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics.

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1(enu/+) mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1(enu/+) mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.

Expression of acrB and ramA in fluoroquinolone resistant mutants from multi-drug resistant Salmonella enterica serovar Haardt.

AIMS: To identify the effect of elevation of resistance in multi-drug resistant Salmonella enterica serovar Haardt with reduced susceptibility to ciprofloxacin, a parental strain SAL59-FC-KF04 was exposed to fluoroquinolone. Sequence of quinolone resistance determining region (QRDR) was analysed and expression level of efflux pump related genes was compared between the parental strain and the derived mutants. METHODS AND RESULTS: The molecular expression levels of efflux pump genes (acrB, acrF) and transcriptional regulatory genes (marA, ramA, robA and soxS) were quantified using qRT-PCR. For the confirmation of the impact of efflux pump on drug-resistance, efflux inhibition test and sequence analysis of QRDR were performed. Two mutants were obtained by point mutation on QRDR and the increased level of expression of acrB and ramA. CONCLUSIONS: As a result of in vitro exposure to fluoroquinolones for parental strain, elevated fluoroquinolone resistance and overexpression of acrB and ramA have been observed. One of the mutants combined with additional QRDR point mutation showed increase of resistance to fluoroquinolone and several antimicrobials in other classes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates how continuous usage of antimicrobials affects the increase of drug-resistance in Salmonella enterica induced by QRDR mutation and efflux pump related genes.

Lack of O-polysaccharide enhances biofilm formation by Bradyrhizobium japonicum.

AIMS: To reveal the effects of the O-polysaccharide antigen of Bradyrhizobium japonicum LPS on biofilm formation and motility. METHODS AND RESULTS: Wild type and O-antigen-deficient mutant strains of B. japonicum were tested for biofilm formation on polyvinyl chloride (PVC) surfaces and motility on semi-solid (0.3%) agar media. After 7 days of incubation, the amount of biofilms formed by the mutant was c. 3.5-fold greater than that of the wild type. Unlike biofilm formation, the motility assay revealed that the mutant strain was less motile than the wild type. CONCLUSIONS: This study shows enhanced biofilm formation and decreased motility by the O-antigen-deficient mutant, suggesting that the lack of the O-polysaccharide of the rhizobial LPS is associated with biofilm-forming ability and movement. SIGNIFICANCE AND IMPACT OF THE STUDY: LPS plays an important role in both pathogenic and beneficial bacteria. It has also been reported that LPS deficiency negatively affects biofilm formation. However, our results demonstrate that the O-antigen-deficient mutant enhances biofilm formation, presumably through a significant increase in hydrophobicity. It is notable that the hydrophobicity of cell walls might be a key regulator in controlling biofilm development in B. japonicum.

Asarone inhibits adipogenesis and stimulates lipolysis in 3T3-L1 adipocytes.

Asarone is a molecule found in certain plants such as Acorus calamus, the root of which is used in traditional medicine to treat diabetes. We determined the molecular mechanism underlying the anti-diabetic activity of asarone. Treatment of asarone significantly inhibited the differentiation of 3T3-L1 preadipocytes through suppression of expression of the transcription factors, CCAAT/enhancer binding protein-alpha and peroxisome proliferator activated receptor-gamma, which activate adipogenesis. Intracellular triglyceride levels were reduced by asarone in a dose-dependent manner and asarone treatment stimulated the phosphorylation of hormone-sensitive lipase. Together, the present findings indicate that asarone inhibits adipogenesis by down-regulation of PPARgamma and C/EBPalpha and reduces lipid accumulation by stimulation of lipolysis through an increase in hormone-sensitive lipase activity.

Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.

