Effects of fermented cottonseed meal inclusions on growth performance, serum biochemical parameters and hepatic lipid metabolism of geese during 28-70 d of age.

The aim of this study was to investigate the effects of solid-state fermented cottonseed meal (FCSM) inclusion levels on the growth performance, serum biochemical parameters and hepatic lipid metabolism in geese from 28 to 70 d of age. A total of 288 twenty-eight-d-old male geese were randomly divided into 4 treatments with FCSM levels of 0, 5, 15 and 25% including 0, 22.74, 67.33, 111.27 mg FG/kg diet, respectively. Each treatment contained 6 replicates and 12 birds per replicate. Treatments of FCSM inclusions from 0 to 25% had no effect on growth rate and feed intake in geese during d 28 to 70. The F/G ratio was increased (P < 0.05) in geese fed the diet with 25% FCSM compared with birds fed the diet with 0% FCSM. Treatment with 25% FCSM levels had no effect on the contents of TC, TG, HDL-C, LDL-C, but increased (P < 0.05) AST and ALT activities in serum of geese at d 70. Treatment with 25% FCSM increased the contents of FG, HDL-C, TC, C18:2n6, C20:4n6 and PUFA and decreased (P < 0.05) the contents of NEFA, SFA, MUFA in liver compared with treatment of 0% FCSM inclusion. Additionally, treatment with 25% FCSM decreased (P < 0.05) the PPARα, AMPK, and LXR mRNA expression related to lipid deposition, and increased (P < 0.05) PPARγ and ACC mRNA expression related to lipolysis in liver compared with birds fed the diet with 0% FCSM. Overall, treatment with 0 to 15% FCSM (<=67.33 mg FG/kg diet) had no adverse effects on the growth performance and lipid metabolism of geese. However, treatment fed 25% FCSM (111.27 mg FG/kg diet) decreased feed efficiency and promoted hepatic lipid deposition associated with the alteration of related gene expression in geese at 28 to 70 d of age.

A binary pulsar in a 53-minute orbit.

Spider pulsars are neutron stars that have a companion star in a close orbit. The companion star sheds material to the neutron star, spinning it up to millisecond rotation periods, while the orbit shortens to hours. The companion is eventually ablated and destroyed by the pulsar wind and radiation1,2. Spider pulsars are key for studying the evolutionary link between accreting X-ray pulsars and isolated millisecond pulsars, pulsar irradiation effects and the birth of massive neutron stars3-6. Black widow pulsars in extremely compact orbits (as short as 62 minutes7) have companions with masses much smaller than 0.1 M⊙. They may have evolved from redback pulsars with companion masses of about 0.1-0.4 M⊙ and orbital periods of less than 1 day8. If this is true, then there should be a population of millisecond pulsars with moderate-mass companions and very short orbital periods9, but, hitherto, no such system was known. Here we report radio observations of the binary millisecond pulsar PSR J1953+1844 (M71E) that show it to have an orbital period of 53.3 minutes and a companion with a mass of around 0.07 M⊙. It is a faint X-ray source and located 2.5 arcminutes from the centre of the globular cluster M71.

Risk factors of common bile duct stones recurrence and nomogram for predicting recurrence after endoscopic retrograde cholangiopancreatography: a dual-center retrospective cohort study.

OBJECTIVE: Common bile duct stone (CBDS) is one of the common diseases in the digestive system, for which endoscopic retrograde cholangiopancreatography (ERCP) is a treatment procedure. However, the risk factors for CBDS recurrence after ERCP remains unclear. This study aims to compare the risk factors of CBDS recurrence after ERCP, and to set up a nomogram model to predict the long-term risk. PATIENTS AND METHODS: A retrospective analysis of 355 patients was reviewed. Univariate and multivariate analyses were performed to identify the risk factors for recurrence. The R packages were used for the model building. The validation set contained 100 patients. RESULTS: The patients were divided into three subgroups: treated by cholecystectomy after ERCP (11.76% recurrence rate), treated without surgery after ERCP (19.70%), and with a prior history of cholecystectomy (43.64%). Each of them has different independent risk factors, and high body mass index (BMI) is correlated with an increased risk among all the subgroups. A prior history of cholecystectomy is a candidate factor that increases the risk of CBDS recurrence in patients older than 60 years, with a greater BMI, or receiving ERCP combined with EPBD. We built a nomogram model to predict the risk of long-term CBDS recurrence based on the risk factors including age, BMI, CBD diameter, the number of CBDS, and the gallbladder- or biliary tract-related events. CONCLUSIONS: CBDS recurrence is related to congenital and anatomical factors. Cholecystectomy would not be helpful to prevent CBDS recurrence, and a prior history of cholecystectomy may indicate a high risk of recurrence.

