Pastoral production is associated with increased peste des petits ruminants seroprevalence in northern Tanzania across sheep, goats and cattle.

Peste des petits ruminants virus (PPRV) causes a contagious disease of high morbidity and mortality in small ruminant populations globally. Using cross-sectional serosurvey data collected in 2016, our study investigated PPRV seroprevalence and risk factors among sheep, goats and cattle in 20 agropastoral (AP) and pastoral (P) villages in northern Tanzania. Overall observed seroprevalence was 21.1% (95% exact confidence interval (CI) 20.1-22.0) with 5.8% seroprevalence among agropastoral (95% CI 5.0-6.7) and 30.7% among pastoral villages (95% CI 29.3-32.0). Seropositivity varied significantly by management (production) system. Our study applied the catalytic framework to estimate the force of infection. The associated reproductive numbers (R0) were estimated at 1.36 (95% CI 1.32-1.39), 1.40 (95% CI 1.37-1.44) and 1.13 (95% CI 1.11-1.14) for sheep, goats and cattle, respectively. For sheep and goats, these R0 values are likely underestimates due to infection-associated mortality. Spatial heterogeneity in risk among pairs of species across 20 villages was significantly positively correlated (R2: 0.59-0.69), suggesting either cross-species transmission or common, external risk factors affecting all species. The non-negligible seroconversion in cattle may represent spillover or cattle-to-cattle transmission and must be investigated further to understand the role of cattle in PPRV transmission ahead of upcoming eradication efforts.

Impacts of long-term precipitation manipulation on hydraulic architecture and xylem anatomy of piñon and juniper in Southwest USA.

Hydraulic architecture imposes a fundamental control on water transport, underpinning plant productivity, and survival. The extent to which hydraulic architecture of mature trees acclimates to chronic drought is poorly understood, limiting accuracy in predictions of forest responses to future droughts. We measured seasonal shoot hydraulic performance for multiple years to assess xylem acclimation in mature piñon (Pinus edulis) and juniper (Juniperus monosperma) after 3+ years of precipitation manipulation. Our treatments consisted of water addition (+20% ambient precipitation), partial precipitation-exclusion (-45% ambient precipitation), and exclusion-structure control. Supplemental watering elevated leaf water potential, sapwood-area specific hydraulic conductivity, and leaf-area specific hydraulic conductivity relative to precipitation exclusion. Shifts in allocation of leaf area to sapwood area enhanced differences between irrigated and droughted KL in piñon but not juniper. Piñon and juniper achieved similar KL under ambient conditions, but juniper matched or outperformed piñon in all physiological measurements under both increased and decreased precipitation treatments. Embolism vulnerability and xylem anatomy were unaffected by treatments in either species. Absence of significant acclimation combined with inferior performance for both hydraulic transport and safety suggests piñon has greater risk of local extirpation if aridity increases as predicted in the southwestern USA.

The slow dynamics of mycoplasma infections in a tortoise host reveal heterogeneity pertinent to pathogen transmission and monitoring.

The epidemiology of infectious diseases depends on many characteristics of disease progression, as well as the consistency of these processes across hosts. Longitudinal studies of infection can thus inform disease monitoring and management, but can be challenging in wildlife, particularly for long-lived hosts and persistent infections. Numerous tortoise species of conservation concern can be infected by pathogenic mycoplasmas that cause a chronic upper respiratory tract disease (URTD). Yet, a lack of detailed data describing tortoise responses to mycoplasma infections obscures our understanding of URTDs role in host ecology. We therefore monitored Mycoplasma agassizii infections in 14 captive desert tortoises and characterised clinical signs of disease, infection intensity, pathogen shedding and antibody production for nearly 4 years after initial exposure to donor hosts. Persistent infections established in all exposed tortoises within 10 weeks, but hosts appeared to vary in resistance, which affected the patterns of pathogen shedding and apparent disease. Delays in host immune response and changes to clinical signs and infection intensity over time resulted in inconsistencies between diagnostic tools and changes in diagnostic accuracy throughout the study. We discuss the implications these results have for URTD epidemiology and past and future research assessing disease prevalence and dynamics in tortoise populations.

