Objective:To assess the application value of low-dose contrast agent combined with bolus tracking technique in radiotherapy localization for nasopharyngeal carcinoma (NPC).Methods:One hundred patients with NPC for radiotherapy localization were enrolled prospectively and randomly divided into observation ( n = 50) and control ( n = 50) group using random number table. All patients in the control group underwent routine computerized tomography (CT) for radiotherapy localization with an intravenous bolus of 80 ml iodinated contrast media. Images were obtained at a fixed delay of 50 s from the beginning of contrast injection. Patients in the observation group underwent CT scan using a bolus tracking technique with an intravenous bolus of 50-60 ml iodinated contrast media. By observing the time-CT value monitoring curve, the scanning was manually triggered when the peak fell. The score of image quality, enhanced CT value, iodine contrast dose, and dose calculation variance of planning target volume were compared between the two different dose groups. The differences of dose were compared using independent sample t-tests and the differences in graph scores were compared using Wilcoxon rank sum tests. Results:The dosage of iodine contrast agent in the observation group was 28.75% less than that in the control group, and the difference achieved statistical significance ( t=-42.11, P<0.001). The enhanced CT value of carotid artery and jugular vein in the control group increased 32.58% and 21.02% compared with patients in the observation group, respectively ( t=-8.11 and -4.82, P<0.001 for both). No statistically significant differences in the enhanced CT value were found for the gross tumor volume (GTV) of nasopharynx or sternocleidomastoid between the two groups ( P>0.05). The images in both groups can met the requirements of target volume delineation, and the difference of subjective score was not statistically significant ( Z=-1.36, P=0.175). There were no significant differences in the signal-to-noise ratio (SNR) or contrast-to-noise ratio (CNR) of nasopharynx between the two groups ( P>0.05). The results of subjective score and GTV target area showed high consistency between the two doctors with a κ value of 0.77. The differences in dose calculation variance of planning target volume between the two different dose groups was also not statistically significant ( P>0.05). Conclusions:In the radiotherapy localization for nasopharyngeal carcinoma, the application of low-dose contrast agent combined with bolus tracking technique could obtain good images for target delineation and effectively reduce the intake of iodine contrast agent and the risk of adverse reactions. Based on these results, low-dose contrast agent combined with bolus tracking technique has wide range of application.

Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.

Objective: To investigate a cluster of coronavirus disease 2019(COVID-19)caused by latent infection in Haikou,so as to provide reference for the prevention and control of COVID-19 clusters. Methods: An epidemiological investigation was conducted according to the COVID-19 Prevention and Control Program(Fourth Edition). The course of diagnosis and treatment,clinical characteristics and field investigation data were collected to analyze the transmission chain and the intergeneration between cases. Results : Among 39 people involved,five confirmed cases and two asymptomatic infections were found,with an attack rate of 17.95%. The cases aged from 40 to 65 years and lived in the dormitories near the farm of Dongshan. The first case(named Case 1)closely contacted with a confirmed case of COVID-19 in Chengmai from January 28 to February 10,was isolated on February 13 and developed symptoms on February 16. The other six cases(Case 2 to Case 7)shared the water source with Case 1 from January 28 to February 13(within the incubation period of Case 1). They needed to open the power switch outside Case 1's room and the water valve in Case 1's washroom before the use of water. They might be infected by contacting the doorknob and the water valve contacted by Case 1,or by the aerosol formed after Case 1 used the washroom,then infected each other when living together. The onset of Case 2 to Case 7 was earlier than Case 1,and they had no travelling history in Hubei Province 14 days before and contacted no confirmed cases except Case 1. Therefore,Case 1 was the source of the cluster during his incubation period of COVID-19. Conclusion This was a cluster of COVID-19 due to latent transmission by living in the same area and touching the same objects indirectly,which indicated that COVID-19 was infectious in the incubation period.

