[Functional analysis of laparoscopic intracorporeal Xing's neobladder in long-term follow-up].

Evaluation of neobladder function in patients with long-term survival and no recurrence after laparoscopic radical cystectomy and intracorporeal Xing's neobladder. The clinical data of laparoscopic radical cystectomy and intracorporeal Xing's neobladder in long-term survival patients with bladder cancer treated in Beijing Chaoyang Hospital from July 2013 to July 2018 were analyzed retrospectively. All 17 patients underwent the surgery by the same surgical team, including 15 males and 2 females, whose mean age at the time of operation was (55.9±7.6) years. Thepostoperative urinary function and renal function were summarized. All operations were successfully completed. The mean operative time was (340±62) min. All patients were followed up for a long time, with a median follow-up time of 80(70, 96) months, Urinary continence was achieved in 17 (100%)casesduring the day and 13 (76.5%) cases at night, with a median bladder volume of 350 (200, 400) ml. All patients had good urinary control after surgery, and no hydronephrosis or creatinine increase was found in reexamination.After the application of Xing's neobladder operation, the patient maintained acceptable urinary control status after the operation, and the long-term follow-up effect was satisfactory.

[Clinical efficacy of dorsal venous complex pre-suture technique in the robot-assisted laparoscopic radical prostatectomy].

The clinical data of 33 patients who underwent robot-assisted laparoscopic Vattikuti Institute prostatectomy (VIP) in Cancer Hospital of Chinese Academy of Medical Sciences from October 2020 to April 2022 were reviewed and analyzed. Among them, 18 patients received traditional VIP technique and 15 patients received VIP technique with pre-sutured dorsal venous complex (DVC). The ages of the traditional VIP group and the pre-sutured DVC VIP group were (66.1±7.3) and (66.6±5.7) years, respectively. The body mass index (BMI) of the traditional VIP group and the pre-sutured DVC VIP group was (24.3±2.9) and (25.3±2.6) kg/m2, respectively. The medical expenses were (91 797.0±7 029.6) and (87 106.6±7 031.4) yuan, respectively, with no statistical significance (all P>0.05). The operations of all the 33 cases were successfully completed, and no cases were transferred to open surgery. The operation time of traditional VIP group was longer than that of pre-sutured DVC VIP group [(193.0±37.2) vs (171.1±16.1) min]. The amount of intraoperative blood loss was higher than that in the pre-sutured DVC VIP group [(68.3±22.8) vs (44.0±9.1) ml)], all P<0.05. No patients had perioperative blood transfusion, and no patients had complications within 30 days after surgery. All the 33 patients had negative incisal margins. DVC presuture technique can reduce intraoperative bleeding and shorten operative time in robot-assisted laparoscopic VIP radical prostatectomy, and has good curative effect.

[Comparative study on metagenomics of esophageal flora in elderly and middle-aged esophageal squamous cell carcinoma patients].

