Cladding pumped few-mode EDFA for mode division multiplexed transmission.

We experimentally demonstrate a few-mode erbium doped fiber amplifier (FM-EDFA) supporting 6 spatial modes with a cladding pumped architecture. Average modal gains are measured to be >20dB between 1534nm-1565nm with a differential modal gain of ~3dB among the mode groups and noise figures of 6-7dB. The cladding pumped FM-EDFA offers a cost effective alternative to core-pumped variant as low cost, high power multimode pumps can be used, and offers performance, scalability and simplicity to FM-EDFA design.

Non-invasive detection of regulatory T cells with Raman spectroscopy.

Regulatory T cells (Tregs) are a type of lymphocyte that is key to maintaining immunological self-tolerance, with great potential for therapeutic applications. A long-standing challenge in the study of Tregs is that the only way they can be unambiguously identified is by using invasive intracellular markers. Practically, the purification of live Tregs is often compromised by other cell types since only surrogate surface markers can be used. We present here a non-invasive method based on Raman spectroscopy that can detect live unaltered Tregs by coupling optical detection with machine learning implemented with regularized logistic regression. We demonstrate the validity of this approach first on murine cells expressing a surface Foxp3 reporter, and then on peripheral blood human T cells. By including methods to account for sample purity, we could generate reliable models that can identify Tregs with an accuracy higher than 80%, which is already comparable with typical sorting purities achievable with standard methods that use proxy surface markers. We could also demonstrate that it is possible to reliably detect Tregs in fully independent donors that are not part of the model training, a key milestone for practical applications.

Population response to information on reversibility of Type 2 diabetes.

AIMS: Following publication of the Counterpoint Study (on the reversibility of Type 2 diabetes using a very low energy diet), the extent of public interest prompted the authors to make available, on a website, general information about reversing diabetes. Shortly thereafter, individuals began to feed back their personal experiences of attempting to reverse their diabetes. We have collated this information on the effects of energy restriction in motivated individuals with Type 2 diabetes that has been achieved outside a research setting. METHODS: Emails, letters and telephone communications received between July 2011 and September 2012 were evaluated (n = 77: 66 men, 11 women). Median diabetes duration was 5.5 years (3 months-28 years). Reversal of diabetes was defined as achieving fasting capillary blood glucose < 6.1 mmol/l and/or, if available, HbA1c less than 43 mmol/mol (6.1%) off treatment. RESULTS: Self-reported weight fell from 96.7 ± 17.5 kg at baseline to 81.9 ± 14.8 kg after weight loss (P < 0.001). Self-reported fasting blood glucose levels fell from 8.3 mmol/l (5.9-33.0) to 5.5 mmol/l (4.0-10.0) after the weight loss period (P < 0.001). Diabetes reversal was considered to have occurred in 61% of the population. Reversal of diabetes was observed in 80, 63 and 53% of those with > 20, 10-20 and < 10 kg weight loss, respectively. There was a significant correlation between degree of weight loss and reported fasting glucose levels (Rs -0.38, P = 0.006). Reversal rates according to diabetes duration were: short (< 4 years) = 73%, medium (4-8 years) = 56% and long (> 8 years) = 43%. CONCLUSION: These data demonstrate that intentional weight loss achieved at home by health-motivated individuals can reverse Type 2 diabetes. Diabetes reversal should be a goal in the management of Type 2 diabetes.

Reversal of type 2 diabetes: normalisation of beta cell function in association with decreased pancreas and liver triacylglycerol.

