Humanized antibody neutralizing 2009 pandemic H1N1 virus

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (K-D = 0.75 +/- 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 mu g/mL), with marginally higher IC50 as compared to MA2077 (0.08 mu g/mL). In addition, 2077Hu2 also retained the epitope specificity for the ``Sa'' antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus.

Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells

Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.

Metallophosphoesterases: structural fidelity with functional promiscuity

Calcineurin-like metallophosphoesterases (MPEs) form a large superfamily of binuclear metal-ion-centre-containing enzymes that hydrolyse phosphomono-, phosphodi-or phosphotri-esters in a metal-dependent manner. The MPE domain is found in Mre11/SbcD DNA-repair enzymes, mammalian phosphoprotein phosphatases, acid sphingomyelinases, purple acid phosphatases, nucleotidases and bacterial cyclic nucleotide phosphodiesterases. Despite this functional diversity, MPEs show a remarkably similar structural fold and active-site architecture. In the present review, we summarize the available structural, biochemical and functional information on these proteins. We also describe how diversification and specialization of the core MPE fold in various MPEs is achieved by amino acid substitution in their active sites, metal ions and regulatory effects of accessory domains. Finally, we discuss emerging roles of these proteins as non-catalytic protein-interaction scaffolds. Thus we view the MPE superfamily as a set of proteins with a highly conserved structural core that allows embellishment to result in dramatic and niche-specific diversification of function.

Engineering a nanostructured ``super surface'' with superhydrophobic and superkilling properties

We present a nanostructured ``super surface'' fabricated using a simple recipe based on deep reactive ion etching of a silicon wafer. The topography of the surface is inspired by the surface topographical features of dragonfly wings. The super surface is comprised of nanopillars 4 mm in height and 220 nm in diameter with random inter-pillar spacing. The surface exhibited superhydrophobicity with a static water contact angle of 154.0 degrees and contact angle hysteresis of 8.3 degrees. Bacterial studies revealed the bactericidal property of the surface against both gram negative (Escherichia coli) and gram positive (Staphylococcus aureus) strains through mechanical rupture of the cells by the sharp nanopillars. The cell viability on these nanostructured surfaces was nearly six-fold lower than on the unmodified silicon wafer. The nanostructured surface also killed mammalian cells (mouse osteoblasts) through mechanical rupture of the cell membrane. Thus, such nanostructured super surfaces could find applications for designing selfcleaning and anti-bacterial surfaces in diverse applications such as microfluidics, surgical instruments, pipelines and food packaging.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly

We have identified a potent antibacterial agent N-(4-sec-butylphenyl)-2-(thiophen-2-yl)-1H-benzod]imidazole-4-carboxa mide (BT-benzo-29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT-benzo-29 disassembled the cytokinetic Z-ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT-benzo-29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid-cell. Further, BT-benzo-29 bound to FtsZ with a dissociation constant of 24 +/- 3 m and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT-benzo-29 may bind to FtsZ at the C-terminal domain near the T7 loop. BT-benzo-29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT-benzo-29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT-benzo-29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT-benzo-29 did not affect the membrane integrity of mammalian red blood cells. BT-benzo-29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

A self-assembling polycationic nanocarrier that exhibits exceptional gene transfection efficiency

The lack of an efficient and safe carrier is a major impediment in the field of gene therapy. Although gelatin (GT), a naturally derived polymer, is widely used in drug delivery applications, it is unable to bind DNA efficiently. In this study, a novel polycationic gene carrier was prepared by conjugation of low molecular weight polyethyleneimine (LPEI) with GT through 4-bromonaphthaleic anhydride as a coupling agent to avoid self crosslinking. Self-assembly of LPEI conjugated GT (GT-LPEI) with plasmid DNA (pDNA) yielded nanoparticles with high gene complexation ability to form similar to 250 nm cylindrical nanoparticles with a zeta potential of similar to 27 mV. GT-LPEI showed exceptionally high transfection efficiency (> 90%) in various mammalian cells including primary stem cells with minimal cytotoxicity. The transfection efficiency of GT-LPEI significantly surpassed that of many commercial reagents. The high gene transfection expression was confirmed in vivo. Thus, GT-LPEI is shown to be a promising nonviral carrier for potential use in gene therapy.

