Therapeutic high affinity T cell receptor targeting a KRASG12D cancer neoantigen.

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen.

A Ten-Year Radiostereometric Analysis of Polyethylene Wear Between Oxidized Zirconium and Cobalt Chrome Articulations in Total Hip Arthroplasty.

BACKGROUND: Oxidized zirconium (OxZi) femoral heads combine the decreased abrasive properties of ceramics with the toughness of metal alloys to lower wear and increase the durability of total hip arthroplasty. The purpose of this study was to compare the 10-year polyethylene wear rates between OxZi/XLPE and cobalt chrome (CoCr)/XLPE articulations. METHODS: A radiostereometric analysis (RSA) was performed on two cohorts of 23 patients who underwent total hip arthroplasty using either OxZi/XLPE or CoCr/XLPE at a minimum of 10-year follow-up. Cohorts were matched for age, gender, body mass index (BMI), and diagnosis. Polyethylene wear was measured using RSA to determine total and steady-state wear rates for both cohorts. Preoperative and postoperative patient-reported outcome measures (SF12, HHS, and Western Ontario and McMaster Universities Arthritis Index scores) were compared. RESULTS: The mean total head penetration rate was found to be statistically different between the entire cohorts (OxZi 0.048 ± 0.021 mm/y, CoCr 0.035 ± 0.017 mm/y, P = .02) but not when 28-mm heads only (OxZi 0.045 ± 0.016 mm/y, CoCr 0.034 ± 0.017 mm/y, P = .066) were directly compared. The mean steady-state wear rate was not significantly different between the entire cohorts (OxZi 0.031 ± 0.021 mm/y, CoCr 0.024 ± 0.019 mm/y, P = .24) or 28-mm head cohorts (OxZi 0.028 ± 0.019 mm/y, CoCr 0.024 ± 0.019 mm/y, P = .574). Outcome measures showed no statistical difference except for the Harris Hip Score where the OxZi cohort demonstrated higher median scores. CONCLUSION: Using RSA to evaluate the 10-year in-vivo head penetration, there was no statistically significant difference in steady-state wear rates between OxZi and CoCr articulations. Both bearing combinations demonstrated wear rates well below the threshold for osteolysis.

Determining whether Public Communications Campaigns Based on the The Social Norms Approach are a Viable COVID-19 Prevention Strategy for College Campuses.

The purpose of this study is to (a) outline the formative steps that universities can follow to determine if a media campaign based on the social norms approach (SNA) is a viable method for increasing COVID-19 prevention behaviors among their students, (b) present formative research data collected at a large public land-grant university in the U.S., and (c) as a test case, apply that data to assess the SNA's viability for promoting COVID-19 prevention behaviors among students at that institution. Over time, a series of fast-track surveys were conducted to determine the descriptive and injunctive norms for four COVID-19 prevention strategies: wearing a mask in public, physical distancing, limiting the size of indoor gatherings, and receiving or planning to get a vaccination. The results demonstrated that, at this particular university, an SNA-based public communications campaign would be a promising strategy for promoting these protective behaviors. First, a clear majority of the survey respondents reported engaging in the behaviors. Second, the respondents perceived the behaviors to be less common than was actually the case, with one exception: wearing a mask. In all four cases, they perceived the behaviors to be less approved of than what the surveys documented.

The long-term effectiveness of a social norming campaign to reduce high-risk drinking: The Michigan State University experience, 2000-2014.

OBJECTIVE: Evaluate effectiveness of a university-wide social norms marketing campaign to reduce high-risk drinking and its consequences among students at MSU. Participants: Campaign messages regarding descriptive and injunctive norms were distributed campus-wide from 2001 to 2014 to correct norm misperceptions. Methods: Random samples of students surveyed most semesters to monitor message saturation, dosage, and believability along with drinking attitudes, behaviors and harm related to celebratory events. NCHA conducted biennially since 2000 to assess overall progress. Results: Perceived drinking norms declined along with measures of actual drinking intensity, frequency, and frequency of intense drinking. Use of protective behaviors most often addressed in campaign messages increased while driving after drinking declined. Importantly, reported adverse effects of drinking on academic performance declined substantially. Conclusions: The evidence suggests the social norms approach has been effective at reducing adverse academic outcomes of drinking. Changes in perceptions, attitudes, behaviors and outcomes appear to be continuing.