Energetic costs of mange in wolves estimated from infrared thermography.

Parasites, by definition, extract energy from their hosts and thus affect trophic and food web dynamics even when the parasite may have limited effects on host population size. We studied the energetic costs of mange (Sarcoptes scabiei) in wolves (Canis lupus) using thermal cameras to estimate heat losses associated with compromised insulation during the winter. We combined the field data of known, naturally infected wolves with a data set on captive wolves with shaved patches of fur as a positive control to simulate mange-induced hair loss. We predict that during the winter in Montana, more severe mange infection increases heat loss by around 5.2-12 MJ per night (1,240-2,850 kcal, or a 65-78% increase) for small and large wolves, respectively, accounting for wind effects. To maintain body temperature would require a significant proportion of a healthy wolf's total daily energy demands (18-22 MJ/day). We also predict how these thermal costs may increase in colder climates by comparing our predictions in Bozeman, Montana to those from a place with lower ambient temperatures (Fairbanks, Alaska). Contrary to our expectations, the 14°C differential between these regions was not as important as the potential differences in wind speed. These large increases in energetic demands can be mitigated by either increasing consumption rates or decreasing other energy demands. Data from GPS-collared wolves indicated that healthy wolves move, on average, 17 km per day, which was reduced by 1.5, 1.8, and 6.5 km for light, medium, and severe hair loss. In addition, the wolf with the most hair loss was less active at night and more active during the day, which is the converse of the movement patterns of healthy wolves. At the individual level, mange infections create significant energy demands and altered behavioral patterns, this may have cascading effects on prey consumption rates, food web dynamics, predator-prey interactions, and scavenger communities.

Social living mitigates the costs of a chronic illness in a cooperative carnivore.

Infection risk is assumed to increase with social group size, and thus be a cost of group living. We assess infection risk and costs with respect to group size using data from an epidemic of sarcoptic mange (Sarcoptes scabiei) among grey wolves (Canis lupus). We demonstrate that group size does not predict infection risk and that individual costs of infection, in terms of reduced survival, can be entirely offset by having sufficient numbers of pack-mates. Infected individuals experience increased mortality hazards with increasing proportions of infected pack-mates, but healthy individuals remain unaffected. The social support of group hunting and territory defence are two possible mechanisms mediating infection costs. This is likely a common phenomenon among other social species and chronic infections, but difficult to detect in systems where infection status cannot be measured continuously over time.

The relative importance of host characteristics and co-infection in generating variation in Heligmosomoides polygyrus fecundity.

We examined the relative importance of intrinsic host factors and microparasite co-infection in generating variation in Heligmosomoides polygyrus fecundity, a parameter that serves as a proxy for infectiousness. We undertook extensive trapping of Apodemus flavicollis, the yellow-necked mouse in the woodlands of the Italian Alps and recorded eggs in utero from the dominant nematode species H. polygyrus, and tested for the presence of five microparasite infections. The results showed that sex and breeding status interact, such that males in breeding condition harboured more fecund nematodes than other hosts; in particular, worms from breeding males had, on average, 52% more eggs in utero than worms from non-breeding males. In contrast, we found a weak relationship between intensity and body mass, and no relationship between intensity and sex or intensity and breeding condition. We did not find any evidence to support the hypothesis that co-infection with microparasites contributed to variation in worm fecundity in this system. The age-intensity profiles for mice singly-infected with H. polygyrus and those co-infected with the nematode and at least one microparasite were both convex and not statistically different from each other. We concluded that intrinsic differences between hosts, specifically with regard to sex and breeding condition, contribute relatively more to the variation in worm fecundity than parasite co-infection status.

Identifying Age Cohorts Responsible for Peste Des Petits Ruminants Virus Transmission among Sheep, Goats, and Cattle in Northern Tanzania.

