Synchronized infection of cell cultures by magnetically controlled virus

To override the diffusion-limited adsorption step of viral infection, we magnetically synchronized cell attachment. Human immunodeficiency virus type 1-based lentivirus preparations were rendered magnetically reactive by association with magnetite nanoparticles, 50 nm in diameter. Application of a magnetic field resulted in immediate redistribution of the viral inoculum to the cell-associated state and completion of the productive adsorption process within 1 min. Independent of adsorption time, viral concentration, and diffusion rate, infection subsequently progressed by the receptor-mediated entry mechanism. Synchronization of this rate-limiting step of infection may now be applied to analyze isolated events in the viral replication sequence.     

Seasonality and seasons out of time--the thermoregulatory effects of light interference

The change in photoperiod is the main environmental cue for seasonal function of the reproductive, thermoregulatory, and immune systems in rodents existing outside of the tropics. In Israel, the social vole Microtus socialis breeds mainly under short photoperiod (SP) conditions. Previous studies showed that exposing voles to light interference (LI) in the field during the winter resulted in death. The aim of the current study was to determine the thermoregulatory response of SP-acclimated voles to LI. Therefore, heat production (VO2) at different ambient temperatures (Ta) and nonshivering thermogenesis (NST) were measured. Results show that LI of 15 min every 4h during the dark period significantly (p < 0.02) decreased VO2 at Ta = 15 degrees C and significantly (p < 0.05) decreased NST-capacity. These results can at least partly explain why LI-voles died during the winter under field conditions, through eliminating winter acclimatization of the thermoregulatory system, or what is considered as "seasons out of time."     

Approaching the CAPRI challenge with an efficient geometry-based docking

The last 3 rounds (3-5) of CAPRI included a wide range of docking targets. Several targets were especially challenging, since they involved large-scale movements and symmetric rearrangement, while others were based on homology models. We have approached the targets with a variety of geometry-based docking algorithms that include rigid docking, symmetric docking, and flexible docking with symmetry constraints. For all but 1 docking target, we were able to submit at least 1 acceptable quality prediction. Here, we detail for each target the prediction methods used and the specific biological data employed, and supply a retrospective analysis of the results. We highlight the advantages of our techniques, which efficiently exploit the geometric shape complementarity properties of the interaction. These enable them to run only few minutes on a standard PC even for flexible docking, thus proving their scalability toward computational genomic scale experiments. We also outline the major required enhancements, such as the introduction of side-chain position refinement and the introduction of flexibility for both docking partners.     

Increased concentration of polyvalent phospholipids in the adsorption domain of a charged protein

We studied the adsorption of a charged protein onto an oppositely charged membrane, composed of mobile phospholipids of differing valence, using a statistical-thermodynamical approach. A two-block model was employed, one block corresponding to the protein-affected region on the membrane, referred to as the adsorption domain, and the other to the unaffected remainder of the membrane. We calculated the protein-induced lipid rearrangement in the adsorption domain as arising from the interplay between the electrostatic interactions in the system and the mixing entropy of the lipids. Equating the electrochemical potentials of the lipids in the two blocks yields an expression for the relations among the various lipid fractions in the adsorption domain, indicating a sensitive dependence of lipid fraction on valence. This expression is a result of the two-block picture but does not depend on further details of the protein-membrane interaction. We subsequently calculated the lipid fractions themselves using the Poisson-Boltzmann theory. We examined the dependence of lipid enrichment, i.e., the ratio between the lipid fractions inside and outside the adsorption domain, on various parameters such as ionic strength and lipid valence. Maximum enrichment was found for lipid valence in the range between -3 and -4 in physiological conditions. Our results are in qualitative agreement with recent experimental studies on the interactions between peptides having a domain of basic residues and membranes containing a small fraction of the polyvalent phosphatidylinositol 4,5-bisphosphate (PIP2). This study provides theoretical support for the suggestion that proteins adsorbed onto membranes through a cluster of basic residues may sequester PIP2 and other polyvalent lipids.     

