Imaging the Meissner effect in hydride superconductors using quantum sensors.

By directly altering microscopic interactions, pressure provides a powerful tuning knob for the exploration of condensed phases and geophysical phenomena1. The megabar regime represents an interesting frontier, in which recent discoveries include high-temperature superconductors, as well as structural and valence phase transitions2-6. However, at such high pressures, many conventional measurement techniques fail. Here we demonstrate the ability to perform local magnetometry inside a diamond anvil cell with sub-micron spatial resolution at megabar pressures. Our approach uses a shallow layer of nitrogen-vacancy colour centres implanted directly within the anvil7-9; crucially, we choose a crystal cut compatible with the intrinsic symmetries of the nitrogen-vacancy centre to enable functionality at megabar pressures. We apply our technique to characterize a recently discovered hydride superconductor, CeH9 (ref. 10). By performing simultaneous magnetometry and electrical transport measurements, we observe the dual signatures of superconductivity: diamagnetism characteristic of the Meissner effect and a sharp drop of the resistance to near zero. By locally mapping both the diamagnetic response and flux trapping, we directly image the geometry of superconducting regions, showing marked inhomogeneities at the micron scale. Our work brings quantum sensing to the megabar frontier and enables the closed-loop optimization of superhydride materials synthesis.

Emergent hydrodynamics in a strongly interacting dipolar spin ensemble.

Conventional wisdom holds that macroscopic classical phenomena naturally emerge from microscopic quantum laws1-7. However, despite this mantra, building direct connections between these two descriptions has remained an enduring scientific challenge. In particular, it is difficult to quantitatively predict the emergent 'classical' properties of a system (for example, diffusivity, viscosity and compressibility) from a generic microscopic quantum Hamiltonian7-14. Here we introduce a hybrid solid-state spin platform, where the underlying disordered, dipolar quantum Hamiltonian gives rise to the emergence of unconventional spin diffusion at nanometre length scales. In particular, the combination of positional disorder and on-site random fields leads to diffusive dynamics that are Fickian yet non-Gaussian15-20. Finally, by tuning the underlying parameters within the spin Hamiltonian via a combination of static and driven fields, we demonstrate direct control over the emergent spin diffusion coefficient. Our work enables the investigation of hydrodynamics in many-body quantum spin systems.

Imaging the Local Charge Environment of Nitrogen-Vacancy Centers in Diamond.

Characterizing the local internal environment surrounding solid-state spin defects is crucial to harnessing them as nanoscale sensors of external fields. This is especially germane to the case of defect ensembles which can exhibit a complex interplay between interactions, internal fields, and lattice strain. Working with the nitrogen-vacancy (NV) center in diamond, we demonstrate that local electric fields dominate the magnetic resonance behavior of NV ensembles at a low magnetic field. We introduce a simple microscopic model that quantitatively captures the observed spectra for samples with NV concentrations spanning more than two orders of magnitude. Motivated by this understanding, we propose and implement a novel method for the nanoscale localization of individual charges within the diamond lattice; our approach relies upon the fact that the charge induces a NV dark state which depends on the electric field orientation.

Probing many-body dynamics in a two-dimensional dipolar spin ensemble.

The most direct approach for characterizing the quantum dynamics of a strongly interacting system is to measure the time evolution of its full many-body state. Despite the conceptual simplicity of this approach, it quickly becomes intractable as the system size grows. An alternate approach is to think of the many-body dynamics as generating noise, which can be measured by the decoherence of a probe qubit. Here we investigate what the decoherence dynamics of such a probe tells us about the many-body system. In particular, we utilize optically addressable probe spins to experimentally characterize both static and dynamical properties of strongly interacting magnetic dipoles. Our experimental platform consists of two types of spin defects in nitrogen delta-doped diamond: nitrogen-vacancy colour centres, which we use as probe spins, and a many-body ensemble of substitutional nitrogen impurities. We demonstrate that the many-body system's dimensionality, dynamics and disorder are naturally encoded in the probe spins' decoherence profile. Furthermore, we obtain direct control over the spectral properties of the many-body system, with potential applications in quantum sensing and simulation.

Imaging stress and magnetism at high pressures using a nanoscale quantum sensor.

Pressure alters the physical, chemical, and electronic properties of matter. The diamond anvil cell enables tabletop experiments to investigate a diverse landscape of high-pressure phenomena. Here, we introduce and use a nanoscale sensing platform that integrates nitrogen-vacancy (NV) color centers directly into the culet of diamond anvils. We demonstrate the versatility of this platform by performing diffraction-limited imaging of both stress fields and magnetism as a function of pressure and temperature. We quantify all normal and shear stress components and demonstrate vector magnetic field imaging, enabling measurement of the pressure-driven [Formula: see text] phase transition in iron and the complex pressure-temperature phase diagram of gadolinium. A complementary NV-sensing modality using noise spectroscopy enables the characterization of phase transitions even in the absence of static magnetic signatures.

