[Significance of TERT promoter mutation in differential diagnosis of non-invasive inverted urothelial lesions of bladder].

Objective: To investigate the gene mutation of telomerase reverse transcriptase (TERT) promoter in inverted urothelial lesions of the bladder and its significance in differential diagnosis. Methods: From March 2016 to February 2022, a total of 32 patients with inverted urothelial lesions diagnosed in Department of Pathology at Qingdao Chengyang People's Hospital and 24 patients at the Affiliated Hospital of Qingdao University were collected, including 7 cases of florid glandular cystitis, 13 cases of inverted urothelial papilloma, 8 cases of inverted urothelial neoplasm with low malignant potential, 17 cases of low-grade non-invasive inverted urothelial carcinoma, 5 cases of high-grade non-invasive inverted urothelial carcinoma, and 6 cases of nested subtype of urothelial carcinoma were retrospectively analyzed for their clinical data and histopathological features. TERT promoter mutations were analyzed by Sanger sequencing in all the cases. Results: No mutations in the TERT promoter were found in the florid glandular cystitis and inverted urothelial papilloma. The mutation rates of the TERT promoter in inverted urothelial neoplasm with low malignant potential, low grade non-invasive inverter urothelial carcinoma, high grade non-invasive inverted urothelial carcinoma and nested subtype urothelial carcinoma were 1/8, 8/17, 2/5 and 6/6, respectively. There was no significant difference in the mutation rate of TERT promoter among inverted urothelial neoplasm with low malignant potential, low-grade non-invasive inverted urothelial carcinoma, and high-grade non-invasive inverted urothelial carcinoma (P>0.05). All 6 cases of nested subtype of urothelial carcinoma were found to harbor the mutation, which was significantly different from inverted urothelial neoplasm with low malignant potential and non-invasive inverted urothelial carcinoma (P<0.05). In terms of mutation pattern, 13/17 of TERT promoter mutations were C228T, 4/17 were C250T. Conclusions: The morphology combined with TERT promoter mutation detection is helpful for the differential diagnosis of bladder non-invasive inverted urothelial lesions.

First report of Alternaria alternata causing leaf yellow spot on Heteropanax fragrans in China.

Heteropanax fragrans (Roxb.) Seem is a common garden landscape tree in China. In December 2020, a leaf disease on H. fragrans was observed in a 2 ha field in Zhanjiang (20.85° N, 109.28° E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned into necrotic tissues with a clear yellow halo and a white center. The disease incidence on plants was 100%. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed thrice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (HFA-1, HFA-2, and HFA-3). The colonies first produced a light-grayish aerial mycelia, which turned dark grayish upon maturity. Conidiophores were branched. Conidia numbered from two to four in chains, were dark brown, ovoid, or ellipsoid and mostly beakless; had 1-4 transverse and 0-3 longitudinal septa; measured within 7.2-17.8 (average = 10.2) × 2.5-7.5 (average = 4.3) µm (n = 30). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify the large subunit (LSU), internal transcribed spacer (ITS) region, translation elongation factor (TEF) , and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with NL1/LR3, ITS1/ITS4, EF-1α-F/EF-1α-R, and GDF1/GDR1 (Walther et al. 2013;Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (LSU, ON088978-ON088980; ITS, MW629797, ON417005 and ON417006; TEF, MW654167, ON497264,and ON497265;GAPDH, MW654166, ON497262,and ON497263). The obtained sequences were 100% identical with those of Alternaria alternata strain CBS 102600 upon BLAST analysis . The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. alternata (CBS 102600, CBS 102598, CBS 118814, CBS 918.96,CBS 106.24, CBS 119543, CBS 916.96). The fungus associated with leaf yellow spot on H. fragrans was thus identified as A. alternata. Pathogenicity tests were conducted in a greenhouse at 24 â„ƒ-30 â„ƒ with 80% relative humidity. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control). The test was performed thrice. Disease symptoms were found on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch's postulates. To our knowledge, this report is the first one on A. alternata causing leaf yellow spot on H. fragrans. Thus, this work provides an important reference for the control of this disease in the future.

Pseudocercospora rhododendricola Causing Leaf dark Spot on Rhododendron pulchrum by the first phylogenetic analyses.