Polymyxin B and Related Cyclic Peptides Facilitate Leanness and Reduce Fat Mass and Triglyceride Content in Ageing Rats: Potential Prototype Drugs Against Obesity

Polymyxin B (PMXB) blocks the action of insulin on glucose uptake in vitro. In vivo, it reverses hypoglycemia induced by exogenous insulin. Here we have treated mature male rats daily with PMXB over a period of two weeks. This therapy has decreased body weight by 11%, adipose fat mass by 46% and triglyceride levels by 39%, with no indication of liver or kidney toxicity. Two suboptimal parameters, however, were a decrease in food intake in the first week of treatment and some increase in fasting glucose levels. We have screened for PMXB-analogs having less associating affinity with rat-muscle phospholipids, and revealed that the same therapy using PMXB-derived peptide (nona-PMXB) is most optimal. This PMXB-analog is devoid of antibacterial activity and is four times less toxic than PMXB. Nona-PMXB therapy lower by 10, 32, 35 and 6% body weight gain, fat mass, circulating triglycerides and fasting glucose levels, respectively, in spite of normal or even elevated food intake in nona-PMXB treated rats. In summary, we found that nona-PMXB therapy is capable if inducing leanness in mature rats, particularly at the expense of decreasing fat-mass in adipose tissue. By and large, we suggest that lowering the action of insulin, on fat build-up solely, may be a therapeutically feasible task to fight with human adiposity in the future.     

The influence of sagittal center of pressure offset on gait kinematics and kinetics

ObjectivesKinetic patterns of the lower extremity joints have been shown to be influenced by modification of the location of the center of pressure (CoP) of the foot. The accepted theory is that a shifted location of the CoP alters the distance between the ground reaction force and the center of the joint, thereby modifying torques during gait. Various footwear designs have been reported to significantly alter the magnitude of sagittal joint torques during gait. However, the relationship between the CoP and the kinetic patterns in the sagittal plane has not been examined. The aim of this study was to evaluate the association between the sagittal location of the CoP and gait patterns during gait in healthy men.MethodsA foot-worn biomechanical device which allows controlled manipulation of the CoP location was utilized. Fourteen healthy men underwent successive gait analysis with the device set to convey three different sagittal locations of the CoP: neutral, anterior offset and posterior offset.ResultsCoP translation in the sagittal plane (i.e., from posterior to anterior) significantly related with an ankle dorsiflexion torque and a knee extension torque shift throughout the stance phase. Likewise, an anterior translation of the CoP significantly reduced the extension torque at the hip during pre-swing.ConclusionsThe study results confirm a direct correlation between sagittal offset of the CoP and the magnitude of joint torques throughout the lower extremity.     