Objective: To evaluate the dosimetric effects of set-up errors on nasal NK/T cell lymphoma by introducing set-up errors into the radiotherapy planning system for dose reconstruction. Methods: Ten patients with nasal NK/T cell lymphoma were recruited. A non-coplanar volumetric modulated arc therapy plan was designed for CT image and clinical target area of each patient. After the completion of the plan, the set-up errors were introduced into the radiotherapy plan by changing the ISO of the treatment, and dose calculation was performed to reconstruct the dose distribution. Results: With the increase of system set-up errors, the dose of target was decreased and the order affected by set-up errors in different directions was: left-right direction> head-foot direction> front-rear direction. When the translational set-up errors in each direction were -3 mm to 3 mm and the rotating set-up errors were -3° to 3°, the range of dose change in all targets was less than ±3%. When the set-up errors in all directions were ≤ 3 mm, the dose of organ at risk was less than or similar to the prescribed dose. When the set-up errors were> 3 mm, the doses of lens, spinal cord, parotid gland and optic nerve gradually exceeded the prescribed dose. Only when the rotating set-up errors were ≥ 3°, the dose of lens exceeded the prescribed dose. Special attention should be paid to the influence of the greater set-up errors in the left and right direction on lens, spinal cord and parotid gland, as well as on the spinal cord due to the larger set-up errors in the front and rear direction. After the actual set-up errors were introduced from our department, it exerted slight effect on the irradiation dose of GTV and CTV, which was less than ±2%. In a few cases, the dose of organ at risk potentially exceeded the prescribed dose limit, and special attention should be diverted to overdose of the lens and optic nerve. Conclusions The set-up errors will result in target dose deficiency and overdose of organ at risk in nasal NK/T cell lymphoma, especially upon the set-up errors in the left and right direction. The effect of 3 mm and 3° set-up errors on target and organ at risk is limited. Therefore, it is recommended to maintain the single direction set-up errors within 3 mm and 3°. The actual set-up errors introduced from our department exert little effect on the target dose, but a small number of organs are at risk of exceeding the prescribed dose limit. It is necessary to increase the evaluation of the extension region of organ at risk.

Objective To investigate the genetic carrier rate of thalassemia and its gene mutation types as well as the distribution characteristics among couples of reproductive age in Dongfang City of Hainan Province,and to provide a basis for making prevention and control strategies against thalassemia.Methods Samples were collected from 1 000 couples undergoing premarital and pregestational screenings for thalassemia in Dongfang City of Hainan Province from September 2012 to March 2013,in which the positive ones in preliminary screening were further tested by genetic diagnoses and the genotypes were retrospectively analyzed.Results Among 1 000 couples,322 spouses were diagnosed with thalassemia gene mutation and the carrying rate was 16.10％ (322/2 000).In those carriers,246 spouses were α-thalassemia and the carrying rate was 12.30％ (246/2 000),accounting for 76.40％(246/322) of all thalassemia carriers,among them,there were 197 cases of α-deficiency genotype,accounting for 61.18％ (197/322),32 carried mutated α-gene,accounting for 9.94％ (32/322),17 carried both deleted and mutated α-gene,accounting for 5.28％ (17/322);43 spouse were β-thalassemia and the carrying rate was 2.15％(43/2 000);33 spouse were both α-and β-thalassemia and the carrying rate was 1.65％ (33/2 000).In spouses diagnosed with α-thalassemia,the major genotype was-α37/αα,accounting for 19.25％ (62/322);the second ranked was-α4.2/αα,accounting for 17.70％ (57/322),and the third ranked was--SEA/αα,accounting for 8.70％ (28/322).In spouses diagnosed with β-thalassemia,the major genotype was CD41-42/N,accounting for 9.63％ (31/322).Conclusions The population carrying rate of thalassemia in Dongfang City of Hainan Province is high,and its major type is α-thalassemia.For the purpose of decreasing the birth rate of thalassemia,major,local public health department should attach great importance to thalassemia prevention,and strengthen premarital and pregestational screening for thalassemia.

