Autocalibration based on dilution of a single concentrated standard is used for the determination of silicate in sea water by the modified molybdenum blue method.

The silicate (Si) molybdenum blue method was modified by combining oxalate and ascorbic acid into a single reagent and was used for determining Si in sea water samples. The first step of this automated assay protocol was designed to perform either a calibration by a single Si standard prepared in deionized (DI) water, or to dilute samples in the range of 0-160 µM Si to fit into 0-20 µM Si calibration range using a 20 cm flow cell. By designing the assay protocol to function in batch mode, the influence of salinity on calibration was eliminated, thus making the method suitable for analysis of samples collected in the open ocean, coastal areas, or rivers. Reproducibility and accuracy of this method were evaluated by analysis of certified sea water reference materials. Phosphate (P) does not interfere significantly if the Si:P ratio is 4:1 or larger. The limit of detection was 514 nM Si, r.s.d. 2.1 %, sampling frequency 40 s/h, reagent consumption 700 µL/sample, and using deionized water as the carrier solution.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley's genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Analysis of existence of multidrug-resistant H58 gene in Salmonella enterica serovar Typhi isolated from typhoid fever patients in Makassar, Indonesia.

The surveillance of multidrug-resistant (MDR) H58 typhoid is highly important, especially in endemic areas. MDR strain detection is needed by using a simple PCR technique that only uses a pair of primers. This is conducted considering the detection of Salmonella Typhi strains that have been carried out so far are only using antimicrobial sensitivity tests to determine microbial resistance phenotypically and to determine genotypically using complex molecular techniques. We aimed to analyse the existence of Salmonella Typhi MDR H58 in patients with typhoid fever in Makassar, Indonesia. A total of 367 blood samples of typhoid fever patients were collected from April 2018 until April 2019. The blood sample was cultured, then confirmed via simple PCR. All of the confirmed samples were tested for susceptibility against antibiotics and molecularly analysed for MDR H58 existence using a simple PCR technique. We found 7% (27/367) of the samples to be positive by both blood culture and PCR. All 27 isolates were found to be sensitive to sulfamethoxazole/trimethoprim. The lowest drug sensitivities were to amoxicillin, at one (3.7%) of 27 isolates, and ampicillin, at 13 (48.1%) of 27 isolates. Salmonella Typhi H58 PCR results showed that one (3.7%) of 27 isolates carried a positive fragment of 993 bp that led to the H58 strain, since the deletion flanks this fragment. The isolate was also found to be resistant to amoxicillin and fluoroquinolone according to a sensitivity test. Further molecular analysis needs to be conducted to examine the single isolate that carried the 933 bp fragment.

Extensive Genetic Variation at the Sr22 Wheat Stem Rust Resistance Gene Locus in the Grasses Revealed Through Evolutionary Genomics and Functional Analyses.

In the last 20 years, severe wheat stem rust outbreaks have been recorded in Africa, Europe, and Central Asia. This previously well controlled disease, caused by the fungus Puccinia graminis f. sp. tritici, has reemerged as a major threat to wheat cultivation. The stem rust (Sr) resistance gene Sr22 encodes a nucleotide-binding and leucine-rich repeat receptor which confers resistance to the highly virulent African stem rust isolate Ug99. Here, we show that the Sr22 gene is conserved among grasses in the Triticeae and Poeae lineages. Triticeae species contain syntenic loci with single-copy orthologs of Sr22 on chromosome 7, except Hordeum vulgare, which has experienced major expansions and rearrangements at the locus. We also describe 14 Sr22 sequence variants obtained from both Triticum boeoticum and the domesticated form of this species, T. monococcum, which have been postulated to encode both functional and nonfunctional Sr22 alleles. The nucleotide sequence analysis of these alleles identified historical sequence exchange resulting from recombination or gene conversion, including breakpoints within codons, which expanded the coding potential at these positions by introduction of nonsynonymous substitutions. Three Sr22 alleles were transformed into wheat cultivar Fielder and two postulated resistant alleles from Schomburgk (hexaploid wheat introgressed with T. boeoticum segment carrying Sr22) and T. monococcum accession PI190945, respectively, conferred resistance to P. graminis f. sp. tritici race TTKSK, thereby unequivocally confirming Sr22 effectiveness against Ug99. The third allele from accession PI573523, previously believed to confer susceptibility, was confirmed as nonfunctional against Australian P. graminis f. sp. tritici race 98-1,2,3,5,6.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Online survey of genital and urinary symptoms among Japanese women aged between 40 and 90 years.