AIMS/HYPOTHESIS: Type 2 diabetes is regarded as inevitably progressive, with irreversible beta cell failure. The hypothesis was tested that both beta cell failure and insulin resistance can be reversed by dietary restriction of energy intake. METHODS: Eleven people with type 2 diabetes (49.5 ± 2.5 years, BMI 33.6 ± 1.2 kg/m(2), nine male and two female) were studied before and after 1, 4 and 8 weeks of a 2.5 MJ (600 kcal)/day diet. Basal hepatic glucose output, hepatic and peripheral insulin sensitivity and beta cell function were measured. Pancreas and liver triacylglycerol content was measured using three-point Dixon magnetic resonance imaging. An age-, sex- and weight-matched group of eight non-diabetic participants was studied. RESULTS: After 1 week of restricted energy intake, fasting plasma glucose normalised in the diabetic group (from 9.2 ± 0.4 to 5.9 ± 0.4 mmol/l; p = 0.003). Insulin suppression of hepatic glucose output improved from 43 ± 4% to 74 ± 5% (p = 0.003 vs baseline; controls 68 ± 5%). Hepatic triacylglycerol content fell from 12.8 ± 2.4% in the diabetic group to 2.9 ± 0.2% by week 8 (p = 0.003). The first-phase insulin response increased during the study period (0.19 ± 0.02 to 0.46 ± 0.07 nmol min(-1) m(-2); p < 0.001) and approached control values (0.62 ± 0.15 nmol min(-1) m(-2); p = 0.42). Maximal insulin response became supranormal at 8 weeks (1.37 ± 0.27 vs controls 1.15 ± 0.18 nmol min(-1) m(-2)). Pancreatic triacylglycerol decreased from 8.0 ± 1.6% to 6.2 ± 1.1% (p = 0.03). CONCLUSIONS/INTERPRETATION: Normalisation of both beta cell function and hepatic insulin sensitivity in type 2 diabetes was achieved by dietary energy restriction alone. This was associated with decreased pancreatic and liver triacylglycerol stores. The abnormalities underlying type 2 diabetes are reversible by reducing dietary energy intake.

Multicenter external validation of the ABCD2 score in triaging TIA patients.

OBJECTIVES: A simple clinical score (ABCD(2) score) has been introduced to triage TIA patients with a high early risk of stroke. External validation studies have yielded inconsistent results regarding the predictive ability of the ABCD(2) score. We aimed to prospectively validate the former score in a multicenter case series study. METHODS: We prospectively calculated the ABCD(2) score (age [> or = 60 years: 1 point]; blood pressure [systolic >140 mm Hg or diastolic >90 mm Hg: 1[; clinical features [unilateral weakness: 2, speech disturbance without weakness: 1, other symptom: 0]; duration of symptoms [ <10 minutes: 0, 10-59 minutes: 1, > or = 60 minutes: 2]; diabetes mellitus [yes: 1]) in consecutive TIA patients hospitalized in 3 tertiary care neurology departments across 2 different racial populations (white and Asian). RESULTS: The 7-day and 90-day risks of stroke in the present case series (n = 148) were 8% (95% CI 4%-12%) and 16% (95% CI 10%-22%). The ABCD(2) score accurately discriminated between TIA patients with high 7-day (c statistic 0.72, 95% CI 0.57-0.88) and 90-day (c statistic 0.75, 95% CI 0.65-0.86) risks of stroke. The 90-day risk of stroke was 7-fold higher in patients with an ABCD(2) score >3 points (28%, 95% CI 18%-38%) than in patients with an ABCD(2) score < or = 3 points (4%, 95% CI 0%-9%). After adjustment for stroke risk factors, race, history of previous TIA, medication use before the index TIA and secondary prevention treatment strategies, an ABCD(2) score of >2 was associated with a nearly 5-fold greater 90-day risk of stroke (hazard ratio 4.65, 95% CI 1.04-20.84, p = 0.045). CONCLUSION: Our findings externally validate the usefulness of the ABCD(2) score in triaging TIA patients with a high risk of early stroke in a multiethnic sample of hospitalized patients. The present data support current guidelines endorsing the immediate hospitalization of patients with an ABCD(2) score >2.

Intrauterine growth rate in pregnancies complicated by type 1, type 2 and gestational diabetes.

Fetal macrosomia is a feature of all subtypes of maternal diabetes. The intrauterine time course of development of macrosomia in type 1, type 2 and gestational diabetes (GDM) could identify the times of more rapid growth, which differ as a result of different influences in subtypes of diabetes. Higher maternal weight in type 2 and GDM may be expected to contribute to macrosomia and the blood glucose control will exert an additional influence. Information was collected prospectively on 217 pregnancies in insulin-treated women at a single centre over a six-year period. All women were managed by a single team of obstetricians and diabetologists at a Joint Obstetric Medical Clinic. The rate of increase in abdominal circumference from 28 weeks was identical in each subtype of diabetes and there were no differences between subtypes at the earliest gestation assessed. Use of customized growth centiles showed rates of macrosomia to be similar in type 1, type 2 and GDM (43.0%, 50.0% and 41.8%, respectively). The intrauterine time course to macrosomia is similar in type 1, type 2 and GDM. The relationship of macrosomia to extent of elevation of mean blood glucose control is weak, implying a low threshold for maximal effect on the rate of fetal growth.