A fifty-locus phylogenetic analysis provides deep insights into the phylogeny of Tricholoma (Tricholomataceae, Agaricales).

As an ectomycorrhizal fungal genus that contains matsutake and other edible mushrooms, Tricholoma has great economic and ecological significance. However, the phylogenetic relationships within the genus remain unsettled. To clarify the infrageneric relationships of Tricholoma, including the identification of monophyletic subgenera and sections, three phylogenetic analyses were conducted employing single-locus (ITS), five-locus (ITS/ RPB2/EF-1α/MCM7/mtSSU) and 50-locus (45 single-copy orthologous genes plus the aforementioned ones) DNA nucleotide sequences. Our data indicated that ITS sequences could serve the species delimitation of Tricholoma in most cases and monophyletic groups recognition in some cases, and the five-locus dataset could resolve a section-level phylogeny of this genus, while the 50-locus dataset could clarify the delimitation of subgenera and settle the relationships among sections within this genus. A fifty-locus dataset was firstly employed to construct a robust phylogeny of Tricholoma. Based on this, a new infrageneric arrangement for the genus Tricholoma, with four subgenera, of which two are in accordance with the previous subgenera Pardinicutis and Sericeicutis, and eleven sections, is suggested. Subgenus Pardinicutis, occupying the basal position, only harbors sect. Pardinicutis, while the subg. Sericeicutis comprises sects. Lasciva and Sericella located at the sub-basal position with good support. Subgenus Terrea is newly erected here and consists of sect. Terrea, sect. Atrosquamosa and two as yet unnamed phylogenetic lineages. Besides an unnamed section-level lineage, subg. Tricholoma consists of sects. Genuina, Muscaria, Rigida, Tricholoma, Fucata and Matsutake, of which the two latter are newly proposed. The previously defined subg. Contextocutis is clustered within subg. Tricholoma and is a synonym of the latter. Tricholoma colossus, T. acerbum and their allies, which used to be allocated in sect. Megatricholoma (or genus Megatricholoma), are relocated to sect. Genuina since they form a strongly supported monophyletic group and share rusty or black spots on lamellae with other species in this section. Taxonomic descriptions of the new infrageneric taxa and a key to subgenera and sections of the genus Tricholoma are presented. Citation: Ding XX, Xu X, Cui YY, et al. 2023. A fifty-locus phylogenetic analysis provides deep insights into the phylogeny of Tricholoma (Tricholomataceae, Agaricales). Persoonia 50: 1-26. https://doi.org/10.3767/persoonia.2023.50.01.

Usefulness of transperineal shear wave elastography of levator ani muscle in women with stress urinary incontinence.

This study aimed to quantitatively assess the quality of levator ani muscle (LAM) using shear wave elastography (SWE) and to evaluate the association between the elasticity of LAM and stress urinary incontinence (SUI). The study population included 32 women with SUI and 34 women with normal pelvic support. The thickness of LAM, bladder neck descent (BND), and urethral funneling (UF) were assessed by transperineal ultrasound. LAM elasticity was measured by SWE at rest and during the maximal Valsalva maneuver. Age, menopause, BND, and UF showed a positive correlation with SUI. There was no significant between-group difference in the elastic modulus values of LAM at rest. The thickness of LAM in women with SUI was greater than that in control group at rest and during the maximal Valsalva maneuver (P < 0.001). The elastic modulus values of Emax and Emean were significantly increased from rest to the maximal Valsalva maneuver in all participants (56.24 vs 82.43 kPa and 47.92 vs 72.37 kPa, P < 0.001). The change of these variables from rest to the maximal Valsalva maneuver in the control group was more obvious than that in the SUI group (34.09 vs 17.87 kPa and 31.55 vs 16.82 kPa, P < 0.05). The elasticity of LAM, as quantified by SWE, may potentially be used as an index for predicting SUI.

Prefertilization Exposure of Rainbow Trout Eggs to Per- and Polyfluoroalkyl Substances to Simulate Accumulation During Oogenesis.