OBJECTIVE: The purpose of this survey was to assess the prevalence of genital and urinary tract symptoms among Japanese women with declining estrogen levels. METHODS: A health-related questionnaire survey was conducted among women in their 40s or older to inquire about their genital, intercourse-related, and urinary symptoms and concern over their symptoms. RESULTS: Of the consecutive 10,000 respondents recruited, 4488 (44.9%) reported having symptoms: 3546 (79.0%) expressed concern over their symptoms. Furthermore, 2173 women (21.7%) had incontinence, 1999 (20.0%) had urinary frequency, 1648 (16.5%) had itching, and 1560 (15.6%) reported odor; these were followed by looseness, dryness, and burning. Of the 2518 (25.2%) sexually active women, 518 (20.6%) reported having dyspareunia and more reported having urinary symptoms than genital symptoms. Of the symptomatic respondents, 33.1% had genital symptoms alone, 28.4% had urinary symptoms alone, and 38.4% had both. More sexually active women had genital symptoms, while more sexually inactive women had urinary symptoms. CONCLUSIONS: Genital and urinary symptoms were shown to be common and coexist in a considerable proportion of the respondents, highlighting the pathology of genitourinary syndrome of menopause. Again, dyspareunia and lower urinary tract symptoms were shown to be quite common among postmenopausal women.

Resistance gene cloning from a wild crop relative by sequence capture and association genetics.

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel.

Effects of Pollination by the Indo-Malaya Stingless Bee (Hymenoptera: Apidae) on the Quality of Greenhouse-Produced Rockmelon.

Rockmelon (Cucumis melo Linnaeus (Cucurbitales: Cucurbitaceae)) is a novel commercialized fruit in Malaysia and has great potential to become an important horticultural crop for the international market. In this study, we investigated the effects of pollination by the Indo-Malaya stingless bee Heterotrigona itama Smith (Hymenoptera: Apidae) on measures of yield and quality of rockmelon cultivated in the greenhouse, compared with hand cross-pollination and self-pollination. Results showed that rockmelon produced from plants pollinated by stingless bees and hand cross-pollination had higher fruit set, were heavier and larger, and contained higher numbers of seed per fruit compared with those produced by self-pollination. Pollination by stingless bees produced fruit with greater sweetness than either hand cross-pollination or self-pollination. This study demonstrated that stingless bee pollination produced rockmelon fruit of similar quality, but better yields compared to the other pollination treatments. We showed that stingless bees should be considered as an alternative, effective pollinator for the improved production of high quality rockmelon in commercial greenhouse cultivation.

The effect of anti-tuberculosis drugs therapy on mRNA efflux pump gene expression of Rv1250 in Mycobacterium tuberculosis collected from tuberculosis patients.

Efflux pumps are transmembrane proteins that vigorously participate in extruding a wide range of substrates, including drugs, outside the bacterial cell. We aimed to investigate the mRNA expression level of the Rv1250 efflux pump gene in Mycobacterium tuberculosis isolated from individuals with tuberculosis who received drug therapy, at the 1st, 3rd and 5th months, and newly diagnosed patients with tuberculosis who will receive drug therapy (0 month). The study was a multiple cross-sectional longitudinal design-50 different M. tuberculosis isolates and a reference strain H37Rv were subcultured in LJ medium and confirmed by multiplex PCR for identification of M. tuberculosis and collected for RNA extraction. Total bacterial mRNA was analysed using real-time quantitative PCR to evaluate mRNA quantification gene expression. There were differences in the level of Rv1250 mRNA expression between sensitive (n = 11) and resistant (n = 40) groups of 5.961 ± 0.414 and 10.192 ± 1.978, respectively (fold changes; p < 0.05). There were significant differences of expression level among M. tuberculosis-resistant groups (p < 0.05) specifically 7.573 ± 0.424 for 0-month drug therapy (n = 10), 9.438 ± 0.644 for 1st month drug therapy (n = 10), 11.057 ± 0.262 for 3rd month drug therapy (n = 10) and 12.701 ± 0.460 for 5th month drug therapy (n = 10). We assume that the extent of Rv1250 gene expression in M. tuberculosis clinical isolates is a result of the exposure to antimicrobials during treatment, which affect the basic expression of the efflux pump Rv1250 gene.