Sequences near the 3' secretion-specific polyadenylation site influence levels of secretion-specific and membrane-specific IgG2b mRNA in myeloma cells.

The expressed immunoglobulin gamma 2b (IgG2b) heavy-chain gene of 4T001 was cloned into the shuttle vector pSV2-gpt and transfected into myeloma J558L and lymphoma A20.2J. Northern blots indicated that the transfected gamma 2b gene was processed in a manner similar to the endogenous heavy chain in both lymphoma and myeloma cells. To identify sequences important for immunoglobulin mRNA processing, we constructed deletions around the secretion-specific polyadenylation site and introduced the deleted genes into J558L cells. The BAL deletion lacked 670 base pairs of intervening sequence between secreted and membrane regions; the Kpn deletion lacked 830 base pairs in this region. J558L cells transfected with either the entire gamma 2b gene or the delta BAL vector produced predominantly secretion-specific gamma 2b mRNA and protein. J558L cells transfected with the delta Kpn vector produced approximately equimolar amounts of secretion-specific and membrane-specific gamma 2b mRNA. Both 55,000-dalton secreted and 62,000-dalton putative surface IgG2b proteins were detected in the delta Kpn transfectants. We conclude that sequences absent in the Kpn deletion but present in the BAL deletion exert an important role in the production of secretion-specific mRNA. The Kpn deletion removes the normal site of cleavage and poly(A) addition, and it is possible that it is the absence of this site which changes the processing pattern. Alternatively, it is possible that sequences absent in the Kpn deletion but present in the BAL deletion function in regulating the production of predominantly secretion-specific mRNA in myeloma cells. The possible role of a highly conserved sequence found in this region is discussed.

Establishment, molecular rescue, and expression of 123AV16-1, a tumor-reactive human monoclonal antibody.

The human monoclonal antibody (mAb) 123AV16-1 was generated by Epstein-Barr virus transformation of peripheral blood lymphocytes from a colorectal patient undergoing active specific immunotherapy with an autologous tumor cell-Bacille Calmette-Guérin vaccine. Direct immunohistochemical staining of tumor and normal pairs of tissues indicated that this human IgA1, lambda 2 mAb preferentially reacted with colon tumor epithelium. To generate a recombinant derivative of this Epstein-Barr virus-transformed cell line, we isolated the expressed complete heavy and light chain genes by a novel strategy and cloned them into modified pSV2-neo and pSV2-gpt expression vectors. The recombinant 123AV16-1 human mAb was expressed in both a murine myeloma and a human-murine heteromyeloma and was secreted as both monomers and dimers. The recombinant 123AV16-1 mAb expressed by both cell lines reacted with human colon tumor xenografts in a manner similar to the mAb derived from the Epstein-Barr virus-transformed cell line, indicating that the antibody specificity was not appreciably altered during the molecular rescue, cloning, or expression.

An immunoglobulin heavy chain gene deletion at direct repeats: nucleotide sequence and effect on mRNA accumulation.

The DNA from the mouse myeloma cell, I17, which produces aberrant gamma 2b heavy chain mRNAs, was cloned and sequenced. The I17 mutant, and its parent line 10.1, share a small deletion at the splice junction of the CH1 domain which results in the absence of CH1 sequences from the mRNA. In addition, the genomic DNA of I17 has a deletion of 253 nucleotides which fuses the CH2 and CH3 exons, causes a frameshift of the next 43 amino acids and results in a truncated protein. The deleted nucleotides are flanked by two direct repeats of the CAGCA pentamer in the normal gene. One copy of the repeat and the interposed DNA is removed in the mutant. The DNA deletion is colinear with the mRNA. Both I17 and 10.1 cells have decreased accumulation of the secretory-specific gamma 2b mRNA. The amounts of membrane-specific gamma 2b mRNA are also affected in the mutants.

In vitro activation of a nonproductive immunoglobulin allele by a single base pair insertion.

We have shown that one of the alleles in a hybridoma was nonproductive because the sequence in the N region of the heavy chain caused it to be out of frame and to terminate prematurely. This allele became productive, and the gamma 1 heavy chain that it encoded was secreted when an adenosine was inserted to produce an open reading frame. This event occurred at a very low frequency following mutagenesis of the cultured cells. This result suggests that similar sorts of events must occur in vivo when both productive and nonproductive alleles undergo frequent mutations and that new genes may be expressed in hybridomas as they are being subcloned.

Cost-effective manufacture of an allogeneic GM-CSF-secreting breast tumor vaccine in an academic cGMP facility.

BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant.

A novel tumor-specific human single-chain Fv selected from an active specific immunotherapy phage display library.

A colon tumor-associated antigen, CTAA 28A32-32K (CTA # 2E), related to the annexin family of proteins, was initially identified by its reactivity with a low affinity human IgM monoclonal antibody (mAb), 28A32. Both in vitro lymphoproliferative assays with human peripheral blood lymphocytes and delayed type hypersensitivity responses in patients immunized with autologous colon tumor cells indicated that CTA # 2E elicits potent T cell mediated responses and may be an important antigen in the development of a generic colorectal vaccine (Pomato et al. Vaccine Res 1994;3:145-161). A CTA # 2E-specific, murine hybridoma-derived mAb, 5-11A, which recognizes the amino-terminus of the tumor-associated antigen, exhibited qualitative human colon tumor-specific immunohistochemical reactivity. To rapidly develop a human mAb with similar antigen specificity and tumor reactivity as the murine 5-11A mAb, antibody phage display technology was employed. Two human antibody phage display libraries with 3.1 x 10(7) and 2.3 x 10(8) members were prepared from the variable region genes expressed by circulating B cells of patients undergoing active specific immunotherapy (ASI) with autologous tumor cells, predominantly from the colon, admixed with Bacille Calmette-Guerin (BCG). A CTA # 2E-reactive human single-chain (sc)Fv was selected by panning the larger library on decreasing concentrations of biotinylated tumor-associated antigen in solution. It exhibited similar antigen specificity as the murine hybridoma-derived 5-11A scFv, requiring the presence of the CTA # 2E amino-terminus for reactivity. This human scFv exhibited qualitative human colon tumor-specific immunohistochemical reactivity when displayed as a gene III fusion protein on phage. When reconstructed and expressed as an intact human IgG1, K mAb, its qualitative colon tumor-specificity was unaltered. Two other CTA # 2E-reactive human scFvs were selected from the smaller library by panning initially on decreasing concentrations of CTA # 2E coated to polystyrene and then on biotinylated CTA # 2E in solution. These human scFvs, which exhibited modest reactivity with different epitopes on the CTA # 2E antigen, did not exhibit human colon tumor-specific immunohistochemical reactivity.

A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors.

Several different techniques were used to determine the apparent half-lives of immunoglobulin gamma 2b heavy chain and kappa light chain mRNA's in mouse myeloma 4T001 and a mutant derived from 4T001, i.e., mutant I17. The mutant I17 Ig heavy chain mRNA lacks CH1 and has fused CH2 and CH3 domains resulting in a truncated protein. By all four techniques the Ig heavy chain mRNA from mutant I17 displays a half-life that is approximately 70% the half-life of Ig mRNA in 4T001 cells. However, the absolute values of apparent half-life varied by greater than twofold for both lines among several of the techniques employed. The half-life of Ig gamma 2b mRNA in 4T001 cells was found to be 6.4 h by measuring decay following administration of the adenosine analog DRB to block new mRNA synthesis and 5.7 hr by measuring accumulation in an approach to steady-state labeling protocol. In contrast, the observed Ig mRNA half-lives determined by measuring decay following administration of actinomycin D to block new mRNA synthesis, or in a pulse-chase analysis were 2.9 and 3.8 h, respectively. The apparent half-life for Ig kappa light chain mRNA was the same in the 4T001 and I17 lines using any one technique but the value varied depending on the technique from a high value of 5.9 h following DRB to a low value of 2.4 h with actinomycin decay. Approach to steady-state is theoretically the most accurate method to measure mRNA half-life when that value is less than the doubling time of the cells. Pulse-chase analyses are accurate for measuring mRNA half-life when that value is longer than the effective chase period. Measuring preformed message decay following administration of drugs to block new mRNA synthesis is adaptable over a range of half-lives, but the cells must be shown to retain correct RNA metabolism over the time frame of the experiment. Determining a correct half-life for a particular mRNA may not be feasible using only one method and may, in fact, require several different approaches until a consensus value emerges.

Sequences 3' of immunoglobulin heavy chain genes influence their expression.