Rhododendron pulchrum Sweet is a famous ornamental flower in China. In December 2020, a leaf spot disease was observed on cv. Maojuan in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The spots were irregular and distributed on both sides of the main vein. They were dark to black, and their borders were obvious. The coalescence of the spots eventually led to leaf wilt. The disease incidence was 100% (n = 100, about 50 ha ). Thirty infected leaves were collected from the field, and the margin of the diseased tissues was cut into 2 mm × 2 mm pieces. Samples were surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively. They were rinsed thrice with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 ℃. After 5 days, fungal colonies appeared on the PDA. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates (RSP-1, RSP-2, and RSP-3) were obtained and the colonies of isolates were preserved in glycerol (15%) at -80 °C deposited at the Museum of Guangdong Ocean University. The morphology of these three isolates was consistent, and their sequences showed 100% homology according to ITS, TEF1, and ACT analysis results. The colonies grew to approximately 5 cm in diameter after 10 days. They showed olive green with off-white aerial mycelia. Stromata and conidia were observed on leaf lesions. Stromata were olivaceous brown. Conidia were solitary, cylindrical to narrowly obclavate, mildly curved, obtuse to rounded at the apex, and 1- to 3-septate; they had dimensions of 20 to 60 × 2.0 to 3.0 µm (n = 30). These morphological characteristics were not different from the description of Pseudocercospora rhododendricola (J.M. Yen) Deighton (Liu et al. 1998). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), and actin (ACT) loci of the isolates using primer pairs ITS4/ITS5, EF1/EF2, and ACT-512F/ACT-783R, respectively (White et al., 1990; O'Donnell et al. 1997). The sequences of the isolate RSP-1 were deposited in the GenBank (ITS, MW629798; TEF1, MW654168; and ACT, MW654170). BLAST analysis showed that the sequences of P. rhododendricola were submitted to GenBank for the first time by the author of this paper. A phylogenetic tree was generated based on the concatenated data of ITS, TEF1, and ACT sequences from GenBank by the Maximum Likelihood method. The isolates were closest to Pseudocercospora sp. CPC 14711 (Crous et al., 2013). Phylogenetic and morphological analyses identified the isolates as P. rhododendricola. Pathogenicity tests were conducted in a greenhouse at 24 °C-30 â„ƒ with 80% relative humidity. Healthy cv. Maojuan were grown in pots. Unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control) (5 leaflets per plant, 3 plants, 2-month-old plants). The test was performed thrice. Disease symptoms were found on the leaves after 2 weeks, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. P. rhododendricola was the cause of leaf spot of Rhododendron sp. from Singapore (Liu et al., 1998). For the first time, this pathogen was identified by combining phylogenetic and morphological analyses. The sequences in this study would be used as the reference sequences for further studies.

Spastic paraplegia as the only symptom in two adult-onset patients carrying a novel pathogenic variant in PYCR2.

A novel pathogenic PYCR2 variant and corresponding brain images in two patients characterized by spastic paraplegia.

LncRNA MALAT1 promotes colorectal cancer development by sponging miR-363-3p to regulate EZH2 expression.

LncRNA MALAT1 is reported to play a potential role in human cancers. Hence, we investigated the effects of MALAT1 on colorectal cancer in vitro and in vivo, and further validated whether MALAT1 affected colorectal cancer development and EZH2 expression via regulating miR-363-3p. The fresh colorectal cancer tissues, adjacent non-tumor tissues, FHC, LOVO, SW620, CL40 and HCT116 cells were analyzed in this study. MALAT1, miR-363-3p and EZH2 expression levels were assessed using qRT-PCR and Western blot. Cell proliferation, migration and invasion were also measured. Binding effects between MALAT1 and miR-363-3p, or miR-363-3p and EZH2 3'UTR were detected by dual luciferase assay. We observed that MALAT1 was highly expressed in colorectal cancer tissues and cells, and MALAT1 knockdown inhibited cell proliferation as well as expression levels of EZH2 by upregulated miR-363-3p in cell models and in vivo. Moreover, miR-363-3p functions as a downstream target of MALAT1, meanwhile EZH2 was a target of miR-363-3p, suggesting MALAT1 might regulate miR-363-3p and/or EZH2 expression. Collectively, we concluded that MALAT1 functioned as a ceRNA to promote colorectal cancer development and EZH2 expression through sponging miR-363-3p in vitro and in vivo.

Effect of weaning age on cortisol release in piglets.