Objective: To explore the flora characteristics and differences of esophageal tissues between elderly esophageal squamous cell carcinoma (ESCC) patients and young and middle-aged ESCC patients, so as to assist in studying the potential biomarkers of elderly ESCC patients. Methods: In this study, a retrospective study was adopted. 72 ESCC patients diagnosed in Taihe Hospital, Shiyan City, Hubei Province from July 2018 to July 2019 were selected, including 49 patients in the elderly group (≥ 60 years old, 40 males and 9 females), 23 patients in the young and middle-aged group (<60 years old, 21 males and 2 females). In the same period, 20 healthy persons without abnormal gastroscopy in endoscopy center were selected as the control group (aged 35-78 years old, median age 57 years old, 16 males and 4 females). The genomic DNA was extracted from the affected esophageal tissues of patients with ESCC and the middle esophageal samples of the control group. The V4 hypervariable region of bacterial 16SrRNA gene sequence was amplified. Illumina HiSeq sequencing technology was adopted. The flora characteristics of elderly, young and middle-aged ESCC patients was compared and analyzed. QIIME and Rstudio software were used to analyze the sequence data, and nonparametric Kruskal-Wallis test or Wilcoxon rank sum test were used for statistical methods. Results: Shannon index [5.17 (4.53, 5.95) vs. 4.79 (3.74, 5.97)], Simpson index [0.94 (0.91, 0.96) vs. 0.92 (0.83, 0.96)] and Chao1 index [343.55 (259.76, 570.59) vs. 329.16 (268.88, 648.00)] were similar in flora of two groups, and there was no significant difference (Z=-0.791, -1.057, -0.380, all P>0.05). There was no significant difference in ß-diversity between the elderly group and the young and middle-aged group (PC1=19.14%, PC2=6.95%, PPC1=0.67, PPC2=0.42). At the phyla level, the top 5 phyla in abundance were as follows: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria in the young and middle-aged group, while the top 5 phyla in abundance were as follows: Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria and Actinobacteria in the elderly group; the significant difference between the two groups was Fusobacteria (Q=0.596, P<0.05). At the genus level, the top 5 genera in the young and middle-aged group in abundance were as follows: Prevotella, Bacteroides, Streptococcus, Selenomonas and Veillonella. In the elderly group, Prevotella, Bacteroides, Streptococcus, Selenomonas and Haemophilus were the top 5 in abundance, and there were significant difference in Fusobacterium between the two groups (Q=0.938, P<0.05). PICRUSt function prediction showed that the abundance of Aminoacyl.tRNA.biosynthesis, Nucleotide.excision.repair, RNA.polymerase, Ribosome, Clavulanic.acid.biosynthesis, Photosynthesis and Photosynthesis. proteins in the elderly group were lower than those in the young and middle-aged group (all Q=0.734, P<0.05). Conclusion: There is no significant difference in α-diversity and ß-diversity between elderly ESCC patients and young and middle-aged patients, but the abundance of Fusobacterium flora increased.

[A homozygous variant in a consanguineous pedigree with inherited protein S deficiency].

Objective: To observe the clinical feature of familiar hereditary protein S deficiency, and to explore the related gene mutation. Methods: The blood samples were obtained from the proband and the family memebers(3 generations,6 persons). PROS1 gene of the proband and the family members was analyzed. The 15 exons and flanking sequence of PROS1 gene were analyzed by PCR and DNA sequencing. Results: Five out of 6 family members were diagnosed as having hereditary protein S deficiency. The proband suffered from pulmonary embolism. The others had no obvious thrombotic event. The gene sequencing revealed that the proband carried a c.-168C>T homozygous variant in the promoter of exon 1. His parents, brother and son all carried c.-168C>T heterozygosis variant at the same position. The gene of his wife was a wild type. Conclusion: A gene variant (c.-168C>T) of PROS1 was discovered in this Chinese family. Gene variant of PROS1 may result in protein S deficiency. Patients with protein S deficiency may suffer from vein thrombosis and(or) pulmonary embolism.

Genetic signature of hybridization between Chinese spot-billed ducks and domesticated ducks.

In this study, we analyzed 93 whole genomes from Chinese spot-billed ducks (CSB), meat-type ducks (MET), and egg and dual purpose-type ducks (EDT) to characterize the genetic material flowing between the CSB and modern ducks. Using a frequency of shared identical-by-descent method, approximately 10.9 Mb introgression segments containing 140 genes were identified showing the signatures of introgression between CSB and EDT. Meanwhile, nearly 10.6 M introgression regions containing 149 genes were identified between CSB and MET. Based on the haplotypes tree of each segment, we found that the introgression between CSB and domesticated ducks was asymmetric with a high level of gene flow from domestic to CSB and a low level of migration in the opposite direction. Moreover, we identified several genes that were introgressions from CSB and showed the signature of positive selection, which may contribute to the breeding of modern ducks. Our results provide new insight into the evolution and breeding history of domestic ducks and may be useful for the future management of wild and domestic duck populations.

[The effect on bleeding volume and postoperative recovery of regional cerebral oxygen saturation guides controlled hypotension in elderly patients with hypertension undergoing spinal surgery].

