Random Mutagenesis by PCR.

Random mutagenesis of DNA sequences has the advantage of generating DNA sequences with novel properties, either directly in the case of aptamers or through subsequent transcription/translation of the mutated sequence in the case of proteins. In both cases no prior structural or mechanistic knowledge of the molecule is required. For sequences greater than 100 bp, one of the easiest methods to introduce the mutations is to use Error-prone PCR (EP-PCR) as discussed in this chapter. When coupled with an appropriate selection or high throughput screening methodology, PCR-based random mutagenesis can provide a powerful tool for modern molecular biologists.

High prevalence of vaterite in sagittal otoliths causes hearing impairment in farmed fish.

The rapid growth of aquaculture raises questions about the welfare status of mass-produced species. Sagittal otoliths are primary hearing structures in the inner ear of all teleost (bony) fishes and are normally composed of aragonite, though abnormal vaterite replacement is sometimes seen in the wild. We provide the first widespread evaluation of the prevalence of vaterite in otoliths, showing that farmed fish have levels of vaterite replacement over 10 times higher than wild fish, regardless of species. We confirm this observation with extensive sampling of wild and farmed Atlantic salmon in Norway, the world's largest producer, and verify that vateritic otoliths are common in farmed salmon worldwide. Using a mechanistic model of otolith oscillation in response to sound, we demonstrate that average levels of vaterite replacement result in a 28-50% loss of otolith functionality across most of a salmonid's known hearing range and throughout its life cycle. The underlying cause(s) of vaterite formation remain unknown, but the prevalence of hearing impairment in farmed fish has important implications for animal welfare, the survival of escapees and their effects on wild populations, and the efficacy of restocking programs based on captive-bred fish.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

SETTING: Hanoi Lung Hospital, Hanoi, Viet Nam. OBJECTIVE: To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. DESIGN: Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. RESULTS: Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. DISCUSSION: CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes.

Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion. The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA). H3 and H4 acetylation was found targeted to the CpG island region at the 5' end of both these genes, falling off in the downstream direction. In contrast, at the beta(A)-globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter. Low level acetylation was observed at the 5' end of the inactive ovalbumin gene. Run-on assays to measure ongoing transcription showed that the GAPDH and CA genes are transcribed at a much lower rate than the adult beta(A)-globin gene. The extensive high level acetylation at the beta(A)-globin gene correlates most simply with its high rate of transcription. The targeted acetylation of histones H3 and H4 at the GAPDH and CA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

Three-dimensional microscopy data exploration by interactive volume visualization.

This paper presents a new volume visualization approach for three-dimensional (3-D) interactive microscopy data exploration. Because of their unique image characteristics, 3-D microscopy data are often not able to be visualized effectively by conventional volume visualization techniques. In our approach, microscopy visualization is carried out in an interactive data exploration environment, based on a combination of interactive volume rendering techniques and image-based transfer function design methods. Interactive volume rendering is achieved by using two-dimensional (2-D) texture mapping in a Shear-Warp volume rendering algorithm. Image processing techniques are employed and integrated into the rendering pipeline for the definition and searching of appropriate transfer functions that best reflect the user's visualization intentions. These techniques have been implemented successfully in a prototype visualization system on low-end and middle-range SGI desktop workstations. Since only 2-D texture mapping is required, the system can also be easily ported to PC platforms.

Moving home: costs associated with different models of accommodation for adults with learning disabilities.

