What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.     

Is the interaction of CNP with tubulin and microtubules required for process extension in oligodendrocytes?

The STOP protein (stable tubule-only polypeptide) is a calmodulin-regulated protein which associates with microtubules and induces cold stabilization. There are different isoforms of this protein that arise from alternative splicing of STOP mRNA. Neurons express two major variants N-STOP (125 kDa) and E-STOP (84 kDa). NIH 3T3 fibroblasts contain a major F-STOP isoform (42 kDa) and two minor STOP variants (48 and 89 kDa). Previously, we demonstrated the presence of N-STOP in the cytoskeleton associated with myelin isolated from animals injected with apotransferrin. Since this protein was only described as a neuronal protein we decided to further investigate the expression of this protein in oligodendrocyte cultures. The analysis of the STOP protein expression in oligodendrocyte shows that STOP protein is expressed in the soma and processes of oligodendrocyte precursors, as well as in immature and mature oligodendroglial cells. In addition, we found that MBP shows a high degree of colocalization with STOP protein. By Western blot analysis, it was found that these cells express a major STOP variant (89 kDa). When the cultures were exposed to cold temperature we found that STOP protein associates with microtubules and induces microtubule cold stabilization. Under these experimental conditions, we found that MBP associates with microtubules too, and maintains its colocalization with STOP protein. At present, we are doing new assays directed to further characterize STOP (89 kDa) protein and to elucidate how this protein participates in the formation of myelin by oligodendrocytes.     

Is the Cerebellum the Optimal Reference Region for Intensity Normalization of Perfusion MR Studies in Early Alzheimer’s Disease?

The cerebellum is the region most commonly used as a reference when normalizing the intensity of perfusion images acquired using magnetic resonance imaging (MRI) in Alzheimer’s disease (AD) studies. In addition, the cerebellum provides unbiased estimations with nuclear medicine techniques. However, no reports confirm the cerebellum as an optimal reference region in MRI studies or evaluate the consequences of using different normalization regions. In this study, we address the effect of using the cerebellum, whole-brain white matter, and whole-brain cortical gray matter in the normalization of cerebral blood flow (CBF) parametric maps by comparing patients with stable mild cognitive impairment (MCI), patients with AD and healthy controls. According to our results, normalization by whole-brain cortical gray matter enables more sensitive detection of perfusion abnormalities in AD patients and reveals a larger number of affected regions than data normalized by the cerebellum or whole-brain white matter. Therefore, the cerebellum is not the most valid reference region in MRI studies for early stages of AD. After normalization by whole-brain cortical gray matter, we found a significant decrease in CBF in both parietal lobes and an increase in CBF in the right medial temporal lobe. We found no differences in perfusion between patients with stable MCI and healthy controls either before or after normalization.     

Is wildlife research useful for wildlife conservation in the tropics? A review for Borneo with global implications

The urgency of the tropical biodiversity crisis continues to be a major justification for wildlife research and its funding. To examine the benefits of this research for on-the-ground conservation, we focused on Borneo, where conservation has a long history and we have direct experience. We compiled, categorized and evaluated 284 publications from a broad variety of sources, 153 from peer-reviewed journals. We found that few studies address threats to species and fewer still provide input for or guidance to effective management. We consider various reasons for these shortcomings. Research is seldom judged on its relevance to pragmatic problem solving. Furthermore, many research programs lack the necessary long-term vision and organizational structure for useful applied research. We consulted conservation leaders about our conclusions and all responses suggest that our concerns are not unique to Borneo but reflect wider problems. We conclude that conservation research across most of the tropics is failing to address conservation needs. We make a number of recommendations based on our findings. Conservation biologists should place a higher priority on addressing practical conservation needs and goals.     

Is the treatment with psychostimulants in children and adolescents with attention deficit hyperactivity disorder harmful for the dopaminergic system?

A major concern regarding psychostimulant medication (amphetamine and methylphenidate) in the treatment of children and adolescents with attention deficit/hyperactivity disorder (ADHD) are the potential adverse effects to the developing brain, particularly in regard to dopaminergic brain function. The present review focuses on the pharmacology of these psychostimulants, their mode of action in the human brain and their potential neurotoxic effects to the developing brain in animals, particularly concerning DA brain function. The potential clinical significance of these findings for the treatment of ADHD in children and adolescents is discussed. Studies on sensitization to psychostimulants’ rewarding effects, which is a process expected to increase the risk of substance abuse in humans, are not included. The available findings in non-human primates support the notion that the administration of amphetamine and methylphenidate with procedures simulating clinical treatment conditions does not lead to long-term adverse effects in regard to development, neurobiology or behaviour as related to the central dopaminergic system.     