Peste des petits ruminants virus (PPRV) causes a contagious disease of high morbidity and mortality in global sheep and goat populations. To better control this disease and inform eradication strategies, an improved understanding of how PPRV transmission risk varies by age is needed. Our study used a piece-wise catalytic model to estimate the age-specific force of infection (FOI, per capita infection rate of susceptible hosts) among sheep, goats, and cattle from a cross-sectional serosurvey dataset collected in 2016 in Tanzania. Apparent seroprevalence increased with age, reaching 53.6%, 46.8%, and 11.6% (true seroprevalence: 52.7%, 52.8%, 39.2%) for sheep, goats, and cattle, respectively. Seroprevalence was significantly higher among pastoral animals than agropastoral animals across all ages, with pastoral sheep and goat seroprevalence approaching 70% and 80%, respectively, suggesting pastoral endemicity. The best fitting piece-wise catalytic models merged age groups: two for sheep, three for goats, and four for cattle. The signal of these age heterogeneities were weak, except for a significant FOI peak among 2.5-3.5-year-old pastoral cattle. The subtle age-specific heterogeneities identified in this study suggest that targeting control efforts by age may not be as effective as targeting by other risk factors, such as production system type. Further research should investigate how specific husbandry practices affect PPRV transmission.

Prevention of population cycles by parasite removal.

The regular cyclic fluctuations in vertebrate numbers have intrigued scientists for more than 70 years, and yet the cause of such cycles has not been clearly demonstrated. Red grouse populations in Britain exhibit cyclic fluctuations in abundance, with periodic crashes. The hypothesis that these fluctuations are caused by the impact of a nematode parasite on host fecundity was tested by experimentally reducing parasite burdens in grouse. Treatment of the grouse population prevented population crashes, demonstrating that parasites were the cause of the cyclic fluctuations.

Heligmosomoides polygyrus reduces infestation of Ixodes ricinus in free-living yellow-necked mice, Apodemus flavicollis.

Free-living animals are usually inhabited by a community of parasitic species that can interact with each other and alter both host susceptibility and parasite transmission. In this study we tested the prediction that an increase in the gastrointestinal nematode Heligmosomoides polygyrus would increase the infestation of the tick Ixodes ricinus, in free-living yellow-necked mice, Apodemus flavicollis. An extensive cross-sectional trapping survey identified a negative relationship between H. polygyrus and I. ricinus counter to the prediction. An experimental reduction of the nematode infection through anthelmintic treatment resulted in an increase in tick infestation, suggesting that this negative association was one of cause and effect. Host characteristics (breeding condition and age) and habitat variables also contributed to affect tick infestation. While these results were counter to the prediction, they still support the hypothesis that interactions between parasite species can shape parasite community dynamics in natural systems. Laboratory models may act differently from natural populations and the mechanism generating the negative association is discussed.

Parasite co-infection and interaction as drivers of host heterogeneity.

We examined the hypothesis that the interaction between concomitant infecting parasites modifies host susceptibility, parasite intensity and the pattern of parasite distribution within the host population. We used a 26 year time series of three common parasites in a natural population of rabbits: two gastrointestinal nematodes (Trichostrongylus retortaeformis and Graphidium strigosum) and the immunosuppressive myxoma virus. The frequency distribution of nematodes in the host population and the relationship between host age and nematode intensity were explored in rabbits with either single or dual nematode infections and rabbits infected with the nematodes and myxoma virus. The aggregation of T. retortaeformis and G. strigosum among the rabbits varied with the nature of the co-infection both in male and female hosts. The two nematodes exhibited different age-intensity profiles: G. strigosum intensity increased exponentially with host age while T. retortaeformis intensity exhibited a convex shape. The presence of a secondary infection did not change the age-intensity profile for G. strigosum but for T. retortaeformis co-infection (either both nematodes or myxoma-nematodes) resulted in significantly greater intensities in adult hosts. Results suggest that multi-species infections contributed to aggregation of parasites in the host population and to seasonal variation in intensity, but also enhanced differences in parasitism between sexes. This effect was apparent for T. retortaeformis, which appears to elicit a strong acquired immune response but not for G. strigosum which does not produce any evident immune reaction. We concluded that concomitant infections mediated by host immunity are important in modifying host susceptibility and influencing heterogeneity amongst individual hosts.