A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations: implications for peptide-based analgesics.

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor.

Larval settlement: the role of surface topography for sessile coral reef invertebrates.

For sessile marine invertebrates with complex life cycles, habitat choice is directed by the larval phase. Defining which habitat-linked cues are implicated in sessile invertebrate larval settlement has largely concentrated on chemical cues which are thought to signal optimal habitat. There has been less effort establishing physical settlement cues, including the role of surface microtopography. This laboratory based study tested whether surface microtopography alone (without chemical cues) plays an important contributing role in the settlement of larvae of coral reef sessile invertebrates. We measured settlement to tiles, engineered with surface microtopography (holes) that closely matched the sizes (width) of larvae of a range of corals and sponges, in addition to surfaces with holes that were markedly larger than larvae. Larvae from two species of scleractinian corals (Acropora millepora and Ctenactis crassa) and three species of coral reef sponges (Luffariella variabilis, Carteriospongia foliascens and Ircinia sp.,) were used in experiments. L. variabilis, A. millepora and C. crassa showed markedly higher settlement to surface microtopography that closely matched their larval width. C. foliascens and Ircinia sp., showed no specificity to surface microtopography, settling just as often to microtopography as to flat surfaces. The findings of this study question the sole reliance on chemical based larval settlement cues, previously established for some coral and sponge species, and demonstrate that specific physical cues (surface complexity) can also play an important role in larval settlement of coral reef sessile invertebrates.

The effects of physical and chemical treatments on Na2S produced feather keratin films.

The industrial utilisation of feather keratin as a biopolymer has proven difficult due to the lack of a viable extraction technique and the poor mechanical properties of the regenerated products. Here, pure keratin films were produced from chicken feathers using sodium sulphide as sole extraction reagent in a scheme that allows films to be formed without residual chemicals. In a comparison to other films, those produced using Na2S extraction were found to be superior to other regenerated protein films and were similar to un-oriented commercial polymers. However, there was considerable variation in tensile properties between twenty repetitions of extracting and casting films which was attributed to variations in chain entanglement caused by the drying conditions. Chemical and physical treatments including crosslinking, dehydration and addition of nano-particles were investigated as means to enhance these properties. Significant increases were achieved by soaking films in isopropyl alcohol or weak acid (13 to 50% increases) or by formaldehyde or glutaraldehyde crosslinking (24 to 40% increases). The wide range of values across the pure keratin films indicates that the best route to further strength improvement may be from optimising self-assembly via controlling drying conditions, rather than from chemical treatment.

Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities.

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1-10 µm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28 days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4 µm PDMS surfaces. Also, field deployments over 28 days showed a significant reduction in biovolume on the 4 and 10 µm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10 µm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.

ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons.

Back in 2003, we published 'MAX' randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. 'MAX' randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner. We have now developed 'ProxiMAX' randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or equipment, and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering.

The fouling hydroid Ectopleura larynx: a lack of effect of next generation antifouling technologies.