Aqueous film-forming foams (AFFFs) are used in firefighting and are sources of per- and polyfluoroalkyl substances (PFAS) to the environment through surface runoff and groundwater contamination at defense and transportation sites. Little is known regarding the toxicity and bioaccumulation of newer AFFF formulations containing novel PFAS. To mimic maternal transfer of PFAS, prefertilization rainbow trout eggs were exposed to three PFAS using novel methodologies. Batches of unfertilized oocytes were exposed for 3 h to 0, 0.01, 0.1, 1, or 10 µg/ml separately to perfluorooctanoic acid, perfluorohexanoic acid, or perfluorooctanesulfonic acid in either coelomic fluid or Cortland's solution. After exposure, the gametes were fertilized and rinsed with dechlorinated water. Egg yolk was aspirated from a subset of fertilized eggs for PFAS quantification. Each PFAS was detected in yolks of eggs exposed to the respective PFAS, and yolk concentrations were directly proportional to concentrations in aqueous media to which they were exposed. Exposure in coelomic fluid or Cortland's solution resulted in similar concentrations of PFAS in egg yolks. Ratios of PFAS concentrations in oocytes to concentrations in exposure media (oocyte fluid ratios) were <0.99 when exposed from 0.01 to 10 µg/ml and <0.45 when exposed from 0.1 to 10 µg/ml for both media and all three PFAS, demonstrating that the water solubility of the chemicals was relatively great. Prefertilization exposure of eggs effectively introduced PFAS into unfertilized egg yolk. This method provided a means of mimicking maternal transfer to evaluate toxicity to developing embryos from an early stage. This method is more rapid and efficient than injection of individual fertilized eggs and avoids trauma from inserting needles into eggs. Environ Toxicol Chem 2021;40:3159-3165. © 2021 SETAC.

Phylogeny and species delimitation of Strobilomyces (Boletaceae), with an emphasis on the Asian species.

Strobilomyces is broadly distributed geographically and serves an important ecological function. However, it has been difficult to delimit species within the genus, primarily due to developmental variations and phenotypic plasticity. To elucidate phylogenetic relationships among species within the genus and to understand its species diversity, especially in Asia, materials of the genus collected from five continents (Africa, Asia, Australia, Europe, and North/Central America) were investigated. The phylogeny of Strobilomyces was reconstructed based on nucleotide sequences of four genes coding for: the largest and the second largest subunits of the RNA polymerase II (RPB1 and RPB2); the translation elongation factor subunit 1-α (TEF1); and the mitochondrial cytochrome oxidase subunit 3 (COX3). The combined results based on molecular phylogenetics, morphological characters, host tree associations, and geographical distribution patterns support a new classification consisting of two sections, sect. Strobilomyces and sect. Echinati. Using the genealogical concordance phylogenetic species recognition (GCPSR) approach, at least 33 phylogenetic species in Asia can be delimited, all of which are supported by morphological features, and five phylogenetic species remain to be described. The mountainous region of Southwest China is especially special, containing at least 21 species and likely represents a centre of diversification. We further compared our specimens with the type specimens of 25 species of Strobilomyces. Our comparisons suggest that, there are a total of 31 distinct species, while S. sanmingensis, S. verruculosus, S. subnigricans, and S. zangii/S. areolatus, are synonyms of S. mirandus, S. giganteus, S. alpinus and S. seminudus, respectively. Eight new species, namely, S. albidus, S. anthracinus, S. calidus, S. cingulatus, S. densisquamosus, S. douformis, S. microreticulatus and S. pinophilus, are described. A dichotomous key to the Asian Strobilomyces species is provided.

[Efficacy comparison between laparoscopy and open surgery in the treatment of gastric gastrointestinal stromal tumors larger than 2 cm using multicenter propensity score matching method].

