Photo-Oxidation Mechanisms in Liquid Pharmaceutical Formulations: The Overlooked Role of Singlet Oxygen Presented as a Case Study.

PURPOSE: Oxidation is one of the most common degradation pathways for active pharmaceutical ingredients (APIs) in pharmaceutical formulations, mostly involving 1-electron processes via peroxy radicals and 2-electron processes by peroxides. In liquid pharmaceutical formulations, several factors can impact oxidative instabilities including pH, excipient impurities, headspace oxygen, and the potential for photo-oxidation. Photo-oxidation can be particularly challenging to characterize given the number of oxidative mechanisms which can occur. This was observed during formulation development of a new chemical entity, MK-1454, where a degradation peak was observed during photostability studies which was not previously observed during peroxide and peroxyradical forced stress studies. METHODS: To gain a fundamental understanding of reactive oxygen species generation and its role in degradation of MK-1454, experiments were performed with materials which either generate or measure reactive oxygen species including organic hydroperoxides, singlet oxygen, and superoxide to fundamentally understand a photodegradation mechanism which was observed in the original formulation. LC-MS experiments further elucidated the structure and mechanism of this observed degradation pathway. RESULTS: A clear relationship between the decrease in dissolved oxygen after light exposure and the loss of MK-1454 was established. The data indicate that singlet oxygen is the most likely contributor of a particular photodegradation product. The singlet oxygen was generated by the inactive ingredients in the formulation, and LC-MS confirm this as the most likely pathway. CONCLUSION: This work highlights the importance of understanding photochemical degradation of APIs in solution formulations and provides approaches which can better elucidate those mechanisms and thereby control strategies.

Direct Drug Analysis in Polymeric Implants Using Desorption Electrospray Ionization - Mass Spectrometry Imaging (DESI-MSI).

PURPOSE: Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) coupled with gas-phase ion mobility spectrometry was used to characterize the drug distribution in polymeric implants before and after exposure to accelerated in vitro release (IVR) media. DESI-MSI provides definitive chemical identification and localization of formulation components, including 2D chemical mapping of individual components with essentially no sample preparation. METHODS: Polymeric implants containing 40% (w/w) entecavir and poly(D,L-lactide) (PLA) were prepared and then exposed to either acidified PBS (pH 2.5) or MeOH:H2O (50:50, v/v) medias during a 7-day IVR test using continuous flow-through (CFT) cell dissolution. The amount of drug released from the polymer matrix during the 7-day IVR test was monitored by online-ultraviolet spectroscopy (UV) and HPLC-UV. After that period, intact implants and radial sections of implants were analyzed by DESI-MSI with ion mobility spectrometry. The active ingredient along with impurities and contaminants were used to generate chemical maps before and after exposure to the release medias. RESULTS: Bi-phasic release profiles were observed for implants during IVR release using both medias. During the second phase of release, implants exposed to PBS, pH 2.5, released the entecavir faster than the implants exposed to MeOH:H2O (50:50, v/v). Radial images of the polymer interior show that entecavir is localized along the central core of the implant after exposure to MeOH:H2O (50:50, v/v) and that the drug is more uniformly distributed throughout the implant after exposure to acidified PBS (pH 2.5). CONCLUSIONS: DESI-MSI coupled with ion mobility analysis produced chemical images of the drug distribution on the exterior and interior of cylindrical polymeric implants before and after exposure to various release medias. These results demonstrated the utility of this technique for rapid characterization of drug and impurity/degradant distribution within polymeric implants with direct implications for formulation development as well as analytical method development activities for various solid parenteral and oral dosage forms. These results are especially meaningful since samples were analyzed with essentially no preparative procedures.

Multiple Pathways in Capsid Assembly.

