P < 0.001), and presepsin was markedly elevated even in the early stage of SIRS . According to the receiver-operating characteristic (ROC) analysis, presepsin demonstrated a high diagnostic value compared with either PCT or CRP. In the early stage of SIRS, presepsin remained a highly sensitive (74.7%) and specific (88.4%) diagnostic marker compared with either PCT, CRP, or WBC. Moreover, the areas under the curve (AUCs) of presepsin (84.6%) were also superior to those seen in either PCT (79.6%) or CRP (71.8%). Thus plasma presepsin levels have comparable performance in SIRS for patients with nephrolithiasis.It is relatively difficult to diagnose bacterial sepsis in nephrolithiasis patients. The aim of the study is to evaluate the diagnostic ability of presepsin in the differential diagnosis including SIRS, infection, or sepsis and to compare its diagnostic value with other markers, mainly as CRP, procalcitonin (PCT), and white blood cell (WBC) in patients of nephrolithiasis presenting with SIRS. 39 patients of nephrolithiasis who were diagnosed as SIRS were prospectively investigated. Plasma presepsin was detected by Pathfast presepsin assay system; CRP and PCT were measured as well. Additionally, 25 nephrolithiasis patients without SIRS were included. At all timing samples, patients were classified as SIRS or non-SIRS group. Median plasma presepsin levels were significantly increased in the SIRS group compared with non-SIRS group (452 pg/mL versus 178 ng/mL,  Percα , 4. Systα , 6. Sincα . In factVarious markers have been studied for diagnosing sepsis. At present, serum levels procalcitonin (PCT) and C-reactive protein (CRP) are the two most common parameters in the diagnosis of SIRS , 9. HoweSoluble cluster of differentiation 14 subtype (sCD14-ST), also called presepsin, is cleaved from the monocyte/macrophage-specific CD14 receptor complex after binding with lipopolysaccharides (LPS) and LPS binding protein (LPB) during systemic infections –15. AspaHere, we intend to evaluate the diagnostic ability of presepsin in the differential diagnosis including SIRS, infection, or sepsis and to compare its diagnostic value with other markers, mainly as CRP, PCT, and WBC in patients of nephrolithiasis presenting with SIRS. Diagnosis of SIRS in the nephrolithiasis patients is sometimes not timely; hence a marker that is able to distinguish inflammatory response to infection from other causes of inflammation should be tried in clinical practice and the advantage of using presepsin as a marker is its accuracy, which will be demonstrated in this work . Besides2 < 32 mmHg and leukocyte count >12 × 109/L or <4 × 109/L, levels > 10% of immature neutrophils; then the simultaneous presence of two or more of these criteria clinical/laboratory signs was defined as SIRS [μmol/L, a percentage increase in serum creatinine of more than or equal to 50%. We have also collected blood urea nitrogen (BUN), serum creatinine (SCr), and urinary WBC at the time when SIRS was evident and once more 48 h after therapy. Approval for the study was received in advance from the local ethics committee of our institution.Between July 2013 and June 2014, all in-patients clinically diagnosed as nephrolithiasis by imaging examination at our department were enrolled in the study. Diagnostic criteria of SIRS were based on clinical manifestations and laboratory findings, according to  ACCP/SCCM guidelines: body temperature >38.5°C or <36°C, heart rate (HR) > 90/min, respiratory rate (RR) > 20/min, or PaCO as SIRS . In thisAfter obtaining informed consent, the serum samples of SIRS patients were obtained immediately when the symptoms occurred before antibiotics intervention. The second and third serum samples were obtained after 6 h and 24 h. Plasma and serum supernatants were obtained after centrifugation of the blood samples for 10 min, following which samples were stored in 0.5 mL aliquots at −80°C in order to avoid multiple freeze-thaw cycles. Plasma presepsin levels were measured by a chemiluminescent enzyme immunoassay  system. PCT levels were performed on an E 170 autoanalyzer  which was operated according to the electrochemiluminescence immunoassay (ECLIA) measurement principle . CRP ana U test. The concentrations of presepsin in SIRS patients when untreated and 48 h following treatment were compared by Student's paired t-test. Concentrations were expressed as median (5–95 percentile) and AUC ROC values as percent (%).Data obtained by measurements were given as mean ± one standard deviation. The main data collected for assessing patient symptoms and laboratory parameters, such as presepsin, serum PCT, CRP and WBC were compared by the Mann-WhitneyP < 0.05 was considered statistically significant.The receiver-operating characteristic (ROC) curve analyses were performed with calculation of area under the curve (AUC) for diagnosis of sepsis, severe sepsis, and septic shock during the first day of SIRS. The area under the curve (AUC) was reported to evaluate the utility of these potential markers in discriminating SIRS patients from the non-SIRS simple nephrolithiasis group. An AUC of 0.5 is considered to be no better than expected by chance, whereas a value of 1.0 signifies a perfect biomarker. Sensitivity, specificity, and cutoff were calculated in accordance with ROC curves for each biochemical marker. SPSS software  and MedCalc  were used for statistical analyses, and P > 0.05) (P > 0.05), which suggested that the stone size was not an impact factor between different groups in our study. With reference to the SIRS criteria, the body temperature and blood WBC value were expressively higher in patients with SIRS as compared to those without SIRS (P < 0.05). In addition, urinary WBC values in the untreated patients in SIRS group were different as compared with non-SIRS group. Body temperature and WBC data were described in P < 0.05), and the length of stay in hospital was also longer in the SIRS group  (This study included 39 nephrolithiasis patients with SIRS in SIRS group and 25 simple nephrolithiasis patients in the non-SIRS group. The demographic parameters are listed in  > 0.05) . The ana < 0.05) .P < 0.001). Furthermore, the levels in the SIRS group were much higher in the reexamination after 6 h and 24 h  (P = 0.26) (The presepsin concentrations were 178 (70–312) pg/mL in the non-SIRS group; 452 (129–880) ng/mL in the untreated SIRS group; 584 (154–1331) pg/mL in the SIRS group reexamination after 6 h and 660 (190–1705) pg/mL after 24 h . The lev < 0.01) . When pr = 0.26) .P < 0.001). Additionally, in the SIRS group the level of PCT in the reexamination was even higher than that found in the group where PCT was determined when SIRS happened (P < 0.001).The serum PCT concentrations were 0.172 (0.017–1.215) ng/mL in the non-SIRS group and 0.973 (0.341–4.794) in the untreated SIRS group; PCT was 1.425 (0.698–9.176) ng/mL and 1.864 (0.712–15.392) ng/mL, respectively, in the SIRS group that was redetermined 6 h and 24 h after treatment. There were also statistically significant differences found between the non-SIRS group and the SIRS group (P < 0.001) , 15.9 (4.9–36.5), 28.4 (7.1–64.2), and 55.2 (13.2–82.5) mg/L, respectively. There were statistically significant differences found between the non-SIRS group and the SIRS group (< 0.001) . Timely To evaluate laboratory markers, we have used the ROC curve method wherein the areas under the curve (AUCs) for plasma presepsin, PCT, and CRP in the SIRS group were 84.6%, 79.6%, and 71.8%, respectively . TherefoSIRS is an important and predictive factor for UTI, especially in urinary sepsis. However, traditional urinary tests could not efficiently reflect the progression of SIRS that had originated from the urinary tract. Mariappan et al. observed 54 patients and found that 42% of patients had a positive upper urinary tract culture (pelvic and stone), whereas only 5.6% had a positive urine culture from the bladder . AccordiPresepsin is a glycoprotein expressed on the membrane surface of monocytes and macrophages and serves as a receptor for lipopolysaccharides (LPSs) and LPS-binding proteins (LPBs). By activating a proinflammatory signaling cascade on contact with infectious agents, CD14 has a role as a recognition molecule in the innate immune response against microorganisms. During inflammation, plasma protease activity generates soluble CD14 (sCD14) fragments. One of them, called sCD14 subtype (sCD14-ST), or presepsin, is normally present in very low concentrations in the serum of healthy individuals and has been shown to be increased in response to bacterial infections . CheneviP < 0.001). This might be due to the accumulation of neutrophils within the tubular lumen during systemic inflammation and sepsis, which could lead to a noticeable increase of presepsin. Our study was concordant with other studies done with children presenting with sepsis [P < 0.001), but was not different with 24 h reexamination (660 (190–1705) and 584 (154–1331) pg/mL, P = 0.26). Thus, in the early stage of SIRS before the occurrence of AKI, determination of the levels of presepsin might be an objective parameter before clinical diagnosis of AKI.In our study, we evaluated presepsin and found that presepsin was noticeably higher in the SIRS group as compared to the non-SIRS group (178 (70–312) and 452 (129–880) pg/mL, h sepsis . Presepsh sepsis . The meah sepsis . We aimeP > 0.05). Therefore, at the early onset of SIRS, the measurements of presepsin, PCT, and CRP were all highly sensitive and specific markers of SIRS. It is noteworthy that this is an initial study in the cohort of nephrolithiasis. However, WBC's ineffective diagnostic performance should be kept in mind.PCT and CRP have been used as classic markers in the clinic for diagnosis and monitoring of SIRS during attempts to cure critically ill patients. Mokart et al. suggested that PCT could be a more reliable marker than CRP in septic patients . HoweverIn addition, we have valued the redetermined data 6 h and 24 h after treatment. The increase of presepsin was lower than PCT and CRP. Analysis revealed that in the early stage of SIRS, the presepsin reached the peak fast compared to PCT and CRP in the value of diagnosis. This observation has further enhanced the diagnostic importance of presepsin. In Shozushima et al.'s study, the usefulness of presepsin for diagnosis of bacterial infections was comparable to PCT, but the clinical specificity of presepsin was much higher than PCT. Higher false positive rate of PCT resulted from a subgroup of patients with secondary trauma, indicating that presepsin levels were less influenced by traumatic situations than the PCT levels .In conclusion, we conclude that in our study, measurement of presepsin was a highly sensitive predictor and a useful monitoring marker in the early stage of SIRS for patients with nephrolithiasis. The ability of biomarkers, such as presepsin, to discern both the onset and resolution of SIRS will further validate use of such biomarkers in clinical diagnosis and greatly enhance our understanding of SIRS in the nephrolithiasis cohort. Further validation of serum presepsin as putative biomarkers of SIRS in this population will require a multicenter randomized study.