Objective: To evaluate the effect on bleeding volume and postoperative recovery of regional cerebral oxygen saturation (rSO(2)) guides controlled hypotension in elderly patients with hypertension undergoing spinal surgery. Methods: One hundred and twenty elderly patients who underwent spinal surgery in the department of anesthesiology of Qingdao Municipal Hospital and the Affiliated Hospital of Qingdao University from January 2017 to December 2019 were selected and divided into 2 groups according to the random number table method (n=60): rSO(2) guides the controlled hypotension group (group A) and control group (group C). Both groups were performed with endotracheal intubation for general anesthesia, maintain anesthesia with sevoflurane and remifentanil, rSO(2) were monitored throughout the procedure. If necessary, sodium nitroprusside or esmolol were used to control blood pressure. In group A, the goal of controlled hypotension was that rSO(2) decreased ≤ 10% of the basic value or maintained at 64±3 and the moderate operative field bleeding. Group C underwent routine anesthesia management. Intraoperative blood loss and urine output, the incidence of hypothermia after operation, postoperative delirium, chills, nausea and vomiting, the PACU residence time, postoperative drainage volume, eating time, postoperative hospital stay were compared between the two groups. Results: Compared with group C, the blood loss [(589±157) vs (764±213) ml] and urine output [(778±121) vs (1 079±239) ml] of group A were decreased (t=-5.120, -8.712, all P<0.05). The rates of hypothermia after operation (26.7% vs 45.0%), postoperative delirium (18.3% vs 36.7%), chills (10.0% vs 25.0%), nausea and vomiting (21.7% vs 40.0%) of group A were decreased (χ(2)=4.385, 5.057, 4.675, 4.728, all P<0.05) . The PACU residence time [(56±9) vs (63±11) min], postoperative drainage volume [(217±66) vs (289±81) ml], eating time [(17.8±2.8) vs (22.3±4.1) h] and numbers of days in hospital [(7.2±2.7) vs (8.2±2.9) d] were decreased of group A (t=-3.399, -5.334, -7.000, -2.031, all P<0.05). Conclusion: The guidance of controlled hypotension with rSO(2) monitoring can reduce the blood loss and infusion volume during spinal surgery in elderly patients with hypertension, reduce postoperative related complications and enhance recovery after surgery.

[Application of Endo-GIA stapler in laparoscopic radicalcystectomy].

Objective: To explore the application of Endo-GIA stapler in laparoscopic radical cystectomy, especially in the treatment of lateral bladder ligament, and to evaluate its clinical feasibility and practicability. Methods: A retrospective analysis of clinical data about 38 cases of laparoscopic radical cystectomy (LRC) treated in the Department of Urology, Chaoyang Hospital of Beijing and Cancer Hospital, Chinese Academy of Medical Sciences from July 2017 to June 2019 were conducted. The patients were divided into Endo-GIA stopler group(18 cases) and non-Endo-GIA stopler group (20 cases) according to whether Endo-GIA stapler were used. The basic clinical data, operation time of bladder lateral ligament, operation time of bladder lateral wall, operation time of bladder resection, amount of bleeding during operation, pathological data after operation and related indicators of recovery after operation were compared between the two groups. Results: All 38 patients underwent radical cystectomy (RC) successfully under 3-D laparoscopy without conversion to open surgery. The operation time of bladder lateral ligament in Endo-GIA stapler group was significantly shorter than that in non-Endo-GIA stapler group [(3.25±0.75) min vs (9.20±2.95) min, P=0.042]; the operation time of bladder lateral wall in Endo-GIA stapler group was significantly shorter than that in non-Endo-GIA stapler group [(8.06±1.66) min vs (14.30±3.37) min, P=0.016]. The operation time of cystectomy in the Endo-GIA stapler group was significantly shorter than that in the non-Endo-GIA stapler group [(47.06±4.70) min vs (61.60±14.91) min,P=0.003]. The amount of bleeding in the Endo-GIA stapler group was significantly shorter than that in the non-Endo-GIA stapler group [(37.77±21.30) ml vs (114.50±39.80) ml, P=0.015]. The time of drainage tube removal in Endo-GIA group was significantly shorter than that in the non-Endo-GIA group [(5.83±1.54) d vs (7.30±3.00) d, P=0.002]. The length of post-hospitalization in Endo-GIA group was significantly shorter than that in the non-Endo-GIA group [(7.67±1.78) d vs (9.60±3.25) d,P=0.036]. However, there was no significant difference in other basic clinical data, post-operative pathology and post-operative recovery related indicators. Conclusions: Laparoscopic radical cystectomy using Endo-GIA stapler device is safe and feasible. It is easy to operate, shorten the operation time significantly, and reduce the amount of bleeding. To a certain extent, it is conducive to the recovery of patients after operation to some extent, and worthy of clinical application.