The effect of weaning age on the adrenal cortex, which plays a vital role in the stress response, is currently unknown. Therefore, plasma adrenocorticotropic hormone (ACTH) and cortisol levels, weights and relative weights of adrenal glands, and steroidogenesis-related protein and enzyme expression levels in piglets weaned on different days were determined. Piglets weaned at 35 days had significantly lower ACTH levels than those weaned at 14 or 21 days, and cortisol levels of piglets weaned at 21, 28, and 35 days were significantly lower than those of piglets weaned on day 14. Adrenal gland weights of piglets weaned at 28 and 35 days and relative adrenal gland weights of piglets weaned at 35 days were significantly lower than those of piglets weaned at 14 days. However, no significant difference was detected in the expression of melanocortin-type 2 receptor mRNA, which is associated with weaning age. Steroidogenic acute-regulatory (StAR) mRNA and cholesterol side-chain cleavage cytochrome P450 mRNA expression levels in piglets weaned at 28 and 35 days were significantly lower than in those weaned at 14 or 21 days, and P450 11ß mRNA expression levels in piglets weaned at 28 and 35 days were significantly lower than in those weaned at 14 days. Therefore, early-weaned piglets exhibited increased adrenal gland weights and StAR and steroidogenic enzyme expression, all of which contributed to high cortisol levels. The high plasma ACTH and cortisol levels in early-weaned piglets indicate that these animals would be greatly affected by stress.

A case-control study indicates that the TRIB1 gene is associated with pancreatic cancer.

Pancreatic cancer is a malignant neoplasm originating from transformed cells arising in tissues that form the pancreas. To investigate whether the tribbles homolog 1 (Drosophila) gene (TRIB1) is associated with pancreatic cancer in the Chinese Han population, we conducted this case-control study and genotyped 3 single nucleotide polymorphisms (rs2980879, rs2980874, and rs2235108) of the TRIB1 gene in 182 patients and 359 normal controls of Chinese Han origin and analyzed their association. The results showed that the rs2980879 polymorphism was associated with pancreatic cancer [allele: P = 0.023434, genotype: P = 0.03005; odds ratio (OR) and 95% confidence interval (CI) = 0.727788 (0.552664-0.958404)], whereas the rs2980874 polymorphism had no association with pancreatic cancer [allele: P = 0.749885, genotype: P = 0.699533; OR and 95%CI = 1.041981 (0.809196-1.341734)], and the rs2235108 polymorphism was not associated with the disease [allele: P = 0.629475, genotype: P = 0.547534, OR and 95%CI = 1.128290 (0.690829-1.842770)]. Haplotype analyses and linkage disequilibrium tests were also conducted, and the results showed that these 3 loci are not in the same block. In conclusion, our study indicated that the TRIB1 gene is associated with pancreatic cancer. More studies with larger samples are needed in order to support this finding.

Relative bioavailability of iron proteinate for broilers fed a casein-dextrose diet.

An experiment was carried out to determine the bioavailability of organic Fe as Fe proteinate (Alltech, Nicholasville, KY) relative to inorganic Fe source (FeSO4•7H2O) for broiler chicks fed a casein-dextrose diet. A total of 448 1-d-old Arbor Acres commercial male broiler chicks were randomly allotted to 1 of 8 replicate cages (8 chicks per cage) for each of 7 treatments in a completely randomized design involving a 2 × 3 factorial arrangement of treatments with 2 Fe sources (Fe proteinate and Fe sulfate) and 3 levels of added Fe (10, 20, or 40 mg of Fe/kg) plus a Fe-unsupplemented control diet containing 4.56 mg of Fe/kg by analysis. Feed and distilled-deionized water were available ad libitum for an experimental phase of 14 d. At 14 d of age, blood samples were collected for testing hemoglobin (Hb) and hematocrit, and calculating total body Hb Fe, whereas liver and kidney samples were excised for Fe analyses. The results showed that ADG, ADFI, blood Hb, hematocrit, and total body Hb Fe and Fe concentrations in liver and kidney increased linearly (P < 0.0001), whereas mortality decreased linearly (P < 0.0001) as dietary Fe level increased. However, only blood Hb concentration and total body Hb Fe differed (P < 0.004) between the 2 Fe sources. Based on slope ratios from the multiple linear regression of Hb concentration and total body Hb Fe on daily intake of analyzed dietary Fe, the bioavailability of Fe proteinate relative to FeSO4•7H2O (100%) was 117 and 114%, respectively (P < 0.009). The results indicated that blood Hb concentration and total body Hb Fe were sensitive indices in reflecting differences in bioavailability among different Fe sources, and Fe proteinate was significantly more available to broilers than inorganic Fe sulfate in enhancing Hb concentration and total body Hb Fe.

Arginine requirements of White Pekin ducks from 1 to 21 days of age.