Objective: To compare the efficacy between laparoscopy and open surgery for gastric gastrointestinal stromal tumor (GIST) larger than 2 cm. Methods: A multicenter retrospective cohort study was performed. Inclusion criteria: long diameter of primary gastric GIST > 2 cm; undergoing laparoscopy or open surgery; diagnosis confirmed by postoperative pathology without distant metastasis; without preoperative targeted therapy. Clinicopathological data of 857 gastric GIST patients, including 320 in PLA General Hospital, 284 in Shanghai Renji Hospital, 175 in Wuhan Union Hospital and 78 in Tianjin Cancer Hospital, from January 2010 to May 2017 were retrospectively collected. There were 418 males and 439 females, mainly aged between 50 and 70 years old. Among 857 patients, 413 were in the laparoscopy group and 444 in the open group. The nearest neighbor matching of propensity score matching method was conducted with 1:1 matching based on tumor location and size between laparoscopy and open group to obtain samples of covariate equilibrium, and the caliper value was 0.04. The t test, χ(2) test and Wilcoxon rank test were used to compare short-term efficacy, and the Kaplan-Meier curve and log rank test were applied to compare long-term outcomes between the two groups. Results: After propensity score matching, laparoscopy group and open group both enrolled 293 cases. The baseline data, including age, gender, tumor location, tumor long diameter, NIH classification, etc. were not significantly different between the two groups (all P>0.05). Compared with the open group, the laparoscopy group had less intraoperative blood loss [<100 ml: 2.9% (155/293) vs. 36.2% (106/293), Z=-12.857, P<0.001], shorter time to postoperative feeding [(4.0±0.2) days vs. (5.3±0.9) days, t=1.505, P=0.003] and to the removal of drainage tube [(4.8±1.0) days vs. (6.5±1.0) days, t=1.847, P=0.008], and shorter postoperative hospital stay [(8.6±0.3) days vs. (10.5±0.3) days, t=4.235, P<0.001]. Subgroups analysis according to anatomical location: (1) Gastric cardia and pylorus: there were no statistically significant differences in perioperative parameters between the two groups (all P>0.05). (2) Stomach base: feeding time after surgery [(4.0±0.2) days vs. (4.5±0.2) days, t=0.512, P=0.038], drainage tube removal time [(5.1±0.4) days vs. (6.4±0.6) days, t=0.517, P=0.044], postoperative hospital stay [(8.0±0.5) days vs. (11.1±0.9) days, t=0.500, P=0.002] were all significantly shorter in the laparoscopy group as compared to the open group, while the differences in other perioperative parameters were not statistically significant (all P>0.05). (3) Lesser curvature of the stomach: the laparoscopy group had less intraoperative blood loss [<100 ml ratio: 58.1% (43/74) vs. 33.7% (25/74), Z=7.632, P=0.034], shorter gastric tube removal time [(2.7±0.2) days vs. (3.2±0.3) days, t=0.503, P=0.007], earlier postoperative passage of gas [(2.8±0.1) days vs. (3.4±0.2) days, t=0.532, P=0.030], earlier postoperative feeding [(3.6±0.2) days vs. (4.3±0.2) days, t=0.508, P=0.020], shorter drainage tube removal time [(4.2±0.4) days vs. (5.7±0.5) days, t=0.508, P=0.020] and postoperative hospital stay [(8.3±0.6) days vs. (10.7±0.3) days, t=0.502, P=0.006] as compared to the open group. (4) Great curvature of the stomach: the laparoscopy group presented less intraoperative blood loss [<100 ml ratio: 52.7% (39/74) vs. 36.5% (27/74), Z=7.681, P=0.032], earlier gastric tube removal [(2.6±0.2) days vs. (3.6±0.2) days, t=0.501, P=0.001], earlier postoperative passage of gas [(2.7±0.2) days vs. (3.4±0.2) days, t=0.501, P=0.016], earlier postoperative feeding [(3.6±0.2) days vs. (4.7±0.2) days, t=0.500, P=0.001], shorter drainage tube removal time [(4.0±0.5) days to (5.9±0.4) days, t=0.508, P=0.002] and postoperative hospital stay [(7.5±0.3) days to (9.5±0.1) days, t=0.500, P=0.001] than the open group. Subgroup analysis according to tumor size: (1) Tumor long diameter 2.0-5.0 cm: the laparoscopy group had earlier passage of gas [(2.9±0.1) days vs. (3.5±0.1) days, t=0.500, P=0.001], earlier postoperative feeding [(4.5±0.1) days vs. (5.0±0.2) days, t=0.501, P=0.013], shorter drainage tube removal time [(4.8±0.3) days vs. (6.0±0.3) days, t=0.511, P=0.008] and postoperative hospital stay [(8.1±0.4) days to (10.1±0.3) days, t=0.513, P=0.001] than the open group. (2) Tumor long diameter 5.1-10.0 cm: in the laparoscopic group, postoperative feeding time [(4.0±0.2) days vs. (4.7±0.2) days, t=0.506, P=0.015], drainage tube removal time [(4.6±0.4) days vs. (6.4±0.5)) days, t=0.501, P=0.004], postoperative hospital stay [(8.2±0.3) days vs. (10.9±0.6) days, t=0.500, P=0.001] were all shorter than those in the open group. No intraoperative and postoperative complications were observed in each group. The 5-year recurrence-free survival rates of the laparoscopy group and the open group were 95.4% and 91.6%, respectively (P=0.734), and the 5-year overall survival rates were 93.8% and 90.8% (P=0.691), respectively, and the differences were not statistically significant. Conclusions: In experienced medical centers, laparoscopic surgery for gastric GIST larger than 2 cm is safe and feasible, and can achieve comparable efficacy with open surgery. For gastric GISTs which do not locate in the greater curvature and the anterior wall of the stomach, and whose long diameter is ≤5 cm, laparoscopic surgery does not increase the risk of recurrence and metastasis, and can accelerate postoperative recovery.