[Effects of double-catheter epidural analgesia by lidocaine injection respectively on the delivery outcomes and maternal-infant complications for persistent posterior or lateral occipital position of protracted active phase].

Objective: To evaluate the effect of dual-tube epidural segmental injection of lidocaine analgesia on the delivery outcome and maternal and infant complications of persistent posterior occipital position postpartum or lateral occipital position postpartum patients with protracted active phase. Methods: The full and single-term primiparas (n=216, 37 to 42 weeks gestation, 22 to 35 years) diagnosed as persistent posterior or lateral occipital position during the active period were selected from the Department of Obstetrics of Qingdao Municipal Hospital from January 2015 to October 2019. The subjects were randomly assigned into two groups: double-tube epidural block group (n=108) and single-tube epidural block group (n=108), 1% lidocaine was used for epidural analgesia respectively under ultrasound guidance. Senior midwife or obstetricians implement new partogram, and guide women to perform position management, and push or rotate the fetal head in a timely manner. Observation indicators: general condition, the use of non-pharmacological analgesic measures, analgesia related conditions and pain visual analogue scale (VAS) score, delivery-related indicator, cesarean section indication, anesthesia-related indicator, maternal and child complications. Results: (1) General condition: the age, weight, height, gestational age, the ratio of persistent lateral or posterior occipital position, cephalic score, and neonatal birth weight between the two groups of women were not statistically significant (all P>0.05). (2) The use of non-pharmacological analgesic measures: the women's Lamaze breathing method, Doula delivery companionship, percutaneous electrical stimulation, and other measures between two groups were compared, and there were not significant differences (all P>0.05). (3) Analgesia related conditions and VAS scores of women undergoing vaginal delivery: compared with the single-tube epidural block group (n=40), the second-partum time of the women in the double-tube epidural block group (n=59) was significantly shortened [(124±44) vs (86±33) minutes, P<0.01]; after 30 minutes of analgesia (4.4±0.5 vs 0.9±0.5, P<0.01), during forced labor in the second stage of labor (5.7±0.6 vs 1.3±0.4, P<0.01), the VAS scores of pain were also significantly reduced (P<0.01). (4) Labor-related indicators: compared with the single-tube epidural block group, the natural delivery rate (21.3% vs 49.1%) and the delivery experience satisfaction rate (51.9% vs 98.1%) of women in the double-tube epidural block group were significantly increased (all P<0.01), cesarean section rate (63.0% vs 45.4%), instrument assisted rate (15.7% vs 5.6%) decreased significantly (all P<0.05). (5) Cesarean section indications: compared with the single-tube epidural block group, the cesarean section rate caused by prolonged labor or protracted active phase of women in the double-tube epidural block group was significantly reduced (38.0% vs 22.2%； P<0.05), and the fetal distress, intrauterine infection, and social factors caused by cesarean section between the two groups were compared, while the differences were not statistically significant (all P>0.05).(6) Anesthesia related indexes: the block planes of the maternal upper tube administration in the double-tube epidural block group were mostly T7, T8, T9-L2 and L3,While,the block planes in the single-tube epidural block group were mostly T10, T11-S1, S2, S3, and the modified Bromage score were all 0. (7) Maternal and child complications: compared with the single-tube epidural block group, the postpartum hemorrhage rate (18.5% vs 7.4%), the perineal lateral cut rate (20.4% vs 5.6%), the neonatal asphyxia rate (12.0% vs 3.7%), ICU rate of transferred neonates (13.9% vs 4.6%) in the double-tube epidural block group were significantly reduced (all P<0.05). Soft birth canal injury rate, puerperal disease rate and neonatal birth rate between two groups were compared, and there were not statistically significant differences (all P>0.05). Conclusion: Dual-tube epidural segmental injection of lidocaine analgesia could increase the natural delivery rate of women with posterior occipital or lateral occipital position with active stagnation, reduce the rate of cesarean section and the rate of transvaginal instruments, and reduce the complications of mother and child.