Objective: To investigate DNA damage in the transformed human bronchial epithelial cells (16HBE) induced by hexavalent chromium (Cr⁶⁺⁾ and further elucidate the potential carcinogenesis mechanism of Cr⁶⁺. Methods: 16HBE were treated with different concentration of Cr^{6+ (}0, 0.625, 1.25, 2.5 μmol/L) for 15 weeks. The malignant degrees of transformed cells were identified by the assays for anchorage-independent growth and tumorigenicity. According to the single cell gel electrophoresis (SCGE) assay, the DNA damage rate was calculated. The expression level of 53BP1 was determined by Western blot. Results: Chromium-treated cells could form colonies in soft agar and tumors in nude mice. Compared with the control group, colony formation efficiency of 1.25μmol/L and 2.5 μmol/L Cr⁶⁺-treated cells in soft agar showed significant increases (p<0.05) . The 2.5 μmol/L Cr⁶⁺-treated cells also formed tumors subcutaneously in nude mice. Cr⁶⁺ could cause different degree of DNA damage to 16HBE cells in a dose-dependent manner. In addition, Western blot analyses showed that 53BP1 was aberrantly down-regulated at 2.5 μmol/L dose and has no significant changes at 0.625 μmol/L and 1.25 μmol/L dose under the treatment of Cr⁶⁺. Conclusion The declined expression of 53BP1 may mediate Cr⁶⁺-induced DNA damage and further involved in the cell malignant transformation.

Objective To construct a lentiviral vector delivering the Nicastrin (NCT) gene-targeted short hairpin RNA (shRNA) and determine gene-silencing efficiency of the vector in the human immortalized keratinocyte cell line HaCaT,and to construct a NCT gene-silenced HaCaT cell model to lay an experimental foundation for subsequently studying effects of NCT gene silencing on biological behavior of keratinocytes.Methods Three NCT gene-targeted shRNAs were designed and inserted into the pGLV3/ H1/GFP + Puro vector to construct three recombinant plasmids,which were then confirmed by sequencing.Recombinant plasmids combined with lentivirus packaging plasmids were co-transfected into 293T cells to obtain lentivirus particles,and the virus titer was determined.Cultured HaCaT cells were divided into 3 groups:blank group receiving no treatment,negative control group infected with the empty vector LV3-shNC,interference groups infected with lentivirus NCT-shRNA1,-shRNA2,-shRNA3,respectively.Flow cytometry was performed to determine transfection efficiency,and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine efficiency of target gene silencing in HaCaT cells,so as to select the most efficient interference sequence.Results Sequencing analysis indicated that recombinant lentiviral vector NCT-shRNA was constructed successfully.After co-transfection of recombinant plasmids and lentivirus packaging plasmids into 293T cells,the titer of recombinant lentivirus particles was about 109 TU/ml.Flow cytometry showed that the transfection efficiency was greater than 95％.qRT-PCR revealed that the NCT mRNA expression was obviously down-regulated in the interference group compared with the negative control group,and NCT-shRNA1 was the most efficient sequence with interference efficiency being 75％.Western blot analysis showed that the inhibition rate of NCT protein expression was 71.7％ in the shRNA1 group compared with the negative control group.Conclusion The most efficient NCT-shRNA interference sequence is screened out,and the recombinant lentiviral vector NCT-shRNA and an NCT gene-silenced HaCaT cell model are both constructed successfully.

Objective To study the correlation between lactate/albumin ratio level and incidence of multiple organ dysfunction syndrome (MODS) as well as mortality in patients with severe sepsis or septic shock.Methods In January 2012-September 2013, 54 patients who were admitted to the intensive care unit(ICU) of a hospital developed severe sepsis and septic shock on the first day of admission, clinical data of patients were analyzed.Results On the first and second days of admission, 30(55.56%)and 26(53.06%)patients developed MODS;lactate/albumin ratio between MODS group and non-MODS group on the first and second days of admission were both significantly different (both P<0.05).Multivariate logistic regression analysis showed that lactate/albumin ratio, oxygenation index (PaO2/FiO2), as well as acute physiology and chronic health evaluation (APACHE Ⅱ) were independent risk factors for predicting MODS in patients with severe sepsis.Lactate/albumin ratios between MODS group and non-MODS group, death group and non-death group were both significantly different (both P<0.05);lactate/albumin ratio was correlated with APACHE Ⅱand PaO2/FiO2, the higher the APACHE Ⅱ score and the lower the PaO2/FiO2, the higher of lactate/albumin ratio.The area under the receiver operating curve (ROC curve) analysis showed that incidence and mortality of MODS on the first day of admission predicted by lactate /albumin ratio were 0.85 and 0.84 respectively;sensitivity, specificity, positive predictive value, and negative predictive value of occurrence of MODS predicted by lactate /albumin ratio>1.735 were 80.00%, 79.17%, 82.67%, and 75.92% respectively, sensitivity, specificity, positive predictive value, and negative predictive value of mortality were 100.00%, 51.02%, 17.23%, and 100.00% respectively.Conclusion Lactate/albumin ratio level is closely correlated with incidence and mortality of MODS in patients with severe sepsis or septic shock.