Is the pollination biology of Bathysa and Schizocalyx consistent with their segregation? A first approach for two sympatric species in southeastern Brazil

Tribe Condamineeae appears to be well supported in recent phylogenetic studies. However, the species of Bathysa were divided into two clades, leading to restoration of Schizocalyx. We studied the reproductive biology of one species from each clade, which occur sympatrically in the montane Brazilian Atlantic forest. Flowering overlap was short (from December to March in B. australis and from February to June in S. cuspidatus). The flowers of both species are protogynous and homostylous and last for about 3 days. The unit of pollination in B. australis is the inflorescence. Its flowers have a greenish hypocrateriform corolla (tube about 5 mm long) and were mainly pollinated by bees and wasps in search of nectar. Schizocalyx cuspidatus has white flowers with an infundibuliform corolla (tube about 8 mm long), and its main pollinators were stingless bees in search of pollen. The pollination systems of the two species did not correspond to their pollination syndromes. Morphological differences between Bathysa and Schizocalyx were reflected in their pollination systems, with greater phenotypic specialization in S. cuspidatus, the flowers of which offer pollen as the main resource, an unusual feature within Rubiaceae. Schizocalyx cuspidatus showed higher reproductive capacity by having more inflorescences per plant, more ovules per flower, and twice the proportion of flowering individuals. However, the reproductive efficiency (fruit set, seed/ovule ratio) did not differ between the species, despite the higher frequency of visits by pollinators to S. cuspidatus. Self-compatibility in B. australis and self-incompatibility in S. cuspidatus seem to explain these results.     

A Field Evaluation of the Hardy TB MODS Kit? for the Rapid Phenotypic Diagnosis of Tuberculosis and Multi-Drug Resistant Tuberculosis

Background

Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory.

Methods & Findings

2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3–99.8%), 98.3% (97.5–98.8%), 95.8% (94.0–97.1%), and 99.7% (99.3–99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9–13) days for conventional MODS and 8.5 (7–11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples).

Conclusions

MODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest.     

Regional Variation in Analytical Techniques used in the Diagnosis and Monitoring of Porphyria: a Case for Harmonisation?

Background:The Royal College of Pathologists of Australasia (RCPA) Porphyrin Quality Assurance Program assesses the measurement of urine, faecal, plasma and whole blood porphyrins and their components plus urinary porphobilinogen and delta aminolaevulinic acid and has laboratories enrolled from around the world. It was observed that there was a wide scatter in results submitted to some subsections of the program.Methods:A detailed questionnaire covering the analytical techniques used in the diagnosis of porphyria was sent to all laboratories enrolled in the RCPA Porphyrin Quality Assurance Program. Additionally, self-enrolment data over a five year period was examined for trends/changes in standardisation, reagent sources and analytical technique.Results:Twenty of the 45 laboratories enrolled in the Porphyrin Quality Assurance Program completed the survey, providing a snapshot of the analytical techniques used world-wide. Post survey self enrolment data indicated only little or no noticeable changes to analytical standardisation of techniques despite the continual lack of agreement of results in subsections of the External Quality Assurance program.Conclusions:While some aspects of porphyria testing are relatively consistent between laboratories, other diagnostic techniques vary widely. A wide variety of individualised reference intervals and reporting techniques is currently in use world-wide. While most of the participants in the survey are regional reference centres specialising in the diagnosis of porphyria and, as such, their diagnostic capability is not in question, international guidelines or global harmonisation of analytical techniques should allow better inter-laboratory comparisons to be made, ultimately improving diagnostic accuracy.     

Monitoring Procalcitonin in Febrile Neutropenia: What Is Its Utility for Initial Diagnosis of Infection and Reassessment in Persistent Fever?

Background

Management of febrile neutropenic episodes (FE) is challenged by lacking microbiological and clinical documentation of infection. We aimed at evaluating the utility of monitoring blood procalcitonin (PCT) in FE for initial diagnosis of infection and reassessment in persistent fever.