An experiment was conducted to estimate optimal dietary arginine requirement in White Pekin ducks from 1 to 21 d of age. Six hundred thirty 1-d-old male White Pekin ducks were randomly allotted to 10 dietary treatments with 7 replicate pens of 9 birds per pen. Birds in each group were fed corn-corn gluten meals diet containing 0.71, 0.84, 0.95, 1.03, 1.17, 1.27, 1.39, 1.47, 1.62, and 1.72% arginine, respectively. At 21 d of age, weight gain, feed intake, feed/gain ratio, and breast meat yield from each treatment were measured. Dietary supplementation of arginine significantly improved weight gain, feed intake, feed/gain, and breast meat yield (P < 0.01). Based on quadratic broken-line regression analysis, the arginine requirement of male White Pekin ducks from 1 to 21 d of age was 0.95, 1.16, and 0.99% for weight gain, feed/gain, and breast meat yield, respectively.

Effect of intravenously injected zinc on tissue zinc and metallothionein gene expression of broilers.

1. The effect of intravenously injected zinc (Zn) on tissue Zn concentrations and pancreas metallothionein (MT) gene expression in broilers was investigated to detect differences in the tissue utilisation of Zn from different Zn sources. 2. A total of 432 male chickens were randomly allotted on d 22 post-hatch to one of nine treatments in a completely randomised design. Chickens were injected with either a 0.9% (w/v) NaCl solution (control) or a saline solution supplemented with Zn sulphate or one of three organic Zn chelates with weak (Zn-AA W), moderate (Zn-Pro M) or strong (Zn-Pro S) chelation strengths at two injected Zn dosages calculated according to two Zn absorbability levels (6 and 12%). 3. Bone and pancreas Zn concentrations, pancreas MT mRNA levels and MT concentrations increased on d 6 and 12 after Zn injections as the injected Zn dosages increased. Chickens injected with the Zn-Pro S had lower bone Zn concentration than those injected with the Zn-Pro M or Zn-AA W on d 6 after injections. However, no differences among Zn sources were observed in bone Zn concentration on d 12 after injections, pancreas Zn concentrations, pancreas MT mRNA levels and MT concentrations on both d 6 and d 12 after injections. 4. It was concluded that the injected Zn-Pro S was the least favourable for bone Zn utilisation of broilers. The pancreas Zn concentration and pancreas MT gene expressions might not be sensitive enough to detect differences in the tissue utilisation of injected Zn in broilers between organic and inorganic Zn sources or among organic Zn sources.

Estimation of standardized phosphorus retention for corn, soybean meal, and corn-soybean meal diet in broilers.

Two experiments were conducted to estimate standardized phosphorus (P) retention (SPR) of corn, soybean meal (SBM), and corn-soybean meal (C-SBM) diet in broilers and verify the additivity of SPR for feed formulation of broilers. In total, ninety-six 22-d-old male broilers with similar BW were used in each experiment. After 3 d of acclimation, chicks were fasted for 24 h and then fed P-free, corn, SBM, or C-SBM diets, respectively for 4 h in experiment 1 or 72 h in experiment 2. In experiment 1, the results showed that the excreta collection time of 52 h (48 h after feed withdrawal) was adequate for the estimation of SPR. The basal endogenous P loss (EPL) of chicks fed the P-free diet was estimated to be 123±7 mg/52 h per bird. The values of SPR corrected by basal EPL were 37.6 and 50.5% for corn and SBM, respectively. The determined value of SPR of the C-SBM diet was very close (P>0.79) to the predicted summation of SPR from corn and SBM (44.4 vs. 43.5%). In experiment 2, the results showed that the excreta collection time of 96 h (24 h after feed withdrawal) was sufficient for the estimation of SPR. The basal EPL of chicks fed the P-free diet was estimated to be 85.4±4.0 mg/96 h per bird. The values of SPR corrected by basal EPL were 40.2 and 52.9% for corn and SBM, respectively. The determined value of SPR of the C-SBM diet was lower (P<0.001) than the predicted summation of SPR from corn and SBM (39.7 vs. 46.0%), which might be due to the effect of higher total P intake. The results from the current study demonstrated that the P-free diet could be used for measuring basal EPL in broilers and then estimating the SPR values of feedstuffs for broilers. However, the additivity of SPR in the diet formulation needs to be studied further.

Comparison of amino acid digestibility and its additivity determined with slaughter or cecectomy method for yellow-feather chicken.