For a three-dimensional structure to spontaneously self-assemble from many identical components, the steps on the pathway must be kinetically accessible. Many virus capsids are icosahedral and assembled from hundreds of identical proteins, but how they navigate the assembly process is poorly understood. Capsid assembly is thought to involve stepwise addition of subunits to a growing capsid fragment. Coarse-grained models suggest that the reaction occurs on a downhill energy landscape, so intermediates are expected to be fleeting. In this work, charge detection mass spectrometry (CDMS) has been used to track assembly of the hepatitis B virus (HBV) capsid in real time. The icosahedral T = 4 capsid of HBV is assembled from 120 capsid protein dimers. Our results indicate that there are multiple pathways for assembly. Under conditions that favor a modest association energy there is no accumulation of large intermediates, which indicates that available pathways include ones on a downhill energy surface. Under higher salt conditions, where subunit interactions are strengthened, around half of the products of the initial assembly reaction have masses close to the T = 4 capsid and the other half are stalled intermediates which emerge abruptly at around 90 dimers, indicating a bifurcation in the ensemble of assembly paths. When incubated at room temperature, the 90-dimer intermediates accumulate dimers and gradually shift to higher mass and merge with the capsid peak. Though free subunits are present in solution, the stalled intermediates indicate the presence of a local minima on the energy landscape. Some intermediates may result from hole closure, where the growing capsid distorts to close the hole due to the missing capsid proteins or from a species where subsequent additions are particularly labile.

Importin ß Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.

Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin ß (Impα and Impß), which bind to the core protein's C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impß without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impß. Impß density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impß per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impß molecules. Cryo-EM of capsids incubated with excess Impß shows a population of damaged particles and a population of "dark" particles with internal density, suggesting that Impß is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impß. Though the in vitro complexes with great excess of Impß are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.

Pseudomonas chlororaphis Produces Multiple R-Tailocin Particles That Broaden the Killing Spectrum and Contribute to Persistence in Rhizosphere Communities.

R-tailocins are high-molecular-weight bacteriocins resembling bacteriophage tails. Pseudomonas chlororaphis 30-84 is a plant growth-promoting rhizobacterial (PGPR) strain that produces two distinct R-tailocin particles with different killing spectra. The two R-tailocins have different evolutionary histories but are released by the same lysis cassette. A previous study showed that both tailocins are important for pairwise competition with susceptible rhizosphere-colonizing strains; however, the broader role of tailocins in competition with the native rhizosphere microbiome was not tested. Genomic analysis of the P. chlororaphis 30-84 R-tailocin gene cluster uncovered the presence of three tail fiber genes in the tailocin 2 genetic module that could potentially result in tailocin 2 particles having different tail fibers and thus a wider killing spectrum. In this study, the tail fibers were found to incorporate onto different tailocin 2 particles, each with a distinct killing spectrum. A loss of production of one or both tailocins resulted in decreased P. chlororaphis 30-84 persistence within the wheat rhizosphere when in competition with the native microflora but not bulk soil. The capacity to produce three different versions of a single tailocin, each having one of three different types of tail fibers, is a previously unreported mechanism that leads to a broader R-tailocin killing spectrum. This study also provides evidence for the function of R-tailocins in competition with rhizosphere microbiome communities but not in bulk soil.IMPORTANCE Although R-tailocin gene clusters typically encode one tail fiber protein, three tail fiber-resembling genes were identified in association with one of the two sets of R-tailocin genes within the tailocin cluster of P. chlororaphis 30-84 and other sequenced P. chlororaphis strain genomes. This study confirmed that P. chlororaphis 30-84 not only produces two distinct tailocins, but that one of them is produced with three different types of tail fibers. This is a previously unreported strategy to increase the breadth of strains targeted by an R-tailocin. Our finding that R-tailocins produced by a PGPR Pseudomonas strain enhanced its persistence within the wheat rhizosphere microbiome confirms that R-tailocin production contributes to the population dynamics of rhizobacterial communities.

Characterization of the Serralysin-Like Gene of 'Candidatus Liberibacter solanacearum' Associated with Potato Zebra Chip Disease.

The nonculturable bacterium 'Candidatus Liberibacter solanacearum' is the causative agent of zebra chip disease in potato. Computational analysis of the 'Ca. L. solanacearum' genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The 'Ca. L. solanacearum' serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional 'Ca. L. solanacearum' serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the 'Ca. L. solanacearum' serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the 'Ca. L. solanacearum' serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-'Ca. L. solanacearum' serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the 'Ca. L. solanacearum' protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the 'Ca. L. solanacearum' serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.

Global gene expression in two potato cultivars in response to 'Candidatus Liberibacter solanacearum' infection.

