Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA₃ within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.     

Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in <Emphasis Type="Italic">Withania coagulans</Emphasis> (Stocks) Dunal

A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 μM kinetin (Kn) and lower levels of 6–benzyladenine (BA) (13.3 μM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3 μM kinetin (Kn) and higher levels of BA (22.2 μM). Shoot buds were transferred to proliferation medium containing 2.2 μM BA, 2.3 μM Kn, and 3.9 μM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 μM choline chloride (CC) and 3.9 μM PG and then transferred to rooting medium containing ½ MS, 1.2 μM IBA, 3.6 μM PAA, and 14.3 μM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.     

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Influence of aging in the thermoregulatory efficiency of man

Studies were carried out to evaluate the effect of aging on the thermoregulatory efficiency in man. The efficiency of thermoregulatory system was assessed on the basis of tolerance to acute cold stress, Tromp test and cold induced vasodilatation (CIVD) response. The experiments were done in 9 groups (15 each) of human volunteers in the age ranges of 20–25, 26–30, 31–35, 36–40, 41–45, 46–50, 51–55, 56–60 and 61–70 years. They were made to relax in a thermoneutral laboratory (27 ± 1C) for one hour; thereafter their resting heart rate, blood pressure, oxygen consumption, oral, mean skin, mean body and peripheral temperatures were measured. Then they were exposed to cold (10C) in a cold chamber wearing shorts and vests for two hours. The above parameters and shivering responses were recorded at 30 min intervals during cold exposures. On other occasions, their thermoregulatory efficiency test (Tromp Test) and cold induced vasodilatation response were also studied in the same thermoneutral laboratory. The results showed comparatively poor cold tolerance and thermoregulatory efficiency in elderly people. The middle aged category (above 40 years) showed a gradual reduction in the thermoregulatory efficiency, but highly significant reduction was noticed in those who exceeded sixty years of age.     

Seasonal variations in the survival index of rats at simulated high altitudes

Albino rats were maintained in an animal house, the air temperature of which fluctuated with the variations in the ambient air temperature Survival index, which is defined as the percentage of rats surviving for 100 min at a given simulated altitude, was determined for several groups of rats in different months covering a period of about one year. The simulated altitudes used were 25,000,27,500, 30,000 and 32,500 ft, with an air temperature of 33°C and RH of over 80%,within the decompression chamber. The survival index showed a progressive decline with the fall in mean air temperature of 32°C in summer to 13°C in winter, and a rise with increase in air temperature during the summer of the succeeding year. In a group of rats removed from an environment at 28°C to one at 19°C a significant fall in survival index was observed at the end of 4 weeks, but not at the end of 12 weeks. It is concluded that exposure to a mild degree of cold reduces the ability of rats to survive hypoxia between 10 and 30 days of cold exposure,,which tends to revert to normal between 30 and 90 days.

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Zusammenfassung Ratten wurden in einem Tierhaus gehalten, dessen Innentemperatur mit der Aussentemperatur schwankte.Der Überlebens-Index das ist der Prozentsatz Ratten, die 100 min bei einer gegebenen simulierten Höhe überleben, wurde für mehrere Gruppen Ratten in verschiedenen Perioden während eines Jahres bestimmt. Die Höhen betrugen 7,600 m, 8,300 m, 9,100 m und 9,850 m bei 33°C und 80% RF.Der Überlebens-Index zeigte eine progressive Senkung beim Fall der Temperatur von 32°C im Sommer auf 13°C im Winter und eine Zunahme beim Wiederanstieg der Temperatur im Sommer des folgenden Jahres. Ratten, die von 28°C in einen Raum von 19°C überführt wurden,wiesen nach 4 Wochen eine siknifikante Verminderung des Überlebens-Index auf, dagegen nicht mehr nach 12 Wochen. Danach ist bereits bei milder Kälteexponierung die Hypoxie-resistenz der Ratte näch 10–30 Tagen in der Kälte reduziert und normalisiert sich wieder zwischen 30 und 90 Tagen.

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Resume Des rats ont été placés dans des cages dont la température variait avec celle de l'air ambiant. On détermina alors l'indice de survie pour plusieurs groupes de ces animaux et pour différentes périodes couvrant presque une année entière. Cet indice est défini par le taux de rats qui restent en vie après avoir été exposés durant 100 min à des altitudes simulées déterminées et cela par 33°C et 80% d'humidité relative à l'intérieur de la chambre de décompression.Ces altitudes étaient de 7.600m, 8.300 m, 9.100 m et 9.850 m. L'indice de survie diminua progressivement lors d'un abaissement de la température ambiante aux cages de 32°C en été à 13°C enhiver pour remonter à nouveau avec la température l'été de l'année suivante.Des rats qui furent transférés d'un milieu de 28°C dans un autre de 19°C présentèrent un abaissement significatif de leur indice de survie après 4 semaines.Cet abaissement avait cependant disparu après 12 semaines. On en conclut qu'une exposition de 10 à 30 jours à un refroidissement modére réduit de façon évidente la capacité de résistance à l'hypoxie des rats, mais que la dite capacité de résistance se normalise de nouveau entre 30 et 90 jours après.

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A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.