The function of sequences 3' of Ig genes in controlling their expression has been investigated by analyzing mutants of Ig-producing cells and by gene transfection experiments. A mutant of an IgA producing myeloma was isolated whose steady-state level of heavy chain mRNA and protein was decreased. Analysis of the mutant showed it had deleted at least 4 kb of DNA immediately 3' of the alpha gene and introduced at least 5 kb of non-Ig sequence in its place. Examination of nuclear RNA showed no accumulation of aberrant transcripts or altered processing patterns. Instead, the transcription rate of heavy chain in the mutant was approximately 1/7 of that in its parent. This mutant suggests that sequences 3' of Ig genes facilitate their transcription; alternatively, the non-Ig sequences may act to depress transcription. A complete gamma 2b heavy chain gene containing both the secreted and membrane exons was transfected into lymphoid cells. The ratio of membrane/secreted Ig mRNA produced by the transfectants was found to reflect the phenotype of the recipient cell; myelomas made mostly secreted mRNA while lymphomas made only a slight excess of secreted mRNA. When a heavy chain was used with a deletion of sequences within the IVS between the secreted and membrane exons which left the AATAA poly A addition signal intact, but removed the site of cleavage and polyadenylation, the processing ratio was altered so that predominantly membrane Ig was produced in transfected myeloma cells. The alteration in processing could be a result of the deletion of the normal site for poly A addition; alternatively it could be a result of the deletion of another sequence which is recognized by processing enzymes. A 13 bp sequence (GTCCTGGTTCTTT), was found to be highly conserved both in position and sequence in human and mouse gamma chain genes. When a gene with a deletion which left the poly A addition site and the conserved sequence intact was used for transfection, the processing pattern was found to be identical to that of a wild type heavy chain gene.

A V region mutation in a phosphocholine-binding monoclonal antibody results in loss of antigen binding.

A V region mutant producing an antibody that had lost the ability to bind phosphocholine was isolated from a hybridoma producing a germline encoded T15 antibody. The mutation resulted in a single aspartic acid to asparagine substitution at residue 95 of the H chain V region. This confirms that the aspartic acid at residue 95 plays a major role in Ag binding. The results also suggest that somatic cell genetic techniques can be used to generate mAb with useful changes in Ag binding.

Bragg grating fabrication in germanosilicate fibers by use of near-UV light: a new pathway for refractive-index changes.

Using 334-nm light, we demonstrate side writing of Bragg gratings with an index change of ~10(-4) in germanium-doped fibers. No hydrogen loading of the fibers was required. These gratings have the same temperature stability as gratings fabricated with 240-nm light. Our results suggest that photoionization is not needed for formation of gratings in Ge-doped glass. We observe an enhancement of the 334-nm photosensitivity in boron-codoped fibers and suggest that B facilitates a structural transformation of the glass.

A well-differentiated B-cell line is permissive for somatic mutation of a transfected immunoglobulin heavy-chain gene.

pSV2neo plasmids containing an IgM heavy-chain gene with nonsense mutations in either the variable (V) or the constant (C) region were transfected into four differentiated mouse plasma cell lines: S107 and the NSO fusion partner (myeloma cell lines) and 2C3 and 36.65 (hybridoma cell lines). The frequencies of reversion of the nonsense mutations in multiple independent transfectants were determined with the spot ELISA and rates of reversion were calculated by fluctuation analysis. Mutations in both V and C regions were confirmed by sequence analyses. In the S107 cell line, spontaneous point mutations occurred in the V region at a rate of approximately 5 x 10(-5)/bp per cell generation, > 400-fold higher than the rate of V-region mutation in the NSO cell line and considerably higher than the rates in 2C3 and 36.65 hybridoma cell lines. These studies suggest that S107 is a relatively permissive cell line in which V-region mutations can occur constitutively, even though it represents a late stage of B-cell differentiation. Further, the results show that the construct used contains sufficient information in its flanking and coding sequences to allow a relatively high rate of V-region mutation, at least in the S107 cell line.

Immunoglobulin variable region hypermutation in hybrids derived from a pre-B- and a myeloma cell line.

Somatic mutation of the variable (V) regions of immunoglobulin genes occurs in vivo at rates that have been estimated to be between 10(-3) and 10(-4) per bp per generation. To study this process in vitro, the 18.81 pre-B-cell line and hybrids derived by fusing 18.81 to the NSO myeloma fusion partner were transfected with a mu heavy-chain construct containing a nonsense mutation in the V region (Vn) or the constant region (Cn). Mutation was quantitated by reversion analysis using the ELISA spot assay to detect single cells secreting IgM. Fluctuation analysis revealed that V-region mutations spontaneously occurred in 18.81 cells at an average rate of 5.8 x 10(-6) per bp per cell generation and in selected 18.81-NSO hybrids at greatly increased rates of 1.6 x 10(-3) to 5.8 x 10(-4) per bp per generation. The Vn construct also reverted frequently in transgenic mice, indicating that it contained sufficient information to mutate at high rates both in vivo and in vitro. Sequence analysis of reverted genes revealed that reversion was due to point mutations. Since the rates and nature of the mutations that are occurring in these transfected genes are similar to those reported in vivo, it should be possible to use this system to identify the cis-acting sequences and trans-acting factors that are responsible for V-region somatic hypermutation.

A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype.

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.