Molecular identification of nanoplanktonic protists based on small subunit ribosomal RNA gene sequences for ecological studies.

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.

Development and field application of a quantitative method for examining natural assemblages of protists with oligonucleotide probes.

A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists in cultures and environmental water samples is described. Filtration, hybridization, and enumeration of fixed cells with biotinylated eukaryote-specific probes and fluorescein isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, Mass.). Counts of various species of cultured protists by this probe hybridization method were not significantly different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. However, counts of total nanoplankton (TNAN) based on probe hybridizations in several field samples and in samples collected from a mesocosm experiment were frequently higher than TNAN counts obtained by staining with DAPI or AO. On the basis of these results, 25 to 70% of the TNAN determined with probes were not detectable by DAPI or AO staining. The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed to the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with distinguishing DAPI- or AO-stained protists attached to or embedded in aggregates. We conclude from samples examined in this study that enumeration of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains. The quantitative in situ hybridization method we have described here enables the direct enumeration of free-living protists in water samples with oligonucleotide probes. When combined with species-specific probes, this method will enable quantitative studies of the abundance and distribution of specific protistan taxa.

Identification of heterotrophic nanoflagellates by restriction fragment length polymorphism analysis of small subunit ribosomal DNA.

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.

Application of rRNA-based probes for observing marine nanoplanktonic protists.

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.

Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells.

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10(4) cells x ml(-1). The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells x ml(-1) by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

Chemical degradation of composite restoratives.

The chemical environment is one aspect of the oral environment, which could have an appreciable influence on the in vivo degradation of composite restoratives. The effects of chemical media on surface hardness of four composite restoratives (Silux [SX], Z100 [ZO], Ariston [AR] and Surefil [SF]) were investigated. The relationship between hardness and the thickness of the degradation layer was also studied. Thirty six specimens (3 x 4 x 2 mm) were made for each material. Following polymerization, the specimens were stored in artificial saliva at 37 degrees C for 24 h. The specimens were then randomly divided into six groups of six, subjected to microhardness testing (load = 500 gf, dwell time = 15 s) and stored in the following chemicals for 1 week at 37 degrees C: artificial saliva (S), distilled water (W), 0.02 N citric acid (C), 0.02 N lactic acid (L), heptane (H) and 75-25% ethanol-water solution (E). After conditioning, the specimens were again subjected to hardness testing and sectioned. Change in hardness (DH) was computed and the thickness of the degradation layer (DL) was measured using a computerized image analysis system at 600x magnification. Results of statistical analysis (ANOVA/Scheffe's [P < 0.05]) of DH based on materials were as follows: SX - E > all other mediums; ZO - W > C; and AR - S, W, E > H (> indicates significantly greater hardness change). No significant difference in DH was observed between the different chemicals for SF. The effects of chemical media on DH were found to be material dependent. A significant but weak positive correlation (Pearson's correlation [P < 0.05]) exists between change in hardness and thickness of the degradation layer.

Light-dependent phagotrophy in the freshwater mixotrophic chrysophyte Dinobryon cylindricum.

The mixotrophic (bacterivorous), freshwater chrysophyte Dinobryon cylindricum was cultured under a variety of light regimes and in bacterized and axenic cultures to investigate the role of phototrophy and phagotrophy for the growth of this alga. D. cylindricum was found to be an obligate phototroph. The alga was unable to survive in continuous darkness even when cultures were supplemented with high concentrations of bacteria, and bacterivory ceased in cultures placed in the dark for a period longer than one day. Axenic growth of the alga was poor even in an optimal light regime. Live bacteria were required for sustained, vigorous growth of the alga in the light. Carbon (C), nitrogen (N), and phosphorus (P) budgets determined for the alga during growth in bacterized cultures indicated that bacterial biomass ingested by the alga may have contributed up to 25% of the organic carbon budget of the alga. Photosynthesis was the source of most ([Symbol: see text]75%) of the organic carbon of the alga. D. cylindricum populations survived but did not grow when cultured in a continuous low light intensity (30 µE m(-2) sec(-1)), or in a light intensity of 150 µE m(-2) sec(-1) for only two hours each day. Net efficiency of incorporation of bacterial C, N, and P into algal biomass under these two conditions was zero (i.e., no net algal population growth). We conclude that the primary function of bacterivorous behavior in D. cylindricum may be to provide essential growth factor(s) or major nutrients for photosynthetic growth, or to allow for the survival of individuals during periods of very low light intensity or short photoperiod.