Maize smart-canopy architecture enhances yield at high densities.

Increasing planting density is a key strategy for enhancing maize yields1-3. An ideotype for dense planting requires a 'smart canopy' with leaf angles at different canopy layers differentially optimized to maximize light interception and photosynthesis4-6, among other features. Here we identified leaf angle architecture of smart canopy 1 (lac1), a natural mutant with upright upper leaves, less erect middle leaves and relatively flat lower leaves. lac1 has improved photosynthetic capacity and attenuated responses to shade under dense planting. lac1 encodes a brassinosteroid C-22 hydroxylase that predominantly regulates upper leaf angle. Phytochrome A photoreceptors accumulate in shade and interact with the transcription factor RAVL1 to promote its degradation via the 26S proteasome, thereby inhibiting activation of lac1 by RAVL1 and decreasing brassinosteroid levels. This ultimately decreases upper leaf angle in dense fields. Large-scale field trials demonstrate that lac1 boosts maize yields under high planting densities. To quickly introduce lac1 into breeding germplasm, we transformed a haploid inducer and recovered homozygous lac1 edits from 20 diverse inbred lines. The tested doubled haploids uniformly acquired smart-canopy-like plant architecture. We provide an important target and an accelerated strategy for developing high-density-tolerant cultivars, with lac1 serving as a genetic chassis for further engineering of a smart canopy in maize.

Validation of p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs in patient-derived breast tumor xenografts.

Blockade of mammalian target of rapamycin (mTOR) is a promising area in breast cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in breast cancer was modest. Biomarker studies designed to identify the responders of rapalogs are of increasing interest. We validated p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs. We also analyzed the correlation between rapamycin activity and p27KIP1 expression in the primary breast cancer cells and the patient-derived breast tumor xenograft models. The cells isolated from the breast tumor tissues expressing high levels of p27KIP1 were sensitive to rapamycin, whereas the cells from the tissues expressing low levels of p27KIP1 exhibited resistance to rapamycin. The correlation between p27KIP1 expression and rapamycin antitumor activity was also observed in the patient-derived breast tumor xenograft models. Moreover, we also found rapamycin significantly decreased phosphorylated p70S6K1 and phosphorylated 4EBP1 in both samples. It seemed that the different sensitivity of tumor cells to rapamycin did not owe to its different potency against mTOR activity. We further propose p27KIP1 expression level may be also a candidate predictive biomarker of rapalogs for breast cancer therapy, which requires additional clinical validation.

What kind of children do kindergarten teachers prefer? -Typology of the preferred types of children.

The teacher's pets are a common occurrence in the field of education. To investigate the preferences teachers exhibit toward certain children, the study focused on kindergarten teachers and employed a mixed research methodology. Initially, qualitative interviews were conducted with 15 kindergarten teachers to identify specific criteria influencing teacher preferences. Subsequently, A comprehensive model of teacher's pets was developed through a questionnaire survey involving 463 participants. This model encapsulated 32 distinct indicators, categorized into 7 types: children with good appearance (GA), exceptional abilities (OA), commendable conduct (GC), proactive and enthusiastic demeanor (PE), compliant and carefree nature (OC), children from vulnerable groups (VC), and those influenced by their parents (PI). The resulting model demonstrated a sound structure. Not only did it validate existing findings, but it also expanded upon the identified types of teacher's pets. An analysis based on game theory revealed the weighted combinations, highlighting the top three types of teacher's pets: children influenced by parental factors (24.3%), proactive and enthusiastic individuals (15.7%), and obedient, carefree children (14.8%), respectively. Conversely, the representation of vulnerable-concerned children (11.1%) was the lowest among the identified types.

Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.

[Preparation and application of the national reference panel for hepatitis B virus DNA].

