Sensitive detection of heavy metal stress in aquatic plants by dissolved oxygen-quenched fluorescence/materials movement-induced beam deflection method.

Sensitive detection of heavy metal (HM) stress in aquatic plants by dissolved oxygen (DO)-quenched fluorescence/materials movement-induced beam deflection method is demonstrated. Egeria densa Planchon and Cu2+ were used as a model aquatic plant and HM ion, respectively. Reproducibility and experimental errors of the method were first investigated in a control culture solution only containing 10-6 M Ru (II) complex (Tris (2,2'-bipyridyl) ruthenium (II) chloride) without addition of any fertilizer and Cu2+. Changes of DO concentration (∆DO) and deflection (∆DE) during the monitoring periods were used as parameters to quantitatively evaluate the experimental errors and detection limits. Averages or means ([Formula: see text], [Formula: see text]) and standard deviations (σ∆DO, σ∆DE) of ∆DO and ∆DE in seven control experiments with different aquatic plants sheets during both the respiration and photosynthesis processes were obtained. Next, DO and deflection at the middle vicinities of the aquatic plant were monitored during 2 h of both respiration and photosynthesis in presence of 10-10 ~ 10-6 M Cu2+. Experimental results showed that the aquatic plant began to suffer from the HM stress in some extent in presence of 10-9 M Cu2+. When the concentration of Cu2+ was higher than 10-8 M, changing trends of both DO and deflection with time were not reversed during the respiration and photosynthesis, implying that the materials movements in the physiological activities had been altered greatly. It is demonstrated that the method could sensitively detect the HM stress in the aquatic plants given by nM HM ions in culture solution without addition of a fertilizer. Furthermore, detection limits of the method were quantitatively discussed by considering [Formula: see text] σ∆DO and [Formula: see text] σ∆DE as the minimum detectable changes of DO and deflection caused by the HM stress, respectively.

Heterozygous/dispermic complete mole confers a significantly higher risk for post-molar gestational trophoblastic disease.

Hydatidiform moles are classified at the genetic level as androgenetic complete mole and diandric-monogynic partial mole. Conflicting data exist whether heterozygous complete moles are more aggressive clinically than homozygous complete moles. We investigated clinical outcome in a large cohort of hydatidiform moles in Chinese patients with an emphasis on genotypical correlation with post-molar gestational trophoblastic disease. Consecutive products of conceptions undergoing DNA genotyping and p57 immunohistochemistry to rule out molar gestations were included from a 5-year period at Beijing Obstetrics and Gynecology Hospital. Patient demographics and clinical follow-up information were obtained. Post-molar gestational trophoblastic disease or gestational trophoblastic neoplasia was determined by the 2002 WHO/FIGO criteria. A total of 1245 products of conceptions were classified based on genotyping results into 219 complete moles, 250 partial moles, and 776 non-molar gestations. Among 219 complete moles, 186 were homozygous/monospermic and 33 were heterozygous/dispermic. Among 250 partial moles, 246 were triploid dispermic, 2 were triploid monospermic, and 2 were tetraploid heterozygous partial moles. Among 776 non-molar gestations, 644 were diploid without chromosomal aneuploidies detectable by STR genotyping and 132 had various genetic abnormalities including 122 cases of various trisomies, 2 triploid digynic-monoandric non-molar gestations, 7 cases of possible chromosomal monosomy or uniparental disomy. Successful follow-up was achieved in 165 complete moles: post-molar gestational trophoblastic disease developed in 11.6% (16/138 cases) of homozygous complete moles and 37.0% (10/27 cases) of heterozygous complete moles. The difference between the two groups was highly significant (p = 0.0009, chi-square). None of the 218 partial moles and 367 non-molar gestations developed post-molar gestational trophoblastic disease. In conclusion, heterozygous/dispermic complete moles are clinically more aggressive with a significantly higher risk for development of post-molar gestational trophoblastic disease compared with homozygous/monospermic complete moles. Therefore, precise genotyping classification of complete moles is important for clinical prognosis and patient management.