Background. Evaluation of Ki-67 index in lung carcinoid tumors (LCTs) has been of interest in order to identify high risk subsets. Our objectives are (1) to evaluate the usefulness of Ki-67 index, mitoses, and tumor size in predicting metastasis and (2) to compare the Manual Conventional Method (MCM) and the Computer Assisted Image Analysis Method (CIAM) for Ki-67 calculation. Methods. We studied 48 patients with LCTs from two academic centres in Canada. For Ki-67 calculation, digital images of 5000 cells were counted using an image processing software and 2000 cells by MCM. Mitoses/10 HPF was counted. Results. We had 37 typical carcinoids (TCs) and 11 atypical carcinoids (ACs). 7/48 patients developed metastasis. There was a positive relationship between metastasis and carcinoid type (P = 0.039) and metastasis and mitoses (≥2) (P = 0.017). Although not statistically significant, the mean Ki-67 index for ACs was higher than for TCs . Similarly, although not statistically significant, the mean Ki-67 index for metastatic group (MG) was higher than for nonmetastatic group (NMG) . However when Ki-67 index data was categorized at various levels, there is suggestion of a useful cutoff (≥0.50%) to predict metastasis . A significantly higher proportion of patients with mitosis ≥2 and Ki-67 index ≥0.50% had metastasis (P = 0.033) compared to other patients. Similarly patients with tumor size ≥3 cm and Ki-67 ≥0.50% had a greater percentage of metastases than others (P = 0.039). Although there was a strong correlation between two (MCM versus CIAM) counting methods , overall the calculated Ki-67 index was slightly higher by MCM  compared to CIAM . Conclusion. This study confirms that mitoses ≥2 is a powerful predictor of metastasis in LCTs. Although this is a small sample size, there is suggestion that analysis of Ki-67 index along with mitoses and tumor size may be a useful adjunct for predicting metastasis in LCTs. Carcinoid is a relatively uncommon neuroendocrine tumor of the lung. These tumors are classified as typical carcinoids (TC) and atypical carcinoids (AC) using Travis criteria who defined TC as a tumor with <2 mitoses/10 HPF and no necrosis, whereas the presence of spotty necrosis or 2–10 mitoses/10 HPF are diagnostic of AC [Traditional staging and histologic grading have been used to predict the biologic behavior of LCTs. Recently there is much interest to evaluate proliferation markers particularly Ki-67 in neuroendocrine tumors arising from various sites in an attempt to identify high risk subsets that may behave more aggressively. At a 2009 European Neuroendocrine Society Consensus Conference, an international group of experts proposed a grading system G1–G3) utilizing a combination of Ki-67 and mitoses to stratify patients with GI neuroendocrine tumours [ utilizinIn this study our objectives are (1) to evaluate the usefulness of Ki-67 index as well as traditional parameters such as mitoses and tumor size for predicting metastasis in LCTs and (2) to develop a systematic counting method for Ki-67 estimation in LCTs and compare the Manual Conventional Method (MCM) and the Computer Assisted Image Analysis Method (CIAM) for calculating Ki-67 index. We hypothesis that in LCTs in addition to mitoses, Ki-67 index might be an important parameter for identifying high risk subsets of patients for whom multimodal therapy may be considered for management.2 of viable tumor), and the average mitotic figure per 10 HPF (2 mm2) was calculated as suggested by Travis et al. [We conducted a computerized search of the pathology records at London Health Sciences Centre (LHSC) and St. Joseph's Health Centre (SJHC) in London, Ontario, Canada, over a 25-year period 1982–2007). Forty-eight consecutive patients who had a histologic diagnosis of LCTs in resected specimens were selected for this study. All cases were reclassified into TC and AC using the criteria by Travis et al. [982–2007.For quantitative evaluation of Ki-67 staining, densely stained areas from tumor were systematically identified on immunostained sections at high power field . Up to ten high power fields were selected from these high density areas for independent evaluation and a minimum total of 5000 tumor cells including the positive stained cells were counted for each case by one of the authors (N. Panjwani) to calculate the Ki-67 index. The number 5000 tumor cells was chosen based on statistical analysis by calculating the optimal number of cells to be counted based on 95% confidence interval (CI) using multiple proportion of cells. Five thousand cells proved to be the optimal number with a relatively tighter CI. Images were digitized as TIF images using a digital camera system  and imported into a Microcad PC Desktop Computer (Model ANT 303x). The stored digital images were analyzed with image-processing software  with a superimposed grid and manual count tool options. Positive staining was observed as dark brown staining of the nuclei in tumor cells . LymphocSlides were scanned in a routine manner and areas of highest density staining were located. Using a Zeiss microscope and 40x objective one author (A. Shibani) manually counted a minimum of 2000 tumor cells to calculate the Ki-67 labeling index as suggested by Rindi et al. . Positivt test for independent variables. Ki-67, mitoses, and tumour size were categorized at various levels. Clinically meaningful combinations of mitoses and Ki-67, size and Ki-67, and mitoses and tumor size were evaluated. For MG and NMG sensitivity, specificity and odds ratios and their 95% confidence intervals were calculated. To evaluate the relationship between the computerized and manual counting methods, statistical analysis was completed using the Pearson Correlation Coefficient. A P value < 0.05 is considered as statistically significant, and r value > 0.8 is considered as strong correlation.The development of metastasis was considered as the end outcome point in our study and therefore we divided the patients into these 2 groups, metastatic (MG) and nonmetastatic (NMG) groups. Between-group comparisons were made using Fisher's exact two-tailed tests. The relationship between Ki-67 indices and carcinoid subtypes was analyzed with an unpaired The age of the patients ranged from 17 to 81 years with a mean age of 52 years. Eighteen patients were male and 30 patients were female. All patients underwent surgical resection as primary treatment . Out of 48 cases, 37 were classified as TCs and 11 ACs. Patient follow-up ranged from 0.5 months to 306.8 months, with median follow-up of 45 for metastatic group (MG) and 35 for nonmetastatic group (NMG). The tumor size ranged from 0.5 cm to 9.5 cm (mean 2.7 cm) in greatest diameter. The mitoses ranged from 0 to 11.6 (mean 2.7). Seven out of 48 patients developed metastasis: 6 in mediastinal lymph nodes, 3 in liver, and 2 in both. Lymphovascular invasion was identified in 4 cases, 3 of which were TCs. Six patients presented with endocrine symptoms: carcinoid syndrome [P = 0.299; 2.32% versus 1.37%, MCM, P = 0.71) than for typical carcinoid by both counting methods. Similarly, when we analyzed the relationship between Ki-67 index and metastasis, although not statistically significant, the mean Ki-67 index for MG was higher than for NMG . P value were calculated. As expected there is a statistically significant correlation between metastasis and carcinoid type (P = 0.039) and mitoses ≥2 (P = 0.017) with relatively high specificity. In addition, when Ki-67 index data was categorized at various levels, although not statistically significant, there was suggestion of a useful cutoff (≥0.50%) to predict metastasis by both counting methods with relatively high sensitivity. Similarly although not statistically significant there was suggestion of a useful cutoff for tumor size (≥3 cm) to predict metastasis with relatively high sensitivity and specificity.P = 0.033) with high specificity. Similarly patients with tumor size ≥3 cm and Ki-6 7 ≥ 0.50% had a greater percentage of metastases (P = 0.039) with high specificity. In this study, there was no statistically significant correlation between metastatic disease and patient age, sex, presence or absence of lymphovascular invasion, endocrine symptoms, or adjuvant therapy.We performed similar statistical analysis combining multiple clinical and pathologic factors . A signir = 0.929, P = 0.001) (We used both MCM and CIAM for counting Ki-67. Although there was a linear relationship with strong correlation between these two counting methods (= 0.001) , overall= 0.001) .Pulmonary neuroendocrine (NE) tumors encompass a spectrum with four tumour categories being identified by morphology, namely, low-grade TC, intermediate-grade AC, and high-grade large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung carcinoma (SCLC) , 5. The Traditionally the biological behavior of carcinoid tumors is predicted by conventional methods such as staging and histologic grading using Travis criteria. Travis et al.  in a stuDuring the past 10–15 years, newer biological markers have been sought to subtype carcinoid tumors and to identify high risk subtypes. Immunohistochemical analysis of the well-established proliferation marker Ki67 (MIB1) has received much attention and has been incorporated into a number of studies on pulmonary and extrapulmonary tumors. This nuclear antigen is a cell cycle associated protein that is expressed throughout the cell cycle in proliferating cells (G1-M) but not in resting (G0) cells –11.At a European Neuroendocrine Society Consensus Conference , an inteIn this study of 48 cases when we assessed the relationship between carcinoid subtype and metastatic disease, we noticed that 36% of patients with AC had metastasis compared to only 8% of patients with TC. This result emphasizes the observation of others that AC of lung is associated with worse prognosis than TC , 2, 12. P = 0.02) than patients without endocrine symptoms at diagnosis.In this study we did not observe a statistically significant association between metastasis and a number of other clinically important factors such as age, sex, lymph vascular invasion, endocrine symptoms, adjuvant therapy, and death. However, it is important to note that the majority of patients who developed endocrine manifestations (6 patients) are those with a Ki-67 index >0.50% . Granberg et al.  in a stuP = 0.0035). The authors concluded that, by using a ≥4% cutoff, there was a significant difference in the 5-year overall survival rate of patients with pulmonary carcinoids and suggested postoperative chemotherapy for high risk subsets of patients. Laitinen et al. [P < 0.01) in this study. The author concluded that the proliferation rate in carcinoid tumors is generally very low but increased in AC and can be related to worse prognosis. Arbiser et al. [P < 