The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines

Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by ∼8- to 10-kb left and right direct repeats, DR_L and DR_R. We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short ∼2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained ∼8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained ∼2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DR_L, including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DR_R sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth “conquering” the population. The DR1 gene and the first DR6 exon are not required for propagation in culture.Human herpesvirus 6 (HHV-6) is a member of the Betaherpesvirus subfamily, as recently reviewed (46). The virus can enter hematopoietic cells, including T cells, B cells, natural killer (NK) cells, monocytes, and dendritic cells (DCs), as well as nonhematopoietic cells, as reviewed in references 8, 17, and 46. In culture, the virus replicates in activated peripheral blood lymphocytes (PBLs), cord blood mononuclear cells (CBMCs), and in T-cell lines (1, 17, 46). HHV-6 isolates fall into two distinct classes designated as HHV-6A and HHV-6B variants. The two variants can be distinguished by their restriction enzyme patterns, antigenicity, DNA sequences, and disease association (1, 36, 46). HHV-6B is the causative agent of roseola infantum, a prevalent children''s disease characterized by high fever and skin rash (47). In rare cases, the virus exhibits neurotropism and has been found in children experiencing convulsions up to lethal encephalitis (1, 21, 46, 48).HHV-6B reactivation from latency was found to occur in patients receiving immunosuppressive treatment in bone marrow and other transplantations. This was associated with febrile illness, delayed transplant engraftment, and neurological involvement, up to lethal encephalitis (5, 13, 34, 46). HHV-6A has thus far no clear disease association, although several studies have suggested central nervous system (CNS) tropism, including aggravation of symptoms in patients with multiple sclerosis (MS) (6, 14, 33, 41).HHV-6A and HHV-6B share general genomic architecture. The unit-length DNA molecules are approximately 160 kb, composed of a 143-kb unique (U) segment flanked by left and right direct repeats (DR_L and DR_R, respectively) (19, 24, 27, 46). The DRs are of sizes 8 to 10 kb in different viral isolates (2, 19, 24, 46). In both the HHV-6A and HHV-6B genomes, the herpesvirus conserved cleavage/packaging signals pac-1 and pac-2 (9, 15, 17) are located at the left and the right termini of the DRs (17, 19, 46). The PubMed sequence for the U1102 strain (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001664","term_id":"1344462938","term_text":"NC_001664"}}NC_001664) starts with the pac-1 signal at positions 1 to 56, followed by multiple copies of perfect and imperfect telomere-like sequences, up to position 418. It was suggested that the telomeric repeats may have originated from host cell chromosomal telomeres (43). Additionally, the DR encodes several open reading frames (ORFs), four of which are dealt with in our paper: (i) the spliced DR1 at positions 501 to 759 and 843 to 2653; (ii) DR5 at positions 3738 to 4164; (iii) the spliced DR6 at positions 4725 to 5028 and 5837 to 6720; and (iv) an ORF of DR7, at positions 5629 to 6720, partially overlapping the DR6 gene (20). Hollsberg and coworkers (37) have recently found that the homologous gene in HHV-6B encodes a nuclear protein that forms a complex with viral DNA processivity factor p41. Gompels and coworkers have also shown that DR1 and DR6 are partly homologous to the human cytomegalovirus (HCMV) US22 gene family. Both have a CXC motif: DR1 with homology to the HCMV US26 gene and DR6 with homology to the HCMV US22 gene (20). The map continues with reiterated perfect hexanucleotide telomeric sequences (GGGTAA)_n at positions 7655 to 8008 (19, 43). The number of telomeric repeats was found to vary in different viral strains (2, 43). The DR terminates with the pac-2 signal.We have recently cloned the intact HHV-6A genome into bacterial artificial chromosomes (BACs), by direct cloning of unit-length DNA produced from circular or head-to-tail replication intermediates into modified BAC vectors containing the green fluorescent protein (GFP) marker and ampicillin-puromycin (Amp-Puro) selection cassette (3). Surprisingly, the HHV-6A BAC clones as well as the parental HHV-6A (U1102) propagated in our laboratory in SupT1 cells were found to contain DRs of ∼2.7 kb instead of the expected ∼8- to 10-kb DRs, as in the early publications (19, 24, 27, 46) and in the PubMed sequence. This has raised the following questions. When did the deleted DRs arise? What was the detailed structure of deleted DRs?HHV-6 was discovered by Gallo and colleagues in 1986 (35), and viral isolates were obtained in multiple laboratories from AIDS patients, patients with lymphoproliferative disorders, and patients with roseola infantum (12, 26, 42, 45, 47). The isolates were propagated initially in activated PBLs and CBMCs and then in continuous T-cell lines, including HSB-2, J-JHAN, SupT1, Molt-3, and MT-4 (11, 45). The U1102 strain isolated by Downing and colleagues (12) was contributed to our laboratory by Robert Honess and was propagated first in activated PBLs and CBMCs (11, 18, 36, 45) and then in J-JHAN and SupT1 T cells (4, 30). To answer the question with regard to the origin of the short DRs and their structure, we have compared earlier viral HHV-6A passages with the currently propagated virus and the HHV-6A BAC clones. We describe here the detailed structure of the DR_L and DR_R of the “new” virus, containing the short ∼2.7-kb DR. We show that the deletion contained the left multiple repeats of telomere-like sequences and the ORFs from DR1 up to the DR6 first exon. Review of viral passaging since 1992 indicated that the deletion occurred spontaneously. The deleted viruses were stably and efficiently propagated in SupT1 T cells, indicating that the DR1 and DR6 first exons are not essential for virus in vitro replication.