The tissue transglutaminase: a potential target regulating MDR in breast cancer.

OBJECTIVE: Multi-drug resistance (MDR) is the main obstacle influencing the anti-tumor effect in breast cancer. To date, no proper potential targets are found to overcome MDR. Here, tTG was explored to show whether it is a potential target to regulate MDR in breast cancer. MATERIALS AND METHODS: tTG was silenced by small interfere siRNA. After that, the mRNA level of CD44, CD24, LRP, MRP and MDR1 were detected by RT-PCR. The Western blot analysis was used to detect the expression of LRP, P-gp and MRP. In addition, the impact of tTG on cell apoptosis, as well as cell proliferation were observed. Finally, to evaluate the role of tTG in BALB/c nude mice, the growth of tumor was performed, and the immunohistochemistry analysis was used to observe the expression of LRP, P-gp and MRP in vivo. RESULTS: In MCF-7/ADR, Compared to MCF-7, tTG expression was highly increased. After silencing tTG, the mRNA level and the protein level of P-gp, MRP, LRP were both differently decreased. The mRNA level of CD44 and CD24 was also down-regulated after silencing tTG. In addition, the cell proliferation was significantly inhibited in the ADR + tTG siRNA+Adriamycin group (p<0.05), and the tumor growth was prevented in a time-dependent situation. Cell apoptosis was significantly strengthened in the ADR+tTG siRNA+Adriamycin group (p<0.05). In vivo, the growth of tumors was reduced after silencing tTG, and the LRP, P-gp and MRP expression were significantly down-regulated in ADR + tTG SiRNA +adriamycin group (p<0.05). CONCLUSIONS: It is concluded that the tTG may be a potential target regulating the MDR by regulating LRP, P-gp and MRP expression as well as the expression of CD44CD24 to improve the MDR in breast cancer.

[Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines].

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

[Clinical significance of syndecan-1 and syndecan-2 expression in gallbladder squamous cell/adenosquamous carcinoma and adenocarcinoma].

Objective: To investigate the expression of syndecan-1 and syndecan-2 and their clinicopathological significance in patients with gallbladder squamous cell (SC)/adenosquamous carcinoma (ASC) and adenocarcinoma (AC). Methods: A total of 126 patients with SC/ASC (n=46) and AC (n=80) were included in this study. The expression levels of syndecan-1 and syndecan-2 were detected by Envison™ immunohistochemistry assay. The clinical and prognostic significance of syndecan-1 and syndecan-2 were analyzed. Results: In the 46 SC/ASC samples, syndecan-1 and syndecan-2 were positively expressed in 29 (63.0%) and 28 (60.9%) tumor tissues, respectively. (Positive expression was defined based on the staining in the component of squamous cell carcinoma. That is to say, the tissue which adenocarcinoma part was positively stained, but squamous cell carcinoma part was negatively stained is also regarded as negative.) In the 80 AC samples, 47 (58.8%) cases showed syndecan-1 positive expression, and 51 (63.8%) showed syndecan-2 positive expression. There was no significant difference in the positive rates of syndecan-1 and syndecan-2 between SC/ASC and AC groups (P>0.05 for all). The levels of syndecan-1 and syndecan-2 were associated with tumor size, TNM staging, lymph node metastasis, invasion of adjacent tissue, and surgical procedures in SC/ASC patients (P<0.05 for all). However, their expression was associated with tumor differentiation, tumor size, TNM staging, lymph node metastasis, invasion of adjacent tissue, and surgical procedures in AC patients (P<0.05 for all). The Kaplan-Meier survival analysis of SC/ASC and AC patients revealed that the average survival time for patients with positive syndecan-1 and syndecan-2 expression was significantly shorter than that of those with negative expression (P<0.01 for all). Cox multivariate analysis indicated that syndecan-1 and syndecan-2 expression were independent unfavorable prognostic factors for SC/ASC and AC patients (P<0.05 for all). Conclusion: The syndecan-1 and syndecan-2 expression are associated with the tumor progression and poor prognosis in patients with gallbladder SC/ASC and AC.

[Establishment and validation of human cancer cell lines with stable Cas9 expression].

Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions: Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.