0.05); however, proliferation rate did not distinguish between TC and AC, nor between SCC and LCNEC. They concluded that increased rates of proliferation, but not angiogenesis, correlate with tumor type. Igarashi et al. [P < 0.01). He also reported a close correlation between Cyclin B1 and Ki-67 expression in these tumors. Pelosi et al. [When we reviewed the literature on Ki-67 index and pulmonary neuroendocrine , 12–22 an et al.  in a stur et al.  studied i et al.  in a stui et al. , in a sti et al.  in a stui et al.  reportedi et al. , using ai et al.  in a stui et al.  in a recAlthough variable methodology has been used by above authors in counting Ki-67, it is clear that most of the studies observed a higher Ki-67 index in AC compared to TC although cutoff thresholds are variable in these studies. Our study is unique in that we utilized 2 counting techniques, namely, CIAM counting 5000 cells and MCM counting 2000 cells, to calculate Ki-67 index. Although time-consuming, we believe that image analysis allows for more objective, reproducible, and precise counts. Although we observed a strong correlation between two counting methods, it is important to note that Ki-67 labeling index calculated by MCM was slightly higher than that by CIAM for individual cases. This is attributed to the fact that MCM uses human eye for scanning hotspot areas compared to the precise CIAM tool, resulting in slightly imprecise estimation of the total number of cells in the background. In addition we counted a relatively larger number of cells (5000) by CIAM to reduce the margin of error and used statistical analysis to determine the minimum number of cells to be counted by CIAM. However, results of our study suggest that, in every day surgical pathology practice when Ki-67 index needs to be calculated, MCM may be used as a substitute. In such instances we recommend manual counting of at least 2000 cells in hotspot areas.In conclusion, this study confirms that mitoses ≥2 is a powerful predictor of metastasis in LCTs. In addition, in this study, mean Ki-67 index is higher in AC compared to TC and in metastatic group compared to nonmetastatic group. Although this is a small sample size, there is suggestion that analysis of Ki-67 index along with mitoses and tumor size may be a useful adjunct for predicting metastasis and for initiating adjuvant multimodal therapy in lung carcinoid tumours. However a standardized methodology is needed to validate limits of Ki-67 index for practical prognostic applications and this would require larger prospective studies and testing on both small biopsies and resected specimens.


Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in ‘Valencia’ C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of ‘Valencia’ C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid ‘Valencia’ C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in ‘Valencia’ sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in ‘Valencia’ sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites. Citrus sinensis [L.] Osbeck), as the most important Citrus species with great economic and health value, is grown in 114 countries and accounts for nearly 70% of the world’s Citrus production [C.sinensis is believed to arise from the crossing between ancestral pommelo and mandarin with polymorphic alleles in the genome [C. sinensis are rather challenging because of its high heterozygosity [C. sinensis [L.] Osbeck) has been generated via anther culture  Osbeck as the control to investigate the chromosomal characteristics of ‘Valencia’ sweet orange. By combining CMA/DAPI banding with repetitive DNA FISH, the cytological heterozygosity, and the number and loci of the fragile sites in ‘Valencia’ sweet orange were revealed. Furthermore, Ag-NOR showed that the distended 45S rDNA regions as fragile sites had transcriptional activity. The number and characteristics of fragile sites in Citrus were proposed and their possible functions were discussed.So far, chromosome fragile sites in woody plants have not been reported, although lightly–staining chromatin depletion decondensation occurred at 45S rDNA regions are common in d genera , 31–35. C. sinensis [L.] Osbeck) were obtained from the National Center of Citrus Breeding, Huazhong Agricultural University, Wuhan, China. Seeds were peeled off and cultivated in pots in the incubator at 28°C. Root tips from the germinated seeds (about 3–5 mm long) were successively collected. The doubled haploid callus line from anther culture of ‘Valencia’ orange was suspension-cultured to be vigorous. To harvest metaphase cells, young tissues were obtained and pretreated in a saturated aqueous solution of P-dichlorobenzene for 2 h at 20°C and fixed in 3:1 ethanol/glacial acetic acid (v/v) for 24 h, and then were stored at -20°C in 70% ethanol solution until use. Rinsed tissues were macerated in an enzyme mixture containing 0.25% pectinase (Sigma), 0.25% pectolyase Y-23 (Yakult) and 0.5% cellulose RS (Onozuka) for 80–90 min at 37°C. Then, the meristem was mashed using a fine needle with a drop of 60% acetic acid, and the slide was flame-dried with a drop of fixation solution.Fruits of ‘Valencia’ sweet orange cv. Rohde Red . FISH was performed following the procedures of Cui et al. [Plasmid 45S rDNA kindly provided by Kang et al.  was isoli et al. . The imm3 in 50% NH4OH: 0.5% formic acid)and observed under a bright field microscope (Olympus BX61) until the NORs turned black. After staining, the slides were washed with running deionized water and air-dried again. Statistical analysis on the number of nucleoli and NORs were conducted in more than 100 interphase cells and 10 metaphase cells.50 μl 50% (W/V) silver nitrate was added to each slide with the spreading of chromosomes and nuclei. The slides were incubated in a moist chamber at 70°C for 1 h and washed with distilled water, and then were air-dried. The slides were covered with a mixing staining solution in a proportion of 1:1 (v/v), and immediately fixed in 5:3:2 ethanol: glacial acetic acid: chloroform for 24h at room temperature. The anther were taken out and rinsed, then macerated in an enzyme mixture containing 0.25% pectinase (Sigma), 0.25% pectolyase Y-23 (Yakult) and 0.5% cellulose RS (Onozuka) for 60–70 min at 37°C. Microsporocytes were squeezed out of anthers and stained in 1% aceto-carmine on a clean microscope slide. The preparations were observed using a microscope (BX61: Olympus), and photos were taken with an attached video camera (DP70: Olympus). At least 30 PMCs late diakinesis used to analyze the characteristics of meiotic chromosome paring were observed, and the numbers of chromosome configurations per PMC were recorded.Metaphase of both diploid and dihaploid were analyzed for the position and distribution of CMA-DAPI bands, 45S rDNA, telomeric sites and for the integrity of chromosome morphology. We measured the relative length and arm ratio of the chromosomes. The centromeres were indicated by the positions of the primary constrictions. Measurements were performed on digital images using Image-Pro Plus 6.0 software. The banding patterns were determined on the basis of the numbers and positions of CMA positive bands. The ideogram was prepared using Microsoft Office Excel 2003.C. sinensis by cytological examination using a routine chromosome preparation procedure. Interestingly, we found that the number of chromosomes plus chromosome fragments was always 19, which was larger than the expected 18 in most mitotic cells, and it is rather different to distinguish whether one “chromosome” is a complete chromosome or merely a chromosome fragment before telomere-FISH , C , D  and F (no band) according to CMA/DAPI banding [C.sinensis cv. Valencia  dividing and then dividing again to form 4 haploid cells. The representatives PMC meiosis process in ‘Valencia’ orange were shown in C. sinensis and its dihaploid line  was often named as Dst in karyotype analysis [Fragile sites are manifested as non-random incomplete breaks or gaps with chromatin fibers on prometaphase and metaphase chromosomes , 49. So omosomes , 29, 30.analysis , which cC. sinensis, and were doubled in its doubled haploid line. Based on the previously reported karyotype formulas of Citrus and related genera [Citrus and related genera species: (1) Af ; (2) Bf ; (3) Cf ; (4) Df , which is suggested to be the most common fragile site. Although the types of fragile sites in Citrus and its genera vary, the number of fragile sites keeps relatively constant. There are two fragile sites in majority of species but three in pomelo (C. maxima). Hexaploid G. pentaphylla has six fragile sites, which may be within the normal range. The exception here is the doubled haploid of C. sinensis, which has four fragile sites. The callus line of ‘Valencia’ derived from anther culture could not generate plantlet [Citrus and related genera may be associated with karyotype evolution and the formation of new species. Yet, the roles of fragile sites in evolution still remain elusive. Thus, further researches are required to know the more detailed functions of fragile sites.Fragile sites were found to be located at very end of D-type chromosome and proximal to B-type chromosome in d genera , 31–35, ifolia);  Df (fragplantlet , suggestplantlet , or reduplantlet . Two morplantlet , 53–55, plantlet . Incidenplantlet , 58. Var