Comprehensive Analysis of Autophagy-Related Genes in Sweet Orange (Citrus sinensis) Highlights Their Roles in Response to Abiotic Stresses.

Autophagy is a highly conserved intracellular degradation pathway that breaks down damaged macromolecules and/or organelles. It is involved in plant development and senescence, as well as in biotic and abiotic stresses. However, the autophagy process and related genes are largely unknown in citrus. In this study, we identified 35 autophagy-related genes (CsATGs-autophagy-related genes (ATGs) of Citrus sinensis, Cs) in a genome-wide manner from sweet orange (Citrus sinensis). Bioinformatic analysis showed that these CsATGs were highly similar to Arabidopsis ATGs in both sequence and phylogeny. All the CsATGs were randomly distributed on nine known (28 genes) and one unknown (7 genes) chromosomes. Ten CsATGs were predicted to be segmental duplications. Expression patterns suggested that most of the CsATG were significantly up- or down-regulated in response to drought; cold; heat; salt; mannitol; and excess manganese, copper, and cadmium stresses. In addition, two ATG18 members, CsATG18a and CsATG18b, were cloned from sweet orange and ectopically expressed in Arabidopsis. The CsATG18a and CsATG18b transgenic plants showed enhanced tolerance to osmotic stress, salt, as well as drought (CsATG18a) or cold (CsATG18b), compared to wild-type plants. These results highlight the essential roles of CsATG genes in abiotic stresses.

Systematic analysis of the basic/helix-loop-helix (bHLH) transcription factor family in pummelo (Citrus grandis) and identification of the key members involved in the response to iron deficiency.

BACKGROUND: Iron (Fe) deficiency is a common problem in citrus production. As the second largest superfamily of transcription factors (TFs), the basic/helix-loop-helix (bHLH) proteins have been shown to participate in the regulation of Fe homeostasis and a series of other biological and developmental processes in plants. However, this family of members in citrus and their functions in citrus Fe deficiency are still largely unknown. RESULTS: In this study, we identified a total of 128 CgbHLHs from pummelo (Citrus grandis) genome that were classified into 18 subfamilies by phylogenetic comparison with Arabidopsis thaliana bHLH proteins. All of these CgbHLHs were randomly distributed on nine known (125 genes) and one unknown (3 genes) chromosomes, and 12 and 47 of them were identified to be tandem and segmental duplicated genes, respectively. Sequence analysis showed detailed characteristics of their intron-exon structures, bHLH domain and conserved motifs. Gene ontology (GO) analysis suggested that most of CgbHLHs were annotated to the nucleus, DNA-binding transcription factor activity, response to abiotic stimulus, reproduction, post-embryonic development, flower development and photosynthesis. In addition, 27 CgbHLH proteins were predicted to have direct or indirect protein-protein interactions. Based on GO annotation, RNA sequencing data in public database and qRT-PCR results, several of CgbHLHs were identified as the key candidates that respond to iron deficiency. CONCLUSIONS: In total, 128 CgbHLH proteins were identified from pummelo, and their detailed sequence and structure characteristics and putative functions were analyzed. This study provides comprehensive information for further functional elucidation of CgbHLH genes in citrus.

Whole-transcriptome RNA sequencing reveals the global molecular responses and ceRNA regulatory network of mRNAs, lncRNAs, miRNAs and circRNAs in response to copper toxicity in Ziyang Xiangcheng (Citrus junos Sieb. Ex Tanaka).