OBJECTIVES: To calibrate the national hepatitis B virus (HBV) DNA standard according to world health organization's standard material and prepare the national reference panel for HBV DNA reagents. METHODS: Sera from blood donors and HBV patients were collected and detected by home-made HBV DNA PCR kits, HBsAg kits, and anti-HBc kits, and then confirmed by HBV DNA PCR kits produced by Roche in German, which was recognized by the world health organization. The stability of the panel was detected by acceleration method. RESULTS: The convinced copies of the sensitivity samples were gotten by seven independent experiments, the coefficients of variation of logarithm of the copies of L0-L5 were all less than 15%. Regarding the national reference panel as the standard, the quality of most domestic HBV DNA PCR kits was improved, while part of the kits should be further qualified. CONCLUSION: The national reference panel for HBV DNA reagents is developed. It contains eight negative, nine positive sera and seven samples for sensitivity test

Cross-protection of hepatitis E virus genotypes 1 and 4 in rhesus macaques.

The purpose of this study was to determine cross-protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 x 10(4) genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti-HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti-HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2-6 weeks post-inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross-genotype and cross-host-species protection.

[Primary evaluation of anti-HEV diagnostic reagent by experimental infection animal model with hepatitis E virus].

OBJECTIVE: To evaluate anti-HEV diagnostic kits by experimental infecting rhesus monkeys with HEV. METHODS: Eight rhesus monkeys were infected with genotype 1 and 4 HEV separately. The alanine aminotransferase (ALT) level of all monkeys were detected before and after the process of infection. HEV RNA in stool specimens was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Anti-HEV IgG in serum was detected by GL-IgG and WT-IgG. RESULTS: HEV RNA presented in the stool of all the 8 monkeys after infection. The ALT level of 1 monkey infected with genotype 1 HEV and 2 monkeys infected with genotype 4 HEV appeared abnormally after infection. Tested by GL-IgG, 2 of the 4 monkeys infected with genotype 1 HEV and 1 of 4 monkeys infected with genotype 4 HEV seroconverted to anti-HEV IgG. However, when tested by WT-IgG, all the infected monkeys seroconverted to anti-HEV IgG. The anti-HEV IgG tested by WT-IgG was positive during the whole observation period,and the anti-HEV IgG measured by GL-IgG only remained 12 weeks after infection. Detected by GL-IgG and WT-IgG, seropositive conversion of the anti-HEV IgG happened almost at the same time. CONCLUSION: Both GL-IgG and WT-IgG could detect the anti-HEV IgG of experimentally infected rhesus monkeys but the WT-IgG had a higher sensitivity for detection of anti-HEV IgG than

Correlation of hepatitis B virus (HBV) genotypes and mutations in basal core promoter/precore with clinical features of chronic HBV infection.

BACKGROUND/AIMS: To investigate the correlation of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations in patients with chronic hepatitis B. METHODS: HBV genotyping, nucleotide mutation, serum HBV DNA level and serological markers were analyzed in 121 patients with chronic HBV infection using INNO-LiPA HBV genotyping, polymerase chain reaction (PCR) product-based sequencing, fluorescence quantitative PCR and enzyme-linked immunosorbent assays respectively. RESULTS: Forty (33.0%), 77 (63.6%), two (1.7%) and two (1.7%) patients had genotypes B, C, B/C and D infections respectively. Significant differences were found in serum HBV DNA levels (log10 copies/ml: 6.18 vs. 5.61, P=0.042) and mutations at nucleotide (nt) 1762/1764 (71.4% vs. 42.5%, P=0.002) between genotypes C- and B-infected patients. There were significant differences in the mean age, serum biochemical parameter levels and mutation rates in BCP/PC among hepatitis e antigen (HBeAg)-positive and -negative chronic hepatitis B (CHB) and liver cirrhosis (LC) groups. CONCLUSION: Genotypes C and B are predominant in China, and the frequent nt 1762/1764 mutation, which occurs commonly in HBeAg-negative CHB, especially in genotype C patients, may be associated with the progress of chronic HBV infection.

Detection of HEV antigen as a novel marker for the diagnosis of hepatitis E.