Biopsies of lesions were performed, which were positive for Varicella antigen. Subsequently, patient was started on Acyclovir. The patient's clinical status improved and rash resolved. Our patient presented with “atypical” clinical picture of disseminated cutaneous zoster with no obvious dermatome involvement. Disseminated zoster is a potentially serious infection that can have an atypical presentation in patients with immunocompromised status. High index of suspicion is needed to make the diagnosis promptly and to initiate therapy to decrease mortality and morbidity.Patients with rheumatoid arthritis (RA) have 2-fold increased risk of herpes zoster. In literature, limited information exists about disseminated cutaneous zoster in RA patients. An 83-year-old African-American female with RA presented with generalized and widespread vesicular rash covering her entire body. Comorbidities include hypertension, type II diabetes, and dyslipidemia. Patient was on methotrexate 12.5 mg and was not receiving any corticosteroids, anti-TNF therapy, or other biological agents. The patient was afebrile (98 F) with no SIRS criteria. Multiple vesicular lesions were present covering patient's entire body including face. Lesions were in different stages, some umbilicated with diameter of 2–7 cm. Many lesions have a rim of erythema with no discharge. On admission, patient was also pancytopenic with leukocyte count of 1.70 k/mm Herpes zoster called shingles is the result of reactivation of latent varicella-zoster virus . More thRheumatoid arthritis patients have a 2-fold increased risk of herpes zoster compared to general population . RA diseAn 83-year-old African-American female with rheumatoid arthritis presented with generalized and widespread vesicular rash covering her entire body. Comorbidities include hypertension, type II diabetes, and dyslipidemia. Patient had no fevers or chills; review of systems was otherwise negative. The patient was diagnosed with RA and is on low-dose Methotrexate 12.5 mg weekly for the past 8 years. Patient did not receive any corticosteroids, anti-TNF, or other biological therapy in last few years.The patient was afebrile (98 F) and did not meet Systemic Inflammatory Response Syndrome criteria . Multipl3. Patient had positive urine culture for Klebsiella pneumoniae and blood cultures were negative. Patient received 3 days of Ceftriaxone for uncomplicated urinary tract infection. Biopsies of lesions were performed on day 3 of admission by dermatology, which were positive for varicella antigen. Subsequently, patient was started on Acyclovir IV 700 mg BID for 3 days and later switched to PO 800 mg BID for 4 more days. Over the next 3 days, the patient's clinical status improved and rash improved. Patient did not have any relapses of herpes zoster infection or any sequela from infection.On admission, patient was also pancytopenic with leukocyte count of 1.70 k/mmThere is an apparent increase in the incidence of herpes zoster in patient with RA relative to general population . There iIn our patient, advance age, diabetes mellitus, rheumatoid arthritis, and Methotrexate could have contributed to dissemination of herpes zoster. Methotrexate have been implicated as a risk factor for developing infection and varicella-zoster infection , 10. HowPatients with dissemination of herpes zoster are at increased risk for end organ involvement, particularly lungs, liver, and brain . Other cTreatment of choice for disseminated zoster is IV Acyclovir 10 mg/kg every 8 hours for 5–7 days. Infectious agents like bacteria and viral infection can induce pancytopenia , 12. CerDisseminated zoster is potentially serious infection that can have an atypical presentation. High index of suspicion is needed to make the diagnosis promptly and to initiate IV Acyclovir to decrease mortality and morbidity.


Monte Carlo sampling partial least squares linear discriminant analysis (PLS-LDA) was used to distinguish full-mass-range mass spectra of cancer samples from the mass spectra of normal tissues. With 5 principal components and 30 – 40 Monte Carlo samplings, the accuracy of cancer identification in matched tissue samples reached 94.42%. Classification of a tissue sample required less than 1 min, which is much faster than the analysis of frozen sections. The rapid, in situ diagnosis with minimal sample consumption provided by TSI-MS is advantageous for surgeons. TSI-MS allows them to make more informed decisions during surgery.Tissue spray ionization mass spectrometry (TSI-MS) directly on small tissue samples has been shown to provide highly specific molecular information. In this study, we apply this method to the analysis of 38 pairs of human lung squamous cell carcinoma tissue (cancer) and adjacent normal lung tissue . The main components of pulmonary surfactants, dipalmitoyl phosphatidylcholine (DPPC,  Lung cancer is one of the most common malignancies worldwide. With the highest rate of morbidity and mortalityChemical analysis by mass spectrometry (MS) may be an alternative to standard frozen-section histology, as mass spectrometry can provide rich molecular information on the chemicals on and in the tissue, which can be correlated with pathology91014198Several MS methods have been demonstrated suitable for the direct and simple analysis of tiny tissue samples3) were analyzed from the tip of an acupuncture needle. A high voltage was applied to the needle, and to the surface of the tissue. Spectra of high-peak abundances were detected, and characteristic ions were directly observed. Using Monte Carlo sampling and partial least squares linear discriminant analysis (PLS-LDA) of TSI mass spectra, the accuracy of cancer identification in matched tissue samples reached 94.42%. The described experimental procedure is comparable with paraffin-section pathology results.In the present work, we extend the application of tissue spray ionization (TSI) MS to identify lung squamous cell carcinoma in humans. Tiny tissue pieces (1 mm3). A small volume of solvent (2 μL methanol) is used, and the spray period lasts approximately 10 s . This time is sufficient to acquire high-quality mass spectral data containing MS and MS/MS scans. The analysis can be repeated five times with the same sample by using different solvents. The total ion intensity can vary about 30% during each spray period over the course of 3 minutes; however, the relative intensities were found to be highly reproducible . This experiment was also repeated using samples from different extractions of the same tissue. The variation in total ion intensity was still within one order of magnitude, with similar relative intensities obtained for each analysis. The relative standard deviation (RSD) was obtained by measuring the abundance of ions at m/z 782.5, 808.5 and 832.5 (MS stage) of the tumor tissues, and the RSDs were 18.17%, 18.84% and 17.83%, respectively (Table S-1). Tandem mass spectrum and Collision Induced Dissociation (CID) identified a wide range of fatty acids and phospholipids in the tissues examined , but after MS analysis, some cells were collapsed, and the space between the cells could be clearly observed . The cells lysed.m/z 700–900 range in positive ion mode. A total of 380 data points from 190 pairs of matched samples (five points per sample) were interpreted in the score graphs of the PLS-LDA. After PLS dimension reduction, the first two-dimensional LDA ingredient was used in the scoring matrix discriminant analysis. When the principal component number is 5 (PLS-LDA of the spectra recorded shows a clear distinction between the samples from human lung squamous cell carcinoma tissue (cancer) and adjacent normal lung tissue . A strong sampling signal was observed with good reproducibility as the solvent dripped onto the tiny tissue sample . At a flber is 5 , the besber is 5 . Competiber is 5 , the besber is 5 .m/z range 700–900 are phospholipids, and the main peaks are identified as protonated and sodium adducts of phosphatidylcholine (PC). A systematic difference was clearly observed for TSI-MS fingerprints collected from cancerous and normal tissues. The relative abundances of m/z 154.03 , m/z 170.06 , m/z 798.90 , m/z 808.49 , and m/z 832.43  were greater in cancerous tissue, and the relative abundances of m/z 203.08 , m/z 757.47  and m/z 782.52  were higher in normal tissue (+m/z 757.47), 1-palmityl 2-oleoyl PC (POPC + Na+m/z 782.52), 2-oleoyl PC (DOPC + Na+m/z 808.49), and arachidonic acid-stearoyl PC  and surfactant-specific proteins (2%). In 1987, Jablonka et al. suggested that pro-surfactant protein B can serve as a biomarker for lung cancer predictionet al. also showed that the lipid profile is useful for diagnosing cancer and, in some cases, for recognizing its stage+ (m/z 757.47), POPC + Na+ (m/z 782.52), DOPC + Na+ (m/z 808.49), and SAPC + Na+ (m/z 832.43) are the main components of pulmonary surfactant. In this study, we provide evidence that there are significant concentration differences in pulmonary surfactant between lung squamous cell carcinoma and normal tissues: DPPC and POPC decreased but DOPC and SAPC increased in lung squamous cell carcinoma tissue relative to normal tissue, respectively. More research is required to confirm that type II alveolar epithelial cells transforming into cancer stem cells are the cause of these pulmonary surfactant differences.Type II alveolar epithelial cells are a type of lung stem cellIn conclusion, our data shows that the direct examination of tissues using TSI-MS analysis can distinguish lung squamous cell carcinoma tissue from normal lung tissues with high reliability. Using tandem MS, we were able to identify various endogenous chemicals in the tested tissues, from small molecules to phospholipids, which can be used for lung squamous cell carcinoma diagnosis. TSI-MS has the necessary characteristics for the surgical theater, such as high speed, simplicity and automation of analysis. MS analysis of tissue biopsies could provide surgeons with critical and previously unavailable information, which could rapidly guide surgical resection and improve treatment of patients with pulmonary cancerThis study was approved by the Medical Ethnics Committee in the Hospital Institutional Review Board of the Second Affiliated Hospital to Nanchang University, Nanchang, P. R. China. Written informed consent was obtained from all the patients in this study. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The criteria for patient enrollment in this study were the diagnosis of non-small cell lung cancer confirmed by pathology Fig. S-2), absence of accompanying malignancies, absence of other lung diseases, and no history of preoperative chemotherapy or radiotherapy. In total, 38 male patients enrolled in this study, with a median age of 60 (40 – 72 years). The group included 3 well–differentiated, 20 moderately–differentiated and 15 poorly–differentiated squamous cell carcinoma cases. In the group, Stage I was observed in 3 cases, I b was observed in 7 cases, II a was observed in 11 cases, II b was observed in 6 cases, IIIa was observed in 10 cases and IIIb was observed in 1 case with the new lung cancer staging system, absence3; A small piece of lung tissue (about 1 mmd1). Extraction solvent  was infused through a stainless steel capillary  at a flow rate of 4 μL min−1 with a syringe pump  and HPLC fittings. The distance between the capillary and the needle tip was 5 mm  equipped with a homemade ion source for tissue sampling . This eq5 mm d2; . A ring Ion source and the LTQ mass spectrometer were operated in positive ion detection modes. The electrospray ionization (ESI) voltage was +3.5 kV, and the temperature of the capillary inlet was 150 °C. The mass range was set at 50–2000 Da. Other parameters were set at instrument default values and no further optimization was performed. Five sampling signals were acquired from each tissue sample as the solvent dripped five times continuously. For collision-induced dissociation (CID), the MS/MS spectrum for ion detection used a maximum injection time of 100 ms. Ion Selection Operation (ISO) width was 1 Da, normalized collision energy was 13%, third grade collision energy was 14.5%, and the other parameters were set at default values of the instrument.m/z 100–1000. Spectral data was binned using a 0.01 Th bin size for high-resolution experiments and a 1.0 Th bin size for low-resolution experiments, and both were stored in a structured query language (SQL) database . The database contained both the full-known classification of every tissue specimen corresponding to the spectra, the World Health Organization (WHO) tumor type, and WHO gradem/z value variables, respectively. The mass spectral data expressed by relative abundance were directly used for the PLS-LDA33t test was performed to prove the significance of those spectra with relative abundance above 20% in positive ion mode between cancerous and noncancerous tissue.Mass spectra were collected in single-stage MS, positive ion mode, and in the mass range of How to cite this article: Wei, Y. et al. Tissue spray ionization mass spectrometry for rapid recognition of human lung squamous cell carcinoma. Sci. Rep.5, 10077; doi: 10.1038/srep10077 (2015).


