Persistence of Methionine Side Chain Mobility at Low Temperatures in a Nine-Residue Low Complexity Peptide, as Probed by 2 H Solid-State NMR.

Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid-state NMR to assess rotameric inter-conversions and other dynamic modes of the methionine in the context of a nine-residue random-coil peptide (RC9) with the low-complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine-containing systems, characterized previously. Namely, the M35 of hydrated amyloid-ß1-40 in the three-fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)-methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant.

The effectiveness of primary series CoronaVac vaccine in preventing COVID-19 illness: A prospective cohort study among healthcare workers in Azerbaijan, May-November 2021.

Background: Healthcare workers (HCWs) have suffered considerable morbidity and mortality during the COVID-19 pandemic. Few studies have evaluated the CoronaVac vaccine effectiveness (VE), particularly in Eastern Europe, where the vaccine has been widely used. Methods: We conducted a prospective cohort study among HCWs in seven hospitals in Baku, Azerbaijan between May 17 and November 30, 2021, to evaluate primary series (two-dose) CoronaVac VE against symptomatic SARS-CoV-2 infection. Participants completed weekly symptom questionnaires, provided nasopharyngeal swabs for SARS-CoV-2 RT-PCR testing when symptomatic, and provided serology samples at enrollment that were tested for anti-spike and anti-nucleocapsid antibodies. We estimated VE as (1 - hazard ratio)*100 using a Cox proportional hazards model with vaccination status as a time-varying exposure, adjusting for hospital and previous SARS-CoV-2 infection status. Results: We enrolled 1582 HCWs. At enrollment, 1040 (66%) had received two doses of CoronaVac; 421 (27%) were unvaccinated. During the study period, 72 PCR-positive SARS-CoV-2 infections occurred; 36/39 (92%) sequenced samples were classified as Delta variants. Primary series VE against COVID-19 illness was 29% (95% CI: -51%; 67%) for the entire analysis period. For the Delta-only period (July 1-November 30, 2021), primary series VE was 19% (95% CI: -81%; 64%). For the entire analysis period, primary series VE was 39% (95% CI: -40%; 73%) for HCWs vaccinated within 14-149 days and 19% (95% CI: -81%; 63%) for those vaccinated ≥150 days. Conclusions: During a period in Azerbaijan characterized by mostly Delta circulation, VE point estimates suggested that primary series CoronaVac protected nearly 1 in 3 HCWs against COVID-19, but 95% confidence intervals were wide, with lower bounds that crossed zero, reflecting the limited precision of our VE estimates. Our findings underscore the need to consider booster doses for individuals who have received the primary series of CoronaVac.

Deuteron off-resonance rotating frame relaxation for the characterization of slow motions in rotating and static solid-state proteins.

We demonstrate the feasibility of deuterium solid-state NMR off-resonance rotating frame relaxation measurements for studies of slow motions in biomolecular solids. The pulse sequence, which includes adiabatic pulses for magnetization alignment, is illustrated for static and magic-angle spinning conditions away from rotary resonances. We apply the measurements for three systems with selective deuterium labels at methyl groups: a) a model compound, Fluorenylmethyloxycarbonyl methionine-D3 amino acid, for which the principles of the measurements and corresponding motional modeling based on rotameric interconversions are demonstrated; b) amyloid-ß1-40 fibrils labeled at a single alanine methyl group located in the disordered N-terminal domain. This system has been extensively studied in prior work and here serves as a test of the method for complex biological systems. The essential features of the dynamics consist of large-scale rearrangements of the disordered N-terminal domain and the conformational exchange between the free and bound forms of the domain, the latter one due to transient interactions with the structured core of the fibrils. and c) a 15-residue helical peptide which belongs to the predicted α-helical domain near the N-terminus of apolipoprotein B. The peptide is solvated with triolein and incorporates a selectively labeled leucine methyl groups. The method permits model refinement, indicating rotameric interconversions with a distribution of rate constants.

