Expression of PROKR1 and PROKR2 in Human Enteric Neural Precursor Cells and Identification of Sequence Variants Suggest a Role in HSCR

Background. The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. Principal Findings. In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. Conclusions. Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.

New Granada Medium for detection and identification of group B streptococci.

A methotrexate-containing medium for the detection of beta-hemolytic group B streptococci from clinical specimens on the basis of detection of pigment is described. The medium contained peptone, starch, serum, MgSO4, glucose, pyruvate, methotrexate (as pigment enhancer), phosphate-morpholine-propanesulfonic acid buffer, and selective agents. The recovery of beta-hemolytic group B streptococci was comparable to that obtained with selective broth.

Effectiveness and costs of phototest in dementia and cognitive impairment screening

BACKGROUND To assess and compare the effectiveness and costs of Phototest, Mini Mental State Examination (MMSE), and Memory Impairment Screen (MIS) to screen for dementia (DEM) and cognitive impairment (CI). METHODS A phase III study was conducted over one year in consecutive patients with suspicion of CI or DEM at four Primary Care (PC) centers. After undergoing all screening tests at the PC center, participants were extensively evaluated by researchers blinded to screening test results in a Cognitive-Behavioral Neurology Unit (CBNU). The gold standard diagnosis was established by consensus of expert neurologists. Effectiveness was assessed by the proportion of correct diagnoses (diagnostic accuracy [DA]) and by the kappa index of concordance between test results and gold standard diagnoses. Costs were based on public prices and hospital accounts. RESULTS The study included 140 subjects (48 with DEM, 37 with CI without DEM, and 55 without CI). The MIS could not be applied to 23 illiterate subjects (16.4%). For DEM, the maximum effectiveness of the MMSE was obtained with different cutoff points as a function of educational level [k = 0.31 (95% Confidence interval [95%CI], 0.19-0.43), DA = 0.60 (95%CI, 0.52-0.68)], and that of the MIS with a cutoff of 3/4 [k = 0.63 (95%CI, 0.48-0.78), DA = 0.83 (95%CI, 0.80-0.92)]. Effectiveness of the Phototest [k = 0.71 (95%CI, 0.59-0.83), DA = 0.87 (95%CI, 0.80-0.92)] was similar to that of the MIS and higher than that of the MMSE. Costs were higher with MMSE (275.9 ± 193.3€ [mean ± sd euros]) than with Phototest (208.2 ± 196.8€) or MIS (201.3 ± 193.4€), whose costs did not significantly differ. For CI, the effectiveness did not significantly differ between MIS [k = 0.59 (95%CI, 0.45-0.74), DA = 0.79 (95%CI, 0.64-0.97)] and Phototest [k = 0.58 (95%CI, 0.45-0.74), DA = 0.78 (95%CI, 0.64-0.95)] and was lowest for the MMSE [k = 0.27 (95%CI, 0.09-0.45), DA = 0.69 (95%CI, 0.56-0.84)]. Costs were higher for MMSE (393.4 ± 121.8€) than for Phototest (287.0 ± 197.4€) or MIS (300.1 ± 165.6€), whose costs did not significantly differ. CONCLUSION MMSE is not an effective instrument in our setting. For both DEM and CI, the Phototest and MIS are more effective and less costly, with no difference between them. However, MIS could not be applied to the appreciable percentage of our population who were illiterate.

Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases.

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

Identification of serum and urine proteins responsible for enhanced pigment production by group B streptococci as amylases.

The serum and urine proteins responsible for enhanced pigment production in Streptococcus agalactiae in culture media were purified by chromatography and were identified as amylases by comparison of their amino acid composition with that calculated for proteins with known sequences. Similar pigment-enhancing activity was displayed by other amylases of nonanimal origin and by maltooligosaccharides.