Peptidylarginine deiminase is involved in maintaining the cornified oral mucosa of rats.

BACKGROUND AND OBJECTIVE: Epithelial cells derived from different regions exhibit marked differences in their differentiation capacity, allowing them to provide a suitable protective barrier. We aimed to clarify the role of peptidylarginine deiminase (PAD) in modifying the key epidermal proteins filaggrin (FLG) and keratin 1 (K1) during stratification of the rat palate and buccal mucosa. MATERIAL AND METHODS: We performed immunofluorescence, immunoblotting, PAD activity assays and 2-dimensional electrophoresis, and developed an organotypic culture model. RESULTS: PAD1 expression was highest in the palate, whereas PAD2, PAD3 and PAD4 expression was highest in the skin, suggesting the tissue-specific expression of PAD isozymes that leads to differences in calcium dependency. Immunoblotting showed that the FLG monomer, as well as its degradation products and precursor (proFLG), were most abundantly expressed in the skin but had low expression in the palate, whereas only faint proFLG expression was detected in the buccal mucosa. FLG and K1 were colocalized with PAD1 and were likely to be citrullinated in the cornified layers of the skin; this colocalization was not detected on the palatal surface, and dot-like presence of proFLG that might be citrullinated and that of PAD1 were found in the granules of the palate. Organotypic models derived from the rat palate revealed that PAD inhibition reduced the breakdown of FLG, increased its association with K1 together with epithelial compaction, and decreased permeability in a dye permeability assay. Conversely, PAD stimulation had the opposite effects. CONCLUSION: Citrullination is likely a protein modification that plays an important role in maintaining the structure and function of oral cornified mucosa in a way that is distinctly different from that of the skin.

Speed breeding is a powerful tool to accelerate crop research and breeding.

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand 1 . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.

Risk assessment of recent Egyptian H5N1 influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014-2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

Impact of psm-mec in the mobile genetic element on the clinical characteristics and outcome of SCCmec-II methicillin-resistant Staphylococcus aureus bacteraemia in Japan.

Over-expression of alpha-phenol-soluble modulins (PSMs) results in high virulence of community-associated methicillin-resistant Staphylococcus aureus (MRSA). The psm-mec gene, located in the mobile genetic element SCCmec-II, suppresses PSMαs production. Fifty-two patients with MRSA bacteraemia were enrolled. MRSA isolates were evaluated with regard to the psm-mec gene sequence, bacterial virulence, and the minimum inhibitory concentration (MIC) of vancomycin and teicoplanin. Fifty-one MRSA isolates were classified as SCCmec-II, and 10 had one point mutation in the psm-mec promoter. We compared clinical characteristics and outcomes between mutant MRSA and wild-type MRSA. Production of PSMα3 in mutant MRSA was significantly increased, but biofilm formation was suppressed. Wild-type MRSA caused more catheter-related bloodstream infections (30/41 vs. 3/10, p 0.0028), whereas mutant MRSA formed more deep abscesses (4/10 vs. 3/41, p 0.035). Bacteraemia caused by mutant MRSA was associated with reduced 30-day mortality (1/10 vs. 13/41, p 0.25), although this difference was not significant. The MIC90 of teicoplanin was higher for wild-type MRSA (1.5 mg/L vs. 1 mg/L), but the MIC of vancomycin was not different between the two groups. The 30-day mortality of MRSA with a high MIC of teicoplanin (≥1.5 mg/L) was higher than that of strains with a lower MIC (≤0.75 mg/L) (6/10 vs. 6/33, p 0.017). Mutation of the psm-mec promoter contributes to virulence of SCCmec-II MRSA, and the product of psm-mec may determine the clinical characteristics of bacteraemia caused by SCCmec-II MRSA, but it does not affect mortality.

Involvement of IP3-receptor activation in endothelin-1-induced Ca(2+) influx in rat pulmonary small artery.