[Research progress of magnetic resonance imaging in hepatocellular carcinoma].

Hepatocellular carcinoma (HCC) is the most common malignant tumors of liver. Rational treatment in early stage of HCC can significantly increase the survival rate of patients. Detection of early-stage liver lesions through diffusion-weighted imaging, diffusion kurtosis imaging, incoherent motion within voxels, magnetic resonance spectroscopy imaging, dynamic contrast-enhanced imaging, or other functional imaging can accurately evaluate grade of malignancy and guide towards the curative effect of different treatment schemes. Volumetric interpolated breath-hold examination (VIBE) with three techniques, namely, CAIPIRINHA, Dixon, and TWIST, i.e., CDT-VIBE sequence can provide multiple sets of arterial phase images in a short time, and detect small foci transient enhancement that cannot be detected by other examinations, and the differences in initial enhancement phase of apparent lesions are of great significance for suggesting hemodynamic characteristics. In addition, magnetic resonance imaging predicts microvascular invasion of HCC, i.e., indirectly predicts HCC lesions recurrence rate, and overall survival time after liver transplantation. This article reviews the current research progress of magnetic resonance imaging technology in HCC.

[Advances in research of odontoid fractures in the elderly].

Odontoid fractures constitute the most common cervical spine fracture in the population aged. The optimal management strategy for odontoid fractures remains controversial, especially in the elderly, because of the high morbidity and mortality with both conservative and operative intervention. This review discusses the management of odontoid fractures among the elderly, with a focus on the various treatment options and their outcomes.

Long-term outcome of telbivudine versus entecavir in treating higher viral load chronic hepatitis B patients without cirrhosis.

Chronic hepatitis B (CHB) patients with higher hepatitis B virus (HBV) load (higher viral load [HVL], HBV DNA ≥1 × 107 copies/mL) require antiviral therapy, but data for evaluating the long-term outcome of this therapy with antiviral agents remain limited. We comparatively evaluated the efficacy and the safety of nucleoside analogues in 179 noncirrhotic CHB patients with HVL over 5 years. The HBeAg-positive (n = 104) or HBeAg-negative (n = 75) patients were treated consecutively with telbivudine (LdT, n = 88) or entecavir (ETV, n = 91) and evaluated for viral response, drug resistance and safety. HBV DNA, viral serology, biochemistries, HBV mutation and off-therapy relapse were determined. The cumulative rates of HBV DNA negativity were 86.4% and 94.5% for LdT and ETV at year 5, respectively. The rates of early viral response (EVR, HBV DNA <103 copies/mL at month 6) under LdT and ETV treatments were 58.0% and 34.1%, respectively (P < .05). Hepatitis B e antigen (HBeAg) and Hepatitis B surface antigen (HBsAg) loss-seroconversions were 47.7% and 18.2% on LdT and 16.5% and 2.2% on ETV (P < .01). Eighteen patients (age 28.2 ± 3.1) experienced HBsAg loss-seroconversion, followed by 33 ± 4.6 month off-therapy without a relapse. Viral mutations and serum creatine kinase elevation were 9.1% and 8.0% on LdT, but only 1.1% and 0% on ETV. Both LdT and ETV suppressed HBV replication in HVL CHB patients within 5 years. LdT therapy achieved a higher EVR, HBeAg and HBsAg seroconversion, especially in the younger patients, whereas ETV caused lower drug resistance and fewer adverse events. This finding might help to identify the optimal treatment for CHB patients with HVL.

Follow-up observational study of "bi-ring method" breast surgery for treating hypermastia and mastoptosis.

