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Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding. [ABSTRACT FROM AUTHOR]

Rationale Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. Objectives Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. Methods Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. Results Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). Conclusions ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status. [ABSTRACT FROM AUTHOR]

Abstract: A protocol relying on Sanger sequencing reactions in combination with mass spectrometry (MS) for sequence confirmation of antisense phosphorothioate oligodeoxynucleotides is described. In this procedure, synthetic phosphorothioate oligodeoxynucleotides are used as reverse primers for extension of matched templates with enough length (∼150–300bp) for well-established Sanger sequencing. Because the complementary strand of modified primer is used directly for sequencing primer extension, the base order shown in the sequencing result is reversely complementary to phosphorothioate oligodeoxynucleotide. This sequencing method can be applied not only to phosphorothioate oligodeoxynucleotides with different lengths (13–21mer) and base composition but also to sequences with bases’ switch, deletion, or insertion. In addition, modified primers incorporate the 5′ end of polymerase chain reaction (PCR) products conveying the characters of phosphorothioate modification. The method requires only common reagents and instruments and so is better suited to routine sequence analysis in quality control of phosphorothioate antisense drugs. [Copyright &y& Elsevier]

Objectives: Primary pulmonary lymphoepithelioma‐like carcinoma (PLELC) is a subtype of lung carcinoma associated with the Epstein–Barr virus (EBV). The clinical predictive biomarkers of immune checkpoint blockade (ICB) in PLELC require further investigation. Methods: We prospectively analysed EBV levels in the blood and immune tumor biomarkers of 31 patients with ICB‐treated PLELC. Viral EBNA‐1 and BamHI‐W DNA fragments in the plasma were quantified in parallel using quantitative polymerase chain reaction. Results: Progression‐free survival (PFS) was significantly longer in EBNA‐1 high or BamHI‐W high groups. A longer PFS was also observed in patients with both high plasma EBNA‐1 or BamHI‐W and PD‐L1 ≥ 1%. Intriguingly, the tumor mutational burden was inversely correlated with EBNA‐1 and BamHI‐W. Plasma EBV load was negatively associated with intratumoral CD8+ immune cell infiltration. Dynamic changes in plasma EBV DNA level were in accordance with the changes in tumor volume. An increase in EBV DNA levels during treatment indicated molecular progression that preceded the imaging progression by several months. Conclusions: Plasma EBV DNA could be a useful and easy‐to‐use biomarker for predicting the clinical activity of ICB in PLELC and could serve to monitor disease progression earlier than computed tomography imaging. [ABSTRACT FROM AUTHOR]

• Strain MEPF0303 inhibited mycelial growth of S. delphinii. • Strain MEPF0303 had a good control against southern blight of D. officinale. • The down-regulated DEGs were related to growth and pathogenicity of S. delphinii. • MEPF0303 may be a potential biocontrol agent against D. officinale southern blight. Southern blight is a common and devastating disease adversely affecting the yield and quality of cultivated Dendrobium officinale plants. The pathogens responsible for this disease are Sclerotium delphinii and S. rolfsii , although S. delphinii is more pathogenic than S. rolfsii. Plant endophytes are potential sources of biological control agents. In this study, 18 microbial strains were isolated from wild Dendrobium shixingense plants (e.g., Streptomyces , Bacillus , and Trichoderma), among which strain MEPF0303 significantly inhibited S. delphinii mycelial growth. Based on 16S rRNA gene sequence, this strain was identified as Streptomyces sp. In addition, the volatile organic compounds and cell-free supernatant of MEPF0303 had significant inhibitory effects on S. delphinii growth. Moreover, MEPF0303 effectively controlled southern blight of D. officinale. A total of 1,797 differentially expressed genes were identified by a comparative transcriptome analysis of S. delphinii mycelia treated with MEPF0303 and the untreated control mycelia. Strain MEPF0303 significantly altered the expression of genes related to cell membrane morphological development and integrity, pathogenicity, energy metabolism, and oxidative stress responses. Furthermore, the transcriptome sequencing results were validated by analyzing gene expression via a quantitative real-time polymerase chain reaction. The study results showed that MEPF0303 can inhibit the growth and pathogenicity of S. delphinii , with potential implications for the biological control of southern blight of D. officinale. [ABSTRACT FROM AUTHOR]

