Biophysics of V(D)J recombination and Genome Packaging: In singulo studies on RAG, HMGB1, and TFAM

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.

Temperature-and pH-Sensitive Nanohydrogels of Poly(N-Isopropylacrylamide) for Food Packaging Applications: Modelling the Swelling-Collapse Behaviour

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPA) nanohydrogels were synthesized by nanoemulsion polymerization in water-in-oil systems. Several cross-linking degrees and the incorporation of acrylic acid as comonomer at different concentrations were tested to produce nanohydrogels with a wide range of properties. The physicochemical properties of PNIPA nanohydrogels, and their relationship with the swelling-collapse behaviour, were studied to evaluate the suitability of PNIPA nanoparticles as smart delivery systems (for active packaging). The swelling-collapse transition was analyzed by the change in the optical properties of PNIPA nanohydrogels using ultraviolet-visible spectroscopy. The thermodynamic parameters associated with the nanohydrogels collapse were calculated using a mathematical approach based on the van't Hoff analysis, assuming a two-state equilibrium (swollen to collapsed). A mathematical model is proposed to predict both the thermally induced collapse, and the collapse induced by the simultaneous action of two factors (temperature and pH, or temperature and organic solvent concentration). Finally, van't Hoff analysis was compared with differential scanning calorimetry. The results obtained allow us to solve the problem of determining the molecular weight of the structural repeating unit in cross-linked NIPA polymers, which, as we show, can be estimated from the ratio of the molar heat capacity (obtained from the van't Hoff analysis) to the specific heat capacity (obtained from calorimetric measurements).

Studies on packaging of fresh fish

Studies conducted in the laboratory and field have shown that iced fish can be preserved for longer periods in fresh and edible condition in conventional bamboo baskets by providing additional insulated linings of double layer of gunny and polythene or bitumen coated kraft paper. The quality of fish is adjudged by chemical and organoleptic evaluations.

Studies on transportation of fish. Pt. 1. Packaging of frozen fish in insulated containers

The paper deals with certain technological aspects of transportation of fish. Frozen fish packed in thermocole insulated plywood box (tea chests) and transported has been found to remain in good condition, for 3 to 4 days. Fish reaching the destination in a thawed state can be stored in crushed ice for a further period of 2-3 days. The effect of initial temperature of frozen fish on the storage life, the maximum storage period for different types of frozen fish packed in the insulated container and the changes in chemical and organoleptic qualities of different varieties of fish under condition of transport are also discussed.

A packaging technique for silicon MEMS strain sensors on steel

A packaging technique suited to applying MEMS strain sensors realized on a silicon chip to a steel flat surface is described. The method is based on adhesive bonding of the silicon chip rear surface on steel using two types of glue normally used for standard piezoresistive strain sensors (Mbond200/ 600), using direct wire bonding of the chip to a Printed Circuit Board, also fixed on steel. In order to protect the sensor from the external environment, and to improve the MEMS performance, the silicon chip is encapsulated with a metal cap hermetically sealed-off under vacuum condition with a vacuum adhesive in which the bonding wires are also protected from possible damage. In order to evaluate the mechanical coupling of the silicon chip with the bar and thestress transfer extent to the silicon surface, commercial strain sensors have been applied on the chip glued on a steel bar in alaboratory setup able to generate strain by inflection, yielding a stress transfer around 70% from steel to silicon. © 2008 IEEE.

Studies on radiation pasteurisation of medium fatty fish 1. Control of radiation induced oxidative changes in white pomfret (Stromateus cinereus) by vacuum packaging

Marked differences were observed in proximate biochemical compositions of the skin and muscle of white pomfret. The skin showed comparatively higher content of extractable lipids and was more susceptible to radiation-induced oxidative changes like development of rancid odours and yellow discolouration than the muscle. Irradiation of skin samples under vacuum suppressed these changes. The present paper also reports on the efficacy of vacuum packaging in controlling oxidative rancidity and yellow discolouration in white pomfret skin subjected to irradiation and subsequent storage at 0-2°C.

Packaging and storage studies of dried lizard fish

Packaging and storage studies of salted and dried lizard fish (Saurida sp.) have been conducted using different synthetic films like low density polyethylene (LDPE) of different gauges, high density polyethylene (HDPE) of 200 gauge, polyvinylidene chloride (PVDC) coated 400 MXXT cellophane, 100 gauge polypropylene (PP) and paper laminate of 100 gauge polythene. The films found most effective in the preliminary studies were subsequently used for packaging and storage of dried fish at atmospheric and lower temperature and humidity conditions for confirming their suitability under these conditions. Polyethylene films of higher gauges showed better results under both sets of conditions. PVDC coated cellophane film also performed satisfactorily under the latter conditions which under the former condition got easily attacked by insects. Lower temperature and humidity conditions in general enhanced the storage life of the dried product.

Use of linear low density polythene film for frozen fish packaging

Physical properties of linear low density polythene film of 60 gauge thickness and its suitability as packaging material in frozen fish industry was studied in comparison with conventional 100 gauge low density polythene film. Both were comparable in water vapor transmission rate both at 38°C and -20°C, overall migration test and :flexibility at low temperatures. ·Even though the tearing strength was inferior to the conventional one, it is of no significance in its use in frozen fish industry. The new film worked out to be more economical than the other one.