BACKGROUND: Copper (Cu) toxicity has become a potential threat for citrus production, but little is known about related mechanisms. This study aims to uncover the global landscape of mRNAs, long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) in response to Cu toxicity so as to construct a regulatory network of competing endogenous RNAs (ceRNAs) and to provide valuable knowledge pertinent to Cu response in citrus. RESULTS: Tolerance of four commonly used rootstocks to Cu toxicity was evaluated, and 'Ziyang Xiangcheng' (Citrus junos) was found to be the most tolerant genotype. Then the roots and leaves sampled from 'Ziyang Xiangcheng' with or without Cu treatment were used for whole-transcriptome sequencing. In total, 5734 and 222 mRNAs, 164 and 5 lncRNAs, 45 and 17 circRNAs, and 147 and 130 miRNAs were identified to be differentially expressed (DE) in Cu-treated roots and leaves, respectively, in comparison with the control. Gene ontology enrichment analysis showed that most of the DEmRNAs and targets of DElncRNAs and DEmiRNAs were annotated to the categories of 'oxidation-reduction', 'phosphorylation', 'membrane', and 'ion binding'. The ceRNA network was then constructed with the predicted pairs of DEmRNAs-DEmiRNAs and DElncRNAs-DEmiRNAs, which further revealed regulatory roles of these DERNAs in Cu toxicity. CONCLUSIONS: A large number of mRNAs, lncRNAs, circRNAs, and miRNAs in 'Ziyang Xiangcheng' were altered in response to Cu toxicity, which may play crucial roles in mitigation of Cu toxicity through the ceRNA regulatory network in this Cu-tolerant rootstock.

Clinicopathological features of ovarian Brenner tumors / 中华病理学杂志

Objective@#To investigate the clinicopathological characteristics and diagnosis of ovarian Brenner tumors.@*Methods@#Forty-seven cases of ovarian Brenner tumors were enrolled from January 2012 to May 2018 at Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Clinical data, imaging examination, histopathological characteristics and immunohistochemical phenotype were analyzed.@*Results@#The age of the patients ranged from 30-73 years and the mean age was 55 years. Thirty-nine patients (83.0%) were postmenopausal. Forty cases (85.1%) of the Brenner tumors were benign, five (10.6%) borderline and two (4.3%) malignant. Usual tumor markers of ovarian carcinoma, including CA199 and CA125 were normal or mild elevated in the 47 cases. Imaging before surgery was not specific to Brenner tumors. Microscopically, benign Brenner tumors were composed of nests of bland, transitional-type cells within a fibromatous stroma. In our 5 cases of borderline Brenner tumors, mildly atypical transitional-type cells were projected into the cyst lumens and lack of stromal invasion. In 2 cases of malignant Brenner tumors, different degrees of nuclear atypial transitional-type cells exhibited stromal invasion. Immunohistochemical stains for CK7, GATA3, p63 and CK5/6 were positive in all cases. Ki-67 was less than 5% in Brenner tumors, and up to 20%-30% in malignant Brenner tumors.@*Conclusion@#Brenner tumors are mostly seen in postmenopausal patients and are usually benign. Imaging examination and usual ovarian tumor markers do not provide diagnostic value. Diagnosis and classification of Brenner tumors depend on histopathological evaluation.

Improvements on the Fluorescence Quenching/Deflection Method for Real-time in situ Simultaneous Monitoring of Dissolved Oxygen and Material Movement-induced Beam Deflection in the Vicinity of an Aquatic Plant.

Although the newly developed beam deflection/fluorescence detection system for real-time in situ simultaneous monitoring of dissolved oxygen (DO) and material movements in the vicinity of aquatic plants was not only much more sensitive but also could be carried out much more closely to real time than conventional analytical methods that monitor the concentration changes at a bulk solution, it could not be applied to the photosynthesis process of aquatic plants. Here, improvements are reported to enable application of the system to the photosynthesis process. A white-light LED, which was used as a light source for photosynthesis in our previous paper, was replaced by a red-blue LED with wavelength of about 660 and 450 nm. Also, an interference filter of 589 ± 25 nm was placed in front of a photomultiplier tube (PMT). Furthermore, the LED and its electric power supply were placed outside of the dark room for preventing great temperature increases in the photosynthetic experiments. Experimental results showed the DO-quenched fluorescence could be sensitively monitored in both the respiration and photosynthesis processes, while only in the respiration process before the improvements. It is successfully demonstrated that the DO change and material movement-induced beam deflection in the vicinity of the plants in both the respiration and photosynthesis processes could be real-time in situ monitored with high sensitivity.