Recent reclassification of the Klebsiella genus to include Klebsiella variicola, and its association with bacteremia and mortality, has raised concerns. We examined Klebsiella spp. infections among battlefield trauma patients, including occurrence of invasive K. variicola disease. Klebsiella isolates collected from 51 wounded military personnel (2009–2014) through the Trauma Infectious Disease Outcomes Study were examined using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis. K. variicola isolates were evaluated for hypermucoviscosity phenotype by the string test. Patients were severely injured, largely from blast injuries, and all received antibiotics prior to Klebsiella isolation. Multidrug-resistant Klebsiella isolates were identified in 23 (45%) patients; however, there were no significant differences when patients with and without multidrug-resistant Klebsiella were compared. A total of 237 isolates initially identified as K. pneumoniae were analyzed, with 141 clinical isolates associated with infections (remaining were colonizing isolates collected through surveillance groin swabs). Using PCR sequencing, 221 (93%) isolates were confirmed as K. pneumoniae, 10 (4%) were K. variicola, and 6 (3%) were K. quasipneumoniae. Five K. variicola isolates were associated with infections. Compared to K. pneumoniae, infecting K. variicola isolates were more likely to be from blood (4/5 versus 24/134, p = 0.04), and less likely to be multidrug-resistant (0/5 versus 99/134, p<0.01). No K. variicola isolates demonstrated the hypermucoviscosity phenotype. Although K. variicola isolates were frequently isolated from bloodstream infections, they were less likely to be multidrug-resistant. Further work is needed to facilitate diagnosis of K. variicola and clarify its clinical significance in larger prospective studies. [ABSTRACT FROM AUTHOR]

[Display omitted] • Strain P32-3 inhibited C. fimbriata mycelial growth and spore germination. • Strain P32-3 effectively controlled postharvest sweet potato black rot. • The down-regulated DEGs were related to C. fimbriata growth and pathogenicity. • Strain P32-3 may be a potential biocontrol agent against postharvest sweet potato black rot. Sweet potato black rot is a common postharvest disease caused by the pathogenic fungus Ceratocystis fimbriata. The toxins produced by infected tubers are potentially harmful to humans. Plant endophytic bacteria may serve as biocontrol agents for protecting crops (including vegetables) during postharvest storage without detrimental effects on humans or other animals. In this study, 11 bacterial strains of Bacillus , Priestia , Blautia , Pseudomonas , and Sphingomonas species were isolated from sweet potato storage roots, of which only one strain (P32-3), which was identified as Bacillus altitudinis , strongly inhibited C. fimbriata mycelial growth and spore germination. Furthermore, the volatile organic compounds and cell-free supernatant of strain P32-3 repressed C. fimbriata growth. Additionally, P32-3 treatments significantly decreased the disease spot diameter and depth on harvested sweet potato roots. A total of 981 differentially expressed genes were identified by comparing the transcriptomes of the control and P32-3-treated mycelia. Strain P32-3 significantly altered the expressions of genes related to cell wall integrity, the cell membrane, spore germination, energy metabolism, pathogenicity, and oxidative stress. In addition, RNA-seq results were validated by a quantitative real-time polymerase chain reaction analysis. Our results indicate that B. altitudinis strain P32-3 can inhibit C. fimbriata growth and development and protect postharvest sweet potato storage roots from C. fimbriata infection. The study findings provide the theoretical basis for future investigations on the utility of strain P32-3 for protecting sweet potato against black rot during the postharvest stage. [ABSTRACT FROM AUTHOR]