Previous studies on the association between the −94ins/del ATTG polymorphism and cancer risk reported conflicting results. To clarify this relationship, we performed a meta-analysis of 21 case-control studies involving 6127 cases and 9238 controls. We used pooled odds ratios (ORs) with their 95% confidence intervals (95% CIs) to assess the association. We found that the NFKB1 promoter −94ins/del ATTG polymorphism was significantly associated with cancer risk in four genetic models . Stratified analyses revealed a significant association between the polymorphism and ovarian, oral, and prostate cancers. Similar results were determined in an Asian population and not in a Caucasian population. Thus, our results suggested that the polymorphism can contribute to cancer risk. Moreover, the polymorphism can exert race- and cancer-specific effects on cancer risk. Further large-scale and functional studies are necessary to elucidate this possible effect.Nuclear factor- Cancer is a major public health problem worldwide; it is the primary and secondary causes of death in economically developed and developing countries, respectively . The gloκB (NF-κB) was initially identified in 1986 as a transcription factor which binds to a 10 bp DNA element in kappa immunoglobulin light-chain enhancer in B cells [κB family consists of p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-Rel (Rel), and RelB. The major form of NF-κB is a heterodimer of the p50 and p65/RelA subunits which are encoded by the NFKB1 and NFKB2 genes, respectively [ NFKB1 gene is mapped to chromosome 4q24 and encodes a 50 kDa DNA-binding protein (p50) that can act as a master regulator of inflammation and cancer development [Nuclear factor- B cells . The NF-ectively . The humelopment . NFKB1 gene elicits a regulatory effect on the NFKB1 gene [ NFKB1 promoter −94ins/del ATTG polymorphism and cancer risk [ NFKB1 promoter −94ins/del ATTG polymorphism and cancer risk.A common insertion/deletion polymorphism  in the promoter region of thecer risk –12. Howehttp://www.ncbi.nlm.nih.gov/pubmed/) was performed with the following key words: , , and . The search was limited to human studies in English. All eligible studies were retrieved. The reviews and references of eligible studies were hand-searched for additional relevant publications. The most recent or complete study was selected when more than one publications contain overlapping data. A flow diagram of the study selection process is presented in A comprehensive literature search for relevant articles published  in PubMed  according to the inclusion criteria listed above, and the result was reviewed by a third investigator (Jun Tao). The following data were collected from each study: surname of first author, year of publication, ethnicity, genotyping method, source of controls, frequencies of the genotypes in cases and controls, cancer type, and Hardy-Weinberg equilibrium (HWE) of genotype distribution among controls. Ethnicity was categorised as “Asian” or “Caucasian.” Studies that investigated more than one type of cancer were regarded as individual datasets only in subgroup analyses according to cancer type. No minimum number of patients was required for this meta-analysis. Articles that reported different ethnic groups and countries or locations were considered different study samples for each category cited above. NFKB1 promoter −94ins/del ATTG polymorphism and cancer risk was estimated through pooled odds ratio (OR) with its corresponding 95% CI. Pooled ORs were calculated for insertion allele versus deletion allele, ins/ins versus del/del, ins/del versus del/del, ins/ins + ins/del versus del/del, and ins/ins versus ins/del + del/del. Subgroup stratification analyses by ethnicity and cancer type were conducted to identify the association of the −94ins/del ATTG polymorphism with cancer susceptibility.The strength of association between theQ and I2 statistic tests, where I2 > 50% indicated heterogeneity [I2 was significant (>50%); otherwise, the fixed-effects model was selected. The allele frequencies of the NFKB1 promoter −94ins/del ATTG polymorphism from the respective study were determined by allele counting. In addition, a chi-square test was used to determine whether or not the observed frequencies of genotypes conform to HWE. Pooled OR in the current meta-analysis was performed by weighting individual ORs by the inverse of their variance. The significance of the pooled OR was determined by the Z-test. In addition to the comparison among all subjects, we performed stratification analyses by cancer type  and ethnicity. Begg's funnel plot and Egger's test were adopted to evaluate the publication bias in our meta-analysis [The between-study heterogeneity of the studies included in this meta-analysis was evaluated using the ogeneity . The rananalysis , 15. All NFKB1 promoter −94ins/del ATTG polymorphism were extracted from all eligible studies. In total, this meta-analysis included 3 bladder cancer studies, 4 colorectal cancer studies, 2 ovarian cancer studies, 2 oral cancer studies, 2 prostate cancer studies, and 8 studies with the “other cancers.” Of the 21 studies, 14 were conducted among Asians and 7 were conducted among Caucasians. All cases were clinically pathologically confirmed.A total of 21 case-control studies involving 6127 cases and 9239 controls were analysed. The characteristics of all studies are presented in The results of HWE test for the genotype distribution in the control population are shown in  NFKB1 promoter −94ins/del ATTG polymorphism and cancer risk across the four genetic models ins/ins versus del/del, OR = 1.47, 95%, CI = 1.11—1.93; dominant model, OR = 1.26, 95%  CI = 1.03—1.53; recessive model, OR = 1.26, 95%  CI = 1.05—1.51; and ins allele versus del allele, OR = 1.19, 95%, CI = 1.05–1.35 (http://dx.doi.org/10.1155/2014/612972).The pooled ORs of the included case-control studies revealed a statistically significant association between the.05–1.35 . StratifP = 0.004).Publication bias was evaluated by Begg's funnel plot and Egger's test, and the visual asymmetry was determined in the funnel plot analysis . We furtκB serves important functions in pathogenetic regulation and influences cancer development and aggressiveness by enhancing tumour angiogenesis, antiapoptosis, and proliferation and by repressing immune response [κB in various malignancies [ NFKB1 encoding the p50 subunit of NF-κB modulates gene activity. This polymorphism has been recently reported to influence cancer risk.NF-response , 33, 34.gnancies , 36, inc NFKB1 promoter −94ins/del ATTG polymorphism and cancer among Asians and Caucasians [A meta-analysis of all eligible studies in 2010 suggested that the deletion allele serves as a protective or risk allele for cancer susceptibility among Asians or Caucasians, respectively . Howeverucasians , 32. Howucasians  found anucasians .κB1). The insertion allele is reportedly associated with the increased promoter activity and enhanced NFKB1 mRNA expression [In this study, we analysed 21 eligible case-control studies with 6127 cases and 9239 controls. The results of this meta-analysis revealed a significant association between insertion allele careers and enhanced cancer risk. The probable mechanism behind the observed association may be linked to the enhanced expression and activity of p50  is mediated by its function as a component of the transcription factor NF-κB, which is among the major signalling pathways involved in the cellular response to environmental stress [κB signalling pathway [κB1) signalling pathways participate in cellular proliferation by increasing IL-5 [NFKB1) [The major effect of p50  , 12, 17, NFKB1 gene might function as a prominent factor in these cancers. Therefore, further investigations are warranted to validate ethnic difference and cancer specificity in the effect of this functional polymorphism on cancer susceptibility.In the stratified analyses, the increased cancer risk remained in subgroups of Asians but not in those of Caucasians. The ethnic differences in the allele frequencies may be caused by natural selection or balance to other related genetic variants. Possible differences in genetic backgrounds and gene environment may also interact with the etiology. The increased cancer risk also remained in the subgroups of ovarian, oral, and prostate cancers. This result suggested that theThis study has several limitations. First, significant between-study heterogeneity was detected in some comparisons and may be distorting the meta-analysis. Second, the genotype distribution among controls did not completely agree with HWE. However, the association between the insertion allele and cancer risk in the overall population and in the Asian population became pronounced when the six studies that deviated from HWE were excluded. Third, the studies included in the analysis used different genotyping methods with different quality control issues that may have also influenced the results. Fourth, publication bias was observed in our study, which may affect the validity of conclusion. In the stratified analysis, we found that the publication bias was significant among the Asian groups and other cancer groups but not significant among the Caucasian, bladder, and colorectal cancer groups. The sample sizes of the included studies were diverse, and most of them were insufficiently large. These conditions might partly interpret the publication bias. Finally, only three controls were population based; thus, they may not represent the general population. Therefore, the results of this study should be interpreted with caution. NFKB1 promoter −94ins/del ATTG polymorphism is associated with cancer risk. Well-designed studies with representative sample sizes are necessary to validate these findings.In conclusion, theThe meta-analysis result of the studies which did not deviated from HWE.Click here for additional data file.