BACKGROUND: Transcriptomic analyses were performed to compare the molecular responses of two potato varieties previously shown to differ in the severity of disease symptoms due to infection by "Candidatus Liberibacter solanacearum" (Lso), the causative agent of Zebra Chip in potato. A factorial design utilizing the two varieties and psyllids either harboring Lso or without bacteria was used to discriminate varietal responses to pathogen infection versus psyllid feeding. Plant response was determined from leaf samples 3 weeks after infection. RESULTS: In response to Lso infection, 397 genes were differentially expressed in the variety Atlantic (most susceptible) as compared to 1027 genes in Waneta. Over 80% of the transcriptionally-changed genes were down-regulated in both varieties, including genes involved in photosynthesis or primary and secondary metabolism. Many of the Lso-responsive genes involved in stress responses or hormonal pathways were regulated differently in the two potato varieties. CONCLUSIONS: This study focused on the time point just prior to the onset of symptom development and provides valuable insight into the mechanisms of Liberibacter pathogenicity, especially the widespread suppression of plant gene expression, including genes involved in plant defenses.

Hepatitis B Virus Capsid Completion Occurs through Error Correction.

Understanding capsid assembly is important because of its role in virus lifecycles and in applications to drug discovery and nanomaterial development. Many virus capsids are icosahedral, and assembly is thought to occur by the sequential addition of capsid protein subunits to a nucleus, with the final step completing the icosahedron. Almost nothing is known about the final (completion) step because the techniques usually used to study capsid assembly lack the resolution. In this work, charge detection mass spectrometry (CDMS) has been used to track the assembly of the T = 4 hepatitis B virus (HBV) capsid in real time. The initial assembly reaction occurs rapidly, on the time scale expected from low resolution measurements. However, CDMS shows that many of the particles generated in this process are defective and overgrown, containing more than the 120 capsid protein dimers needed to form a perfect T = 4 icosahedron. The defective and overgrown capsids self-correct over time to the mass expected for a perfect T = 4 capsid. Thus, completion is a distinct phase in the assembly reaction. Capsid completion does not necessarily occur by inserting the last building block into an incomplete, but otherwise perfect icosahedron. The initial assembly reaction can be predominently imperfect, and completion involves the slow correction of the accumulated errors.

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84.

Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots.

R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere.IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.

Charge detection mass spectrometry: weighing heavier things.

Charge detection mass spectrometry (CDMS) is a single molecule method where the mass of each ion is directly determined from individual measurements of its mass-to-charge ratio and charge. CDMS is particularly valuable for the analysis of high mass and heterogeneous analytes, where conventional MS methods are often confounded. In the last few years, CDMS has received a renaissance. Technical developments have improved the resolution and dramatically increased the breadth of problems that can be addressed. These improvements have moved CDMS more into the mainstream as interest in the application of mass spectrometry to high molecular weight species has grown. In the article, the three main variants of CDMS are described, along with an overview of recent applications.

Resolving Adeno-Associated Viral Particle Diversity With Charge Detection Mass Spectrometry.

Recombinant adeno-associated viruses (AAVs) are promising vectors for human gene therapy. However, current methods for evaluating AAV particle populations and vector purity are inefficient and low resolution. Here, we show that charge detection mass spectrometry (CDMS) can resolve capsids that contain the entire vector genome from those that contain partial genomes and from empty capsids. Measurements were performed for both single-stranded and self-complementary genomes. The self-complementary AAV vector preparation appears to contain particles with partially truncated genomes averaging at half the genome length. Comparison to results from electron microscopy with manual particle counting shows that CDMS has no significant mass discrimination in the relevant mass range (after a correction for the ion velocity is taken into account). Empty AAV capsids are intrinsically heterogeneous, and capsids from different sources have slightly different masses. However, the average masses of both the empty and full capsids are in close agreement with expected values. Mass differences between the empty and full capsids for both single-stranded and self-complementary AAV vectors indicate that the genomes are largely packaged without counterions.

Structurally similar woodchuck and human hepadnavirus core proteins have distinctly different temperature dependences of assembly.

UNLABELLED: Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 µM and 43.3 µM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ∼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host. IMPORTANCE: In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

Adaptation genomics of a small-colony variant in a Pseudomonas chlororaphis 30-84 biofilm.

The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Silverleaf Nightshade (Solanum elaeagnifolium), a Reservoir Host for 'Candidatus Liberibacter solanacearum', the Putative Causal Agent of Zebra Chip Disease of Potato.