Introduction: Urotensin II (UII) is an important vasoactive peptide involved in the pathogenesis of atherosclerosis. Monocytes/macrophages play important roles in every step of atherosclerosis. Although UII has a chemoattractant effect on monocytes, it is unclear whether UII regulates inflammatory responses in macrophages. The present study sought to explore whether UII can promote leukotriene B4 (LTB4) production by macrophages.Material and Methods: The mRNA expression level of LTB4 and 5-lipoxygenase were determined by real-time polymerase chain reaction. The protein level of LTB4 and 5-lipoxygenase expression was assayed by enzyme-linked immunosorbent assay and Western blot, respectively. Western blot analysis was also employed to determine the phosphorylated forms of Akt. Reactive oxygen species (ROS) level was detected by the fluorescent probe 2',7'-dichlorofluorescin diacetate and fluorescence intensity was measured with a multiwell fluorescence plate reader.Results: Urotensin II promoted LTB4 release and increased 5-lipoxygenase expression in a concentration- and time-dependent manner in RAW264.7 cells. Leukotriene B4 production and 5-lipoxygenase expression were decreased by blocking the UII receptor (UT) with urantide, eliminating ROS with N-acetylcysteine and diphenyliodonium, and inhibiting Akt phosphorylation with LY294002. UII significantly elevated ROS production, whereas urantide, N-acetylcysteine and diphenyliodonium substantially attenuated this effect. UII also enhanced Akt phosphorylation significantly, and this effect was potently inhibited by urantide, N-acetylcysteine, diphenyliodonium and LY294002.Conclusions: Urotensin II may promote 5-lipoxygenase expression and LTB4 release in RAW264.7 macrophages via UT-ROS-Akt pathways. These results indicate that UII may participate in macrophage activation and suggest a potential new mechanism underlying atherosclerosis. [ABSTRACT FROM AUTHOR]

Aim: The X-ray repair cross-complementing group 7 (XRCC7) plays an important role in the repair of DNA double-strand breaks by nonhomologous end-joining repair (NEJR) pathway. However, the role of XRCC7 polymorphisms (rs#7003908 and rs#10109984) possibly influencing NEJR capacity in hepatocellular carcinoma (HCC) induced by aflatoxin B1 (AFB1) has not been well elaborated. Methods: This hospital-based case-control study, including 348 patients with newly diagnosed HCC and 597 controls without any evidence of liver diseases, was conducted to elucidate the association between these two polymorphisms and the risk of HCC related to AFB1 exposure among a Guangxi population from a high AFB1-exposure area by means of TaqMAN-polymerase chain reaction technique. Results: We observed that HCC patients featured higher AFB1 exposure than control group (odds ratios [OR] = 6.49 and 6.75 for exposure years and exposure levels, respectively). Furthermore, these individuals with the genotypes of XRCC7 rs#7003908 G alleles (namely XRCC7-TG or -GG), compared the homozygote of XRCC7 rs#7003908 T alleles (XRCC7-TT), faced increasing risk of HCC (OR, 3.45 and 5.04; 95% confidence intervals [CIs], 2.40-4.94 and 3.28-7.76, respectively). We also found some evidence that this polymorphism interacted with AFB1-expousure years or levels in the process of HCC carcinogenesis. Additionally, XRCC7 rs#7003908 polymorphism was correlated with the levels of AFB1-DNA adducts (r = 0.142, P < 0.001). XRCC7 rs#10109984 polymorphism, however, did not modify the risk of HCC related to AFB1 exposure ( P > 0.05). Conclusion: These data suggest that XRCC7 rs#7003908 polymorphism may be one of the genetic modifiers for AFB1-related HCC among Guangxi population. [ABSTRACT FROM AUTHOR]