The effects of social structure and sex-biased transmission on macroparasite infection.

Mathematical models of disease dynamics tend to assume that individuals within a population mix at random and so transmission is random, and yet, in reality social structure creates heterogeneous contact patterns. We investigated the effect of heterogeneity in host contact patterns on potential macroparasite transmission by first quantifying the level of assortativity in a socially structured wild rodent population (Apodemus flavicollis) with respect to the directly-transmitted macroparasitic helminth, Heligmosomoides polygyrus. We found the population to be disassortatively mixed (i.e. male mice mixing with female mice more often than same sex mixing) at a constant level over time. The macroparasite H. polygyrus has previously been shown to exhibit male-biased transmission so we used a Susceptible-Infected (SI) mathematical model to simulate the effect of increasing strengths of male-biased transmission on the prevalence of the macroparasite using empirically-derived transmission networks. When transmission was equal between the sexes the model predicted macroparasite prevalence to be 73% and infection was male biased (82% of infection in the male mice). With a male-bias in transmission ten times that of the females, the expected macroparasite prevalence was 50% and was equally prevalent in both sexes, results that both most closely resembled empirical dynamics. As such, disassortative mixing alone did not produce macroparasite dynamics analogous to those from empirical observations; a strong male-bias in transmission was also required. We discuss the relevance of our results in the context of network models for transmission dynamics and control.

Recombinant antibody constructs in cancer therapy.

Recombinant antibodies and their fragments now represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. The focus on antibodies as the ideal cancer-targeting reagents recently culminated in approval by the Food and Drugs Administration for the first engineered therapeutic antibodies. In the past year, important advances have been made in the design, selection and production of new types of engineered antibodies. Innovative selection methods have enabled the isolation of high-affinity cancer-targeting and antiviral antibodies, the latter capable of redirecting viruses for gene therapy applications. In other strategies for cancer diagnosis and therapy, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. Bispecific antibodies and related fusion proteins have been produced for cancer immunotherapy, effectively enhancing the human immune response in anticancer vaccines and T cell recruitment strategies.

The role of sex in parasite dynamics: model simulations on transmission of Heligmosomoides polygyrus in populations of yellow-necked mice, Apodemus flavicollis.

We investigated possible mechanisms that could cause sex-biased parasite transmission of the helminth Heligmosomoides polygyrus in its rodent host, Apodemus flavicollis, using a modelling approach. Two, not mutually exclusive, hypotheses were examined: that sex-biased parasite transmission is caused by differences in immunity that influence the success of free-living stages and/or is caused by sex differences in host behaviour and the dissemination of infective stages. Model simulations were compared with results from a field manipulation experiment of H. polygyrus in replicated populations of A. flavicollis. Simulations predicted the experimental field results, and both hypotheses explained the pattern observed. Transmission is male-biased if a male immune response increases fertility, hatching or survival of free-living stages. Alternatively, transmission is male-biased if their behavioural characteristics allow them to spread infective larvae in areas more frequently used by females. These results highlight that host sex is not only responsible for differences in parasite susceptibility, but may profoundly influence host-parasite interactions, resulting in a sex bias in parasite transmission.

Three-dimensional structures of single-chain Fv-neuraminidase complexes.