Zebra chip disease of potato is caused by the bacterial pathogen 'Candidatus Liberibacter solanacearum' and is a growing concern for commercial potato production in several countries in North and Central America and New Zealand. 'Ca. L. solanacearum' is vectored by the potato psyllid Bactericera cockerelli, which transmits the pathogen to several cultivated and wild solanaceaous host plants. Silverleaf nightshade (SLN), Solanum elaeagnifolium, is a common weed in the Lower Rio Grande Valley of Texas and a host for both the potato psyllid and 'Ca. L. solanacearum'. SLN plants were successfully inoculated with 'Ca. L. solanacearum' under laboratory conditions. Retention studies demonstrated that 'Ca. L. solanacearum'-infected SLN planted in the field in January 2013, concurrent with commercial potato planting, retained the pathogen under field conditions throughout the year despite extensive dieback during summer. The presence of 'Ca. L. solanacearum' was confirmed in leaves, roots, and stolons of SLN plants collected the following year using polymerase chain reaction. Acquisition assays using B. cockerelli adults also revealed that SLN retained the pathogen. Transmission studies determined that B. cockerelli can acquire 'Ca. L. solanacearum' within a 2-week acquisition access period on 'Ca. L. solanacearum'-infected SLN and subsequently transmit the pathogen to potato. These results demonstrate that SLN plants can serve as a reservoir for 'Ca. L. solanacearum', providing a source of inoculum for B. cockerelli adults colonizing potato the next season. The presence of SLN plants all year round in the LRGV makes the weed an epidemiologically important host. These findings underscore the importance of eradicating or managing SLN plants growing in the vicinity of potato fields to prevent spread of 'Ca. L. solanacearum' and damage caused by zebra chip.

Detection of late intermediates in virus capsid assembly by charge detection mass spectrometry.

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Charge detection mass spectrometry of bacteriophage P22 procapsid distributions above 20 MDa.

RATIONALE: Charge state resolution is required to determine the masses of ions in electrospray mass spectrometry, a feat which becomes increasingly difficult as the mass increases. Charge detection mass spectrometry (CDMS) circumvents this limitation by simultaneously measuring the charge and the m/z of individual ions. In this work, we have used electrospray CDMS to determine the number of scaffolding proteins associated with bacteriophage P22 procapsids. METHODS: P22 procapsids containing a native cargo of scaffolding protein were assembled in E. coli and purified via differential centrifugation. Electrospray CDMS was used to measure their mass distribution. RESULTS: The procapsid peak was centered at 23.60 MDa, which indicates that they contain an average of ~112 scaffolding proteins. The distribution is relatively narrow, less than 31 scaffolding proteins wide. In addition, a peak at 19.84 MDa with a relative abundance of ~15% is attributed to empty capsids. Despite having the same sizes in solution, the empty capsid and the procapsid have significantly different average charges. CONCLUSIONS: The detection of empty capsids is unexpected and the process that leads to them is unknown. The average charge on the empty capsids is significantly lower than expected from the charge residue model, which probably indicates that the empty capsids have contracted in the gas phase. The scaffolding protein presumably limits the contraction of the procapsids. This work shows that electrospray CDMS can provide valuable information for masses greater than 20 MDa.

Correlation between "Candidatus Liberibacter solanacearum" infection levels and fecundity in its psyllid vector.

The potato/tomato psyllid, Bactericera cockerelli (Sulc) transmits the bacterium, "Candidatus (Ca.) Liberibacter solanacearum" (Lso), also known as "Ca. Liberibacter psyllaurous", which causes zebra chip disease in potato and other solanaceous crops. The authors previously showed that fecundity and nymph survival is significantly reduced in Lso-infected psyllids compared to uninfected psyllids on tomato. However, it is not known whether the level of the pathogen is correlated with concomitant reduction in fitness of the psyllid vector. Using quantitative PCR assays, Lso levels were determined in adult female founders of isofemale lines for whom several life history traits were previously recorded. Analysis of psyllid isofemale lines revealed that Lso infection levels in founders or mothers was negatively correlated with 7-day fecundity, nymph survival percentage, and number of F1 progeny including eggs, nymphs and adults. There was a significant negative density-dependent relationship between Lso level and fecundity. That is, psyllids experienced decreasing levels in fecundity with increasing bacterial titer. There was no apparent negative density-dependent relationship between Lso copies and number of nymphs, nymph survival percentage and number of adults. The negative effect of Lso on psyllid fecundity is likely due to direct effects of the bacteria on the insect host and not via the host plant. Taken together, these findings suggest that the level of Lso in its psyllid vector correlates with reduction in psyllid fitness.