Background Recent work has argued that genus Klebsiella is best divided into 3 clades: K. pneumoniae (Kp) , K. quasipneumoniae (Kq), and K. variicola (Kv). Kv has drawn attention from reports of higher mortality and virulence. We evaluated a previously defined group of military trauma patients with Klebsiella infections for the presence of Kv, described clinical and isolate characteristics, and compared Kv and Kp groups. Methods All initial and serial (≥7 days from prior isolate) infecting Kp isolates (identified by clinical laboratories without the ability to speciate Kq and Kv) were collected from the Trauma Infectious Disease Outcomes Study (6/09–12/14). Additionally, a previously defined group of colonizing isolates linked to the infecting isolates and a selection of random colonizers were included from groin swabs. DNA extraction and PCR targeting Kv per published methods was performed. Antimicrobial susceptibilities were determined using the BD Phoenix Automated Microbiology System and CLSI criteria. Multidrug resistance was defined as either resistance to ≥3 classes of aminoglycosides, β-lactams, carbapenems and/or fluoroquinolones or production of ESBL or KPC. Results Of 237 archived Kp isolates (from 122 patients), 10 (4%) were identified as Kv by PCR (from 8 [7%] patients). The Kv sources were 4 from blood (40%), 1 intra-abdominal (10%) and 5 from groin (50%). Six (3%) isolates were identified as Kq (4 from groin and 2 from respiratory specimens). The Kv and Kp patients were all males, with a median age of 25 (IQR 21–46) and 23 (IQR 21–28), length of hospital stay of 24 days (IQR: 5–106) and 53 days (IQR 36–74), and Injury Severity Score of 21 (IQR: 10–50) and 38 (IQR: 30–45), respectively. There were no deaths in the Kv group compared with 4 with Kp. Infecting Kv isolates were more likely to be from blood compared with Kp (80% vs. 17%, P = 0.04). No infecting Kv isolates were multidrug-resistant compared with 70% of infecting Kp isolates (P < 0.01). Conclusion Kv represented 4% of the previously identified Kp isolates in this population. Patient characteristics were similar in both groups. While Kv was less resistant than Kp, it was more likely to be associated with invasive disease in this group. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

Background Biocides play an integral role in infection control. Paralleling concern about rising incidence of multidrug-resistant (MDR) organisms is a concern for resistance to biocides. In small studies, several genes involved in the production of efflux pump proteins have been identified as markers of biocide resistance in Klebsiella spp. namely cepA , qacA , qacE , qac∆E , and acrA. This study aimed to analyze the Klebsiella spp. isolates of a previously defined military trauma group with a high incidence of MDR organisms for the presence of these genes and their correlation with other resistance. Methods All infecting K. pneumoniae, K. variicola , and K. quasipneumoniae isolates archived by the Trauma Infectious Disease Outcomes Study (June 2009–December 2014) were selected. Additionally, all colonizing isolates linked with infecting isolates were included; the remainder to total 50 MDR and 46 non-MDR colonizing isolates were chosen randomly. Antimicrobial identification and susceptibilities were determined by CLSI criteria using the BD Phoenix Automated Microbiology System. PCR according to published methods for cepA , qacA , qacE , qac∆E , and acrA was accomplished in duplicate. MDR was defined as either resistance to ≥3 classes of aminoglycosides, β-lactams, carbapenems and/or fluoroquinolones or production of an ESBL or KPC. Results A total of 237 isolates (221 K. pneumoniae , 10 K. variicola , 6 K. quasipneumoniae) met inclusion criteria, of which 149 (63%) were MDR. All isolates had been exposed to antimicrobials prior to isolation. Of all isolates, 234 (98%) carried cepA : 218 (98%) K. pneumoniae carried cepA, 10 (100%) K. variicola carried cepA, and 6 (100%) of K. quasipneumoniae carried cepA. In addition, 148 (62%) isolates with cepA were MDR. One (10%) K. variicola isolate carried qacE along with cepA. This isolate was the only MDR K. variicola. None of the isolates carried qacA, qac∆E, or acrA. Conclusion We confirmed the near universal presence of the cepA biocide resistance gene in Klebsiella spp. isolated from trauma patients in Iraq and Afghanistan. In the largest evaluation of biocide resistance genes in Klebsiella spp. to our knowledge, the presence of qacA , qacE , qac∆E , and acrA was less common than has been reported elsewhere. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]