We examined the endothelin-1 (ET-1)-induced increase in the intracellular free Ca(2+) concentration ([Ca(2+)]i) in fura-2-loaded rat pulmonary small arteries. ET-1 (30 nM) elicited a long-lasting increase in [Ca(2+)]i in physiological salt solution (PSS). In subsequent experiments, arteries were pretreated with BQ-788, an ETB-specific blocker, to allow us to focus on responses mediated via the ETA receptor, the existence of which was confirmed by immunohistochemistry. In Ca(2+)-free PSS, ET-1 evoked a small transient increase in [Ca(2+)]i, indicating Ca(2+) release from the SR (sarcoplasmic reticulum). After a switch to PSS (containing 2mM CaCl2), ET-1 elicited a long-lasting increase in [Ca(2+)]i that was not inhibited by 1 µM nicardipine, an L-type Ca(2+)-channel inhibitor, suggesting involvement of a Ca(2+)-influx pathway independent of that channel. In arteries preincubated with 30 µM cyclopiazonic acid (CPA) or 2 µM thapsigargin (TG), the ET-1-induced Ca(2+)-release was greatly reduced, and the induced Ca(2+)-influx was attenuated. U-73122, a phospholipase C (PLC) inhibitor, had inhibitory effects similar to those of CPA and TG on the ET-1-induced Ca(2+)-release and Ca(2+)-influx, whereas U-73343, an inactive analogue of U-73122, had no such effects. Two putative membrane-permeable IP3-receptor blockers, 2-aminoethoxydiphenyl borate (2APB, 50 µM) and Xestospongin C (20 µM), (a) almost completely inhibited the ET-1-induced Ca(2+)-release and Ca(2+)-influx, and (b) reduced the ET-1-induced contraction. These results indicate that in rat pulmonary small arteries, ET-1 induces receptor-operated Ca(2+) influx via the ETA receptor, and that this influx interacts with InsP3-receptor activation.

Outcome of molar pregnancies in Malaysia: a tertiary centre experience.

Gestational trophoblastic disease (GTD) is a common problem among Asian ethnics. A total of 102 women with molar pregnancies between 1 January 2005 and 31 December 2010, were analysed. The aim of the study was to determine the outcome of all molar pregnancies in our institution. The local incidence of molar pregnancy was 2.6 per 1,000 deliveries. A total of 48 women (47.1%) had complete hydatidiform mole and another 54 (52.9%) had partial mole. The mean age of the women with molar pregnancies was 32.0 ± 7.9 years. The mean gestational age at initial diagnosis was 11 weeks ± 3 days. The majority (97 women, 95.1%) had symptoms of vaginal bleeding and 18 (17.6%) women had a uterus larger than dates. A total of 48 (47.1%) women had ultrasound scan findings of 'snow-storm' appearance. None of the women with uncomplicated molar pregnancy had evidence of relapse following one undetectable serum ß-hCG level. Four out of the 102 women (3.9%) developed persistent trophoblastic disease before attaining one undetectable serum ß-hCG level. All four women required single agent methotrexate and they remained in remission. The prognosis for uncomplicated molar pregnancy is good. Establishment of a National Trophoblastic Centre is recommended to maintain optimal outcome.

Acetylation curtails nucleosome binding, not stable nucleosome remodeling, by FoxO1.

Transcriptional activity of FoxO factors is controlled through the actions of multiple growth factors signaling through protein kinase B, whereby phosphorylation of FoxO factors inhibits FoxO-mediated transactivation by promoting nuclear export. Phosphorylation of FoxO factors is enhanced by p300-mediated acetylation, which decreases their affinity for DNA. The negative effect of acetylation on FoxO DNA binding, together with nuclear FoxO mobility, is eliminated by over-expression of the de-acetylase Sirt1, suggesting that acetylation mobilizes FoxO factors in chromatin for inducible gene expression. Here, we show that acetylation significantly curtails the affinity of FoxO1 for its binding sites in nucleosomal DNA but has no effect on either stable nucleosome binding or remodeling by this factor. We suggest that, while acetylation provides a first, essential step toward mobilizing FoxO factors for inducible gene repression, additional mechanisms exist for overcoming their inherent capacity to stably bind and remodel nuclear chromatin.

Is there an increased risk of TB relapse in patients treated with fixed-dose combination drugs in Indonesia?