[Effects of IL10-592 locus of AA genotype on the incidence of aGVHD and survival after HLA-matched unrelated allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the impact of IL10-592 (rs1800872) single nucleic acid polymorphism (SNP) on the prognosis of HLA matched unrelated hematopoietic stem cell transplantation (HSCT). METHODS: The polymorphism of IL10-592 in 104 recipient-donor pairs and 100 healthy volunteers was analyzed with sequence based typing (SBT). RESULTS: When the genotype of IL10-592 in donors and recipients matched, AA/AA genotype had higher incidence of Ⅲ-Ⅳ aGVHD than AC/AC or CC/CC genotype (47.1%, 3.7%, 0, P=0.002). When the genotype of IL10-592 in donors and recipients mismatched, recipients with AC genotype or donors with AA genotype, there was significant different incidence of Ⅲ-ⅣaGVHD among donors or recipients with different genotype (P=0.046, P=0.041). The recipients with AA genotype had higher incidence of Ⅲ-Ⅳ aGVHD than AC or CC genotype (27.8% vs 10.2%, 11.1%; P=0.072), and higher incidence of intestinal aGVHD (22.2% vs 5.1%,11.1%; P=0.040) , lower incidence of 2-year overall survival (OS: 48.2% vs 75.1%, 85.7%; P=0.002), lower incidence of 2 year disease free survival (DFS: 48.5% vs 66.3%, 76.2%; P=0.045). Patients had higher incidence of Ⅲ-Ⅳ aGVHD with donors of AA genotype than with donors of AC or CC genotype (26.5% vs 8.9%, 0; P= 0.024), and higher incidence of intestinal aGVHD (20.4% vs 4.4%, 0; P=0.026). In multivariate analysis, the genotype of IL10-592AA in recipients and donors had increased risk of Ⅲ-Ⅳ aGVHD (OR=3.3, P= 0.049; OR=3.9, P=0.043). There were no statistical differences on the incidence of cGVHD and relapse. CONCLUSION: In HLA-10/10 matched unrelated HSCT, the presence of IL10-592 AA genotype in recipients and/or donors is an adverse factor for Ⅲ-ⅣaGVHD, worse OS and 2-year DFS.

Determination of the potential of induced pluripotent stem cells to differentiate into mouse nucleus pulposus cells in vitro.

We determined the potential for induced pluripotent stem (iPS) cells to differentiate into nucleus pulposus (NP)-like cells in mice. iPS cells were generated from tail-tip fibroblasts. We used a pellet culture model with the aim of determining the applicability of iPS cell-based therapy to intervertebral disc degeneration (IVD). The cell pellet was cultured in an NP cell basal medium comprising Dulbecco's modified Eagle's medium supplemented with transforming growth factor beta 1, dexamethasone, ascorbate-2-phosphate, and 1% ITS-Premix. The pellet was evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemical staining, and biochemical composition. The differentiation of iPS cells into NP cells was demonstrated by the protein and mRNA expression levels of proteoglycan, collagen II, aggrecan, and CD24. Furthermore, increased hydroxyproline content and dimethylmethylene blue staining demonstrated that the collagen II and glycosaminoglycan content in the NP cells increased with time. We have shown that cultured mouse iPS cells can be induced to differentiate into NP cells. Such proof-of-concept opens up the possibility of producing patient-specific NP cells in a relatively simple and straightforward manner with high efficiency. We are confident that such cells could be immediately useful for the study of IVD disease.

Differential microRNA expression in signet-ring cell carcinoma compared with tubular adenocarcinoma of human gastric cancer.

Gastric cancer is a disease with a heterogeneous pathology; its pathological mechanisms remain unclear because there is a poor understanding of its etiology. In this study, we identified differentially expressed microRNAs (miRNAs) among various gastric cancer subtypes. miRNA microarray analysis and bioinformatic analysis were used to compare miRNA expression between the signet-ring cell carcinoma and tubular adenocarcinoma subtypes of gastric cancer. Thirteen dysregulated miRNAs were identified in signet-ring cell carcinoma compared with tubular adenocarcinoma: miR-30a, miR-26b, miR-381, let-7i, miR-29c, miR-543, miR-499-3p, miR-628-3p, miR-524-5p, miR-181b, miR-1914, miR-663b, and miR-676. This is the first time that miR-499-3p, miR-628-3p, miR-524-5p, and miR-1914 have been identified in gastric cancer tissues. Bioinformatic analysis using target prediction algorithms indicated that these miRNAs are directly involved in gastric cancer pathogenesis and have different pathological mechanisms in various subtypes of signet-ring cell carcinoma and tubular adenocarcinoma. The miRNA expression patterns in different gastric adenocarcinoma subtypes may help discriminate between signet-ring cell and tubular gland cancer or other gastric cancer subtypes that would otherwise be difficult to identify using routine histological and immunohistochemical analyses. These preliminary data should be verified in further prospective studies.