KRAS mutations analysis in mucinous epithelial lesions of the endometrium / 中华病理学杂志

Objective@#To investigate the frequency of KRAS mutation in mucinous epithelial lesions of the endometrium, and analyze the correlation between KRAS mutation and the clinicopathologic features.@*Methods@#The cohort included forty-three cases of mucinous epithelial lesions of the endometrium selected from July 2015 to October 2017 from Beijing Obstetrics and Gynecology Hospital, and 22 control cases. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue sections. Polymerase chain reaction amplification for KRAS exons 2 and 3 was performed, followed by sequencing using capillary electrophoresis. The Fisher exact test was used to compare the prevalence of KRAS mutation among the different groups.@*Results@#The patients′age ranged from 33 to 77 years [mean (55.12±9.34) years, median 55 years]. None of the eight cases of endometrial hyperplasia with mucinous differentiation without atypia showed KRAS mutation. The frequency of KRAS mutations was 1/10 in endometrial atypical hyperplasia, 1/12 in endometrioid carcinoma, 4/11 in endometrial atypical hyperplasia with mucinous differentiation (EAHMD), 6/15 in endometrioid carcinoma with mucinous differentiation (ECMD) and 8/9 in mucinous carcinoma (MC), respectively. The differences were statistically significant between MC versus EC (P<0.01) and MC versus ECMD (P<0.05).@*Conclusion@#The high frequency of KRAS mutation in EAHMD, ECMD and MC indicates that KRAS mutational activation is implicated in the pathogenesis of endometrial mucinous carcinoma.

Clinical application of STR genotyping diagnosis for hydatidiform mole and nonmolar gestation / 中华病理学杂志

Objective@#To investigate the value of short tandem repeat (STR) genotyping in the diagnostic workup of molar and non-molar gestations with correlation of histological characteristics.@*Methods@#Six hundred and fifty-six cases were selected based on clinically suspected hydropic abortion and/or molar pregnancy from July 2015 to September 2017 at Beijing Obstetrics and Gynecology Hospital. DNA was extracted from dissected chorionic villi and paired maternal endometrial FFPE tissue samples by Simplex OUP™ FFPE DNA Tissue Kit. STR genotyping was performed by PowerPlex 16 HS system.@*Results@#DNA genotyping was informative in 649 of 656 cases, leading to identification of 215 hydatidiform mole gestations and 434 non-molar gestations. Most of non-molar gestations (375 cases, 86.4%) were diploid hydropic abortion. Various trisomy syndromes were found (53 cases, 12.2%), including trisomy 2, 3, 4, 7, 8, 13, 16 and 21. Only 2(0.5%) digynic triploid gestations were detected. Moreover, 4 cases (0.9%) of uniparental disomies (homologous or heterologous) were found. There were 196 cases with histologic diagnostic suspicious of hydatidiform moles were accurate sub-classified. Among them, 59 cases hydatidiform moles were under-diagnosed as diploid hydropic abortions, and 28 cases diploid hydropic abortions were over-diagnosed as hydatidiform moles.Compared with partial moles(PHM), there were no specific histomorphological features between the various types of non-molar gestations and partial moles for definitive diagnostic separation. There was no significant difference in the expression of p57kip2 among PHM, trisomy and diploid hydropic abortions group (P=0.247).@*Conclusions@#STR genotyping can distinguish non-molar gestations from early hydatidiform moles, and efficiently avoid misdiagnosis based only on histological evaluation. Therefore, using STR genotyping, not only can the overdiagnosis of non-molar pregnancy be avoided, but also individualized management can be offered to patients including monitoring of serum hCG.

