Occurrence and distribution of rhizobiophages in Indian soils.

Soil samples from 30 fields in neighbouring districts of Varanasi were screened for rhizobiophages on 60 Rhizobium strains. Plaques were observed on five strains: P1, P5, SU391 (R. leguminosarum), CB756 and 32H1 (Rhizobium sp.). Rhizobiophages infective on one or more of the five strains were present in all fields. There seems to be no correlation between presence of phage and the standing crop or the pH (7.1-8.2) of the soil. Eight distinct rhizobiophages have been isolated and characterized for host specificity, plaque morphology and maximum titer in broth.     

Current focus of stem cell application in retinal repair

The relevance of retinal diseases, both in society’s economy and in the quality of people’s life who suffer with them, has made stem cell therapy an interesting topic forresearch. Embryonic stem cells(ESCs), induced pluripotent stem cells(i PSCs) and adipose derived mesenchymal stem cells(ADMSCs) are the focus in current endeavors as a source of different retinal cells, such as photoreceptors and retinal pigment epithelial cells. The aim is to apply them for cell replacement as an option for treating retinal diseases which so far are untreatable in their advanced stage. ESCs, despite the great potential for differentiation, have the dangerous risk of teratoma formation as well as ethical issues, which must be resolved before starting a clinical trial. i PSCs, like ESCs, are able to differentiate in to several types of retinal cells. However, the process to get them for personalized cell therapy has a high cost in terms of time and money. Researchers are working to resolve this since i PSCs seem to be a realistic option for treating retinal diseases. ADMSCs have the advantage that the procedures to obtain them are easier. Despite advancements in stem cell application, there are still several challenges that need to be overcome before transferring the research results to clinical application. This paper reviews recent research achievements of the applications of these three types of stem cells as well as clinical trials currently based on them.     

Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.DNA double-strand breaks (DSBs) are the most deleterious to the genome among various lesions. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways in higher eukaryotes.^{1, 2, 3} In the absence of key NHEJ factors, another distinct but error-prone pathway known as alternative NHEJ (A-NHEJ) has been described to have an important role in DSB repair.^{4, 5, 6, 7} It has been shown that majority of A-NHEJ-mediated repair of DSBs utilize distinct microhomology regions, hence termed microhomology-mediated end joining (MMEJ).^{4, 8, 9}A-NHEJ has been proposed as a possible cause for chromosomal translocations. Studies have shown co-amplification of c-MYC and IgH locus from pro-B lymphomas in mice deficient for p53 and NHEJ.¹⁰ A reduced level of class switch recombination (CSR) and increased number of chromosomal rearrangements at IgH locus have been shown in XRCC4- and LIGASE IV-deficient murine B cells.⁸ The occurrence of robust alternative end joining has been reported in the absence of NHEJ proteins, when murine RAG proteins were absent.¹¹Unraveling the enzymatic machinery involved in alternative end joining is currently an active area of research. Recently, it was shown that MRE11-RAD50-NBS1 complex may be involved in a subset of alternative NHEJ,^{5, 12, 13, 14} whereas ATM has a regulatory role.¹⁵ Role of PARP1 in repairing switch regions through a microhomology-mediated pathway leading to IgH/c-MYC translocations during immunoglobulin CSR has been described.¹⁶ Besides, studies have also suggested a role for DNA LIGASE IIIα and WRN in A-NHEJ.¹⁷ Interestingly, XRCC1 was shown to be dispensable in A-NHEJ during CSR, whereas functional relevance of Ligase I, III and Pol λ have been established.^{18, 19, 20} Hence, it can be concluded that canonical NHEJ (C-NHEJ) requires LIGASE IV–XRCC4 complex, while A-NHEJ is predominant in the absence of C-NHEJ proteins and is mainly characterized by joining utilizing microhomology (MMEJ). Further, it has been demonstrated that RPA, when bound to single-stranded DNA can antagonize MMEJ.²¹ Very recently, a genetic system was reported in budding yeast to detect microhomology-mediated repair.²² However, little is known whether alternative NHEJ can be operative when classical NHEJ machinery is intact.²³ A recent study suggested that MMEJ is also functional in normal mammalian cells. Besides, HR and MMEJ share the initial steps of end resection for DSB repair in mammalian cells.²⁴ However, it appears that there is not much consensus among different research groups over its presence and relevance in normal cells.²³ Therefore, several aspects of alternative NHEJ still need to be resolved. For example, its precise mechanism and microhomology length requirements are yet to be fully uncovered. Its occurrence in normal cells needs to be proved beyond doubt. Although there are independent studies showing the role of multiple proteins using gene knockdown or knockout strategies, their involvement needs to be confirmed.In the present study, we have established a cell-free repair assay system using which we show that MMEJ is operative even in the presence of classical NHEJ machinery. Further, our data suggest that MMEJ operates not only in cancer cells but also in normal cells. We show that a minimum of 5 nt microhomology is required for MMEJ and is independent of classical NHEJ proteins such as KU70, KU80 and LIGASE IV. Finally, we show that MRN complex, XRCC1, FEN1, PARP1 and LIGASE III are the factors responsible for joining mediated through microhomology.     

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.     

Establishment and maintenance of an axenic culture of <Emphasis Type="Italic">Ettlia</Emphasis> sp. using a species-specific approach

The establishment of an axenic culture of microalgae is essential step in understanding its physiology, genetics, and ecology. However, culturing of microalgae is usually accompanied by complex and variable associated prokaryotic and eukaryotic microorganisms. Conventional approaches used for obtaining axenic cultures of microalgae are time-consuming and often involve difficulties in maintaining and preserving axenicity. In this study, we developed a procedure for establishing an axenic culture of Ettlia sp. YC001 and demonstrate that we maintained the axenic culture through subculture in the long term. Three sequential treatments, an antibiotic cocktail, serial dilution, and plate spreading, were applied to strain YC001 and we confirmed axenicity using molecular and physiological methods. The bacterial community associated with strain YC001 was investigated to select antibiotics for their specific elimination. The xenic culture (1 × 10⁶ cells/mL) was treated with the antibiotic cocktail-5 (AC-5), carbendazim, chloramphenicol, imipenem, rifampicin, and tetracycline for 3 days, followed by serial dilution up to 1 × 102 cells and spreading on agar plates. The pure colonies were analyzed using denaturing gradient gel electrophoresis (DGGE), fluorescence-activated cell sorting (FACS), and scanning electron microscopy (SEM). The procedure we developed can be applied to other strains of microalgae for the establishment of axenic cultures.