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knock-down studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the beta H-beta I loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-a-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Time-Instant Sampling Based Encoding of Time-Varying Acoustic Spectrum

The inner ear has been shown to characterize an acoustic stimuli by transducing fluid motion in the inner ear to mechanical bending of stereocilia on the inner hair cells (IHCs). The excitation motion/energy transferred to an IHC is dependent on the frequency spectrum of the acoustic stimuli, and the spatial location of the IHC along the length of the basilar membrane (BM). Subsequently, the afferent auditory nerve fiber (ANF) bundle samples the encoded waveform in the IHCs by synapsing with them. In this work we focus on sampling of information by afferent ANFs from the IHCs, and show computationally that sampling at specific time instants is sufficient for decoding of time-varying acoustic spectrum embedded in the acoustic stimuli. The approach is based on sampling the signal at its zero-crossings and higher-order derivative zero-crossings. We show results of the approach on time-varying acoustic spectrum estimation from cricket call signal recording. The framework gives a time-domain and non-spatial processing perspective to auditory signal processing. The approach works on the full band signal, and is devoid of modeling any bandpass filtering mimicking the BM action. Instead, we motivate the approach from the perspective of event-triggered sampling by afferent ANFs on the stimuli encoded in the IHCs. Though the approach gives acoustic spectrum estimation but it is shallow on its complete understanding for plausible bio-mechanical replication with current mammalian auditory mechanics insights.

Telomerase Activity and Microgravity-Dependent Apoptosis in Human Leukemia HL-60 Cells

Mammalian cells subjected to conditions of spaceflight and the microgravity environment ofspace; manifest a number of alterations in structure and function. Among the most notable changes incells flown on the Space Shuttle are reduced growth activation and decline in growth rate in the totalpopulation. Other changes include chromosomal aberrations, inhibited locomotion, alteredcytokine production, changes in PKC distribution, and increased apoptos.

Caspase-3-dependent phagocyte death during systemic Salmonella enterica serovar Typhimurium infection of mice.

Growth of Salmonella enterica in mammalian tissues results from continuous spread of bacteria to new host cells. Our previous work indicated that infective S. enterica are liberated from host cells via stochastic necrotic burst independently of intracellular bacterial numbers. Here we report that liver phagocytes can undergo apoptotic caspase-3-mediated cell death in vivo, with apoptosis being a rare event, more prevalent in heavily infected cells. The density-dependent apoptotic cell death is likely to constitute an alternative mechanism of bacterial spread as part of a bet-hedging strategy, ensuring an ongoing protective intracellular environment in which some bacteria can grow and persist.

Modelling within-host spatiotemporal dynamics of invasive bacterial disease

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics. © 2008 Grant et al.

Investigation of unusual mortalities of bottlenose dolphins along the mid-Texas coastal bay ecosystem during 1992

An investigation was conducted into the deaths of more than 220 bottlenose dolphins (Tursiops truncatus) that occurred within the coastal bay ecosystem of mid-Texas between January and May 1992. The high mortality rate was unusual in that it was limited to a relatively small geographical area, occurred primarily within an inshore bay system separated from the Gulf of Mexico by barrier islands, and coincided with deaths of other taxa including birds and fish. Factors examined to determine the potential causes of the dolphin mortalities included microbial pathogens, natural biotoxins, industrial pollutants, other environmental contaminants, and direct human interactions. Emphasis was placed on nonpoint source pesticide runoff from agricultural areas, which had resulted from record rainfall that occurred during the period of increased mortality. Analytical results from sediment, water, and biota indicated that biotoxins, trace metals, and industrial chemical contamination were not likely causative factors in this mortality event. Elevated concentrations of pesticides (atrazine and aldicarb) were detected in surface water samples from bays within the region, and bay salinities were reduced to <10 ppt from December 1991 through April 1992 due to record rainfall and freshwater runoff exceeding any levels since 1939. Prolonged exposure to low salinity could have played a significant role in the unusual mortalities because low salinity exposure may cause disruption of the permeability barrier in dolphin skin. The lack of established toxicity data for marine mammals, particularly dermal absorption and bioaccumulation, precludes accurate toxicological interpretation of results beyond a simple comparison to terrestrial mammalian models. Results clearly indicated that significant periods of agricultural runoff and accompanying low salinities co-occurred with the unusual mortality event in Texas, but no definitive cause of the mortalities was determined. (PDF file contains 25 pages.)