PURPOSE: This study investigated the efficacy and patient satisfaction of "bi-ring method" breast surgery in 46 patients with hypermastia and/or mastoptosis. MATERIALS AND METHODS: A questionnaire survey, objective data measurements, visual analysis system survey, and various scoring scales were used to qualitatively and quantitatively assess the patients' indicators before and after surgery. RESULTS: Statistical analysis showed the following: symptoms and signs in patients with macromastia improved significantly; all patients' breast shapes improved significantly and became more symmetrical and durable; all patients had minor hidden scars; the nipples and areolas had good feeling postoperatively, and there were relatively few mild complications. CONCLUSIONS: The patients' overall satisfaction was high, indicating that the "bi-ring method" of breast plastic surgery could not only improve the breast shape and boast concealed scars but could significantly improve the patients' signs and symptoms of hypermastia, but the nipples and areolas had good postoperative feeling and there were few complications. Thus, this is a reasonable surgical approach that is worthy of promotion.

[Association between the C46T polymorphism of coagulation factor â « gene and the involvement of factor â « activity in patients with unexplained recurrent spontaneous abortion].

OBJECTIVE: To explore the association between the C46T polymorphism of coagulation factor Ⅻ (FⅫ) gene and the involvement of FⅫ activity (FⅫ:C) in patients with unexplained recurrent spontaneous abortion (URSA), and to elucidate its role in the pathogenesis of URSA. METHODS: This study included 203 patients with URSA (URSA group) and 171 healthy women with at least one child and no history of infertility or miscarriage (control group) in the southern area of Zhejiang Province. The C46T polymorphism of the FⅫ gene was analyzed with matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) in all subjects. The values of prothrombin time, activated partial thromboplastin time (APTT), fibrinogen, FⅫ:C and other coagulant parameters were determined. The frequency distribution of the wild-type (CC), heterozygote (CT), homozygote (TT) genotypes and C and T alleles were compared between the patients and controls. A comprehensive analysis of association was conducted between C46T genotypes and the FⅫ:C levels in URSA patients. RESULTS: The CC, CT, TT genotypes of the FⅫ gene were observed in 7 (3.4%, 7/203), 83 (40.9%, 83/203) and 113 (55.7%, 113/203) patients with URSA versus 7 (4.1%, 7/171), 46 (26.9%, 46/171) and 118 (69.0%, 118/171) controls. The frequency of CT in the patients with URSA was significantly higher than that in controls, but the frequency of TT in the patients was lower than that in controls (χ(2)=7.939, OR=1.884, 95%CI: 1.210-2.935, P<0.05). The frequencies of allele C and allele T were observed in 97 (23.9%, 97/406) and 309 (76.1%, 309/406) patients with URSA versus 60 (17.5%, 60/342) and 282 (82.5%, 282/342) controls. The distribution frequency of allele T in URSA group was lower than that in control group (χ(2)=4.510, OR=1.475, 95%CI: 1.029-2.115, P<0.05). The FⅫ: C levels in the patients were (102±13)% in CC genotype, (78±11)% in CT genotype and (59±9)% in TT genotype, respectively. The differences of the FⅫ: C levels between the CC and CT, CT and TT, CC and TT genotypes in the patients were significant (all P<0.05). CONCLUSIONS: The low level of FⅫ:C maybe result from the T allele of the FⅫ gene in URSA patients. The CT genotype might be relative to the pathogenesis of URSA in a Chinese Han female population from the southern area of Zhejiang province.

[Impact of age-related cataract on regulation of circadian rhythm in elderly].

This review presented an introduction of the visual pathway related circadian rhythm regulation system: the intrinsically photosensitive retinal ganglion cells-suprachiasmatic nucleus-pineal gland-melatonin axis, and discussed the impact of light with different wave length and irradiation received by retina on circadian rhythm and sleep habit. A hypothesis was proposed consequently that the high morbidity of sleep disorder in elderly might be partially attributable to the long-term blue light blocking status induced by age-related cataract. A number of relative literatures were reviewed and a novel research direction was advanced on improving circadian rhythm and sleep condition in elderly based on the current knowledge. (Chin J Ophthalmol, 2016, 52: 309-314).

Identification and characterization of the duck enteritis virus (DEV) US2 gene.