The hydroid Ectopleura larynx is one of the main fouling organisms on salmon aquaculture cages in Norway; this study investigated novel surface materials and microtopographies to deter its settlement. The settlement preferences of hydroid larvae for 12 materials with wettabilities ranging from hydrophobic (54°) to hydrophilic (112°) were tested in a no-choice bioassay. Although settlement differed between materials, with the highest average settlement on polytetrafluoroethylene (95%) and the lowest on untreated polyurethane (53%), no trend regarding the tested wettabilities could be found and none of the tested materials was able to reduce average settlement below 50%. Furthermore, nine high-density polyethylene (HDPE, 100-600 µm microtopographies) and seven polydimethylsiloxane (PDMS; 40-400 µm microtopographies) microtextured surfaces were tested. There was no systematic effect of microtopography on the settlement of E. larynx larvae. However, there was a preference for settlement in channels on PDMS microtopographies between 80 and 300 µm. Similarly, there were no preferences for any of the examined microtopographies in a 12-day field test using PDMS surfaces at a commercial fish farm. The study indicated that neither surface wettability (hydrophilicity-phobicity) nor microtopographies were effective at deterring the settlement of the hydroid E. larynx. The high plasticity of the aboral pole and the hydrorhiza of the hydroids may explain settlement even under unfavourable conditions, highlighting the successful colonisation traits of this dominant biofouling species.

Reducing costly falls of total knee replacement patients.

The objective of this study was to determine whether the use of a knee immobilizer brace reduces patient falls associated with the recent use of femoral nerve blocks for pain control after total knee arthroplasty (TKA). The authors conducted a retrospective study to investigate fall rates before and after the introduction of an immobilizer brace. The demographics of patients and total cost of care were examined. Of the 600 TKA patients who did not receive a knee immobilizer, 22 (3.7%) experienced a fall. In contrast, of the 502 patients who received knee immobilizers, only 8 patients (1.6%) fell. This difference achieves statistical significance (P = .04). Given the considerable costs associated with hospital falls and the significant reduction of these falls related to knee immobilizer use shown in this study, the authors recommend that knee immobilizers be given to TKA patients as standard practice.

Where to settle--settlement preferences of Mytilus galloprovincialis and choice of habitat at a micro spatial scale.

The global mussel aquaculture industry uses specialised spat catching and nursery culture ropes made of multi-filament synthetic and natural fibres to optimise settlement and retention of mussels for on-growing. However, the settlement ecology and preferences of mussels are poorly understood and only sparse information exists in a commercial context. This study quantified the settlement preferences of pediveligers and plantigrades of Mytilus galloprovincialis on increasingly complex surfaces and settlement locations at a micro spatial scale on and within ropes under commercial hatchery operating conditions using optical microscopy and X-ray micro-computed tomography (µCT). M. galloprovincialis has clear settlement preferences for more complex materials and high selectivity for settlement sites from the pediveliger through to the plantigrade stage. Pediveligers of M. galloprovincialis initially settle inside specialised culture ropes. Larger pediveligers were located close to the exterior of ropes as they increased in size over time. In contrast, smaller individuals were located deeper inside of the ropes over time. This study demonstrates that X-ray µCT is an excellent non-destructive technique for mapping settlement and attachment sites of individuals as early as one day post settlement, and quantifies the number and location of settled individuals on and within ropes as a tool to understand and optimise settlement in complex multi-dimensional materials and environments.

Environmentally sustainable fibers from regenerated protein.

Concerns for the environment and consumer demand are driving research into environmentally friendly fibers as replacements for part of the 38 million tonnes of synthetic fiber produced annually. While much current research focuses on cellulosic fibers, we highlight that protein fibers regenerated from waste or byproduct sources should also be considered. Feather keratin and wheat gluten may both be suitable. They are annually renewable, commercially abundant, of consistent quality, and have guaranteed supply. They contain useful amino acids for fiber making, with interchain cross-linking possible via cysteine residues or through the metal-catalyzed photocrosslinking of tyrosine residues. Previous commercially produced fibers suffered from poor wet strength. Contemporary nanoparticle and cross-linking technology has the potential to overcome this, allowing commercial production to resume. This would bring together two existing large production and processing pipelines, agricultural protein production and textile processing, to divert potential waste streams into useful products.

A mutation affecting ASCORBATE PEROXIDASE 2 gene expression reveals a link between responses to high light and drought tolerance.