The structure of the complex between a recombinant single-chain Fv construct of antibody NC10 with a five-residue peptide linker between VH and VL (termed scFv(5)), and its antigen, tetrameric neuraminidase from influenza virus (NA), has been determined and refined at 2.5 A resolution. The antibody-antigen binding interface is very similar to that of a similar NC10 scFv-NA complex in which the scFv has a 15-residue peptide linker (scFv(15)), and the NC10 Fab-NA complex. However, scFv(5) and scFv(15) have different stoichiometries in solution. While scFv(15) is predominantly monomeric in solution, scFv(5) forms dimers exclusively, because the five-residue linker is not long enough to permit VH and VL domains from the same polypeptide associating and forming an antigen-binding site. Upon forming a complex with NA, scFv(15) forms a approximately 300 kDa complex corresponding to one NA tetramer binding four scFv(15) monomers, while scFv(5) forms a approximately 590 kDa complex, corresponding to two NA tetramers crosslinked by four bivalent scFv(5) dimers. However, the dimeric scFv(5) in the scFv(5)-NA crystals does not crosslink NA tetramers, and modelling studies indicate that it is not possible to pack four dimeric and simultaneously bivalent scFvs between the NA tetramers with only a five-residue linker between VH and VL. The inability arises from the exacting requirement to orient the two antigen-binding surfaces to bind the tetrameric NA antigen while avoiding steric clashes with NC10 scFv(5) dimers bound to other sites on the NA tetramer. The utility of bivalent or bifunctional scFvs with short linkers may therefore be restricted by the steric constraints imposed by binding multivalent antigens.

Peak shift and epidemiology in a seasonal host-nematode system.

Insight into the dynamics of parasite-host relationships of higher vertebrates requires an understanding of two important features: the nature of transmission and the development of acquired immunity in the host. A dominant hypothesis proposes that acquired immunity develops with the cumulative exposure to infection, and consequently predicts a negative relationship between peak intensity of infection and host age at this peak. Although previous studies have found evidence to support this hypothesis through between-population comparisons, these results are confounded by spatial effects. In this study, we examined the dynamics of infection of the nematode Trichostrongylus retortaeformis within a natural population of rabbits sampled monthly for 26 years. The rabbit age structure was reconstructed using body mass as a proxy for age, and the host age-parasite intensity relationship was examined for each rabbit cohort born from February to August. The age-intensity curves exhibited a typical concave shape, and a significant negative relationship was found between peak intensity of infection and host age at this peak. Adult females showed a distinct periparturient rise in T. retortaeformis infection, with higher intensities in breeding adult females than adult males and non-breeding females. These findings are consistent with the hypothesis of an acquired immune response of the host to a parasite infection, supporting the principle that acquired immunity can be modelled using the cumulative exposure to infection. These findings also show that seasonality can be an important driver of host-parasite interactions.

The effect of single and concomitant pathogen infections on condition and fecundity of the wild rabbit (Oryctolagus cuniculus).

We assessed the effect of two pathogens (myxoma virus and Eimeria stiedae) and five macroparasites (gastrointestinal helminth species) of the wild rabbit (Oryctolagus cuniculus) upon total host body mass and abdominal fat level. Additionally, we assessed the effects of these organisms on the number of foetuses in adult females during the peak breeding period. Both mass of abdominal fat and total body mass of the rabbit were negatively associated with myxoma virus infection and increasing helminth species richness. Total body mass was also negatively associated with the protozoan parasite E. steidae. No relationship was found between any of the parasites/pathogens and the number of foetuses in adult females, although only relatively small sample sizes were available for this section of the analysis. Increasing host body mass was positively associated with number of foetuses and we propose that mass reduction caused by the pathogen and parasite species could also have the consequence of reducing foetal number.

Recombinant antibody fragments.

Recombinant antibodies and their fragments now represent over 30% of all biological proteins undergoing clinical trials, which recently culminated in FDA approval for the first engineered cancer therapeutic antibody. Other successful applications include both the effective enhancement of the human immune response for cancer therapy and the reduction of unwanted immune rejections following transplantation and antibody therapy. Important advances have been made in the methods for design, selection and production of these new types of engineered antibodies. Innovative selection methods have enabled the isolation of catalytic antibodies and high-affinity viral-specific antibodies, the latter capable of redirecting viruses for gene therapy applications. In numerous practical applications, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces.

Biodistribution properties of (111)indium-labeled C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid humanized 3S193 diabody and F(ab')(2) constructs in a breast carcinoma xenograft model.