Slow methyl axes motions in perdeuterated villin headpiece subdomain probed by cross-correlated NMR relaxation measurements.

Protein methyl groups can participate in multiple motional modes on different time scales. Sub-nanosecond to nano-second time scale motions of methyl axes are particularly challenging to detect for small proteins in solutions. In this work we employ NMR relaxation interference between the methyl H-H/H-C dipole-dipole interactions [Sun&Tugarinov, J. Magn. Reason. 2012] to characterize methyl axes motions as a function of temperature in a small model protein villin headpiece subdomain (HP36), in which all non-exchangeable protons are deuterated with the exception of methyl groups of leucine and valine residues. The data points to the existence of slow motional modes of methyl axes on sub-nanosecond to nanosecond time scales. Further, at high temperatures for which the overall tumbling of the protein is on the order of 2 ns, we observe a coupling between the slow internal motion and the overall molecular tumbling, based on the anomalous order parameters and their temperature-dependent trends. The addition of 28%(w/w) glycerol-d8 increases the viscosity of the solvent and separates the timescales of internal and overall tumbling, thus permitting for another view of the necessity of the coupling assumption for these sites at high temperatures.

Efficacy of Cefazolin versus Ceftriaxone for Extremity Open Fracture Management at a Level 1 Trauma Center.

Background: Antibiotic agents have been shown to improve outcomes in open extremity fractures. The first-generation cephalosporins, which are used most often, are often under-dosed based on weight and recommended frequency. Ceftriaxone offers a broader coverage and a decreased frequency of administration. Our institution began utilizing ceftriaxone for open fracture management in 2017 to address those concerns. Objective: To examine the efficacy of cefazolin versus ceftriaxone for open fracture management of extremity trauma. Patients and Methods: Retrospective study from 2015-2019 of patients who sustained open extremity fractures. Patients were stratified by antibiotic administered and Gustilo-Anderson grade. Outcomes included non-union/malunion, superficial surgical site infection (SSI), deep SSI, osteomyelitis, re-operation after index hospital visit, re-admission due to prior injury, limb loss, and death. Subgroup analysis stratified each antibiotic group by Gustilo-Anderson grade 1 or 2 and grade 3. Results: Data was collected from 2015 to 2019. Of the 1,149 patients, 619 patients met inclusion criteria. Three hundred fifty-five patients received cefazolin and 264 patients received ceftriaxone. There were no statistically significant differences between groups on specified outcomes. No statistically significant differences existed during subgroup analysis for the specified outcomes. Multivariable analysis demonstrated increased Gustilo-Anderson grade increased risk of infectious outcome. Conclusions: Ceftriaxone is a safe and effective alternative for open fracture extremity management that offers the advantage of 24-hour dosing and single antibiotic coverage for grade 3 open fractures. It does not increase infectious complications and offers benefits of resource efficiency.

Impact of coronavirus disease 2019 (COVID-19) on trauma surgical education at a level I trauma center.

BACKGROUND: During the coronavirus disease 2019 pandemic, trauma presentations to the emergency room decreased across the country. The goal of this study is to analyze the educational impact of coronavirus disease 2019 on trauma education and training at a level I trauma center. METHODS: Trauma patient presentations were analyzed 6 months before a Tennessee executive stay-at-home order and 6 months after the state executive order. To control for the seasonal trauma volumes, an additional 6 months before the executive order was then analyzed comparing month to month. Total number of presentations, demographics, procedures, airway management, and coronavirus disease 2019 status of patients and residents were analyzed. RESULTS: The number of trauma presentations were sustained after executive orders at our level I trauma center. There was no significant difference in intubations, central line placements, and chest tube placements before and during the pandemic. Blunt trauma decreased after stay-at-home orders. Of the 36 residents, no residents tested positive during the study period. CONCLUSION: Trauma-focused surgical education was not affected at an academic level I trauma center. Understanding that it is region, city, and hospital specific, this study shows that quality trauma education can continue throughout the coronavirus disease 2019 pandemic while keeping trainees safe. Proper airway management, personal protective equipment, social distancing, and coronavirus disease 2019-preventative protocols seem to protect residents from potential harm while allowing them to participate and continue in quality trauma education and training.

