Monitoring the biofertilizing potential and establishment of inoculated cyanobacteria in soil using physiological and molecular markers

This paper aims to develop methods for quantifying their establishment; using physiological activity (chlorophyll as a growth index and nitrogen-fixing potential as a measure of their biofertilizing capacity), along with evaluation based on DNA fingerprints generated using repeat sequences/palindromes. Time course studies were undertaken in liquid and soil microcosm experiments inoculated with a set of four rhizosphere cyanobacterial strains (BF1 Anabaena sp., BF2 Nostoc sp., BF3 Nostoc sp., BF4 Anabaena sp.). Observations revealed the synergistic effect of three-membered combinations (especially the i.e. BF1 + 2 + 3, 1 + 2 + 4, 1 + 3 + 4) in terms of enhancing chlorophyll and acetylene reducing activity. PCR-based amplification profiles (using short tandemly repetitive repeat (STRR) 1A, STRR_mod, and HIP_AT sequences) proved discriminative in monitoring the presence of the inoculated cyanobacteria in soil microcosm. Future work is in progress to assess the utility of the selected markers/primers in pot experiments, followed by field-level experiments with crop.     

Translational products of adipose tissue-derived mesenchymal stem cells: Bench to bedside applications

With developments in the field of tissue engineering and regenerative medicine, the use of biological products for the treatment of various disorders has come into the limelight among researchers and clinicians. Among all the available biological tissues, research and exploration of adipose tissue have become more robust. Adipose tissue engineering aims to develop by-products and their substitutes for their regenerative and immunomodulatory potential. The use of biodegradable scaffolds along with adipose tissue products has a major role in cellular growth, proliferation, and differentiation. Adipose tissue, apart from being the powerhouse of energy storage, also functions as the largest endocrine organ, with the release of various adipokines. The progenitor cells among the heterogeneous population in the adipose tissue are of paramount importance as they determine the capacity of regeneration of these tissues. The results of adipose-derived stemcell assisted fat grafting to provide numerous growth factors and adipokines that improve vasculogenesis, fat graft integration, and survival within the recipient tissue and promote the regeneration of tissue are promising. Adipose tissue gives rise to various by-products upon processing. This article highlights the significance and the usage of various adipose tissue by-products, their individual characteristics, and their clinical applications.     

Variation in heat stress-induced antioxidant enzyme activities among three mulberry cultivars

The effects of high temperature on antioxidant enzymes were investigatedin three mulberry (Morus alba L.) cultivars (cv. K-2, MR-2and BC2-59). High temperature was imposed by maintaining the plants at 40°Cfor 120, 240 and 360 min in an environmentalplant growth chamber.The activities of superoxide disumutase (SOD), catalase (CAT), guaiacolperoxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR)wereassayed in the leaf extracts of control and high temperature-treated plants.Antioxidant enzyme activities were high in all the mulberry cultivars inresponse to high temperature treatment. However, cv. BC2-59 showedsignificantlyhigher activities of all the five antioxidant enzymes in response to hightemperature compared to those from the leaves of K-2, and MR-2 mulberrycultivars. The present study suggested that the cv. BC2-59 has an efficientantioxidant system among the three cultivars, which could prevent the oxidativedamage in the leaves caused by high temperature stress.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.     

Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic- like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1⁺, Rab5⁺ early endosomes. It does appear in Rab4⁺ early/sorting endosome, but, in the time period examined, fails to reach Rab11⁺ recycling or Rab7⁺ late endosomes or lysosome-associated membrane protein-1⁺ lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.     

Gastrointestinal Delivery of Baculovirus Displaying Influenza Virus Hemagglutinin Protects Mice against Heterologous H5N1 Infection

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD₅₀) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.The recent outbreaks of H5N1 avian flu and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV) are clear indications of the urgent need for effective vaccines against influenza A viruses (31). Preventive and therapeutic measures against influenza A viruses have received much interest and effort globally to combat the current pandemic and to prevent such a situation in the future. Currently used vaccines for influenza are administered mainly parenterally and include live attenuated reassortant viruses, conventional inactivated whole viral antigens, or split-virus vaccines. Although some of these vaccines have proven to be quite effective, the manufacturing of these vaccines involves several technical and safety issues (21). Furthermore, the production of currently available influenza vaccines often requires high-level biocontainment facilities, an additional hurdle that limits the advancement of present vaccines.Vaccines containing purified recombinant viral proteins have recently gained special attention due to their ease of production without any safety concerns (25). Recombinant hemagglutinin (rHA) subunit vaccines produced in baculovirus-insect cell expression systems have been extensively tested and evaluated in humans (29, 30). Baculovirus-derived rHA subunit vaccines administered parenterally are safe and immunogenic in animals and humans. Along with its success in recombinant protein vaccines, baculovirus surface display technology allows us to present large complex proteins on the baculovirus envelope in its native antigenic conformation, resulting in good stability and a longer half-life in the host (18, 14, 8).Along with a suitable antigen, the route of administration of the vaccine has a profound effect in controlling mucosally acquired infections such as influenza. Vaccination via the mucosal route stimulates both systemic and mucosal immune responses (16). Oral and intranasal vaccines are the two main options for mucosal administration. Intranasal vaccines would have a detrimental effect on persons with asthma, reactive airway disease, and other chronic pulmonary or cardiovascular disorders (4). Oral vaccines therefore seem to be the safest alternative (13). Moreover, there is evidence to prove the ability of oral vaccination to prevent infection of the lungs (23) and cause transcytosis of the molecule across the cells into the circulation (24).In this report, we describe the construction of recombinant baculovirus under control of the immediate-early promoter 1 (ie1) derived from the white spot syndrome virus (WSSV) genome, which enables the expression of hemagglutinin at the early stage of infection in insect cells, thereby enhancing the display of HA on the baculovirus envelope. Incorporation of more HA into the budding baculovirus particles would improve their efficacy as immunogens. We have studied the efficacy of WSSV ie1-based baculovirus displaying hemagglutinin (BacHA) as an oral vaccine in a mouse model of infection. We have also assessed its efficacy with recombinant cholera toxin B (rCTB) as a mucosal adjuvant. This strategy will enable rapid production of prepandemic vaccines with minimal infrastructure around the world, alleviating the need for high-biosafety facilities, risky inactivation of virulent viruses, and meticulous protein purification procedures.     

An ICEBs1-like element may be associated with the extreme radiation and desiccation resistance of Bacillus pumilus SAFR-032 spores

Comparisons of the genomes of Bacillus pumilus SAFR-032 and the closely related type strain, B. pumilus ATCC7061^T, exposed an extended region of non-homologous genes. A detailed examination of this region revealed the presence of an ICEBs1-like integrative conjugative element in SAFR-032. A similar element was subsequently located elsewhere in the ATCC7061^T genome. A detailed comparison of these elements and the ICEBs1 of B. subtilis revealed extremely rapid flux in gene content, genome organization and sequence similarity. It is not clear if the B. pumilus elements as they are currently structured are functional. However, it is clear that the past involvement of these elements has brought multiple genes of unknown function to the SAFR-032 genome and these genes may be responsible for the rapid evolution that led to the extreme radiation and desiccation resistance of this organism’s spores.     

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.