The humanized complementarity determining region-grafted anti-Lewis Y (Le(y)) monoclonal antibody [humanized 3S193 (hu3S193)] was developed for targeting Le(y)-expressing epithelial tumors such as breast, colon, lung, prostate, and ovarian carcinoma. We are exploring the potential use of smaller molecular size, bivalent analogues of hu3S193, because the faster blood clearance of M(r) approximately 54,000 diabody and M(r) approximately 110,000 F(ab')(2) molecules may be advantageous in achieving optimal and rapid tumor uptake for diagnostic and potential therapeutic applications. The single-chain variable fragment-5 residue linker construct (diabody) was expressed using the bacterial secretion vector pPOW3, and soluble product was purified without refolding processes. The F(ab')(2) fragment was obtained by pepsin digest of parental hu3S193. To facilitate evaluations, the radiometal (111)In was used to label C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid chelated diabody and F(ab')(2). The immunoreactivity of the radiolabeled constructs was 41.3 and 58.6%, and the K(a) was 1.68 x 10(6) M(-1) and 5.33 x 10(6) M(-1) for the diabody and F(ab')(2), respectively. Radioconjugates were injected into mice bearing Le(y)-positive MCF-7 tumors, and biodistribution properties were determined at various time points after injection. The uptake of radiolabeled diabody in xenografts was maximal at 1 h after injection (4.7 +/- 0.6% injected dose/g), whereas the F(ab')(2) peaked at 8 h after injection (14.2 +/- 2.4% injected dose/g). The tumor:blood ratio at 4 h for the diabody and F(ab')(2) was 5:1 and 2:1, which increased to 20:1 and 5:1, respectively, at 8 h and increased further to 40:1 and 130:1, respectively, at 48 h. These results demonstrate that the diabody construct may have applications as a diagnostic imaging reagent, whereas F(ab')(2) displayed effective tumor targeting and may have potential as a therapeutic molecule in patients with Le(y)-expressing tumors.

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase.

We have designed and produced a stable bispecific scFv dimer (bisFv) by non-covalent association of two hybrid VH-VL pairs derived from an anti-neuraminidase antibody (NC10) and an anti-glycophorin antibody (1C3). The bisFv dimer was demonstrated to have binding activity to the two respective target antigens and was evaluated as a reagent for rapid whole blood agglutination assays. The bisFv was expressed in the periplasm of Escherichia coli, from a secretion vector which comprised two cistrons in tandem under the control of a single lac promoter, inducible with IPTG. Each cistron encoded one of the hybrid VH-VL pairs, with V domains separated by a linker region encoding the five amino acids, Gly4Ser. The short linker region was designed to prevent association of VH and VL regions of the same molecule and favour the formation of dimers. The protein synthesized from each hybrid scFv cistron was directed to the E. coli periplasm by the inclusion of distinctive signal secretion sequences preceding each hybrid gene; from pel B of Erwinia cartovora and from gene III of fd phage. The bisFv was affinity-purified from culture supernatants via the C-terminal tag epitope FLAG and was shown, by FPLC on a Superose 6 column, to be consistent in size with that of a scFv dimer. The bisFv was stable for more than 4 months at 4 degrees C and was shown by BIAcore analysis to bind to either target antigen, human glycophorin, or tern N9 neuraminidase. Simultaneous binding to both target antigens was demonstrated when a pre-formed bisFv-neuraminidase complex was shown to bind to immobilized glycophorin. In whole blood agglutination assays, the bisFv dimer was able to agglutinate red blood cells when crosslinked with an anti-idiotype antibody (3-2G12) binding to the NC10 combining site, but no agglutination occurred on binding the antigen neuraminidase. These results are a function of the topology of the epitopes on neuraminidase and have implications for the use of relatively rigid bifunctional molecules (as bisFv dimers) to cross link two large membrane-anchored moieties, in this case, red blood cell glycophorin and neuraminidase, an M(r) 190,000 tetramer.