Toward the Interpretation of Positive Testing for Fentanyl and Its Analogs in Real Hair Samples: Preliminary Considerations.

The detection of new psychoactive substances (NPS) in hair has become extensively researched in recent years. Although most NPS fall into the classes of synthetic cannabinoids and designer cathinones, novel synthetic opioids (NSO) have appeared with increasing frequency in the illicit drug supply. While the detection of NSO in hair is now well documented, interpretation of results presents several controversial issues, as is quite common in hair analysis. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair was applied to 293 real samples. Samples were collected in the USA between November 2016 and August 2018 from subjects who had reported heroin use in the past year or had already tested positive to hair testing for common opiates. The range, mean and median concentrations were calculated for each analyte, in order to draw a preliminary direction for a possible cut-off to discriminate between exposure to either low or high quantities of the drug. Over two-thirds (68%) of samples tested positive for fentanyl at concentrations between LOQ and 8600 pg/mg. The mean value was 382 pg/mg and the median was 95 pg/mg. The metabolites norfentanyl and 4-ANPP were also quantified and were found between LOQ and 320 pg/mg and between LOQ and 1400 pg/mg, respectively. The concentration ratios norfentanyl/fentanyl, 4-ANPP/fentanyl and norfentanyl/4-ANPP were also tested as potential markers of active use and to discriminate the intake of fentanyl from other analogs. The common occurrence of samples positive for multiple drugs may suggest that use is equally prevalent among consumers, which is not the case, as correlations based on quantitative results demonstrated. We believe this set of experimental observations provides a useful starting point for a wide discussion aimed to better understand positive hair testing for fentanyl and its analogs in hair samples.

Refeeding Syndrome in the Critically Ill: a Literature Review and Clinician's Guide.

PURPOSE OF REVIEW: To provide an overview of current methods of diagnosis and management of refeeding syndrome in the critically ill patient population. RECENT FINDINGS: Despite recent publications indicating refeeding syndrome (RFS) is an ongoing problem in critically ill patients, there is no standard for the diagnosis and management of this life-threatening condition. There is not a "gold standard" nutrition assessment tool for the critically ill. Currently, the National Institute for Health and Clinical Excellence criteria represent the best clinical assessment tool for RFS. Diagnosis and management with the help of a multidisciplinary metabolic team can decrease morbidity and mortality. Although a universal definition of RFS has yet to be defined, the diagnosis is made in patients with moderate to severe malnutrition who develop electrolyte imbalance after beginning nutritional support. The imbalances potentially can lead to cardiac, pulmonary, and gastrointestinal complications and failure. Standardizing a multidisciplinary nutrition care plan and formulating a protocol for critically ill patients who develop RFS can potentially decrease complication rates and overall mortality.

Geographic distribution of risk ("Hotspots") for HIV, HCV, and drug overdose among persons who use drugs in New York City: the importance of local history.