The objective of this experiment was to compare the slaughter and cecectomy methods to determine amino acid (AA) digestibility of corn and soybean meal and their additivity in a corn-soybean meal diet. A completely randomized design was adopted to determine endogenous AA losses (EAAL) and AA digestibility in each of corn, soybean meal, and a corn-soybean meal diet using either slaughter or cecectomy methods. Each treatment contained 6 replicates with 3 chickens per replicate. The endogenous loss (EL) of histidine and glycine was lower and the EL of methionine and phenylalanine was greater when determined by slaughter vs. cecectomy (P < 0.05). The EL of arginine, isoleucine, leucine, lysine, methionine, phenylalanine, valine, alanine, aspartic acid, glutamic acid, and serine determined by slaughter were 1.2 to 3.2 times of those from cecectomy. The standard error (SE) of EL of 14 AA (excluding histidine and glycine) obtained by slaughter method was 2.1 to 9.6 times of those by cecectomy method. The apparent and standardized digestibility was not affected by methods for most AA except apparent digestibility of methionine, phenylalanine and glycine, and standardized digestibility of glycine in corn. The apparent and standardized digestibility of most AA except apparent digestibility of glycine and standardized digestibility of lysine, cysteine and glycine were less for slaughter versus cecectomy methods in soybean meal (P < 0.05). Using slaughter method resulted in reduced apparent digestibility of 15 AA (except glycine) and reduced standardized digestibility of 7 AA (arginine, isoleucine, leucine, valine, aspartic acid, glutamic acid, and proline) relative to cecectomy method (P < 0.05), but the standardized digestibility of glycine was greater when determined by slaughter vs. cecectomy methods in corn-soybean meal diet (P < 0.05). The mean value of SE of 16 AA digestibility in slaughter method was 2.9 times of that by cecectomy method. The apparent digestibility of 2 and 9 of 16 AA and the standardized digestibility of 15 and 7 of 16 AA were additive when using slaughter and cecectomy determinations, respectively. In conclusion, compared to the slaughter method, cecectomy method had less SE and EAAL but greater apparent digestibility of methionine and phenylalanine in corn, and the apparent digestibility of 15 AA (except glycine) in soybean meal and corn-soybean meal diet. Additivity in apparent and standardized AA digestibility was more inconsistent when determined with slaughter vs. cecectomy methods. These findings suggest that the cecectomy method is more suitable than the slaughter method to determine the digestibility of AA.

[Preliminary study on the effect of B7H3 interaction with fibronectin on apoptosis of human chronic myeloid leukemia cells].

Objective: To explore the effect of the interaction between B7H3 and fibronectin (FN) on the apoptosis of human chronic myeloid leukemia K562 cells. Methods: The expression of B7H3 molecules in K562 cells was detected using flow cytometry and B7H3 overexpressing cells were constructed. The interaction between B7H3 and FN was detected using the co-immunoprecipitation technology. After adding exogenous FN, cell experiments were performed to detect changes in adhesion and cell apoptosis. The changes in apoptosis-related proteins and PI3K/AKT signaling pathway were detected using Western blot. Results: The expression of B7H3 was low in K562, and the cell line K562 OE (overexpression) -B7H3 and the control cell line K562 NC (negative control) -B7H3 were obtained after lentivirus transfection. There is an interaction between B7H3 and FN (P=0.036) , and this interaction promoted cell adhesion (P<0.05) , inhibited cell apoptosis (P<0.05) , and activated the PI3K/AKT signaling pathway (P<0.05) . Conclusion: B7H3 interacts with FN to promote cell adhesion and may inhibit K562 cell apoptosis by activating the PI3K/AKT signaling pathway.

Experimental study on early protective effect of ischemic preconditioning on rat kidney graft.

OBJECTIVE: Our aim was to investigate the mechanisms of early protection by ischemic preconditioning (IPC) of transplanted rat kidneys. MATERIALS AND METHODS: Thirty-six male donor and recipient Sprague-Dawley (SD) rats were randomly divided into 3 groups: sham-operated (A), transplantation (B), and IPC treatment (C) groups. Group A underwent exploratory laparotomy, group B received orthotopic transplantation, and group C, 15 minutes of ischemia followed by 10 minutes of reperfusion before transplantation. We measured the serum creatinine (SCr), blood urea nitrogen (BUN), and degree of kidney graft ischemic/reperfusion injury (IRI) by tumor necrosis factor-alpha (TNF-alpha) IkappaB kinase beta (IKK-beta) nuclear factor-kappa beta (NF-kappabeta) p65 subunit mRNA expressions. RESULTS: The SCr and BUN levels in groups C and B were higher than those in the sham-operated group (P < .01), without a significant difference between groups C and B at 24 hours after transplantation (P > .05). The degree of renal graft tubular injury in group C was significantly lower than that in group B (P < .01). TNF-alpha transcription levels at 24 hours after transplantation were significantly lower compared with the non-IPC group (P < .01). However, we failed to note a significant difference in IKK-beta or p65 mRNA expressions between groups C and B (P > .05). CONCLUSIONS: One cycle of preconditioning (15 min/10 min) attenuated renal graft IRI in the early phase. The inhibiting effects on TNF-alpha and the positive feedback signaling of TNF-alpha/NF-KB pathways may play important roles in renal graft protection.