BACKGROUND: Doxorubicin (DOX) is a widely used anticancer drug for solid tumors and hematologic malignancy, but its active use is hampered by serious adverse effects, including thrombocytopenia. Although bone marrow toxicity of DOX has been suggested to be the sole mechanism underlying the reduced platelet counts, the direct effects of DOX on platelets have never been examined. OBJECTIVE: Here, we investigated the DOX-induced platelet cytotoxicity and its underlying mechanism in an effort to elucidate the contribution of platelet cytotoxicity to DOX-induced thrombocytopenia. RESULTS: In freshly isolated human platelets, DOX induced platelet cytotoxicity in a time-dependent and concentration-dependent manner. Reactive oxygen species (ROS) generation, decreased glutathione levels and subsequent protein thiol depletion were shown to underlie the DOX-induced platelet cytotoxicity. Conspicuously, DOX-treated platelets displayed apoptotic features such as caspase-3 activation, reduced mitochondrial transmembrane potential, and phosphatidylserine exposure. Decreased glutathiolation of procaspase-3 was shown to be a link between protein thiol depletion and caspase-3 activation. It is of note that DOX-mediated platelet cytotoxicity was significantly enhanced by shear stress, a common complicating factor in cancer patients. These in vitro results were further confirmed by an in vivo animal model, where administration of DOX induced a platelet count decrease, ROS generation, caspase-3 activation, protein thiol depletion, and damaged platelet integrity. CONCLUSION: We demonstrated that DOX can directly induce platelet cytotoxicity through ROS generation, decreased glutathione levels, and protein thiol depletion. We believe that this study provides important evidence for the role of DOX-induced platelet cytotoxicity in the development of thrombocytopenia in DOX-treated patients.

The heat-shock protein 90 inhibitor, geldanamycin, induces apoptotic cell death in Epstein-Barr virus-positive NK/T-cell lymphoma by Akt down-regulation.

NK/T-cell lymphoma (NKTL) is strongly associated with latent Epstein-Barr virus (EBV) infection. Recently, latent membrane protein 1 (LMP1), an EBV oncoprotein, was reported to activate the phosphatidylinositol-3 kinase (PI3K)/Akt pathway for cell survival. Because geldanamycin (GA) and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG), exhibit anti-tumour activity by degrading HSP90 client proteins, including Akt, we investigated the effect of GA and 17-AAG on the survival of NKTL cell lines. EBV-positive NKTL cell lines, Hank-1 and NK-YS, and an EBV-negative NK leukaemia cell line, NK-L, were treated with PI3K and Akt inhibitors, GA, and 17-AAG, and were subjected to apoptosis and cell viability assays, and immunoblot analysis. EBV-positive B-lymphoblastoid cell lines IM9 and LMP1-transfected IM9 (IM9-LMP1) were also included. Hank-1 and NK-YS cell viability was compromised and apoptosis was induced by LY294002 (PI3K inhibitor) or Akt inhibitor II. GA or 17-AAG administration resulted in the apoptosis of NKTL cells, accompanied by Akt and pAkt down-regulation, caspase 3 activation, and mitochondrial membrane potential disruption. The intrinsic level of pAkt was higher in EBV-positive NKTL cells than in EBV-negative NK-L, and GA or 17-AAG decreased the viability of NKTL cells more efficiently than NK-L. Moreover, IM9-LMP1 was more sensitive to Akt inhibitor II or HSP90 inhibitors than IM9. Importantly, GA showed little effect on the viability of normal peripheral NK cells as non-neoplastic counterparts for comparison. In conclusion, this study suggests that the PI3K/Akt pathway is frequently activated in EBV-positive NKTL and that therapeutic modalities based on targeting the PI3K/Akt pathway with HSP90 inhibitors could be useful for achieving NKTL control.

A 37-year-old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood, with improved sensory perception and mobility, both functionally and morphologically: a case study.

HLA-matched UC blood-derived multipotent stem cells were directly transplanted into the injured spinal cord site of a 37-year-old female patient suffering from spinal cord injury (SPI). In this case, human cord blood (UCB)-derived multipotent stem cells improved sensory perception and movement in the SPI patient's hips and thighs within 41 days of cell transplantation. CT and MRI results also showed regeneration of the spinal cord at the injured site and some of the cauda equina below it. Therefore, it is suggested that UCB multipotent stem cell transplantation could be a good treatment method for SPI patients.

Gamma-glutamyltransferase: an effect modifier in the association between age and hypertension in a 4-year follow-up study.

