Somatic embryogenesis inCicer arietinum L: Influence of genotype and auxins

Plant regeneration through somatic embryogenesis was obtained in chickpea (Cicer arietinum L.) using immature cotyledons and immature embryonal axes as explants. 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3.5-6-trichloropicolinic acid (picloram) and 3.6-dichloro-0-anisc, acid (dicamba) in concentrations 1, 2, 5, 10 mg dm^?3 were found better than naphthaleneacetic acid (NAA) (10–20 mg dm^?3) for the induction of globular and heart-shaped somatic embryos. The embryos developed upto the dicotyledonary stage on medium supplemented with saccharose, mannitol and silver nitrate (AgNO₃) and developed further into plantlets on medium containing gibberellic acid (GA₃) and abscisic acid (ABA). The frequency of somatic embryogenesis was dependent on the genotype and auxins used.     

Two NAD-dependent alcohol dehydrogenases (E.C. 1.1.1.1) in callus cultures of wheat,rye and triticale

Summary Two NAD-dependent alcohol dehydrogenases ADH-1 and ADH-2, under independent genetic control of genes designated as Adh-1 and Adh-2 located on chromosomes 4A, 4B and 4D, have been reported in aestivum wheat (Hart 1980). Only ADH-1 is expressed in developing seeds, dry seeds, pollen and germinating seedlings. ADH-2 can be induced in seedling roots or shoots under conditions of partial anaerobiosis or by certain chemicals. Expression of ADH-1 and ADH-2 isoenzymes was investigated in undifferentiated calli from aestivum and durum wheats, rye, triticale and also in in vitro regenerated roots and leaves from aestivum cultures. Wheat callus cultures originating from seed, mature and immature embryos, mesocotyl and root, as well as cultures grown on media containing different supplements did not show any variation in the overall expression of ADH-1 or ADH-2, although differences in the band intensities were observed. The callus isoenzyme pattern was similar to that observed in roots under anaerobic conditions. Both ADH-1 and ADH-2 were expressed in in vitro regenerated roots but were absent in regenerated leaves. Expression of ADH-1 and ADH-2 in wheat calli seems to be related to the type of differentiation.     

Callus induction and plant regeneration from immature embryos of rye and triticale

Callus cultures were established from the scutellum, scutellar node and radicle region of immature embryos of rye and octoploid triticale on modified Murashige-Skoog basal medium supplemented with various growth regulators. 2, 4-D, 2, 4, 5-T and 2, 4, 5-Cl, POP were found suitable for initiation and maintenance of callus cultures. Cytokinins had no or inhibitory effect on callus induction and growth. On basal medium containing 5 mg/l of 2,4,5-Cl₃ POP, 16% of triticale and 17% of rye primary cultures exhibited shoot bud regeneration after 3–4 weeks. Transfer of such cultures to basal medium supplemented with zeatin or zeatin in combination with IAA further promoted shoot elongation and plantlet formation. Plantlets were rooted on basal medium containing 1 mg/l NAA and were eventually transferred to soil. Chlorophyll variants were observed in about 6% of triticale cultures.     

Kinetic studies on the diaqua form of cis-platin and various nucleobases

Kinetic studies on cis-[Pt(NH3)2(OH2)2]2+ and various nucleobases show that this ion reacts more quickly with guanosine than with adenosine, cytidine, and thymidine, and that a monophosphoric acid unit considerably enhances the rate of reaction of guanosine; the kinetic preference of 5'-GMP over 5'-AMP may point to a greater thermodynamic selectivity.     

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct.MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations.ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R² >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations).ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.     

DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression

Budding yeast cells suffering a single unrepaired DNA double-strand break (DSB) trigger the ATR (Mec1)-dependent DNA damage checkpoint and arrest prior to anaphase for 12–15 h, following which they adapt and resume cell division. When the DNA lesion can be repaired, the checkpoint is extinguished and cells “recover” and resume mitosis. In this autophagic punctum, we report that hyperactivation of autophagy—specifically via the cytoplasm-to-vacuole targeting (Cvt) pathway—prevents both adaptation to, and recovery from, DNA damage, resulting in the permanent arrest of cells in G₂/M. We show that Saccharomyces cerevisiae deleted for genes encoding the Golgi-associated retrograde protein transport (GARP) complex are both adaptation- and recovery-defective. GARP mutants such as vps51Δ exhibit mislocalization of the key mitotic regulator, securin (Pds1), and its degradation by the vacuolar protease Prb1. In addition, separase (Esp1), is excluded from the nucleus, accounting for pre-anaphase arrest. Pds1 is degraded via the Cvt pathway. Many of the same defects seen by deleting GARP genes can be mimicked by hyperactivation of the Cvt pathway by overexpressing an unphosphorylatable form of ATG13 or by adding the TORC1 inhibitor rapamycin. These results suggest that nuclear events such as DNA damage can have profound effects on cytoplasmic processes and further expand the burgeoning connections between DNA damage and autophagy.     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Thidiazuron-induced shoot regeneration in pigeonpea (Cajanus cajan L.)

Thidiazuron either alone or in combination with IAA induced high frequency shoot regeneration from primary leaf segments of three pigeonpea cultivars. Transfer of the cultures to medium with reduced concentration of thidiazuron resulted in further development of the shoots. The regenerated shoots were subsequently transferred to medium supplemented with BA, IAA and gibberellic acid where 5-10% of the shoots elongated further. Rooting of shoots could be obtained on half strength MS medium supplemented with NAA. Histological studies confirmed the mode of regeneration as shoot organogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.