Purpose: To evaluate overall survival (OS), progression-free survival (PFS), loco-regional recurrence (LRR), and toxicities for early breast-cancer patients with one to three positive axillary lymph nodes, by the addition of radiotherapy to adjuvant chemotherapy.Patients and methods: Patients were eligible for enrollment into the study if they had pathologically proven stages II breast cancer, with one to three positive axillary lymph nodes. Patients were assigned to one of the two groups; Group 1; adjuvant chemotherapy then radiotherapy, and group 2; adjuvant chemotherapy only.Results: Between September 2008 and August 2014, 75 patients were enrolled. Forty patients group 1, and 35 group 2. The 4-year OS for group 1, and two were 77.5 and 71.4%, respectively. The 4-year PFS for group 1 and 2 were 72.5 and 60%, respectively. During the 54 months follow-up period, 11 patients from group 1 had recurrence , and 14 patients from group 2 had recurrence . The distant metastasis rate was the same in the two groups. However, the metastasis sites were different in the two groups.Conclusion: The addition of radiotherapy in stage II breast cancer with one to three positive lymph nodes improved the PFS, and LRR. Radiotherapy improved OS in patients with high-risk features. Breast cancer is the most frequent malignant tumor in women worldwide. In Egypt, it is the most common cancer among women, representing 18.9% of total cancer cases. More than 50% of patients present as early stages, thanks to advances that had recently made in the strategies of screening and early detection. A study by Salem et al. .Radiation therapy (RT) is frequently recommended for patients with early breast cancer who had undergone mastectomy. Guidelines from the American Society of Clinical Oncology (ASCO), the National Comprehensive Cancer Network (NCCN), and the American College of Radiology (ACR), all recommend for post-mastectomy RT for patients with four positive lymph nodes. However, these patients account for only a minority of node-positive disease. Unfortunately, all the three guidelines reported that insufficient evidence still exist to make any evidence-based post-mastectomy recommendations for patients with one to three positive nodes –5.Considering RT in early breast cancer with one to three positive axillary lymph nodes remains unclear. Till recently, studies were unable to provide definite answers regarding this controversial issue –8.To evaluate the overall survival (OS), progression-free survival (PFS), loco-regional recurrence (LRR), and toxicities for early breast-cancer patients with one to three positive axillary lymph nodes, who underwent mastectomy, and axillary lymph node dissection, followed by either adjuvant chemotherapy and radiotherapy, or only adjuvant chemotherapy.The primary end point was survivals benefits (PFS and OS). The secondary end points were LRR and toxicities.Female patients were eligible for enrollment into the study if they had pathologically proven breast cancer with one to three positive axillary lymph nodes on pathological examination. Status post mastectomy and axillary lymph node dissection. Patients should have stages II breast cancer , with tumor size ≤5cm (pT1-2).Further, for enrollment, patients should be between 17 and 65 years. Performance state had to be from 0 to 2 (ECOG). Patients should have adequate hematological, renal, and liver functions. Written informed consent from patients needed before proceeding in the trial. Female patients of childbearing potential must have a negative pregnancy test (serum β-HCG); and both the patient and her husband must employ effective contraceptive measures prior to the start of therapy until 4 weeks after the last dose of chemotherapy. Hormone receptor status has to be either ER or PR + ve, with HER 2 neu either positive or negative. Adjuvant chemotherapy should include anthracycline. Additionally, patients must have no concurrent malignancy.Settings: The study was run in 2 educational institutes in Egypt; Ain Shams University Hospital, and Ismailia oncology teaching hospital.Study design: Patients were assigned to one of the following groups;Group 1: patients underwent adjuvant chemotherapy then radiotherapy,Group 2: patients underwent adjuvant chemotherapy only.Radiotherapy was delivered by 3D conformal radiotherapy (CRT).Positioning: The patient was placed supine with both arms above her head. Immobilization was maintained by the commercially available devices.Simulation: Simulation was done using CT simulation technique.Surgery scar, borders, and reference points were marked with radio-opaque wire before CT scanning. Clinical target volume (CTV), (OAR) definition as the following Figure ;CTV was defined as the excision cavity plus 1.5 cm free resection margin.The definitions of chest wall borders were guided by the following landmarks;-Cranial: the caudal border of the clavicle head,-Caudal: clinical reference, loss of CT apparent contralateral breast,-Anterior: skin,-Posterior: deep fascia,-Lateral: clinical reference, mid axillary line,-Medial: clinical reference.The definitions of supraclavicular field borders are guided by the following landmarks;-Cranial: inferior border of cricoid cartilage,-Caudal: caudal edge of clavicular head/junction of brachiocephalic axillary veins,-Anterior: sterocleidomastoid muscle,-Posterior: anterior edge of the scalene muscle,-Lateral: cranially: lateral edge of sterocleidomastoid muscle, and caudally: the first rib-clavicle junction,-Medial: excludes thyroid and trachea.Planning target volume (PTV) was equal the CTV plus 0.5–1 cm. The target was considered to be appropriately treated if the PTV was enclosed within the 95–105% isodose line.The organs at risk (OAR) included the heart major blood vessels, ipsilateral, contolateral lungs, contralateral breast, cervical cord, and brachial plexus. Radiotherapy planning had to be adjusted to avoid OAR .Radiotherapy was delivered to the chest wall, and the supraclavicular fields using the same isocenter (mono-isocentric technique). Chest wall irradiation was done using two tangential fields, with the use of wedges as indicated. Multi-leaf collimator applied for optimally adjusting the therapeutic field to the tumor area. Supraclavicular Field irradiation was done by an anterior oblique field angled slightly away from the spinal cord. A humeral head block was added to block the humeral head and acromioclavicular joint.The intended dose was 50 Gy, given in 25 fractions over a period of 5 weeks by conventional fractionation (2Gy/fraction).RT machine was Linear accelerator 6-9 MV , 11.After RT, all the patients were put on hormonal therapy. Additionally, those who had HER2 + ve disease continued on trastuzumab for 1 year.After finish of the treatment protocol, patients were followed-up according to the NCCN recommendations; by regular clinic visits every 4–6 months for the first 5 years, then annually thereafter. In each visit, patients were evaluated by history, physical examination. Annual X ray mammography, annual gynecological assessment for those on tamoxifin, and frequent DEXA scanning for those on aromatase inhibitors  without distant metastases.Between September 2008 and August 2014, 75 patients were enrolled in the current study. Forty patients were assigned to treatment group 1, and 35 patients were assigned to group 2. All the 75 patients fulfilled our eligibility criteria. The mean age was 52.4 years (range 29–65 years). The median performance status was 0 (range 0–2). All the 75 patients had stage II   . Axillary lymph node dissection was done after sentinel lymph node examination in 50 patients by frozen section during the operation. A median of 10 lymph nodes were removed (range 5–16).Following mastectomy, all our patients underwent adjuvant chemotherapy at mean time of 4 weeks. Chemotherapy regimes used included FAC (12 patients of group 1 and 13 patients group 2), AC (6 patients of group 1 and 2 patients group 2), and sequential AC, taxene (20 patients of group 1 and 20 patients group 2).Additionally, two patients of group 1 received adjuvant CMF after the first cycle of FAC. The first patient developed prolonged febrile neutropenia grade IV, prolonged grade IV vomiting after cycle 1, and the second patient, who had history of rheumatic mitral regurgitation, developed low ejection fraction (EF) after cycle 1.Further, 2 patients received adjuvant trastuzumab for HER-2 neu positive status .Radiotherapy was initiated at mean time of 2 weeks (range 2–3 weeks) after the last chemotherapy cycle for group 1 patients. Radiotherapy was given to all patients by 3D CRT, with CT simulation. The median RT dose was 50 Gy, in 25 fractions for all patients of group 1 (2Gy/fraction) (range 45–50 Gy).Following adjuvant treatment, the two patients with HER + ve disease continued the full year of adjuvant Trastuzumab. Adjuvant hormonal therapy was given to our patients in the form of tamoxifin (25 patients) and sequential tamoxifin, armatase inhibitors (50 patients).The median follow-up period was 54 (range 50–60 months).The 4-year OS for group 1 and 2 were 77.5 and 71.4%, respectively , and 14 patients from group 2 had recurrence  (Tables The distant metastasis rate was the same in the two groups (20% in both). However, the metastasis sites were different in the two groups. The sites of distant metastasis for group 1 were bone, liver, and lung, respectively . The sites of distant metastasis for group 2 were liver, lung, and bone, respectively .Radiotherapy delay happened in four patients (10%). The mean delay time was 1 week (range 5–14 days). No patient discontinued from RT for side effect. No deaths occurred related to treatment Table .During the 54 months follow-up period, four patients from group 1 developed lymphedema grade I–II (10%). No patient developed lung toxicity or brachial plexopathy. One patient from group 1 died from congestive cardiac failure at 50 months, to note that patient was known to have long history of rheumatic mitral regurgitation, and her EF was 45% before any treatment. EF dropped to 40% after the first cycle of FAC chemotherapy. EF was maintained at 40% after radiotherapy. Three years later, her EF was maintained at 40%. At 48 months, her EF dropped to 30%.During the same follow-up period, two patients from group 2 had lymphedema grade I 5.7%). One of them developed cellulitis on top of lymphedema. No other events were noticed to suggest delayed toxicities Tables  and 7.