The US2 protein has been reported to contribute to duck enteritis virus (DEV) infection; however, its kinetics and localization during infection, and whether it is a component of virion, have not been previously reported. To elucidate the function of DEV US2, US2 was amplified by polymerase chain reaction (PCR) and inserted into pET-32a(+); this was expressed, the recombinant US2 protein was purified, and a polyclonal antibody generated. In addition, the kinetics and localization of the US2 gene and protein were determined by quantitative real-time fluorescent PCR, ganciclovir (GCV), and cycloheximide (CHX) treatment, western-blot, and indirect immunofluorescence assay. The packaging of US2 into DEV virions was revealed by a protease protection assay. US2 was found to be transcribed 24 h post-infection (pi) and peaked at 72 h pi; the US2 protein was detected 48 h pi, except in the presence of GCV or CHX. US2 was packed into virions and also localized to the plasma membrane and cytoplasm in infected cells. The results showed that the DEV US2 is a late gene, and that its encoding protein could be a tegument component localized mainly in the cytoplasm. This study provides useful data for the further analysis of DEV US2, including an explanation for the genetic conservation among alphaherpesviruses.

Bioinformatic analysis and characteristics of glycoprotein C encoded by the newly identified UL44 gene of duck plague virus.

Glycoprotein C is one of the duck plague virus (DPV) glycoproteins and is encoded by the DPV UL44 gene. DPV glycoprotein C (DPV-gC) comprises 431 amino acids with a putative molecular mass of 47.35 kDa. Sequence analysis indicated that the protein possesses typical characteristics of type-I membrane glycoproteins, containing an N-terminal signal sequence, an external domain, a C-terminal membrane anchor region, and a short cytoplasmic domain. Comparisons of 22 alphaherpesvirus-gC protein sequences revealed eight conservative Cys-residue sites, which may play a crucial role in the biological functions and structural stabilization of the DPV-gC protein. Estimates of potential antigenic epitopes and secondary structure identified four B cell dominant epitopes, which are located at amino acids 68-71, 87-91, 369-352, and 372-374. A model for the structure of DPV-gC was derived by associating its predicted secondary and three-dimensional structures. In conclusion, these results will provide a basis for further functional studies of DPV-gC, establishing novel clinical diagnoses of DPV, and in the development of a new DPV vaccine.

Characterization of Riemerella anatipestifer CH-1 gldJ gene and GldJ protein.

Riemerella anatipestifer (RA) CH-1, a highly virulent field strain, was isolated and identified by our laboratory. The gldJ gene was conserved in RA, and it had a typical TATA promoter region and AU-rich sequence within the 5' untranslated region. The GldJ protein was an outer-membrane lipoprotein with a signal peptide cleavage site between amino acids 20 and 21. GldJ was also a member of proteins involved in gliding motility. The RA GldJ protein had 16 phosphorylation sites and 4 N-glycosylation sites. These functional sites played an important role in the posttranslational modification of the GldJ protein. In addition, the GldJ protein of RA CH-1 had strong immunogenicity. Fifteen B-cell epitopes were identified in the GldJ protein, which might be a good biological material for the development of a subunit vaccine and diagnostic reagents. The GldJ protein also had the activity of formylglycine-generating sulfatase enzyme. The 3-dimensional structure models of GldJ were constructed based on 2 templates using the SwissModel automatic modeling mode in the SWISS-MODEL workplace. Here, we characterized the RA gldJ gene and GldJ protein to contribute to the functional annotation for the GldJ protein.

Different impact of two mutations of a novel compound heterozygous protein C deficiency with late onset thrombosis.

We investigated the alteration of coagulation state in a protein C (PC) deficiency pedigree and the impact of the PC gene mutations. The pedigree of a proband with cerebral hemorrhagic infarction had sixteen members with four generations. The plasma levels of PC activity (PC:A), protein S activity (PS:A), factor V:C and factor VIII:C, and routine coagulation tests were measured. Nine exons of the PC gene (PROC) were sequenced. Plasma PC:A and PC antigen (PC:Ag) of the proband were 26 and 18%, respectively, which was significantly lower than normal ranges. Two heterozygous missense mutations of PC in the proband were identified, T>G at site 6128 (exon 7) and G>C at site 8478 (exon 9) resulting in F139V and D255H, respectively. The family members with F139V (N = 4) or D255H (N = 4) had lower levels of PC:A and PC:Ag than members with wild-type PROC (N = 6). D255H mutation caused a more significant decrease in the levels of PC:A, PC:Ag and factor V:C as compared to F139V mutation (P < 0.05). Two independent mutations, F139V and D255H, of PROC reduce PC function. Compound heterozygous condition of the two mutations can cause synergistic PC deficiency, but resulting in later onset of cerebral thrombosis.