Molecular analyses of plants have revealed a number of genes whose expression changes in response to high light (HL), including the H2O2 scavenger, ASCORBATE PEROXIDASE 2 (APX2). We carried out a screen in Arabidopsis thaliana for lesions that alter HL-induced expression of APX2 to identify components in abiotic stress signalling pathways. High light was used as it can be instantaneously applied or removed and accurately measured. We identified a number of alx mutations causing altered APX2 expression. Here we describe the gain-of-function mutant, alx8, which has constitutively higher APX2 expression and higher levels of foliar abscisic acid (ABA) than wild type. In fact, exogenous ABA increased APX2 expression and the APX2 promoter contains ABA response elements. Furthermore, we have shown that HL stress increases ABA in wild-type plants, implicating ABA in the regulation of HL-inducible genes. The alx8 mutant is drought tolerant, exhibits improved water-use efficiency and a number of drought-tolerance genes are upregulated. Additionally, alx8 demonstrates the complexity of ABA-dependent and ABA-independent transcriptional networks as some components in both pathways are upregulated in alx8. This study provides evidence for common steps in drought and HL stress response pathways.

Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase.

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.

An unstable base alters limb and abdominal activation strategies during the flexionrelaxation response.

The flexion-relaxation phenomenon consisting of an erector spinae silent period occurring with trunk flexion can place considerable stress upon tissues. Since individuals often flex their trunks while unstable, the purpose of the study was to examine the effect of an unstable base on the flexion-relaxation response. Fourteen participants flexed at the hips and back while standing on a stable floor or an unstable dyna-disc. Hip and trunk flexion were repeated four times each with one-minute rest. Electromyographic (EMG) electrodes were placed over the right lumbo-sacral erector spinae (LSES), upper lumbar erector spinae (ULES), lower abdominals (LA), biceps femoris and soleus. In addition to the flexion-relaxation phenomenon of the ES, a quiescence of biceps femoris and a burst of LA EMG activity was observed with the majority of stable trials. There was no effect of instability on the flexion-relaxation phenomenon of the ULES or LSES. The incidence of a biceps femoris silent period while stable was diminished with an unstable base. Similarly, the incidence of a LA EMG burst was curtailed with instability. Soleus EMG activity increased 29.5% with an unstable platform. An unstable base did not significantly affect LSES and ULES EMG flexion-relaxation, but did result in more persistent lower limb and LA activity. Key PointsAn unstable base did not affect the flexion relaxation response of the erector spinae.An unstable base decreased the incidence of biceps femoris quiescent period.An unstable base diminished the incidence of the lower abdominals EMG burst.

Mechanism of aerobic biological destabilisation of wool scour effluent emulsions.

Wool scouring effluent is a highly polluted industrial wastewater in which the main pollutant, wool wax, is held in a stable oil-in-water emulsion by non-ionic detergent. The use of microbial action to cause emulsion destabilisation has been proposed as a new treatment strategy for this effluent stream. This strategy aims at improving aerobic treatment performance by physically removing the high-COD, slowly bio-degradable wool wax from the system without bio-degradation. The mechanism by which an aerobic-mixed culture destabilises the wool scouring effluent emulsion was investigated. Our results show that destabilisation is due to partial bio-degradation of both the scouring detergent and the wool wax. Cleavage of the wool wax esters was the first stage in wax degradation, when 40-50% of wax was de-emulsified. Over the same period, detergent degradation was low, at 7-21%. With further incubation, detergent degradation increased, aiding further breakdown of the emulsion. The degradation of the detergent, a nonylphenol ethoxylate, resulted in both a reduction in molar concentration (of up to 82%) and a shortening of the ethoxylate chain length. The latter reduced the hydrophile-lipophile balance (HLB) from 12 to approximately 7, thereby reducing the ability of the residual detergent to stabilise the emulsion. Analysis of the emulsified and de-emulsified wax fractions could not identify a group of compounds that were preferentially de-emulsified based on molecular weight or polarity. These findings will assist in using a de-emulsification strategy in both existing and new treatment systems in order to save on aeration costs and treatment times for biological treatment of this highly polluted wastewater.