%. One ofRadiotherapy in early breast cancer has been debatable for decades. While studies had showed its role in improving loco-regional control, its impact on OS is still unclear , 14.p = 0.087), and the later showed improvement by 6% at 5 years, that was as well non-statistically significant (p = 0.1)  .Beside these individual studies, number of systemic reviews and metaanalyses also demonstrate an absolute survival benefit of approximately 5–10% with the addition of RT , 16, 17.A recent metanalaysis conducted by the Early Breast-Cancer Trialists’ Collaborative Group (EBCTCG) in women who had one to three positive nodes. They reported that post-mastectomy RT reduced the recurrence rate by 32% and reduced the breast-cancer mortality rate by 20% .On the other hand, an important meta-analysis by EBCTCG , that inMany reasons can explain why the improvement in PFS was not reflected on definite improvement in OS. Cuzik et al. , reportep < 0.001)  .Additional supporting evidence of the benefits of RT on OS came from the hypothesis that, if the burden of distant metastasis can be reduced by systemic therapy, then RT given to the loco-regional sites might prevent secondary dissemination , 21.p = 0.04). These results had led to the general acceptance that RT given after breast conservative surgery had favorable effects on survival . However, the sites of metastasis were different between the two groups (Bone group 1 and liver group 2). The significance for that, as well as the correlation of that with RT is not yet clear.Regarding the toxicity profile, our study confirmed that, RT by conformal 3D radiotherapy was generally tolerated well, and associated with mild toxicity profile.The addition of radiotherapy to adjuvant chemotherapy in stage II breast cancer with one to three positive lymph nodes improved the PFS, and LRR. Radiotherapy improved OS only in patients with high-risk features including those with PS 2, two to three positive axillary lymph nodes, and those with lymphatic invasion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Z. officinale cv. Ogawa Umare or “Ogawa Umare” (OG), contains more active ingredients, showing properties as a new resource for the production of herbal medicines derived from ginger in terms of its chemical constituents and rhizome yield.In traditional herbal medicine, comprehensive understanding of bioactive constituent is important in order to analyze its true medicinal function. We investigated the chemical properties of medicinal and edible ginger cultivars using a liquid-chromatography mass spectrometry (LC-MS) approach. Our PCA results indicate the importance of acetylated derivatives of gingerol, not gingerol or shogaol, as the medicinal indicator. A newly developed ginger cultivar,  Traditional medicine embodies accumulation of knowledge, skills, and practices on the maintenance of health as well as the prevention, diagnosis, improvement, or treatment of physical and mental illness. World Health Organization reported that, even today, more than 80% of the world's population utilizes traditional medicine for primary health care . Such meCurcuma longa cv. Okinawa Ougon) + ion at m/z 277.1798. [6]-Gingerdiol predominantly provided the [(M+H)–2H2O]+ ion at m/z 261.1849 together with the weak (M+H)+ and [(M+H)–H2O]+ ions. In the case of diacetoxy-[6]-gingerdiol, intense signals for the [(M+H)–CH3COOH]+ and [(M+H)–2CH3COOH]+ ions were observed at m/z 321.2046 and 261.1843, respectively. Furthermore, three characteristic adduct ions, (M+NH4)+, (M+Na)+, and (M+K)+, were detected. These results indicate that gingerol, shogaol, and their related compounds could be annotated by ESI mass spectral patterns together with high-resolution mass data.The major pungent principles of ginger are gingerols and shogaols (dehydrated form of gingerols). The conversion of gingerols to shogaols is favored at higher temperature , and shoLC-MS chromatograms of OG and “Shoga” are shown in In order to clarify the medicinal properties of ginger, LC-MS chromatograms of the extracts of all six ginger types are shown in  Z. officinale var. rubens and Z. officinale var. amarum) showed higher contents of [10]-gingerol and lower contents of acetylated compounds. Although Z. officinale var. rubens is used as the raw material in the production of Kampo medicine (“Shokyo” and “Kankyo”), only the most pungent fresh ginger is selected and utilized -gingerol and [6]-shogaol to many biological activities of ginger. Prasad and Tyagi summarized many molecular targets of the compounds . HoweverThe molecular targets of the certain compounds have gradually been made clear through several omics approaches, whereas knowledge on chemical system is still limited. Integration of the knowledge of  “chemical system” as described in this paper may help understand the action between “chemical system” and “body system” in traditional medicines.


A 3-year-old boy presented with acute corneal hydrops on the left eye and spontaneous corneal rupture on the right eye. A diagnosis of brittle cornea syndrome was confirmed by molecular analysis. A novel mutation, the homozygous variant c.17T>G, p.V6G, was found in the gene for PR-domain-containing protein 5 (PRDM5) in exon 1. Brittle cornea syndrome is a rare connective tissue disease with typical ocular, auditory, musculoskeletal, and cutaneous disorders. Almost all patients suffer from declined vision due to corneal scarring, thinning, and rupture. The most common ophthalmologic findings include keratoconus, progressive central corneal thinning, high myopia, irregular astigmatism, retinal detachment, and high risk for spontaneous corneal or scleral rupture. In addition to describing the case with a novel mutation here we review the current literature on brittle cornea syndrome pathogenesis, clinical findings, and therapy. Heterozygous mutations are usually associated with milder corneal thinning and keratoconus with blue sclera [Brittle cornea syndrome is a rare autosomal recessive disease with generalized connective tissue damage . Classicμm) , irregulμm) –3. Mutate type 2 –4. Cornee type 2 , 3, 5. Ie type 2 . Alterate type 2 . Homozyge sclera . Due to e sclera –3. SinceA 3-year-old boy presented with a whitish-clouded cornea and loss of vision on the left eye for one week and a physiologically appearing right eye. There was no trauma remembered or pain, family history showed no conspicuous eye disorders, no infections, or abnormalities in pregnancy or birth, and no genetic disorders were known. General pediatric physical examination was normal for all systems. The parents had Turkish origin, and history of parental consanguinity is known.The clinical examination under general anesthesia showed a blue discoloration of sclera in both eyes. Intraocular pressure was within normal levels (8 mmHg in the left eye and 10 mmHg in the right eye). The posterior segment was examined by standardized echography and showed no retinal anomalies or retinal detachment. Axial length was 21.7 mm in the right eye and 22.1 mm in the left eye, suggesting myopic eyes. Retinoscopy in cycloplegia showed −4.0 diopter and astigmatism of −4.5 at 168 degrees on the right side. Retinoscopy in the left eye was not possible.μm in the left eye and the mean value of thickness peripherally was 550 μm as measured by ultrasound pachymetry .The cornea of the left eye revealed complete corneal edema with obvious stromal and bullous epithelial keratopathy. The epithelial layer was closed. Central corneal thickness was 745 μm and a peripheral thickness of 308 μm with corneal astigmatism in topography (−4.5D at 169 degrees). The inferior paracentral cornea showed a deep stromal scar with the remaining corneal surface clear. Funduscopy showed normal optic nerve and retina.The right eye showed corneal thinning with a central thickness of 212 Reduced central corneal thickness with deep stromal scar on the right eye and the acute corneal edema suggested the diagnosis of keratoconus on both eyes, with acute keratoconus of the left eye. In addition, a connective tissue disorder causative for the blue discolored sclera was suspected. ZNF469 and PRDM5 was performed by polymerase chain reaction amplification and direct DNA sequencing. No mutation was found in ZNF469, but the homozygous variant c.17T>G, p.V6G, was detected in PRDM5. To exclude a dosage effect, a quantitative real-time PCR analysis of PRDM5 showed no larger deletion or duplication. This variant c.17T>G, p.V6G, was not found in the 2504 control subjects in the “1000 genomes project” [Molecular analysis of the genesproject” . It doesμm , Ehlers Danlos syndrome especially type VI, and osteogenesis imperfecta , 12.Also genetic testing in family members should be performed, especially to detect heterozygous mutation carriers to calculate the risk for further children . AudiomeSurgical repair of spontaneous corneal ruptures in brittle cornea syndrome is difficult. Here we achieved corneal sealing by a combination of cheese wiring sutures with bandage contact lens, pressure patches for one week, and systemic carboanhydrase inhibitors. Alternatively repair of corneal perforations has been described using tissue adhesives in dry conditions, viscoelastic agents, onlay epikeratoplasty, and gas/air tamponade . IntraopEarly diagnosis of brittle cornea syndrome is the most important step in the treatment of individuals suffering from this syndrome. Early treatment with protective glasses and observing the rules of conduct can prevent corneal rupture and so minimizes the risk for vision loss. Ophthalmic long-term follow-up and general follow-up examinations are needed to prevent systemic, extraocular disorders by this multisystemic connective tissue disorder.