This paper reports on variation in the cost and social outcomes of provision for clients with learning disabilities, resettled across different models of community accommodation; identifying personal and service-related characteristics influential on such variation. The study was conducted to inform the community reprovisioning strategy associated with the phased closure of the Gogarburn and Tornaveen learning disability hospitals in the Lothian region, Scotland. Total mean service costs, quality of life and community integration outcome data were collected and compared. Total mean service costs ranged between pound16 438 and pound74 097 per year (mean pound42 023; SD = pound16 712). Cost estimates varied by age and dependency group, with costs for elderly clients comparatively low. Overall, there was an inverse relationship between total mean costs and size of accommodation. There was no evidence of a direct relationship between costs and changes in social outcomes. Services with the lowest mean costs were, however, associated with the smallest increases in social outcomes. The most expensive services did not realise proportionally greater gains in social outcomes. The paper concludes that clients generally benefited from the transition from hospital to community accommodation. However, some experienced better outcomes than others. In the absence of a clear link between levels and type of resource use and social outcomes, it is difficult to identify which service features are more or less efficacious in achieving positive outcomes. Broader evaluation perspectives, embracing a fuller range of costs and benefits, will be required to unpack exactly what it is about different models of community care provision that leads to positive outcomes, or otherwise, for learning disability clients. A more sound evidence base is required before effective strategies can be designed to ensure that key policy outcomes are realised and social integration truly achieved.

Relationship between social rhythms and mood in patients with rapid cycling bipolar disorder.

Disruptions in the sleep-wake cycle frequently characterize affective illness and have led to a number of theories linking sleep-wake and/or circadian rhythm disturbance to affective illness. Recently, researchers have expanded these chronobiological theories to include the role of lifestyle regularity, or daily social rhythms. In this study, the Social Rhythm Metric (SRM) was used to explore the relationship between social rhythms and mood in patients with rapid cycling bipolar disorder and to compare the social rhythms of patients with those of healthy control subjects. Patients' SRM scores and activity level indices were significantly lower than those of control subjects. In addition, the timing of five, mostly morning, activities was phase delayed in patients compared to control subjects. Patients also demonstrated a phase delay in the timing of morning activities during depression compared to hypomania or euthymia. The phase changes in the timing of morning activities are consistent with other data implicating morning zeitgebers in the pathophysiology of rapid cycling bipolar disorder.

Idiopathic reactive hyperplasia of the retinal pigment epithelium.

OBJECTIVES: To present and discuss 2 patients with acquired peripapillary pigmented lesions. METHODS: We reviewed the patients' clinical records and histopathologic findings. RESULTS: The first patient was diagnosed with a pigmented papillary lesion that was followed up for 38 years. The second patient was a child with neurofibromatosis type 1 who developed a pigmented peripapillary lesion following excision of an optic nerve glioma. Histologic findings in both cases demonstrated hyperplasia of the retinal pigment epithelium with associated findings. CONCLUSIONS: The lesions presented an idiopathic reactive hyperplasia of the retinal pigment epithelium. The clinical and histopathologic findings resemble findings reported with the combined hamartoma. We suggest that such lesions are reactive in nature, rather than hamartomatous.

Greater improvement in summer than with light treatment in winter in patients with seasonal affective disorder.

OBJECTIVE: The authors sought to compare the degree of mood improvement after light treatment with mood improvement in the subsequent summer in patients with seasonal affective disorder. METHOD: By using the Seasonal Affective Disorder Version of the Hamilton Depression Rating Scale, the authors rated 15 patients with seasonal affective disorder on three occasions: during winter when the patients were depressed, during winter following 2 weeks of light therapy, and during the following summer. They compared the three conditions by using Friedman's analysis of variance and the Wilcoxon signed ranks test. RESULTS: The patients' scores on the depression scale were significantly higher after 2 weeks of light therapy in winter than during the following summer. CONCLUSIONS: Light treatment for 2 weeks in winter is only partially effective when compared to summer. Further studies will be necessary to assess if summer's light or other factors are the main contributors to this difference.

Validation of the hypomania interview guide-seasonal affective disorder (HIGH-SAD) version in patients with rapid cycling bipolar disorder.