Objective: To assess the feasibility of coronary lfow reserve (CFR) detection by SPECT myocardial perfusion imaging using a self developed software with preliminary clinical veriifcation. Methods: CFR calculation software was developed according to Mat lab guide. A total of 16 patients were enrolled including 13 male and 3 female at the mean age of (58±11) years . CAG conifrmed that 25 coronary branches were with stenosis>50% and 23 branches were without stenosis. 2-day ATP/rest99mTc-sestamibi dynamic SPECT myocardial perfusion imaging was conducted to detect CFR. First transit counts were used to sketch the interested pulmonary artery segments and to obtain the arterial input curve of contrast agent as total PAC reached to heart. Reconstructed short-axis images were divided into 3 sections to sketch interested territories (ROI) and to obtain RMC at each territory. Estimated CFR was expressed by the ratio of MBF=RMC/PAC followed by calculating the ratio of MFR=MBFstress/MBFrest. Results: The difference between simulated value and true value could be ignored which conifrmed that our program may accurately measure CFR. The reproducibility by different operators (r=0.986) and the same operator (r=0.983) was good. CFR value in non-stenosis branches were higher than stenosis branches (1.28 ± 0.19) vs (1.10 ± 0.27),P=0.008 and CFR value in stenosis branches was negatively related to stenosis degree (r=-0.5,P=0.02). Conclusion: Our self developed software is reliable for CFR detection by SPECT myocardial perfusion imaging; preliminary study showed good application prospect in clinical practice.

Objective: To assess the impact of viable myocardium in left ventricular aneurysm (LVA) and ventricular arrhythmia on prognosis of LVA patients.
Methods: A total of one hundred and sixty LVA patients who received99Tcm-MIBI SPECT and18F-FDG PET were enrolled, including 139 male and 21 female with the mean age of (58 ± 10) years.There were 42 (26.3%) patients combining ventricular arrhythmia. LVEDV, LVESV and LVEF were detected. Semi-quantitative analysis of myocardium perfusion imaging was conducted, viable myocardium in aneurysm was deifned as the perfusion-metabolism mismatch score (MMS) ≥ 2.0. According to myocardium viability, the patients were divided into 2 groups: No viability group,n=97 and With viability group,n=63;based on ventricular arrhythmia, the patients were divided into another 4 groups: Group①, viability-, ventricular arrhythmia-, n=68, Group②, viability-, ventricular arrhythmias+,n=29, Group③, viability+, ventricular arrhythmias-,n=50 and Group④, viability+,ventricular arrhythmias+,n=13. The average follow-up time was (50 ± 7) months, the end point was cardiac death. The survival curve was obtained by Kaplan-Meier method and survival rates were compared by Log-rank analysis.
Results: The mean LVEF in 160 patients was (34 ± 11) %, cardiac death occurred in 19 (11.9%) patients. Long-term survival rates in Groups①,② and③ were 94.1%, 89.7% and 86.0%, respectively,P>0.05; while in Group④, the survival rate was 61.5%, which was lower than the other 3 groups,P=0.004. Multivariate Cox regression analysis showed that female (HR=5.101, 95% CI 1.853-14.044, P=0.002), GPET-ESV (HR=1.009, 95% CI 1.002-1.015,P=0.013), interaction between MMS and ventricular arrhythmia (HR=1.368, 95%CI 1.113-1.681,P=0.003) were independent risk factors for cardiac death;while surgical treatment (HR=0.199, 95% CI 0.054-0.742,P=0.016) could decrease the risk of cardiac death.
Conclusion: Patients with viable aneurysm and ventricular arrhythmia had poor long-term prognosis; while early and active treatment is needed for them (surgery with anti-arrhythmic therapy).