Identification of a major quantitative trait locus for ear size induced by space flight in sweet corn.

The development of molecular markers has contributed to progress in identifying the gene(s) responsible for favorable variations in maize studies. In this study, quantitative trait locus (QTL) mapping was conducted using simple sequence repeat markers in an F2 sweet corn population from a cross between parental line 1132 and space flight-induced mutant line 751 to identify the loci contributing to an increase in some yield traits. A primary mutated genomic region was located on chromosome 9. In total, 26 QTL were detected for eight yield-related traits and assembled into three clusters on chromosome 9. The largest QTL cluster at bin 9.02/03, primarily contributing to >10% of the phenotypic variation in ear and cob diameters, was likely due to a major QTL. Desired alleles of these QTL were provided by the mutant line 751. The primary action of the major mutant allele was an additive effect. Another mutant locus, which was induced in bin 9.01, increased cob and ear diameters by dominant genetic action.

Comparative study of clinicopathological significance, BIRC7, and STC2 expression between squamous cell/adenosquamous carcinomas and adenocarcinoma of gallbladder.

Gallbladder cancers (GBCs) are uncommon, but highly aggressive cancers. The majority of GBCs are adenocarcinomas (ACs), but rare subtypes of GBCs such as squamous cell carcinoma (SC) and adenosquamous carcinoma (ASC) are observed as well. The clinicopathological characteristics of SC/ASC have not been well documented. Expressions of BIRC7 and STC2 were observed in some tumors. However, BIRC7 and STC2 expressions and clinical significances in gallbladder cancer have not been reported.In this study, the protein expressions of BIRC7 and STC2 in 46 SCs/ASCs and 80 ACs were measured using immunohistochemistry. We demonstrated that positive BIRC7 and STC2 expressions were significantly associated with large tumor mass (>3cm), high TNM stage and lymph node metastasis in SC/ASC and AC (p<0.05). Positive expression of BIRC7 was significantly associated with invasion of around tissues and organs in both SC/ASC and AC. Additionally, negative BIRC7 and STC2 expressions were significantly associated with surgical curability in AC. Univariate Kaplan-Meier analysis showed that BIRC7 and STC2 expressions, differentiation, tumor size, TNM stages, invasion, lymph node metastasis, and surgical curability were significantly associated with post-operative survival in both SC/ASC and AC patients(p < 0.001). Multivariate Cox regression analysis showed that positive BIRC7 and STC2 expressions are independent poor-prognostic factors in both SC/ASC and AC patients. Our study suggested that positive BIRC7 and STC2 expressions are closely correlated with clinical, pathological, and biological behaviors as well as poor-prognosis of gallbladder cancer.

Changes in peripheral blood Th1 and Th2 cells in rat liver transplantation under different immune statuses.

In this study, early expressions of peripheral blood Th1 and Th2 cells were documented following rat liver transplantation and related to immune status. Rats were divided into 3 groups: group A (control): syngeneic transplantation (Brown Norway (BN) → BN); group B: allogeneic transplantation + cyclosporine A (CsA); group C: allogeneic transplantation (Lewis → BN). Flow cytometry was used to analyze peripheral blood CD4(+)CD45RC percentage on days 1, 3, 5, 7, and 14 following transplantation, and were compared to graft rejection pathological grades and receptor survival times. The average survival of groups A and B exceeded 100 days, which was significantly longer than that of group C (3.56 ± 34.3 days). With the exception of the first day, rejection grades were significantly higher in groups C and B compared to group A, and group C rejection grades were significantly higher than those of group B. Three days after transplantation, the CD4(+)CD45RC(+) to CD4(+)CD45RC(-) ratio of group C was significantly higher than that of groups A and B. In group B, the CD4(+)CD45RC(+) to CD4(+)CD45RC(-) ratio was negatively correlated to the rejection grade (r = -0.565, P < 0.01), whereas this relationship was positive in group C (r = 0.745, P < 0.01). In conclusion, peripheral blood Th1 was highly expressed during rejection in rat liver grafts. Peripheral blood Th2 tended to increase early under rejection inhibition with CsA, and its high expression level may correlate with long-term acceptance or tolerance of transplanted livers.