We validated the Hypomania Interview Guide-Seasonal Affective Disorder version (HIGH-SAD) in patients with rapid cycling bipolar disorder (RCBD). Fourteen outpatients were rated on six separate occasions (total = 84 visits). On each visit the patients were rated with the HIGH-SAD and the Young Mania Rating Scale (YMRS) in a counterbalanced order. Clinical assessment was completed at the end of the visit by the treating psychiatrist. Patients were assessed as hypomanic/manic on 22 of the visits. Pearson correlation coefficient between the YMRS total scores and the HIGH-SAD total scores for those 22 visits in which patients were hypomanic/manic was r = 0.629 (P < 0.05) and for all visits was r = 0.769 (P < 0.0001). Analysis with only one rating per patient yielded a Pearson correlation coefficient of r = 0.792 (P < 0.0004). We found that the HIGH-SAD was a valid scale for the measurement of hypomania in patients with RCBD. However, the scale does not differentiate hypomania from mania in this group of patients.

Subretinal fibrosis in diabetic macular edema. ETDRS report 23. Early Treatment Diabetic Retinopathy Study Research Group.

OBJECTIVE: To describe the characteristics of and risk factors for subretinal fibrosis (SRF) in patients with diabetic macular edema. PATIENTS AND METHODS: A total of 109 eyes (in 96 persons) with SRF, defined as a mound or sheet of gray to white tissue beneath the retina at or near the center of the macula, were identified during the Early Treatment Diabetic Retinopathy Study, which is a randomized clinical trial of photocoagulation and aspirin treatment in patients with mild to severe nonproliferative or early proliferative diabetic retinopathy. The patients and the ocular characteristics of these 109 eyes, all of which had clinically significant macular edema, were compared with those of 5653 eyes in which clinically significant macular edema, but not SRF, was observed during the trial. RESULTS: In 9 of 109 eyes, the development of SRF may have been directly related to focal photocoagulation. Seventy-four percent of the eyes in which SRF developed had very severe hard exudates in the macula prior to the development of SRF, while this level of hard exudates was seen in only 2.5% of the eyes with clinically significant macular edema in which SRF did not develop (P < .001). Of the 264 eyes with this level of hard exudates at baseline (n = 29) or during follow-up (n = 235), SRF developed in 30.7% of the eyes, while this complication developed in only 0.05% of 5498 eyes with clinically significant macular edema without this level of hard exudates. CONCLUSIONS: Subretinal fibrosis is an infrequent complication of diabetic macular edema. Although it has been reported to be associated with photocoagulation burn intensity, in only 9 of 109 eyes in which SRF developed was it located adjacent to a photocoagulation-related scar (among 4823 eyes that received focal photocoagulation for treatment of macular edema). The strongest risk factor for the development of SRF is very severe hard exudate.

Agreement between face-to-face and telephone-administered mood ratings in patients with rapid cycling bipolar disorder.

We examined the reliability and level of agreement between the telephone and face-to-face administration of two mood-rating scales (HIGH-SAD and SIGH-SAD) in patients with rapid cycling bipolar disorder (RCBD). Two clinicians administered the HIGH-SAD and SIGH-SAD to 14 outpatients with RCBD. Patients received consecutive phone and face-to-face mood ratings in a randomized order. Using a paired t-test, no significant differences were found when comparing HIGH-SAD and SIGH-SAD scores administered face-to-face and over the phone. There was a high correlation between the face-to-face and phone administration of both scales as measured by intraclass correlation (r = 0.94 for SIGH-SAD; r = 0.85 for HIGH-SAD). Our results support the use of phone-administered mood ratings as a reliable and convenient method to monitor patients with RCBD.

Immunocytochemical features of retinoblastoma in an adult.

