Direct haplotyping of kilobase-size DNA using carbon nanotube probes

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.     

Desmin knockout muscles generate lower stress and are less vulnerable to injury compared with wild-type muscles

The functional roleof the skeletal muscle intermediate filament system was investigated bymeasuring the magnitude of muscle force loss after cyclic eccentriccontraction (EC) in normal and desmin null mouse extensor digitorumlongus muscles. Isometric stress generated was significantly greater inwild-type (313 ± 8 kPa) compared with knockout muscles (276 ± 13 kPa) before EC (P < 0.05), but 1 h after 10 ECs, both muscle types generated identical levels of stress (~250kPa), suggesting less injury to the knockout. Differences in injurysusceptibility were not explained by the different absolute stresslevels imposed on wild-type versus knockout muscles (determined bytesting older muscles) or by differences in fiber length or mechanicalenergy absorbed. Morphometric analysis of longitudinal electronmicrographs indicated that Z disks from knockout muscles were morestaggered (0.36 ± 0.03 µm) compared with wild-type muscles(0.22 ± 0.03 µm), which may indicate that the knockoutcytoskeleton is more compliant. These data demonstrate that lack of theintermediate filament system decreases isometric stress production andthat the desmin knockout muscle is less vulnerable to mechanical injury.

     

Dependence of adenovirus infectivity on length of the fiber shaft domain

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/alphav integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/alphav integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and alphav integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.     

Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob

Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K_D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.     

The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Mammalian collagenase increases in early alcoholic liver disease and decreases with cirrhosis

To determine if alterations in collagen degradation may contribute to the pathogenesis of fibrosis and cirrhosis, we studied the hepatic collagenase activity of 20 baboons given alcohol containing diets or control diets under carefully controlled experimental conditions. We also studied 28 patients whose livers were biopsied for diagnostic purposes. Hepatic collagenase activity was significantly increased in baboons with fatty liver compared to levels in normal, control fed animals [(1.98 +/- 0.19 vs 1.20 +/- 0.08 units (microgram collagen digested/hour/mg liver protein) +/- S.E.M., p less than 0.001)]. The increase in hepatic collagenase activity persisted at the stage of fibrosis when compared to the activity in control baboons (2.16 +/- 0.07 vs 1.20 +/- 0.08 units +/- S.E.M., p less than 0.001). A single cirrhotic baboon available for study had an hepatic collagenase activity of 1.58 units. Patients with hepatic fibrosis had significantly higher hepatic collagenase activity than those with fatty livers [(9.12 +/- 0.94 (n =14) vs 4.52 +/- 0.54 (n = 7) units +/- S.E.M., p less than 0.001)]. However, in the group with cirrhosis, hepatic collagenase was lower [(3.92 +/- 0.61 (n = 7) units +/- S.E.M., p less than 0.001)] than in the group with fibrosis. Our findings suggest that the development of cirrhosis is coincident with, or favored by a failure of hepatic collagen degradative enzymes to keep pace with hepatic collagen synthesis.     

Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-beta-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-beta-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-beta-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

Evidence That the DNA Endonuclease ARTEMIS also Has Intrinsic 5′-Exonuclease Activity

ARTEMIS is a member of the metallo-β-lactamase protein family. ARTEMIS has endonuclease activity at DNA hairpins and at 5′- and 3′-DNA overhangs of duplex DNA, and this endonucleolytic activity is dependent upon DNA-PKcs. There has been uncertainty about whether ARTEMIS also has 5′-exonuclease activity on single-stranded DNA and 5′-overhangs, because this 5′-exonuclease is not dependent upon DNA-PKcs. Here, we show that the 5′-exonuclease and the endonuclease activities co-purify. Second, we show that a point mutant of ARTEMIS at a putative active site residue (H115A) markedly reduces both the endonuclease activity and the 5′-exonuclease activity. Third, divalent cation effects on the 5′-exonuclease and the endonuclease parallel one another. Fourth, both the endonuclease activity and 5′-exonuclease activity of ARTEMIS can be blocked in parallel by small molecule inhibitors, which do not block unrelated nucleases. We conclude that the 5′-exonuclease is intrinsic to ARTEMIS, making it relevant to the role of ARTEMIS in nonhomologous DNA end joining.     

Muscle geometry affects accuracy of forearm volume determination by magnetic resonance imaging (MRI)

Upper extremity musculoskeletal modeling is becoming increasingly sophisticated, creating a growing need for subject-specific muscle size parameters. One method for determining subject-specific muscle volume is magnetic resonance imaging (MRI). The purpose of this study was to determine the validity of MRI-derived muscle volumes in the human forearm across a variety of muscle sizes and shapes. Seventeen cadaveric forearms were scanned using a fast-spoiled gradient echo pulse sequence with high isotropic spatial resolution (1mm(3) voxels) on a 3T MR system. Pronator teres (PT), extensor carpi radialis brevis (ECRB), extensor pollicis longus (EPL), flexor carpi ulnaris (FCU), and brachioradialis (BR) muscles were manually segmented allowing volume to be calculated. Forearms were then dissected, muscles isolated, and muscle masses obtained, which allowed computation of muscle volume. Intraclass correlation coefficients (ICC(2,1)) and absolute volume differences were used to compare measurement methods. There was excellent agreement between the anatomical and MRI-derived muscle volumes (ICC = 0.97, relative error = 12.8%) when all 43 muscles were considered together. When individual muscles were considered, there was excellent agreement between measurement methods for PT (ICC = 0.97, relative error = 8.4%), ECRB (ICC = 0.93, relative error = 7.7%), and FCU (ICC = 0.91, relative error = 9.8%), and fair agreement for EPL (ICC = 0.68, relative error = 21.6%) and BR (ICC = 0.93, relative error = 17.2%). Thus, while MRI-based measurements of muscle volume produce relatively small errors in some muscles, muscles with high surface area-to-volume ratios may predispose them to segmentation error, and, therefore, the accuracy of these measurements may be unacceptable.