An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T₀ transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T₁ progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.     

Antibody-Induced Lysis of Isolated Rat Epididymal Adipocytes and Complement Activation In Vivo

Objective: To identify human monoclonal antibodies selectively binding to human adipocytes and to evaluate their ability to induce lysis of isolated rat adipocytes in vitro and to reduce rat complement levels in vivo. Research Methods and Procedures: Using phage display technology, human monoclonal antibodies binding to human adipocyte plasma membranes were identified. Three antibodies (Fat 13, Fat 37, and Fat 41) were selected based on their additional cross-reaction with rat adipocytes and reformatted as a rat chimeric IgG2bs. The ability of these antibodies, both singly and in combination, to induce lysis of rat epididymal adipocytes in vitro and the reduction of serum complement levels in vivo in the rat was evaluated. Results: All antibodies caused similar time- and dose-dependent lysis of isolated rat adipocytes. Calculated mean EC₅₀ values (maximum percentage of lysis in parentheses) were 0.680 μg/mL (63.2%), 0.546 μg/mL (72.4%), and 0.391 μg/mL (73.7%) for Fat 13, Fat 37, and Fat 41, respectively. Combinations were no more effective than individual antibodies in inducing lysis. Anti-adipocyte antibodies (both singly and in combination) were also similarly effective in vivo. In rats, doses of monoclonal antibody up to 10 mg/kg intraperitoneal generally caused almost complete depletion of serum complement up to 24 hours after dosing recovering to baseline values by day 5. Discussion: Individual and combinations of monoclonal anti-adipocyte antibodies produced a complement-dependent and concentration-dependent activity to lyse adipocytes in vitro and in vivo as measured by a dramatic depletion in serum complement.     

Eastern equine encephalomyelitis virus in experimentally infected bats.

Colonial bats (Myotis supp. and Eptesicus sp.) were infected with eastern equine encephalomyelitis virus by subcutaneous inoculation or by the bite of infected mosquitoes. Bats were maintained in an environment simulating conditions encountered in hibernacula or in summer maternal colonies. Virus was detected in the blood of hibernating bats at irregular intervals over a 42-day observation period; viremia perhaps was influenced by the amount of disturbance (arousal) involved in the blood sampling process. Target organs included brown fat, spleen, lung, kidneys, pancreas, and liver. Neutralizing antibody was not detected in sera collected from these bats between days 4 and 42 post-inoculation. In nonhibernating bats, virus was recovered from mammary glands, brown fat, pancreas, lungs, kidneys, and liver, in addition to blood. Attempts to infect bats orally or to transmit virus to suckling mice by the bite of viremic bats were unsuccessful. Virus was transmitted from viremic chickens to E. fuscus by the bite of Culiseta melanura and Aedes aegypti.     

Effect of sex, size, and age of commensal rat hosts on the infestation parameters of their ectoparasites in a rural area of Egypt.

The present study was carried out in the Bilbeis area, Sharqiya Governorate, Egypt. A total of 127 male and 115 female black rats Rattus rattus and 124 male and 153 female Norway rats R. norvegicus was collected during the study period. A total of 20,643 and 40,997 ectoparasites was recovered from R. rattus and R. norvegicus, respectively. Three common mite species were recovered from both rat hosts: Ornithonyssus bacoti, Radfordia ensifera, and Laelaps nuttalli. Three common flea species were also recovered from both rat hosts: Echidnophaga gallinacea, Leptopsylla segnis, and Xenopsylla cheopis. Polyplax spinulosa was the only louse species that infested both rat hosts. Rats that lived more than 12 mo formed about 24 and 14% of the populations of the black and Norway rats, respectively. The prevalences and general indices of many ectoparasites were significantly higher on males than on females of both rat hosts. Seasonal differences in prevalences and general indices of many ectoparasites were recorded between the 2 sexes of both rat species. Prevalences and general indices of most ectoparasites on both rat species showed a tendency to increase with increasing size and age of rat hosts. The type and degree of correlation between prevalences and general indices on the one hand and each of body size and age on the other hand differed among the species of ectoparasites.     

Use of Clq enzyme-linked immunosorbent assay to detect plant viruses and their serologically different strains

Clq was prepared from bovine serum using a simple method involving repeated dialysis at low ionic strength in the presence of chelating agents (yield c. 3 mg/100 ml serum). It was viable when stored at -18°C for up to 2 months, and at 4°C for at least 10 wk in a storage buffer containing 10% sucrose. When used in Clq ELISA this test was as sensitive as the direct double antibody sandwich form of ELISA (direct ELISA) in detecting purified potato virus Y (PVY), with a limit of detection in both methods of c. 15 ng/ml, and slightly more sensitive in detecting purified cocksfoot mild mosaic virus (CMMV), with limits of detection of c. 15 ng/ml and c. 15–60 ng/ml respectively. Using an antiserum to one strain of each virus, Clq ELISA readily detected strains of PVY, CMMV, Andean potato latent virus (APLV) and barley yellow dwarf virus (BYDV). This included detection of APLV-Hu by APLV-Caj antibodies and CMMV(G) by PMV(S) antibodies, neither of which system gives detection in direct ELISA. Clq ELISA was therefore less specific than direct ELISA in detecting serologically different virus strains. Virus detection by Clq ELISA was inhibited when sap of tobacco, Nicotiana clevelandii and Setaria italica was used at low dilution. Inhibition by N. clevelandii sap was alleviated by using increased concentrations of virus specific antibody to detect APLV and plum pox virus. Also, extracting APLV infective N. clevelandii or CMMV infective S. italica saps in a minimum of buffer, centrifuging at low speed and diluting the supernatant before testing, partially overcame the inhibition. The inhibitory substance(s) in sap may act by preventing the binding of Clq to virus-antibody aggregates. Sap of wheat, oat and barley did not appear to have an inhibitory effect and BYDV was readily detected in naturally infected field grown plants of these species.     

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.     

Insulin-like growth factor I (IGF-I) and IGF-binding protein 3 as diagnostic markers of growth hormone deficiency in infancy

BACKGROUND: The diagnosis of growth hormone deficiency (GHD) in infancy is difficult, and no specific cutoff value during GH provocative testing is recommended in early life. METHODS: Serum insulin-like growth factor I (IGF-I) and serum IGF-binding protein 3 (IGFBP-3) levels were evaluated as diagnostic markers of GHD. Measurements of IGF-I and IGFBP-3 during the 1st year of life were analyzed in 11 patients clinically suspected of having GHD (neonatal hypoglycemia, micropenis, or evidence of other pituitary hormone deficiencies), in whom the diagnosis was later verified. A prospective cohort of 51 healthy infants served as controls. RESULTS: The sensitivity of IGF-I as a diagnostic marker of GHD was 90% (10 out of 11 patients) with a cutoff value of -2 standard deviations (SD), and the sensitivity of IGFBP-3 measurements was 81% (9 out of 11 patients) with a cutoff value below -2 SD. One patient had serial measurements before initiation of GH treatment where the IGF-I was fluctuating (3 of 6 slightly above -2 SD), whereas all IGFBP-3 measurements were below -2 SD. CONCLUSIONS: The IGF-I had a high sensitivity in detecting infants with GHD. The combination of IGF-I and IGFBP-3 increased the diagnostic sensitivity. We speculate that assessment of IGF-I and IGFBP-3 may add diagnostic value in infants suspected of having GHD and furthermore that values below -2 SD are highly suggestive of GHD.