Infection with hepatitis E virus (HEV) may be diagnosed by the presence of HEV RNA or anti-HEV antibodies. An enzyme immunoassay (EIA) was developed for the detection of antigen. Twenty-four monoclonal antibodies (mAbs) were produced. An indirect sandwich EIA was developed to detect HEV antigen using a combination of three mAbs as coating antibodies. Approximately 44.6% (33/74), 28.6% (50/175), and none (0/27) of sera positive for anti-HEV IgM alone, both anti-HEV IgM and IgG, and anti-HEV IgG alone also were positive for HEV antigen using this EIA. Forty-two HEV antibody-positive sera were tested for HEV RNA and antigen in parallel and the concordance was 81.0% (34/42). All PCR products were found to belong to HEV genotype 4. In order to evaluate the temporal relationship between HEV antigen positivity and HEV RNA, anti-HEV IgG and IgM, and ALT concentrations, macaques were infected with HEV genotypes 1 and 4 and serial samples were collected. The results showed that the antigen EIA can detect the capsid proteins of both genotypes. HEV antigen was detectable prior to ALT elevation and the appearance of anti-HEV antibodies in the infected monkeys and lasted for several weeks in all cases. HEV antigen became detectable in the serum at almost the same time as HEV RNA in feces but persisted for 4 weeks less than HEV RNA. This assay should be valuable for the diagnosis of acute hepatitis E, particularly in the window period prior to seroconversion to anti-HEV.

[Expression of HBV S and preS1 fusion gene in Pichia pastoris expression system].

BACKGROUND: To clone and express the ss1 recombinant gene containing S gene and preS1 (10-50 AA) gene in P. pastoris expression system. METHODS: The fusion gene ss1 containing the S (1-222 AA) gene and preS1 (10-50 AA) gene was constructed with PCR method. The fusion ss1 gene was cloned into the expression vector of pPIC3.5k. The linear vector DNA was transformed into the host cell of GS115 with electroporation method. After screening with G418, the product was induced to express with methanol and its antigenicity was analyzed. RESULTS: The molecular weight of expressed ss1 protein was about 30,000 dalton. The product was reactive to anti-HBs and anti-preS1 mAb. CONCLUSION: The fusion gene was efficiently expressed in P. pastoris expression system.The expressed products have the antigenicity of both S and preS1 protein.

[Genomic sequence of hepatitis A virus L-A-1 vaccine strain].

OBJECTIVE: To study the genome sequence of hepatitis A virus L-A-1 strain which has been applied for live attenuated vaccine production in China, to compare with other HAV strains, to understand some characteristics of L-A-1 strain, and to find the mechanism of attenuation and cell adaptation. METHODS: Genome fragments were prepared by antigen-capture PCR from infected cell (2BS), PCR products were cloned into T vector, sequenced and analyzed by using bioinformatics program. RESULTS: Analysis of the genomic sequences(nt 25-7,418) showed that the open reading frame contains 6,675 nucleotides in length encoding 2,225 amino acids. Sequence homology comparison showed 98.00% and 94.00% homology at nucleotide level, and 98.51% and 98.65% homology at amino acid level with international strains MBB and HM 175, respectively. Through comparison with other attenuated, cell adapted and cytopathic effect (CPE) strains, L-A-1 strain had mutation at nt 152, 591, 646, 687 and insertion at nt 180-181 in 5?NTR and had mutation at nt 3,889 (aa 1 052-Val) in 2B region, these mutations and insertion are molecular basis for cell adaptation; mutation at nt 4,185 (aa 1 152-Lys) in 2C region should be attenuated marker; deletion in 3A region (nt 5,020-5,025) that caused two amino acids deletion is virus fast growth basis. CONCLUSION: Through analyzing L-A-1 strain genomic sequence, certain sites related to cell adaptation and attenuation were found.

[Human hepatitis B virus infection of tree shrews and Macaca assamensis in vivo].