We performed a prospective study to assess whether the relationship of age with hypertension was stronger in men with high normal serum gamma glutamyltransferase (GGT) than in those with lower GGT levels. The study population included 8170 healthy male workers in a steel manufacturing company who had undergone health examinations in both 1994 and 1998. The higher the baseline GGT level, the effect of age on the development of hypertension was stronger. The incidence of hypertension among those aged 25-34, 35-44 and 45-50 years was 0.9, 2.2, 3.8% in those with GGT<20 U/l; 1.0, 4.1, 12.5% in those with GGT between 20 and 39 U/l; and 1.9, 6.3, 17.2% in those with GGT> or =40 U/l, respectively. All relationships persisted after adjusting for baseline values of body mass index, alcohol intake, smoking, exercise, family history of hypertension, systolic and diastolic blood pressure, and changes of body mass index during 4 years (P for interaction=0.03). Our data supported the hypothesis that the effect of age on the development of hypertension differed by baseline GGT level, although the underlying mechanism for this interaction is unclear.

Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina.

AIMS/HYPOTHESIS: Neurodegenerative changes in the diabetic retina occurring before diabetic retinopathy could be inevitable by the altered energy (glucose) metabolism, in the sense that dynamic image-processing activity of the retinal neurons is exclusively dependent on glucose. We therefore investigated the morphological changes in the neural retina, including neuronal cell death, of a streptozotocin-induced model of diabetes. METHODS: Streptozotocin was intravenously injected. Rats were maintained hyperglycaemic without insulin treatment for 1 week and 4, 8, 12, and 24 weeks, respectively. Diabetic retinas were processed for histology, electron microscopy, and immunohistochemistry using the TUNEL method. RESULTS: A slight reduction in the thickness of the inner retina was observed throughout the diabetic retinas and a remarkable reduction was seen in the outer nuclear layer 24 weeks after the onset of diabetes. The post-synaptic processes of horizontal cells in the deep invaginations of the photoreceptors showed degeneration changes from 1 week onwards. A few necrotic ganglion cells were observed after 4 weeks. At 12 weeks, some amacrine cells and a few horizontal cells showed necrotic features. Three to seven cellular layers in the outer nuclear layer and nerve terminals, rolled by the fine processes of the Müller cells near the somata of the degenerated ganglion cells, were apparent at 24 weeks. Apoptosis appeared in a few photoreceptor cells at 4 weeks, and the number of apoptotic photoreceptors increased thereafter. CONCLUSION/INTERPRETATION: These findings suggest that the visual loss associated with diabetic retinopathy could be attributed to an early phase of substantial photoreceptor loss, in addition to later microangiopathy.

Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch.

A DO-stat control strategy for two variables was introduced to the rGuamerin production process in Pichia pastoris and applied to repeated fed-batch culture. Two interrelated variables, namely the ratio of partial pressure of pure O2 in the inlet air-stream and the methanol feed rate, were controlled simultaneously. By using this control strategy, methanol feeding for induction could be controlled automatically while efficiently controlling the dissolved oxygen level. As a result, the cell concentration reached more than 140 g l(-1) and rGuamerin expression level 450 iu l(-1). rGuamerin was secreted into the culture medium and reached a level that was 40% higher than achieved in a fed-batch process using manual control of the methanol feeding rate. Repeated rGuamerin induction was achieved by repeating the methanol feeding and withdrawing the culture broth during extended production. During more than 250 h of culture, expression of rGuamerin was maintained at an average of about 430 iu l(-1 )(473 mg l(-1)), without causing the cell density to decrease. In addition to the rGuamerin production process, the proposed control system might be applied to cultivation of other methylotrophic yeasts in the production of therapeutic proteins.

X-ray and extreme ultraviolet emission induced by variable pulse-width irradiation of Ar and Kr clusters and droplets

Measurements are presented of x-ray (>1.5 keV) and extreme ultraviolet (EUV, lambda=2-44 nm) emission from argon and krypton supersonic gas jets at room (T=300 K) and cryogenic (T=173 K) temperatures irradiated with constant energy (50 mJ), variable width laser pulses ranging from 100 fs to 10 ns. Two regimes of jet operation are explored: cluster formation (radius<100 nm) and droplet formation (radius>1 &mgr;m). The results for both clusters and droplets can be understood in terms of two time scales: a short time scale for optimal resonant absorption at the critical density layer in the expanding plasma, and a longer time scale for the plasma to drop below critical density.