AIMS: To identify geographic "hotspots" for potential transmission of HIV and HCV and for drug overdose among persons who use heroin and cocaine in New York City and to examine historical continuities in problem drug use hotspots in the city. METHODS: A total of 2714 study participants were recruited among persons entering Beth Israel substance use treatment programs. A structured questionnaire was administered and blood samples for HIV and HCV testing were collected. Hotspots for potential virus transmission were defined as ZIP codes with 10+ participants, 2+ persons infected with the virus and engaging in transmission behavior, and 2+ persons not infected and engaging in acquisition behavior. ZIP codes with 3+ persons with previous overdoses were considered potential hotspots for future overdoses. RESULTS: Participants resided in 166/178 (93%) of the ZIP codes in New York City. Injecting drug use was reported in 150/178 (84%) of the ZIP codes. No zip codes were identified for injecting-related HIV transmission, 5 zip codes were identified for sexual HIV transmission, 3 for HCV transmission, and 8 for drug overdose. Many of the ZIP code potential hotspots were in neighborhoods long associated with drug use: Lower Eastside and Harlem in Manhattan, the South Bronx, and Central Brooklyn. DISCUSSION: Heroin and cocaine use requiring treatment were reported from almost all ZIP codes in New York City, indicating needs for widely dispersed harm reduction services. Identified hotspots should be targeted for reducing sexual transmission of HIV, transmission of HCV, and drug overdoses. Some of the hotspots have persisted as problem drug use areas for 40 to over 100 years. Monitoring of drug use patterns in historical hotspot neighborhoods may permit early identification of and response to emerging drug use-related health problems. Persistent historical hotspots for problem drug use present a complex problem for implementing harm reduction services that deserve additional research.

Being "hooked up" during a sharp increase in the availability of illicitly manufactured fentanyl: Adaptations of drug using practices among people who use drugs (PWUD) in New York City.

Illicitly manufactured fentanyl (IMF), a category of synthetic opioids 50-100 times more potent than morphine, is increasingly being added to heroin and other drugs in the United States (US). Persons who use drugs (PWUD) are frequently unaware of the presence of fentanyl in drugs. Use of heroin and other drugs containing fentanyl has been linked to sharp increases in opioid mortality. In New York City (NYC), opioid-related mortality increased from 8.2 per 100,000 residents in 2010 to 19.9 per 100,000 residents in 2016; and, in 2016, fentanyl accounted for 44% of NYC overdose deaths. Little is known about how PWUD are adapting to the increase in fentanyl and overdose mortality. This study explores PWUDs' adaptations to drug using practices due to fentanyl. In-depth qualitative interviews were conducted with 55 PWUD at three NYC syringe services programs (SSP) about perceptions of fentanyl, overdose experiences and adaptations of drug using practices. PWUD utilized test shots, a consistent drug dealer, fentanyl test strips, naloxone, getting high with or near others and reducing drug use to protect from overdose. Consistent application of these methods was often negated by structural level factors such as stigma, poverty and homelessness. To address these, multi-level overdose prevention approaches should be implemented in order to reduce the continuing increase in opioid mortality.

Hepatitis C virus prevalence and estimated incidence among new injectors during the opioid epidemic in New York City, 2000-2017: Protective effects of non-injecting drug use.

OBJECTIVE: Assess hepatitis C virus (HCV) prevalence and incidence among person who began injecting drugs during the opioid epidemic in New York City (NYC) and identify possible new directions for reducing HCV infection among persons who inject drugs. METHODS: 846 persons who began injecting drugs between 2000 and 2017 were recruited from persons entering Mount Sinai Beth Israel substance use treatment programs. A structured interview was administered and HCV antibody testing conducted. Protective effects of non-injecting drug use were examined among persons who "reversed transitioned" to non-injecting drug use and persons who used non-injected heroin in addition to injecting. RESULTS: Participants were 79% male, 41% White, 15% African-American, 40% Latinx, with a mean age of 35. Of those who began injecting in 2000 or later, 97 persons (11%) "reverse transitioned" back to non-injecting drug use. Reverse transitioning was strongly associated with lower HCV seroprevalence (30% versus 47% among those who continued injecting, p < 0.005). Among those who continued injecting, HCV seropositivity was inversely associated with current non-injecting heroin use (AOR = 0.72, 95%CI 0.52-0.99). HCV incidence among persons continuing to inject was estimated as 13/100 person-years. HCV seropositive persons currently injecting cocaine were particularly likely to report behavior likely to transmit HCV. CONCLUSIONS: Similar to other locations in the US, NYC is experiencing high rates of HCV infection among persons who have begun injecting since 2000. New interventions that facilitate substitution of non-injecting for injecting drug use and that reduce transmission behavior among HCV seropositives may provide additional methods for reducing HCV transmission.