Epithelial-mesenchymal transition biomarkers and support vector machine guided model in preoperatively predicting regional lymph node metastasis for rectal cancer.

BACKGROUND: Current imaging modalities are inadequate in preoperatively predicting regional lymph node metastasis (RLNM) status in rectal cancer (RC). Here, we designed support vector machine (SVM) model to address this issue by integrating epithelial-mesenchymal-transition (EMT)-related biomarkers along with clinicopathological variables. METHODS: Using tissue microarrays and immunohistochemistry, the EMT-related biomarkers expression was measured in 193 RC patients. Of which, 74 patients were assigned to the training set to select the robust variables for designing SVM model. The SVM model predictive value was validated in the testing set (119 patients). RESULTS: In training set, eight variables, including six EMT-related biomarkers and two clinicopathological variables, were selected to devise SVM model. In testing set, we identified 63 patients with high risk to RLNM and 56 patients with low risk. The sensitivity, specificity and overall accuracy of SVM in predicting RLNM were 68.3%, 81.1% and 72.3%, respectively. Importantly, multivariate logistic regression analysis showed that SVM model was indeed an independent predictor of RLNM status (odds ratio, 11.536; 95% confidence interval, 4.113-32.361; P<0.0001). CONCLUSION: Our SVM-based model displayed moderately strong predictive power in defining the RLNM status in RC patients, providing an important approach to select RLNM high-risk subgroup for neoadjuvant chemoradiotherapy.

First Report of Leaf Spot Caused by Alternaria longipes on Atractylodes macrocephala in China.

Atractylodes macrocephala Koidz. is a perennial herb that has been used in Chinese herbal medicine for centuries. During the summer of 2011, leaf spots were observed on leaves of A. macrocephala in Panan County of Zhejiang Province, China. Approximately 40% of the plants surveyed showed severe symptoms of leaf spot. At the initial stage of the infection, small, light brown spots appeared on the leaves that gradually became semicircular, oval, or irregular-shaped with dark brown or black centers surrounded by brown or light brown margins. The lesions enlarged and coalesced to form large areas of necrosis on leaves until entire leaves died. Eight fungal isolates were obtained from diseased A. macrocephala leaves by a tissue isolation method (1). Isolates were cultured on potato dextrose agar (PDA) plates at 25°C in darkness. Colonies on PDA were initially white and became grayish brown over time. Conidiophores were light brown with one or a few regular septa, mostly unbranched. Conidia were obclavate, dark brown, with three to eight transverse and zero to two longitudinal or oblique septa, and on average measured 35.1 (20 to 53) × 9.8 (5.8 to 13.3) µm (n = 20). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1F/ITS4 and sequenced (GenBank Accession No. JQ004404). The ITS sequence had 99% identity over 545 bp with Alternaria longipes (Ellis & Everh.) E. W. Mason (GenBank Accession No. AY278835), a fungal pathogen reported to cause leaf spot on Smilax china in China (3). To further identify the fungus, we chose the Alternaria allergen gene (Alt a 1 gene), which aids in identifying species of Alternaria (2). Amplification of the Alt a 1 gene was conducted using primers Alt-for/Alt-rev and sequenced (GenBank Accession No. JQ004405). Sequence comparisons showed there was 97% sequence identity with Alternaria longipes (GenBank Accession No. AY563304). Pathogenicity tests were performed on detached healthy A. macrocephala leaves. Nine leaves were inoculated by placing a PDA plug (0.5 cm2) of mycelia on upper surfaces of the leaves. Another nine leaves treated with sterile PDA plugs served as a control. Leaves were incubated in three petri dishes with a 12-h photoperiod at 25°C and 95% relative humidity. After 7 days, the symptoms described above were observed on all inoculated leaves, whereas no symptoms developed on control leaves. Reisolation of the fungus from symptomatic leaf tissues on PDA confirmed that the causal agent was Alternaria longipes. This pathogenicity test was performed three times. The genus Alternaria is ubiquitous and includes both plant pathogenic and saprophytic species that may cause leaf spot and blight on numerous plants (3,4), but to our knowledge, our research demonstrated this is the first report of leaf spot on A. macrocephalae caused by Alternaria longipes in China. References: (1) O. D. Dhingra and J. B. Sinclair. Basic Plant Pathology Methods. CRC Press, Boca Raton, FL, 1995. (2) S. G. Hong et al. Fungal Genet. Biol. 42:119, 2005. (3) Y. Long et al. Plant Pathol. 58:800, 2009. (4) S. X. Zang et al. J. Hebei Normal Univ. Sci. Tech. 19:46, 2005.