A new chemorheological analysis of highly filled thermosets used in integrated circuit packaging

Chemorheology (and thus process modeling) of highly filled thermosets used in integrated circuit (IC) packaging has been complicated by their highly filled nature, fast kinetics of curing, and viscoelastic nature. This article summarizes a more thorough chemorheological analysis of a typical IC packaging thermoset material, including novel isothermal and nonisothermal multiwave parallel-plate chemorheology. This new chemorheological analysis may be used to optimize existing and design new IC packaging processes. (C) 1997 John Wiley & Sons, Inc.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1

Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (or) and an E. coli on were introduced into Cos-1 cells by DNA transfection. PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coil as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1+L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1+L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BW sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BW VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb. (C) 1998 Academic Press.

STD syndrome packets: Improving syndromic management of sexually transmitted diseases in developing countries

Objective: To design, introduce, and evaluate STD syndrome packets containing recommended drugs for each syndrome, four condoms, a partner treatment card, and a patient information leaflet, with the goal of improving sexually transmitted disease (STD) case management. Methods: Packet design evolved around available packaging technology, informed by pilot testing with nurses working in primary care clinics, doctors in private medical practices, and patients with an STD, in Hlabisa, South Africa. Evaluation 1 year later included analysis of distribution records and interviews with 16 nurses and 61 patients. Results: A cheap packet (2 U, S, cents each, excluding contents) compatible with current legislation was designed and introduced to six public sector clinics and as a short pilot to five private medical practices, Four thousand eighty-five packets were distributed to the clinics, equivalent to approximately 115% of the STDs reported over that period. All 16 nurses reported using the packets, but only 63% did so all the time because of occasional supply problems, All believed the packets improved treatment by saving time (75%), improving supply of condoms and partner cards (44%), and making treatment easier (56%), Patients also responded positively, and most said they would buy a packet (up to $5) at a pharmacy (84%) or store (63%) if available. Conclusions: The STD syndrome packets have the potential to improve STD syndromic management by standardizing therapy and improving the supply of condoms, partner cards, and information leaflets. Packets are popular with practitioners and patients, but consistent supply is essential for maximal impact, There may be scope for social marketing of the packets, which could further increase use.

Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican

BACKGROUND. Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS. Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS. The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65-95 kDa. The KS moiety was susceptible to digestion with keratanase 11 and peptide N-glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS. The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation. (C) 2003 Wiley-Liss, Inc.

Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus

In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in PKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles, Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C, I. Nugent et al,, J, Virol, 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses, This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

The work of fracture in uniaxial and biaxial oriented unplasticised polyvinylchloride pipes

In oriented unplasticised polyvinylchloride (uPVC) pipes, cracks propagate tangentially rather than through the wall as in conventional pipe. Notched impact, a modified peel test and the specific work of fracture approach have been used to measure fracture toughness of a conventionally extruded, a uniaxially oriented and a biaxially oriented uPVC pipe in different directions. The different failure mode for the oriented pipes was found to result from an order of magnitude increase in the fracture toughness for cracks propagating perpendicular to the orientation direction. Differences in the fracture toughness between the oriented pipes were also related to their molecular orientation. (C) 2002 Elsevier Science Ltd. All rights reserved.

Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L(-/-) germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L(-/-) meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Effect of modified atmosphere packaging on the quality of minimally processed pineapple cv.'Smooth Cayenne' fruit

The effects of modified atmosphere (MA) conditions on the quality of minimally processed pineapple slices were determined. Commercial pineapple slice packs sealed with 40 pm thick polyester film were kept at 4.5 degrees C for 14 d. The oxygen transmission rate of the film was 23 ml m(-2) day(-1) atm(-1) (at 25 degrees C, 75% RH). In-built atmospheres and the quality of the products were determined. O-2 concentrations within the packs stabilised at 2%, while CO2 concentrations increased to 70% by day 14. The high CO2 level suggested an inappropriate lidding film permeability for the product, and hence affected its quality. Three batches of pineapple slices were packed in the laboratory using lidding films with oxygen transmission rate of 75, 2790 or 5000 ml m(-2) day(-1) atm(-1) (at 23 degrees C, 0% RH). Headspace atmospheres from laboratory-packed pineapple slices suggested an optimum equilibrium modified atmosphere of ca. 2% O-2 and 15% CO2. Respiration data from the laboratory-prepared packs were pooled together and used to develop a correlation model relating respiration rates to O-2 and CO2 concentrations. The model showed a decrease in respiration rate with decreasing O-2 and increasing CO2 concentrations. Respiration rate stabilised at 2% 02 and 10% CO2. The high concentrations of CO2 observed in the commercial packs did not fit the range in the respiration model. The model could aid in selection of MA conditions for minimally processed pineapple fruit.