Correlated motions of C'-N and Cα-Cß pairs in protonated and per-deuterated GB3.

We investigated correlated µs-ms time scale motions of neighboring 13C'-15N and 13Cα-13Cß nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100-400 s-1 range) for the 13C'-15N pairs and into the slow to intermediate regime for the 13Cα-13Cß pairs (about 150 s-1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the 13Cα-13Cß pairs.

Potential geographic "hotspots" for drug-injection related transmission of HIV and HCV and for initiation into injecting drug use in New York City, 2011-2015, with implications for the current opioid epidemic in the US.

OBJECTIVE: We identified potential geographic "hotspots" for drug-injecting transmission of HIV and hepatitis C virus (HCV) among persons who inject drugs (PWID) in New York City. The HIV epidemic among PWID is currently in an "end of the epidemic" stage, while HCV is in a continuing, high prevalence (> 50%) stage. METHODS: We recruited 910 PWID entering Mount Sinai Beth Israel substance use treatment programs from 2011-2015. Structured interviews and HIV/ HCV testing were conducted. Residential ZIP codes were used as geographic units of analysis. Potential "hotspots" for HIV and HCV transmission were defined as 1) having relatively large numbers of PWID 2) having 2 or more HIV (or HCV) seropositive PWID reporting transmission risk-passing on used syringes to others, and 3) having 2 or more HIV (or HCV) seronegative PWID reporting acquisition risk-injecting with previously used needles/syringes. Hotspots for injecting drug use initiation were defined as ZIP codes with 5 or more persons who began injecting within the previous 6 years. RESULTS: Among PWID, 96% injected heroin, 81% male, 34% White, 15% African-American, 47% Latinx, mean age 40 (SD = 10), 7% HIV seropositive, 62% HCV seropositive. Participants resided in 234 ZIP codes. No ZIP codes were identified as potential hotspots due to small numbers of HIV seropositive PWID reporting transmission risk. Four ZIP codes were identified as potential hotspots for HCV transmission. 12 ZIP codes identified as hotspots for injecting drug use initiation. DISCUSSION: For HIV, the lack of potential hotspots is further validation of widespread effectiveness of efforts to reduce injecting-related HIV transmission. Injecting-related HIV transmission is likely to be a rare, random event. HCV prevention efforts should include focus on potential hotspots for transmission and on hotspots for initiation into injecting drug use. We consider application of methods for the current opioid epidemic in the US.

Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin.

Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences. It is unknown to what extent the plant villin headpiece structure and function resemble those of the well-studied vertebrate counterparts. Here we present the first solution NMR structure and backbone dynamics characterization of a headpiece from plants, villin isoform 4 from Arabidopsis thaliana. The villin 4 headpiece is a 63-residue domain (V4HP63) that adopts a typical headpiece fold with an aromatics core and a tryptophan-centered hydrophobic cap within its C-terminal subdomain. However, V4HP63 has a distinct N-terminal subdomain fold as well as a novel, high mobility loop due to the insertion of serine residue in the canonical sequence that follows the variable length loop in headpiece sequences. The domain binds actin filaments with micromolar affinity, like the vertebrate analogues. However, the V4HP63 surface charge pattern is novel and lacks certain features previously thought necessary for high-affinity F-actin binding. Utilizing the updated criteria for strong F-actin binding, we predict that the headpiece domains of all other villin isoforms in A. thaliana have high affinity for F-actin.

Management and closure of multiple large cutaneous lesions in a juvenile cat with severe acquired skin fragility syndrome secondary to iatrogenic hyperadrenocorticism.