Placental site nodule: a clinicopathological study of 20 cases / 临床与实验病理学杂志

Purpose To investigate the clinical manifesta-tions and morphologic features of placental site nodules (PSNs), and its clinical significance. Methods Twenty patients diag-nosed as PSNs were collected, then a retrospective analysis was conducted, and the characteristics of clinical data and follow-up results were analyzed,including of clinical manifestations, ultra-sonographic evaluation, morphologic and immunohistochemical features. Results The age of patients ranged from 25 to 41 years (32. 48 ± 4. 77 years in average). Three fifths of patients had pregnancy history for at least two times and the interval time to the last pregnancy ranged from 5 to 37 months (15. 33 ± 8. 05 months on average). 15 (75% ) patients went to the hospital because of abnormal vaginal bleeding. In our study, most of the samples showed a membrane-like structure without definite nod-ule. Microscopically, single or multiple, well-circumscribed and oval small nodules were found in endometrial tissue. In most ca- ses, the hyalinization was generally uniform in the center of the nodules, more or less intermediate trophoblasts appeared on the edge of the nodules. Immunohistochemically, the strong diffuse expressed CK (AE1/AE3), CAM5. 2, EMA, GATA-3, Cyclin E and p63 were detected in most of all cases, and PLAP showed strong focal expression, α-inhibin and hPL showed faint focal expression, Ki-67 staining for proliferative index was less than 4% . Conclusion PSN is a benign lesion of the intermediate trophoblast at the chorionic leave. Some diseases including hya-linized decidua, epithelioid trophoblastic tumor, and squamous cell carcinoma with hyalinization need to be identified. Some im-munohistochemical markers may be certain helpful in distinguis-hing as necessary.

Clinicopathological analysis of 5 cases of placental mesenchymal dysplasia / 临床与实验病理学杂志

Purpose To explore the clinicopathological features, diagnosis, differential diagnosis and prognosis of placental mesenchymal dysplasia. Method The clinicopathological data of 5 cases with placental mesenchymal dysplasia were retrospectively analysed and related literatures were also re-viewed. Results All of 5 patients were consciously fetal movement disappeared or found abnormal ultrasound results at routine examination of the pregnancy. The placentas were enlarged, partly with oedematous "grape-like" cysts. On histologic exami-nation, enlarged villi with varying degrees of edema contained abnormal thick walled fetal blood vessels. The chorionic vessels were expanded and congested, and some chorionic villi showed mesenchymal cell hyperplasia. In immunohistochemical staining, p57 was positive, and Ki-67 showed low expression. There was no the trophoblastic proliferation. It's mainly differential diagnosis was hydatidiform mole.2 cases were accompanied with stillbirth. Conclusion The diagnosis of placental mesenchymal dysplasia can be confirmed by pathology examination. When a cystic placenta is detected by ultrasound examination, placental mesenchymal dysplasia should be considered in the differential diagnosis.

Genome-wide identification of sweet orange (Citrus sinensis) metal tolerance proteins and analysis of their expression patterns under zinc, manganese, copper, and cadmium toxicity.

Plant metal tolerance proteins (MTPs) play important roles in heavy metal homeostasis; however, related information in citrus plants is limited. Citrus genome sequencing and assembly have enabled us to perform a systematic analysis of the MTP gene family. We identified 12 MTP genes in sweet orange, which we have named as CitMTP1 and CitMTP3 to CitMTP12 based on their sequence similarity to Arabidopsis thaliana MTPs. The CitMTPs were predicted to encode proteins of 864 to 2556 amino acids in length that included 4 to 6 putative transmembrane domains (TMDs). Furthermore, all the CitMTPs contained a highly conserved signature sequence encompassing the TMD-II and the start of the TMD-III. Phylogenetic analysis further classified the CitMTPs into Fe/Zn-MTP, Mn-MTP, and Zn-MTP subgroups, which coincided with the MTPs of A. thaliana and rice. The closely clustered CitMTPs shared a similar gene structure. Expression analysis indicated that most CitMTP transcripts were upregulated to various extents under heavy metal stress. Among these, CitMTP5 in the roots and CitMTP11 in the leaves during Zn stress, CitMTP8 in the roots and CitMTP8.1 in the leaves during Mn stress, CitMTP12 in the roots and CitMTP1 in the leaves during Cu stress, and CitMTP11 in the roots and CitMTP1 in the leaves during Cd stress showed the highest extent of upregulation. These findings are suggestive of their individual roles in heavy metal detoxification.

Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.).