Methods

PCT kinetics was prospectively monitored in 194 consecutive FE (1771 blood samples): 65 microbiologically documented infections (MDI, 33.5%; 49 due to non-coagulase-negative staphylococci, non-CNS), 68 clinically documented infections (CDI, 35%; 39 deep-seated), and 61 fever of unexplained origin (FUO, 31.5%).

Results

At fever onset median PCT was 190 pg/mL (range 30–26''800), without significant difference among MDI, CDI and FUO. PCT peak occurred on day 2 after onset of fever: non-CNS-MDI/deep-seated-CDI (656, 80–86350) vs. FUO (205, 33–771; p<0.001). PCT >500 pg/mL distinguished non-CNS-MDI/deep-seated-CDI from FUO with 56% sensitivity and 90% specificity. PCT was >500 pg/ml in only 10% of FUO (688, 570–771). A PCT peak >500 pg/mL (1196, 524–11950) occurred beyond 3 days of persistent fever in 17/21 (81%) invasive fungal diseases (IFD). This late PCT peak identified IFD with 81% sensitivity and 57% specificity and preceded diagnosis according to EORTC-MSG criteria in 41% of cases. In IFD responding to therapy, median days to PCT <500 pg/mL and defervescence were 5 (1–23) vs. 10 (3–22; p = 0.026), respectively.

Conclusion

While procalcitonin is not useful for diagnosis of infection at onset of neutropenic fever, it may help to distinguish a minority of potentially severe infections among FUOs on day 2 after onset of fever. In persistent fever monitoring procalcitonin contributes to early diagnosis and follow-up of invasive mycoses.     

PCR-Based Methods for the Diagnosis of Invasive Candidiasis: Are They Ready for Use in the Clinic?

Purpose of Review

We review the performance of Candida PCR and the T2Candida panel (T2Biosystems, Lexington, MA) in diagnosing invasive candidiasis, consider how these tests may be incorporated into patient care, and determine if they are ready to be used in the clinic.

Recent Findings

PCR and T2Candida sensitivity/specificity for diagnosing candidemia are ~?90%/90% and ~?90%/98%, respectively. Limited data for intra-abdominal candidiasis suggest PCR sensitivity of ~?85–90%, but specificity has varied from 33 to 97%. T2Candida data are lacking for infections other than candidemia.

Summary

PCR and T2Candida will have the greatest value if their use is restricted to cases in which positive and negative predictive values differ in a clinically meaningful way from the pre-test likelihood. Studies are needed to establish that patient care and stewardship strategies incorporating Candida PCR or T2Candida improve patients’ outcomes, reduce unnecessary antifungal usage, limit emergence of resistance, and are cost-effective. The development and validation of standardized PCR assays is a top priority.

     

Is pathological metabolism in the parental organism responsible for defective and monstrous development of the offspring?

Ohne Zusammenfassung

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Is pathological metabolism in the parental organism responsible for defective and monstrous development of the offspring?

     

Is Inositol Bisphosphate the Product of A23187 and Carbachol-Mediated Polyphosphoinositide Breakdown in Synaptosomes?

Synaptosomes have been isolated from rat cerebral cortex and labelled in vitro with [32P]orthophosphate and myo-[2-3H]inositol. Subsequent addition of the Ca2+ ionophore A23187 in the presence of 2 mM extrasynaptosomal Ca2+ raised intrasynaptosomal free [Ca2+] to greater than 2 microM from a resting level of 200 nM and led to rapid breakdown of polyphosphoinositides. This was accompanied by a small increase in the level of inositol monophosphate, greatly enhanced accumulation in inositol bisphosphate, but no detectable increase in inositol trisphosphate. Depolarising (25 mM) extrasynaptosomal K+ produced a smaller increase in intrasynaptosomal free [Ca2+] (to around 400 nM) and a proportional increase in inositol bisphosphate radioactivity. Carbachol (1 mM) alone elicited only limited polyphosphoinositide breakdown and inositol mono- and bisphosphate formation, but this was greatly increased in the presence of 25 mM K+. The effect of carbachol in the presence of depolarising K+ was time- and dose-dependent and was antagonised by atropine (10 microM). There was no detectable accumulation of inositol trisphosphate in the presence of carbachol, K+, or carbachol plus K+, even after short (30 s.) incubations. The lack of inositol trisphosphate accumulation does not appear to result from rapid formation of inositol tetrakisphosphate or from enhanced breakdown of the trisphosphate in synaptosomes.     

Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective

Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.

Materials and Methods

Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.

Results

Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.

Conclusions

Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.