CASE DESCRIPTION A 13-month-old castrated male cat was evaluated for a large, spontaneously developed cutaneous laceration over the left scapular region. The cat had a history of severe gingivostomatitis, conjunctivitis, giardiasis, and feline herpesvirus infection and had received systemic glucocorticoid treatment for 7 weeks prior to evaluation. CLINICAL FINDINGS Physical examination revealed a 10 × 7-cm full-thickness cutaneous laceration over the left scapular region, extremely thin skin, crusts over the dorsal aspect of the neck and base of the skull, medially curling pinnae, and moderate gingivostomatitis. TREATMENT AND OUTCOME Staged wound closure was performed with a combination of daily wound cleaning and debridement, tension and appositional sutures, and wet-to-dry and nonadherent dressings initially with a bacitracin, neomycin, and polymyxin B ointment and then with a 30:1 mixture of silver sulfadiazine and insulin. Multiple additional lesions developed and were treated in the same manner. Complete closure and resolution of all cutaneous lesions was achieved in 9 weeks. CLINICAL RELEVANCE Cats are fairly resistant to the adverse effects of glucocorticoid treatment, and iatrogenic hyperadrenocorticism is rarely reported. This case demonstrated that acquired skin fragility syndrome secondary to iatrogenic hyperadrenocorticism can develop following short-term systemic glucocorticoid administration and that large cutaneous wounds associated with this condition can be successfully managed and closed by means of the reported methods. The prognosis for skin recovery in cats with acquired skin fragility syndrome may be more favorable than previously reported.

Optimized purification of a fusion protein by reversed-phase high performance liquid chromatography informed by the linear solvent strength model.

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography.

The unusual internal motion of the villin headpiece subdomain.

The thermostable 36-residue subdomain of the villin headpiece (HP36) is the smallest known cooperatively folding protein. Although the folding and internal dynamics of HP36 and close variants have been extensively studied, there has not been a comprehensive investigation of side-chain motion in this protein. Here, the fast motion of methyl-bearing amino acid side chains is explored over a range of temperatures using site-resolved solution nuclear magnetic resonance deuterium relaxation. The squared generalized order parameters of methyl groups extensively spatially segregate according to motional classes. This has not been observed before in any protein studied using this methodology. The class segregation is preserved from 275 to 305 K. Motions detected in Helix 3 suggest a fast timescale of conformational heterogeneity that has not been previously observed but is consistent with a range of folding and dynamics studies. Finally, a comparison between the order parameters in solution with previous results based on solid-state nuclear magnetic resonance deuterium line shape analysis of HP36 in partially hydrated powders shows a clear disagreement for half of the sites. This result has significant implications for the interpretation of data derived from a variety of approaches that rely on partially hydrated protein samples.

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target.

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.

The Physiological Molecular Shape of Spectrin: A Compact Supercoil Resembling a Chinese Finger Trap.

The primary, secondary, and tertiary structures of spectrin are reasonably well defined, but the structural basis for the known dramatic molecular shape change, whereby the molecular length can increase three-fold, is not understood. In this study, we combine previously reported biochemical and high-resolution crystallographic data with structural mass spectroscopy and electron microscopic data to derive a detailed, experimentally-supported quaternary structure of the spectrin heterotetramer. In addition to explaining spectrin's physiological resting length of ~55-65 nm, our model provides a mechanism by which spectrin is able to undergo a seamless three-fold extension while remaining a linear filament, an experimentally observed property. According to the proposed model, spectrin's quaternary structure and mechanism of extension is similar to a Chinese Finger Trap: at shorter molecular lengths spectrin is a hollow cylinder that extends by increasing the pitch of each spectrin repeat, which decreases the internal diameter. We validated our model with electron microscopy, which demonstrated that, as predicted, spectrin is hollow at its biological resting length of ~55-65 nm. The model is further supported by zero-length chemical crosslink data indicative of an approximately 90 degree bend between adjacent spectrin repeats. The domain-domain interactions in our model are entirely consistent with those present in the prototypical linear antiparallel heterotetramer as well as recently reported inter-strand chemical crosslinks. The model is consistent with all known physical properties of spectrin, and upon full extension our Chinese Finger Trap Model reduces to the ~180-200 nm molecular model currently in common use.