Zinc (Zn) and iron (Fe) deficiency are widespread among citrus plants, but the molecular mechanisms regarding uptake and transport of these two essential metal ions in citrus are still unclear. In the present study, 12 members of the Zn/Fe-regulated transporter (ZRT/IRT)-related protein (ZIP) gene family were identified and isolated from a widely used citrus rootstock, trifoliate orange (Poncirus trifoliata L. Raf.), and the genes were correspondingly named as PtZIPs according to the sequence and functional similarity to Arabidopsis thaliana ZIPs. The 12 PtZIP genes were predicted to encode proteins of 334-419 amino acids, harboring 6-9 putative transmembrane (TM) domains. All of the PtZIP proteins contained the highly conserved ZIP signature sequences in TM-IV, and nine of them showed a variable region rich in histidine residues between TM-III and TM-IV. Phylogenetic analysis subdivided the PtZIPs into four groups, similar as found for the ZIP family of A. thaliana, with clustered PtZIPs sharing a similar gene structure. Expression analysis showed that the PtZIP genes were very differently induced in roots and leaves under conditions of Zn, Fe and Mn deficiency. Yeast complementation tests indicated that PtIRT1, PtZIP1, PtZIP2, PtZIP3, and PtZIP12 were able to complement the zrt1zrt2 mutant, which was deficient in Zn uptake; PtIRT1 and PtZIP7 were able to complement the fet3fet4 mutant, which was deficient in Fe uptake, and PtIRT1 was able to complement the smf1 mutant, which was deficient in Mn uptake, suggesting their respective functions in Zn, Fe, and Mn transport. The present study broadens our understanding of metal ion uptake and transport and functional divergence of the various PtZIP genes in citrus plants.

Real-time in-situ Simultaneous Monitoring of Dissolved Oxygen and Materials Movements at a Vicinity of Micrometers from an Aquatic Plant by Combining Deflection of a Probe Beam and Fluorescence Quenching.

It is desirable to be able to monitor the intake or release of the components at different organs of aquatic plants in real time and in-situ. Here, we report a novel optical detection system that allows for real-time in-situ simultaneous monitoring of the dissolved oxygen and material movements at a vicinity of micrometers from the aquatic plant surface. A blue semiconductor diode-laser was used as the light source of both the probe beam and excitation light for fluorescence. The laser light reflected by a dichroic mirror was focused to a vicinity of the plant/water interface in a culture dish by an objective lens. The distance between the focused laser beam and the plant surface was adjusted by an X-Y-Z micro-stage. Deflection of the probe beam was detected by a position sensor, and fluorescence from the vicinity was monitored by a PMT. A commercial fluorescent DO sensor, which simultaneously monitored temperature, was immersed into the culture dish at about 1 cm away from the aquatic plants. A white-light LED was used to illuminate the aquatic plants in the dish in photosynthesis process. A Ru-complex (tris (2,2'-bipyridyl)ruthenium(II) chloride) was used as a fluorescent probe, and Egeria densa Planch. was used as a model aquatic plant. The DO-quenched fluorescence and material movement-induced deflection signals are compared at different distances from the aquatic plant surface. The results show that the optical detection system can monitor DO and the material movements at a vicinity of the aquatic plants not only much more sensitively, but also much more closely to real time than analytical methods that monitor concentration changes at a bulk solution.

Mulberry trees conserved soil and protected water quality in the riparian zone of the Three Gorges Reservoir, China.

China's Sloping Land Conversion Program (SLCP) was designed to restore perennial plant cover on sloping land in western China, in part to protect the Three Gorges Reservoir (TGR). In this study, we examined use of white mulberry (Morus alba L.) in the SLCP to protect water quality and conserve soil. We established nine runoff monitoring plots divided among three categories (vegetable farming, fallow control, and mulberry plantation) on a bank of the Liangtan River situated at the western margin of the TGR. The land had been used previously by farmers for growing vegetables. We found that soil loss and surface water runoff were lowest in the mulberry plots and highest in the vegetable plots. We used inductively coupled plasma atomic emission spectroscopy (ICP-AES) to assess the concentration of selected heavy metal pollution indicators (Zn, Hg, As, Ni, Pb, Cr, Cd, and Cu) in the monitoring plot soils at the beginning of the experiment in May 2009. The heavy metals were assessed again at the end of the experiment in October 2012, and we found that the concentrations of these pollutants had been reduced in all fallow and mulberry plots, and to the greatest extent in the mulberry plots. We found that levels of Hg, Pb, and Cu increased in the vegetable plots. For these reasons, we conclude that riparian mulberry plantations are useful for reducing rapid runoff of storm water, conserving soil, and sequestering heavy metal pollutants in the TGR region.

