Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes

Despite the identification of SRY as the testis-determining gene in mammals, the genetic interactions controlling the earliest steps of male sex determination remain poorly understood. In particular, the molecular lesions underlying a high proportion of human XY gonadal dysgenesis, XX maleness and XX true hermaphroditism remain undiscovered. A number of screens have identified candidate genes whose expression is modulated during testis or ovary differentiation in mice, but these screens have used whole gonads, consisting of multiple cell types, or stages of gonadal development well beyond the time of sex determination. We describe here a novel reporter mouse line that expresses enhanced green fluorescent protein under the control of an Sf1 promoter fragment, marking Sertoli and granulosa cell precursors during the critical period of sex determination. These cells were purified from gonads of male and female transgenic embryos at 10.5 dpc (shortly after Sry transcription is activated) and 11.5 dpc (when Sox9 transcription begins), and their transcriptomes analysed using Affymetrix genome arrays. We identified 266 genes, including Dhh, Fgf9 and Ptgds, that were upregulated and 50 genes that were downregulated in 11.5 dpc male somatic gonad cells only, and 242 genes, including Fst, that were upregulated in 11.5 dpc female somatic gonad cells only. The majority of these genes are novel genes that lack identifiable homology, and several human orthologues were found to map to chromosomal loci implicated in disorders of sexual development. These genes represent an important resource with which to piece together the earliest steps of sex determination and gonad development, and provide new candidates for mutation searching in human sexual dysgenesis syndromes.

Minimising cold damage during reproductive development among temperate rice genotypes. I. Avoiding low temperature with the use of appropriate sowing time and photoperiod-sensitive varieties

Multiple-sown field trials in 4 consecutive years in the Riverina region of south-eastern Australia provided 24 different combinations of temperature and day length, which enabled the development of crop phenology models. A crop model was developed for 7 cultivars from diverse origins to identify if photoperiod sensitivity is involved in determining phenological development, and if that is advantageous in avoiding low-temperature damage. Cultivars that were mildly photoperiod-sensitive were identified from sowing to flowering and from panicle initiation to flowering. The crop models were run for 47 years of temperature data to quantify the risk of encountering low temperature during the critical young microspore stage for 5 different sowing dates. Cultivars that were mildly photoperiod-sensitive, such as Amaroo, had a reduced likelihood of encountering low temperature for a wider range of sowing dates compared with photoperiod-insensitive cultivars. The benefits of increased photoperiod sensitivity include greater sowing flexibility and reduced water use as growth duration is shortened when sowing is delayed. Determining the optimal sowing date also requires other considerations, e. g. the risk of cold damage at other sensitive stages such as flowering and the response of yield to a delay in flowering under non-limiting conditions. It was concluded that appropriate sowing time and the use of photoperiod-sensitive cultivars can be advantageous in the Riverina region in avoiding low temperature damage during reproductive development.

A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNIA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADM), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type I virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Real-time PCR in the microbiology laboratory

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: Investigations of the cyclotide kalata B1 in biological fluids

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B I in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R-2 > 0.99) in the concentration range 0.05-10 mu g/mL with a limit of detection of 0.05 mu g/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures. (c) 2005 Elsevier B.V. All rights reserved.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy (R) or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to I infected in 800 samples with pepper but never detecting more than I infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Use of pulse transit time to distinguish respiratory events from tidal breathing in sleeping children

Study objectives: Currently, esophageal pressure monitoring is the "gold standard" measure for inspiratory efforts, hut its invasive nature necessitates a better tolerated and noninvasive method to be used on children. Pulse transit time (PTT) has demonstrated its potential as a noninvasive surrogate marker for inspiratory efforts. The principle velocity determinant of PTT is the change in stiffness of the arterial wall and is inversely correlated to BP. Moreover, PTT has been shown to identify changes in inspiratory effort via the BP fluctuations induced by negative pleural pressure swings. In this study, the capability of PTT to classify respiratory, events during sleep as either central or obstructive in nature was investigated. Setting and participants: PTT measure was used in adjunct to routine overnight polysomnographic studies performed on 33 children (26 boys and 7 girls; mean +/- SD age, 6.7 +/- 3.9 years). The accuracy of PTT measurements was then evaluated against scored corresponding respiratory events in the polysomnography recordings. Results: Three hundred thirty-four valid respiratory events occurred and were analyzed. One hundred twelve obstructive events (OEs) showed a decrease in mean PTT over a 10-sample window that had a probability of being correctly ranked below the baseline PTT during tidal breathing of 0.92 (p < 0.005); 222 central events (CEs) showed a decrease in the variance of PTT over a 10-sample window that had a probability of being ranked below the baseline PTT of 0.94 (p < 0.005). This indicates that, at a sensitivity of 0.90, OEs can be detected with a specificity of 0.82 and CEs can be detected with a specificity of 0.80. Conclusions: PTT is able to categorize CEs and OEs accordingly in the absence of motion artifacts, including hypopneas. Hence, PTT shows promise to differentiate respiratory, events accordingly and can be an important diagnostic tool in pediatric respiratory sleep studies.< 0.005); 222 central events (CEs) showed a decrease in the variance of PTT over a 10-sample window that had a probability of being ranked below the baseline PTT of 0.94 (p < 0.005). This indicates that, at a sensitivity of 0.90, OEs can be detected with a specificity of 0.82 and CEs can be detected with a specificity of 0.80. Conclusions: PTT is able to categorize CEs and OEs accordingly in the absence of motion artifacts, including hypopneas. Hence, PTT shows promise to differentiate respiratory, events accordingly and can be an important diagnostic tool in pediatric respiratory sleep studies.');"

Real-time PCR assays for detection of bocavirus in human specimens

The recently discovered human bocavirus (HBoV) is the first member of the family Parpoviridae, genus Bocavirus, to be potentially associated with human disease. Several studies have identified HBoV in respiratory specimens from children with acute respiratory disease, but the full spectrum of clinical disease and the epidemiology of HBoV infection remain unclear. The availability of rapid and reliable molecular diagnostics would therefore aid future studies of this novel virus. To address this, we developed two sensitive and specific real-time TaqMan PCR assays that target the HBoV NS1 and NP-1 genes. Both assays could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV genome, with a dynamic range of 8 log units (10(1) to 10(8) copies). Eight blinded clinical specimen extracts positive for HBoV by an independent PCR assay were positive by both real-time assays. Among 1,178 NP swabs collected from hospitalized pneumonia patients in Sa Kaeo Province, Thailand, 53 (4.5%) were reproducibly positive for HBoV by one or both targets. Our data confirm the possible association of HBoV infection with pneumonia and demonstrate the utility of these real-time PCR assays for HBoV detection.

Specific detection of enterovirus 71 directly from clinical specimens using real-time RT-PCR hybridization probe assay

Enterovirus 71 (EV71) is one of the main causative agents of hand, foot and mouth disease (HFMD) in young children. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. Thus, rapid detection of the virus is required to enable measures to be implemented in preventing widespread transmission. Based on primers and probes targeting at the VP1 region, a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) hybridization probe assay was developed for specific detection of EV71 from clinical specimens. Quantitative analysis showed that the assay was able to detect as low as 5 EV71 viral copies and EV71 was detected from 46 of the 55 clinical specimens obtained from pediatric patients suffering from HFMD during the period from 2000 to 2003 in Singapore. This study showed that the single tube real-time RT-PCR assay developed in this study can be applied as a rapid and sensitive method for specific detection of EV71 directly from clinical specimens. (c) 2005 Elsevier Ltd. All rights reserved.