OBJECTIVE: To describe the clinical course and immunocytochemical characteristics of an unusual intraocular tumor. METHODS: Immunocytochemical analysis of the enucleated eye with an intraocular mass that markedly waxed and waned in size during 1 year of close observation of a 29-year-old woman. RESULTS: Most of the tumor was composed of either dying or rapidly proliferating cells. One area located near the retina consisted mostly of well-differentiated cells in uniform sheets (bacillettes) with lacelike glial processes between the tumor cells. Almost all of the differentiated tumor cells were positive for S antigen. In particular, the dominant cell type stained positively for both antibodies known to be specific for those isoforms of S antigen found only in blue cones and rods but not in red or green cones. Only a few of these cells labeled positively with an anti-rhodopsin antibody. CONCLUSIONS: This is the first case of adult retinoblastoma to be confirmed immunocytochemically. The tumor was unusual because the differentiated regions contained bacillettes composed mostly of blue cones. It is possible that this and other adult retinoblastomas may arise from previously existing retinocytomas.

Effects of blood alcohol concentrations on negative punishment: implications for decision making.

OBJECTIVE: A series of studies examined how various blood ethanol concentrations influenced the effectiveness of a negative punishment procedure in a risk-taking/decision-making task. Based on the animal punishment literature plus the impression that ethanol diminishes the effects of punishment and impairs decision making, it was predicted that individuals receiving alcohol would increase their risk- taking behavior despite the presence of a negative punishment stimulus (point loss) given on a variable schedule. METHODS: In Experiment 1, a within-subjects design with repeated measures was used where 10 male subjects played a modified computer-generated choice game under three levels of punishment certainty, three levels of punishment severity and four ranges of blood ethanol (0.00%, 0.01-0.049%, 0.050-0.099%, 0.10-0.15%) RESULTS: Results indicated significant decreases in risk taking as either punishment certainty or severity was increased. However, no significant effects were found for ethanol, or interactions of ethanol with punishment certainty or punishment severity. Two additional studies examining aspects of the experimental procedure produced similar results CONCLUSIONS: Overall, the results of these studies do not support the hypothesis that high concentrations of blood alcohol are sufficient in and of themselves to eliminate the suppression function of negative punishment contingencies leading to increased risk-taking behavior. The implications of these results for understanding blood alcohol effects are discussed.

Effects of season on electro-oculographic ratio in winter seasonal affective disorder.

Low electro-oculographic (EOG) ratios have been reported in patients with seasonal affective disorder (SAD) during the winter. This study evaluated the effects of the changing seasons on EOG ratios in SAD patients. Sixteen outpatients with SAD and 16 age-, sex-, and medication-matched normal volunteers had EOG testing during the winter and again during the summer. There was a significant season x group interaction in EOG ratios, with normal subjects showing higher ratios in winter than in summer--a seasonal variation not observed in SAD patients. SAD patients may have a subsensitivity to environmental light that leads them to experience symptoms during the winter.

Degradation of maternal string mRNA is controlled by proteins encoded on maternally contributed transcripts.

In Drosophila, maternal string mRNAs are stable for the first few hours of development, but undergo specific timed degradation at the cellularisation stage. To determine whether the proteins that control this degradation are maternally or zygotically transcribed, we have used in situ hybridisation to determine the fate of maternal string transcripts in mutant embryos which lack individual chromosome arms. Our data indicate that maternal string mRNA persists for the normal period in all these mutants. Using alpha-amanitin to inhibit zygotic transcription we have shown that degradation of maternal mRNA is unaffected. Therefore, the proteins required to activate the degradation of string mRNA are encoded on a maternally contributed mRNA. We discuss possible models to explain the degradation pathway.

The reproducibility of depressive and hypomanic symptoms across repeated episodes in patients with rapid-cycling bipolar disorder.

The purpose of this study is to determine the stability of symptoms of hypomania and depression across repeated affective episodes in patients with rapid-cycling bipolar disorder. Nine patients had a total of 30 depressive episodes and 31 hypomanic episodes during the period of observation. Standardized observer ratings indicated that the three symptoms most consistently reported during depressive episodes were fatiguability, decreased work activities and hypersomnia. These results as well as those from the standardized observer ratings of hypomania indicate that depression in this population consists of a lethargic, hypoactive state while hypomania may be a heightened state of activation. The clinical and theoretical implications of these findings are discussed.