Antisense suppression of a (+)-delta-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression.

In cotton (Gossypium hirsutum) the enzyme (+)-delta-cadinene synthase (CDNS) catalyzes the first committed step in the biosynthesis of cadinane-type sesquiterpenes, such as gossypol, that provide constitutive and inducible protection against pests and diseases. A cotton cDNA clone encoding CDNS (cdn1-C4) was isolated from developing embryos and functionally characterized. Southern analysis showed that CDNS genes belong to a large multigene family, of which five genomic clones were studied, including three pseudogenes and one gene that may represent another subfamily of CDNS. CDNS expression was shown to be induced in cotton infected with either the bacterial blight or verticillium wilt pathogens. Constructs for the constitutive or seed-specific antisense suppression of cdn1-C4 were introduced into cotton by Agrobacterium-mediated transformation. Gossypol levels were not reduced in the seeds of transformants with either construct, nor was the induction of CDNS expression affected in stems of the constitutive antisense plants infected with Verticillium dahliae Kleb. However, the induction of CDNS mRNA and protein in response to bacterial blight infection of cotyledons was completely blocked in the constitutive antisense plants. These results suggest that cdn1-C4 may be involved specifically in the bacterial blight response and that the CDNS multigene family comprises a complex set of genes differing in their temporal and spatial regulation and responsible for different branches of the cotton sesquiterpene pathway.

Plants with increased expression of ent-kaurene oxidase are resistant to chemical inhibitors of this gibberellin biosynthesis enzyme.

The gibberellin (GA) biosynthetic pathway includes the three-step oxidation of ent-kaurene to ent-kaurenoic acid, catalyzed by the enzyme ent-kaurene oxidase (KO). Arabidopsis plants overexpressing the KO cDNA under the control of the cauliflower mosaic virus 35S promoter, with or without a translational fusion to a modified green fluorescent protein (GFP), are very similar to wild-type (WT) plants under normal growth conditions. In contrast, when WT and 35S:KO (or 35S:KO-GFP) seeds, seedlings or pollen tubes are grown in the presence of chemical inhibitors of KO, such as paclobutrazol and uniconazole, plants with increased KO expression are partially resistant to the effects of these inhibitors. In combination with the observation that decreased KO levels increase the sensitivity to KO inhibitors, the 35S:KO phenotypes demonstrate that the modification of KO enzyme levels could be used to create transgenic crop plants with altered KO inhibitor response. These results also suggest that the KO gene could be used as a selectable marker for plant regeneration based on resistance to KO inhibitors. Finally, the observation that pollen tubes expressing 35S:KO or 35S:KO-GFP have decreased sensitivity to KO inhibitors provides further evidence for a physiological role for GAs in pollen tube elongation.

Taurodontism and learning disabilities in patients with Klinefelter syndrome.

PURPOSE: The purpose of this descriptive clinical study was to determine the prevalence rates of taurodontism and learning disabilities in a sample of patients with Klinefelter syndrome. METHODS: Questionnaires and dental radiographs of Klinefelter syndrome patients were obtained and analyzed using previously published methods. Prevalence rates were determined for taurodontism and learning disabilities in the sample population and compared to the general population. Statistical analyses included a Fisher's exact 2-sided test to compare the prevalence rates to that found in the general population and subsequent determination of the positive predictive value. RESULTS: Taurodontism was found in 75% of the 24 participants. Eighty-three percent of the participants reported having a learning disability. These rates are significantly higher than the general population, as reported in the literature. CONCLUSIONS: The positive predictive value for Klinefelter syndrome, given a male patient with taurodontism and a learning disability, is 84%. In this case, the dentist should recommend karyotyping to the patient, parent, or physician. This demonstrates how important it is for dentists to understand and assist physicians in the diagnosis of genetic disorders.