Comparative Studies on Effects of Acid Solutions on Aquatic Plants by Beam Deflection and Absorbance Spectroscopy Methods.

The beam deflection method and absorbance spectroscopy were applied to study effects of acid solutions on aquatic plants, and their results were compared. Aquatic plants Egeria densa and Ceratophyllum demersum L were used as model plants. In absorbance experiments, a piece of the plants was put in a beaker with 20 mL HCl solution, and absorbance of the HCl solution was measured every 30 min. In beam deflection experiments, a probe beam from a He-Ne laser was focused to a vicinity of the plants in a culture dish with HCl solution by an objective lens, and deflection signals of the probe beam were monitored by a position sensor. Absorbance spectra of the HCl solutions with immersing of the plants showed absorbance below 410 nm, suggesting that some compounds leaked from the plants into the HCl solutions. Changes of absorbance and deflection signals with immersion time were examined for different pH levels. The changing trends of the absorbance and deflection signals with time were similar, but the absorbance changes were delayed for about 2 - 3 h. The absorbance method could not detect the effect of the pH 5.0 HCl solutions on the aquatic plants, while the deflection method could.

Direct detection of methicillin-resistant Staphylococcus aureus in sputum specimens from patients with hospital-associated pneumonia using a novel multilocus PCR assay.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of hospital-associated pneumonia (HAP). The rapid identification of MRSA would be beneficial for early diagnosis. The study aimed to evaluate a multilocus, fluorescence-based PCR assay based on the detection of mecA and nuc genes for identification of S. aureusin lower respiratory tract (LRT) specimens. Sensitivity and specificity of the PCR assay were analyzed. Clinical evaluation for the assay was performed using LRT specimens from patients with HAP, and the sensitivity, specificity, positive and negative predictive values (PPV and NPV) were evaluated in comparison with semi-quantitative culture methods. The result showed the assay provided positive identification of all MRSA reference strains with a limit of detection for MRSA of 4 × 103 CFU/mL. Compared with semi-quantitative culture, the sensitivity, specificity, PPV and NPV were 100%, 89.6%, 75.0%, and 100%, respectively. A positive correlation between MRSA bacterial colonies and PCR copy number was found. The specificity and PPV reached 96.6% and 89.7% respectively, if the PCR copy number reached a definite positive threshold of 5.96 × 105. It suggested that this novel multilocus, fluorescence-based PCR assay proved to be a fast, sensitive and specific tool for direct detection of MRSA from LRT specimens.

Value of detection of DNA mismatch repair proteins deficiency by immunohistochemistry in predicting tumor microsatellite status / 中华病理学杂志