Cladosporium cladosporioides Identified in China on Tobacco Seeds.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China is the biggest single tobacco market and accounts for more than 40% of the global tobacco consumption (1). Tobacco seed harvested in Guiyang, Guizhou Province, China, are commonly contaminated or infected by various fungal pathogens, which can cause abnormal seedlings with dark brown lesions and stunting of roots and decayed seeds. In 2013, five samples of 500 seeds from tobacco cv. Guiyan 4 were tested for germination on moistened paper on petri dishes. On average, 35% of the seeds from all five samples developed into abnormal seedlings or were decayed and were plated onto potato dextrose agar media and grown for 5 days at 25°C in darkness to confirm the presence of a pathogen. However, one fungus was isolated from an average of 10% of the 500 seeds sampled. It was identified morphologically as Cladosporium cladosporioides (Fresen.) de Vries based on the velvety olive-brown with almost black reverse colony color and dimensions and color of conidia and conidiophores. Conidia formed in long branched chains that readily disarticulate, single celled, elliptical to limoniform, 2 to 8 (avg. 4.3) × 2 to 3 (avg. 2.1) µm. Conidia were pale to olive brown and smooth to verruculose. Ramoconidia were 0 to 1 septate, 7 to 14 (avg. 9.2) × 2 to 4 (avg. 2.6) µm, smooth or sometimes minutely verruculose. Conidiophores were pale to olive brown, macro- and micronemateus, smooth or sometimes verruculose, and of various lengths up to 320 µm long and 2 to 5 µm wide. Primer pair ITS1 and ITS4 was employed to amplify the regions of ITS1-5.8s-ITS2 of the pathogens. Sequences of all three isolates (G3, G10, and G18) (Accession Nos. KF841547, KF841554, and KF841560) were identical to each other and to four sequences in GenBank (JX230994.1, JQ768317.1, JQ768322.1, and AB763555.1). Pathogenicity of the three isolates of C. cladosporioides was verified on tobacco seedlings of 3-week-old grown on wet filter paper in the petri dishes (9 cm in diameter). For each isolate, 20 seedlings incubated in one plate were inoculated with 0.5 ml of a suspension of 105 conidia/ml. Twenty seedlings were treated with sterile water as control treatment. After inoculation, the petri dishes were incubated at 25°C, 100 to 120 µEm-2 S-1, RH > 80%, and 16 h light per day for disease development. At 96 h after inoculation, symptoms comprising medium brown to black lesions on the roots were clearly visible on inoculated plants but not on the control plants. All seedlings inoculated died 9 days after inoculation whereas control seedlings remained symptomless. Re-isolation attempts on PDA from roots demonstrated C. cladosporioides to be present in symptomatic seedlings but not in roots of the control plants. Moreover, the characteristics of the cultured fungi were exactly the same as those originally isolated. Isolates G3, G10, and G18 (KF841547, KF841554, and KF841560) were deposited with the Tobacco Diseased Fungi, Guizhou Academy of Tobacco Sciences, Guizhou, China. Previously, C. cladosporioides has also been isolated from macadamia (Macadamia integrifolia Maiden & Betche) racemes in South Africa (4), from diseased papaya (Carica papaya L.) in Taiwan province of China (2), and from seeds of Amaranthus spp. in Poland (3). To the best of our knowledge, this is the first report of C. cladosporioides causing seed disease on tobacco in China and the disease should be considered in existing disease management practices. References: (1) British American Tobacco Annual Report, 8, 2012. (2) R. S. Chen, et al. Plant Dis. 93:426, 2009. (3) W. Pusz. Phytopathologia 54:15, 2009. (4) N. van den Berg et al. Plant Dis. 92:484, 2008.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.