Quantitative electrocardiographic (ECG) waveform analysis provides a noninvasive reflection of the metabolic milieu of the myocardium during resuscitation and is a potentially useful tool to optimize the defibrillation strategy. However, whether combining multiple ECG features can improve the capability of defibrillation outcome prediction in comparison to single feature analysis is still uncertain.A total of 3828 defibrillations from 1617 patients who experienced out-of-hospital cardiac arrest were analyzed. A 2.048-s ECG trace prior to each defibrillation without chest compressions was used for the analysis. Sixteen predictive features were optimized through the training dataset that included 2447 shocks from 1050 patients. Logistic regression, neural network and support vector machine were used to combine multiple features for the prediction of defibrillation outcome. Performance between single and combined predictive features were compared by area under receiver operating characteristic curve (AUC), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and prediction accuracy (PA) on a validation dataset that consisted of 1381 shocks from 567 patients.Among the single features, mean slope (MS) outperformed other methods with an AUC of 0.876. Combination of complementary features using neural network resulted in the highest AUC of 0.874 among the multifeature-based methods. Compared to MS, no statistical difference was observed in AUC, sensitivity, specificity, PPV, NPV and PA when multiple features were considered.In this large dataset, the amplitude-related features achieved better defibrillation outcome prediction capability than other features. Combinations of multiple electrical features did not further improve prediction performance. Early cardiopulmonary resuscitation (CPR) and early defibrillation are the key points in the chain of survival in cardiac arrest patients with shockable rhythms , 2. HoweQuantitative electrocardiogram (ECG) waveform analysis provides a noninvasive reflection of the metabolic status of the myocardium during resuscitation and is a potential tool to guide and optimize CPR interventions, i.e., chest compression or defibrillation . During The purpose of the present study was to investigate whether combination of multiple VF features, by different machine learning strategies, including logistical regression (LR), artificial neural network and support vector machine (SVM), could improve the prediction capacity of defibrillation outcome using a large multicenter database of OHCA patients.This study was approved by the ethics committee of the coordinating center, San Gerardo University Hospital, Monza, Italy. The institutional review board waived the requirement of informed consent since the data were already collected for administrative and statistical reasons by the National Health System.A total of 3828 defibrillation shocks from 1617 patients who experienced OHCA were analyzed. The detailed descriptions of the multicenter database and population characteristics have been previously reported . Data inSuccessful defibrillation was defined as the achievement of an organized rhythm with heart rate ≥ 40 beats/min within 60 s postdefibrillation, while shocks resulting in VF, ventricular tachycardia (VT), asystole or pulseless electrical activity with pauses > 3 s were regarded as unsuccessful defibrillations . In the Sixteen predictive features of ECG waveforms with good prediction power in previous clinical studies , 26 wereOptimum frequency range of bandpass filters for each feature was obtained with a criterion of maximum area under the receiver operating characteristic (ROC) curve (AUC) using the training data. The boundaries of the lower and upper frequencies for calculating the optimum frequency range were 2–5 Hz and 20–48 Hz, respectively.Three different machine learning techniques, including LR, neural network and SVM were used to combine different VF features for the prediction of defibrillation outcome.p values all less than 0.0001) were automatically selected from the 16 features employing the training data by forward stepwise using the likelihood ratio test. The LR equation for prediction was β0 is the regression constant and βn is the nth regression coefficient of the selected feature yn. The predictive values of validation data were obtained according to the corresponding LR equations by a threshold (for successful or unsuccessful decisions) with equal sensitivity and specificity for the training data.In the LR model, optimal features (with The back propagation (BP) neural network with a feed forward structure was used in the training set to achieve an optimal outcome. The training processing adopted the Bayesian regularization training function, two hidden layers, and sigmoid and linear transfer functions. All features in the training and validation sets were normalized by minus of mean and division of standard deviation values. The AUC of direct outcomes of the BP neural network was calculated as these outcomes were not binary decisions (0 or 1). For compatibility, a threshold with equal sensitivity and specificity for training data was used to result in a binary decision. Combination of all features (BP-C1), combination of features with a high predictive power (with AUC > 0.8) (BP-C2) and combination of complementary features (correlation coefficient r < 0.3) (BP-C3) were tested by the BP neural network, respectively.In the SVM model, a Gaussian radial basis function was selected as the kernel function with an error penalty factor (C = 1) and a scaling factor (σ = 0.01). Choosing small values for the error penalty factor and the scaling factor was intended to make the risk function of SVM have solutions for large training data. Combinations of all features (SVM-C1), high predictive power features (SVM-C2) and complementary features (SVM-C3) were also adopted in the training and validation processes of SVM.The prediction power was assessed by ROC curves, AUC, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and prediction accuracy (PA) , 26. Forp value < 0.05 was considered statistically significant.Pearson's correlation coefficients were calculated among single features for correlation analysis. AUCs were compared using Z-test. Chi-squared test was employed to distinguish differences among sensitivity, specificity, PPV, NPV and PA of the different predictive features. A final two-tailed p was not significant vs. MS for all and/or for the first defibrillations). Considering all the defibrillation attempts, AUCs for spectrum entropy  and max power  were relatively lower when compared with MS, but no significant differences were observed when the first defibrillations were considered. Additionally, AUCs for PF (0.619/0.607), CF (0.565/0.547), SFM (0.489/0.401) and Hu (0.478/0.445) were significantly lower compared with MS (p < 0.001), both for all and first defibrillations.ROC curves and AUCs of the candidate features for all and the first defibrillations in training and validation datasets are reported in Fig. p < 0.001). For frequency-related methods, CF was highly correlated with PF  and SFM . Poor correlations were observed among the other measures.Correlation analysis demonstrated that most of the features were significantly correlated with each other Table . AmplituThe performance of combined features in the validation set for all and first defibrillations are listed in Tables p < 0.001), specificity  and NPV . It also showed higher sensitivity , PPV  and PA  compared to SVM-C3 when the first defibrillations were considered.Combining MS and SFM with BP neural network (BP-C3) resulted in the highest AUC (0.875/0.873) and accuracy (80.9 %/80.0 %) for all and first defibrillations, but no statistical differences were observed when compared with the combined LR, BP-C1 and BP-C2, for all and first defibrillations. Compared with SVM-C3, BP-C3 predicted outcome of all defibrillations with higher sensitivity , sensitivity (p = 0.281 and 0.330), specificity (p = 0.254 and 0.790), PPV (p = 0.568 and 0.955), NPV (p = 0.453 and 0.422) and PA (p = 0.771 and 0.765) between BP-C3 and MS for all  arrival time. Additional clinically relevant attributes, independent from ECG waveform metrics, such as end-tidal carbon dioxide, blood pressure, blood oxygen saturation and compression depths, might be considered to further improve prediction power . From anWe recognized that several limitations need to be considered in the study. First, this was a retrospective study on prospectively collected data. Sixteen predictive features were calculated only during the predefibrillation hands-off time and not in real time during chest compression. Second, the successful defibrillation was defined as sustained ROSC, but long-term survival was not considered. Peri-arrest factors such as age, sex, presenting rhythm, EMS arrival time, drug treatments, comorbidities, were not analyzed in this study. Third, further studies including independent ECG waveform metrics should to be tested in future prospective evaluations.In this large population of OHCA patients, amplitude-related features such as MS, AMSA and MdS, achieved better prediction power of defibrillation outcome than other features. Combining multiple electrical features did not further improve prediction performance in comparison to the single features.The electrical features obtained from ECG waveform are promising in prediction of defibrillation outcome. However, most of these features are highly correlated with each other.The amplitude related features achieve better defibrillation outcome prediction capability than other features.Combinations of multiple electrical features using machine learning strategies does not further improve prediction performance.