OBJECTIVE: To examine sensitivity of the tree shrews and Macaca assamensis to human hepatitis B virus (HHBV) by serologic methods. METHODS: Totally 233 tree shrews and 28 Macaca assamensis were inoculated with human sera containing HBV. After inoculation, the sera were collected weekly from them and HBV markers were detected with HBV ditecting ELISA kits. RESULTS: Ninety percent of the tree shrews developed acute infection, among them, 44.4 % persisted for over one year, 33.3% of them developed chronic infection persisted for 2 years and one month; the persistence of HBV in Macaca assamensis was much shorter. CONCLUSION: These data clearly indicated that tree shrew may be used as an animal model for study of chronic HBV infection, whereas, Macaca assamensis, showed only a transient sensitivity to HHBV.

Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China.

Evidence that hepatitis E is zoonotic is accumulating. Serum samples were collected from pigs, cattle, and goats from various regions of China to determine whether they had been infected with hepatitis E virus (HEV). An in-house enzyme immunoassay (EIA) and reverse transcriptase-polymerase chain reaction (RT-PCR) with primers from open reading frame (ORF) 2 were used to detect anti-HEV antibodies and HEV RNA. The mean positivity rates of anti-HEV antibody for pigs and cattle were 78.8% and 6.3% but none of the goat sera were positive. Pigs may be more susceptible to infection with HEV than cattle or goats. Five of 263 pig sera were positive for HEV RNA and four of these five were also positive for anti-HEV. The PCR products (nt 6007-6354) were cloned and sequenced and compared to other HEV sequences in the nucleotide databases. The five sequences shared 83-93% identity to each other at the nucleotide level and 74-79%, 73-74%, 73-78%, and 83-99% identity to HEV genotypes 1, 2, 3, and 4, respectively. They were closely related to human isolates of HEV genotype 4. Phylogenetic analyses also place these swine sequences in HEV genotype 4, resembling most closely viruses isolated from Chinese patients with acute hepatitis. These data support the hypothesis that sporadic hepatitis E in China is zoonotic.

[Safety and immunogenicity of Bilive combined hepatitis A and B vaccine].

OBJECTIVE: To study the safety and immunogenicity of the Bilive combined hepatitis A and B vaccine produced by Sinovac Biotech Co., Ltd. METHODS: Samples were selected from first year students of a senior high school (adults group) and first to fifth grade 1-5 students of 3 primary schools (children group). Those who were susceptible to both hepatitis A virus (HAV) and hepatitis B virus (HBV), HAV only or HBV only were assigned to group AB, A and B respectively and were vaccinated with three doses (0, 1 and 6 month schedule) of Bilive combined hepatitis A and B vaccine, inactivated hepatitis A vaccine and recombined hepatitis B vaccine respectively. The dosage for adult group was 500 U hepatitis A antigen and/or 10 micro g hepatitis B surface antigen and the dosage for children group was half the dosage of adult group. The potential adverse effects were observed within 72 hours after vaccination. Serum samples were collected for testing anti-HAV and anti-HBs at month 2 and 7 after the initial dose. RESULTS: The rates of local adverse effects were 0.58% and 2.56% in children AB group and adults AB group and the general adverse effects rates were 9.88% and 5.45% respectively. Both local and general adverse effect rates were not significantly different to the control group. The sero-conversion rate of anti-HAV in children and adults AB group reached 100%, one month after 3 doses. The geometric mean titer (GMTs) reached 33,910 mIU/ml and 23,435 mIU/ml respectively, significant higher than that in control group (group A). The sero-conversion rates of anti-HBs were 97.30% and 96.63%, and GMTs were 103 mIU/ml and 102 mIU/ml in children and adults AB group respectively. No significant difference on sero-conversion and GMT was observed when compared with control group. CONCLUSION: The Bilive combined hepatitis A and B vaccine had good safety profile, and the immunogenicity both on anti-HAV and anti-HBs was similar to that of separated components.