<p><b>OBJECTIVE</b>To evaluate the sensitivity and specificity of immunohistochemical (IHC) staining of DNA mismatch repair (MMR) protein for the screening of microsatellite instability (MSI) colorectal cancer (CRC).</p><p><b>METHODS</b>A total of 255 CRC cases were studied, including 140 cases of routine paraffin-embedded tissue samples and 115 cases constructed on tissue microarray. Expressions of 4 MMR proteins including MHL1, MSH2, MSH6 and PMS2 were investigated by IHC. Negative protein expression was defined as complete absence of nuclear staining within tumor cells in the presence of positively labeled internal non-neoplastic cells. Focal staining was defined as the presence of staining in < 5% of the tumor cells. CRCs showing negative staining for any MMR proteins were interpreted as MMR deficient tumors. PCR-genescan MSI analysis was performed in each case by a five marker panel including Bat26, Bat25, NR-21, NR-24 and MONO-27.</p><p><b>RESULTS</b>Among the 140 CRCs with routine formalin-fixed paraffin embedded tissue sections, concordance rate between IHC and PCR-genescan was 98.6% (138/140), the sensitivity and specificity of IHC in detecting MSI tumors were 94.9% (37/39) and 100.0% (101/101), respectively. The 2 disconcordant cases showed focal staining in at least one of the MMR proteins but were confirmed to be MSI-H CRCs by PCR-genescan assay. On tissue microarray, 91.3% (105/115) of the cases had informative results. The concordance rate between IHC and PCR-genescan was 100.0% (105/105). Both the specificity and sensitivity of IHC in detecting MSI tumors on available tissue microarray samples were 100.0%. Ten cases were inclusive due to the presence of negative stains of MMR proteins in both the tumor and internal control cells.</p><p><b>CONCLUSIONS</b>Detection of 4 MMR proteins expression by IHC is reliable for identifying MSI CRCs and is recommended for routine practice. Tumors with focal MMR protein staining are highly suspected for the presence of MSI-H and PCR-genescan based MSI analysis should be performed to confirm.</p>

Diagnostic utility of STR genotyping for partial hydatidiform moles / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the clinical utility of short tandem repeats(STR) genotyping technique for diagnosis of partial hydatidiform moles (PHM).</p><p><b>METHODS</b>Ten cases with the original diagnosis of PHM and six cases diagnosed as "favour PHM" or "abnormal villous, PHM not excluded" were selected for the study. The clinical information and follow-up data were reviewed. Histopathologic features were evaluated along with p57 immunohistochemistry. After DNA extraction from each sample, genotyping was performed by AmpFlSTR(®) Identifiler™ PCR kit to amplify 15 STR polymorphism loci plus the amelogenin gender-determining in a single robust PCR.</p><p><b>RESULTS</b>The age of patients ranged from 18 to 49 years (mean=29 years, median=29 years). Two villous populations (7/16), irregular villous contour (13/16), at least moderate trophoblastic hyperplasia (2/16), cistern formation (8/16), syncytiotrophoblastic knuckles (14/16), trophoblastic pseudoinclusions (6/16) and nucleated fetal red blood cells (8/16) were presented in these cases. Of the cases in the study, STR genotyping identified 4 monospermic complete hydatidiform moles (MCM), 3 dispermic partial hydatidiform moles (DPM) and 9 hydropic abortions (HA). The misdiagnosis rate was 13/16 only relied on morphology evaluation. Immunostaining of p57 showed 3/4 of MCM were focally positive (<5%-20%+), 1/4 of MCM were diffusely positive (70%+), 3/3 of DPM were diffusely positive (≥50%+), 7/9 of HA were diffusely positive (≥50%+), and 2/9 of HA were focally positive (10%+).</p><p><b>CONCLUSIONS</b>Combination of histomorphologic evaluation and p57 immunostaining is insufficient for a definitive diagnosis of PHM. STR genotyping offers an accurate diagnosis of PHM.</p>

Real-time noninvasive monitoring of UV light-induced cell death by the deflection of a probe beam.

Cell death and its deregulation characterize numerous human diseases. Here, we report on real-time noninvasive monitoring of UV light-induced cell death by the deflection of a probe beam. UV light of 330-370 nm from a high-pressure Hg lamp illuminated cultured HepG2 cells, and at the same time a probe beam from a diode laser was passed through a vicinity of the HepG2 cell. The deflection signal of the probe beam, which was induced by changes of the concentration gradients in processes of the active materials movements across the cell membrane, was monitored. It was found that the deflection signals changed greatly after UV illumination, suggesting that the materials movements across the cell membrane were greatly affected by the UV illumination. After UV illumination of about 5400-7400 s at a light power of 0.028 W/cm(2), the deflection signals became little changed with time, suggesting that the living cells had been killed by the UV illumination. This conclusion agreed well with cell viability determinations of the traditional trypan blue method.