SETTING: South Sulawesi Province, Republic of Indonesia. OBJECTIVE: To compare relapse rates among tuberculosis (TB) patients treated with fixed-dose combination drugs (FDCs) and patients treated with the same regimen using loose drugs. METHODOLOGY: Between 1999 and 2001, new smear-positive TB patients were randomly allocated to treatment with four-drug FDCs or loose drugs to study differences in treatment outcomes. Although it was not in the original study design, in 2004-2005 we performed a follow-up study by home visit of cured patients. We conducted an interview and tried to collect a sputum sample from each patient. If the patient was absent or had died, a proxy interview was conducted. The sputum samples were examined by microscopy and culture. RESULTS: The overall relapse rate was 7.0% in patients who were able to produce a sputum sample. Relapse appeared to be more frequent in the FDC group compared to the loose drug group (10.1% vs. 2.7%, P = 0.074). CONCLUSION: This is the first documented long-term follow-up study of patients treated with four-drug FDCs. There is an indication that treatment of new sputum smear-positive TB patients with FDCs provides an increased risk of relapse compared to treatment with loose drugs. The long-term results of treatment with FDCs should be carefully evaluated in other settings.

Serum levels of interferon-gamma, tumour necrosis factor-alpha, soluble interleukin-6R and soluble cell activation markers for monitoring response to treatment of leprosy reactions.

Identifying pathogen and host-related laboratory parameters are essential for the early diagnosis of leprosy reactions. The present study aimed to clarify the validity of measuring the profiles of serum cytokines [interleukin (IL)-4, IL-6, IL-10, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha], the soluble IL-6 receptor (sIL-6R), soluble T cell (sCD27) and macrophage (neopterin) activation markers and Mycobacterium leprae-specific anti-PGL-I IgM antibodies in relation to the leprosy spectrum and reactions. Serum samples from 131 Indonesian leprosy patients (82 non-reactional leprosy patients and 49 reactional) and 112 healthy controls (HC) from the same endemic region were investigated. Forty-four (89.8%) of the reactional patients had erythema nodosum leprosum (ENL) while only five (10.2%) had reversal reaction (RR). Follow-up serum samples after corticosteroid treatment were also obtained from 17 of the patients with ENL and one with RR. A wide variability in cytokine levels was observed in the patient groups. However, IFN-gamma and sIL-6R were elevated significantly in ENL compared to non-ENL patients. Levels of IFN-gamma, TNF-alpha and sIL-6R declined significantly upon corticosteroid treatment of ENL. Thus, although the present study suggests limited applicability of serial measurement of IFN-gamma, TNF-alpha and sIL-6R in monitoring treatment efficacy of ENL, reactions it recommends a search for a wider panel of more disease-specific markers in future studies.

An increased incidence of Enterobacter cloacae in a cardiovascular ward.

Routine surveillance in a cardiovascular ward showed that the incidence of Enterobacter cloacae isolated from sputum and oropharyngeal cultures in June 2004 increased to 27.6% (8/29) compared to 5.5% (12/219) from the rest of the hospital during the same period (OR=13.2; 95% CI 2.97-58.7; P<0.05). While an increase in E. cloacae pneumonia was not verified, an investigation was undertaken by the infection control team to prevent an outbreak. The estimate of relative risk for E. cloacae infection was based on a case-control study which measured exposure to intubation, history of a stay in the intensive care unit (ICU) and oral care between patients with E. cloacae and those negative for E. cloacae. An odds ratio of 13.2 suggested cross-contamination via the transoesophageal echocardiography (TOE) probe in the ICU prior to transfer to the cardiovascular ward. Pulsed-field gel electrophoresis and antibiogram patterns were also consistent with this hypothesis. Intervention was undertaken in the form of enforcing the disinfection of TOE probes using a 0.55% phtharal solution and the use of a single-use sheath to protect the probe from recontamination. Following intervention, the incidence rate returned to previous levels. This report illustrates the limitations in the effectiveness of current nosocomial surveillance strategies due to the retrospective nature of analysis. Improved surveillance methods such as data-mining tools specifically applicable to the institution, patient population, region and country are needed to increase the sensitivity of detecting unrecognized outbreaks, including cross-contamination.

Evaluation of a fully automated system (RAISUS) for rapid identification and antimicrobial susceptibility testing of Staphylococci.

RAISUS is a system for rapid bacterial identification and antimicrobial susceptibility testing. RAISUS and VITEK showed 97.8% and 75.9% agreement in identification of 45 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CoNS), respectively, and RAISUS and CLSI (formerly NCCLS) methods showed 87.2% and 87.9% agreement in the MICs for S. aureus and CoNS, respectively. RAISUS provided these data within 3.75 h, suggesting its utility for clinical bacteriological laboratories.